CD70 is downregulated by interaction with CD27

PubMed Central

Kuka, Mirela; Munitic, Ivana; Torchia, Maria Letizia Giardino; Ashwell, Jonathan D.

2013-01-01

Engagement of the receptor CD27 by CD70 affects the magnitude and quality of T cell responses in a variety of infection models, and exaggerated signaling via this pathway results in enhanced immune responses and autoimmunity. One means by which signaling is regulated is tight control of cell surface CD70, which is expressed on dendritic, T, and B cells only upon activation. Here we show that there is a second level of regulation. First, although undetectable on the cell surface by flow cytometry, immature dendritic cells (DC) have a small pool of CD70 that continuously recycles from the plasma membrane. In addition, surface levels of CD70 on DC and T cells were higher in mice deficient in CD27, or on DC for which the interaction between CD70 and CD27 was precluded by blocking antibodies. Binding of CD70 by its receptor resulted in downregulation of CD70 transcription and protein levels, suggesting that CD70-mediated “reverse signals” regulate its own levels. Therefore, the ability of CD70 to trigger costimulation is self-regulated when it binds its complementary receptor. PMID:23913967

Involvement of up-regulated Necl-5/Tage4/PVR/CD155 in the loss of contact inhibition in transformed NIH3T3 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Minami, Yukiko; Department of Surgery and Clinical Oncology, Osaka University Graduate School of Medicine/Faculty of Medicine, Suita 565-0871, Osaka; Ikeda, Wataru

2007-01-26

Normal cells show contact inhibition of cell movement and proliferation, but this is lost following transformation. We found that Necl-5, originally identified as a poliovirus receptor and up-regulated in many cancer cells, enhances growth factor-induced cell movement and proliferation. We showed that when cells contact other cells, Necl-5 interacts in trans with nectin-3 and is removed by endocytosis from the cell surface, resulting in a reduction of cell movement and proliferation. We show here that up-regulation of the gene encoding Necl-5 by the oncogene V12-Ki-Ras causes enhanced cell movement and proliferation. Upon cell-cell contact, de novo synthesis of Necl-5 exceedsmore » the rate of Necl-5 endocytosis, eventually resulting in a net increase in the amount of Necl-5 at the cell surface. In addition, expression of the gene encoding nectin-3 is markedly reduced in transformed cells. Thus, up-regulation of Necl-5 following transformation contributes to the loss of contact inhibition in transformed cells.« less

Rapid down-regulation of γc on T cells in early SIV infection correlates with impairment of T-cell function

PubMed Central

Xu, Huanbin; Wang, Xiaolei; Pahar, Bapi; Alvarez, Xavier; Rasmussen, Kelsi K.; Lackner, Andrew A.; Veazey, Ronald S.

2012-01-01

The common γc subunit molecule is shared among all γc cytokines and clearly involved in T-cell function, but its role in HIV infection and immunity is not well understood. Here, we examined expression and function of γc on T cells during SIV infection in Rhesus macaques. Surface γc distribution was differentially expressed on CD4+ and CD8+ T cells, and CD4+ naive/memory cell populations in various lymphoid tissues of normal macaques. However, surface γc expression was rapidly and significantly down-regulated on T cells in acute infection with pathogenic SIV, compared to infection with a less virulent SHIV or controls and did not recover on CD8+ T cells in the chronic stage. Moreover, the peripheral and CD4+T cell loss was inversely correlated with γc+ CD8+ T cells in individual tissues. γc+ T cells were mainly functional as evidenced by higher cytokine secretion and proliferative capacity. Further in vitro experiments found that surface γc expression could be down-regulated following high level of IL-7 treatment by both internalization and shedding. Down-regulation of γc during early HIV/SIV infection may inhibit T-cell function, particularly of CD8+ T cells, and, may be linked with immune failure and loss of viral containment.—Xu, H., Wang, X., Pahar, B., Alvarez, X., Rasmussen, K. K., Lackner, A. A., Veazey, R. S. Rapid down-regulation of γc on T cells in early SIV infection correlates with impairment of T-cell function. PMID:22375017

Rapid down-regulation of γc on T cells in early SIV infection correlates with impairment of T-cell function.

PubMed

Xu, Huanbin; Wang, Xiaolei; Pahar, Bapi; Alvarez, Xavier; Rasmussen, Kelsi K; Lackner, Andrew A; Veazey, Ronald S

2012-06-01

The common γ(c) subunit molecule is shared among all γ(c) cytokines and clearly involved in T-cell function, but its role in HIV infection and immunity is not well understood. Here, we examined expression and function of γ(c) on T cells during SIV infection in Rhesus macaques. Surface γ(c) distribution was differentially expressed on CD4(+) and CD8(+) T cells, and CD4(+) naive/memory cell populations in various lymphoid tissues of normal macaques. However, surface γ(c) expression was rapidly and significantly down-regulated on T cells in acute infection with pathogenic SIV, compared to infection with a less virulent SHIV or controls and did not recover on CD8(+) T cells in the chronic stage. Moreover, the peripheral and CD4(+)T cell loss was inversely correlated with γ(c)(+) CD8(+) T cells in individual tissues. γ(c)(+) T cells were mainly functional as evidenced by higher cytokine secretion and proliferative capacity. Further in vitro experiments found that surface γ(c) expression could be down-regulated following high level of IL-7 treatment by both internalization and shedding. Down-regulation of γ(c) during early HIV/SIV infection may inhibit T-cell function, particularly of CD8(+) T cells, and, may be linked with immune failure and loss of viral containment.

Tumor-derived exosomes regulate expression of immune function-related genes in human T cell subsets.

PubMed

Muller, Laurent; Mitsuhashi, Masato; Simms, Patricia; Gooding, William E; Whiteside, Theresa L

2016-02-04

Tumor cell-derived exosomes (TEX) suppress functions of immune cells. Here, changes in the gene profiles of primary human T lymphocytes exposed in vitro to exosomes were evaluated. CD4(+) Tconv, CD8(+) T or CD4(+) CD39(+) Treg were isolated from normal donors' peripheral blood and co-incubated with TEX or exosomes isolated from supernatants of cultured dendritic cells (DEX). Expression levels of 24-27 immune response-related genes in these T cells were quantified by qRT-PCR. In activated T cells, TEX and DEX up-regulated mRNA expression levels of multiple genes. Multifactorial data analysis of ΔCt values identified T cell activation and the immune cell type, but not exosome source, as factors regulating gene expression by exosomes. Treg were more sensitive to TEX-mediated effects than other T cell subsets. In Treg, TEX-mediated down-regulation of genes regulating the adenosine pathway translated into high expression of CD39 and increased adenosine production. TEX also induced up-regulation of inhibitory genes in CD4(+) Tconv, which translated into a loss of CD69 on their surface and a functional decline. Exosomes are not internalized by T cells, but signals they carry and deliver to cell surface receptors modulate gene expression and functions of human T lymphocytes.

Time-Resolved Analysis of Cytosolic and Surface-Associated Proteins of Staphylococcus aureus HG001 under Planktonic and Biofilm Conditions.

PubMed

Moche, Martin; Schlüter, Rabea; Bernhardt, Jörg; Plate, Kristina; Riedel, Katharina; Hecker, Michael; Becher, Dörte

2015-09-04

Staphylococcal biofilms are associated with persistent infections due to their capacity to protect bacteria against the host's immune system and antibiotics. Cell-surface-associated proteins are of great importance during biofilm formation. In the present study, an optimized biotinylation approach for quantitative GeLC-MS-based analysis of the staphylococcal cell-surface proteome was applied and the cytoplasmic protein fraction was analyzed to elucidate proteomic differences between colony biofilms and planktonic cells. The experimental setup enabled a time-resolved monitoring of the proteome under both culture conditions and the comparison of biofilm cells to planktonic cells at several time points. This allowed discrimination of differences attributed to delayed growth phases from responses provoked by biofilm conditions. Biofilm cells expressed CcpA-dependent catabolic proteins earlier than planktonic cells and strongly accumulated proteins that belong to the SigB stress regulon. The amount of the cell-surface protein and virulence gene regulator Rot decreased within biofilms and MgrA-dependent regulations appeared more pronounced. Biofilm cells simultaneously up-regulated activators (e.g., SarZ) as well as repressors (e.g., SarX) of RNAIII. A decreased amount of high-affinity iron uptake systems and an increased amount of the iron-storage protein FtnA possibly indicated a lower demand of iron in biofilms.

Sustained neurotensin exposure promotes cell surface recruitment of NTS2 receptors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Perron, Amelie; Sharif, Nadder; Gendron, Louis

2006-05-12

In this study, we investigated whether persistent agonist stimulation of NTS2 receptors gives rise to down-regulation, in light of reports that their activation induced long-lasting effects. To address this issue, we incubated COS-7 cells expressing the rat NTS2 with neurotensin (NT) for up to 24 h and measured resultant cell surface [{sup 125}I]-NT binding. We found that NTS2-expressing cells retained the same surface receptor density despite efficient internalization mechanisms. This preservation was neither due to NTS2 neosynthesis nor recycling since it was not blocked by cycloheximide or monensin. However, it appeared to involve translocation of spare receptors from internal stores,more » as NT induced NTS2 migration from trans-Golgi network to endosome-like structures. This stimulation-induced regulation of cell surface NTS2 receptors was even more striking in rat spinal cord neurons. Taken together, these results suggest that sustained NTS2 activation promotes recruitment of intracellular receptors to the cell surface, thereby preventing functional desensitization.« less

A conserved WW domain-like motif regulates invariant chain-dependent cell-surface transport of the NKG2D ligand ULBP2.

PubMed

Uhlenbrock, Franziska; van Andel, Esther; Andresen, Lars; Skov, Søren

2015-08-01

Malignant cells expressing NKG2D ligands on their cell surface can be directly sensed and killed by NKG2D-bearing lymphocytes. To ensure this immune recognition, accumulating evidence suggests that NKG2D ligands are trafficed via alternative pathways to the cell surface. We have previously shown that the NKG2D ligand ULBP2 traffics over an invariant chain (Ii)-dependent pathway to the cell surface. This study set out to elucidate how Ii regulates ULBP2 cell-surface transport: We discovered conserved tryptophan (Trp) residues in the primary protein sequence of ULBP1-6 but not in the related MICA/B. Substitution of Trp to alanine resulted in cell-surface inhibition of ULBP2 in different cancer cell lines. Moreover, the mutated ULBP2 constructs were retained and not degraded inside the cell, indicating a crucial role of this conserved Trp-motif in trafficking. Finally, overexpression of Ii increased surface expression of wt ULBP2 while Trp-mutants could not be expressed, proposing that this Trp-motif is required for an Ii-dependent cell-surface transport of ULBP2. Aberrant soluble ULBP2 is immunosuppressive. Thus, targeting a distinct protein module on the ULBP2 sequence could counteract this abnormal expression of ULBP2. Copyright © 2015 Elsevier Ltd. All rights reserved.

Functional roles of cell surface peptidases in reproductive organs

PubMed Central

2004-01-01

A number of biologically active peptides have been proposed to regulate function and differentiation of reproductive organs in an autocrine and/or paracrine fashion. Regulation of the local concentrations of these peptides is one of the important factors influencing their physiological effects on target cells. Membrane‐bound cell surface peptidases can activate or inactivate biologically active peptides before peptide factors access their receptors on the cell surface. Aminopeptidase A (EC 3.4.11.7), placental leucine aminopeptidase (EC 3.4.11.3), aminopeptidase‐N/CD13 (EC 3.4.11.2), dipeptidyl peptidases IV/CD26 (EC.3.4.14.5), carboxypeptidase‐M (EC 3.4.17.12), neutral endopeptidase/CD10 (EC 3.4.24.11) and endothelin converting enzyme‐1 (EC 3.4.23) are differentially expressed on the ovary, endometrium and placenta. The inhibition of enzyme activity affects steroid hormone production by granulosa and thecal cells, decidualization of endometrium and migration of extravillous trophoblasts. These findings suggest that membrane‐bound cell surface peptidases are local regulators for cellular growth and differentiation in reproductive organs by controlling extracellular concentration of peptide factors. (Reprod Med Biol 2004; 3: 165 –176) PMID:29662383

Cell Surface Galectin-9 Expressing Th Cells Regulate Th17 and Foxp3+ Treg Development by Galectin-9 Secretion

PubMed Central

Niki, Toshiro; Kadowaki, Takeshi; Ueno, Masaki; Nishi, Nozomu; Yamauchi, Akira; Hattori, Toshio; Masaki, Tsutomu; Hirashima, Mitsuomi

2012-01-01

Galectin-9 (Gal-9), a β-galactoside binding mammalian lectin, regulates immune responses by reducing pro-inflammatory IL-17-producing Th cells (Th17) and increasing anti-inflammatory Foxp3+ regulatory T cells (Treg) in vitro and in vivo. These functions of Gal-9 are thought to be exerted by binding to receptor molecules on the cell surface. However, Gal-9 lacks a signal peptide for secretion and is predominantly located in the cytoplasm, which raises questions regarding how and which cells secrete Gal-9 in vivo. Since Gal-9 expression does not necessarily correlate with its secretion, Gal-9-secreting cells in vivo have been elusive. We report here that CD4 T cells expressing Gal-9 on the cell surface (Gal-9+ Th cells) secrete Gal-9 upon T cell receptor (TCR) stimulation, but other CD4 T cells do not, although they express an equivalent amount of intracellular Gal-9. Gal-9+ Th cells expressed interleukin (IL)-10 and transforming growth factor (TGF)-β but did not express Foxp3. In a co-culture experiment, Gal-9+ Th cells regulated Th17/Treg development in a manner similar to that by exogenous Gal-9, during which the regulation by Gal-9+ Th cells was shown to be sensitive to a Gal-9 antagonist but insensitive to IL-10 and TGF-β blockades. Further elucidation of Gal-9+ Th cells in humans indicates a conserved role of these cells through evolution and implies the possible utility of these cells for diagnosis or treatment of immunological diseases. PMID:23144904

CD40 expression in Wehi-164 cell line

PubMed Central

Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-01-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body’s defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system. PMID:20496113

CD40 expression in Wehi-164 cell line.

PubMed

Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Soheili, Zahra Soheila; Moazzeni, Seyed Mohammad

2010-07-01

CD40-CD154 interaction is an important process for cellular and humoral immunity regulation and can be effective in the body's defense against tumors. In the present study, we evaluated the expression of CD40 in Wehi-164 cell line. CD40 expressions on the cell surface and in the cytoplasm were assessed by flow cytometry and intracellular staining assay, respectively. Also, the mRNA expression was identified by real time-PCR. The obtained results showed the high mRNA and cytoplasmic protein expression of CD40 but no surface expression. These results suggest that the Wehi-164 cell line down regulates expression of CD40 on the surface for evasion of immune system.

Controlled surface topography regulates collective 3D migration by epithelial-mesenchymal composite embryonic tissues.

PubMed

Song, Jiho; Shawky, Joseph H; Kim, YongTae; Hazar, Melis; LeDuc, Philip R; Sitti, Metin; Davidson, Lance A

2015-07-01

Cells in tissues encounter a range of physical cues as they migrate. Probing single cell and collective migratory responses to physically defined three-dimensional (3D) microenvironments and the factors that modulate those responses are critical to understanding how tissue migration is regulated during development, regeneration, and cancer. One key physical factor that regulates cell migration is topography. Most studies on surface topography and cell mechanics have been carried out with single migratory cells, yet little is known about the spreading and motility response of 3D complex multi-cellular tissues to topographical cues. Here, we examine the response to complex topographical cues of microsurgically isolated tissue explants composed of epithelial and mesenchymal cell layers from naturally 3D organized embryos of the aquatic frog Xenopus laevis. We control topography using fabricated micropost arrays (MPAs) and investigate the collective 3D migration of these multi-cellular systems in these MPAs. We find that the topography regulates both collective and individual cell migration and that dense MPAs reduce but do not eliminate tissue spreading. By modulating cell size through the cell cycle inhibitor Mitomycin C or the spacing of the MPAs we uncover how 3D topographical cues disrupt collective cell migration. We find surface topography can direct both single cell motility and tissue spreading, altering tissue-scale processes that enable efficient conversion of single cell motility into collective movement. Copyright © 2015 Elsevier Ltd. All rights reserved.

GARP-TGF-β complexes negatively regulate regulatory T cell development and maintenance of peripheral CD4+ T cells in vivo.

PubMed

Zhou, Angela X; Kozhaya, Lina; Fujii, Hodaka; Unutmaz, Derya

2013-05-15

The role of surface-bound TGF-β on regulatory T cells (Tregs) and the mechanisms that mediate its functions are not well defined. We recently identified a cell-surface molecule called Glycoprotein A Repetitions Predominant (GARP), which is expressed specifically on activated Tregs and was found to bind latent TGF-β and mediate a portion of Treg suppressive activity in vitro. In this article, we address the role of GARP in regulating Treg and conventional T cell development and immune suppression in vivo using a transgenic mouse expressing GARP on all T cells. We found that, despite forced expression of GARP on all T cells, stimulation through the TCR was required for efficient localization of GARP to the cell surface. In addition, IL-2 signals enhanced GARP cell surface expression specifically on Tregs. GARP-transgenic CD4(+) T cells and Tregs, especially those expressing higher levels of GARP, were significantly reduced in the periphery. Mature Tregs, but not conventional CD4(+) T cells, were also reduced in the thymus. CD4(+) T cell reduction was more pronounced within the effector/memory subset, especially as the mouse aged. In addition, GARP-overexpressing CD4(+) T cells stimulated through the TCR displayed reduced proliferative capacity, which was restored by inhibiting TGF-β signaling. Furthermore, inhibiting TGF-β signals greatly enhanced surface expression of GARP on Tregs and blocked the induction of Foxp3 in activated CD4(+) T cells overexpressing GARP. These findings suggest a role for GARP in natural and induced Treg development through activation of bound latent TGF-β and signaling, which negatively regulates GARP expression on Tregs.

Chemical and Enzymatic Strategies for Bacterial and Mammalian Cell Surface Engineering.

PubMed

Bi, Xiaobao; Yin, Juan; Chen Guanbang, Ashley; Liu, Chuan-Fa

2018-06-07

The cell surface serves important functions such as the regulation of cell-cell and cell-environment interactions. The understanding and manipulation of the cell surface is important for a wide range of fundamental studies of cellular behavior and for biotechnological and medical applications. With the rapid advance of biology, chemistry and materials science, many strategies have been developed for the functionalization of bacterial and mammalian cell surfaces. Here, we review the recent development of chemical and enzymatic approaches to cell surface engineering with particular emphasis on discussing the advantages and limitations of each of these strategies. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

ER-mitochondria contacts control surface glycan expression and sensitivity to killer lymphocytes in glioma stem-like cells.

PubMed

Bassoy, Esen Yonca; Kasahara, Atsuko; Chiusolo, Valentina; Jacquemin, Guillaume; Boydell, Emma; Zamorano, Sebastian; Riccadonna, Cristina; Pellegatta, Serena; Hulo, Nicolas; Dutoit, Valérie; Derouazi, Madiha; Dietrich, Pierre Yves; Walker, Paul R; Martinvalet, Denis

2017-06-01

Glioblastoma is a highly heterogeneous aggressive primary brain tumor, with the glioma stem-like cells (GSC) being more sensitive to cytotoxic lymphocyte-mediated killing than glioma differentiated cells (GDC). However, the mechanism behind this higher sensitivity is unclear. Here, we found that the mitochondrial morphology of GSCs modulates the ER-mitochondria contacts that regulate the surface expression of sialylated glycans and their recognition by cytotoxic T lymphocytes and natural killer cells. GSCs displayed diminished ER-mitochondria contacts compared to GDCs. Forced ER-mitochondria contacts in GSCs increased their cell surface expression of sialylated glycans and reduced their susceptibility to cytotoxic lymphocytes. Therefore, mitochondrial morphology and dynamism dictate the ER-mitochondria contacts in order to regulate the surface expression of certain glycans and thus play a role in GSC recognition and elimination by immune effector cells. Targeting the mitochondrial morphology, dynamism, and contacts with the ER could be an innovative strategy to deplete the cancer stem cell compartment to successfully treat glioblastoma. © 2017 The Authors.

Cell surface dynamics - how Rho GTPases orchestrate the interplay between the plasma membrane and the cortical cytoskeleton.

PubMed

de Curtis, Ivan; Meldolesi, Jacopo

2012-10-01

Small GTPases are known to regulate hundreds of cell functions. In particular, Rho family GTPases are master regulators of the cytoskeleton. By regulating actin nucleation complexes, Rho GTPases control changes in cell shape, including the extension and/or retraction of surface protrusions and invaginations. Protrusion and invagination of the plasma membrane also involves the interaction between the plasma membrane and the cortical cytoskeleton. This interplay between membranes and the cytoskeleton can lead to an increase or decrease in the plasma membrane surface area and its tension as a result of the fusion (exocytosis) or internalization (endocytosis) of membranous compartments, respectively. For a long time, the cytoskeleton and plasma membrane dynamics were investigated separately. However, studies from many laboratories have now revealed that Rho GTPases, their modulation of the cytoskeleton, and membrane traffic are closely connected during the dynamic remodeling of the cell surface. Arf- and Rab-dependent exocytosis of specific vesicles contributes to the targeting of Rho GTPases and their regulatory factors to discrete sites of the plasma membrane. Rho GTPases regulate the tethering of exocytic vesicles and modulate their subsequent fusion. They also have crucial roles in the different forms of endocytosis, where they participate in the sorting of membrane domains as well as the sculpting and sealing of membrane flasks and cups. Here, we discuss how cell surface dynamics depend on the orchestration of the cytoskeleton and the plasma membrane by Rho GTPases.

Cell Surface Trafficking of TLR1 Is Differentially Regulated by the Chaperones PRAT4A and PRAT4B*

PubMed Central

Hart, Bryan E.; Tapping, Richard I.

2012-01-01

The subcellular localization of Toll-like receptors (TLRs) is critical to their ability to function as innate immune sensors of microbial infection. We previously reported that an I602S polymorphism of human TLR1 is associated with aberrant trafficking of the receptor to the cell surface, loss of responses to TLR1 agonists, and differential susceptibility to diseases caused by pathogenic mycobacteria. Through an extensive analysis of receptor deletion and point mutants we have discovered that position 602 resides within a short 6 amino acid cytoplasmic region that is required for TLR1 surface expression. This short trafficking motif, in conjunction with the adjacent transmembrane domain, is sufficient to direct TLR1 to the cell surface. A serine at position 602 interrupts this trafficking motif and prevents cell surface expression of TLR1. Additionally, we have found that ER-resident TLR chaperones, PRAT4A and PRAT4B, act as positive and negative regulators of TLR1 surface trafficking, respectively. Importantly, either over-expression of PRAT4A or knock-down of PRAT4B rescues cell surface expression of the TLR1 602S variant. We also report that IFN-γ treatment of primary human monocytes derived from homozygous 602S individuals rescues TLR1 cell surface trafficking and cellular responses to soluble agonists. This event appears to be mediated by PRAT4A whose expression is strongly induced in human monocytes by IFN-γ. Collectively, these results provide a mechanism for the differential trafficking of TLR1 I602S variants, and highlight the distinct roles for PRAT4A and PRAT4B in the regulation of TLR1 surface expression. PMID:22447933

Leukocyte Cell Surface Proteinases: Regulation of Expression, Functions, and Mechanisms of Surface Localization

PubMed Central

Owen, Caroline A.

2008-01-01

A number of proteinases are expressed on the surface of leukocytes including members of the serine, metallo-, and cysteine proteinase superfamilies. Some proteinases are anchored to the plasma membrane of leukocytes by a transmembrane domain or a glycosyl phosphatidyl inositol (GPI) anchor. Other proteinases bind with high affinity to classical receptors, or with lower affinity to integrins, proteoglycans, or other leukocyte surface molecules. Leukocyte surface levels of proteinases are regulated by: 1) cytokines, chemokines, bacterial products, and growth factors which stimulate synthesis and/or release of proteinase by cells; 2) the availability of surface binding sites for proteinases; and/or 3) internalization or shedding of surface-bound proteinases. The binding of proteinases to leukocyte surfaces serves many functions including: 1) concentrating the activity of proteinases to the immediate pericellular environment; 2) facilitating pro-enzyme activation; 3) increasing proteinase stability and retention in the extracellular space; 4) regulating leukocyte function by proteinases signaling through cell surface binding sites or other surface proteins; and 5) protecting proteinases from inhibition by extracellular proteinase inhibitors. There is strong evidence that membrane-associated proteinases on leukocytes play critical roles in wound healing, inflammation, extracellular matrix remodeling, fibrinolysis, and coagulation. This review will outline the biology of membrane-associated proteinases expressed by leukocytes and their roles in physiologic and pathologic processes. PMID:18329945

Ubiquitination by the Membrane-associated RING-CH-8 (MARCH-8) Ligase Controls Steady-state Cell Surface Expression of Tumor Necrosis Factor-related Apoptosis Inducing Ligand (TRAIL) Receptor 1*

PubMed Central

van de Kooij, Bert; Verbrugge, Inge; de Vries, Evert; Gijsen, Merel; Montserrat, Veronica; Maas, Chiel; Neefjes, Jacques; Borst, Jannie

2013-01-01

The eleven members of the membrane-associated RING-CH (MARCH) ubiquitin ligase family are relatively unexplored. Upon exogenous (over)expression, a number of these ligases can affect the trafficking of membrane molecules. However, only for MARCH-1 endogenous functions have been demonstrated. For the other endogenous MARCH proteins, no functions or substrates are known. We report here that TRAIL-R1 is a physiological substrate of the endogenous MARCH-8 ligase. Human TRAIL-R1 and R2 play a role in immunosurveillance and are targets for cancer therapy, because they selectively induce apoptosis in tumor cells. We demonstrate that TRAIL-R1 is down-regulated from the cell surface, with great preference over TRAIL-R2, by exogenous expression of MARCH ligases that are implicated in endosomal trafficking, such as MARCH-1 and -8. MARCH-8 attenuated TRAIL-R1 cell surface expression and apoptosis signaling by virtue of its ligase activity. This suggested that ubiquitination of TRAIL-R1 was instrumental in its down-regulation by MARCH-8. Indeed, in cells with endogenous MARCH expression, TRAIL-R1 was ubiquitinated at steady-state, with the conserved membrane-proximal lysine 273 as one of the potential acceptor sites. This residue was also essential for the interaction of TRAIL-R1 with MARCH-1 and MARCH-8 and its down-regulation by these ligases. Gene silencing identified MARCH-8 as the endogenous ligase that ubiquitinates TRAIL-R1 and attenuates its cell surface expression. These findings reveal that endogenous MARCH-8 regulates the steady-state cell surface expression of TRAIL-R1. PMID:23300075

Burn-injury affects gut-associated lymphoid tissues derived CD4+ T cells.

PubMed

Fazal, Nadeem; Shelip, Alla; Alzahrani, Alhusain J

2013-01-01

After scald burn-injury, the intestinal immune system responds to maintain immune balance. In this regard CD4+T cells in Gut-Associated Lymphoid Tissues (GALT), like mesenteric lymph nodes (MLN) and Peyer's patches (PP) respond to avoid immune suppression following major injury such as burn. Therefore, we hypothesized that the gut CD4+T cells become dysfunctional and turn the immune homeostasis towards depression of CD4+ T cell-mediated adaptive immune responses. In the current study we show down regulation of mucosal CD4+ T cell proliferation, IL-2 production and cell surface marker expression of mucosal CD4+ T cells moving towards suppressive-type. Acute burn-injury lead to up-regulation of regulatory marker (CD25+), down regulation of adhesion (CD62L, CD11a) and homing receptor (CD49d) expression, and up-regulation of negative co-stimulatory (CTLA-4) molecule. Moreover, CD4+CD25+ T cells of intestinal origin showed resistance to spontaneous as well as induced apoptosis that may contribute to suppression of effector CD4+ T cells. Furthermore, gut CD4+CD25+ T cells obtained from burn-injured animals were able to down-regulate naïve CD4+ T cell proliferation following adoptive transfer of burn-injured CD4+CD25+ T cells into sham control animals, without any significant effect on cell surface activation markers. Together, these data demonstrate that the intestinal CD4+ T cells evolve a strategy to promote suppressive CD4+ T cell effector responses, as evidenced by enhanced CD4+CD25+ T cells, up-regulated CTLA-4 expression, reduced IL-2 production, tendency towards diminished apoptosis of suppressive CD4+ T cells, and thus lose their natural ability to regulate immune homeostasis following acute burn-injury and prevent immune paralysis.

Fluorogenic Green-Inside Red-Outside (GIRO) Labeling Approach Reveals Adenylyl Cyclase-Dependent Control of BKα Surface Expression

PubMed Central

2015-01-01

The regulation of surface levels of protein is critical for proper cell function and influences properties including cell adhesion, ion channel contributions to current flux, and the sensitivity of surface receptors to ligands. Here we demonstrate a two-color labeling system in live cells using a single fluorogen activating peptide (FAP) based fusion tag, which enables the rapid and simultaneous quantification of surface and internal proteins. In the nervous system, BK channels can regulate neural excitability and neurotransmitter release, and the surface trafficking of BK channels can be modulated by signaling cascades and assembly with accessory proteins. Using this labeling approach, we examine the dynamics of BK channel surface expression in HEK293 cells. Surface pools of the pore-forming BKα subunit were stable, exhibiting a plasma membrane half-life of >10 h. Long-term activation of adenylyl cyclase by forskolin reduced BKα surface levels by 30%, an effect that could not be attributed to increased bulk endocytosis of plasma membrane proteins. This labeling approach is compatible with microscopic imaging and flow cytometry, providing a solid platform for examining protein trafficking in living cells. PMID:26301573

Cell surface localization of the 78 kD glucose regulated protein (GRP 78) induced by thapsigargin.

PubMed

Delpino, A; Piselli, P; Vismara, D; Vendetti, S; Colizzi, V

1998-01-01

In the present study it was found that the synthesis of the 78 kD glucose-regulated protein (GRP 78 or BIP) is vigorously induced in human rabdomiosarcoma cells (TE 671/RD) following both short-term (1 h) and prolonged (18 h) exposure to 100 nM thapsigargin (Tg). Flow cytometric analysis with a specific anti-GRP 78 polyclonal antibody showed that Tg-treated cells express the GRP 78 on the plasma membrane. Cell surface localization of the Tg-induced GRP 78 was confirmed by biotinylation of membrane-exposed proteins and subsequent isolation of the biotin-labelled proteins by streptavidin/agarose affinity chromatography. It was found that a fraction of the Tg-induced GRP 78 is present among the biotin-labelled, surface-exposed, proteins. Conversely, the GRP 78 immunoprecipitated from unfractionated lysates of Tg-treated and biotin-reacted cells was found to be biotinylated. This is the first report demonstrating surface expression of GRP 78 in cells exposed to a specific GRP 78-inducing stimulus.

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion

PubMed Central

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-01-01

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry. PMID:22328521

Myo1c regulates lipid raft recycling to control cell spreading, migration and Salmonella invasion.

PubMed

Brandstaetter, Hemma; Kendrick-Jones, John; Buss, Folma

2012-04-15

A balance between endocytosis and membrane recycling regulates the composition and dynamics of the plasma membrane. Internalization and recycling of cholesterol- and sphingolipid-enriched lipid rafts is an actin-dependent process that is mediated by a specialized Arf6-dependent recycling pathway. Here, we identify myosin1c (Myo1c) as the first motor protein that drives the formation of recycling tubules emanating from the perinuclear recycling compartment. We demonstrate that the single-headed Myo1c is a lipid-raft-associated motor protein that is specifically involved in recycling of lipid-raft-associated glycosylphosphatidylinositol (GPI)-linked cargo proteins and their delivery to the cell surface. Whereas Myo1c overexpression increases the levels of these raft proteins at the cell surface, in cells depleted of Myo1c function through RNA interference or overexpression of a dominant-negative mutant, these tubular transport carriers of the recycling pathway are lost and GPI-linked raft markers are trapped in the perinuclear recycling compartment. Intriguingly, Myo1c only selectively promotes delivery of lipid raft membranes back to the cell surface and is not required for recycling of cargo, such as the transferrin receptor, which is mediated by parallel pathways. The profound defect in lipid raft trafficking in Myo1c-knockdown cells has a dramatic impact on cell spreading, cell migration and cholesterol-dependent Salmonella invasion; processes that require lipid raft transport to the cell surface to deliver signaling components and the extra membrane essential for cell surface expansion and remodeling. Thus, Myo1c plays a crucial role in the recycling of lipid raft membrane and proteins that regulate plasma membrane plasticity, cell motility and pathogen entry.

Roles of proteolysis in regulation of GPCR function

PubMed Central

Cottrell, GS

2013-01-01

The enzymatic activity of peptidases must be tightly regulated to prevent uncontrolled hydrolysis of peptide bonds, which could have devastating effects on biological systems. Peptidases are often generated as inactive propeptidases, secreted with endogenous inhibitors, or they are compartmentalized. Propeptidases become active after proteolytic removal of N-terminal activation peptides by other peptidases. Some peptidases only become active towards substrates only at certain pHs, thus confining activity to specific compartments or conditions. This review discusses the different roles proteolysis plays in regulating GPCRs. At the cell-surface, certain GPCRs are regulated by the hydrolytic inactivation of bioactive peptides by membrane-anchored peptidases, which prevent signalling. Conversely, cell-surface peptidases can also generate bioactive peptides, which directly activate GPCRs. Alternatively, cell-surface peptidases activated by GPCRs, can generate bioactive peptides to cause transactivation of receptor tyrosine kinases, thereby promoting signalling. Certain peptidases can signal directly to cells, by cleaving GPCR to initiate intracellular signalling cascades. Intracellular peptidases also regulate GPCRs; lysosomal peptidases destroy GPCRs in lysosomes to permanently terminate signalling and mediate down-regulation; endosomal peptidases cleave internalized peptide agonists to regulate GPCR recycling, resensitization and signalling; and soluble intracellular peptidases also participate in GPCR function by regulating the ubiquitination state of GPCRs, thereby altering GPCR signalling and fate. Although the use of peptidase inhibitors has already brought success in the treatment of diseases such as hypertension, the discovery of new regulatory mechanisms involving proteolysis that control GPCRs may provide additional targets to modulate dysregulated GPCR signalling in disease. PMID:23043558

Formation of a physiological complex between TRPV2 and RGA protein promotes cell surface expression of TRPV2.

PubMed

Stokes, Alexander J; Wakano, Clay; Del Carmen, Kimberly A; Koblan-Huberson, Murielle; Turner, Helen

2005-03-01

The transient receptor potential, sub-family Vanilloid (TRPV)(2) cation channel is activated in response to extreme temperature elevations in sensory neurons. However, TRPV2 is widely expressed in tissues with no sensory function, including cells of the immune system. Regulation of GRC, the murine homolog of TRPV2 has been studied in insulinoma cells and myocytes. GRC is activated in response to certain growth factors and neuropeptides, via a mechanism that involves regulated access of the channel to the plasma membrane. This is likely to be an important primary control mechanism for TRPV2 outside the CNS. Here, we report that a regulated trafficking step controls the access of TRPV2 to the cell surface in mast cells. In mast cells, elevations in cytosolic cAMP are sufficient to drive plasma membrane localization of TRPV2. We have previously proposed that the recombinase gene activator protein (RGA), a four-transmembrane domain, intracellular protein, associates with TRPV2 during the biosynthesis and early trafficking of the channel. We use a polyclonal antibody to RGA to confirm the formation of a physiological complex between RGA and TRPV2. Finally, we show that over-expression of the RGA protein potentiates the basal surface localization of TRPV2. We propose that trafficking and activation mechanisms intersect for TRPV2, and that cAMP mobilizing stimuli may regulate TRPV2 localization in non-sensory cells. RGA participates in the control of TRPV2 surface levels, and co-expression of RGA may be a key component of experimental systems that seek to study TRPV2 physiology.

The role of heparins and nano-heparins as therapeutic tool in breast cancer.

PubMed

Afratis, Nikos A; Karamanou, Konstantina; Piperigkou, Zoi; Vynios, Demitrios H; Theocharis, Achilleas D

2017-06-01

Glycosaminoglycans are integral part of the dynamic extracellular matrix (ECM) network that control crucial biochemical and biomechanical signals required for tissue morphogenesis, differentiation, homeostasis and cancer development. Breast cancer cells communicate with stromal ones to modulate ECM mainly through release of soluble effectors during cancer progression. The intracellular cross-talk between cell surface receptors and estrogen receptors is important for the regulation of breast cancer cell properties and production of ECM molecules. In turn, reorganized ECM-cell surface interface modulates signaling cascades, which regulate almost all aspects of breast cell behavior. Heparan sulfate chains present on cell surface and matrix proteoglycans are involved in regulation of breast cancer functions since they are capable of binding numerous matrix molecules, growth factors and inflammatory mediators thus modulating their signaling. In addition to its anticoagulant activity, there is accumulating evidence highlighting various anticancer activities of heparin and nano-heparin derivatives in numerous types of cancer. Importantly, heparin derivatives significantly reduce breast cancer cell proliferation and metastasis in vitro and in vivo models as well as regulates the expression profile of major ECM macromolecules, providing strong evidence for therapeutic targeting. Nano-formulations of the glycosaminoglycan heparin are possibly novel tools for targeting tumor microenvironment. In this review, the role of heparan sulfate/heparin and its nano-formulations in breast cancer biology are presented and discussed in terms of future pharmacological targeting.

Regulation of Memory T Cells by Interleukin-23.

PubMed

Li, Yanchun; Wang, Hongbo; Lu, Honghua; Hua, Shucheng

2016-01-01

Interleukin-23 (IL-23), a member of the IL-12 family of cytokines, is a heterodimeric cytokine. It is composed of subunits p40 (shared with IL-12) and p19 (an IL-12 p35-related subunit) and is secreted by several types of immune cells, such as natural killer cells and dendritic cells. The IL-23 receptor is composed of the subunit IL-12Rβ1 and the IL-23-specific subunit IL-23R. The binding of IL-23 to its specific cell surface receptor regulates a number of functions, including proliferation and differentiation of cells and secretion of cell factors. Memory T cells are a subset of T cells that secrete numerous important cell factors, and they function in the immune response to infection and diseases like cancer, autoimmune disease and bronchial asthma. IL-23R is expressed on the surface of memory T cells, which suggests that it can specifically regulate memory T cell function. IL-23 has been widely used as a clinical indicator in immune-related diseases and shows potential for use in disease treatment. Here we review the current progress in the study of the role of IL-23 in the regulation of memory T cells. © 2016 S. Karger AG, Basel.

Chlorine stress mediates microbial surface attachment in drinking water systems.

PubMed

Liu, Li; Le, Yang; Jin, Juliang; Zhou, Yuliang; Chen, Guowei

2015-03-01

Microbial attachment to drinking water pipe surfaces facilitates pathogen survival and deteriorates disinfection performance, directly threatening the safety of drinking water. Notwithstanding that the formation of biofilm has been studied for decades, the underlying mechanisms for the origins of microbial surface attachment in biofilm development in drinking water pipelines remain largely elusive. We combined experimental and mathematical methods to investigate the role of environmental stress-mediated cell motility on microbial surface attachment in chlorination-stressed drinking water distribution systems. Results show that at low levels of disinfectant (0.0-1.0 mg/L), the presence of chlorine promotes initiation of microbial surface attachment, while higher amounts of disinfectant (>1.0 mg/L) inhibit microbial attachment. The proposed mathematical model further demonstrates that chlorination stress (0.0-5.0 mg/L)-mediated microbial cell motility regulates the frequency of cell-wall collision and thereby controls initial microbial surface attachment. The results reveal that transport processes and decay patterns of chlorine in drinking water pipelines regulate microbial cell motility and, thus, control initial surface cell attachment. It provides a mechanistic understanding of microbial attachment shaped by environmental disinfection stress and leads to new insights into microbial safety protocols in water distribution systems.

Differential signaling and regulation of apical vs. basolateral EGFR in polarized epithelial cells.

PubMed

Kuwada, S K; Lund, K A; Li, X F; Cliften, P; Amsler, K; Opresko, L K; Wiley, H S

1998-12-01

Overexpression of the epidermal growth factor receptors (EGFR) in polarized kidney epithelial cells caused them to appear in high numbers at both the basolateral and apical cell surfaces. We utilized these cells to look for differences in the regulation and signaling of apical vs. basolateral EGFR. Apical and basolateral EGFR were biologically active and mediated EGF-induced cell proliferation to similar degrees. Receptor downregulation and endocytosis were less efficient at the apical surface, resulting in prolonged EGF-induced tyrosine kinase activity at the apical cell membrane. Tyrosine phosphorylation of EGFR substrates known to mediate cell proliferation, Src-homologous and collagen protein (SHC), extracellularly regulated kinase 1 (ERK1), and ERK2 could be induced similarly by activation of apical or basolateral EGFR. Focal adhesion kinase was tyrosine phosphorylated more by basolateral than by apical EGFR; however, beta-catenin was tyrosine phosphorylated to a much greater degree following the activation of mislocalized apical EGFR. Thus EGFR regulation and EGFR-mediated phosphorylation of certain substrates differ at the apical and basolateral cell membrane domains. This suggests that EGFR mislocalization could result in abnormal signal transduction and aberrant cell behavior.

Requirements for cell rounding and surface protein down-regulation by Ebola virus glycoprotein.

PubMed

Francica, Joseph R; Matukonis, Meghan K; Bates, Paul

2009-01-20

Ebola virus causes an acute hemorrhagic fever that is associated with high morbidity and mortality. The viral glycoprotein is thought to contribute to pathogenesis, though precise mechanisms are unknown. Cellular pathogenesis can be modeled in vitro by expression of the Ebola viral glycoprotein (GP) in cells, which causes dramatic morphological changes, including cell rounding and surface protein down-regulation. These effects are known to be dependent on the presence of a highly glycosylated region of the glycoprotein, the mucin domain. Here we show that the mucin domain from the highly pathogenic Zaire subtype of Ebola virus is sufficient to cause characteristic cytopathology when expressed in the context of a foreign glycoprotein. Similarly to full length Ebola GP, expression of the mucin domain causes rounding, detachment from the extracellular matrix, and the down-regulation of cell surface levels of beta1 integrin and major histocompatibility complex class 1. These effects were not seen when the mucin domain was expressed in the context of a glycophosphatidylinositol-anchored isoform of the foreign glycoprotein. In contrast to earlier analysis of full length Ebola glycoproteins, chimeras carrying the mucin domains from the Zaire and Reston strains appear to cause similar levels of down-modulation and cell detachment. Cytopathology associated with Ebola glycoprotein expression does not occur when GP expression is restricted to the endoplasmic reticulum. In contrast to a previously published report, our results demonstrate that GP-induced surface protein down-regulation is not mediated through a dynamin-dependent pathway. Overall, these results support a model in which the mucin domain of Ebola GP acts at the cell surface to induce protein down modulation and cytopathic effects.

Platelets regulate lymphatic vascular development through CLEC-2-SLP-76 signaling.

PubMed

Bertozzi, Cara C; Schmaier, Alec A; Mericko, Patricia; Hess, Paul R; Zou, Zhiying; Chen, Mei; Chen, Chiu-Yu; Xu, Bin; Lu, Min-min; Zhou, Diane; Sebzda, Eric; Santore, Matthew T; Merianos, Demetri J; Stadtfeld, Matthias; Flake, Alan W; Graf, Thomas; Skoda, Radek; Maltzman, Jonathan S; Koretzky, Gary A; Kahn, Mark L

2010-07-29

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.

Golgi sorting regulates organization and activity of GPI-proteins at apical membranes

PubMed Central

Tivodar, Simona; Formiggini, Fabio; Ossato, Giulia; Gratton, Enrico; Tramier, Marc; Coppey-Moisan, Maïté; Zurzolo, Chiara

2014-01-01

Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homo-clusters are required for their subsequent plasma membrane organization into cholesterol-dependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface. PMID:24681536

Getting to the Outer Leaflet: Physiology of Phosphatidylserine Exposure at the Plasma Membrane.

PubMed

Bevers, Edouard M; Williamson, Patrick L

2016-04-01

Phosphatidylserine (PS) is a major component of membrane bilayers whose change in distribution between inner and outer leaflets is an important physiological signal. Normally, members of the type IV P-type ATPases spend metabolic energy to create an asymmetric distribution of phospholipids between the two leaflets, with PS confined to the cytoplasmic membrane leaflet. On occasion, membrane enzymes, known as scramblases, are activated to facilitate transbilayer migration of lipids, including PS. Recently, two proteins required for such randomization have been identified: TMEM16F, a scramblase regulated by elevated intracellular Ca(2+), and XKR8, a caspase-sensitive protein required for PS exposure in apoptotic cells. Once exposed at the cell surface, PS regulates biochemical reactions involved in blood coagulation, and bone mineralization, and also regulates a variety of cell-cell interactions. Exposed on the surface of apoptotic cells, PS controls their recognition and engulfment by other cells. This process is exploited by parasites to invade their host, and in specialized form is used to maintain photoreceptors in the eye and modify synaptic connections in the brain. This review discusses what is known about the mechanism of PS exposure at the surface of the plasma membrane of cells, how actors in the extracellular milieu sense surface exposed PS, and how this recognition is translated to downstream consequences of PS exposure. Copyright © 2016 the American Physiological Society.

Surface association and the MreB cytoskeleton regulate pilus production, localization and function in Pseudomonas aeruginosa.

PubMed

Cowles, Kimberly N; Gitai, Zemer

2010-06-01

Spatial organization of bacterial proteins influences many cellular processes, including division, chromosome segregation and motility. Virulence-associated proteins also localize to specific destinations within bacterial cells. However, the functions and mechanisms of virulence factor localization remain largely unknown. In this work, we demonstrate that polar assembly of the Pseudomonas aeruginosa PAO1 type IV pilus is regulated by surface association in a manner that affects gene transcription, protein levels and protein localization. We also uncover one mechanism for this regulation that acts through the actin homologue MreB. Inactivation of MreB leads to mislocalization of the pilus retraction ATPase PilT, mislocalization of the pili themselves and a reduction in motility. Furthermore, the role of MreB in polar localization of PilT is modulated by surface association, corroborating our results that environmental factors influence the regulation of pilus production. Specifically, MreB mediates both the initiation and maintenance of PilT localization when cells are grown in suspension but only affects the initiation of localization when cells are grown on a surface. Together, these results suggest that the bacterial cytoskeleton provides a mechanism for the polar localization of P. aeruginosa pili and demonstrate that protein localization may represent an important aspect of virulence factor regulation in bacterial pathogens.

Insulin receptor substrate-2 regulates aerobic glycolysis in mouse mammary tumor cells via glucose transporter 1.

PubMed

Pankratz, Shannon L; Tan, Ernest Y; Fine, Yumiko; Mercurio, Arthur M; Shaw, Leslie M

2009-01-23

The insulin receptor substrate (IRS) proteins are cytoplasmic adaptor molecules that function as signaling intermediates downstream of activated cell surface receptors. Based on data implicating IRS-2 but not IRS-1 in breast cancer invasion, survival, and metastasis, we assessed the contribution of IRS-1 and IRS-2 to aerobic glycolysis, which is known to impact tumor growth and progression. For this purpose, we used tumor cell lines derived from transgenic mice that express the polyoma virus middle T antigen (PyV-MT) in the mammary gland and that are wild-type (WT) or null for either Irs-1 (Irs-1-/-) or Irs-2 (Irs-2-/-). Aerobic glycolysis, as assessed by the rate of lactic acid production and glucose consumption, was diminished significantly in Irs-2-/- cells when compared with WT and Irs-1-/- cells. Expression of exogenous Irs-2 in Irs-2-/- cells restored the rate of glycolysis to that observed in WT cells. The transcription factor FoxO1 does not appear to be involved in Irs-2-mediated glycolysis. However, Irs-2 does regulate the surface expression of glucose transporter 1 (Glut1) as assessed by flow cytometry using a Glut1-specific ligand. Suppression of Glut1 expression inhibits Irs-2-dependent invasion, which links glycolysis to mammary tumor progression. Irs-2 was shown to be important for mammalian target of rapamycin (mTor) activation, and Irs-2-dependent regulation of Glut1 surface expression is rapamycin-sensitive. Collectively, our data indicate that Irs-2, but not Irs-1, promotes invasion by sustaining the aerobic glycolysis of mouse mammary tumor cells and that it does so by regulating the mTor-dependent surface expression of Glut1.

Maintaining the pluripotency of mouse embryonic stem cells on gold nanoparticle layers with nanoscale but not microscale surface roughness

NASA Astrophysics Data System (ADS)

Lyu, Zhonglin; Wang, Hongwei; Wang, Yanyun; Ding, Kaiguo; Liu, Huan; Yuan, Lin; Shi, Xiujuan; Wang, Mengmeng; Wang, Yanwei; Chen, Hong

2014-05-01

Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr01540a

Ephrinb1 and Ephrinb2 Are Associated with Interleukin-7 Receptor α and Retard Its Internalization from the Cell Surface*

PubMed Central

Luo, Hongyu; Wu, Zenghui; Qi, Shijie; Jin, Wei; Han, Bing; Wu, Jiangping

2011-01-01

IL-7 plays vital roles in thymocyte development, T cell homeostasis, and the survival of these cells. IL-7 receptor α (IL-7Rα) on thymocytes and T cells is rapidly internalized upon IL-7 ligation. Ephrins (Efns) are cell surface molecules and ligands of the largest receptor kinase family, Eph kinases. We discovered that T cell-specific double gene knock-out (dKO) of Efnb1 and Efnb2 in mice led to reduced IL-7Rα expression in thymocytes and T cells, and that IL-7Rα down-regulation was accelerated in dKO CD4 cells upon IL-7 treatment. On the other hand, Efnb1 and Efnb2 overexpression on T cell lymphoma EL4 cells retarded IL-7Rα down-regulation. dKO T cells manifested compromised STAT5 activation and homeostatic proliferation, an IL-7-dependent process. Fluorescence resonance energy transfer and immunoprecipitation demonstrated that Efnb1 and Efnb2 interacted physically with IL-7Rα. Such interaction likely retarded IL-7Rα internalization, as Efnb1 and Efnb2 were not internalized. Therefore, we revealed a novel function of Efnb1 and Efnb2 in stabilizing IL-7Rα expression at the post-translational level, and a previously unknown modus operandi of Efnbs in the regulation of expression of other vital cell surface receptors. PMID:22069310

Multidisciplinary Analysis of Cyclophilin A Function in Human Breast Cancer

DTIC Science & Technology

2011-03-01

4 INTRODUCTION The growth and progression of human breast cancer is regulated by several cell surface receptors, including the...substantively to the biology of human breast cancer through its regulation of cell surface signaling, including that of the PRLr. We believe that the knowledge... dynamic structure of CypA in complex with PRLr and its proximal molecule Jak2. We have purified recombinant CypA, the intracellular domain (ICD) of

Select α-arrestins control cell-surface abundance of the mammalian Kir2.1 potassium channel in a yeast model.

PubMed

Hager, Natalie A; Krasowski, Collin J; Mackie, Timothy D; Kolb, Alexander R; Needham, Patrick G; Augustine, Andrew A; Dempsey, Alison; Szent-Gyorgyi, Christopher; Bruchez, Marcel P; Bain, Daniel J; Kwiatkowski, Adam V; O'Donnell, Allyson F; Brodsky, Jeffrey L

2018-05-21

Protein composition at the plasma membrane is tightly regulated, with rapid protein internalization and selective targeting to the cell surface occurring in response to environmental changes. For example, ion channels are dynamically relocalized to or from the plasma membrane in response to physiological alterations, allowing cells and organisms to maintain osmotic and salt homeostasis. To identify additional factors that regulate the selective trafficking of a specific ion channel, we used a yeast model for a mammalian potassium channel, the K+ inwardly rectifying channel Kir2.1. Kir2.1 maintains potassium homeostasis in heart muscle cells, and Kir2.1 defects lead to human disease. By examining the ability of Kir2.1 to rescue the growth of yeast cells lacking endogenous potassium channels, we discovered that specific α-arrestins regulate Kir2.1 localization. Specifically, we found that the Ldb19/Art1, Aly1/Art6, and Aly2/Art3 α-arrestin adaptor proteins promote Kir2.1 trafficking to the cell surface, increase Kir2.1 activity at the plasma membrane, and raise intracellular potassium levels. To better quantify the intracellular and cell-surface populations of Kir2.1, we created fluorescence-activating protein fusions and for the first time used this technique to measure the cell-surface residency of a plasma membrane protein in yeast. Our experiments revealed that two α-arrestin effectors also control Kir2.1 localization. In particular, both the Rsp5 ubiquitin ligase and the protein phosphatase calcineurin facilitated the α-arrestin-mediated trafficking of Kir2.1. Together, our findings implicate α-arrestins in regulating an additional class of plasma membrane proteins and establish a new tool for dissecting the trafficking itinerary of any membrane protein in yeast. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Dipeptidyl peptidase IV (DPPIV) enzyme activity on immature T-cell line R1.1 is down-regulated by dynorphin-A(1-17) as a non-substrate inhibitor.

PubMed

Gabrilovac, Jelka; Abramić, Marija; Uzarević, Branka; Andreis, Ana; Poljak, Ljiljana

2003-05-30

In this study we examined surface expression of CD26 and the corresponding enzyme activity of dipeptidyl peptidase IV (DPPIV) on the cells of immature murine T-cell line, R1.1. The data obtained have shown that R1.1 cells express high density of surface CD26 as compared to normal thymus cells. This was associated with strong enzyme activity, which, based on substrates and inhibitor specificity, corresponded to DPPIV. The DPPIV enzyme activity of R1.1 cells was 10 times stronger than that found on normal murine thymus cells (V(max) = 39 micromol/min/10(6) cells, vs 3.7 micromol/min/10(6) cells, respectively). Upon activation with anti-CD3, up-regulation of both membrane CD26, as well as of DPPIV enzyme activity on R1.1 cells were observed. The finding of strong DPPIV on R1.1 cells makes them suitable model for testing putative substrates/inhibitors of the enzyme in its natural microenvironment. Since in addition to strong DPPIV, R1.1 cells also express kappa opioid receptors (KOR) [European Journal of Pharmacology 227 (1992) 257], we tested the effect of dynorphin-A(1-17), an endogenous opioid peptide with KOR selectivity, on DPPIV of R1.1 cells. Dynorphin-A(1-17) down-regulated DPPIV in a dose-dependent manner, with the potency similar to that of substance P, a known natural DPPIV substrate [Journal of Pharmacology and Experimental Therapeutics 260 (1992) 1257]. DPPIV down-regulation was resistant to bestatin and thiorphan, the inhibitors of two cell surface peptidases (APN and NEP, respectively) with potential of dynorphin-A(1-17) degradation, suggesting that the mechanism underlying the observed effect does not involve degradative products of dynorphin-A(1-17). DPPIV down-regulation was also resistent to KOR antagonist, NBI, suggesting that the mechanism underlying the observed phenomenon involves neither cointernalization of KOR and DPPIV. Collectively, cells of immature T cell line, R1.1 exert strong DPPIV enzyme activity, which could be down-regulated in the presence of dynorphin-A(1-17) by mechanism that presumably includes non-substrate inhibition. By down-regulating DPPIV, dynorphin-A(1-17) may indirectly affect activity and/or specificity of natural substrates of DPPIV, such as substance P, RANTES, and endomorphins.

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Ying; Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023; Huang, Xiaohua

2013-10-25

Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study,more » we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.« less

Directional Spread of Surface-Associated Retroviruses Regulated by Differential Virus-Cell Interactions▿ †

PubMed Central

Sherer, Nathan M.; Jin, Jing; Mothes, Walther

2010-01-01

The spread of viral infections involves the directional progression of virus particles from infected cells to uninfected target cells. Prior to entry, the binding of virus particles to specific cell surface receptors can trigger virus surfing, an actin-dependent lateral transport of viruses toward the cell body (M. J. Lehmann et al., J. Cell Biol. 170:317-325, 2005; M. Schelhaas, et al., PLoS Pathog. 4:e1000148, 2008; J. L. Smith, D. S. Lidke, and M. A. Ozbun, Virology 381:16-21, 2008). Here, we have used live-cell imaging to demonstrate that for cells chronically infected with the gammaretrovirus murine leukemia virus in which receptor has been downregulated, a significant portion of completely assembled virus particles are not immediately released into the supernatant but retain long-term association with the cell surface. Retention can be attributed, at least in part, to nonspecific particle attachment to cell surface glycosylaminoglycans. In contrast to virus surfing, viruses retained at the surface of infected cells undergo a lateral motility that is random and actin independent. This diffusive motility can be abruptly halted and converted into inward surfing after treatment with Polybrene, a soluble cation that increases virus-cell adsorption. In the absence of Polybrene, particle diffusion allows for an outward flow of viruses to the infected cell periphery. Peripheral particles are readily captured by and transmitted to neighboring uninfected target cells in a directional fashion. These data demonstrate a surface-based mechanism for the directional spread of viruses regulated by differential virus-cell interactions. PMID:20089647

Surface Curvature Differentially Regulates Stem Cell Migration and Differentiation via Altered Attachment Morphology and Nuclear Deformation

PubMed Central

Werner, Maike; Blanquer, Sébastien B. G.; Haimi, Suvi P.; Korus, Gabriela; Dunlop, John W. C.; Duda, Georg N.; Grijpma, Dirk. W.

2016-01-01

Signals from the microenvironment around a cell are known to influence cell behavior. Material properties, such as biochemical composition and substrate stiffness, are today accepted as significant regulators of stem cell fate. The knowledge of how cell behavior is influenced by 3D geometric cues is, however, strongly limited despite its potential relevance for the understanding of tissue regenerative processes and the design of biomaterials. Here, the role of surface curvature on the migratory and differentiation behavior of human mesenchymal stem cells (hMSCs) has been investigated on 3D surfaces with well‐defined geometric features produced by stereolithography. Time lapse microscopy reveals a significant increase of cell migration speed on concave spherical compared to convex spherical structures and flat surfaces resulting from an upward‐lift of the cell body due to cytoskeletal forces. On convex surfaces, cytoskeletal forces lead to substantial nuclear deformation, increase lamin‐A levels and promote osteogenic differentiation. The findings of this study demonstrate a so far missing link between 3D surface curvature and hMSC behavior. This will not only help to better understand the role of extracellular matrix architecture in health and disease but also give new insights in how 3D geometries can be used as a cell‐instructive material parameter in the field of biomaterial‐guided tissue regeneration. PMID:28251054

Clathrin-Independent Endocytosis Suppresses Cancer Cell Blebbing and Invasion.

PubMed

Holst, Mikkel Roland; Vidal-Quadras, Maite; Larsson, Elin; Song, Jie; Hubert, Madlen; Blomberg, Jeanette; Lundborg, Magnus; Landström, Maréne; Lundmark, Richard

2017-08-22

Cellular blebbing, caused by local alterations in cell-surface tension, has been shown to increase the invasiveness of cancer cells. However, the regulatory mechanisms balancing cell-surface dynamics and bleb formation remain elusive. Here, we show that an acute reduction in cell volume activates clathrin-independent endocytosis. Hence, a decrease in surface tension is buffered by the internalization of the plasma membrane (PM) lipid bilayer. Membrane invagination and endocytosis are driven by the tension-mediated recruitment of the membrane sculpting and GTPase-activating protein GRAF1 (GTPase regulator associated with focal adhesion kinase-1) to the PM. Disruption of this regulation by depleting cells of GRAF1 or mutating key phosphatidylinositol-interacting amino acids in the protein results in increased cellular blebbing and promotes the 3D motility of cancer cells. Our data support a role for clathrin-independent endocytic machinery in balancing membrane tension, which clarifies the previously reported role of GRAF1 as a tumor suppressor. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

CD4-CCR5 interaction in intracellular compartments contributes to receptor expression at the cell surface

PubMed Central

Achour, Lamia; Scott, Mark G.H.; Shirvani, Hamasseh; Thuret, Alain; Bismuth, Georges; Labbé-Jullié, Catherine; Marullo, Stefano

2009-01-01

The association of CD4, a glycoprotein involved in T cell development and antigen recognition, and CCR5, a chemotactic G protein-coupled receptor, which regulates trafficking and effector functions of immune cells, forms the main receptor for the human immunodeficiency virus HIV. We observed that the vast majority of CCR5 is maintained within the intracellular compartments of primary T lymphocytes and in a monocytic cell line, contrasting with its relative low density at the cell surface. The CCR5-CD4 association, which occurs in the endoplasmic reticulum, enhanced CCR5 export to the plasma membrane in a concentration–dependent manner, whereas inhibition of endogenous CD4 with small interfering RNAs decreased cell surface expression of endogenous CCR5. This effect was specific for CCR5, as CD4 did not affect cell distribution of CXCR4, the other HIV co-receptor. These results reveal a previously unappreciated role of CD4, which contributes to regulate CCR5 export to the plasma membrane. PMID:19064722

Regulation of STIM1 and SOCE by the ubiquitin-proteasome system (UPS).

PubMed

Keil, Jeffrey M; Shen, Zhouxin; Briggs, Steven P; Patrick, Gentry N

2010-10-18

The ubiquitin proteasome system (UPS) mediates the majority of protein degradation in eukaryotic cells. The UPS has recently emerged as a key degradation pathway involved in synapse development and function. In order to better understand the function of the UPS at synapses we utilized a genetic and proteomic approach to isolate and identify novel candidate UPS substrates from biochemically purified synaptic membrane preparations. Using these methods, we have identified Stromal interacting molecule 1 (STIM1). STIM1 is as an endoplasmic reticulum (ER) calcium sensor that has been shown to regulate store-operated Ca(2+) entry (SOCE). We have characterized STIM1 in neurons, finding STIM1 is expressed throughout development with stable, high expression in mature neurons. As in non-excitable cells, STIM1 is distributed in a membranous and punctate fashion in hippocampal neurons. In addition, a population of STIM1 was found to exist at synapses. Furthermore, using surface biotinylation and live-cell labeling methods, we detect a subpopulation of STIM1 on the surface of hippocampal neurons. The role of STIM1 as a regulator of SOCE has typically been examined in non-excitable cell types. Therefore, we examined the role of the UPS in STIM1 and SOCE function in HEK293 cells. While we find that STIM1 is ubiquitinated, its stability is not altered by proteasome inhibitors in cells under basal conditions or conditions that activate SOCE. However, we find that surface STIM1 levels and thapsigargin (TG)-induced SOCE are significantly increased in cells treated with proteasome inhibitors. Additionally, we find that the overexpression of POSH (Plenty of SH3's), an E3 ubiquitin ligase recently shown to be involved in the regulation of Ca(2+) homeostasis, leads to decreased STIM1 surface levels. Together, these results provide evidence for previously undescribed roles of the UPS in the regulation of STIM1 and SOCE function.

Proximity-Based Differential Single-Cell Analysis of the Niche to Identify Stem/Progenitor Cell Regulators.

PubMed

Silberstein, Lev; Goncalves, Kevin A; Kharchenko, Peter V; Turcotte, Raphael; Kfoury, Youmna; Mercier, Francois; Baryawno, Ninib; Severe, Nicolas; Bachand, Jacqueline; Spencer, Joel A; Papazian, Ani; Lee, Dongjun; Chitteti, Brahmananda Reddy; Srour, Edward F; Hoggatt, Jonathan; Tate, Tiffany; Lo Celso, Cristina; Ono, Noriaki; Nutt, Stephen; Heino, Jyrki; Sipilä, Kalle; Shioda, Toshihiro; Osawa, Masatake; Lin, Charles P; Hu, Guo-Fu; Scadden, David T

2016-10-06

Physiological stem cell function is regulated by secreted factors produced by niche cells. In this study, we describe an unbiased approach based on the differential single-cell gene expression analysis of mesenchymal osteolineage cells close to, and further removed from, hematopoietic stem/progenitor cells (HSPCs) to identify candidate niche factors. Mesenchymal cells displayed distinct molecular profiles based on their relative location. We functionally examined, among the genes that were preferentially expressed in proximal cells, three secreted or cell-surface molecules not previously connected to HSPC biology-the secreted RNase angiogenin, the cytokine IL18, and the adhesion molecule Embigin-and discovered that all of these factors are HSPC quiescence regulators. Therefore, our proximity-based differential single-cell approach reveals molecular heterogeneity within niche cells and can be used to identify novel extrinsic stem/progenitor cell regulators. Similar approaches could also be applied to other stem cell/niche pairs to advance the understanding of microenvironmental regulation of stem cell function. Copyright © 2016 Elsevier Inc. All rights reserved.

Roles of Heparan Sulfate Sulfation in Dentinogenesis*

PubMed Central

Hayano, Satoru; Kurosaka, Hiroshi; Yanagita, Takeshi; Kalus, Ina; Milz, Fabian; Ishihara, Yoshihito; Islam, Md. Nurul; Kawanabe, Noriaki; Saito, Masahiro; Kamioka, Hiroshi; Adachi, Taiji; Dierks, Thomas; Yamashiro, Takashi

2012-01-01

Cell surface heparan sulfate (HS) is an essential regulator of cell signaling and development. HS traps signaling molecules, like Wnt in the glycosaminoglycan side chains of HS proteoglycans (HSPGs), and regulates their functions. Endosulfatases Sulf1 and Sulf2 are secreted at the cell surface to selectively remove 6-O-sulfate groups from HSPGs, thereby modifying the affinity of cell surface HSPGs for its ligands. This study provides molecular evidence for the functional roles of HSPG sulfation and desulfation in dentinogenesis. We show that odontogenic cells are highly sulfated on the cell surface and become desulfated during their differentiation to odontoblasts, which produce tooth dentin. Sulf1/Sulf2 double null mutant mice exhibit a thin dentin matrix and short roots combined with reduced expression of dentin sialophosphoprotein (Dspp) mRNA, encoding a dentin-specific extracellular matrix precursor protein, whereas single Sulf mutants do not show such defective phenotypes. In odontoblast cell lines, Dspp mRNA expression is potentiated by the activation of the Wnt canonical signaling pathway. In addition, pharmacological interference with HS sulfation promotes Dspp mRNA expression through activation of Wnt signaling. On the contrary, the silencing of Sulf suppresses the Wnt signaling pathway and subsequently Dspp mRNA expression. We also show that Wnt10a protein binds to cell surface HSPGs in odontoblasts, and interference with HS sulfation decreases the binding affinity of Wnt10a for HSPGs, which facilitates the binding of Wnt10a to its receptor and potentiates the Wnt signaling pathway, thereby up-regulating Dspp mRNA expression. These results demonstrate that Sulf-mediated desulfation of cellular HSPGs is an important modification that is critical for the activation of the Wnt signaling in odontoblasts and for production of the dentin matrix. PMID:22351753

Receptor-like kinases as surface regulators for RAC/ROP-mediated pollen tube growth and interaction with the pistil

PubMed Central

Zou, Yanjiao; Aggarwal, Mini; Zheng, Wen-Guang; Wu, Hen-Ming; Cheung, Alice Y.

2011-01-01

Background RAC/ROPs are RHO-type GTPases and are known to play diverse signalling roles in plants. Cytoplasmic RAC/ROPs are recruited to the cell membrane and activated in response to extracellular signals perceived and mediated by cell surface-located signalling assemblies, transducing the signals to regulate cellular processes. More than any other cell types in plants, pollen tubes depend on continuous interactions with an extracellular environment produced by their surrounding tissues as they grow within the female organ pistil to deliver sperm to the female gametophyte for fertilization. Scope We review studies on pollen tube growth that provide compelling evidence indicating that RAC/ROPs are crucial for regulating the cellular processes that underlie the polarized cell growth process. Efforts to identify cell surface regulators that mediate extracellular signals also point to RAC/ROPs being the molecular switches targeted by growth-regulating female factors for modulation to mediate pollination and fertilization. We discuss a large volume of work spanning more than two decades on a family of pollen-specific receptor kinases and some recent studies on members of the FERONIA family of receptor-like kinases (RLKs). Significance The research described shows the crucial roles that two RLK families play in transducing signals from growth regulatory factors to the RAC/ROP switch at the pollen tube apex to mediate and target pollen tube growth to the female gametophyte and signal its disintegration to achieve fertilization once inside the female chamber. PMID:22476487

GLUT4 Retention in Adipocytes Requires Two Intracellular Insulin-regulated Transport Steps

PubMed Central

Zeigerer, Anja; Lampson, Michael A.; Karylowski, Ola; Sabatini, David D.; Adesnik, Milton; Ren, Mindong; McGraw, Timothy E.

2002-01-01

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level. PMID:12134080

GLUT4 retention in adipocytes requires two intracellular insulin-regulated transport steps.

PubMed

Zeigerer, Anja; Lampson, Michael A; Karylowski, Ola; Sabatini, David D; Adesnik, Milton; Ren, Mindong; McGraw, Timothy E

2002-07-01

Insulin regulates glucose uptake into fat and muscle by modulating the distribution of the GLUT4 glucose transporter between the surface and interior of cells. The GLUT4 trafficking pathway overlaps with the general endocytic recycling pathway, but the degree and functional significance of the overlap are not known. In this study of intact adipocytes, we demonstrate, by using a compartment-specific fluorescence-quenching assay, that GLUT4 is equally distributed between two intracellular pools: the transferrin receptor-containing endosomes and a specialized compartment that excludes the transferrin receptor. These pools of GLUT4 are in dynamic communication with one another and with the cell surface. Insulin-induced redistribution of GLUT4 to the surface requires mobilization of both pools. These data establish a role for the general endosomal system in the specialized, insulin-regulated trafficking of GLUT4. Trafficking through the general endosomal system is regulated by rab11. Herein, we show that rab11 is required for the transport of GLUT4 from endosomes to the specialized compartment and for the insulin-induced translocation to the cell surface, emphasizing the importance of the general endosomal pathway in the specialized trafficking of GLUT4. Based on these findings we propose a two-step model for GLUT4 trafficking in which the general endosomal recycling compartment plays a specialized role in the insulin-regulated traffic of GLUT4. This compartment-based model provides the framework for understanding insulin-regulated trafficking at a molecular level.

Maintaining the pluripotency of mouse embryonic stem cells on gold nanoparticle layers with nanoscale but not microscale surface roughness.

PubMed

Lyu, Zhonglin; Wang, Hongwei; Wang, Yanyun; Ding, Kaiguo; Liu, Huan; Yuan, Lin; Shi, Xiujuan; Wang, Mengmeng; Wang, Yanwei; Chen, Hong

2014-06-21

Efficient control of the self-renewal and pluripotency maintenance of embryonic stem cell (ESC) is a prerequisite for translating stem cell technologies to clinical applications. Surface topography is one of the most important factors that regulates cell behaviors. In the present study, micro/nano topographical structures composed of a gold nanoparticle layer (GNPL) with nano-, sub-micro-, and microscale surface roughnesses were used to study the roles of these structures in regulating the behaviors of mouse ESCs (mESCs) under feeder-free conditions. The distinctive results from Oct-4 immunofluorescence staining and quantitative real-time polymerase chain reaction (qPCR) demonstrate that nanoscale and low sub-microscale surface roughnesses (Rq less than 392 nm) are conducive to the long-term maintenance of mESC pluripotency, while high sub-microscale and microscale surface roughnesses (Rq greater than 573 nm) result in a significant loss of mESC pluripotency and a faster undirectional differentiation, particularly in long-term culture. Moreover, the likely signalling cascades engaged in the topological sensing of mESCs were investigated and their role in affecting the maintenance of the long-term cell pluripotency was discussed by analyzing the expression of proteins related to E-cadherin mediated cell-cell adhesions and integrin-mediated focal adhesions (FAs). Additionally, the conclusions from MTT, cell morphology staining and alkaline phosphatase (ALP) activity assays show that the surface roughness can provide a potent regulatory signal for various mESC behaviors, including cell attachment, proliferation and osteoinduction.

Nanomaterials modulate stem cell differentiation: biological interaction and underlying mechanisms.

PubMed

Wei, Min; Li, Song; Le, Weidong

2017-10-25

Stem cells are unspecialized cells that have the potential for self-renewal and differentiation into more specialized cell types. The chemical and physical properties of surrounding microenvironment contribute to the growth and differentiation of stem cells and consequently play crucial roles in the regulation of stem cells' fate. Nanomaterials hold great promise in biological and biomedical fields owing to their unique properties, such as controllable particle size, facile synthesis, large surface-to-volume ratio, tunable surface chemistry, and biocompatibility. Over the recent years, accumulating evidence has shown that nanomaterials can facilitate stem cell proliferation and differentiation, and great effort is undertaken to explore their possible modulating manners and mechanisms on stem cell differentiation. In present review, we summarize recent progress in the regulating potential of various nanomaterials on stem cell differentiation and discuss the possible cell uptake, biological interaction and underlying mechanisms.

Roles of the multifunctional glycoprotein, emmprin (basigin; CD147), in tumour progression.

PubMed

Yan, Li; Zucker, Stanley; Toole, Bryan P

2005-02-01

Emmprin (basigin;CD147) is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily and is highly enriched on the surface of malignant tumour cells. Emmprin is involved in numerous physiological and pathological systems and exhibits several molecular and cellular characteristics, but a major function of emmprin is stimulation of synthesis of several matrix metalloproteinases. In tumours, emmprin most likely stimulates matrix metalloproteinase production in stromal fibroblasts and endothelial cells as well as in tumour cells themselves by a mechanism involving homophilic interactions between emmprin molecules on apposing cells or on neighbouring cells after membrane vesicle shedding. Membrane-associated cofactors, including caveolin-1 and annexin II, regulate emmprin activity. Emmprin induces angiogenesis via stimulation of VEGF production, invasiveness via stimulation of matrix metalloproteinase production and multidrug resistance via hyaluronan-mediated up-regulation of ErbB2 signaling and cell survival pathway activities. Although the detailed mechanisms whereby it regulates these numerous phenomena are not yet known, it is clear that emmprin is a major mediator of malignant cell behavior.

Surface free energy predominates in cell adhesion to hydroxyapatite through wettability.

PubMed

Nakamura, Miho; Hori, Naoko; Ando, Hiroshi; Namba, Saki; Toyama, Takeshi; Nishimiya, Nobuyuki; Yamashita, Kimihiro

2016-05-01

The initial adhesion of cells to biomaterials is critical in the regulation of subsequent cell behaviors. The purpose of this study was to investigate a mechanism through which the surface wettability of biomaterials can be improved and determine the effects of biomaterial surface characteristics on cellular behaviors. We investigated the surface characteristics of various types of hydroxyapatite after sintering in different atmospheres and examined the effects of various surface characteristics on cell adhesion to study cell-biomaterial interactions. Sintering atmosphere affects the polarization capacity of hydroxyapatite by changing hydroxide ion content and grain size. Compared with hydroxyapatite sintered in air, hydroxyapatite sintered in saturated water vapor had a higher polarization capacity that increased surface free energy and improved wettability, which in turn accelerated cell adhesion. We determined the optimal conditions of hydroxyapatite polarization for the improvement of surface wettability and acceleration of cell adhesion. Copyright © 2016 Elsevier B.V. All rights reserved.

The cysteine-rich domain regulates ADAM protease function in vivo.

PubMed

Smith, Katherine M; Gaultier, Alban; Cousin, Helene; Alfandari, Dominique; White, Judith M; DeSimone, Douglas W

2002-12-09

ADAMs are membrane-anchored proteases that regulate cell behavior by proteolytically modifying the cell surface and ECM. Like other membrane-anchored proteases, ADAMs contain candidate "adhesive" domains downstream of their metalloprotease domains. The mechanism by which membrane-anchored cell surface proteases utilize these putative adhesive domains to regulate protease function in vivo is not well understood. We address this important question by analyzing the relative contributions of downstream extracellular domains (disintegrin, cysteine rich, and EGF-like repeat) of the ADAM13 metalloprotease during Xenopus laevis development. When expressed in embryos, ADAM13 induces hyperplasia of the cement gland, whereas ADAM10 does not. Using chimeric constructs, we find that the metalloprotease domain of ADAM10 can substitute for that of ADAM13, but that specificity for cement gland expansion requires a downstream extracellular domain of ADAM13. Analysis of finer resolution chimeras indicates an essential role for the cysteine-rich domain and a supporting role for the disintegrin domain. These and other results reveal that the cysteine-rich domain of ADAM13 cooperates intramolecularly with the ADAM13 metalloprotease domain to regulate its function in vivo. Our findings thus provide the first evidence that a downstream extracellular adhesive domain plays an active role in regulating ADAM protease function in vivo. These findings are likely relevant to other membrane-anchored cell surface proteases.

Microfabricated Nanotopological Surfaces for Study of Adhesion-dependent Cell mechanosensitivity**

PubMed Central

Chen, Weiqiang; Sun, Yubing

2014-01-01

Cells display high sensitivity and exhibit diverse responses to the intrinsic nanotopography of the extracellular matrix through their nanoscale cellular sensing machinery. Here, we reported a simple microfabrication method for precise control and spatial patterning of the local nanoroughness on glass surfaces using photolithography and reactive ion etching (RIE). Using RIE-generated nanorough glass surfaces, we demonstrated that local nanoroughness could provide a potent biophysical signal to regulate a diverse array of NIH/3T3 fibroblast behaviors, including cell morphology, adhesion, proliferation and migration. We further showed that cellular responses to nanotopography might be regulated by cell adhesion signaling and actin cytoskeleton remodeling. To further investigate the role of cytoskeleton contractility in nanoroughness sensing, we applied the RIE method to generate nanoroughness on the tops of an array of elastomeric poly-dimethylsiloxane (PDMS) microposts. We utilized the PDMS microposts as force sensors and demonstrated that nanoroughness could indeed regulate the cytoskeleton contractility of NIH/3T3 fibroblasts. Our results suggested that a feedback regulation and mechano-chemical integration mechanism involving adhesion signaling, actin cytoskeleton, and intracellular mechanosensory components might play an important role in regulating mechanosensitive behaviors of NIH/3T3 fibroblasts. The capability to control and further predict cellular responses to nanoroughness might suggest novel methods for developing biomaterials mimicking nanotopographic structures in vivo and suitable local cellular microenvironments for functional tissue engineering. PMID:22887768

Nano-patterned SU-8 surface using nanosphere-lithography for enhanced neuronal cell growth

NASA Astrophysics Data System (ADS)

Kim, Eunhee; Yoo, Seung-Jun; Kim, Eunjung; Kwon, Tae-Hwan; Zhang, Li; Moon, Cheil; Choi, Hongsoo

2016-04-01

Mimicking the nanoscale surface texture of the extracellular matrix can affect the regulation of cellular behavior, including adhesion, differentiation, and neurite outgrowth. In this study, SU-8-based polymer surfaces with well-ordered nanowell arrays were fabricated using nanosphere lithography with polystyrene nanoparticles. We show that the SU-8 surface with nanowells resulted in similar neuronal development of rat pheochromocytoma (PC12) cells compared with an unpatterned poly-L-lysine (PLL)-coated SU-8 surface. Additionally, even after soaking the substrate in cell culture medium for two weeks, cells on the nanowell SU-8 surface showed long-term neurite outgrowth compared to cells on the PLL-coated SU-8 surface. The topographical surface modification of the nanowell array demonstrates potential as a replacement for cell adhesive material coatings such as PLL, for applications requiring long-term use of polymer-based implantable devices.

Autophagy mediates cell cycle response by regulating nucleocytoplasmic transport of PAX6 in limbal stem cells under ultraviolet-A stress

PubMed Central

Laggner, Maria; Pollreisz, Andreas; Schmidinger, Gerald; Schmidt-Erfurth, Ursula; Chen, Ying-Ting

2017-01-01

Limbal stem cells (LSC) account for homeostasis and regeneration of corneal epithelium. Solar ultraviolet A (UVA) is the major source causing oxidative damage in the ocular surface. Autophagy, a lysosomal degradation mechanism, is essential for physiologic function and stress defense of stem cells. PAX6, a master transcription factor governing corneal homeostasis by regulating cell cycle and cell fate of LSC, responds to oxidative stress by nucleocytoplasmic shuttling. Impaired autophagy and deregulated PAX6 have been reported in oxidative stress-related ocular surface disorders. We hypothesize a functional role for autophagy and PAX6 in LSC’s stress response to UVA. Therefore, human LSC colonies were irradiated with a sub-lethal dose of UVA and autophagic activity and intracellular reactive oxygen species (ROS) were measured by CYTO-ID assay and CM-H2DCFDA live staining, respectively. Following UVA irradiation, the percentage of autophagic cells significantly increased in LSC colonies while intracellular ROS levels remained unaffected. siRNA-mediated knockdown (KD) of ATG7 abolished UVA-induced autophagy and led to an excessive accumulation of ROS. Upon UVA exposure, LSCs displayed nuclear-to-cytoplasmic translocation of PAX6, while ATG7KD or antioxidant pretreatment largely attenuated the intracellular trafficking event. Immunofluorescence showing downregulation of proliferative marker PCNA and induction of cell cycle regulator p21 indicates cell cycle arrest in UVA-irradiated LSC. Abolishing autophagy, adenoviral-assisted restoration of nuclear PAX6 or antioxidant pretreatment abrogated the UVA-induced cell cycle arrest. Adenoviral expression of an ectopic PAX gene, PAX7, did not affect UVA cell cycle response. Furthermore, knocking down PAX6 attenuated the cell cycle progression of irradiated ATG7KD LSC by de-repressing p21 expression. Collectively, our data suggest a crosstalk between autophagy and PAX6 in regulating cell cycle response of ocular progenitors under UVA stress. Autophagy deficiency leads to impaired intracellular trafficking of PAX6, perturbed redox balance and uncurbed cell cycle progression in UVA-stressed LSCs. The coupling of autophagic machinery and PAX6 in cell cycle regulation represents an attractive therapeutic target for hyperproliferative ocular surface disorders associated with solar radiation. PMID:28700649

VEGFR-3 signaling is regulated by a G-protein activator, activator of G-protein signaling 8, in lymphatic endothelial cells.

PubMed

Sakima, Miho; Hayashi, Hisaki; Mamun, Abdullah Al; Sato, Motohiko

2018-07-01

Vascular endothelial growth factor C (VEGFC) and its cognate receptor VEGFR-3 play a key role in lymphangiogenesis. We previously reported that an ischemia-inducible Gβγ signal regulator, activator of G-protein signaling 8 (AGS8), regulated the subcellular distribution of vascular endothelial growth factor receptor-2 (VEGFR-2) and influenced VEGFA-induced signaling in vascular endothelial cells. Here, we report that AGS8 regulates VEGFR-3, which is another subtype of the VEGF receptor family, and mediates VEGFC signaling in human dermal lymphatic endothelial cells (HDLECs). VEGFC stimulated the proliferation of HDLECs and tube formation by HDLECs, which were inhibited by knocking down AGS8 by small interfering RNA (siRNA). AGS8 siRNA inhibited VEGFC-mediated phosphorylation of VEGFR-3 and its downstream molecules, including ERK1/2 and AKT. Analysis of fluorescence-activated cell sorting and immunofluorescence staining demonstrated that AGS8 knockdown was associated with a reduction of VEGFR-3 at the cell surface. Endocytosis inhibitors did not rescue the decrease of cell-surface VEGFR-3, suggesting that AGS8 regulated the trafficking of VEGFR-3 to the plasma membrane. An immunoprecipitation assay indicated that VEGFR-3 formed a complex including AGS8 and Gβγ in cells. These data suggest the novel regulation of VEGFC-VEGFR-3 by AGS8 in HDLECs and a potential role for AGS8 in lymphangiogenesis. Copyright © 2018 Elsevier Inc. All rights reserved.

Surfactant protein D delays Fas- and TRAIL-mediated extrinsic pathway of apoptosis in T cells.

PubMed

Djiadeu, Pascal; Kotra, Lakshmi P; Sweezey, Neil; Palaniyar, Nades

2017-05-01

Only a few extracellular soluble proteins are known to modulate apoptosis. We considered that surfactant-associated protein D (SP-D), an innate immune collectin present on many mucosal surfaces, could regulate apoptosis. Although SP-D is known to be important for immune cell homeostasis, whether SP-D affects apoptosis is unknown. In this study we aimed to determine the effects of SP-D on Jurkat T cells and human T cells dying by apoptosis. Here we show that SP-D binds to Jurkat T cells and delays the progression of Fas (CD95)-Fas ligand and TRAIL-TRAIL receptor induced, but not TNF-TNF receptor-mediated apoptosis. SP-D exerts its effects by reducing the activation of initiator caspase-8 and executioner caspase-3. SP-D also delays the surface exposure of phosphatidylserine. The effect of SP-D was ablated by the presence of caspase-8 inhibitor, but not by intrinsic pathway inhibitors. The binding ability of SP-D to dying cells decreases during the early stages of apoptosis, suggesting the release of apoptotic cell surface targets during apoptosis. SP-D also delays FasL-induced death of primary human T cells. SP-D delaying the progression of the extrinsic pathway of apoptosis could have important implications in regulating immune cell homeostasis at mucosal surfaces.

SNARE proteins underpin insulin-regulated GLUT4 traffic.

PubMed

Bryant, Nia J; Gould, Gwyn W

2011-06-01

Delivery of the glucose transporter type 4 (GLUT4) from an intracellular location to the cell surface in response to insulin represents a specialized form of membrane traffic, known to be impaired in the disease states of insulin resistance and type 2 diabetes. Like all membrane trafficking events, this translocation of GLUT4 requires members of the SNARE family of proteins. Here, we discuss two SNARE complexes that have been implicated in insulin-regulated GLUT4 traffic: one regulating the final delivery of GLUT4 to the cell surface in response to insulin and the other controlling GLUT4's intracellular trafficking. © 2011 John Wiley & Sons A/S.

Feedback Regulation of Intracellular Hydrostatic Pressure in Surface Cells of the Lens

PubMed Central

Gao, Junyuan; Sun, Xiurong; White, Thomas W.; Delamere, Nicholas A.; Mathias, Richard T.

2015-01-01

In wild-type lenses from various species, an intracellular hydrostatic pressure gradient goes from ∼340 mmHg in central fiber cells to 0 mmHg in surface cells. This gradient drives a center-to-surface flow of intracellular fluid. In lenses in which gap-junction coupling is increased, the central pressure is lower, whereas if gap-junction coupling is reduced, the central pressure is higher but surface pressure is always zero. Recently, we found that surface cell pressure was elevated in PTEN null lenses. This suggested disruption of a feedback control system that normally maintained zero surface cell pressure. Our purpose in this study was to investigate and characterize this feedback control system. We measured intracellular hydrostatic pressures in mouse lenses using a microelectrode/manometer-based system. We found that all feedback went through transport by the Na/K ATPase, which adjusted surface cell osmolarity such that pressure was maintained at zero. We traced the regulation of Na/K ATPase activity back to either TRPV4, which sensed positive pressure and stimulated activity, or TRPV1, which sensed negative pressure and inhibited activity. The inhibitory effect of TRPV1 on Na/K pumps was shown to signal through activation of the PI3K/AKT axis. The stimulatory effect of TRPV4 was shown in previous studies to go through a different signal transduction path. Thus, there is a local two-legged feedback control system for pressure in lens surface cells. The surface pressure provides a pedestal on which the pressure gradient sits, so surface pressure determines the absolute value of pressure at each radial location. We speculate that the absolute value of intracellular pressure may set the radial gradient in the refractive index, which is essential for visual acuity. PMID:26536260

BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein modulates ABCA1 trafficking and function

PubMed Central

Lin, Sisi; Zhou, Chun; Neufeld, Edward; Wang, Yu-Hua; Xu, Suo-Wen; Lu, Liang; Wang, Ying; Liu, Zhi-Ping; Li, Dong; Li, Cuixian; Chen, Shaorui; Le, Kang; Huang, Heqing; Liu, Peiqing; Moss, Joel; Vaughan, Martha; Shen, Xiaoyan

2013-01-01

Objective Cell surface localization and intracellular trafficking of ATP-binding cassette transporter A-1 (ABCA1) are essential for its function. However, regulation of these activities is still largely unknown. Brefeldin A (BFA), a uncompetitive inhibitor of brefeldin A-inhibited guanine nucleotide-exchange proteins (BIGs), disturbs the intracellular distribution of ABCA1, and thus inhibits cholesterol efflux. This study aimed to define the possible roles of BIGs in regulating ABCA1 trafficking and cholesterol efflux, and further to explore the potential mechanism. Methods and Results By vesicle immunoprecipitation, we found that BIG1 was associated with ABCA1 in vesicles preparation from rat liver. BIG1 depletion reduced surface ABCA1 on HepG2 cells and inhibited by 60% cholesterol release. In contrast, BIG1 over-expression increased surface ABCA1 and cholesterol secretion. With partial restoration of BIG1 through over-expression in BIG1-depleted cells, surface ABCA1 was also restored. Biotinylation and glutathione cleavage revealed that BIG1 siRNA dramatically decreased the internalization and recycling of ABCA1. This novel function of BIG1 was dependent on the guanine nucleotide-exchange activity and achieved through activation of ADP-ribosylation factor 1 (ARF1). Conclusions BIG1, through its ability to activate ARF1, regulates cell surface levels and function of ABCA1, indicating a transcription-independent mechanism for controlling ABCA1 action. PMID:23220274

THE AP-2 CLATHRIN ADAPTOR MEDIATES ENDOCYTOSIS OF AN INHIBITORY KILLER CELL Ig-LIKE RECEPTOR (KIR) IN HUMAN NK CELLS1

PubMed Central

Purdy, Amanda K.; Alvarez-Arias, Diana A.; Oshinsky, Jennifer; James, Ashley M.; Serebriiskii, Ilya; Campbell, Kerry S.

2014-01-01

Stable surface expression of human inhibitory killer cell immunoglobulin-like receptors (KIR) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC-I+ cells. Our recent experiments, however, have found that antibody-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the μ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/μ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of μ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon antibody engagement with the receptor, as compared to untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIR though their ITIM domains. Based upon our results, we propose a model in which non-engaged KIR are internalized by this mechanism, whereas engagement with MHC-I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors. PMID:25238755

Regulation of cell protrusions by small GTPases during fusion of the neural folds

PubMed Central

Rolo, Ana; Savery, Dawn; Escuin, Sarah; de Castro, Sandra C; Armer, Hannah EJ; Munro, Peter MG; Molè, Matteo A; Greene, Nicholas DE; Copp, Andrew J

2016-01-01

Epithelial fusion is a crucial process in embryonic development, and its failure underlies several clinically important birth defects. For example, failure of neural fold fusion during neurulation leads to open neural tube defects including spina bifida. Using mouse embryos, we show that cell protrusions emanating from the apposed neural fold tips, at the interface between the neuroepithelium and the surface ectoderm, are required for completion of neural tube closure. By genetically ablating the cytoskeletal regulators Rac1 or Cdc42 in the dorsal neuroepithelium, or in the surface ectoderm, we show that these protrusions originate from surface ectodermal cells and that Rac1 is necessary for the formation of membrane ruffles which typify late closure stages, whereas Cdc42 is required for the predominance of filopodia in early neurulation. This study provides evidence for the essential role and molecular regulation of membrane protrusions prior to fusion of a key organ primordium in mammalian development. DOI: http://dx.doi.org/10.7554/eLife.13273.001 PMID:27114066

HFE interacts with the BMP type I receptor ALK3 to regulate hepcidin expression

PubMed Central

Wu, Xing-gang; Wang, Yang; Wu, Qian; Cheng, Wai-Hang; Liu, Wenjing; Zhao, Yueshui; Mayeur, Claire; Schmidt, Paul J.; Yu, Paul B.; Wang, Fudi

2014-01-01

Mutations in HFE are the most common cause of hereditary hemochromatosis (HH). HFE mutations result in reduced expression of hepcidin, a hepatic hormone, which negatively regulates iron absorption from the duodenum and iron release from macrophages. However, the mechanism by which HFE regulates hepcidin expression in hepatocytes is not well understood. It is known that the bone morphogenetic protein (BMP) pathway plays a central role in controlling hepcidin expression in the liver. Here we show that HFE overexpression increased Smad1/5/8 phosphorylation and hepcidin expression, whereas inhibition of BMP signaling abolished HFE-induced hepcidin expression in Hep3B cells. HFE was found to associate with ALK3, inhibiting ALK3 ubiquitination and proteasomal degradation and increasing ALK3 protein expression and accumulation on the cell surface. The 2 HFE mutants associated with HH, HFE C282Y and HFE H63D, regulated ALK3 protein ubiquitination and trafficking differently, but both failed to increase ALK3 cell-surface expression. Deletion of Hfe in mice resulted in a decrease in hepatic ALK3 protein expression. Our results provide evidence that HFE induces hepcidin expression via the BMP pathway: HFE interacts with ALK3 to stabilize ALK3 protein and increase ALK3 expression at the cell surface. PMID:24904118

Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

PubMed

Lim, HooiCheng; Yu, Chun-Ying; Jou, Tzuu-Shuh

2017-11-01

Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O -glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O -glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells. © FASEB.

Pigment Epithelial-derived Factor (PEDF)-triggered Lung Cancer Cell Apoptosis Relies on p53 Protein-driven Fas Ligand (Fas-L) Up-regulation and Fas Protein Cell Surface Translocation*

PubMed Central

Li, Lei; Yao, Ya-Chao; Fang, Shu-Huan; Ma, Cai-Qi; Cen, Yi; Xu, Zu-Min; Dai, Zhi-Yu; Li, Cen; Li, Shuai; Zhang, Ting; Hong, Hong-Hai; Qi, Wei-Wei; Zhou, Ti; Li, Chao-Yang; Yang, Xia; Gao, Guo-Quan

2014-01-01

Pigment epithelium-derived factor (PEDF), a potent antiangiogenesis agent, has recently attracted attention for targeting tumor cells in several types of tumors. However, less is known about the apoptosis-inducing effect of PEDF on human lung cancer cells and the underlying molecular events. Here we report that PEDF has a growth-suppressive and proapoptotic effect on lung cancer xenografts. Accordingly, in vitro, PEDF apparently induced apoptosis in A549 and Calu-3 cells, predominantly via the Fas-L/Fas death signaling pathway. Interestingly, A549 and Calu-3 cells are insensitive to the Fas-L/Fas apoptosis pathway because of the low level of cell surface Fas. Our results revealed that, in addition to the enhancement of Fas-L expression, PEDF increased the sensitivity of A549 and Calu-3 cells to Fas-L-mediated apoptosis by triggering the translocation of Fas protein to the plasma membrane in a p53- and FAP-1-dependent manner. Similarly, the up-regulation of Fas-L by PEDF was also mediated by p53. Furthermore, peroxisome proliferator-activated receptor γ was determined to be the upstream regulator of p53. Together, these findings uncover a novel mechanism of tumor cell apoptosis induced by PEDF and provide a potential therapeutic strategy for tumors that are insensitive to Fas-L/Fas-dependent apoptosis because of a low level of cell surface Fas. PMID:25225287

New approaches for solving old problems in neuronal protein trafficking.

PubMed

Bourke, Ashley M; Bowen, Aaron B; Kennedy, Matthew J

2018-04-10

Fundamental cellular properties are determined by the repertoire and abundance of proteins displayed on the cell surface. As such, the trafficking mechanisms for establishing and maintaining the surface proteome must be tightly regulated for cells to respond appropriately to extracellular cues, yet plastic enough to adapt to ever-changing environments. Not only are the identity and abundance of surface proteins critical, but in many cases, their regulated spatial positioning within surface nanodomains can greatly impact their function. In the context of neuronal cell biology, surface levels and positioning of ion channels and neurotransmitter receptors play essential roles in establishing important properties, including cellular excitability and synaptic strength. Here we review our current understanding of the trafficking pathways that control the abundance and localization of proteins important for synaptic function and plasticity, as well as recent technological advances that are allowing the field to investigate protein trafficking with increasing spatiotemporal precision. Copyright © 2018 Elsevier Inc. All rights reserved.

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

DOE Office of Scientific and Technical Information (OSTI.GOV)

Curbo, Sophie; Gaudin, Raphael; Carlsten, Mattias

2009-12-25

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4R{alpha} receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown tomore » be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.« less

Epimorphin Regulates the Intestinal Stem Cell Niche via Effects on the Stromal Microenvironment.

PubMed

Vishy, Courtney E; Swietlicki, Elzbieta A; Gazit, Vered; Amara, Suneetha; Heslop, Gabriela; Lu, Jianyun; Levin, Marc S; Rubin, Deborah C

2018-04-06

Stem cell therapy is a potential therapeutic approach for disorders characterized by intestinal injury or loss of functional surface area. Stem cell function and proliferation are mediated by the stem cell niche. Stromal cells such as intestinal subepithelial myofibroblasts (ISEMFs) are important but poorly studied components of the stem cell niche. To examine the role of ISEMFs, we have previously generated mice with deletion of epimorphin (Epim), an ISEMF protein and member of the syntaxin family of intracellular vesicle docking proteins that regulate cell secretion. Herein we explore the mechanisms for previous observations that Epim deletion increases gut crypt cell proliferation, crypt fission and small bowel length in vivo. Stem cell derived crypt culture techniques were used to explore the interaction between enteroids and myofibroblasts from Epim -/- and WT mice. Enteroids co-cultured with ISEMFS had increased growth and crypt-like budding compared to enteroids cultured without stromal support. Epim deletion in ISEMFs resulted in increased enteroid budding and surface area compared to co-cultures with WT ISEMFs. In primary crypt cultures, Epim -/- enteroids had significantly increased surface area and budding compared WTs. However stem cell assays comparing the number of Epim -/- vs WT colony forming units after first passage showed no differences in the absence of ISEMF support. Epim -/- vs. WT ISEMFs had increased Wnt4 expression and addition of Wnt4 to WT co-cultures enhanced budding. We conclude that ISEMFs play an important role in the stem cell niche. Epim regulates stem cell proliferation and differentiation via stromal contributions to the niche microenvironment.

[Effect of Shexiang Baoxin Pills on isoprenaline-induced myocardial cell hypertrophy and Cx43 expression].

PubMed

Tang, Fen; Jiang, Zhentao; Tan, Wenting; Long, Junrong; Liu, Shengquan; Chu, Chun

2017-08-28

To observe the effects of Shexiang Baoxin Pill (SBP) on isoprenaline (Iso)-induced changes in myocardial cell volume, shape, and connexin 43 (Cx43) expression.
 Methods: H9C2 myocardial cells were randomly divided into a control group, a Iso group and a Iso+SBP group. After 72 h of culture, the average surface area of H9C2 cells was measured under phase contrast microscope. Bicinchoninic acid (BCA) protein assay was carried out to determine the concentration of proteins. The survival rate of myocardial cells was measured by methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, and the Cx43 expression was detected by Western blot.
 Results: The mean surface area and Cx43 concentration in Iso-treated myocardial cells were increased under the phase contrast microscope (P<0.05). Compared with the Iso group, the mean surface area was decreased, and the Cx43 concentration was reduced in the Iso+SBP group (both P<0.05). Compared with the control group, the Cx43 expression was obviously down-regulated in the H9C2 cells of the Iso group (P<0.05); while compared with the Iso group, the Cx43 expression was obviously up-regulated in the Iso+SBP group (P<0.05).
 Conclusion: Shexiang Baoxin Pills can prevent Iso-induced myocardial hypertrophy and down-regulate Cx43 expression.

SHINE transcription factors act redundantly to pattern the archetypal surface of Arabidopsis flower organs.

PubMed

Shi, Jian Xin; Malitsky, Sergey; De Oliveira, Sheron; Branigan, Caroline; Franke, Rochus B; Schreiber, Lukas; Aharoni, Asaph

2011-05-01

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions.

SHINE Transcription Factors Act Redundantly to Pattern the Archetypal Surface of Arabidopsis Flower Organs

PubMed Central

Shi, Jian Xin; Malitsky, Sergey; De Oliveira, Sheron; Branigan, Caroline; Franke, Rochus B.; Schreiber, Lukas; Aharoni, Asaph

2011-01-01

Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ surface patterning and the broader control of flower development and biological functions. PMID:21637781

Regulation of surface expression of TRPV2 channels in the retinal pigment epithelium.

PubMed

Reichhart, Nadine; Keckeis, Susanne; Fried, Frederik; Fels, Gabriele; Strauss, Olaf

2015-06-01

The retinal pigment epithelium (RPE) interacts closely with the photoreceptors in fulfilling tasks of visual function. Since an understanding of the RPE function is essential for understanding the patho-mechanisms involved in vision loss, we explored the regulation of the vanilloid receptor subtype transient receptor potential TRPV2 channels that trigger insulin-like growth factor-1 (IGF-1)-induced vascular endothelial growth factor A (VEGF-A) secretion. Immunohistochemistry was used to assess TRPV2 expression in retinal cross-sections or ARPE-19 cells, and surface expression of TRPV2 was quantified using confocal microscopy. Membrane currents of ARPE-19 cells were recorded using a whole-cell configuration of the patch-clamp technique. TRPV2 expression was detected in the RPE of the mouse retina as well as in ARPE-19 cells. Increasing the temperature to 45 °C activated membrane conductance sensitive to SKF-96365 and ruthenium red in 60 % of cells. Preincubation with either cannabidiol (CBD) or IGF-1 led to a three- or fourfold increase in current density, respectively, in all cells, which was blocked by SKF-96365. In contrast to IGF-1, CBD stimulation of membrane conductance was further increased by heat. TRPV2 surface expression was increased by both IGF-1 and CBD, with the increase by CBD twice as large as that by IGF-1. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 abolished the effects on membrane conductance and surface expression. Both CBD and IGF-1 enhance TRPV2 channel activity by specific proportions of both channel activation and PI 3-kinase-dependent surface expression: IGF-1 predominantly increases ion channel activity, whereas CBD is more active in increasing TRPV2 surface expression. Thus, differential regulation of TRPV2 surface expression is an important mechanism for modulating the responsiveness of the RPE to growth factors.

Regulation of Biofilm Formation in Escherichia coli O157:H7

USDA-ARS?s Scientific Manuscript database

Escherichia coli O157:H7 encodes a variety of genetic factors for adherence to epithelial cells and to abiotic surfaces. While adherence to epithelial cells culminates in the formation of characteristic attaching and effacing (A/E) lesions, adherence to abiotic surfaces represents a prelude to the f...

The Class III Kinase Vps34 Promotes T Lymphocyte Survival through Regulating IL-7Rα Surface Expression

PubMed Central

McLeod, Ian X.; Zhou, Xiang; Li, Qi-Jing; Wang, Fan; He, You-Wen

2011-01-01

IL-7Rα mediated signals are essential for naive T lymphocyte survival. Recent studies show that IL-7Rα is internalized and either recycled to cell surface or degraded. However, how the intracellular process of IL-7Rα trafficking is regulated is unclear. Here we show that Vps34, the class III phosphatidylinositol 3-kinase, plays a critical role in proper IL-7Rα intracellular trafficking. Mice lacking Vps34 in T lymphocytes had a severely reduced T lymphocyte compartment. Vps34-deficient T lymphocytes exhibit increased death and reduced IL-7Rα surface expression, though three major forms of autophagy remain intact. Intracellular IL-7Rα in normal T lymphocytes at steady-state is trafficked through either early endosome/multivesicular bodies (MVB) to the late endosome-Golgi for surface expression or to the lysosome for degradation. However, Vps34-deficient T cells have mislocalized intracellular Eea1, HRS, and Vps36 protein levels, the combined consequence of which is the inability to mobilize internalized IL-7Rα into the retromer pathway for surface display. Our studies reveal that Vps34, though dispensible for autophagy induction, is a critical regulator of naïve T cell homeostasis, modulating IL-7Rα trafficking, signaling, and recycling. PMID:22021616

Regulation of cytokine receptors by Golgi N-glycan processing and endocytosis.

PubMed

Partridge, Emily A; Le Roy, Christine; Di Guglielmo, Gianni M; Pawling, Judy; Cheung, Pam; Granovsky, Maria; Nabi, Ivan R; Wrana, Jeffrey L; Dennis, James W

2004-10-01

The Golgi enzyme beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is up-regulated in carcinomas and promotes the substitution of N-glycan with poly N-acetyllactosamine, the preferred ligand for galectin-3 (Gal-3). Here, we report that expression of Mgat5 sensitized mouse cells to multiple cytokines. Gal-3 cross-linked Mgat5-modified N-glycans on epidermal growth factor and transforming growth factor-beta receptors at the cell surface and delayed their removal by constitutive endocytosis. Mgat5 expression in mammary carcinoma was rate limiting for cytokine signaling and consequently for epithelial-mesenchymal transition, cell motility, and tumor metastasis. Mgat5 also promoted cytokine-mediated leukocyte signaling, phagocytosis, and extravasation in vivo. Thus, conditional regulation of N-glycan processing drives synchronous modification of cytokine receptors, which balances their surface retention against loss via endocytosis.

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains.

PubMed

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS.

The Endogenous GRP78 Interactome in Human Head and Neck Cancers: A Deterministic Role of Cell Surface GRP78 in Cancer Stemness.

PubMed

Chen, Hsin-Ying; Chang, Joseph Tung-Chieh; Chien, Kun-Yi; Lee, Yun-Shien; You, Guo-Rung; Cheng, Ann-Joy

2018-01-11

Cell surface glucose regulated protein 78 (GRP78), an endoplasmic reticulum (ER) chaperone, was suggested to be a cancer stem cell marker, but the influence of this molecule on cancer stemness is poorly characterized. In this study, we developed a mass spectrometry platform to detect the endogenous interactome of GRP78 and investigated its role in cancer stemness. The interactome results showed that cell surface GRP78 associates with multiple molecules. The influence of cell population heterogeneity of head and neck cancer cell lines (OECM1, FaDu, and BM2) according to the cell surface expression levels of GRP78 and the GRP78 interactome protein, Progranulin, was investigated. The four sorted cell groups exhibited distinct cell cycle distributions, asymmetric/symmetric cell divisions, and different relative expression levels of stemness markers. Our results demonstrate that cell surface GRP78 promotes cancer stemness, whereas drives cells toward a non-stemlike phenotype when it chaperones Progranulin. We conclude that cell surface GRP78 is a chaperone exerting a deterministic influence on cancer stemness.

Cloning of B cell-specific membrane tetraspanning molecule BTS possessing B cell proliferation-inhibitory function.

PubMed

Suenaga, Tadahiro; Arase, Hisashi; Yamasaki, Sho; Kohno, Masayuki; Yokosuka, Tadashi; Takeuchi, Arata; Hattori, Takamichi; Saito, Takashi

2007-11-01

Lymphocyte proliferation is regulated by signals through antigen receptors, co-stimulatory receptors, and other positive and negative modulators. Several membrane tetraspanning molecules are also involved in the regulation of lymphocyte growth and death. We cloned a new B cell-specific tetraspanning (BTS) membrane molecule, which is similar to CD20 in terms of expression, structure and function. BTS is specifically expressed in the B cell line and its expression is increased after the pre-B cell stage. BTS is expressed in intracellular granules and on the cell surface. Overexpression of BTS in immature B cell lines induces growth retardation through inhibition of cell cycle progression and cell size increase without inducing apoptosis. This inhibitory function is mediated predominantly by the N terminus of BTS. The development of mature B cells is inhibited in transgenic mice expressing BTS, suggesting that BTS is involved in the in vivo regulation of B cells. These results indicate that BTS plays a role in the regulation of cell division and B cell growth.

Dominant-Negative Regulation of Cell Surface Expression by a Pentapeptide Motif at the Extreme COOH Terminus of an Slo1 Calcium-Activated Potassium Channel Splice Variant

PubMed Central

Chiu, Yu-Hsin; Alvarez-Baron, Claudia; Kim, Eun Young

2010-01-01

Large-conductance Ca2+-activated K+ (BKCa) channels regulate the physiology of many cell types. A single vertebrate gene variously known as Slo1, KCa1.1, or KCNMA1 encodes the pore-forming subunits of BKCa channel but is expressed in a potentially very large number of alternative splice variants. Two splice variants of Slo1, Slo1VEDEC and Slo1QEERL, which differ at the extreme COOH terminus, show markedly different steady-state expression levels on the cell surface. Here we show that Slo1VEDEC and Slo1QEERL can reciprocally coimmunoprecipitate, indicating that they form heteromeric complexes. Moreover, coexpression of even small amounts of Slo1VEDEC markedly reduces surface expression of Slo1QEERL and total Slo1 as indicated by cell-surface biotinylation assays. The effects of Slo1VEDEC on steady-state surface expression can be attributed primarily to the last five residues of the protein based on surface expression of motif-swapped constructs of Slo1 in human embryonic kidney (HEK) 293T cells. In addition, the presence of the VEDEC motif at the COOH terminus of Slo1 channels is sufficient to confer a dominant-negative effect on cell surface expression of itself or other types of Slo1 subunits. Treating cells with short peptides containing the VEDEC motif increased surface expression of Slo1VEDEC channels transiently expressed in HEK293T cells and increased current through endogenous BKCa channels in mouse podocytes. Slo1VEDEC and Slo1QEERL channels are removed from the HEK293T cell surface with similar kinetics and to a similar extent, which suggests that the inhibitory effect of the VEDEC motif is exerted primarily on forward trafficking into the plasma membrane. PMID:20051533

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation.

PubMed

Reis, Carlos R; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L

2017-01-17

Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand-death receptor (TRAIL-DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL-DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL-DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors.

TRAIL-death receptor endocytosis and apoptosis are selectively regulated by dynamin-1 activation

PubMed Central

Reis, Carlos R.; Chen, Ping-Hung; Bendris, Nawal; Schmid, Sandra L.

2017-01-01

Clathrin-mediated endocytosis (CME) constitutes the major pathway for uptake of signaling receptors into eukaryotic cells. As such, CME regulates signaling from cell-surface receptors, but whether and how specific signaling receptors reciprocally regulate the CME machinery remains an open question. Although best studied for its role in membrane fission, the GTPase dynamin also regulates early stages of CME. We recently reported that dynamin-1 (Dyn1), previously assumed to be neuron-specific, can be selectively activated in cancer cells to alter endocytic trafficking. Here we report that dynamin isoforms differentially regulate the endocytosis and apoptotic signaling downstream of TNF-related apoptosis-inducing ligand–death receptor (TRAIL–DR) complexes in several cancer cells. Whereas the CME of constitutively internalized transferrin receptors is mainly dependent on the ubiquitously expressed Dyn2, TRAIL-induced DR endocytosis is selectively regulated by activation of Dyn1. We show that TRAIL stimulation activates ryanodine receptor-mediated calcium release from endoplasmic reticulum stores, leading to calcineurin-mediated dephosphorylation and activation of Dyn1, TRAIL–DR endocytosis, and increased resistance to TRAIL-induced apoptosis. TRAIL–DR-mediated ryanodine receptor activation and endocytosis is dependent on early caspase-8 activation. These findings delineate specific mechanisms for the reciprocal crosstalk between signaling and the regulation of CME, leading to autoregulation of endocytosis and signaling downstream of surface receptors. PMID:28049841

Protein Kinase WNK1 Promotes Cell Surface Expression of Glucose Transporter GLUT1 by Regulating a Tre-2/USP6-BUB2-Cdc16 Domain Family Member 4 (TBC1D4)-Rab8A Complex*

PubMed Central

Mendes, Ana Isabel; Matos, Paulo; Moniz, Sónia; Jordan, Peter

2010-01-01

One mechanism by which mammalian cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular stores through inactivation of the Rab GTPase activating protein Tre-2/USP6-BUB2-Cdc16 domain family member 4 (TBC1D4) (also known as AS160). Here we describe that TBC1D4 forms a protein complex with protein kinase WNK1 in human embryonic kidney (HEK293) cells. We show that WNK1 phosphorylates TBC1D4 in vitro and that the expression levels of WNK1 in these cells regulate surface expression of the constitutive glucose transporter GLUT1. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. PMID:20937822

Design and fabrication of miniaturized PEM fuel cell combined microreactor with self-regulated hydrogen mechanism

NASA Astrophysics Data System (ADS)

Balakrishnan, A.; Frei, M.; Kerzenmacher, S.; Reinecke, H.; Mueller, C.

2015-12-01

In this work we present the design and fabrication of the miniaturized PEM fuel cell combined microreactor system with hydrogen regulation mechanism and testing of prototype microreactor. The system consists of two components (i) fuel cell component and (ii) microreactor component. The fuel cell component represents the miniaturized PEM fuel cell system (combination of screen printed fuel cell assembly and an on-board hydrogen storage medium). Hydrogen production based on catalytic hydrolysis of chemical hydride takes place in the microreactor component. The self-regulated hydrogen mechanism based on the gaseous hydrogen produced from the catalytic hydrolysis of sodium borohydride (NaBH4) gets accumulated as bubbles at the vicinity of the hydrophobic coated hydrogen exhaust holes. When the built up hydrogen bubbles pressure exceeds the burst pressure at the hydrogen exhaust holes the bubble collapses. This collapse causes a surge of fresh NaBH4 solution onto the catalyst surface leading to the removal of the reaction by-products formed at the active sites of the catalyst. The catalyst used in the system is platinum deposited on a base substrate. Nickel foam, carbon porous medium (CPM) and ceramic plate were selected as candidates for base substrate for developing a robust catalyst surface. For the first time the platinum layer fabricated by pulsed electrodeposition and dealloying (EPDD) technique is used for hydrolysis of NaBH4. The major advantages of such platinum catalyst layers are its high surface area and their mechanical stability. Prototype microreactor system with self-regulated hydrogen mechanism is demonstrated.

UV laser-ablated surface textures as potential regulator of cellular response.

PubMed

Chandra, Prafulla; Lai, Karen; Sung, Hak-Joon; Murthy, N Sanjeeva; Kohn, Joachim

2010-06-01

Textured surfaces obtained by UV laser ablation of poly(ethylene terephthalate) films were used to study the effect of shape and spacing of surface features on cellular response. Two distinct patterns, cones and ripples with spacing from 2 to 25 μm, were produced. Surface features with different shapes and spacings were produced by varying pulse repetition rate, laser fluence, and exposure time. The effects of the surface texture parameters, i.e., shape and spacing, on cell attachment, proliferation, and morphology of neonatal human dermal fibroblasts and mouse fibroblasts were studied. Cell attachment was the highest in the regions with cones at ∼4 μm spacing. As feature spacing increased, cell spreading decreased, and the fibroblasts became more circular, indicating a stress-mediated cell shrinkage. This study shows that UV laser ablation is a useful alternative to lithographic techniques to produce surface patterns for controlling cell attachment and growth on biomaterial surfaces.

CD44 staining of cancer stem-like cells is influenced by down-regulation of CD44 variant isoforms and up-regulation of the standard CD44 isoform in the population of cells that have undergone epithelial-to-mesenchymal transition.

PubMed

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44(high) cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44(high) population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44(high) population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44(high) population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed.

Physical and biological regulation of neuron regenerative growth and network formation on recombinant dragline silks

DOE PAGES

An, Bo; Tang-Schomer, Min D.; Huang, Wenwen; ...

2015-02-11

In this paper, recombinant spider silks produced in transgenic goat milk were studied as cell culture matrices for neuronal growth. Major ampullate spidroin 1 (MaSp1) supported neuronal growth, axon extension and network connectivity, with cell morphology comparable to the gold standard poly-lysine. In addition, neurons growing on MaSp1 films had increased neural cell adhesion molecule (NCAM) expression at both mRNA and protein levels. The results indicate that MaSp1 films present useful surface charge and substrate stiffness to support the growth of primary rat cortical neurons. Moreover, a putative neuron-specific surface binding sequence GRGGL within MaSp1 may contribute to the biologicalmore » regulation of neuron growth. These findings indicate that MaSp1 could regulate neuron growth through its physical and biological features. Finally, this dual regulation mode of MaSp1 could provide an alternative strategy for generating functional silk materials for neural tissue engineering.« less

Physical and biological regulation of neuron regenerative growth and network formation on recombinant dragline silks

DOE Office of Scientific and Technical Information (OSTI.GOV)

An, Bo; Tang-Schomer, Min D.; Huang, Wenwen

In this paper, recombinant spider silks produced in transgenic goat milk were studied as cell culture matrices for neuronal growth. Major ampullate spidroin 1 (MaSp1) supported neuronal growth, axon extension and network connectivity, with cell morphology comparable to the gold standard poly-lysine. In addition, neurons growing on MaSp1 films had increased neural cell adhesion molecule (NCAM) expression at both mRNA and protein levels. The results indicate that MaSp1 films present useful surface charge and substrate stiffness to support the growth of primary rat cortical neurons. Moreover, a putative neuron-specific surface binding sequence GRGGL within MaSp1 may contribute to the biologicalmore » regulation of neuron growth. These findings indicate that MaSp1 could regulate neuron growth through its physical and biological features. Finally, this dual regulation mode of MaSp1 could provide an alternative strategy for generating functional silk materials for neural tissue engineering.« less

Ecto-F1-ATPase: A moonlighting protein complex and an unexpected apoA-I receptor

PubMed Central

Vantourout, Pierre; Radojkovic, Claudia; Lichtenstein, Laeticia; Pons, Véronique; Champagne, Eric; Martinez, Laurent O

2010-01-01

Mitochondrial ATP synthase has been recently detected at the surface of different cell types, where it is a high affinity receptor for apoA-I, the major protein component in high density lipoproteins (HDL). Cell surface ATP synthase (namely ecto-F1-ATPase) expression is related to different biological effects, such as regulation of HDL uptake by hepatocytes, endothelial cell proliferation or antitumor activity of Vγ9/Vδ2 T lymphocytes. This paper reviews the recently discovered functions and regulations of ecto-F1-ATPase. Particularly, the role of the F1-ATPase pathway(s) in HDL-cholesterol uptake and apoA-I-mediated endothelial protection suggests its potential importance in reverse cholesterol transport and its regulation might represent a potential therapeutic target for HDL-related therapy for cardiovascular diseases. Therefore, it is timely for us to better understand how this ecto-enzyme and downstream pathways are regulated and to develop pharmacologic interventions. PMID:21157968

Physical and biological regulation of neuron regenerative growth and network formation on recombinant dragline silks.

PubMed

An, Bo; Tang-Schomer, Min; Huang, Wenwen; He, Jiuyang; Jones, Justin; Lewis, Randolph V; Kaplan, David L

2015-04-01

Recombinant spider silks produced in transgenic goat milk were studied as cell culture matrices for neuronal growth. Major ampullate spidroin 1 (MaSp1) supported neuronal growth, axon extension and network connectivity, with cell morphology comparable to the gold standard poly-lysine. In addition, neurons growing on MaSp1 films had increased neural cell adhesion molecule (NCAM) expression at both mRNA and protein levels. The results indicate that MaSp1 films present useful surface charge and substrate stiffness to support the growth of primary rat cortical neurons. Moreover, a putative neuron-specific surface binding sequence GRGGL within MaSp1 may contribute to the biological regulation of neuron growth. These findings indicate that MaSp1 could regulate neuron growth through its physical and biological features. This dual regulation mode of MaSp1 could provide an alternative strategy for generating functional silk materials for neural tissue engineering. Copyright © 2015 Elsevier Ltd. All rights reserved.

The Cryptococcus neoformans Capsule: a Sword and a Shield

PubMed Central

O'Meara, Teresa R.

2012-01-01

Summary: The human fungal pathogen Cryptococcus neoformans is characterized by its ability to induce a distinct polysaccharide capsule in response to a number of host-specific environmental stimuli. The induction of capsule is a complex biological process encompassing regulation at multiple steps, including the biosynthesis, transport, and maintenance of the polysaccharide at the cell surface. By precisely regulating the composition of its cell surface and secreted polysaccharides, C. neoformans has developed intricate ways to establish chronic infection and dormancy in the human host. The plasticity of the capsule structure in response to various host conditions also underscores the complex relationship between host and parasite. Much of this precise regulation of capsule is achieved through the transcriptional responses of multiple conserved signaling pathways that have been coopted to regulate this C. neoformans-specific virulence-associated phenotype. This review focuses on specific host stimuli that trigger the activation of the signal transduction cascades and on the downstream transcriptional responses that are required for robust encapsulation around the cell. PMID:22763631

Differential effect of rebamipide on transmembrane mucin biosynthesis in stratified ocular surface epithelial cells.

PubMed

Uchino, Yuichi; Woodward, Ashley M; Argüeso, Pablo

2016-12-01

Mucins are a group of highly glycosylated glycoproteins responsible for the protection of wet-surfaced epithelia. Recent data indicate that transmembrane mucins differ in their contribution to the protective function of the ocular surface, with MUC16 being the most effective barrier on the apical surface glycocalyx. Here, we investigated the role of the mucoprotective drug rebamipide in the regulation of transmembrane mucin biosynthesis using stratified cultures of human corneal and conjunctival epithelial cells. We find that the addition of rebamipide to corneal, but not conjunctival, epithelial cells increased MUC16 protein biosynthesis. Rebamipide did not affect the levels of MUC1, 4 and 20 compared to control. In these experiments, rebamipide had no effect on the expression levels of Notch intracellular domains, suggesting that the rebamipide-induced increase in MUC16 biosynthesis in differentiated corneal cultures is not regulated by Notch signaling. Overall these findings indicate that rebamipide induces the differential upregulation of MUC16 in stratified cultures of human corneal epithelial cells, which may have implications to the proper restoration of barrier function in ocular surface disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

Intracellular Acidosis Promotes Mitochondrial Apoptosis Pathway: Role of EMMPRIN Down-regulation via Specific Single-chain Fv Intrabody

PubMed Central

Thammasit, Patcharin; Sangboonruang, Sirikwan; Suwanpairoj, Supattara; Khamaikawin, Wannisa; Intasai, Nutjeera; Kasinrerk, Watchara; Tayapiwatana, Chatchai; Tragoolpua, Khajornsak

2015-01-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a human leukocyte surface molecule that is enriched on the surface of many cancer cells, and it plays an important role in proliferation and metastasis. In this study, we utilized the chimeric adenoviral vector Ad5/F35 carrying gene encoding scFv against EMMPRIN (scFv-M6-1B9) to down-regulate EMMPRIN cell surface expression and investigated programmed cell death response in colorectal cancer (CRC) cell, Caco-2. The scFv-M6-1B9 intrabody exhibits robust activity in reducing EMMPRIN cell surface expression. This approach led to the inducing of apoptosis, which was relative to the increasing of apoptotic bodies in sub-G1 peak, phosphatidylserine externalization, as well as TUNEL-positive cells. In addition, real-time RT-PCR and western blotting analysis indicated that apoptosis was enhanced through the mitochondrial pathway, a marked reduction of Bcl-2, leading to the translocation of cytochrome c and also the dramatic activation of caspase-3. Moreover, carcinoembryonic antigen (CEA), a tumor marker for CRC, was found to have significantly diminished in both secreted protein and mRNA levels. In conclusion, these findings suggest that EMMPRIN down-regulation by scFv-M6-1B9 intrabody has great potential in enhancing the efficacy of apoptosis induction through the mitochondrial pathway and in effecting a decline in the CEA level. Thus, its benefits could be applied to project the future prospects for targeted gene therapy and therapeutic application in monitoring colorectal cancer. PMID:25663946

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

PubMed Central

Shipp, M A; Tarr, G E; Chen, C Y; Switzer, S N; Hersh, L B; Stein, H; Sunday, M E; Reinherz, E L

1991-01-01

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung. Images PMID:1660144

CD10/neutral endopeptidase 24.11 hydrolyzes bombesin-like peptides and regulates the growth of small cell carcinomas of the lung.

PubMed

Shipp, M A; Tarr, G E; Chen, C Y; Switzer, S N; Hersh, L B; Stein, H; Sunday, M E; Reinherz, E L

1991-12-01

Bombesin-like peptides are essential autocrine growth factors for many small cell carcinomas (SCCas) of the lung. Herein, we demonstrate that these malignant pulmonary neuroendocrine cells express low levels of the cell surface metalloendopeptidase CD10/neutral endopeptidase 24.11 (CD10/NEP, common acute lymphoblastic leukemia antigen) and that this enzyme hydrolyzes bombesin-like peptides. The growth of bombesin-like peptide-dependent SCC as is inhibited by CD10/NEP and potentiated by CD10/NEP inhibition. The results provide evidence that CD10/NEP is involved in the regulation of tumor cell proliferation. Since SCCa of the lung occurs almost exclusively in cigarette smokers and cigarette smoke inactivates CD10/NEP, decreased cell surface CD10/NEP enzymatic activity may be causally related to the development of SCCa of the lung.

CMTM3 (CKLF-Like Marvel Transmembrane Domain 3) Mediates Angiogenesis by Regulating Cell Surface Availability of VE-Cadherin in Endothelial Adherens Junctions.

PubMed

Chrifi, Ihsan; Louzao-Martinez, Laura; Brandt, Maarten; van Dijk, Christian G M; Burgisser, Petra; Zhu, Changbin; Kros, Johan M; Duncker, Dirk J; Cheng, Caroline

2017-06-01

Decrease in VE-cadherin adherens junctions reduces vascular stability, whereas disruption of adherens junctions is a requirement for neovessel sprouting during angiogenesis. Endocytosis plays a key role in regulating junctional strength by altering bioavailability of cell surface proteins, including VE-cadherin. Identification of new mediators of endothelial endocytosis could enhance our understanding of angiogenesis. Here, we assessed the function of CMTM3 (CKLF-like MARVEL transmembrane domain 3), which we have previously identified as highly expressed in Flk1 + endothelial progenitor cells during embryonic development. Using a 3-dimensional coculture of human umbilical vein endothelial cells-GFP (green fluorescent protein) and pericytes-RFP (red fluorescent protein), we demonstrated that siRNA-mediated CMTM3 silencing in human umbilical vein endothelial cells impairs angiogenesis. In vivo CMTM3 inhibition by morpholino injection in developing zebrafish larvae confirmed that CMTM3 expression is required for vascular sprouting. CMTM3 knockdown in human umbilical vein endothelial cells does not affect proliferation or migration. Intracellular staining demonstrated that CMTM3 colocalizes with early endosome markers EEA1 (early endosome marker 1) and Clathrin + vesicles and with cytosolic VE-cadherin in human umbilical vein endothelial cells. Adenovirus-mediated CMTM3 overexpression enhances endothelial endocytosis, shown by an increase in Clathrin + , EEA1 + , Rab11 + , Rab5 + , and Rab7 + vesicles. CMTM3 overexpression enhances, whereas CMTM3 knockdown decreases internalization of cell surface VE-cadherin in vitro. CMTM3 promotes loss of endothelial barrier function in thrombin-induced responses, shown by transendothelial electric resistance measurements in vitro. In this study, we have identified a new regulatory function for CMTM3 in angiogenesis. CMTM3 is involved in VE-cadherin turnover and is a regulator of the cell surface pool of VE-cadherin. Therefore, CMTM3 mediates cell-cell adhesion at adherens junctions and contributes to the control of vascular sprouting. © 2017 American Heart Association, Inc.

Cellular localization of the activated EGFR determines its effect on cell growth in MDA-MB-468 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hyatt, Dustin C.; Ceresa, Brian P.

2008-11-01

The epidermal growth factor (EGF) receptor (EGFR) is a ubiquitously expressed receptor tyrosine kinase that regulates diverse cell functions that are dependent upon cell type, the presence of downstream effectors, and receptor density. In addition to activating biochemical pathways, ligand stimulation causes the EGFR to enter the cell via clathrin-coated pits. Endocytic trafficking influences receptor signaling by controlling the duration of EGFR phosphorylation and coordinating the receptor's association with downstream effectors. To better understand the individual contributions of cell surface and cytosolic EGFRs on cell physiology, we used EGF that was conjugated to 900 nm polystyrene beads (EGF-beads). EGF-beads canmore » stimulate the EGFR and retain the activated receptor at the plasma membrane. In MDA-MB-468 cells, a breast cancer cell line that over-expresses the EGFR, only internalized, activated EGFRs stimulate caspase-3 and induce cell death. Conversely, signaling cascades triggered from activated EGFR retained at the cell surface inhibit caspase-3 and promote cell proliferation. Thus, through endocytosis, the activated EGFR can differentially regulate cell growth in MDA-MB-468 cells.« less

Surface localization of the nuclear receptor CAR in influenza A virus-infected cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Takahashi, Tadanobu; Department of Biochemistry, School of Pharmaceutical Sciences, University of Shizuoka, CREST, JST, and COE Program in the 21st Century, Shizuoka 422-8526; Moriyama, Yusuke

Constitutive active/androstane receptor CAR is a member of the nuclear receptors which regulate transcription of xenobiotic metabolism enzymes. CAR is usually localized in the cytosol and nucleus. Here, we found that CAR was localized at the cell surface of influenza A virus (IAV)-infected cells. Additionally, we demonstrated that expression of a viral envelope glycoprotein, either hemagglutinin (HA) or neuraminidase (NA), but not viral nucleoprotein (NP), was responsible for this localization. This report is the first demonstration of CAR at the surface of tissue culture cells, and suggests that CAR may exert the IAV infection mechanism.

Regulation of Eosinophil Recruitment and Activation by Galectins in Allergic Asthma.

PubMed

Rao, Savita P; Ge, Xiao Na; Sriramarao, P

2017-01-01

Eosinophils are differentiated granulocytes that are recruited from the bone marrow to sites of inflammation via the vascular system. Allergic asthma is characterized by the presence of large numbers of eosinophils in the lungs and airways. Due to their capacity to rapidly release inflammatory mediators such as cytokines, chemokines, growth factors, and cytotoxic granule proteins upon stimulation, eosinophils play a critical role in pro-inflammatory processes in allergen-exposed lungs. Identifying key players and understanding the molecular mechanisms directing eosinophil trafficking and recruitment to inflamed airways is a key to developing therapeutic strategies to limit their influx. Recent studies have brought to light the important role of glycans and glycan binding proteins in regulating recruitment of eosinophils. In addition to the role of previously identified eosinophil- and endothelial-expressed adhesion molecules in mediating eosinophil trafficking and recruitment to the inflamed airways, studies have also indicated a role for galectins (galectin-3) in this process. Galectins are mammalian lectins expressed by various cell types including eosinophils. Intracellularly, they can regulate biological processes such as cell motility. Extracellularly, galectins interact with β-galactosides in cell surface-expressed glycans to regulate cellular responses like production of inflammatory mediators, cell adhesion, migration, and apoptosis. Eosinophils express galectins intracellularly or on the cell surface where they interact with cell surface glycoconjugate receptors. Depending on the type (galectin-1, -3, etc.) and location (extracellular or intracellular, endogenous or exogenously delivered), galectins differentially regulate eosinophil recruitment, activation, and apoptosis and thus exert a pro- or anti-inflammatory outcome. Here, we have reviewed information pertaining to galectins (galectin-1, -3 -9, and -10) that are expressed by eosinophils themselves and/or other cells that play a role in eosinophil recruitment and function in the context of allergic asthma and their potential use as disease biomarkers or therapeutic targets for immunomodulation.

Rbt1 protein domains analysis in Candida albicans brings insights into hyphal surface modifications and Rbt1 potential role during adhesion and biofilm formation.

PubMed

Monniot, Céline; Boisramé, Anita; Da Costa, Grégory; Chauvel, Muriel; Sautour, Marc; Bougnoux, Marie-Elisabeth; Bellon-Fontaine, Marie-Noëlle; Dalle, Frédéric; d'Enfert, Christophe; Richard, Mathias L

2013-01-01

Cell wall proteins are central to the virulence of Candida albicans. Hwp1, Hwp2 and Rbt1 form a family of hypha-associated cell surface proteins. Hwp1 and Hwp2 have been involved in adhesion and other virulence traits but Rbt1 is still poorly characterized. To assess the role of Rbt1 in the interaction of C. albicans with biotic and abiotic surfaces independently of its morphological state, heterologous expression and promoter swap strategies were applied. The N-terminal domain with features typical of the Flo11 superfamily was found to be essential for adhesiveness to polystyrene through an increase in cell surface hydrophobicity. A 42 amino acid-long domain localized in the central part of the protein was shown to enhance the aggregation function. We demonstrated that a VTTGVVVVT motif within the 42 amino acid domain displayed a high β-aggregation potential and was responsible for cell-to-cell interactions by promoting the aggregation of hyphae. Finally, we showed through constitutive expression that while Rbt1 was directly accessible to antibodies in hyphae, it was not so in yeast. Similar results were obtained for another cell wall protein, namely Iff8, and suggested that modification of the cell wall structure between yeast and hyphae can regulate the extracellular accessibility of cell wall proteins independently of gene regulation.

A high-throughput study on endothelial cell adhesion and growth mediated by adsorbed serum protein via signaling pathway PCR array

PubMed Central

Qu, Yayun; Hong, Ying; Huang, Yan; Zhang, Yiwen; Yang, Dayun; Zhang, Fudan; Xi, Tingfei; Zhang, Deyuan

2018-01-01

Abstract The purpose of this paper is to utilize the signaling pathway polymerase chain reaction (PCR) arrays to investigate the activation of two important biological signaling pathways in endothelial cell adhesion and growth mediated by adsorbed serum protein on the surface of bare and titanium nitride (TiN)-coated nickel titanium (NiTi) alloys. First, the endothelial cells were cultured on the bare and TiN-coated NiTi alloys and chitosan films as control for 4 h and 24 h, respectively. Then, the total RNA of the cells was collected and the PCR arrays were performed. After that, the differentially expressed genes in the transforming growth factor beta (TGF-β) signaling pathway and the regulation of actin cytoskeleton pathway were screened out; and the further bioinformatics analyses were performed. The results showed that both TGF-β signaling pathway and regulation of actin cytoskeleton pathway were activated in the cells after 4 h and 24 h culturing on the surface of bare and TiN-coated NiTi alloys compared to the chitosan group. The activated TGF-β signaling pathway promoted cell adhesion; the activated regulation of actin cytoskeleton pathway promoted cell adhesion, spreading, growth and motility. In addition, the activation of both pathways was much stronger in the cells cultured for 24 h versus 4 h, which indicated that cell adhesion and growth became more favorable with longer time on the surface of two NiTi alloy materials. PMID:29423265

In vivo regulation of Bcl6 and T follicular helper cell development1

PubMed Central

Poholek, Amanda C.; Hansen, Kyle; Hernandez, Sairy G.; Eto, Danelle; Chandele, Anmol; Weinstein, Jason S.; Dong, Xuemei; Odegard, Jared M.; Kaech, Susan M.; Dent, Alexander L.; Crotty, Shane; Craft, Joe

2010-01-01

Follicular helper T (TFH) cells, defined by expression of the surface markers CXCR5 and PD-1 and synthesis of IL-21, require upregulation of the transcriptional repressor Bcl6 for their development and function in B cell maturation in germinal centers. We have explored the role of B cells, and the cytokines IL-6 and IL-21, in the in vivo regulation of Bcl6 expression and TFH cell development. We found that TFH cells are characterized by a Bcl6-dependent downregulation of P-selectin glycoprotein ligand-1 (PSGL1, a CCL19- and CCL21-binding protein), indicating that, like CXCR5 and PD-1 upregulation, modulation of PSGL1 expression is part of the TFH cell program of differentiation. B cells were neither required for initial upregulation of Bcl6 nor PSGL1 downregulation, suggesting these events preceded T-B cell interactions, although they were required for full development of the TFH cell phenotype, including CXCR5 and PD-1 upregulation, and IL-21 synthesis. Bcl6 upregulation and TFH cell differentiation were independent of IL-6 and IL-21, revealing that either cytokine is not absolutely required for development of Bcl6+ TFH cells in vivo. These data increase our understanding of Bcl6 regulation in TFH cells and their differentiation in vivo, and identifies a new surface marker that may be functionally relevant in this subset. PMID:20519643

Differential regulation of membrane-associated mucins in the human ocular surface epithelium.

PubMed

Hori, Yuichi; Spurr-Michaud, Sandra; Russo, Cindy Leigh; Argüeso, Pablo; Gipson, Ilene K

2004-01-01

Membrane-associated mucins present in the apical cells of the ocular surface epithelium (MUC1, -4, and -16) are believed to contribute to the maintenance of a hydrated and wet-surfaced epithelial phenotype. Serum and retinoic acid (RA) have been used to treat drying ocular surface diseases. The goal of this study was to determine whether serum or RA regulates the production of membrane-associated mucins in human conjunctival epithelial cells. A telomerase-immortalized human conjunctival epithelial cell line (HCjE) was used. Cells were cultured in serum-free medium to confluence and then cultured with either 10% calf serum or with 100 nM RA for 0 to 72 hours. Conventional RT-PCR was used to determine the expression of retinoic acid receptors (RARs) and quantitative real-time PCR was used to investigate the mRNA expression of MUC1, -4, and -16. Protein levels were assayed by immunoblot analysis, using the antibodies HMFG-2, 1G8, or OC125, which are specific to MUC1, -4 and -16, respectively. To determine whether RA-associated MUC4 mRNA induction is a direct or indirect effect, HCjE cells were treated with RA and the protein synthesis inhibitor cycloheximide (1.0 microg/mL) for 12 hours. MUC1 and -16, but not -4, mRNAs were detectable in HCjE cells grown in serum-free medium. Real-time PCR revealed that MUC4 mRNA was significantly induced by serum 3 hours after its addition, and that MUC1 and MUC16 mRNA levels were significantly upregulated at 72 hours. Western blot analysis demonstrated that the MUC1, -4, and -16 proteins increased over time after addition of serum. Conventional RT-PCR analysis demonstrated that RAR-alpha and -gamma mRNA were expressed in native human conjunctival tissue as well as in the HCjE cells. Treatment with RA upregulated the expression of both MUC4 and -16 mRNA and protein, but MUC1 was unaffected. Because the protein synthesis inhibitor cycloheximide did not prevent the RA-associated induction of MUC4 mRNA, the action of RA on the MUC4 promoter may be direct. The membrane-associated mucins of the ocular surface epithelia, MUC1, -4, and -16, are differentially regulated by serum and RA in the telomerase-immortalized human conjunctival epithelial cell line. Serum derived from vessels in the conjunctiva may play an important role in mucin regulation in the ocular surface epithelia. These data also support the clinical efficacy of autologous serum and RA application in patients with ocular surface diseases. Furthermore, the data suggest that MUC4 and -16 are particularly important hydrophilic molecules involved in maintenance of a healthy ocular surface.

Mapping cell surface adhesion by rotation tracking and adhesion footprinting

NASA Astrophysics Data System (ADS)

Li, Isaac T. S.; Ha, Taekjip; Chemla, Yann R.

2017-03-01

Rolling adhesion, in which cells passively roll along surfaces under shear flow, is a critical process involved in inflammatory responses and cancer metastasis. Surface adhesion properties regulated by adhesion receptors and membrane tethers are critical in understanding cell rolling behavior. Locally, adhesion molecules are distributed at the tips of membrane tethers. However, how functional adhesion properties are globally distributed on the individual cell’s surface is unknown. Here, we developed a label-free technique to determine the spatial distribution of adhesive properties on rolling cell surfaces. Using dark-field imaging and particle tracking, we extract the rotational motion of individual rolling cells. The rotational information allows us to construct an adhesion map along the contact circumference of a single cell. To complement this approach, we also developed a fluorescent adhesion footprint assay to record the molecular adhesion events from cell rolling. Applying the combination of the two methods on human promyelocytic leukemia cells, our results surprisingly reveal that adhesion is non-uniformly distributed in patches on the cell surfaces. Our label-free adhesion mapping methods are applicable to the variety of cell types that undergo rolling adhesion and provide a quantitative picture of cell surface adhesion at the functional and molecular level.

Reversible epigenetic down-regulation of MHC molecules by devil facial tumour disease illustrates immune escape by a contagious cancer

PubMed Central

Siddle, Hannah V.; Kreiss, Alexandre; Tovar, Cesar; Yuen, Chun Kit; Cheng, Yuanyuan; Belov, Katherine; Swift, Kate; Pearse, Anne-Maree; Hamede, Rodrigo; Jones, Menna E.; Skjødt, Karsten; Woods, Gregory M.; Kaufman, Jim

2013-01-01

Contagious cancers that pass between individuals as an infectious cell line are highly unusual pathogens. Devil facial tumor disease (DFTD) is one such contagious cancer that emerged 16 y ago and is driving the Tasmanian devil to extinction. As both a pathogen and an allograft, DFTD cells should be rejected by the host–immune response, yet DFTD causes 100% mortality among infected devils with no apparent rejection of tumor cells. Why DFTD cells are not rejected has been a question of considerable confusion. Here, we show that DFTD cells do not express cell surface MHC molecules in vitro or in vivo, due to down-regulation of genes essential to the antigen-processing pathway, such as β2-microglobulin and transporters associated with antigen processing. Loss of gene expression is not due to structural mutations, but to regulatory changes including epigenetic deacetylation of histones. Consequently, MHC class I molecules can be restored to the surface of DFTD cells in vitro by using recombinant devil IFN-γ, which is associated with up-regulation of the MHC class II transactivator, a key transcription factor with deacetylase activity. Further, expression of MHC class I molecules by DFTD cells can occur in vivo during lymphocyte infiltration. These results explain why T cells do not target DFTD cells. We propose that MHC-positive or epigenetically modified DFTD cells may provide a vaccine to DFTD. In addition, we suggest that down-regulation of MHC molecules using regulatory mechanisms allows evolvability of transmissible cancers and could affect the evolutionary trajectory of DFTD. PMID:23479617

EMMPRIN Regulates Cytoskeleton Reorganization and Cell Adhesion in Prostate Cancer

PubMed Central

Zhu, Haining; Zhao, Jun; Zhu, Beibei; Collazo, Joanne; Gal, Jozsef; Shi, Ping; Liu, Li; Ström, Anna-Lena; Lu, Xiaoning; McCann, Richard O.; Toborek, Michal; Kyprianou, Natasha

2011-01-01

Background Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment. Methods In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) vs. malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization. Results EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins. Conclusions Our results suggest that EMMPRIN regulates cell adhesion, invasion and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis. PMID:21563192

EMMPRIN regulates cytoskeleton reorganization and cell adhesion in prostate cancer.

PubMed

Zhu, Haining; Zhao, Jun; Zhu, Beibei; Collazo, Joanne; Gal, Jozsef; Shi, Ping; Liu, Li; Ström, Anna-Lena; Lu, Xiaoning; McCann, Richard O; Toborek, Michal; Kyprianou, Natasha

2012-01-01

Proteins on cell surface play important roles during cancer progression and metastasis via their ability to mediate cell-to-cell interactions and navigate the communication between cells and the microenvironment. In this study a targeted proteomic analysis was conducted to identify the differential expression of cell surface proteins in human benign (BPH-1) versus malignant (LNCaP and PC-3) prostate epithelial cells. We identified EMMPRIN (extracellular matrix metalloproteinase inducer) as a key candidate and shRNA functional approaches were subsequently applied to determine the role of EMMPRIN in prostate cancer cell adhesion, migration, invasion as well as cytoskeleton organization. EMMPRIN was found to be highly expressed on the surface of prostate cancer cells compared to BPH-1 cells, consistent with a correlation between elevated EMMPRIN and metastasis found in other tumors. No significant changes in cell proliferation, cell cycle progression, or apoptosis were detected in EMMPRIN knockdown cells compared to the scramble controls. Furthermore, EMMPRIN silencing markedly decreased the ability of PC-3 cells to form filopodia, a critical feature of invasive behavior, while it increased expression of cell-cell adhesion and gap junction proteins. Our results suggest that EMMPRIN regulates cell adhesion, invasion, and cytoskeleton reorganization in prostate cancer cells. This study identifies a new function for EMMPRIN as a contributor to prostate cancer cell-cell communication and cytoskeleton changes towards metastatic spread, and suggests its potential value as a marker of prostate cancer progression to metastasis. Copyright © 2011 Wiley Periodicals, Inc.

Cell Signaling Pathways that Regulate Ag Presentation

PubMed Central

Brutkiewicz, Randy R.

2016-01-01

Cell signaling pathways regulate much in the life of a cell: from shuttling cargo through intracellular compartments and onto the cell surface, how it should respond to stress, protecting itself from harm (environmental insults or infections), to ultimately, death by apoptosis. These signaling pathways are important for various aspects of the immune response as well. However, not much is known in terms of the participation of cell signaling pathways in Ag presentation--a necessary first step in the activation of innate and adaptive T cells. In this brief review, I will discuss the known signaling molecules (and pathways) that regulate how Ags are presented to T cells and the mechanism(s) if identified. Studies in this area have important implications in vaccine development and new treatment paradigms against infectious diseases, autoimmunity and cancer. PMID:27824592

Tumor Cells Surviving Exposure to Proton or Photon Radiation Share a Common Immunogenic Modulation Signature, Rendering Them More Sensitive to T Cell–Mediated Killing

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gameiro, Sofia R.; Malamas, Anthony S.; Bernstein, Michael B.

Purpose: To provide the foundation for combining immunotherapy to induce tumor antigen–specific T cells with proton radiation therapy to exploit the activity of those T cells. Methods and Materials: Using cell lines of tumors frequently treated with proton radiation, such as prostate, breast, lung, and chordoma, we examined the effect of proton radiation on the viability and induction of immunogenic modulation in tumor cells by flow cytometric and immunofluorescent analysis of surface phenotype and the functional immune consequences. Results: These studies show for the first time that (1) proton and photon radiation induced comparable up-regulation of surface molecules involved in immune recognition (histocompatibilitymore » leukocyte antigen, intercellular adhesion molecule 1, and the tumor-associated antigens carcinoembryonic antigen and mucin 1); (2) proton radiation mediated calreticulin cell-surface expression, increasing sensitivity to cytotoxic T-lymphocyte killing of tumor cells; and (3) cancer stem cells, which are resistant to the direct cytolytic activity of proton radiation, nonetheless up-regulated calreticulin after radiation in a manner similar to non-cancer stem cells. Conclusions: These findings offer a rationale for the use of proton radiation in combination with immunotherapy, including for patients who have failed radiation therapy alone or have limited treatment options.« less

High cell surface death receptor expression determines type I versus type II signaling.

PubMed

Meng, Xue Wei; Peterson, Kevin L; Dai, Haiming; Schneider, Paula; Lee, Sun-Hee; Zhang, Jin-San; Koenig, Alexander; Bronk, Steve; Billadeau, Daniel D; Gores, Gregory J; Kaufmann, Scott H

2011-10-14

Previous studies have suggested that there are two signaling pathways leading from ligation of the Fas receptor to induction of apoptosis. Type I signaling involves Fas ligand-induced recruitment of large amounts of FADD (FAS-associated death domain protein) and procaspase 8, leading to direct activation of caspase 3, whereas type II signaling involves Bid-mediated mitochondrial perturbation to amplify a more modest death receptor-initiated signal. The biochemical basis for this dichotomy has previously been unclear. Here we show that type I cells have a longer half-life for Fas message and express higher amounts of cell surface Fas, explaining the increased recruitment of FADD and subsequent signaling. Moreover, we demonstrate that cells with type II Fas signaling (Jurkat or HCT-15) can signal through a type I pathway upon forced receptor overexpression and that shRNA-mediated Fas down-regulation converts cells with type I signaling (A498) to type II signaling. Importantly, the same cells can exhibit type I signaling for Fas and type II signaling for TRAIL (TNF-α-related apoptosis-inducing ligand), indicating that the choice of signaling pathway is related to the specific receptor, not some other cellular feature. Additional experiments revealed that up-regulation of cell surface death receptor 5 levels by treatment with 7-ethyl-10-hydroxy-camptothecin converted TRAIL signaling in HCT116 cells from type II to type I. Collectively, these results suggest that the type I/type II dichotomy reflects differences in cell surface death receptor expression.

Promoting Thiol Expression Increases The Durability of Antitumor T cell Functions

PubMed Central

Scurti, Gina; Thyagarajan, Krishnamurthy; Kaur, Navtej; Husain, Shahid; Fang, Quan; Naga, Osama S.; Simms, Patricia; Beeson, Gyda; Voelkel-Johnson, Christina; Garrett-Mayer, Elizabeth; Beeson, Craig C.; Nishimura, Michael I.; Mehrotra, Shikhar

2014-01-01

Ex vivo-expanded CD8+ T cells used for adoptive immunotherapy generally acquire an effector memory-like phenotype (TEM cells). With regard to therapeutic applications, two undesired features of this phenotype in vivo are limited persistence and reduced anti-tumor efficacy, relative to CD8+ T cells with a central memory-like phenotype (TCM cells). Further, there is incomplete knowledge about all the differences between TEM and TCM cells that may influence tumor treatment outcomes. Given that TCM cells survive relatively longer in oxidative tumor microenvironments, we investigated the hypothesis that TCM possess relatively greater anti-oxidative capacity than TEM cells. Here we report that TCM cells exhibit a relative increase compared to TEM cells in expression of cell surface thiols, a key target of cellular redox controls, along with other antioxidant molecules. Increased expression of redox regulators in TCM cells inversely correlated with the generation of reactive oxygen and nitrogen species, proliferative capacity and glycolytic enzyme levels. Notably, TCR-transduced T cells pretreated with thiol donors, such as N-acetyl cysteine or rapamycin, up-regulated thiol levels and antioxidant genes. A comparison of anti-tumor CD8+ T cell populations on the basis of surface thiol expression showed that thiol-high cells persisted longer in vivo and exerted superior tumor control. Our results suggest that higher levels of reduced cell surface thiols are a key characteristic of T cells that can control tumor growth, and that profiling this biomarker may have benefits to T cell adoptive immunotherapy protocols. PMID:25164014

Flagellum Density Regulates Proteus mirabilis Swarmer Cell Motility in Viscous Environments

PubMed Central

Tuson, Hannah H.; Copeland, Matthew F.; Carey, Sonia; Sacotte, Ryan

2013-01-01

Proteus mirabilis is an opportunistic pathogen that is frequently associated with urinary tract infections. In the lab, P. mirabilis cells become long and multinucleate and increase their number of flagella as they colonize agar surfaces during swarming. Swarming has been implicated in pathogenesis; however, it is unclear how energetically costly changes in P. mirabilis cell morphology translate into an advantage for adapting to environmental changes. We investigated two morphological changes that occur during swarming—increases in cell length and flagellum density—and discovered that an increase in the surface density of flagella enabled cells to translate rapidly through fluids of increasing viscosity; in contrast, cell length had a small effect on motility. We found that swarm cells had a surface density of flagella that was ∼5 times larger than that of vegetative cells and were motile in fluids with a viscosity that inhibits vegetative cell motility. To test the relationship between flagellum density and velocity, we overexpressed FlhD4C2, the master regulator of the flagellar operon, in vegetative cells of P. mirabilis and found that increased flagellum density produced an increase in cell velocity. Our results establish a relationship between P. mirabilis flagellum density and cell motility in viscous environments that may be relevant to its adaptation during the infection of mammalian urinary tracts and movement in contact with indwelling catheters. PMID:23144253

Switching of the positive feedback for RAS activation by a concerted function of SOS membrane association domains

PubMed Central

Nakamura, Yuki; Hibino, Kayo; Yanagida, Toshio; Sako, Yasushi

2016-01-01

Son of sevenless (SOS) is a guanine nucleotide exchange factor that regulates cell behavior by activating the small GTPase RAS. Recent in vitro studies have suggested that an interaction between SOS and the GTP-bound active form of RAS generates a positive feedback loop that propagates RAS activation. However, it remains unclear how the multiple domains of SOS contribute to the regulation of the feedback loop in living cells. Here, we observed single molecules of SOS in living cells to analyze the kinetics and dynamics of SOS behavior. The results indicate that the histone fold and Grb2-binding domains of SOS concertedly produce an intermediate state of SOS on the cell surface. The fraction of the intermediated state was reduced in positive feedback mutants, suggesting that the feedback loop functions during the intermediate state. Translocation of RAF, recognizing the active form of RAS, to the cell surface was almost abolished in the positive feedback mutants. Thus, the concerted functions of multiple membrane-associating domains of SOS governed the positive feedback loop, which is crucial for cell fate decision regulated by RAS. PMID:27924253

Thiols decrease cytokine levels and down-regulate the expression of CD30 on human allergen-specific T helper (Th) 0 and Th2 cells

PubMed Central

Bengtsson, Å; Lundberg, M; Avila-Cariño, J; Jacobsson, G; Holmgren, A; Scheynius, A

2001-01-01

The thiol antioxidant N-acetyl-l-cysteine (NAC), known as a precursor of glutathione (GSH), is used in AIDS treatment trials, as a chemoprotectant in cancer chemotherapy and in treatment of chronic bronchitis. In vitro, GSH and NAC are known to enhance T cell proliferation, production of IL-2 and up-regulation of the IL-2 receptor. The 120-kD CD30 surface antigen belongs to the tumour necrosis factor (TNF) receptor superfamily. It is expressed by activated T helper (Th) cells and its expression is sustained in Th2 cells. We have analysed the effect of GSH and NAC on the cytokine profile and CD30 expression on human allergen-specific T cell clones (TCC). TCC were stimulated with anti-CD3 antibodies in the presence of different concentrations of GSH and NAC. Both thiols caused a dose dependent down-regulation of IL-4, IL-5 and IFN-γ levels in Th0 and Th2 clones, with the most pronounced decrease of IL-4. Furthermore, they down-regulated the surface expression of CD30, and the levels of soluble CD30 (sCD30) in the culture supernatants were decreased. In contrast, the surface expression of CD28 or CD40 ligand (CD40L) was not significantly changed after treatment with 20 mm NAC. These results indicate that GSH and NAC favour a Th1 response by a preferential down-regulation of IL-4. In addition, the expression of CD30 was down regulated by GSH and NAC, suggesting that CD30 expression is dependent on IL-4, or modified by NAC. In the likely event that CD30 and its soluble counterpart prove to contribute to the pathogenesis in Th2 related diseases such as allergy, NAC may be considered as a future therapeutic agent in the treatment of these diseases. PMID:11298119

Nanotechnology in the regulation of stem cell behavior

NASA Astrophysics Data System (ADS)

Wu, King-Chuen; Tseng, Ching-Li; Wu, Chi-Chang; Kao, Feng-Chen; Tu, Yuan-Kun; So, Edmund C.; Wang, Yang-Kao

2013-10-01

Stem cells are known for their potential to repair damaged tissues. The adhesion, growth and differentiation of stem cells are likely controlled by the surrounding microenvironment which contains both chemical and physical cues. Physical cues in the microenvironment, for example, nanotopography, were shown to play important roles in stem cell fate decisions. Thus, controlling stem cell behavior by nanoscale topography has become an important issue in stem cell biology. Nanotechnology has emerged as a new exciting field and research from this field has greatly advanced. Nanotechnology allows the manipulation of sophisticated surfaces/scaffolds which can mimic the cellular environment for regulating cellular behaviors. Thus, we summarize recent studies on nanotechnology with applications to stem cell biology, including the regulation of stem cell adhesion, growth, differentiation, tracking and imaging. Understanding the interactions of nanomaterials with stem cells may provide the knowledge to apply to cell-scaffold combinations in tissue engineering and regenerative medicine.

Visible light controls cell adhesion on a photoswitchable biointerface.

PubMed

Ming, Zunzhen; Hua, Xin; Xue, Yuan; Lin, Qiuning; Bao, Chunyan; Zhu, Linyong

2018-05-04

Bioactive surfaces with specific interactions with cells have been greatly interested due to their potential applications in biosensors and tissue engineering. Herein, we fabricated a dopamine contained photoswitch molecule (compound 1) which could form self-assembled monolayer (SAM) on substrates. The SAM showed a good photoswitch ability and manifested excellent fatigue resistance, which displayed its potential application as a biologically friendly surface coating. Contact angle analysis and cell experiments exhibited that the SAM surface was hydrophobic before irradiation which favored cell adhesion, while, it turned hydrophilic and induced cell unfouling or detachment after light irradiation. The uses of visible light stimulation (λ ex  = 530 nm) and the reversible regulation on cell adhesion and detachment should open up new avenues for bioacitve surfaces in biomedical applications. Copyright © 2018 Elsevier B.V. All rights reserved.

Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration.

PubMed

Schram, Kristin; Ganguly, Riya; No, Eun Kyung; Fang, Xiangping; Thong, Farah S L; Sweeney, Gary

2011-05-01

Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response to leptin, again via a Rho-dependent signaling mechanism. Our results suggest that leptin regulates myocardial matrix remodeling by regulating the cell surface localization of MT1-MMP in adult cardiac fibroblasts via Rho/ROCK-dependent actin cytoskeleton reorganization. Subsequent pro-MMP-2 activation then contributes to stimulation of cell migration.

CD147, CD44, and the epidermal growth factor receptor (EGFR) signaling pathway cooperate to regulate breast epithelial cell invasiveness.

PubMed

Grass, G Daniel; Tolliver, Lauren B; Bratoeva, Momka; Toole, Bryan P

2013-09-06

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777-788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer.

Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces – A Novel Method of Enhancing Flow-Resistant Cell Adhesion

PubMed Central

Tran, Phat L.; Gamboa, Jessica R.; McCracken, Katherine E.; Riley, Mark R.

2014-01-01

Assuring cell adhesion to an underlying biomaterial surface is vital in implant device design and tissue engineering, particularly under circumstances where cells are subjected to potential detachment from overriding fluid flow. Cell-substrate adhesion is a highly regulated process involving the interplay of mechanical properties, surface topographic features, electrostatic charge, and biochemical mechanisms. At the nanoscale level the physical properties of the underlying substrate are of particular importance in cell adhesion. Conventionally, natural, pro-adhesive, and often thrombogenic, protein biomaterials are frequently utilized to facilitate adhesion. In the present study nanofabrication techniques are utilized to enhance the biological functionality of a synthetic polymer surface, polymethymethacrylate, with respect to cell adhesion. Specifically we examine the effect on cell adhesion of combining: 1. optimized surface texturing, 2. electrostatic charge and 3. cell adhesive ligands, uniquely assembled on the substrata surface, as an ensemble of nanoparticles trapped in nanowells. Our results reveal that the ensemble strategy leads to enhanced, more than simply additive, endothelial cell adhesion under both static and flow conditions. This strategy may be of particular utility for enhancing flow-resistant endothelialization of blood-contacting surfaces of cardiovascular devices subjected to flow-mediated shear. PMID:23225491

Ferric reductase genes involved in high-affinity iron uptake are differentially regulated in yeast and hyphae of Candida albicans.

PubMed

Jeeves, Rose E; Mason, Robert P; Woodacre, Alexandra; Cashmore, Annette M

2011-09-01

The pathogenic yeast Candida albicans possesses a reductive iron uptake system which is active in iron-restricted conditions. The sequestration of iron by this mechanism initially requires the reduction of free iron to the soluble ferrous form, which is catalysed by ferric reductase proteins. Reduced iron is then taken up into the cell by a complex of a multicopper oxidase protein and an iron transport protein. Multicopper oxidase proteins require copper to function and so reductive iron and copper uptake are inextricably linked. It has previously been established that Fre10 is the major cell surface ferric reductase in C. albicans and that transcription of FRE10 is regulated in response to iron levels. We demonstrate here that Fre10 is also a cupric reductase and that Fre7 also makes a significant contribution to cell surface ferric and cupric reductase activity. It is also shown, for the first time, that transcription of FRE10 and FRE7 is lower in hyphae compared to yeast and that this leads to a corresponding decrease in cell surface ferric, but not cupric, reductase activity. This demonstrates that the regulation of two virulence determinants, the reductive iron uptake system and the morphological form of C. albicans, are linked. Copyright © 2011 John Wiley & Sons, Ltd.

MicroRNA miR-328 Regulates Zonation Morphogenesis by Targeting CD44 Expression

PubMed Central

Wang, Chia-Hui; Lee, Daniel Y.; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B.

2008-01-01

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion. PMID:18560585

MicroRNA miR-328 regulates zonation morphogenesis by targeting CD44 expression.

PubMed

Wang, Chia-Hui; Lee, Daniel Y; Deng, Zhaoqun; Jeyapalan, Zina; Lee, Shao-Chen; Kahai, Shireen; Lu, Wei-Yang; Zhang, Yaou; Yang, Burton B

2008-06-18

Morphogenesis is crucial to initiate physiological development and tumor invasion. Here we show that a microRNA controls zonation morphogenesis by targeting hyaluronan receptor CD44. We have developed a novel system to study microRNA functions by generating constructs expressing pre-miRNAs and mature miRNAs. Using this system, we have demonstrated that expression of miR-328 reduced cell adhesion, aggregation, and migration, and regulated formation of capillary structure. Protein analysis indicated that miR-328 repressed CD44 expression. Activities of luciferase constructs harboring the target site in CD44, but not the one containing mutation, were repressed by miR-328. Zonation morphogenesis appeared in cells transfected by miR-328: miR-328-transfected cells were present on the surface of zonating structures while the control cells stayed in the middle. MiR-328-mediated CD44 actions was validated by anti-CD44 antibody, hyaluronidase, CD44 siRNA, and CD44 expression constructs. In vivo experiments showed that CD44-silencing cells appeared as layers on the surfaces of nodules or zonating structures. Immuno-histochemistry also exhibited CD44-negative cells on the surface layers of normal rat livers and the internal zones of Portal veins. Our results demonstrate that miR-328 targets CD44, which is essential in regulating zonation morphogenesis: silencing of CD44 expression is essential in sealing the zonation structures to facilitate their extension and to inhibit complex expansion.

A novel phenoxazine derivative suppresses surface IgM expression in DT40 B cell line

PubMed Central

Gao, Sanyang; Takano, Tomoko; Sada, Kiyonao; He, Jinsong; Noda, Chiseko; Hori-Tamura, Naoko; Tomoda, Akio; Yamamura, Hirohei

2002-01-01

2-amino-4, 4α-dihydro-4α, 7-dimethyl-3H-phenoxazine-3-one (Phx) has been demonstrated to be an actinomycin D-like phenoxazine, and to display anti-tumour activity. In this study, we report on the effect of Phx on B cell antigen receptor (BCR) and receptor-mediated signalling in DT40 B cells. Treatment of B cells with Phx for 12 h inhibited BCR-stimulated tyrosine phosphorylation of cellular proteins. B cells exposed to Phx exhibited down-regulation of surface IgM which is part of BCR. In contracts with actinomycin D, Phx rapidly reduced the expression of IgM without decreasing the expression of other signalling molecules. Analysis with confocal microscopy demonstrated that Phx treatment reduced IgM expression both at the cell surface and inside the cell. Treatment of B cells with Phx resulted in the reduction of IgM secretion. Since MG-132, a proteasomal inhibitor, restored IgM contents to the control levels, Phx has the specific effect of accelerating IgM degradation. These results suggest that Phx down-regulates the expression of IgM and inhibits BCR-mediated signalling and IgM secretion. Phx may be useful as an immunosuppressive agent for therapeutic purposes. PMID:12411404

Human Cytomegalovirus UL18 Utilizes US6 for Evading the NK and T-Cell Responses

PubMed Central

Kim, Youngkyun; Park, Boyoun; Cho, Sunglim; Shin, Jinwook; Cho, Kwangmin; Jun, Youngsoo; Ahn, Kwangseog

2008-01-01

Human cytomegalovirus (HCMV) US6 glycoprotein inhibits TAP function, resulting in down-regulation of MHC class I molecules at the cell surface. Cells lacking MHC class I molecules are susceptible to NK cell lysis. HCMV expresses UL18, a MHC class I homolog that functions as a surrogate to prevent host cell lysis. Despite a high level of sequence and structural homology between UL18 and MHC class I molecules, surface expression of MHC class I, but not UL18, is down regulated by US6. Here, we describe a mechanism of action by which HCMV UL18 avoids attack by the self-derived TAP inhibitor US6. UL18 abrogates US6 inhibition of ATP binding by TAP and, thereby, restores TAP-mediated peptide translocation. In addition, UL18 together with US6 interferes with the physical association between MHC class I molecules and TAP that is required for optimal peptide loading. Thus, regardless of the recovery of TAP function, surface expression of MHC class I molecules remains decreased. UL18 represents a unique immune evasion protein that has evolved to evade both the NK and the T cell immune responses. PMID:18688275

Phenotypic Heterogeneity and the Evolution of Bacterial Life Cycles.

PubMed

van Gestel, Jordi; Nowak, Martin A

2016-02-01

Most bacteria live in colonies, where they often express different cell types. The ecological significance of these cell types and their evolutionary origin are often unknown. Here, we study the evolution of cell differentiation in the context of surface colonization. We particularly focus on the evolution of a 'sticky' cell type that is required for surface attachment, but is costly to express. The sticky cells not only facilitate their own attachment, but also that of non-sticky cells. Using individual-based simulations, we show that surface colonization rapidly evolves and in most cases leads to phenotypic heterogeneity, in which sticky and non-sticky cells occur side by side on the surface. In the presence of regulation, cell differentiation leads to a remarkable set of bacterial life cycles, in which cells alternate between living in the liquid and living on the surface. The dominant life stage is formed by the surface-attached colony that shows many complex features: colonies reproduce via fission and by producing migratory propagules; cells inside the colony divide labour; and colonies can produce filaments to facilitate expansion. Overall, our model illustrates how the evolution of an adhesive cell type goes hand in hand with the evolution of complex bacterial life cycles.

Function of OPG as a traffic regulator for RANKL is crucial for controlled osteoclastogenesis.

PubMed

Aoki, Shigeki; Honma, Masashi; Kariya, Yoshiaki; Nakamichi, Yuko; Ninomiya, Tadashi; Takahashi, Naoyuki; Udagawa, Nobuyuki; Suzuki, Hiroshi

2010-09-01

The amount of the receptor activator of NF-κB ligand (RANKL) on the osteoblastic cell surface is considered to determine the magnitude of the signal input to osteoclast precursors and the degree of osteoclastogenesis. Previously, we have shown that RANKL is localized predominantly in lysosomal organelles, but little is found on the osteoblastic cell surface, and consequently, the regulated subcellular trafficking of RANKL in osteoblastic cells is important for controlled osteoclastogenesis. Here we have examined the involvement of osteoprotegerin (OPG), which is currently recognized as a decoy receptor for RANKL, in the regulation of RANKL behavior. It was suggested that OPG already makes a complex with RANKL in the Golgi apparatus and that the complex formation is necessary for RANKL sorting to the secretory lysosomes. It was also shown that each structural domain of OPG is indispensable for exerting OPG function as a traffic regulator. In particular, the latter domains of OPG, whose physiologic functions have been unclear, were indicated to sort RANKL molecules to lysosomes from the Golgi apparatus. In addition, the overexpression of RANK-OPG chimeric protein, which retained OPG function as a decoy receptor but lost the function as a traffic regulator, inhibited endogenous OPG function as a traffic regulator selectively in osteoblastic cells and resulted in the upregulation of osteoclastogenic ability despite the increased number of decoy receptor molecules. Conclusively, OPG function as a traffic regulator for RANKL is crucial for regulating osteoclastogenesis at least as well as that as a decoy receptor. © 2010 American Society for Bone and Mineral Research.

Library Screen Identifies Enterococcus faecalis CcpA, the Catabolite Control Protein A, as an Effector of Ace, a Collagen Adhesion Protein Linked to Virulence

PubMed Central

Gao, Peng; Pinkston, Kenneth L.; Bourgogne, Agathe; Cruz, Melissa R.; Garsin, Danielle A.; Murray, Barbara E.

2013-01-01

The Enterococcus faecalis cell wall-anchored protein Ace is an important virulence factor involved in cell adhesion and infection. Expression of Ace on the cell surface is affected by many factors, including stage of growth, culture temperature, and environmental components, such as serum, urine, and collagen. However, the mechanisms that regulate or modulate Ace display are not well understood. With interest in identifying genes associated with Ace expression, we utilized a whole-cell enzyme-linked immunosorbent assay (ELISA)-based screening method to identify mutants from a transposon insertion mutant library which exhibited distinct Ace surface expression profiles. We identified a ccpA insertion mutant which showed significantly decreased levels of Ace surface expression at early growth phase versus those of wild-type OG1RF. Confirmation of the observation was achieved through flow cytometry and complementation analysis. Compared to the wild type, the E. faecalis ccpA mutant had an impaired ability to adhere to collagen when grown to early exponential phase, consistent with the lack of Ace expression in the early growth phase. As a key component of carbon catabolite regulation, CcpA has been previously reported to play a critical role in regulating expression of proteins involved in E. faecalis carbohydrate uptake and utilization. Our discovery is the first to associate CcpA with the production of a major E. faecalis virulence factor, providing new insights into the regulation of E. faecalis pathogenesis. PMID:23974022

N-CADHERIN PRODOMAIN CLEAVAGE REGULATES SYNAPSE FORMATION IN VIVO

PubMed Central

Latefi, Nazlie S.; Pedraza, Liliana; Schohl, Anne; Li, Ziwei; Ruthazer, Edward S.

2009-01-01

Cadherins are initially synthesized bearing a prodomain that is thought to limit adhesion during early stages of biosynthesis. Functional cadherins lack this prodomain, raising the intriguing possibility that cells may utilize prodomain cleavage as a means to temporally or spatially regulate adhesion after delivery of cadherin to the cell surface. In support of this idea, immunostaining for the prodomain of zebrafish N-cadherin revealed enriched labeling at neuronal surfaces at the soma and along axonal processes. To determine whether post-translational cleavage of the prodomain affects synapse formation, we imaged Rohon-Beard cells in zebrafish embryos expressing GFP-tagged wild-type N-cadherin (NCAD-GFP) or a GFP-tagged N-cadherin mutant expressing an uncleavable prodomain (PRON-GFP) rendering it non-adhesive. NCAD-GFP accumulated at synaptic microdomains in a developmentally regulated manner, and its overexpression transiently accelerated synapse formation. PRON-GFP was much more diffusely distributed along the axon and its overexpression delayed synapse formation. Our results support the notion that N-cadherin serves to stabilize pre- to postsynaptic contacts early in synapse development and suggests that regulated cleavage of the N-cadherin prodomain may be a mechanism by which the kinetics of synaptogenesis are regulated. PMID:19365814

IL-10 down-regulates T cell activation by antigen-presenting liver sinusoidal endothelial cells through decreased antigen uptake via the mannose receptor and lowered surface expression of accessory molecules.

PubMed

Knolle, P A; Uhrig, A; Hegenbarth, S; Löser, E; Schmitt, E; Gerken, G; Lohse, A W

1998-12-01

Our study demonstrates that antigen-presenting liver sinusoidal endothelial cells (LSEC) induce production of interferon-gamma (IFN-gamma) from cloned Th1 CD4+ T cells. We show that LSEC used the mannose receptor for antigen uptake, which further strengthened the role of LSEC as antigen-presenting cell (APC) population in the liver. The ability of LSEC to activate cloned CD4+ T cells antigen-specifically was down-regulated by exogenous prostaglandin E2 (PGE2) and by IL-10. We identify two separate mechanisms by which IL-10 down-regulated T cell activation through LSEC. IL-10 decreased the constitutive surface expression of MHC class II as well as of the accessory molecules CD80 and CD86 on LSEC. Furthermore, IL-10 diminished mannose receptor activity in LSEC. Decreased antigen uptake via the mannose receptor and decreased expression of accessory molecules may explain the down-regulation of T cell activation through IL-10. Importantly, the expression of low numbers of antigen on MHC II in the absence of accessory signals on LSEC may lead to induction of anergy in T cells. Because PGE2 and IL-10 are released from LSEC or Kupffer cells (KC) in response to those concentrations of endotoxin found physiologically in portal venous blood, it is possible that the continuous presence of these mediators and their negative effect on the local APC may explain the inability of the liver to induce T cell activation and to clear chronic infections. Our results support the notion that antigen presentation by LSEC in the hepatic microenvironment contributes to the observed inability to mount an effective cell-mediated immune response in the liver.

Beneficial Autoimmunity at Body Surfaces – Immune Surveillance and Rapid Type 2 Immunity Regulate Tissue Homeostasis and Cancer

PubMed Central

Dalessandri, Tim; Strid, Jessica

2014-01-01

Epithelial cells (ECs) line body surface tissues and provide a physicochemical barrier to the external environment. Frequent microbial and non-microbial challenges such as those imposed by mechanical disruption, injury or exposure to noxious environmental substances including chemicals, carcinogens, ultraviolet-irradiation, or toxins cause activation of ECs with release of cytokines and chemokines as well as alterations in the expression of cell-surface ligands. Such display of epithelial stress is rapidly sensed by tissue-resident immunocytes, which can directly interact with self-moieties on ECs and initiate both local and systemic immune responses. ECs are thus key drivers of immune surveillance at body surface tissues. However, ECs have a propensity to drive type 2 immunity (rather than type 1) upon non-invasive challenge or stress – a type of immunity whose regulation and function still remain enigmatic. Here, we review the induction and possible role of type 2 immunity in epithelial tissues and propose that rapid immune surveillance and type 2 immunity are key regulators of tissue homeostasis and carcinogenesis. PMID:25101088

The effect of hydrophilic titanium surface modification on macrophage inflammatory cytokine gene expression.

PubMed

Hamlet, Stephen; Alfarsi, Mohammed; George, Roy; Ivanovski, Saso

2012-05-01

Chemical modification of microrough titanium dental implants to produce a hydrophilic surface with increased wettability and improved surface energy has been demonstrated clinically to achieve superior bone wound healing and osseointegration compared to that achieved with a microrough titanium surface alone. As the recruitment of the necessary osseoinductive precursors involved in bone wound healing and osseointegration to the wound site is facilitated by the action of cytokines, this study sought to determine the in vitro effect of hydrophilic surface modification on the expression of pro-inflammatory cytokines from adherent macrophages. The surface topography and composition of the titanium surfaces was characterized by scanning electron microscopy and X-ray photoelectron spectroscopy. Macrophage attachment and proliferation was assessed using an MTT assay. The expression of 84 pro-inflammatory cytokines and chemokines by adherent RAW 264.7 cells, a murine leukaemic monocyte cell line, was assessed by PCR array after 24 h culture on either smooth polished, sand-blasted acid-etched (SLA) or hydrophilic-modified SLA (SLActive) titanium surfaces. Following 24 h culture on titanium, surface microroughness activated pro-inflammatory cytokine gene transcription in RAW 264.7 cells. Although there was no significant difference in the degree of cellular attachment or proliferation of RAW 264.7 cells to the different titanium surfaces, by 24 h the hydrophilic surface elicited a gene expression profile with significant down-regulation of the key pro-inflammatory cytokines Tnfα, IL-1α, IL-1β and the chemokine Ccl-2. Down-regulation of the expression of pro-inflammatory cytokine genes may thus modulate the inflammatory response and may facilitate the enhanced bone wound healing and osseointegration observed clinically using implants with a microrough hydrophilic surface. © 2011 John Wiley & Sons A/S.

Computational modelling of biomaterial surface interactions with blood platelets and osteoblastic cells for the prediction of contact osteogenesis.

PubMed

Amor, N; Geris, L; Vander Sloten, J; Van Oosterwyck, H

2011-02-01

Surface microroughness can induce contact osteogenesis (bone formation initiated at the implant surface) around oral implants, which may result from different mechanisms, such as blood platelet-biomaterial interactions and/or interaction with (pre-)osteoblast cells. We have developed a computational model of implant endosseous healing that takes into account these interactions. We hypothesized that the initial attachment and growth factor release from activated platelets is crucial in achieving contact osteogenesis. In order to investigate this, a computational model was applied to an animal experiment [7] that looked at the effect of surface microroughness on endosseous healing. Surface-specific model parameters were implemented based on in vitro data (Lincks et al. Biomaterials 1998;19:2219-32). The predicted spatio-temporal patterns of bone formation correlated with the histological data. It was found that contact osteogenesis could not be predicted if only the osteogenic response of cells was up-regulated by surface microroughness. This could only be achieved if platelet-biomaterial interactions were sufficiently up-regulated as well. These results confirmed our hypothesis and demonstrate the added value of the computational model to study the importance of surface-mediated events for peri-implant endosseous healing. Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

CD22 is a recycling receptor that can shuttle cargo between the cell surface and endosomal compartments of B cells.

PubMed

O'Reilly, Mary K; Tian, Hua; Paulson, James C

2011-02-01

CD22 is a member of the sialic acid-binding Ig-like lectin (Siglec) family that is known to be a regulator of B cell signaling. Its B cell-specific expression makes it an attractive target for immunotoxin-mediated B cell depletion therapy for the treatment of B cell lymphomas and autoimmune diseases. Although CD22 is well documented to be an endocytic receptor, it is believed that after internalization, it is targeted for degradation. We show in this study that CD22 is instead constitutively recycled to the cell surface. We also find that glycan ligand-based cargo is released from CD22 and accumulates intracellularly as CD22 recycles between the cell surface and endosomal compartments. In contrast, Abs to CD22 do not accumulate but remain bound to CD22 and recycle to the cell surface. The results have implications for development of agents that target CD22 as an endocytic receptor for delivery of cytotoxic cargo to B cells.

Regulation of B cell functions by the sialic acid-binding receptors siglec-G and CD22.

PubMed

Jellusova, Julia; Nitschke, Lars

2011-01-01

B cell antigen receptor (BCR) engagement can lead to many different physiologic outcomes. To achieve an appropriate response, the BCR signal is interpreted in the context of other stimuli and several additional receptors on the B cell surface participate in the modulation of the signal. Two members of the Siglec (sialic acid-binding immunoglobulin-like lectin) family, CD22 and Siglec-G have been shown to inhibit the BCR signal. Recent findings indicate that the ability of these two receptors to bind sialic acids might be important to induce tolerance to self-antigens. Sialylated glycans are usually absent on microbes but abundant in higher vertebrates and might therefore provide an important tolerogenic signal. Since the expression of the specific ligands for Siglec-G and CD22 is tightly regulated and since Siglecs are not only able to bind their ligands in trans but also on the same cell surface this might provide additional mechanisms to control the BCR signal. Although both Siglec-G and CD22 are expressed on B cells and are able to inhibit BCR mediated signaling, they also show unique biological functions. While CD22 is the dominant regulator of calcium signaling on conventional B2 cells and also seems to play a role on marginal zone B cells, Siglec-G exerts its function mainly on B1 cells and influences their lifespan and antibody production. Both Siglec-G and CD22 have also recently been linked to toll-like receptor signaling and may provide a link in the regulation of the adaptive and innate immune response of B cells.

CD44 Staining of Cancer Stem-Like Cells Is Influenced by Down-Regulation of CD44 Variant Isoforms and Up-Regulation of the Standard CD44 Isoform in the Population of Cells That Have Undergone Epithelial-to-Mesenchymal Transition

PubMed Central

Biddle, Adrian; Gammon, Luke; Fazil, Bilal; Mackenzie, Ian C.

2013-01-01

CD44 is commonly used as a cell surface marker of cancer stem-like cells in epithelial tumours, and we have previously demonstrated the existence of two different CD44high cancer stem-like cell populations in squamous cell carcinoma, one having undergone epithelial-to-mesenchymal transition and the other maintaining an epithelial phenotype. Alternative splicing of CD44 variant exons generates a great many isoforms, and it is not known which isoforms are expressed on the surface of the two different cancer stem-like cell phenotypes. Here, we demonstrate that cancer stem-like cells with an epithelial phenotype predominantly express isoforms containing the variant exons, whereas the cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition down-regulate these variant isoforms and up-regulate expression of the standard CD44 isoform that contains no variant exons. In addition, we find that enzymatic treatments used to dissociate cells from tissue culture or fresh tumour specimens cause destruction of variant CD44 isoforms at the cell surface whereas expression of the standard CD44 isoform is preserved. This results in enrichment within the CD44high population of cancer stem-like cells that have undergone an epithelial-to-mesenchymal transition and depletion from the CD44high population of cancer stem-like cells that maintain an epithelial phenotype, and therefore greatly effects the characteristics of any cancer stem-like cell population isolated based on expression of CD44. As well as effecting the CD44high population, enzymatic treatment also reduces the percentage of the total epithelial cancer cell population staining CD44-positive, with potential implications for studies that aim to use CD44-positive staining as a prognostic indicator. Analyses of the properties of cancer stem-like cells are largely dependent on the ability to accurately identify and assay these populations. It is therefore critical that consideration be given to use of multiple cancer stem-like cell markers and suitable procedures for cell isolation in order that the correct populations are assayed. PMID:23437366

Signal Regulatory Protein α Negatively Regulates β2 Integrin-Mediated Monocyte Adhesion, Transendothelial Migration and Phagocytosis

PubMed Central

Liu, Dan-Qing; Li, Li-Min; Guo, Ya-Lan; Bai, Rui; Wang, Chen; Bian, Zhen; Zhang, Chen-Yu; Zen, Ke

2008-01-01

Background Signal regulate protein α (SIRPα) is involved in many functional aspects of monocytes. Here we investigate the role of SIRPα in regulating β2 integrin-mediated monocyte adhesion, transendothelial migration (TEM) and phagocytosis. Methodology/Principal Findings THP-1 monocytes/macropahges treated with advanced glycation end products (AGEs) resulted in a decrease of SIRPα expression but an increase of β2 integrin cell surface expression and β2 integrin-mediated adhesion to tumor necrosis factor-α (TNFα)–stimulated human microvascular endothelial cell (HMEC-1) monolayers. In contrast, SIRPα overexpression in THP-1 cells showed a significant less monocyte chemotactic protein-1 (MCP-1)–triggered cell surface expression of β2 integrins, in particular CD11b/CD18. SIRPα overexpression reduced β2 integrin-mediated firm adhesion of THP-1 cells to either TNFα–stimulated HMEC-1 monolayers or to immobilized intercellular adhesion molecule-1 (ICAM-1). SIRPα overexpression also reduced MCP-1–initiated migration of THP-1 cells across TNFα–stimulated HMEC-1 monolayers. Furthermore, β2 integrin-mediated THP-1 cell spreading and actin polymerization in response to MCP-1, and phagocytosis of bacteria were both inhibited by SIRPα overexpression. Conclusions/Significance SIRPα negatively regulates β2 integrin-mediated monocyte adhesion, transendothelial migration and phagocytosis, thus may serve as a critical molecule in preventing excessive activation and accumulation of monocytes in the arterial wall during early stage of atherosclerosis. PMID:18820737

Gibberellin DELLA signaling targets the retromer complex to redirect protein trafficking to the plasma membrane

PubMed Central

Salanenka, Yuliya; Verstraeten, Inge; Löfke, Christian; Tabata, Kaori; Naramoto, Satoshi; Glanc, Matouš; Friml, Jiří

2018-01-01

The plant hormone gibberellic acid (GA) is a crucial regulator of growth and development. The main paradigm of GA signaling puts forward transcriptional regulation via the degradation of DELLA transcriptional repressors. GA has also been shown to regulate tropic responses by modulation of the plasma membrane incidence of PIN auxin transporters by an unclear mechanism. Here we uncovered the cellular and molecular mechanisms by which GA redirects protein trafficking and thus regulates cell surface functionality. Photoconvertible reporters revealed that GA balances the protein traffic between the vacuole degradation route and recycling back to the cell surface. Low GA levels promote vacuolar delivery and degradation of multiple cargos, including PIN proteins, whereas high GA levels promote their recycling to the plasma membrane. This GA effect requires components of the retromer complex, such as Sorting Nexin 1 (SNX1) and its interacting, microtubule (MT)-associated protein, the Cytoplasmic Linker-Associated Protein (CLASP1). Accordingly, GA regulates the subcellular distribution of SNX1 and CLASP1, and the intact MT cytoskeleton is essential for the GA effect on trafficking. This GA cellular action occurs through DELLA proteins that regulate the MT and retromer presumably via their interaction partners Prefoldins (PFDs). Our study identified a branching of the GA signaling pathway at the level of DELLA proteins, which, in parallel to regulating transcription, also target by a nontranscriptional mechanism the retromer complex acting at the intersection of the degradation and recycling trafficking routes. By this mechanism, GA can redirect receptors and transporters to the cell surface, thus coregulating multiple processes, including PIN-dependent auxin fluxes during tropic responses. PMID:29463731

Estrogen upregulates MICA/B expression in human non-small cell lung cancer through the regulation of ADAM17.

PubMed

Ren, Jing; Nie, Yunzhong; Lv, Mingming; Shen, Sunan; Tang, Ruijing; Xu, Yujun; Hou, Yayi; Zhao, Shuli; Wang, Tingting

2015-11-01

Estrogen is involved in promoting lung cancer cell division and metastasis. MICA and MICB function as ligands for NKG2D, an important immunoreceptor expressed on natural killer (NK) cells. However, whether estrogen regulates MICA/B expression and affects tumor immune escape remains unknown. In this study, we measured the mRNA levels of MICA, MICB and ADAM17in non-small cell lung cancer (NSCLC) cell lines treated with estrogen. Surface expression of MICA/B on LTEP-a2 and A549 was detected using flow cytometry. We demonstrate that both mRNA and secretory protein levels of MICA/B in lung adenocarcinoma cell lines were upregulated by estradiol. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. This secretion of MICA/B downregulated the NKG2D receptor on the surface of NK92 cells and impaired the cytotoxic activity of NK cells. Estradiol enhanced the expression of ADAM17, which was associated with the secretion of MICA/B. Furthermore, a significant correlation between the concentration of estradiol and the expression of MICA was found in tumor tissues of NSCLC patients. Therefore, we conclude that estrogen can regulate the expression and secretion of MICA/B through ADAM17, which helps lung cancer cells escape NKG2D-mediated immune surveillance.

Induction of neural differentiation by electrically stimulated gene expression of NeuroD2.

PubMed

Mie, Masayasu; Endoh, Tamaki; Yanagida, Yasuko; Kobatake, Eiry; Aizawa, Masuo

2003-02-13

Regulation of cell differentiation is an important assignment for cellular engineering. One of the techniques for regulation is gene transfection into undifferentiated cells. Transient expression of NeuroD2, one of neural bHLH transcription factors, converted mouse N1E-115 neuroblastoma cells into differentiated neurons. The regulation of neural bHLH expression should be a novel strategy for cell differentiation. In this study, we tried to regulate neural differentiation by NeuroD2 gene inserted under the control of heat shock protein-70 (HSP) promoter, which can be activated by electrical stimulation. Mouse neuroblastoma cell line, N1E-115, was stably transfected with expression vector containing mouse NeuroD2 cDNA under HSP promoter. Transfected cells were cultured on the electrode surface and applied electrical stimulation. After stimulation, NeuroD2 expression was induced, and transfected cells adopt a neuronal morphology at 3 days after stimulation. These results suggest that neural differentiation can be induced by electrically stimulated gene expression of NeuroD2.

A solid-state NMR study of the dynamics and interactions of phenylalanine rings in a statherin fragment bound to hydroxyapatite crystals.

PubMed

Gibson, James M; Popham, Jennifer M; Raghunathan, Vinodhkumar; Stayton, Patrick S; Drobny, Gary P

2006-04-26

Extracellular matrix proteins regulate hard tissue growth by acting as adhesion sites for cells, by triggering cell signaling pathways, and by directly regulating the primary and/or secondary crystallization of hydroxyapatite, the mineral component of bone and teeth. Despite the key role that these proteins play in the regulation of hard tissue growth in humans, the exact mechanism used by these proteins to recognize mineral surfaces is poorly understood. Interactions between mineral surfaces and proteins very likely involve specific contacts between the lattice and the protein side chains, so elucidation of the nature of interactions between protein side chains and their corresponding inorganic mineral surfaces will provide insight into the recognition and regulation of hard tissue growth. Isotropic chemical shifts, chemical shift anisotropies (CSAs), NMR line-width information, (13)C rotating frame relaxation measurements, as well as direct detection of correlations between (13)C spins on protein side chains and (31)P spins in the crystal surface with REDOR NMR show that, in the peptide fragment derived from the N-terminal 15 amino acids of salivary statherin (i.e., SN-15), the side chain of the phenylalanine nearest the C-terminus of the peptide (F14) is dynamically constrained and oriented near the surface, whereas the side chain of the phenylalanine located nearest to the peptide's N-terminus (F7) is more mobile and is oriented away from the hydroxyapatite surface. The relative dynamics and proximities of F7 and F14 to the surface together with prior data obtained for the side chain of SN-15's unique lysine (i.e., K6) were used to construct a new picture for the structure of the surface-bound peptide and its orientation to the crystal surface.

Identification of a response regulator involved in surface attachment, cell-cell aggregation, exopolysaccharide production and virulence in the plant pathogen Xylella fastidiosa.

PubMed

Voegel, Tanja M; Doddapaneni, Harshavardhan; Cheng, Davis W; Lin, Hong; Stenger, Drake C; Kirkpatrick, Bruce C; Roper, M Caroline

2013-04-01

Xylella fastidiosa, the causal agent of Pierce's disease of grapevine, possesses several two-component signal transduction systems that allow the bacterium to sense and respond to changes in its environment. Signals are perceived by sensor kinases that autophosphorylate and transfer the phosphate to response regulators (RRs), which direct an output response, usually by acting as transcriptional regulators. In the X. fastidiosa genome, 19 RRs were found. A site-directed knockout mutant in one unusual RR, designated XhpT, composed of a receiver domain and a histidine phosphotransferase output domain, was constructed. The resulting mutant strain was analysed for changes in phenotypic traits related to biofilm formation and gene expression using microarray analysis. We found that the xhpT mutant was altered in surface attachment, cell-cell aggregation, exopolysaccharide (EPS) production and virulence in grapevine. In addition, this mutant had an altered transcriptional profile when compared with wild-type X. fastidiosa in genes for several biofilm-related traits, such as EPS production and haemagglutinin adhesins. © 2012 BSPP AND BLACKWELL PUBLISHING LTD.

RhoA-Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography Dependent.

PubMed

Ogino, Yoichiro; Liang, Ruiwei; Mendonça, Daniela B S; Mendonça, Gustavo; Nagasawa, Masako; Koyano, Kiyoshi; Cooper, Lyndon F

2016-03-01

Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho-family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA-mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and -mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano-scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity. © 2015 Wiley Periodicals, Inc.

Regulation of AMPA receptor localization in lipid rafts

PubMed Central

Hou, Qingming; Huang, Yunfei; Amato, Stephen; Snyder, Solomon H.; Huganir, Richard L.; Man, Heng-Ye

2009-01-01

Lipid rafts are special microdomains enriched in cholesterol, sphingolipids and certain proteins, and play important roles in a variety of cellular functions including signal transduction and protein trafficking. We report that in cultured cortical and hippocampal neurons the distribution of lipid rafts is development-dependent. Lipid rafts in mature neurons exist on the entire cell-surface and display a high degree of mobility. AMPA receptors co-localize and associate with lipid rafts in the plasma membrane. The association of AMPARs with rafts is under regulation; through the NOS–NO pathway, NMDA receptor activity increases AMPAR localization in rafts. During membrane targeting, AMPARs insert into or at close proximity of the surface raft domains. Perturbation of lipid rafts dramatically suppresses AMPA receptor exocytosis, resulting in significant reduction in AMPAR cell-surface expression. PMID:18411055

The Nav1.2 channel is regulated by GSK3

PubMed Central

James, Thomas F.; Nenov, Miroslav N.; Wildburger, Norelle C.; Lichti, Cheryl; Luisi, Jonathan; Vergara, Fernanda; Panova-Electronova, Neli I.; Nilsson, Carol L.; Rudra, Jai; Green, Thomas A.; Labate, Demetrio; Laezza, Fernanda

2015-01-01

Background Phosphorylation plays an essential role in regulating the voltage-gated sodium (Nav) channels and excitability. Yet, a surprisingly limited number of kinases have been identified as regulators of Nav channels. Herein, we posited that glycogen synthase kinase 3 (GSK3), a critical kinase found associated with numerous brain disorders, might directly regulate neuronal Nav channels. Methods We used patch-clamp electrophysiology to record sodium currents from Nav1.2 channels stably expressed in HEK-293 cells. mRNA and protein levels were quantified with RT-PCR, Western blot, or confocal microscopy, and in vitro phosphorylation and mass spectrometry to identify phosphorylated residues. Results We found that exposure of cells to GSK3 inhibitor XIII significantly potentiates the peak current density of Nav1.2, a phenotype reproduced by silencing GSK3 with siRNA. Contrarily, overexpression of GSK3β suppressed Nav1.2-encoded currents. Neither mRNA nor total protein expression were changed upon GSK3 inhibition. Cell surface labeling of CD4-chimeric constructs expressing intracellular domains of the Nav1.2 channel indicates that cell surface expression of CD4-Nav1.2-Ctail was up-regulated upon pharmacological inhibition of GSK3, resulting in an increase of surface puncta at the plasma membrane. Finally, using in vitro phosphorylation in combination with high resolution mass spectrometry, we further demonstrate that GSK3β phosphorylates T1966 at the C-terminal tail of Nav1.2. Conclusion These findings provide evidence for a new mechanism by which GSK3 modulate Nav channel function via its C-terminal tail. General Significance These findings provide fundamental knowledge in understanding signaling dysfunction common in several neuropsychiatric disorders. PMID:25615535

Regulation and Gene Expression Profiling of NKG2D Positive Human Cytomegalovirus-Primed CD4+ T-Cells

PubMed Central

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8+ T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4+ T-cells, however recently a subset of NKG2D+ CD4+ T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D+ CD4+ T-cells possesses effector-like functions, thus resembling the subsets of NKG2D+ CD4+ T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4+ T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D+ CD4+ T-cells, generated from HCMV-primed CD4+ T-cells. We show that the HCMV-primed NKG2D+ CD4+ T-cells possess a higher differentiated phenotype than the NKG2D– CD4+ T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4+ T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4+ T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4+ T-cells, whereas it is produced de novo in resting CD4+ T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D+ CD4+ T-cells, as well as the mechanisms regulating NKG2D cell surface expression. PMID:22870231

Regulation and gene expression profiling of NKG2D positive human cytomegalovirus-primed CD4+ T-cells.

PubMed

Jensen, Helle; Folkersen, Lasse; Skov, Søren

2012-01-01

NKG2D is a stimulatory receptor expressed by natural killer (NK) cells, CD8(+) T-cells, and γδ T-cells. NKG2D expression is normally absent from CD4(+) T-cells, however recently a subset of NKG2D(+) CD4(+) T-cells has been found, which is specific for human cytomegalovirus (HCMV). This particular subset of HCMV-specific NKG2D(+) CD4(+) T-cells possesses effector-like functions, thus resembling the subsets of NKG2D(+) CD4(+) T-cells found in other chronic inflammations. However, the precise mechanism leading to NKG2D expression on HCMV-specific CD4(+) T-cells is currently not known. In this study we used genome-wide analysis of individual genes and gene set enrichment analysis (GSEA) to investigate the gene expression profile of NKG2D(+) CD4(+) T-cells, generated from HCMV-primed CD4(+) T-cells. We show that the HCMV-primed NKG2D(+) CD4(+) T-cells possess a higher differentiated phenotype than the NKG2D(-) CD4(+) T-cells, both at the gene expression profile and cytokine profile. The ability to express NKG2D at the cell surface was primarily determined by the activation or differentiation status of the CD4(+) T-cells and not by the antigen presenting cells. We observed a correlation between CD94 and NKG2D expression in the CD4(+) T-cells following HCMV stimulation. However, knock-down of CD94 did not affect NKG2D cell surface expression or signaling. In addition, we show that NKG2D is recycled at the cell surface of activated CD4(+) T-cells, whereas it is produced de novo in resting CD4(+) T-cells. These findings provide novel information about the gene expression profile of HCMV-primed NKG2D(+) CD4(+) T-cells, as well as the mechanisms regulating NKG2D cell surface expression.

Regulation of Cell Wall Biogenesis in Saccharomyces cerevisiae: The Cell Wall Integrity Signaling Pathway

PubMed Central

Levin, David E.

2011-01-01

The yeast cell wall is a strong, but elastic, structure that is essential not only for the maintenance of cell shape and integrity, but also for progression through the cell cycle. During growth and morphogenesis, and in response to environmental challenges, the cell wall is remodeled in a highly regulated and polarized manner, a process that is principally under the control of the cell wall integrity (CWI) signaling pathway. This pathway transmits wall stress signals from the cell surface to the Rho1 GTPase, which mobilizes a physiologic response through a variety of effectors. Activation of CWI signaling regulates the production of various carbohydrate polymers of the cell wall, as well as their polarized delivery to the site of cell wall remodeling. This review article centers on CWI signaling in Saccharomyces cerevisiae through the cell cycle and in response to cell wall stress. The interface of this signaling pathway with other pathways that contribute to the maintenance of cell wall integrity is also discussed. PMID:22174182

Select Rab GTPases Regulate the Pulmonary Endothelium via Endosomal Trafficking of Vascular Endothelial-Cadherin.

PubMed

Chichger, Havovi; Braza, Julie; Duong, Huetran; Boni, Geraldine; Harrington, Elizabeth O

2016-06-01

Pulmonary edema occurs in settings of acute lung injury, in diseases, such as pneumonia, and in acute respiratory distress syndrome. The lung interendothelial junctions are maintained in part by vascular endothelial (VE)-cadherin, an adherens junction protein, and its surface expression is regulated by endocytic trafficking. The Rab family of small GTPases are regulators of endocytic trafficking. The key trafficking pathways are regulated by Rab4, -7, and -9. Rab4 regulates the recycling of endosomes to the cell surface through a rapid-shuttle process, whereas Rab7 and -9 regulate trafficking to the late endosome/lysosome for degradation or from the trans-Golgi network to the late endosome, respectively. We recently demonstrated a role for the endosomal adaptor protein, p18, in regulation of the pulmonary endothelium through enhanced recycling of VE-cadherin to adherens junction. Thus, we hypothesized that Rab4, -7, and -9 regulate pulmonary endothelial barrier function through modulating trafficking of VE-cadherin-positive endosomes. We used Rab mutants with varying activities and associations to the endosome to study endothelial barrier function in vitro and in vivo. Our study demonstrates a key role for Rab4 activation and Rab9 inhibition in regulation of vascular permeability through enhanced VE-cadherin expression at the interendothelial junction. We further showed that endothelial barrier function mediated through Rab4 is dependent on extracellular signal-regulated kinase phosphorylation and activity. Thus, we demonstrate that Rab4 and -9 regulate VE-cadherin levels at the cell surface to modulate the pulmonary endothelium through extracellular signal-regulated kinase-dependent and -independent pathways, respectively. We propose that regulating select Rab GTPases represents novel therapeutic strategies for patients suffering with acute respiratory distress syndrome.

Insulin promotes cell migration by regulating PSA-NCAM

DOE Office of Scientific and Technical Information (OSTI.GOV)

Monzo, Hector J.; Coppieters, Natacha; Department of Anatomy and Medical Imaging, Faculty of Medical and Health Sciences, The University of Auckland, Private Bag, 92019, Auckland

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cellmore » migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. - Highlights: • Insulin modulates PSA-NCAM turnover through upregulation of p-FAK. • P-FAK modulates αv-integrin/PSA-NCAM clustering. • αv-integrin acts as a carrier for PSA-NCAM endocytosis. • Cell migration is promoted by cell surface PSA. • Insulin promotes PSA-dependent migration in vitro.« less

The molecular mechanism of mediation of adsorbed serum proteins to endothelial cells adhesion and growth on biomaterials.

PubMed

Yang, Dayun; Lü, Xiaoying; Hong, Ying; Xi, Tingfei; Zhang, Deyuan

2013-07-01

To explore molecular mechanism of mediation of adsorbed proteins to cell adhesion and growth on biomaterials, this study examined endothelial cell adhesion, morphology and viability on bare and titanium nitride (TiN) coated nickel titanium (NiTi) alloys and chitosan film firstly, and then identified the type and amount of serum proteins adsorbed on the three surfaces by proteomic technology. Subsequently, the mediation role of the identified proteins to cell adhesion and growth was investigated with bioinformatics analyses, and further confirmed by a series of cellular and molecular biological experiments. Results showed that the type and amount of adsorbed serum proteins associated with cell adhesion and growth was obviously higher on the alloys than on the chitosan film, and these proteins mediated endothelial cell adhesion and growth on the alloys via four ways. First, proteins such as adiponectin in the adsorbed protein layer bound with cell surface receptors to generate signal transduction, which activated cell surface integrins through increasing intracellular calcium level. Another way, thrombospondin 1 in the adsorbed protein layer promoted TGF-β signaling pathway activation and enhanced integrins expression. The third, RGD sequence containing proteins such as fibronectin 1, vitronectin and thrombospondin 1 in the adsorbed protein layer bound with activated integrins to activate focal adhesion pathway, increased focal adhesion formation and actin cytoskeleton organization and mediated cell adhesion and spreading. In addition, the activated focal adhesion pathway promoted the expression of cell growth related genes and resulted in cell proliferation. The fourth route, coagulation factor II (F2) and fibronectin 1 in the adsorbed protein layer bound with cell surface F2 receptor and integrin, activated regulation of actin cytoskeleton pathway and regulated actin cytoskeleton organization. Copyright © 2013 Elsevier Ltd. All rights reserved.

Sorting receptor Rer1 controls surface expression of muscle acetylcholine receptors by ER retention of unassembled alpha-subunits.

PubMed

Valkova, Christina; Albrizio, Marina; Röder, Ira V; Schwake, Michael; Betto, Romeo; Rudolf, Rüdiger; Kaether, Christoph

2011-01-11

The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor α-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled α-subunits to the early secretory pathway.

Functional and genomic analyses of FOXP3-transduced Jurkat-T cells as regulatory T (Treg)-like cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Joon-Young; Kim, Han-Jong; Hurt, Elaine M.

2007-10-12

FOXP3, a forkhead transcription factor is essential for the development and function of CD4{sup +}CD25{sup +} regulatory T cells (Tregs). In contrast to conversion of murine naive T cells to Tregs by transduction of Foxp3, it is controversial whether ectopic expression of FOXP3 in human CD4{sup +} T cells is sufficient for acquisition of suppressive activity. Here, we show that retroviral transduction of FOXP3 induces a Treg phenotype in human leukemic CD4{sup +} Jurkat-T cells, evidenced by increased expression of Treg-associated cell surface markers as well as inhibition of cytokine production. Furthermore, FOXP3-transduced Jurkat-T cells suppress the proliferation of humanmore » CD4{sup +}CD25{sup -} T cells. Additionally, DNA microarray analysis identifies Treg-related genes regulated by FOXP3. Our study demonstrates that enforced expression of FOXP3 confers Treg-like properties on Jurkat-T cells, which can be a convenient and efficient Treg-like cell model for further study to identify Treg cell surface markers and target genes regulated by FOXP3.« less

Endocytosis and Vacuolar Degradation of the Yeast Cell Surface Glucose Sensors Rgt2 and Snf3*

PubMed Central

Roy, Adhiraj; Kim, Jeong-Ho

2014-01-01

Sensing and signaling the presence of extracellular glucose is crucial for the yeast Saccharomyces cerevisiae because of its fermentative metabolism, characterized by high glucose flux through glycolysis. The yeast senses glucose through the cell surface glucose sensors Rgt2 and Snf3, which serve as glucose receptors that generate the signal for induction of genes involved in glucose uptake and metabolism. Rgt2 and Snf3 detect high and low glucose concentrations, respectively, perhaps because of their different affinities for glucose. Here, we provide evidence that cell surface levels of glucose sensors are regulated by ubiquitination and degradation. The glucose sensors are removed from the plasma membrane through endocytosis and targeted to the vacuole for degradation upon glucose depletion. The turnover of the glucose sensors is inhibited in endocytosis defective mutants, and the sensor proteins with a mutation at their putative ubiquitin-acceptor lysine residues are resistant to degradation. Of note, the low affinity glucose sensor Rgt2 remains stable only in high glucose grown cells, and the high affinity glucose sensor Snf3 is stable only in cells grown in low glucose. In addition, constitutively active, signaling forms of glucose sensors do not undergo endocytosis, whereas signaling defective sensors are constitutively targeted for degradation, suggesting that the stability of the glucose sensors may be associated with their ability to sense glucose. Therefore, our findings demonstrate that the amount of glucose available dictates the cell surface levels of the glucose sensors and that the regulation of glucose sensors by glucose concentration may enable yeast cells to maintain glucose sensing activity at the cell surface over a wide range of glucose concentrations. PMID:24451370

Surface topography regulates wnt signaling through control of primary cilia structure in mesenchymal stem cells

PubMed Central

McMurray, R. J.; Wann, A. K. T.; Thompson, C. L.; Connelly, J. T.; Knight, M. M.

2013-01-01

The primary cilium regulates cellular signalling including influencing wnt sensitivity by sequestering β-catenin within the ciliary compartment. Topographic regulation of intracellular actin-myosin tension can control stem cell fate of which wnt is an important mediator. We hypothesized that topography influences mesenchymal stem cell (MSC) wnt signaling through the regulation of primary cilia structure and function. MSCs cultured on grooves expressed elongated primary cilia, through reduced actin organization. siRNA inhibition of anterograde intraflagellar transport (IFT88) reduced cilia length and increased active nuclear β-catenin. Conversely, increased primary cilia assembly in MSCs cultured on the grooves was associated with decreased levels of nuclear active β-catenin, axin-2 induction and proliferation, in response to wnt3a. This negative regulation, on grooved topography, was reversed by siRNA to IFT88. This indicates that subtle regulation of IFT and associated cilia structure, tunes the wnt response controlling stem cell differentiation. PMID:24346024

Pigment Epithelium-Derived Factor Blocks Tumor Extravasation by Suppressing Amoeboid Morphology and Mesenchymal Proteolysis12

PubMed Central

Ladhani, Omar; Sánchez-Martinez, Cristina; Orgaz, Jose L; Jimenez, Benilde; Volpert, Olga V

2011-01-01

Metastatic melanoma cells are highly adaptable to their in vivo microenvironment and can switch between protease-dependent mesenchymal and protease-independent amoeboid invasion to facilitate metastasis. Such adaptability can be visualized in vitro, when cells are cultured in conditions that recapitulate three-dimensional microenvironments. Using thick collagen layers in cell culture and in vivo extravasation assays, we found that pigment epithelium-derived factor (PEDF) suppressed lung extravasation of aggressive melanoma by coordinated regulation of cell shape and proteolysis. In cells grown on a thick collagen bed, PEDF overexpression and exogenous PEDF blocked the rapidly invasive, rounded morphology, and promoted an elongated, mesenchymal-like phenotype associated with reduced invasion. These changes in cell shape depended on decreased RhoA and increased Rac1 activation and were mediated by the up-regulation of Rac1-GEF, DOCK3 and down-regulation of Rac1-GAP, ARHGAP22. Surprisingly, we found that PEDF overexpression also blocked the trafficking of membrane-tethered, MT1-MMP to the cell surface through RhoA inhibition and Rac1 activation. In vivo, knockdown of Rac1 and DOCK3 or overexpression of MT1-MMP was sufficient to reverse the inhibitory effect of PEDF on extravasation. Using functional studies, we demonstrated that PEDF suppressed the rounded morphology and MT1-MMP surface localization through its antiangiongenic, 34-mer epitope and the recently identified PEDF receptor candidate, PNPLA2. Our findings unveil the coordinated regulation of cell shape and proteolysis and identify an unknown mechanism for PEDF's antimetastatic activity. PMID:21750657

Pigment epithelium-derived factor blocks tumor extravasation by suppressing amoeboid morphology and mesenchymal proteolysis.

PubMed

Ladhani, Omar; Sánchez-Martinez, Cristina; Orgaz, Jose L; Jimenez, Benilde; Volpert, Olga V

2011-07-01

Metastatic melanoma cells are highly adaptable to their in vivo microenvironment and can switch between protease-dependent mesenchymal and protease-independent amoeboid invasion to facilitate metastasis. Such adaptability can be visualized in vitro, when cells are cultured in conditions that recapitulate three-dimensional microenvironments. Using thick collagen layers in cell culture and in vivo extravasation assays, we found that pigment epithelium-derived factor (PEDF) suppressed lung extravasation of aggressive melanoma by coordinated regulation of cell shape and proteolysis. In cells grown on a thick collagen bed, PEDF overexpression and exogenous PEDF blocked the rapidly invasive, rounded morphology, and promoted an elongated, mesenchymal-like phenotype associated with reduced invasion. These changes in cell shape depended on decreased RhoA and increased Rac1 activation and were mediated by the up-regulation of Rac1-GEF, DOCK3 and down-regulation of Rac1-GAP, ARHGAP22. Surprisingly, we found that PEDF overexpression also blocked the trafficking of membrane-tethered, MT1-MMP to the cell surface through RhoA inhibition and Rac1 activation. In vivo, knockdown of Rac1 and DOCK3 or overexpression of MT1-MMP was sufficient to reverse the inhibitory effect of PEDF on extravasation. Using functional studies, we demonstrated that PEDF suppressed the rounded morphology and MT1-MMP surface localization through its antiangiongenic, 34-mer epitope and the recently identified PEDF receptor candidate, PNPLA2. Our findings unveil the coordinated regulation of cell shape and proteolysis and identify an unknown mechanism for PEDF's antimetastatic activity.

Keratinocytes negatively regulate the N-cadherin levels of melanoma cells via contact-mediated calcium regulation.

PubMed

Chung, Heesung; Jung, Hyejung; Jho, Eek-Hoon; Multhaupt, Hinke A B; Couchman, John R; Oh, Eok-Soo

2018-06-14

In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, -3 and -6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation. Copyright © 2018. Published by Elsevier Inc.

The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer.

PubMed

Zimmermann, Kerstin; Eells, Rebecca; Heinrich, Frank; Rintoul, Stefanie; Josey, Brian; Shekhar, Prabhanshu; Lösche, Mathias; Stern, Lawrence J

2017-10-27

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζ cyt ) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζ cyt is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζ cyt with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζ cyt to the bilayers in dynamic equilibrium: one in which ζ cyt is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζ cyt apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

Proliferation of prostate cancer cells and activity of neutral endopeptidase is regulated by bombesin and IL-1beta with IL-1beta acting as a modulator of cellular differentiation.

PubMed

Albrecht, Martin; Doroszewicz, Jolanta; Gillen, Sonja; Gomes, Iara; Wilhelm, Beate; Stief, Thomas; Aumüller, Gerhard

2004-01-01

Neutral endopeptidase (NEP) is a cell-surface bound enzyme that cleaves and inactivates neuropeptides such as bombesin and substance P and is involved in the transition from hormonally regulated androgen-dependent prostate cancer (PC) to androgen-independent PC. Neuropeptides are implicated in growth regulation of different cell types and function as transmitters between the neuroendocrine and the immune system. NEP-expression, enzymatic activity of the membrane bound protein, cell proliferation, procalcitonin (PCT) production, and secretion as well as changes in cell morphology of prostatic cells were evaluated after treatment with the immunomodulatory cytokine interleukin-1beta (IL-1beta), neuropeptides (bombesin, substance P), and neuropeptide-conditioned media derived from a human neuroendocrine cell line. Incubation of LNCaP tumor cells with IL-1beta resulted in a diminished proliferative activity, induction of neurite-like outgrowth which was accompanied by the formation of tubular-type mitochondria typical for neuronal/neuroendocrine cells, and an increased production and secretion of PCT. Conversely, proliferation of prostatic stromal cells was enhanced by the cytokine coming along with an increased number of Golgi-apparatuses and ER-cisternae. Bombesin had an antimitotic effect on LNCaP, but not on stromal cells. Substance P did not influence the growth of any of the cell types investigated, whereas neuropeptide-conditioned media exerted a slightly mitogenic effect on both cell types. The activity of LNCaP cell-surface bound NEP was enhanced by bombesin, but was diminished by substance P and neuropeptide-conditioned media. Proliferation and activity of neuropeptide degrading NEP is regulated differently by immunomodulatory substances in PC cells and cells derived from the prostatic stroma with IL-1beta being a potent modulator of cellular differentiation and a potential target for anticancer drug design in PC cells. Copyright 2003 Wiley-Liss, Inc.

Regulation of expression of the ligand for CD40 on T helper lymphocytes.

PubMed

Castle, B E; Kishimoto, K; Stearns, C; Brown, M L; Kehry, M R

1993-08-15

Activated Th cells deliver contact-dependent signals to resting B lymphocytes that initiate and drive B cell proliferation. Recently, a ligand for the B lymphocyte membrane protein, CD40, has been identified that delivers contact-dependent Th cell signals to B cells. A dimeric soluble form of CD40 was produced and used to further characterize the regulation of expression of the CD40 ligand. Expression of the CD40 ligand was rapidly induced after Th lymphocyte activation, and its stability depended upon whether Th cells were activated with soluble or plastic-bound stimuli. Th cells activated with soluble stimuli rapidly turned over cell-surface CD40 ligand whereas Th cells activated with plastic-bound stimuli exhibited more stable CD40 ligand expression for up to 48 h. Removal of activated Th cells from the plastic-bound stimulus resulted in a rapid turnover of CD40 ligand, suggesting that continuous stimulation could maintain CD40 ligand expression. Ligation by soluble CD40 could also stabilize expression of CD40 ligand on the Th cell surface. Both CD40 ligand and IL-2 were transiently synthesized from 1 to 12 h after Th cell activation and had similar kinetics of synthesis. In Con A-activated Th cells newly synthesized CD40 ligand exhibited an initial high turnover (1.5 h t1/2) and after 5 h of Th cell activation became more stable (10-h t1/2). In Th cells activated with plastic-bound anti-CD3, CD40 ligand exhibited a similar biphasic turnover except that the rapid turnover phase began significantly later. This delay could allow more time for newly synthesized CD40 ligand to assemble or associate with other molecules and thus become stabilized on the cell surface. Newly synthesized CD40 ligand in Con A-activated Th cells appeared to not be efficient in delivering Th cell-dependent contact signals to resting B cells, implying the need for assembly or accessory proteins. Regulation of CD40 ligand expression was consistent with all the characteristics of Th cell-delivered contact signals to B cells and may contribute to the high degree of specificity in B cell responses.

PI3K class II α regulates δ-opioid receptor export from the trans-Golgi network.

PubMed

Shiwarski, Daniel J; Darr, Marlena; Telmer, Cheryl A; Bruchez, Marcel P; Puthenveedu, Manojkumar A

2017-08-01

The interplay between signaling and trafficking by G protein-coupled receptors (GPCRs) has focused mainly on endocytic trafficking. Whether and how surface delivery of newly synthesized GPCRs is regulated by extracellular signals is less understood. Here we define a signaling-regulated checkpoint at the trans -Golgi network (TGN) that controls the surface delivery of the delta opioid receptor (δR). In PC12 cells, inhibition of phosphoinositide-3 kinase (PI3K) activity blocked export of newly synthesized δR from the Golgi and delivery to the cell surface, similar to treatment with nerve growth factor (NGF). Depletion of class II phosphoinositide-3 kinase α (PI3K C2A), but not inhibition of class I PI3K, blocked δR export to comparable levels and attenuated δR-mediated cAMP inhibition. NGF treatment displaced PI3K C2A from the Golgi and optogenetic recruitment of the PI3K C2A kinase domain to the TGN-induced δR export downstream of NGF. Of importance, PI3K C2A expression promotes export of endogenous δR in primary trigeminal ganglion neurons. Taken together, our results identify PI3K C2A as being required and sufficient for δR export and surface delivery in neuronal cells and suggest that it could be a key modulator of a novel Golgi export checkpoint that coordinates GPCR delivery to the surface. © 2017 Shiwarski et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Electrochemical Responsive Superhydrophilic Surfaces of Polythiophene Derivatives towards Cell Capture and Release.

PubMed

Hao, Yuwei; Li, Yingying; Zhang, Feilong; Cui, Haijun; Hu, Jinsong; Meng, Jingxin; Wang, Shutao

2018-03-23

Highly efficient cell capture and release with low background are urgently required for early diagnosis of diseases such as cancer. Herein, we report an electrochemical responsive superhydrophilic surface exhibiting specific cell capture and release with high yields and extremely low nonspecific adhesion. Through electrochemical deposition, 3-substituted thiophene derivatives are deposited onto indium tin oxide (ITO) nanowire arrays with 4-n-nonylbenzeneboronic acid (BA) as dopant, fabricating the electrochemical responsive superhydrophilic surfaces. The molecular recognition between sialic acids over-expressed on the cell membrane and doped BAs endows the electrochemical responsive surfaces with the ability to capture and release targeted cancer cells. By adjusting the substituent group of thiophene derivatives, the surface wettability can be readily regulated and further utilized for reducing nonspecific cell adhesion. Significantly, the released cells still maintain a high proliferation ability, which indicates that the applied potential does not significantly harm the cells. Therefore, these results may provide a new strategy to achieve advanced functions of biomedical materials, such as low nonspecific adhesion. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Determinants in the β and δ subunit cytoplasmic loop regulate Golgi trafficking and surface expression of the muscle acetylcholine receptor.

PubMed

Rudell, Jolene Chang; Borges, Lucia S; Rudell, John B; Beck, Kenneth A; Ferns, Michael J

2014-01-03

The molecular determinants that govern nicotinic acetylcholine receptor (AChR) assembly and trafficking are poorly defined, and those identified operate largely during initial receptor biogenesis in the endoplasmic reticulum. To identify determinants that regulate later trafficking steps, we performed an unbiased screen using chimeric proteins consisting of CD4 fused to the muscle AChR subunit cytoplasmic loops. In C2 mouse muscle cells, we found that CD4-β and δ subunit loops were expressed at very low levels on the cell surface, whereas the other subunit loops were robustly expressed on the plasma membrane. The low surface expression of CD4-β and δ loops was due to their pronounced retention in the Golgi apparatus and also to their rapid internalization from the plasma membrane. Both retention and recovery were mediated by the proximal 25-28 amino acids in each loop and were dependent on an ordered sequence of charged and hydrophobic residues. Indeed, βK353L and δK351L mutations increased surface trafficking of the CD4-subunit loops by >6-fold and also decreased their internalization from the plasma membrane. Similarly, combined βK353L and δK351L mutations increased the surface levels of assembled AChR expressed in HEK cells to 138% of wild-type levels. This was due to increased trafficking to the plasma membrane and not decreased AChR turnover. These findings identify novel Golgi retention signals in the β and δ subunit loops that regulate surface trafficking of assembled AChR and may help prevent surface expression of unassembled subunits. Together, these results define molecular determinants that govern a Golgi-based regulatory step in nicotinic AChR trafficking.

Protein synthesis of the pro-inflammatory S100A8/A9 complex in plasmacytoid dendritic cells and cell surface S100A8/A9 on leukocyte subpopulations in systemic lupus erythematosus

PubMed Central

2011-01-01

Introduction Systemic lupus erythematosus (SLE) is an autoimmune disease with chronic or episodic inflammation in many different organ systems, activation of leukocytes and production of pro-inflammatory cytokines. The heterodimer of the cytosolic calcium-binding proteins S100A8 and S100A9 (S100A8/A9) is secreted by activated polymorphonuclear neutrophils (PMNs) and monocytes and serves as a serum marker for several inflammatory diseases. Furthermore, S100A8 and S100A9 have many pro-inflammatory properties such as binding to Toll-like receptor 4 (TLR4). In this study we investigated if aberrant cell surface S100A8/A9 could be seen in SLE and if plasmacytoid dendritic cells (pDCs) could synthesize S100A8/A9. Methods Flow cytometry, confocal microscopy and real-time PCR of flow cytometry-sorted cells were used to measure cell surface S100A8/A9, intracellular S100A8/A9 and mRNA levels of S100A8 and S100A9, respectively. Results Cell surface S100A8/A9 was detected on all leukocyte subpopulations investigated except for T cells. By confocal microscopy, real-time PCR and stimulation assays, we could demonstrate that pDCs, monocytes and PMNs could synthesize S100A8/A9. Furthermore, pDC cell surface S100A8/A9 was higher in patients with active disease as compared to patients with inactive disease. Upon immune complex stimulation, pDCs up-regulated the cell surface S100A8/A9. SLE patients had also increased serum levels of S100A8/A9. Conclusions Patients with SLE had increased cell surface S100A8/A9, which could be important in amplification and persistence of inflammation. Importantly, pDCs were able to synthesize S100A8/A9 proteins and up-regulate the cell surface expression upon immune complex-stimulation. Thus, S100A8/A9 may be a potent target for treatment of inflammatory diseases such as SLE. PMID:21492422

Surface functionalization of nanobiomaterials for application in stem cell culture, tissue engineering, and regenerative medicine.

PubMed

Rana, Deepti; Ramasamy, Keerthana; Leena, Maria; Jiménez, Constanza; Campos, Javier; Ibarra, Paula; Haidar, Ziyad S; Ramalingam, Murugan

2016-05-01

Stem cell-based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial-based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell-based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554-567, 2016. © 2016 American Institute of Chemical Engineers.

Poly(Dopamine)-Assisted Immobilization of Xu Duan on 3D Printed Poly(Lactic Acid) Scaffolds to Up-Regulate Osteogenic and Angiogenic Markers of Bone Marrow Stem Cells.

PubMed

Yeh, Chia-Hung; Chen, Yi-Wen; Shie, Ming-You; Fang, Hsin-Yuan

2015-07-14

Three-dimensional printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid) (PLA) scaffolds and use a mussel-inspired surface coating and Xu Duan (XD) immobilization to regulate cell adhesion, proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBMSCs). We prepared PLA scaffolds and coated with polydopamine (PDA). The chemical composition and surface properties of PLA/PDA/XD were characterized by XPS. PLA/PDA/XD controlled hBMSCs' responses in several ways. Firstly, adhesion and proliferation of hBMSCs cultured on PLA/PDA/XD were significantly enhanced relative to those on PLA. In addition, the focal adhesion kinase (FAK) expression of cells was increased and promoted cell attachment depended on the XD content. In osteogenesis assay, the osteogenesis markers of hBMSCs cultured on PLA/PDA/XD were significantly higher than seen in those cultured on a pure PLA/PDA scaffolds. Moreover, hBMSCs cultured on PLA/PDA/XD showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hBMSCs.

Poly(Dopamine)-Assisted Immobilization of Xu Duan on 3D Printed Poly(Lactic Acid) Scaffolds to Up-Regulate Osteogenic and Angiogenic Markers of Bone Marrow Stem Cells

PubMed Central

Yeh, Chia-Hung; Chen, Yi-Wen; Shie, Ming-You; Fang, Hsin-Yuan

2015-01-01

Three-dimensional printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid) (PLA) scaffolds and use a mussel-inspired surface coating and Xu Duan (XD) immobilization to regulate cell adhesion, proliferation and differentiation of human bone-marrow mesenchymal stem cells (hBMSCs). We prepared PLA scaffolds and coated with polydopamine (PDA). The chemical composition and surface properties of PLA/PDA/XD were characterized by XPS. PLA/PDA/XD controlled hBMSCs’ responses in several ways. Firstly, adhesion and proliferation of hBMSCs cultured on PLA/PDA/XD were significantly enhanced relative to those on PLA. In addition, the focal adhesion kinase (FAK) expression of cells was increased and promoted cell attachment depended on the XD content. In osteogenesis assay, the osteogenesis markers of hBMSCs cultured on PLA/PDA/XD were significantly higher than seen in those cultured on a pure PLA/PDA scaffolds. Moreover, hBMSCs cultured on PLA/PDA/XD showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hBMSCs. PMID:28793441

Transcriptional and posttranscriptional regulation of class I major histocompatibility complex genes following transformation with human adenoviruses.

PubMed Central

Shemesh, J; Rotem-Yehudar, R; Ehrlich, R

1991-01-01

Transformation of rodent cells by human adenoviruses is a well-established model system for studying the expression, regulation, and function of class I antigens. In this report, we demonstrate that the highly oncogenic adenovirus type 12 operates at the transcriptional and posttranscriptional levels in regulating the activity of major histocompatibility complex class I genes and products in transformed cells. Adenovirus type 12 suppresses the cell surface expression of class I antigens in most cell lines. Nevertheless, in a number of cell lines suppression is the result of reduction in the amount of stable specific mRNA, while in another group of cell lines suppression involves interference with processing of a posttranscriptional product. The two mechanisms operate both for the endogenous H-2 genes and for a miniature swine class I transgene that is expressed in the cells. Images PMID:1895404

Actin Engine in Immunological Synapse

PubMed Central

Piragyte, Indre

2012-01-01

T cell activation and function require physical contact with antigen presenting cells at a specialized junctional structure known as the immunological synapse. Once formed, the immunological synapse leads to sustained T cell receptor-mediated signalling and stabilized adhesion. High resolution microscopy indeed had a great impact in understanding the function and dynamic structure of immunological synapse. Trends of recent research are now moving towards understanding the mechanical part of immune system, expanding our knowledge in mechanosensitivity, force generation, and biophysics of cell-cell interaction. Actin cytoskeleton plays inevitable role in adaptive immune system, allowing it to bear dynamic and precise characteristics at the same time. The regulation of mechanical engine seems very complicated and overlapping, but it enables cells to be very sensitive to external signals such as surface rigidity. In this review, we focus on actin regulators and how immune cells regulate dynamic actin rearrangement process to drive the formation of immunological synapse. PMID:22916042

Gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow

PubMed Central

Kim, Su-Hwan; Kim, Young-Sung; Lee, Su-Yeon; Kim, Kyoung-Hwa; Lee, Yong-Moo; Kim, Won-Kyung

2011-01-01

Purpose The aim of this study is to compare the gene expression profile in mesenchymal stem cells derived from dental tissues and bone marrow for characterization of dental stem cells. Methods We employed GeneChip analysis to the expression levels of approximately 32,321 kinds of transcripts in 5 samples of bone-marrow-derived mesenchymal stem cells (BMSCs) (n=1), periodontal ligament stem cells (PDLSCs) (n=2), and dental pulp stem cells (DPSCs) (n=2). Each cell was sorted by a FACS Vantage Sorter using immunocytochemical staining of the early mesenchymal stem cell surface marker STRO-1 before the microarray analysis. Results We identified 379 up-regulated and 133 down-regulated transcripts in BMSCs, 68 up-regulated and 64 down-regulated transcripts in PDLSCs, and 218 up-regulated and 231 down-regulated transcripts in DPSCs. In addition, anatomical structure development and anatomical structure morphogenesis gene ontology (GO) terms were over-represented in all three different mesenchymal stem cells and GO terms related to blood vessels, and neurons were over-represented only in DPSCs. Conclusions This study demonstrated the genome-wide gene expression patterns of STRO-1+ mesenchymal stem cells derived from dental tissues and bone marrow. The differences among the expression profiles of BMSCs, PDLSCs, and DPSCs were shown, and 999 candidate genes were found to be definitely up- or down-regulated. In addition, GOstat analyses of regulated gene products provided over-represented GO classes. These data provide a first step for discovering molecules key to the characteristics of dental stem cells. PMID:21954424

The TspanC8 Subgroup of Tetraspanins Interacts with A Disintegrin and Metalloprotease 10 (ADAM10) and Regulates Its Maturation and Cell Surface Expression*

PubMed Central

Haining, Elizabeth J.; Yang, Jing; Bailey, Rebecca L.; Khan, Kabir; Collier, Richard; Tsai, Schickwann; Watson, Steve P.; Frampton, Jon; Garcia, Paloma; Tomlinson, Michael G.

2012-01-01

A disintegrin and metalloprotease 10 (ADAM10) is a ubiquitous transmembrane metalloprotease that cleaves the extracellular regions from over 40 different transmembrane target proteins, including Notch and amyloid precursor protein. ADAM10 is essential for embryonic development and is also important in inflammation, cancer, and Alzheimer disease. However, ADAM10 regulation remains poorly understood. ADAM10 is compartmentalized into membrane microdomains formed by tetraspanins, which are a superfamily of 33 transmembrane proteins in humans that regulate clustering and trafficking of certain other transmembrane “partner” proteins. This is achieved by specific tetraspanin-partner interactions, but it is not clear which tetraspanins specifically interact with ADAM10. The aims of this study were to identify which tetraspanins interact with ADAM10 and how they regulate this metalloprotease. Co-immunoprecipitation identified specific ADAM10 interactions with Tspan5, Tspan10, Tspan14, Tspan15, Tspan17, and Tspan33/Penumbra. These are members of the largely unstudied TspanC8 subgroup of tetraspanins, all six of which promoted ADAM10 maturation. Different cell types express distinct repertoires of TspanC8 tetraspanins. Human umbilical vein endothelial cells express relatively high levels of Tspan14, the knockdown of which reduced ADAM10 surface expression and activity. Mouse erythrocytes express predominantly Tspan33, and ADAM10 expression was substantially reduced in the absence of this tetraspanin. In contrast, ADAM10 expression was normal on Tspan33-deficient mouse platelets in which Tspan14 is the major TspanC8 tetraspanin. These results define TspanC8 tetraspanins as essential regulators of ADAM10 maturation and trafficking to the cell surface. This finding has therapeutic implications because focusing on specific TspanC8-ADAM10 complexes may allow cell type- and/or substrate-specific ADAM10 targeting. PMID:23035126

CD147, CD44, and the Epidermal Growth Factor Receptor (EGFR) Signaling Pathway Cooperate to Regulate Breast Epithelial Cell Invasiveness*

PubMed Central

Grass, G. Daniel; Tolliver, Lauren B.; Bratoeva, Momka; Toole, Bryan P.

2013-01-01

The immunoglobulin superfamily glycoprotein CD147 (emmprin; basigin) is associated with an invasive phenotype in various types of cancers, including malignant breast cancer. We showed recently that up-regulation of CD147 in non-transformed, non-invasive breast epithelial cells is sufficient to induce an invasive phenotype characterized by membrane type-1 matrix metalloproteinase (MT1-MMP)-dependent invadopodia activity (Grass, G. D., Bratoeva, M., and Toole, B. P. (2012) Regulation of invadopodia formation and activity by CD147. J. Cell Sci. 125, 777–788). Here we found that CD147 induces breast epithelial cell invasiveness by promoting epidermal growth factor receptor (EGFR)-Ras-ERK signaling in a manner dependent on hyaluronan-CD44 interaction. Furthermore, CD147 promotes assembly of signaling complexes containing CD147, CD44, and EGFR in lipid raftlike domains. We also found that oncogenic Ras regulates CD147 expression, hyaluronan synthesis, and formation of CD147-CD44-EGFR complexes, thus forming a positive feedback loop that may amplify invasiveness. Last, we showed that malignant breast cancer cells are heterogeneous in their expression of surface-associated CD147 and that high levels of membrane CD147 correlate with cell surface EGFR and CD44 levels, activated EGFR and ERK1, and activated invadopodia. Future studies should evaluate CD147 as a potential therapeutic target and disease stratification marker in breast cancer. PMID:23888049

Cranial neural crest recycle surface integrins in a substratum-dependent manner to promote rapid motility.

PubMed

Strachan, Lauren R; Condic, Maureen L

2004-11-08

Cell migration is essential for proper development of numerous structures derived from embryonic neural crest cells (NCCs). Although the migratory pathways of NCCs have been determined, the molecular mechanisms regulating NCC motility remain unclear. NCC migration is integrin dependent, and recent work has shown that surface expression levels of particular integrin alpha subunits are important determinants of NCC motility in vitro. Here, we provide evidence that rapid cranial NCC motility on laminin requires integrin recycling. NCCs showed both ligand- and receptor-specific integrin regulation in vitro. On laminin, NCCs accumulated internalized laminin but not fibronectin receptors over 20 min, whereas on fibronectin neither type of receptor accumulated internally beyond 2 min. Internalized laminin receptors colocalized with receptor recycling vesicles and were subsequently recycled back to the cell surface. Blocking receptor recycling with bafilomycin A inhibited NCC motility on laminin, indicating that substratum-dependent integrin recycling is essential for rapid cranial neural crest migration.

Surface design of antibody-immobilized thermoresponsive cell culture dishes for recovering intact cells by low-temperature treatment.

PubMed

Kobayashi, Jun; Hayashi, Masaki; Ohno, Takahiro; Nishi, Masanori; Arisaka, Yoshinori; Matsubara, Yoshinori; Kakidachi, Hiroshi; Akiyama, Yoshikatsu; Yamato, Masayuki; Horii, Akihiro; Okano, Teruo

2014-11-01

Antibody-immobilized thermoresponsive poly(N-isopropylacrylamide-co-2-carboxyisopropylacrylamide) [poly(IPAAm-co-CIPAAm)]-grafted cell culture surfaces were designed to enhance both the initial adhesion of weakly adhering cells and the ability of cells to detach in response to low temperature through the regulation of affinity binding between immobilized antibodies and antigens on the cellular surface. Ty-82 cells and neonatal normal human dermal fibroblasts (NHDFs), which express CD90 on the cell surface, adhered to anti-CD90 antibody-immobilized thermoresponsive surfaces at 37°C, a condition at which the grafted thermoresponsive polymer chains shrank. Adherent Ty-82 cells were detached from the surfaces by lowering the temperature to 20°C and applying external forces, such as pipetting, whereas cultured NHDF sheets spontaneously detached themselves from the surface in response to reduced temperature alone. When the temperature was decreased to 20°C, the swelling of grafted thermoresponsive polymer chains weakened the affinity binding between immobilized antibody and antigen on the cells due to the increasing steric hindrance of the polymer chains around the antigen-recognition site of the immobilized antibodies. No contamination was detected on cells harvested from covalently immobilized antibodies on the culture surfaces by low-temperature treatment, whereas a carryover of the antibody and avidin from the avidin-biotin binding surface was observed. Furthermore, the initial adhesion of adipose tissue-derived cells, which adhere weakly to PIPAAm-grafted surfaces, was enhanced on the antibody-immobilized thermoresponsive surfaces. © 2013 Wiley Periodicals, Inc.

Physical Biology in Cancer. 3. The role of cell glycocalyx in vascular transport of circulating tumor cells

PubMed Central

Mitchell, Michael J.

2013-01-01

Circulating tumor cells (CTCs) in blood are known to adhere to the luminal surface of the microvasculature via receptor-mediated adhesion, which contributes to the spread of cancer metastasis to anatomically distant organs. Such interactions between ligands on CTCs and endothelial cell-bound surface receptors are sensitive to receptor-ligand distances at the nanoscale. The sugar-rich coating expressed on the surface of CTCs and endothelial cells, known as the glycocalyx, serves as a physical structure that can control the spacing and, thus, the availability of such receptor-ligand interactions. The cancer cell glycocalyx can also regulate the ability of therapeutic ligands to bind to CTCs in the bloodstream. Here, we review the role of cell glycocalyx on the adhesion and therapeutic treatment of CTCs in the bloodstream. PMID:24133067

Blood-testis barrier dynamics are regulated by testosterone and cytokines via their differential effects on the kinetics of protein endocytosis and recycling in Sertoli cells

PubMed Central

Yan, Helen H. N.; Mruk, Dolores D.; Lee, Will M.; Cheng, C. Yan

2009-01-01

During spermatogenesis in the mammalian testis, preleptotene/leptotene spermatocytes differentiate from type B spermatogonia and traverse the blood-testis barrier (BTB) at stage VIII of the seminiferous epithelial cycle for further development. This timely movement of germ cells involves extensive junction restructuring at the BTB. Previous studies have shown that these events are regulated by testosterone (T) and cytokines [e.g., the transforming growth factor (TGF) -βs], which promote and disrupt the BTB assembly, respectively. However, the mechanisms underlying the “opening” of the BTB above a migrating preleptotene/leptotene spermatocyte and the “resealing” of the barrier underneath this cell remain obscure. We now report findings on a novel mechanism utilized by the testes to regulate these events. Using cell surface protein biotinylation coupled with immunoblotting and immunofluorescent microscopy, we assessed the kinetics of endocytosis and recycling of BTB-associated integral membrane proteins: occludin, JAM-A, and N-cadherin. It was shown that these proteins were continuously endocytosed and recycled back to the Sertoli cell surface via the clathrin-mediated but not the caveolin-mediated pathway. When T or TGF-β2 was added to Sertoli cell cultures with established functional BTB, both factors accelerated the kinetics of internalization of BTB proteins from the cell surface, perhaps above the migrating preleptotene spermatocyte, thereby opening the BTB. Likewise, T also enhanced the kinetics of recycling of internalized biotinylated proteins back to the cell surface, plausibly relocating these proteins beneath the migrating spermatocyte to reassemble the BTB. In contrast, TGF-β2 targeted internalized biotinylated proteins to late endosomes for degradation, destabilizing the BTB. In summary, the transient opening of the BTB that facilitates germ cell movement is mediated via the differential effects of T and cytokines on the kinetics of endocytosis and recycling of integral membrane proteins at the BTB. The net result of these interactions, in turn, determines the steady-state protein levels at the Sertoli-Sertoli cell interface at the BTB. PMID:18192323

Phenotypic Heterogeneity and the Evolution of Bacterial Life Cycles

PubMed Central

van Gestel, Jordi; Nowak, Martin A.

2016-01-01

Most bacteria live in colonies, where they often express different cell types. The ecological significance of these cell types and their evolutionary origin are often unknown. Here, we study the evolution of cell differentiation in the context of surface colonization. We particularly focus on the evolution of a ‘sticky’ cell type that is required for surface attachment, but is costly to express. The sticky cells not only facilitate their own attachment, but also that of non-sticky cells. Using individual-based simulations, we show that surface colonization rapidly evolves and in most cases leads to phenotypic heterogeneity, in which sticky and non-sticky cells occur side by side on the surface. In the presence of regulation, cell differentiation leads to a remarkable set of bacterial life cycles, in which cells alternate between living in the liquid and living on the surface. The dominant life stage is formed by the surface-attached colony that shows many complex features: colonies reproduce via fission and by producing migratory propagules; cells inside the colony divide labour; and colonies can produce filaments to facilitate expansion. Overall, our model illustrates how the evolution of an adhesive cell type goes hand in hand with the evolution of complex bacterial life cycles. PMID:26894881

Insulin promotes cell migration by regulating PSA-NCAM.

PubMed

Monzo, Hector J; Coppieters, Natacha; Park, Thomas I H; Dieriks, Birger V; Faull, Richard L M; Dragunow, Mike; Curtis, Maurice A

2017-06-01

Cellular interactions with the extracellular environment are modulated by cell surface polysialic acid (PSA) carried by the neural cell adhesion molecule (NCAM). PSA-NCAM is involved in cellular processes such as differentiation, plasticity, and migration, and is elevated in Alzheimer's disease as well as in metastatic tumour cells. Our previous work demonstrated that insulin enhances the abundance of cell surface PSA by inhibiting PSA-NCAM endocytosis. In the present study we have identified a mechanism for insulin-dependent inhibition of PSA-NCAM turnover affecting cell migration. Insulin enhanced the phosphorylation of the focal adhesion kinase leading to dissociation of αv-integrin/PSA-NCAM clusters, and promoted cell migration. Our results show that αv-integrin plays a key role in the PSA-NCAM turnover process. αv-integrin knockdown stopped PSA-NCAM from being endocytosed, and αv-integrin/PSA-NCAM clusters co-labelled intracellularly with Rab5, altogether indicating a role for αv-integrin as a carrier for PSA-NCAM during internalisation. Furthermore, inhibition of p-FAK caused dissociation of αv-integrin/PSA-NCAM clusters and counteracted the insulin-induced accumulation of PSA at the cell surface and cell migration was impaired. Our data reveal a functional association between the insulin/p-FAK-dependent regulation of PSA-NCAM turnover and cell migration through the extracellular matrix. Most importantly, they identify a novel mechanism for insulin-stimulated cell migration. Copyright © 2017 Elsevier Inc. All rights reserved.

Novel Mechanisms in the Regulation of G Protein-coupled Receptor Trafficking to the Plasma Membrane*

PubMed Central

Tholanikunnel, Baby G.; Joseph, Kusumam; Kandasamy, Karthikeyan; Baldys, Aleksander; Raymond, John R.; Luttrell, Louis M.; McDermott, Paul J.; Fernandes, Daniel J.

2010-01-01

β2-Adrenergic receptors (β2-AR) are low abundance, integral membrane proteins that mediate the effects of catecholamines at the cell surface. Whereas the processes governing desensitization of activated β2-ARs and their subsequent removal from the cell surface have been characterized in considerable detail, little is known about the mechanisms controlling trafficking of neo-synthesized receptors to the cell surface. Since the discovery of the signal peptide, the targeting of the integral membrane proteins to plasma membrane has been thought to be determined by structural features of the amino acid sequence alone. Here we report that localization of translationally silenced β2-AR mRNA to the peripheral cytoplasmic regions is critical for receptor localization to the plasma membrane. β2-AR mRNA is recognized by the nucleocytoplasmic shuttling RNA-binding protein HuR, which silences translational initiation while chaperoning the mRNA-protein complex to the cell periphery. When HuR expression is down-regulated, β2-AR mRNA translation is initiated prematurely in perinuclear polyribosomes, leading to overproduction of receptors but defective trafficking to the plasma membrane. Our results underscore the importance of the spatiotemporal relationship between β2-AR mRNA localization, translation, and trafficking to the plasma membrane, and establish a novel mechanism whereby G protein-coupled receptor (GPCR) responsiveness is regulated by RNA-based signals. PMID:20739277

Identification of cell surface glycoprotein markers for glioblastoma-derived stem-like cells using a lectin microarray and LC-MS/MS approach

PubMed Central

He, Jintang; Liu, Yashu; Xie, Xiaolei; Zhu, Thant; Soules, Mary; DiMeco, Francesco; Vescovi, Angelo L.; Fan, Xing; Lubman, David M.

2010-01-01

Despite progress in the treatment of glioblastoma, more than 95% of patients suffering from this disease still die within two years. Recent findings support the belief that cancer stem-like cells are responsible for tumor formation and ongoing growth. Here a method combining lectin microarray and LC-MS/MS was used to discover the cell surface glycoprotein markers of a glioblastoma-derived stem-like cell line. Lectin microarray analysis of cell surface glycans showed that two galactose-specific lectins Trichosanthes kirilowii agglutinin (TKA) and Peanut agglutinin (PNA) could distinguish the stem-like glioblastoma neurosphere culture from a traditional adherent glioblastoma cell line. Agarose-bound TKA and PNA were used to capture the glycoproteins from the two cell cultures, which were analyzed by LC-MS/MS. The glycoproteins were quantified by spectral counting, resulting in the identification of 12 and 11 potential glycoprotein markers from the TKA and PNA captured fractions respectively. Almost all of these proteins were membrane proteins. Differential expression was verified by Western blotting analysis of 6 interesting proteins, including the up-regulated Receptor-type tyrosine-protein phosphatase zeta, Tenascin-C, Chondroitin sulfate proteoglycan NG2, Podocalyxin-like protein 1 and CD90, and the down-regulated CD44. An improved understanding of these proteins may be important for earlier diagnosis and better therapeutic targeting of glioblastoma. PMID:20235609

PDZK1 binding and serine phosphorylation regulate subcellular trafficking of organic anion transport protein 1a1

PubMed Central

Choi, Jo H.; Murray, John W.

2011-01-01

Although perturbation of organic anion transport protein (oatp) cell surface expression can result in drug toxicity, little is known regarding mechanisms regulating its subcellular distribution. Many members of the oatp family, including oatp1a1, have a COOH-terminal PDZ consensus binding motif that interacts with PDZK1, while serines upstream of this site (S634 and S635) can be phosphorylated. Using oatp1a1 as a prototypical member of the oatp family, we prepared plasmids in which these serines were mutated to glutamic acid [E634E635 (oatp1a1EE), phosphomimetic] or alanine [A634A635 (oatp1a1AA), nonphosphorylatable]. Distribution of oatp1a1AA and oatp1a1EE was largely intracellular in transfected human embryonic kidney (HEK) 293T cells. Cotransfection with a plasmid encoding PDZK1 revealed that oatp1a1AA was now expressed largely on the cell surface, while oatp1a1EE remained intracellular. To quantify these changes, studies were performed in HuH7 cells stably transfected with these oatp1a1 plasmids. These cells endogenously express PDZK1. Surface biotinylation at 4°C followed by shift to 37°C showed that oatp1a1EE internalizes quickly compared with oatp1a1AA. To examine a physiological role for phosphorylation in oatp1a1 subcellular distribution, studies were performed in rat hepatocytes exposed to extracellular ATP, a condition that stimulates serine phosphorylation of oatp1a1 via activity of a purinergic receptor. Internalization of oatp1a1 under these conditions was rapid. Thus, although PDZK1 binding is required for optimal cell surface expression of oatp1a1, phosphorylation provides a mechanism for fast regulation of the distribution of oatp1a1 between the cell surface and intracellular vesicular pools. Identification of the proteins and motor molecules that mediate these trafficking events represents an important area for future study. PMID:21183661

Analysis of immune activation and clinical events in acute infectious mononucleosis.

PubMed

Williams, Hilary; Macsween, Karen; McAulay, Karen; Higgins, Craig; Harrison, Nadine; Swerdlow, Anthony; Britton, Kate; Crawford, Dorothy

2004-07-01

The symptoms of infectious mononucleosis (IM) are thought to be caused by T cell activation and cytokine production. Surface lymphocyte activation marker (SLAM)-associated protein (SAP) regulates lymphocyte activation via signals from cell-surface CD244 (2B4) and SLAM (CD150). We followed T cell activation via this SAP/SLAM/CD244 pathway in IM and analyzed whether the results were associated with clinical severity. At diagnosis, SAP, SLAM, and CD244 were significantly up-regulated on CD4 and CD8 T cells; expression decreased during IM, but CD244 and SLAM levels remained higher on CD8 cells 40 days later. There were significantly more lymphocytes expressing CD8 and CD244/CD8 in patients with severe sore throat. The expression of CD8 alone and CD244 on CD8 cells correlated with increased virus load. We suggest that T cells expressing CD244 and SLAM are responsible for the clinical features of IM but that the control of activation is maintained by parallel increased expression of SAP.

Regulation of B Cell Functions by the Sialic Acid-Binding Receptors Siglec-G and CD22

PubMed Central

Jellusova, Julia; Nitschke, Lars

2011-01-01

B cell antigen receptor (BCR) engagement can lead to many different physiologic outcomes. To achieve an appropriate response, the BCR signal is interpreted in the context of other stimuli and several additional receptors on the B cell surface participate in the modulation of the signal. Two members of the Siglec (sialic acid-binding immunoglobulin-like lectin) family, CD22 and Siglec-G have been shown to inhibit the BCR signal. Recent findings indicate that the ability of these two receptors to bind sialic acids might be important to induce tolerance to self-antigens. Sialylated glycans are usually absent on microbes but abundant in higher vertebrates and might therefore provide an important tolerogenic signal. Since the expression of the specific ligands for Siglec-G and CD22 is tightly regulated and since Siglecs are not only able to bind their ligands in trans but also on the same cell surface this might provide additional mechanisms to control the BCR signal. Although both Siglec-G and CD22 are expressed on B cells and are able to inhibit BCR mediated signaling, they also show unique biological functions. While CD22 is the dominant regulator of calcium signaling on conventional B2 cells and also seems to play a role on marginal zone B cells, Siglec-G exerts its function mainly on B1 cells and influences their lifespan and antibody production. Both Siglec-G and CD22 have also recently been linked to toll-like receptor signaling and may provide a link in the regulation of the adaptive and innate immune response of B cells. PMID:22566885

Misfolding of major histocompatibility complex class I molecules in activated T cells allows cis-interactions with receptors and signaling molecules and is associated with tyrosine phosphorylation.

PubMed

Santos, Susana G; Powis, Simon J; Arosa, Fernando A

2004-12-17

Knowledge of the origin and biochemical status of beta(2)-microglobulin-free or misfolded major histocompatibility complex (MHC)-I molecules is essential for understanding their pleiotropic properties. Here we show that in normal human T cells, misfolding of MHC-I molecules is turned on upon activation and cell division and is proportional to the level of proliferation. Immunoprecipitation showed that a number of proteins are associated with MHC-I heavy chains at the surface of activated T cells, including the CD8alphabeta receptor and the chaperone tandem calreticulin/ERp57, associations that rely upon the existence of a pool of HC-10-reactive molecules. Biochemical analysis showed that misfolded MHC-I molecules present at the cell surface are fully glycosylated mature molecules. Importantly, misfolded MHC-I molecules are tyrosine phosphorylated and are associated with kinase activity. In vitro kinase assays followed by reprecipitation indicated that tyrosine phosphorylation of the class I heavy chain is probably mediated by a Src tyrosine kinase because Lck was found associated with HC-10 immunocomplexes. Finally, we show that inhibition of tyrosine phosphorylation by using the Src-family tyrosine kinase inhibitor PP2 resulted in enhanced release of MHC-I heavy chains from the cell surface of activated T cells and a slight down-regulation of cell surface W6/32-reactive molecules. This study provides new insights into the biology of MHC-I molecules and suggests that tyrosine phosphorylation may be involved in the regulation of MHC-I misfolding and expression.

EphA2 cleavage by MT1-MMP triggers single cancer cell invasion via homotypic cell repulsion

PubMed Central

Sugiyama, Nami; Gucciardo, Erika; Tatti, Olga; Varjosalo, Markku; Hyytiäinen, Marko; Gstaiger, Matthias

2013-01-01

Changes in EphA2 signaling can affect cancer cell–cell communication and motility through effects on actomyosin contractility. However, the underlying cell–surface interactions and molecular mechanisms of how EphA2 mediates these effects have remained unclear. We demonstrate here that EphA2 and membrane-anchored membrane type-1 matrix metalloproteinase (MT1-MMP) were selectively up-regulated and coexpressed in invasive breast carcinoma cells, where, upon physical interaction in same cell–surface complexes, MT1-MMP cleaved EphA2 at its Fibronectin type-III domain 1. This cleavage, coupled with EphA2-dependent Src activation, triggered intracellular EphA2 translocation, as well as an increase in RhoA activity and cell junction disassembly, which suggests an overall repulsive effect between cells. Consistent with this, cleavage-prone EphA2-D359I mutant shifted breast carcinoma cell invasion from collective to rounded single-cell invasion within collagen and in vivo. Up-regulated MT1-MMP also codistributed with intracellular EphA2 in invasive cells within human breast carcinomas. These results reveal a new proteolytic regulatory mechanism of cell–cell signaling in cancer invasion. PMID:23629968

Empty conformers of HLA-B preferentially bind CD8 and regulate CD8+ T cell function.

PubMed

Geng, Jie; Altman, John D; Krishnakumar, Sujatha; Raghavan, Malini

2018-05-09

When complexed with antigenic peptides, human leukocyte antigen (HLA) class I (HLA-I) molecules initiate CD8 + T cell responses via interaction with the T cell receptor (TCR) and co-receptor CD8. Peptides are generally critical for the stable cell surface expression of HLA-I molecules. However, for HLA-I alleles such as HLA-B*35:01, peptide-deficient (empty) heterodimers are thermostable and detectable on the cell surface. Additionally, peptide-deficient HLA-B*35:01 tetramers preferentially bind CD8 and to a majority of blood-derived CD8 + T cells via a CD8-dependent binding mode. Further functional studies reveal that peptide-deficient conformers of HLA-B*35:01 do not directly activate CD8 + T cells, but accumulate at the immunological synapse in antigen-induced responses, and enhance cognate peptide-induced cell adhesion and CD8 + T cell activation. Together, these findings indicate that HLA-I peptide occupancy influences CD8 binding affinity, and reveal a new set of regulators of CD8 + T cell activation, mediated by the binding of empty HLA-I to CD8. © 2018, Geng et al.

CD1d-restricted immunoglobulin G formation to GPI-anchored antigens mediated by NKT cells.

PubMed

Schofield, L; McConville, M J; Hansen, D; Campbell, A S; Fraser-Reid, B; Grusby, M J; Tachado, S D

1999-01-08

Immunoglobulin G (IgG) responses require major histocompatibility complex (MHC)-restricted recognition of peptide fragments by conventional CD4(+) helper T cells. Immunoglobulin G responses to glycosylphosphatidylinositol (GPI)- anchored protein antigens, however, were found to be regulated in part through CD1d-restricted recognition of the GPI moiety by thymus-dependent, interleukin-4-producing CD4(+), natural killer cell antigen 1.1 [(NK1.1)+] helper T cells. The CD1-NKT cell pathway regulated immunogobulin G responses to the GPI-anchored surface antigens of Plasmodium and Trypanosoma and may be a general mechanism for rapid, MHC-unrestricted antibody responses to diverse pathogens.

The Effect of Size and Species on Lens Intracellular Hydrostatic Pressure

PubMed Central

Gao, Junyuan; Sun, Xiurong; Moore, Leon C.; Brink, Peter R.; White, Thomas W.; Mathias, Richard T.

2013-01-01

Purpose. Previous experiments showed that mouse lenses have an intracellular hydrostatic pressure that varied from 335 mm Hg in central fibers to 0 mm Hg in surface cells. Model calculations predicted that in larger lenses, all else equal, pressure should increase as the lens radius squared. To test this prediction, lenses of different radii from different species were studied. Methods. All studies were done in intact lenses. Intracellular hydrostatic pressures were measured with a microelectrode-manometer–based system. Membrane conductances were measured by frequency domain impedance analysis. Intracellular Na+ concentrations were measured by injecting the Na+-sensitive dye sodium-binding benzofuran isophthalate. Results. Intracellular hydrostatic pressures were measured in lenses from mice, rats, rabbits, and dogs with radii (cm) 0.11, 0.22, 0.49, and 0.57, respectively. In each species, pressure varied from 335 ± 6 mm Hg in central fiber cells to 0 mm Hg in surface cells. Further characterization of transport in lenses from mice and rats showed that the density of fiber cell gap junction channels was approximately the same, intracellular Na+ concentrations varied from 17 mM in central fiber cells to 7 mM in surface cells, and intracellular voltages varied from −45 mV in central fiber cells to −60 mV in surface cells. Fiber cell membrane conductance was a factor of 2.7 times larger in mouse than in rat lenses. Conclusions. Intracellular hydrostatic pressure is an important physiological parameter that is regulated in lenses from these different species. The most likely mechanism of regulation is to reduce the density of open Na+-leak channels in fiber cells of larger lenses. PMID:23211824

Antibody neutralization of cell-surface gC1qR/HABP1/SF2-p32 prevents lamellipodia formation and tumorigenesis

PubMed Central

Kim, Beom-Chan; Hwang, Hyun-Jung; An, Hyoung-Tae; Lee, Hyun; Park, Jun-Sub; Hong, Jin; Ko, Jesang; Kim, Chungho; Lee, Jae-Seon; Ko, Young-Gyu

2016-01-01

We previously demonstrated that cell-surface gC1qR is a key regulator of lamellipodia formation and cancer metastasis. Here, we screened a monoclonal mouse antibody against gC1qR to prevent cell migration by neutralizing cell-surface gC1qR. The anti-gC1qR antibody prevented growth factor-stimulated lamellipodia formation, cell migration and focal adhesion kinase activation by inactivating receptor tyrosine kinases (RTKs) in various cancer cells such as A549, MDA-MB-231, MCF7 and HeLa cells. The antibody neutralization of cell-surface gC1qR also inhibited angiogenesis because the anti-gC1qR antibody prevented growth factor-stimulated RTK activation, lamellipodia formation, cell migration and tube formation in HUVEC. In addition, we found that A549 tumorigenesis was reduced in a xenograft mouse model by following the administration of the anti-gC1qR antibody. With these data, we can conclude that the antibody neutralization of cell-surface gC1qR could be a good therapeutic strategy for cancer treatment. PMID:27363031

Proteomic and genetics insights on the response of the bacteriocinogenic Lactobacillus sakei CRL1862 during biofilm formation on stainless steel surface at 10°C.

PubMed

Pérez-Ibarreche, Mariana; Mendoza, Lucía M; Vignolo, Graciela; Fadda, Silvina

2017-10-03

Some lactic acid bacteria have the ability to form biofilms on food-industry surfaces and this property could be used to control food pathogens colonization. Lactobacillus sakei CR1862 was selected considering its bacteriocinogenic nature and ability to adhere to abiotic surfaces at low temperatures. In this study, the proteome of L. sakei CRL1862 grown either under biofilm on stainless steel surface and planktonic modes of growth at 10°C, was investigated. Using two-dimensional gel electrophoresis, 29 out of 43 statistically significant spots were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Ten proteins resulted up-regulated whereas 16 were down-regulated during biofilm formation. Differentially expressed proteins were found to belong to carbohydrate, nucleotide, aminoacid and lipid metabolisms as well as translation, peptide hydrolysis, cell envelope/cell wall biosynthesis, adaption to atypical conditions and protein secretion. Some proteins related to carbohydrate and nucleotide metabolisms, translation and peptide degradation were overexpressed whereas those associated to stress conditions were synthesized in lower amounts. It seems that conditions for biofilm development would not imply a stressful environment for L. sakei CRL1862 cells, directing its growth strategy towards glycolytic flux regulation and reinforcing protein synthesis. In addition, L. sakei CRL1862 showed to harbor nine out of ten assayed genes involved in biofilm formation and protein anchoring. By applying qRT-PCR analysis, four of these genes showed to be up regulated, srtA2 being the most remarkable. The results of this study contribute to the knowledge of the physiology of L. sakei CRL1862 growing in biofilm on a characteristic food contact surface. The use of this strain as green biocide preventing L. monocytogenes post-processing contamination on industrial surfaces may be considered. Copyright © 2017 Elsevier B.V. All rights reserved.

Tyrosine phosphorylation–dependent activation of TRPC6 regulated by PLC-γ1 and nephrin: effect of mutations associated with focal segmental glomerulosclerosis

PubMed Central

Kanda, Shoichiro; Harita, Yutaka; Shibagaki, Yoshio; Sekine, Takashi; Igarashi, Takashi; Inoue, Takafumi; Hattori, Seisuke

2011-01-01

Transient receptor potential canonicals (TRPCs) play important roles in the regulation of intracellular calcium concentration. Mutations in the TRPC6 gene are found in patients with focal segmental glomerulosclerosis (FSGS), a proteinuric disease characterized by dysregulated function of renal glomerular epithelial cells (podocytes). There is as yet no clear picture for the activation mechanism of TRPC6 at the molecular basis, however, and the association between its channel activity and pathogenesis remains unclear. We demonstrate here that tyrosine phosphorylation of TRPC6 induces a complex formation with phospholipase C (PLC)-γ1, which is prerequisite for TRPC6 surface expression. Furthermore, nephrin, an adhesion protein between the foot processes of podocytes, binds to phosphorylated TRPC6 via its cytoplasmic domain, competitively inhibiting TRPC6–PLC-γ1 complex formation, TRPC6 surface localization, and TRPC6 activation. Importantly, FSGS-associated mutations render the mutated TRPC6s insensitive to nephrin suppression, thereby promoting their surface expression and channel activation. These results delineate the mechanism of TRPC6 activation regulated by tyrosine phosphorylation, and imply the cell type–specific regulation, which correlates the FSGS mutations with deregulated TRPC6 channel activity. PMID:21471003

Insulin-induced exocytosis regulates the cell surface level of low-density lipoprotein-related protein-1 in Müller Glial cells.

PubMed

Actis Dato, Virginia; Grosso, Rubén A; Sánchez, María C; Fader, Claudio M; Chiabrando, Gustavo A

2018-05-15

Low-density lipoprotein (LDL) receptor-related protein-1 (LRP1) is expressed in retinal Müller glial cells (MGCs) and regulates intracellular translocation to the plasma membrane (PM) of the membrane proteins involved in cellular motility and activity. Different functions of MGCs may be influenced by insulin, including the removal of extracellular glutamate in the retina. In the present work, we investigated whether insulin promotes LRP1 translocation to the PM in the Müller glial-derived cell line MIO-M1 (human retinal Müller glial cell-derived cell line). We demonstrated that LRP1 is stored in small vesicles containing an approximate size of 100 nm (mean diameter range of 100-120 nm), which were positive for sortilin and VAMP2, and also incorporated GLUT4 when it was transiently transfected. Next, we observed that LRP1 translocation to the PM was promoted by insulin-regulated exocytosis through intracellular activation of the IR/PI 3 K/Akt axis and Rab-GTPase proteins such as Rab8A and Rab10. In addition, these Rab-GTPases regulated both the constitutive and insulin-induced LRP1 translocation to the PM. Finally, we found that dominant-negative Rab8A and Rab10 mutants impaired insulin-induced intracellular signaling of the IR/PI3K/Akt axis, suggesting that these GTPase proteins as well as the LRP1 level at the cell surface are involved in insulin-induced IR activation. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

The HTLV-I tax protein transcriptionally modulates OX40 antigen expression.

PubMed

Pankow, R; Dürkop, H; Latza, U; Krause, H; Kunzendorf, U; Pohl, T; Bulfone-Paus, S

2000-07-01

OX40 is a member of the TNF receptor family, expressed on activated T cells. It is the only costimulatory T cell molecule known to be specifically up-regulated in human T cell leukemia virus type-I (HTLV-I)-producing cells. In a T cell line, OX40 surface expression was shown to be induced by HTLV-I Tax alone. To understand molecular mechanisms of OX40 gene regulation and modulation by HTLV-I Tax, we have cloned the human OX40 gene and analyzed its 5'-flanking region. By reporter gene analysis with progressive 5' deletions from nucleotides -1259 to -64, we have defined a 157-bp DNA fragment as a minimal promoter for constitutive expression. In addition, we show that in the OX40+ cell line, Co, Tax is able to further increase OX40 surface expression. Up-regulation of OX40 promoter activity by Tax requires two upstream NF-kappaB sites, which are not active in the constitutive OX40 expression. Their deletion abrogates Tax responsiveness in reporter gene analysis. The site-directed mutagenesis of each NF-kappaB site demonstrates that cooperative NF-kappaB binding is a prerequisite for Tax-directed activity as neither site alone is sufficient for a full Tax responsiveness of the OX40 promoter. Upon Tax expression, both sites bind p65 and c-Rel. These data provide new insight into the direct regulation of OX40 by Tax and add to our understanding of the possible role of the OX40/OX40 ligand system in the proliferation of HTLV-I+ T cells.

iRhom2 regulates CSF1R cell surface expression and non-steady state myelopoiesis in mice.

PubMed

Qing, Xiaoping; Rogers, Lindsay; Mortha, Arthur; Lavin, Yonit; Redecha, Patricia; Issuree, Priya D; Maretzky, Thorsten; Merad, Miriam; McIlwain, David; Mak, Tak W; Overall, Christopher M; Blobel, Carl P; Salmon, Jane E

2016-12-01

CSF1R (colony stimulating factor 1 receptor) is the main receptor for CSF1 and has crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by ADAM17 (A disintegrin and metalloprotease 17). Here, we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2-/- mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2-/- BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild-type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2-/- Lin - SCA-1 + c-Kit + (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

iRhom2 regulates cell surface expression of CSF1R and non-steady state myelopoiesis in mice

PubMed Central

Qing, Xiaoping; Lavin, Yonit; Redecha, Patricia; Issuree, Priya D.; Maretzky, Thorsten; Merad, Miriam; McIlwain, David; Mak, Tak W.; Overall, Christopher M.

2016-01-01

The colony stimulating factor 1 receptor (CSF1R) functions as the major receptor for macrophage colony stimulating factor (CSF1) with crucial roles in regulating myelopoeisis. CSF1R can be proteolytically released from the cell surface by A disintegrin and metalloprotease 17 (ADAM17). Here we identified CSF1R as a major substrate of ADAM17 in an unbiased degradomics screen. We explored the impact of CSF1R shedding by ADAM17 and its upstream regulator, inactive rhomboid protein 2 (iRhom2, gene name Rhbdf2), on homeostatic development of mouse myeloid cells. In iRhom2−/− mice, we found constitutive accumulation of membrane-bound CSF1R on myeloid cells at steady state, although cell numbers of these populations were not altered. However, in the context of mixed bone marrow (BM) chimera, under competitive pressure, iRhom2−/− BM progenitor-derived monocytes, tissue macrophages and lung DCs showed a repopulation advantage over those derived from wild type (WT) BM progenitors, suggesting enhanced CSF1R signaling in the absence of iRhom2. In vitro experiments indicate that iRhom2−/− Lin−SCA-1+c-Kit+ (LSKs) cells, but not granulocyte-macrophage progenitors (GMPs), had faster growth rates than WT cells in response to CSF1. Our results shed light on an important role of iRhom2/ADAM17 pathway in regulation of CSF1R shedding and repopulation of monocytes, macrophages and DCs. PMID:27601030

Understanding the Capsanthin Tails in Regulating the Hydrophilic-Lipophilic Balance of Carbon Dots for a Rapid Crossing Cell Membrane.

PubMed

Chen, Jing; Zhang, Xiang; Zhang, Ye; Wang, Wei; Li, Shuya; Wang, Yucai; Hu, Mengyue; Liu, Li; Bi, Hong

2017-10-03

Here we use natural Chinese paprika to prepare a new kind of amphiphilic carbon dot (A-Dot) that exhibits bright, multicolored fluorescence and contains hydrophilic groups as well as lipophilic capsanthin tails on the surface. It is found that the capsanthin tails in a phospholipid-like structure can promote cell internalization of the A-Dots via crossing cell membranes rapidly in an energy-independent fashion. Compared to highly hydrophilic carbon dots (H-Dots), a control sample prepared from the microwave thermolysis of citric acid and ethylenediamine, our synthesized A-Dots can be taken up by CHO, HeLa, and HFF cells more easily. More importantly, we develop a method to calibrate the hydrophilic-lipophilic balance (HLB) values of various kinds of carbon dots (C-Dots). HLB values of A-Dots and H-Dots are determined to be 6.4 and 18.4, respectively. Moreover, we discover that the cellular uptake efficiency of C-Dots is closely related to their HLBs, and the C-Dots with an HLB value of around 6.4 cross the cell membrane easier and faster. As we regulate the HLB value of the A-Dots from 6.4 to 15.3 by removing the capsanthin tails from their surfaces via alkali refluxing, it is found that the refluxed A-Dots can hardly cross HeLa cell membranes. Our work is an essential step toward understanding the importance of regulating the HLB values as well as the surface polarity of the C-Dots for their practical use in bioimaging and also provides a simple but effective way to judge whether the C-Dots in hand are appropriate for cell imaging.

Altered Expression of TAP-1 and Major Histocompatibility Complex Class I in Laryngeal Papillomatosis: Correlation of TAP-1 with Disease

PubMed Central

Vambutas, Andrea; Bonagura, Vincent R.; Steinberg, Bettie M.

2000-01-01

Recurrent respiratory papillomatosis (RRP) is an insidious disease caused by human papillomavirus (HPV) infection. It is characterized by a variable clinical course that can include frequent disease recurrence, significant morbidity, and occasional mortality. The mechanisms responsible for the variability in the clinical course and the persistence of latent HPV infection remain unknown. Effective T-cell-mediated clearance of HPV-infected cells may be defective in patients with RRP, leading to recurrent disease and failure to suppress latent HPV reactivation. This study describes the down-regulation of the transporter associated with antigen presentation (TAP-1) and the major histocompatibility complex (MHC) class I protein expression in laryngeal papilloma tissue biopsies and cell culture of primary explants. There was a statistically significant correlation between reduction of TAP-1 expression in biopsy tissues and rapid recurrence of disease. Patients with RRP had less frequent recurrence if their papillomas expressed TAP-1 at levels close to that of normal tissue, compared with those with very low expression of TAP-1, who had frequent recurrence (32 versus 5 weeks to the next surgical intervention). These findings suggest that HPV may evade immune recognition by down-regulating class I MHC cell surface expression via decreased TAP-1 levels. Expression of TAP-1 could be used for prognostic evaluation of disease severity. Gamma interferon was able to restore class I MHC expression at the surfaces of laryngeal papilloma cells in culture. This up-regulation of class I MHC antigen at the cell surface potentially allows the infected cell to become a target for the immune system again. This finding provides some promise for nonsurgical treatment of laryngeal papillomas. PMID:10618282

IL-27 induces the production of IgG1 by human B cells.

PubMed

Boumendjel, Amel; Tawk, Lina; Malefijt, René de Waal; Boulay, Vera; Yssel, Hans; Pène, Jérôme

2006-12-01

It has been reported that IL-27 specifically induces the production of IgG2a by mouse B cells and inhibits IL-4-induced IgG1 synthesis. Here, we show that human naïve cord blood expresses a functional IL-27 receptor, consisting of the TCCR and gp130 subunits, although at lower levels as compared to naïve and memory splenic B cells. IL-27 does not induce proliferative responses and does not increase IgG1 production by CD19(+)CD27(+) memory B cells. However, it induces a low, but significant production of IgG1 by naïve CD19(+)CD27(-)IgD(+)IgG(-) spleen and cord blood B cells, activated via CD40, whereas it has no effect on the production of the other IgG subclasses. In addition, IL-27 induces the differentiation of a population of B cells that express high levels of CD38, in association with a down-regulation of surface IgD expression, and that are surface IgG(+/int), CD20(low), CD27(high), indicating that IL-27 promotes isotype switching and plasma cell differentiation of naive B cells. However, as compared to the effects of IL-21 and IL-10, both switch factors for human IgG1 and IgG3, those of IL-27 are modest and regulate exclusively the production of IgG1. Finally, although IL-27 has no effect on IL-4 and anti-CD40-induced Cepsilon germline promoter activity, it up-regulates IL-4-induced IgE production by naive B cells. These results point to a partial redundancy of switch factors regulating the production of IgG1 in humans, and furthermore indicate the existence of a common regulation of the human IgG1and murine IgG2a isotypes by IL-27.

Aft2, a Novel Transcription Regulator, Is Required for Iron Metabolism, Oxidative Stress, Surface Adhesion and Hyphal Development in Candida albicans

PubMed Central

Xu, Ning; Cheng, Xinxin; Yu, Qilin; Qian, Kefan; Ding, Xiaohui; Liu, Ruming; Zhang, Biao; Xing, Laijun; Li, Mingchun

2013-01-01

Morphological transition and iron metabolism are closely relevant to Candida albicans pathogenicity and virulence. In our previous study, we demonstrated that C. albicans Aft2 plays an important role in ferric reductase activity and virulence. Here, we further explored the roles of C. albicans Aft2 in numerous cellular processes. We found that C. albicans Aft2 exhibited an important role in iron metabolism through bi-directional regulation effects on iron-regulon expression. Deletion of AFT2 reduced cellular iron accumulation under iron-deficient conditions. Furthermore, both reactive oxygen species (ROS) generation and superoxide dismutase (SOD) activity were remarkably increased in the aft2Δ/Δ mutant, which were thought to be responsible for the defective responses to oxidative stress. However, we found that over-expression of C. albicans AFT2 under the regulation of the strong PGK1 promoter could not effectively rescue Saccharomyces cerevisiae aft1Δ mutant defects in some cellular processes, such as cell-wall assembly, ion homeostasis and alkaline resistance, suggesting a possibility that C. albicans Aft2 weakened its functional role of regulating some cellular metabolism during the evolutionary process. Interestingly, deletion of AFT2 in C. albicans increased cell surface hydrophobicity, cell flocculation and the ability of adhesion to polystyrene surfaces. In addition, our results also revealed that C. albicans Aft2 played a dual role in regulating hypha-specific genes under solid and liquid hyphal inducing conditions. Deletion of AFT2 caused an impaired invasive growth in solid medium, but an increased filamentous aggregation and growth in liquid conditions. Moreover, iron deficiency and environmental cues induced nuclear import of Aft2, providing additional evidence for the roles of Aft2 in transcriptional regulation. PMID:23626810

Noncontact Viscoelastic Imaging of Living Cells Using a Long-Needle Atomic Force Microscope with Dual-Frequency Modulation

NASA Astrophysics Data System (ADS)

Guan, Dongshi; Charlaix, Elisabeth; Qi, Robert Z.; Tong, Penger

2017-10-01

Imaging of surface topography and elasticity of living cells can provide insight into the roles played by the cells' volumetric and mechanical properties and their response to external forces in regulating the essential cellular events and functions. Here, we report a unique technique of noncontact viscoelastic imaging of live cells using atomic force microscopy (AFM) with a long-needle glass probe. Because only the probe tip is placed in a liquid medium near the cell surface, the AFM cantilever in air functions well under dual-frequency modulation, retaining its high-quality resonant modes. The probe tip interacts with the cell surface through a minute hydrodynamic flow in the nanometer-thin gap region between them without physical contact. Quantitative measurements of the cell height, volume, and Young's modulus are conducted simultaneously. The experiment demonstrates that the long-needle AFM has a wide range of applications in the study of cell mechanics.

Inflammatory cytokine response to titanium chemical composition and nanoscale calcium phosphate surface modification.

PubMed

Hamlet, Stephen; Ivanovski, Saso

2011-05-01

Nanoscale surface modification of titanium dental implants with calcium phosphate (CaP) has been shown to achieve superior bone wound healing and osseointegration compared with smooth or microrough titanium surfaces alone. As bone healing has been shown to be influenced by the action of cytokines, this study examined whether changes in cytokine gene expression from RAW 264.7 cells cultured on commercially pure and titanium alloy (Ti-6Al-4V) microrough or nanoscale crystalline CaP-modified surfaces, may influence downstream events in bone wound healing and osseointegration. Whilst no significant difference in the attachment or proliferation of RAW 264.7 cells was observed, the nanoscale CaP-modified surface elicited a gene expression profile with marked down-regulation of a number of pro-inflammatory cytokines and chemokines. Inflammatory cytokine gene expression was further influenced by chemical composition, with lower levels of pro-inflammatory markers noted following exposure of the macrophage-like cells to titanium alloy (Ti-6Al-4V) compared with the commercially pure titanium surface. Down-regulation of pro-inflammatory cytokine gene expression (confirmed at the protein level for TNFα and CCL5), may thus facilitate the enhanced bone wound healing and osseointegration observed clinically with nanoscale calcium phosphate-modified implant surfaces. Copyright © 2011 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Modulating macrophage polarization with divalent cations in nanostructured titanium implant surfaces

NASA Astrophysics Data System (ADS)

Lee, Chung-Ho; Kim, Youn-Jeong; Jang, Je-Hee; Park, Jin-Woo

2016-02-01

Nanoscale topographical modification and surface chemistry alteration using bioactive ions are centrally important processes in the current design of the surface of titanium (Ti) bone implants with enhanced bone healing capacity. Macrophages play a central role in the early tissue healing stage and their activity in response to the implant surface is known to affect the subsequent healing outcome. Thus, the positive modulation of macrophage phenotype polarization (i.e. towards the regenerative M2 rather than the inflammatory M1 phenotype) with a modified surface is essential for the osteogenesis funtion of Ti bone implants. However, relatively few advances have been made in terms of modulating the macrophage-centered early healing capacity in the surface design of Ti bone implants for the two important surface properties of nanotopography and and bioactive ion chemistry. We investigated whether surface bioactive ion modification exerts a definite beneficial effect on inducing regenerative M2 macrophage polarization when combined with the surface nanotopography of Ti. Our results indicate that nanoscale topographical modification and surface bioactive ion chemistry can positively modulate the macrophage phenotype in a Ti implant surface. To the best of our knowledge, this is the first demonstration that chemical surface modification using divalent cations (Ca and Sr) dramatically induces the regenerative M2 macrophage phenotype of J774.A1 cells in nanostructured Ti surfaces. In this study, divalent cation chemistry regulated the cell shape of adherent macrophages and markedly up-regulated M2 macrophage phenotype expression when combined with the nanostructured Ti surface. These results provide insight into the surface engineering of future Ti bone implants that are harmonized between the macrophage-governed early wound healing process and subsequent mesenchymal stem cell-centered osteogenesis function.

BTK inhibition results in impaired CXCR4 chemokine receptor surface expression, signaling and function in chronic lymphocytic leukemia.

PubMed

Chen, S-S; Chang, B Y; Chang, S; Tong, T; Ham, S; Sherry, B; Burger, J A; Rai, K R; Chiorazzi, N

2016-04-01

Bruton's tyrosine kinase (BTK) is involved in the regulation of B-cell growth, migration and adhesion. The importance of BTK in cell trafficking is emphasized by the clonal contraction proceeded by lymphocytosis typical for the enzyme inhibitor, ibrutinib, in B-cell malignancies, including chronic lymphocytic leukemia (CLL). Here, we investigated BTK regulation of leukemic B-cell trafficking in a mouse model of aggressive TCL1 CLL-like disease. Inhibiting BTK by ibrutinib reduced surface membrane (sm) levels of CXCR4 but not CXCR5, CD49d and other adhesion/homing receptors. Decreased smCXCR4 levels resulted from blocking receptor signal transduction, which in turn aborted cycling from and to the membrane. This resulted in rapid re-distribution of CLL cells from spleens and lymph nodes into the circulation. CLL cells with impaired smCXCR4 from BTK inhibition failed to home to spleens. These functional changes mainly resulted from inhibition of CXCR4 phosphorylation at Ser339, mediated directly by blocking BTK enzymatic activity and indirectly by affecting the function of downstream targets PLCγ2 and PKCμ, and eventually synthesis of PIM-1 and BTK itself. Our data identify CXCR4 as a key regulator in BTK-mediated CLL-cell retention and have elucidated a complex set of not previously described mechanisms responsible for these effects.

Regulated internalization of caveolae

PubMed Central

1994-01-01

Caveolae are specialized invaginations of the plasma membrane which have been proposed to play a role in diverse cellular processes such as endocytosis and signal transduction. We have developed an assay to determine the fraction of internal versus plasma membrane caveolae. The GPI-anchored protein, alkaline phosphatase, was clustered in caveolae after antibody-induced crosslinking at low temperature and then, after various treatments, the relative amount of alkaline phosphatase on the cell surface was determined. Using this assay we were able to show a time- and temperature-dependent decrease in cell-surface alkaline phosphatase activity which was dependent on antibody-induced clustering. The decrease in cell surface alkaline phosphatase activity was greatly accelerated by the phosphatase inhibitor, okadaic acid, but not by a protein kinase C activator. Internalization of clustered alkaline phosphatase in the presence or absence of okadaic acid was blocked by cytochalasin D and by the kinase inhibitor staurosporine. Electron microscopy confirmed that okadaic acid induced removal of caveolae from the cell surface. In the presence of hypertonic medium this was followed by the redistribution of groups of caveolae to the center of the cell close to the microtubule-organizing center. This process was reversible, blocked by cytochalasin D, and the centralization of the caveolar clusters was shown to be dependent on an intact microtubule network. Although the exact mechanism of internalization remains unknown, the results show that caveolae are dynamic structures which can be internalized into the cell. This process may be regulated by kinase activity and require an intact actin network. PMID:7962085

Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes*

PubMed Central

Park, Dayoung; Brune, Kristin A.; Mitra, Anupam; Marusina, Alina I.; Maverakis, Emanual; Lebrilla, Carlito B.

2015-01-01

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. PMID:26355101

LDL receptor-related protein 1 regulates the abundance of diverse cell-signaling proteins in the plasma membrane proteome.

PubMed

Gaultier, Alban; Simon, Gabriel; Niessen, Sherry; Dix, Melissa; Takimoto, Shinako; Cravatt, Benjamin F; Gonias, Steven L

2010-12-03

LDL receptor-related protein 1 (LRP1) is an endocytic receptor, reported to regulate the abundance of other receptors in the plasma membrane, including uPAR and tissue factor. The goal of this study was to identify novel plasma membrane proteins, involved in cell-signaling, that are regulated by LRP1. Membrane protein ectodomains were prepared from RAW 264.7 cells in which LRP1 was silenced and control cells using protease K. Peptides were identified by LC-MS/MS. By analysis of spectral counts, 31 transmembrane and secreted proteins were regulated in abundance at least 2-fold when LRP1 was silenced. Validation studies confirmed that semaphorin4D (Sema4D), plexin domain-containing protein-1 (Plxdc1), and neuropilin-1 were more abundant in the membranes of LRP1 gene-silenced cells. Regulation of Plxdc1 by LRP1 was confirmed in CHO cells, as a second model system. Plxdc1 coimmunoprecipitated with LRP1 from extracts of RAW 264.7 cells and mouse liver. Although Sema4D did not coimmunoprecipitate with LRP1, the cell-surface level of Sema4D was increased by RAP, which binds to LRP1 and inhibits binding of other ligands. These studies identify Plxdc1, Sema4D, and neuropilin-1 as novel LRP1-regulated cell-signaling proteins. Overall, LRP1 emerges as a generalized regulator of the plasma membrane proteome.

Reconstructing human pancreatic differentiation by mapping specific cell populations during development.

PubMed

Ramond, Cyrille; Glaser, Nicolas; Berthault, Claire; Ameri, Jacqueline; Kirkegaard, Jeannette Schlichting; Hansson, Mattias; Honoré, Christian; Semb, Henrik; Scharfmann, Raphaël

2017-07-21

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2 + population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3 , a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated.

Haemagglutinin-neuraminidase from HPIV3 mediates human NK regulation of T cell proliferation via NKp44 and NKp46.

PubMed

McQuaid, Samantha; Loughran, Sinead; Power, Patrick; Maguire, Paula; Walls, Dermot; Grazia Cusi, Maria; Orvell, Claes; Johnson, Patricia

2018-06-01

HPIV3 is a respiratory virus causing airway diseases, including pneumonia, croup, and bronchiolitis, during infancy and childhood. Currently there is no effective vaccine or anti-viral therapy for this virus. Studies have suggested that poor T cell proliferation following HPIV3 infection is responsible for impaired immunological memory associated with this virus. We have previously demonstrated that NK cells mediate regulation of T cell proliferation during HPIV3 infection. Here we add to these studies by demonstrating that the regulation of T cell proliferation during HPIV3 infection is mediated via NK receptors NKp44 and NKp46 and involves the surface glycoprotein haemagglutinin-neuraminidase but not the fusion protein of the virus. These studies extend our knowledge of the regulatory repertoire of NK cells and provide mechanistic insights which may explain reoccurring failures of vaccines against this virus.

Nuclear Migration During Retinal Development

PubMed Central

Baye, Lisa M.; Link, Brian A.

2009-01-01

In this review we focus on the mechanisms, regulation, and cellular consequences of nuclear migration in the developing retina. In the nervous system, nuclear migration is prominent during both proliferative and post-mitotic phases of development. Interkinetic nuclear migration is the process where the nucleus oscillates from the apical to basal surfaces in proliferative neuroepithelia. Proliferative nuclear movement occurs in step with the cell cycle, with M-phase being confined to the apical surface and G1-, S-, and G2-phases occurring at more basal locations. Later, following cell cycle exit, some neuron precursors migrate by nuclear translocation. In this mode of cellular migration, nuclear movement is the driving force for motility. Following discussion of the key components and important regulators for each of these processes, we present an emerging model where interkinetic nuclear migration functions to distinguish cell fates among retinal neuroepithelia. PMID:17560964

The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR

PubMed Central

Boncompain, Gaelle; Laketa, Vibor; Poser, Ina; Beck, Martin; Bork, Peer

2016-01-01

Stimulation of cells with epidermal growth factor (EGF) induces internalization and partial degradation of the EGF receptor (EGFR) by the endo-lysosomal pathway. For continuous cell functioning, EGFR plasma membrane levels are maintained by transporting newly synthesized EGFRs to the cell surface. The regulation of this process is largely unknown. In this study, we find that EGF stimulation specifically increases the transport efficiency of newly synthesized EGFRs from the endoplasmic reticulum to the plasma membrane. This coincides with an up-regulation of the inner coat protein complex II (COPII) components SEC23B, SEC24B, and SEC24D, which we show to be specifically required for EGFR transport. Up-regulation of these COPII components requires the transcriptional regulator RNF11, which localizes to early endosomes and appears additionally in the cell nucleus upon continuous EGF stimulation. Collectively, our work identifies a new regulatory mechanism that integrates the degradation and transport of EGFR in order to maintain its physiological levels at the plasma membrane. PMID:27872256

Effect of therapeutic concentration of lithium on live HEK293 cells; increase of Na+/K+-ATPase, change of overall protein composition and alteration of surface layer of plasma membrane.

PubMed

Vosahlikova, Miroslava; Ujcikova, Hana; Chernyavskiy, Oleksandr; Brejchova, Jana; Roubalova, Lenka; Alda, Martin; Svoboda, Petr

2017-05-01

The effect of long-term exposure of live cells to lithium cations (Li) was studied in HEK293 cells cultivated in the presence of 1mM LiCl for 7 or 21days. The alteration of Na + /K + -ATPase level, protein composition and biophysical state of plasma membrane was determined with the aim to characterize the physiological state of Li-treated cells. Na + /K + -ATPase level was determined by [ 3 H]ouabain binding and immunoblot assays. Overall protein composition was determined by 2D electrophoresis followed by proteomic analysis by MALDI-TOF MS/MS and LFQ. Li interaction with plasma membrane was characterized by fluorescent probes DPH, TMA-DPH and Laurdan. Na + /K + -ATPase was increased in plasma membranes isolated from cells exposed to Li. Identification of Li-altered proteins by 2D electrophoresis, MALDI-TOF MS/MS and LFQ suggests a change of energy metabolism in mitochondria and cytosol and alteration of cell homeostasis of calcium. Measurement of Laurdan generalized polarization indicated a significant alteration of surface layer of isolated plasma membranes prepared from both types of Li-treated cells. Prolonged exposure of HEK293 cells to 1mM LiCl results in up-regulation of Na + /K + -ATPase expression, reorganization of overall cellular metabolism and alteration of the surface layer/polar head-group region of isolated plasma membranes. Our findings demonstrate adaptation of live HEK293 cell metabolism to prolonged exposure to therapeutic concentration of Li manifested as up-regulation of Na + /K + -ATPase expression, alteration of protein composition and change of the surface layer of plasma membrane. Copyright © 2017 Elsevier B.V. All rights reserved.

Topography of calcium phosphate ceramics regulates primary cilia length and TGF receptor recruitment associated with osteogenesis.

PubMed

Zhang, Jingwei; Dalbay, Melis T; Luo, Xiaoman; Vrij, Erik; Barbieri, Davide; Moroni, Lorenzo; de Bruijn, Joost D; van Blitterswijk, Clemens A; Chapple, J Paul; Knight, Martin M; Yuan, Huipin

2017-07-15

The surface topography of synthetic biomaterials is known to play a role in material-driven osteogenesis. Recent studies show that TGFβ signalling also initiates osteogenic differentiation. TGFβ signalling requires the recruitment of TGFβ receptors (TGFβR) to the primary cilia. In this study, we hypothesize that the surface topography of calcium phosphate ceramics regulates stem cell morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation. We developed a 2D system using two types of tricalcium phosphate (TCP) ceramic discs with identical chemistry. One sample had a surface topography at micron-scale (TCP-B, with a bigger surface structure dimension) whilst the other had a surface topography at submicron scale (TCP-S, with a smaller surface structure dimension). In the absence of osteogenic differentiation factors, human bone marrow stromal cells (hBMSCs) were more spread on TCP-S than on TCP-B with alterations in actin organization and increased primary cilia prevalence and length. The cilia elongation on TCP-S was similar to that observed on glass in the presence of osteogenic media and was followed by recruitment of transforming growth factor-β RII (p-TGFβ RII) to the cilia axoneme. This was associated with enhanced osteogenic differentiation of hBMSCs on TCP-S, as shown by alkaline phosphatase activity and gene expression for key osteogenic markers in the absence of additional osteogenic growth factors. Similarly, in vivo after a 12-week intramuscular implantation in dogs, TCP-S induced bone formation while TCP-B did not. It is most likely that the surface topography of calcium phosphate ceramics regulates primary cilia length and ciliary recruitment of p-TGFβ RII associated with osteogenesis and bone formation. This bioengineering control of osteogenesis via primary cilia modulation may represent a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery applications. The surface topography of synthetic biomaterials plays important roles in material-driven osteogenesis. The data presented herein have shown that the surface topography of calcium phosphate ceramics regulates mesenchymal stromal cells (e.g., human bone marrow mesenchymal stromal cells, hBMSCs) with respect to morphology, primary cilia structure and TGFβR recruitment to the cilium associated with osteogenic differentiation in vitro. Together with bone formation in vivo, our results suggested a new type of biomaterial-based ciliotherapy for orthopedic, dental and maxillofacial surgery by the bioengineering control of osteogenesis via primary cilia modulation. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Myosin-II controls cellular branching morphogenesis and migration in 3D by minimizing cell surface curvature

PubMed Central

Elliott, Hunter; Fischer, Robert A.; Myers, Kenneth A.; Desai, Ravi A.; Gao, Lin; Chen, Christopher S.; Adelstein, Robert; Waterman, Clare M.; Danuser, Gaudenz

2014-01-01

In many cases cell function is intimately linked to cell shape control. We utilized endothelial cell branching morphogenesis as a model to understand the role of myosin-II in shape control of invasive cells migrating in 3D collagen gels. We applied principles of differential geometry and mathematical morphology to 3D image sets to parameterize cell branch structure and local cell surface curvature. We find that Rho/ROCK-stimulated myosin-II contractility minimizes cell-scale branching by recognizing and minimizing local cell surface curvature. Utilizing micro-fabrication to constrain cell shape identifies a positive feedback mechanism in which low curvature stabilizes myosin-II cortical association, where it acts to maintain minimal curvature. The feedback between myosin-II regulation by and control of curvature drives cycles of localized cortical myosin-II assembly and disassembly. These cycles in turn mediate alternating phases of directionally biased branch initiation and retraction to guide 3D cell migration. PMID:25621949

Cationic polystyrene nanospheres induce autophagic cell death through the induction of endoplasmic reticulum stress

NASA Astrophysics Data System (ADS)

Chiu, Hui-Wen; Xia, Tian; Lee, Yu-Hsuan; Chen, Chun-Wan; Tsai, Jui-Chen; Wang, Ying-Jan

2014-12-01

Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design.Nanoparticles (NPs) have been used to produce a wide range of products that have applications in imaging and drug delivery in medicine. Due to their chemical stability, well-controlled sizes and surface charges, polystyrene (PS) NPs have been developed as biosensors and drug delivery carriers. However, the possible adverse biological effects and underlying mechanisms are still unclear. Recently, autophagy has been implicated in the regulation of cell death. In this study, we evaluated a library of PS NPs with different surface charges. We found that NH2-labeled polystyrene (NH2-PS) nanospheres were highly toxic with enhanced uptake in macrophage (RAW 264.7) and lung epithelial (BEAS-2B) cells. Furthermore, NH2-PS could induce autophagic cell death. NH2-PS increased autophagic flux due to reactive oxygen species (ROS) generation and endoplasmic reticulum (ER) stress caused by misfolded protein aggregation. The inhibition of ER stress decreased cytotoxicity and autophagy in the NH2-PS-treated cells. In addition, the Akt/mTOR and AMPK signaling pathways were involved in the regulation of NH2-PS-triggered autophagic cell death. These results suggest an important role of autophagy in cationic NP-induced cell death and provide mechanistic insights into the inhibition of the toxicity and safe material design. Electronic supplementary information (ESI) available. See DOI: 10.1039/c4nr05509h

Phosphatidylserine as an anchor for plasminogen and its plasminogen receptor, Histone H2B, to the macrophage surface

PubMed Central

DAS, R.; PLOW, E. F.

2013-01-01

Summary Background Plasminogen (Plg) binding to cell surface Plg receptors (Plg-Rs) on the surface of macrophages facilitates Plg activation and migration of these cells. Histone H2B (H2B) acts as a Plg-R and its cell surface expression is upregulated when monocytes are differentiated to macrophages via a pathway dependent on L-type Ca2+ channels and intracellular Ca2+. Objectives We sought to investigate the mechanism by which H2B, a protein without a transmembrane domain, is retained on themacrophage surface. Methods THP-1 monocytoid cells were induced to differentiate with interferon gamma + Vitamin D3 or to undergo apoptosis by treatment with camptothecin. Flow cytometry and cell surface biotinylation followed by Western blotting were used to measure the interrelationship between Plg binding, cell surface expression of H2B and outermembrane exposure of phosphatidylserine (PS). Results H2B interacted directly with PS via an electrostatic interaction. Anti-PS or PS binding proteins, annexin V and protein S, diminished H2B interaction with PS on the surface of differentiated or apoptotic cells and these same reagents inhibited Plg binding to these cells. L-type Ca2+ channels played a significant role in PS exposure, H2B surface expression and Plg binding induced either by differentiation or apoptosis. Conclusions These data suggest that H2B tethers to the surface of cells by interacting with PS on differentiated or apoptotic monocytoid cells. L-type Ca2+ channels regulate PS exposure on the surface of these cells. The exposed PS interacts directly with H2B and hence provides sites for Plg to bind to. PMID:21040449

The solid state environment orchestrates embryonic development and tissue remodeling

NASA Technical Reports Server (NTRS)

Damsky, C. H.; Moursi, A.; Zhou, Y.; Fisher, S. J.; Globus, R. K.

1997-01-01

Cell interactions with extracellular matrix and with other cells play critical roles in morphogenesis during development and in tissue homeostasis and remodeling throughout life. Extracellular matrix is information-rich, not only because it is comprised of multifunctional structural ligands for cell surface adhesion receptors, but also because it contains peptide signaling factors, and proteinases and their inhibitors. The functions of these groups of molecules are extensively interrelated. In this review, three primary cell culture models are described that focus on adhesion receptors and their roles in complex aspects of morphogenesis and remodeling: the regulation of proteinase expression by fibronectin and integrins in synovial fibroblasts; the regulation of osteoblast differentiation and survival by fibronectin, and the regulation of trophoblast differentiation and invasion by integrins, cadherins and immunoglobulin family adhesion receptors.

Molecular cloning and chromosomal mapping of bone marrow stromal cell surface gene, BST2, that may be involved in pre-B-cell growth

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ishikawa, Jun; Kaisho, Tsuneyasu; Tomizawa, Hitoshi

1995-04-10

Bone marrow stromal cells regulate B-cell growth and development through their surface molecules and cytokines. In this study, we generated a mAb, RS38, that recognized a novel human membrane protein, BST-2, expressed on bone marrow stromal cell lines and synovial cell lines. We cloned a cDNA encoding BST-2 from a rheumatoid arthritis-derived synovial cell line. BST-2 is a 30- to 36-kDa type II transmembrane protein, consisting of 180 amino acids. The BST-2 gene (HGMW-approved symbol BST2) is located on chromosome 19p13.2. BST-2 is expressed not only on certain bone marrow stromal cell lines but also on various normal tissues, althoughmore » its expression pattern is different from that of another bone marrow stromal cell surface molecule, BST-1. BST-2 surface expression on fibroblast cell lines facilitated the stromal cell-dependent growth of a murine bone marrow-derived pre-B-cell line, DW34. The results suggest that BST-2 may be involved in pre-B-cell growth. 45 refs., 7 figs., 2 tabs.« less

Anti-B-Cell Therapies in Autoimmune Neurological Diseases: Rationale and Efficacy Trials.

PubMed

Alexopoulos, Harry; Biba, Angie; Dalakas, Marinos C

2016-01-01

B cells have an ever-increasing role in the etiopathology of a number of autoimmune neurological disorders, acting as antibody-producing cells and, most importantly, as sensors, coordinators, and regulators of the immune response. B cells, among other functions, regulate the T-cell activation process through their participation in antigen presentation and production of cytokines. The availability of monoclonal antibodies or fusion proteins against B-cell surface molecules or B-cell trophic factors bestows a rational approach for treating autoimmune neurological disorders, even when T cells are the main effector cells. This review summarizes basic aspects of B-cell biology, discusses the role(s) of B cells in neurological autoimmunity, and presents anti-B-cell drugs that are either currently on the market or are expected to be available in the near future for treating neurological autoimmune disorders.

A Stable Human-Cell System Overexpressing Cystic Fibrosis Transmembrane Conductance Regulator Recombinant Protein at the Cell Surface

PubMed Central

Dai, Qun; Aleksandrov, Andrei A.; Bajrami, Bekim; Diego, Pamela Ann; Wu, Xing; Ray, Marjorie; Naren, Anjaparavanda P.; Riordan, John R.; Yao, Xudong; DeLucas, Lawrence J.; Urbatsch, Ina L.; Kappes, John C.

2015-01-01

Recent human clinical trials results demonstrated successful treatment for certain genetic forms of cystic fibrosis (CF). To extend treatment opportunities to those afflicted with other genetic forms of CF disease, structural and biophysical characterization of CF transmembrane conductance regulator (CFTR) is urgently needed. In this study, CFTR was modified with various tags, including a His10 purification tag, the SUMOstar (SUMO*) domain, an extracellular FLAG epitope, or an enhanced green fluorescent protein (EGFP), each alone or in various combinations. Expressed in HEK293 cells, recombinant CFTR proteins underwent complex glycosylation, compartmentalized with the plasma membrane, and exhibited regulated chloride-channel activity with only modest alterations in channel conductance and gating kinetics. Surface CFTR expression level was enhanced by the presence of SUMO* on the N-terminus. Quantitative mass-spectrometric analysis indicated approximately 10% of the total recombinant CFTR (SUMO*-CFTRFLAG-EGFP) localized to the plasma membrane. Trial purification using dodecylmaltoside for membrane protein extraction reproducibly recovered 178 ± 56 μg SUMO*-CFTRFLAG-EGFP per billion cells at 80% purity. Fluorescence size-exclusion chromatography indicated purified CFTR was monodisperse. These findings demonstrate a stable mammalian cell expression system capable of producing human CFTR of sufficient quality and quantity to augment futrure CF drug discovery efforts, including biophysical and structural studies. PMID:25577540

An Iron-Regulated Autolysin Remodels the Cell Wall To Facilitate Heme Acquisition in Staphylococcus lugdunensis

PubMed Central

Farrand, Allison J.; Haley, Kathryn P.; Lareau, Nichole M.; Heilbronner, Simon; McLean, John A.; Foster, Timothy

2015-01-01

Bacteria alter their cell surface in response to changing environments, including those encountered upon invasion of a host during infection. One alteration that occurs in several Gram-positive pathogens is the presentation of cell wall-anchored components of the iron-regulated surface determinant (Isd) system, which extracts heme from host hemoglobin to fulfill the bacterial requirement for iron. Staphylococcus lugdunensis, an opportunistic pathogen that causes infective endocarditis, encodes an Isd system. Unique among the known Isd systems, S. lugdunensis contains a gene encoding a putative autolysin located adjacent to the Isd operon. To elucidate the function of this putative autolysin, here named IsdP, we investigated its contribution to Isd protein localization and hemoglobin-dependent iron acquisition. S. lugdunensis IsdP was found to be iron regulated and cotranscribed with the Isd operon. IsdP is a specialized peptidoglycan hydrolase that cleaves the stem peptide and pentaglycine crossbridge of the cell wall and alters processing and anchoring of a major Isd system component, IsdC. Perturbation of IsdC localization due to isdP inactivation results in a hemoglobin utilization growth defect. These studies establish IsdP as an autolysin that functions in heme acquisition and describe a role for IsdP in cell wall reorganization to accommodate nutrient uptake systems during infection. PMID:26123800

JACKDAW controls epidermal patterning in the Arabidopsis root meristem through a non-cell-autonomous mechanism.

PubMed

Hassan, Hala; Scheres, Ben; Blilou, Ikram

2010-05-01

In Arabidopsis, specification of the hair and non-hair epidermal cell types is position dependent, in that hair cells arise over clefts in the underlying cortical cell layer. Epidermal patterning is determined by a network of transcriptional regulators that respond to an as yet unknown cue from underlying tissues. Previously, we showed that JACKDAW (JKD), a zinc finger protein, localizes in the quiescent centre and the ground tissue, and regulates tissue boundaries and asymmetric cell division by delimiting SHORT-ROOT movement. Here, we provide evidence that JKD controls position-dependent signals that regulate epidermal-cell-type patterning. JKD is required for appropriately patterned expression of the epidermal cell fate regulators GLABRA2, CAPRICE and WEREWOLF. Genetic interaction studies indicate that JKD operates upstream of the epidermal patterning network in a SCRAMBLED (SCM)-dependent fashion after embryogenesis, but acts independent of SCM in embryogenesis. Tissue-specific induction experiments indicate non-cell-autonomous action of JKD from the underlying cortex cell layer to specify epidermal cell fate. Our findings are consistent with a model where JKD induces a signal in every cortex cell that is more abundant in the hair cell position owing to the larger surface contact of cells located over a cleft.

Dynamic balance between master transcription factors determines the fates and functions of CD4 T cell and innate lymphoid cell subsets

PubMed Central

2017-01-01

CD4 T cells, including T regulatory cells (Treg cells) and effector T helper cells (Th cells), and recently identified innate lymphoid cells (ILCs) play important roles in host defense and inflammation. Both CD4 T cells and ILCs can be classified into distinct lineages based on their functions and the expression of lineage-specific genes, including those encoding effector cytokines, cell surface markers, and key transcription factors. It was first recognized that each lineage expresses a specific master transcription factor and the expression of these factors is mutually exclusive because of cross-regulation among these factors. However, recent studies indicate that the master regulators are often coexpressed. Furthermore, the expression of master regulators can be dynamic and quantitative. In this review, we will first discuss similarities and differences between the development and functions of CD4 T cell and ILC subsets and then summarize recent literature on quantitative, dynamic, and cell type–specific balance between the master transcription factors in determining heterogeneity and plasticity of these subsets. PMID:28630089

Down-regulation of Pax6 is associated with abnormal differentiation of corneal epithelial cells in severe ocular surface diseases

PubMed Central

Li, W; Chen, Y-T; Hayashida, Y; Blanco, G; Kheirkah, A; He, H; Chen, S-Y; Liu, C-Y; Tseng, SCG

2010-01-01

Pax6 is the universal master control gene for eye morphogenesis. Other than retina and lens, Pax6 also expressed in the ocular surface epithelium from early gestation until the postnatal stage, in which little is known about the function of Pax6. In this study, corneal pannus tissues from patients with ocular surface diseases such as Stevens–Johnson syndrome (SJS), chemical burn, aniridia and recurrent pterygium were investigated. Our results showed that normal ocular surface epithelial cells expressed Pax6. However, corneal pannus epithelial cells from the above patients showed a decline or absence of Pax6 expression, accompanied by a decline or absence of K12 keratin but an increase of K10 keratin and filaggrin expression. Pannus basal epithelial cells maintained nuclear p63 expression and showed activated proliferation, evidenced by positive Ki67 and K16 keratin staining. On 3T3 fibroblast feeder layers, Pax6 immunostaining was negative in clones generated from epithelial cells harvested from corneal pannus from SJS or aniridia, but positive in those from the normal limbal epithelium; whereas western blots showed that some epithelial clones expanded from pannus retained Pax6 expression. Transient transfection of an adenoviral vector carrying EGFP–Pax6 transgenes into these Pax6− clones increased both Pax6 and K12 keratin expression. These results indicate that Pax6 helps to maintain the normal corneal epithelial phenotype postnatally, and that down-regulation of Pax6 is associated with abnormal epidermal differentiation in severe ocular surface diseases. Reintroduction of activation of the Pax6 gene might be useful in treating squamous metaplasia of the ocular surface epithelium. PMID:18027901

Sialyl-lactotetra, a novel cell surface marker of undifferentiated human pluripotent stem cells.

PubMed

Barone, Angela; Säljö, Karin; Benktander, John; Blomqvist, Maria; Månsson, Jan-Eric; Johansson, Bengt R; Mölne, Johan; Aspegren, Anders; Björquist, Petter; Breimer, Michael E; Teneberg, Susann

2014-07-04

Cell surface glycoconjugates are used as markers for undifferentiated pluripotent stem cells. Here, antibody binding and mass spectrometry characterization of acid glycosphingolipids isolated from a large number (1 × 10(9) cells) of human embryonic stem cell (hESC) lines allowed identification of several novel acid glycosphingolipids, like the gangliosides sialyl-lactotetraosylceramide and sialyl-globotetraosylceramide, and the sulfated glycosphingolipids sulfatide, sulf-lactosylceramide, and sulf-globopentaosylceramide. A high cell surface expression of sialyl-lactotetra on hESC and human induced pluripotent stem cells (hiPSC) was demonstrated by flow cytometry, immunohistochemistry, and electron microscopy, whereas sulfated glycosphingolipids were only found in intracellular compartments. Immunohistochemistry showed distinct cell surface anti-sialyl-lactotetra staining on all seven hESC lines and three hiPSC lines analyzed, whereas no staining of hESC-derived hepatocyte-like or cardiomyocyte-like cells was obtained. Upon differentiation of hiPSC into hepatocyte-like cells, the sialyl-lactotetra epitope was rapidly down-regulated and not detectable after 14 days. These findings identify sialyl-lactotetra as a promising marker of undifferentiated human pluripotent stem cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Rapid Steroid Hormone Actions Initiated at the Cell Surface and the Receptors that Mediate Them with an Emphasis on Recent Progress in Fish Models

PubMed Central

Thomas, Peter

2011-01-01

In addition to the classic genomic mechanism of steroid action mediated by activation of intracellular nuclear receptors, there is now extensive evidence that steroids also activate receptors on the cell surface to initiate rapid intracellular signaling and biological responses that are often nongenomic. Recent progress in our understanding of rapid, cell surface-initiated actions of estrogens, progestins, androgens and corticosteroids and the identities of the membrane receptors that act as their intermediaries is briefly reviewed with a special emphasis on studies in teleost fish. Two recently discovered novel proteins with seven-transmembrane domains, G protein-coupled receptor 30 (GPR30), and membrane progestin receptors (mPRs) have the ligand binding and signaling characteristics of estrogen and progestin membrane receptors, respectively, but their functional significance is disputed by some researchers. GPR30 is expressed on the cell surface of fish oocytes and mediates estrogen inhibition of oocyte maturation. mPRα is also expressed on the oocyte cell surface and is the intermediary in progestin induction of oocyte maturation in fish. Recent results suggest there is cross-talk between these two hormonal pathways and that there is reciprocal down-regulation of GPR30 and mPRα expression by estrogens and progestins at different phases of oocyte development to regulate the onset of oocyte maturation. There is also evidence in fish that mPRs are involved in progestin induction of sperm hypermotility and anti-apoptotic actions in ovarian follicle cells. Nonclassical androgen and corticosteroid actions have also been described in fish models but the membrane receptors mediating these actions have not been identified. PMID:22154643

Site-specific O-glycosylation of N-terminal serine residues by polypeptide GalNAc-transferase 2 modulates human δ-opioid receptor turnover at the plasma membrane.

PubMed

Lackman, Jarkko J; Goth, Christoffer K; Halim, Adnan; Vakhrushev, Sergey Y; Clausen, Henrik; Petäjä-Repo, Ulla E

2018-01-01

G protein-coupled receptors (GPCRs) are an important protein family of signalling receptors that govern a wide variety of physiological functions. The capacity to transmit extracellular signals and the extent of cellular response are largely determined by the amount of functional receptors at the cell surface that is subject to complex and fine-tuned regulation. Here, we demonstrate that the cell surface expression level of an inhibitory GPCR, the human δ-opioid receptor (hδOR) involved in pain and mood regulation, is modulated by site-specific N-acetylgalactosamine (GalNAc) -type O-glycosylation. Importantly, we identified one out of the 20 polypeptide GalNAc-transferase isoforms, GalNAc-T2, as the specific regulator of O-glycosylation of Ser6, Ser25 and Ser29 in the N-terminal ectodomain of the receptor. This was demonstrated by in vitro glycosylation assays using peptides corresponding to the hδOR N-terminus, Vicia villosa lectin affinity purification of receptors expressed in HEK293 SimpleCells capable of synthesizing only truncated O-glycans, GalNAc-T edited cell line model systems, and site-directed mutagenesis of the putative O-glycosylation sites. Interestingly, a single-nucleotide polymorphism, at residue 27 (F27C), was found to alter O-glycosylation of the receptor in efficiency as well as in glycosite usage. Furthermore, flow cytometry and cell surface biotinylation assays using O-glycan deficient CHO-ldlD cells revealed that the absence of O-glycans results in decreased receptor levels at the plasma membrane due to enhanced turnover. In addition, mutation of the identified O-glycosylation sites led to a decrease in the number of ligand-binding competent receptors and impaired agonist-mediated inhibition of cyclic AMP accumulation in HEK293 cells. Thus, site-specific O-glycosylation by a selected GalNAc-T isoform can increase the stability of a GPCR, in a process that modulates the constitutive turnover and steady-state levels of functional receptors at the cell surface. Copyright © 2017 Elsevier Inc. All rights reserved.

Silver-nanoparticles-modified biomaterial surface resistant to staphylococcus: new insight into the antimicrobial action of silver

PubMed Central

Wang, Jiaxing; Li, Jinhua; Guo, Geyong; Wang, Qiaojie; Tang, Jin; Zhao, Yaochao; Qin, Hui; Wahafu, Tuerhongjiang; Shen, Hao; Liu, Xuanyong; Zhang, Xianlong

2016-01-01

Titanium implants are widely used clinically, but postoperative implant infection remains a potential severe complication. The purpose of this study was to investigate the antibacterial activity of nano-silver(Ag)-functionalized Ti surfaces against epidemic Staphylococcus from the perspective of the regulation of biofilm-related genes and based on a bacteria-cell co-culture study. To achieve this goal, two representative epidemic Staphylococcus strains, Staphylococcus epidermidis (S. epidermidis, RP62A) and Staphylococcus aureus (S. aureus, USA 300), were used, and it was found that an Ag-nanoparticle-modified Ti surface could regulate the expression levels of biofilm-related genes (icaA and icaR for S. epidermidis; fnbA and fnbB for S. aureus) to inhibit bacterial adhesion and biofilm formation. Moreover, a novel bacteria-fibroblast co-culture study revealed that the incorporation of Ag nanoparticles on such a surface can help mammalian cells to survive, adhere and spread more successfully than Staphylococcus. Therefore, the modified surface was demonstrated to possess a good anti-infective capability against both sessile bacteria and planktonic bacteria through synergy between the effects of Ag nanoparticles and ion release. This work provides new insight into the antimicrobial action and mechanism of Ag-nanoparticle-functionalized Ti surfaces with bacteria-killing and cell-assisting capabilities and paves the way towards better satisfying the clinical needs. PMID:27599568

DSG2 Is a Functional Cell Surface Marker for Identification and Isolation of Human Pluripotent Stem Cells.

PubMed

Park, Jongjin; Son, Yeonsung; Lee, Na Geum; Lee, Kyungmin; Lee, Dong Gwang; Song, Jinhoi; Lee, Jaemin; Kim, Seokho; Cho, Min Ji; Jang, Ju-Hong; Lee, Jangwook; Park, Jong-Gil; Kim, Yeon-Gu; Kim, Jang-Seong; Lee, Jungwoon; Cho, Yee Sook; Park, Young-Jun; Han, Baek Soo; Bae, Kwang-Hee; Han, Seungmin; Kang, Byunghoon; Haam, Seungjoo; Lee, Sang-Hyun; Lee, Sang Chul; Min, Jeong-Ki

2018-06-08

Pluripotent stem cells (PSCs) represent the most promising clinical source for regenerative medicine. However, given the cellular heterogeneity within cultivation and safety concerns, the development of specific and efficient tools to isolate a pure population and eliminate all residual undifferentiated PSCs from differentiated derivatives is a prerequisite for clinical applications. In this study, we raised a monoclonal antibody and identified its target antigen as desmoglein-2 (DSG2). DSG2 co-localized with human PSC (hPSC)-specific cell surface markers, and its expression was rapidly downregulated upon differentiation. The depletion of DSG2 markedly decreased hPSC proliferation and pluripotency marker expression. In addition, DSG2-negative population in hPSCs exhibited a notable suppression in embryonic body and teratoma formation. The actions of DSG2 in regulating the self-renewal and pluripotency of hPSCs were predominantly exerted through the regulation of β-catenin/Slug-mediated epithelial-to-mesenchymal transition. Our results demonstrate that DSG2 is a valuable PSC surface marker that is essential for the maintenance of PSC self-renewal. Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.

The type II cGMP dependent protein kinase regulates GluA1 levels at the plasma membrane of developing cerebellar granule cells

PubMed Central

Incontro, Salvatore; Ciruela, Francisco; Ziff, Edward; Hofmann, Franz; Sánchez-Prieto, José; Torres, Magdalena

2014-01-01

Trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) is regulated by specific interactions with other proteins and by post-translational mechanisms, such as phosphorylation. We have found that the type II cGMP-dependent protein kinase (cGKII) phosphorylates GluA1 (formerly GluR1) at S845, augmenting the surface expression of AMPARs at both synaptic and extrasynaptic sites. Activation of cGKII by 8-Br-cGMP enhances the surface expression of GluA1, whereas its inhibition or suppression effectively diminished the expression of this protein at the cell surface. In granule cells, NMDA receptor activation (NMDAR) stimulates nitric oxide and cGMP production, which in turn activates cGKII and induces the phosphorylation of GluA1, promoting its accumulation in the plasma membrane. GluA1 is mainly incorporated into calcium permeable AMPARs as exposure to 8-Br-cGMP or NMDA activation enhanced AMPA-elicited calcium responses that are sensitive to NASPM inhibition. We summarize evidence for an increase of calcium permeable AMPA receptors downstream of NMDA receptor activation that might be relevant for granule cell development and plasticity. PMID:23545413

Antithetical effects of hemicellulase-treated Agaricus blazei on the maturation of murine bone-marrow-derived dendritic cells

PubMed Central

Kawamura, Masaki; Kasai, Hirotake; He, Limin; Deng, Xuewen; Yamashita, Atsuya; Terunuma, Hiroshi; Horiuchi, Isao; Tanabe, Fuminori; Ito, Masahiko

2005-01-01

We report the effects of hemicellulase-treated Agaricus blazei (ABH) on the maturation of bone-marrow-derived dendritic cells (BMDCs). ABH activated immature BMDCs, inducing up-regulation of surface molecules, such as CD40, CD80 and major histocompatibility complex class I antigens, as well as inducing allogeneic T-cell proliferation and T helper type 1 cell development. However, unlike lipopolysaccharide (LPS), ABH did not stimulate the BMDCs to produce proinflammatory cytokines, such as interleukin-12 (IL-12) p40, tumour necrosis factor-α, or IL-1β. In addition, ABH suppressed LPS-induced DC responses. Pretreatment of DCs with ABH markedly reduced the levels of LPS-induced cytokine secretion, while only slightly decreasing up-regulation of the surface molecules involved in maturation. ABH also had a significant impact on peptidoglycan-induced or CpG oligodeoxynucleotide-induced IL-12p40 production in DCs. The inhibition of LPS-induced responses was not associated with a cytotoxic effect of ABH nor with an anti-inflammatory effect of IL-10. However, ABH decreased NF-κB-induced reporter gene expression in LPS-stimulated J774.1 cells. Interestingly, DCs preincubated with ABH and then stimulated with LPS augmented T helper type 1 responses in culture with allogeneic T cells as compared to LPS-stimulated but non-ABH-pretreated DCs. These observations suggest that ABH regulates DC-mediated responses. PMID:15720441

Directing Stem Cell Differentiation via Electrochemical Reversible Switching between Nanotubes and Nanotips of Polypyrrole Array.

PubMed

Wei, Yan; Mo, Xiaoju; Zhang, Pengchao; Li, Yingying; Liao, Jingwen; Li, Yongjun; Zhang, Jinxing; Ning, Chengyun; Wang, Shutao; Deng, Xuliang; Jiang, Lei

2017-06-27

Control of stem cell behaviors at solid biointerfaces is critical for stem-cell-based regeneration and generally achieved by engineering chemical composition, topography, and stiffness. However, the influence of dynamic stimuli at the nanoscale from solid biointerfaces on stem cell fate remains unclear. Herein, we show that electrochemical switching of a polypyrrole (Ppy) array between nanotubes and nanotips can alter surface adhesion, which can strongly influence mechanotransduction activation and guide differentiation of mesenchymal stem cells (MSCs). The Ppy array, prepared via template-free electrochemical polymerization, can be reversibly switched between highly adhesive hydrophobic nanotubes and poorly adhesive hydrophilic nanotips through an electrochemical oxidation/reduction process, resulting in dynamic attachment and detachment to MSCs at the nanoscale. Multicyclic attachment/detachment of the Ppy array to MSCs can activate intracellular mechanotransduction and osteogenic differentiation independent of surface stiffness and chemical induction. This smart surface, permitting transduction of nanoscaled dynamic physical inputs into biological outputs, provides an alternative to classical cell culture substrates for regulating stem cell fate commitment. This study represents a general strategy to explore nanoscaled interactions between stem cells and stimuli-responsive surfaces.

Immune regulation by CD40-CD40-l interactions - 2; Y2K update.

PubMed

van Kooten, C

2000-11-01

CD40 is a cell surface receptor, which belongs to the TNF-R family, and which was first identified and functionally characterized on B lymphocytes. However, in recent years it has become clear that CD40 is expressed much broader, including expression on monocytes, dendritic cells, endothelial cells and epithelial cells. Therefore it is now thought that CD40 plays a more general role in immune regulation. The present paper reviews recent developments in this field of research, with main emphasis on CD40 signal transduction and on in vivo functions of CD40/CD40-L interactions.

Regulation of the Epithelial-Mesenchymal Transition in Prostate Cancer

DTIC Science & Technology

2013-06-01

removed by a cotton swab, and the cells on the lower surface of the membrane were stained by crystal violet. BD BioCoat Matrigel Invasion Chambers...PTK6 downstream players, including AKT, p130CAS, FAK, and ERK5. AKT signaling pro- motes the EMT in different cancer cell lines (23, 45, 46). PTK6...Yang R, Crawford SE, Vasioukhin V, Fuchs E, et al. Protein tyrosine kinase 6 negatively regulates growth and pro- motes enterocyte differentiation in

A cdk1 gradient guides surface contraction waves in oocytes.

PubMed

Bischof, Johanna; Brand, Christoph A; Somogyi, Kálmán; Májer, Imre; Thome, Sarah; Mori, Masashi; Schwarz, Ulrich S; Lénárt, Péter

2017-10-11

Surface contraction waves (SCWs) in oocytes and embryos lead to large-scale shape changes coupled to cell cycle transitions and are spatially coordinated with the cell axis. Here, we show that SCWs in the starfish oocyte are generated by a traveling band of myosin II-driven cortical contractility. At the front of the band, contractility is activated by removal of cdk1 inhibition of the RhoA/RhoA kinase/myosin II signaling module, while at the rear, contractility is switched off by negative feedback originating downstream of RhoA kinase. The SCW's directionality and speed are controlled by a spatiotemporal gradient of cdk1-cyclinB. This gradient is formed by the release of cdk1-cyclinB from the asymmetrically located nucleus, and progressive degradation of cyclinB. By combining quantitative imaging, biochemical and mechanical perturbations with mathematical modeling, we demonstrate that the SCWs result from the spatiotemporal integration of two conserved regulatory modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.Surface contraction waves (SCWs) are prominent shape changes coupled to cell cycle transitions in oocytes. Here the authors show that SCWs are patterned by the spatiotemporal integration of two conserved modules, cdk1-cyclinB for cell cycle regulation and RhoA/Rok/NMYII for actomyosin contractility.

Notch and PKC are involved in formation of the lateral region of the dorso-ventral axis in Drosophila embryos.

PubMed

Tremmel, Daniel M; Resad, Sedat; Little, Christopher J; Wesley, Cedric S

2013-01-01

The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis.

Notch and PKC Are Involved in Formation of the Lateral Region of the Dorso-Ventral Axis in Drosophila Embryos

PubMed Central

Tremmel, Daniel M.; Resad, Sedat; Little, Christopher J.; Wesley, Cedric S.

2013-01-01

The Notch gene encodes an evolutionarily conserved cell surface receptor that generates regulatory signals based on interactions between neighboring cells. In Drosophila embryos it is normally expressed at a low level due to strong negative regulation. When this negative regulation is abrogated neurogenesis in the ventral region is suppressed, the development of lateral epidermis is severely disrupted, and the dorsal aminoserosa is expanded. Of these phenotypes only the anti-neurogenic phenotype could be linked to excess canonical Notch signaling. The other phenotypes were linked to high levels of Notch protein expression at the surface of cells in the lateral regions indicating that a non-canonical Notch signaling activity normally functions in these regions. Results of our studies reported here provide evidence. They show that Notch activities are inextricably linked to that of Pkc98E, the homolog of mammalian PKCδ. Notch and Pkc98E up-regulate the levels of the phosphorylated form of IκBCactus, a negative regulator of Toll signaling, and Mothers against dpp (MAD), an effector of Dpp signaling. Our data suggest that in the lateral regions of the Drosophila embryos Notch activity, in conjunction with Pkc98E activity, is used to form the slopes of the opposing gradients of Toll and Dpp signaling that specify cell fates along the dorso-ventral axis. PMID:23861806

IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling.

PubMed

Yoneyama, Yosuke; Lanzerstorfer, Peter; Niwa, Hideaki; Umehara, Takashi; Shibano, Takashi; Yokoyama, Shigeyuki; Chida, Kazuhiro; Weghuber, Julian; Hakuno, Fumihiko; Takahashi, Shin-Ichiro

2018-04-11

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)-1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling. © 2018, Yoneyama et al.

IRS-1 acts as an endocytic regulator of IGF-I receptor to facilitate sustained IGF signaling

PubMed Central

Yoneyama, Yosuke; Lanzerstorfer, Peter; Niwa, Hideaki; Umehara, Takashi; Shibano, Takashi; Yokoyama, Shigeyuki; Chida, Kazuhiro; Weghuber, Julian

2018-01-01

Insulin-like growth factor-I receptor (IGF-IR) preferentially regulates the long-term IGF activities including growth and metabolism. Kinetics of ligand-dependent IGF-IR endocytosis determines how IGF induces such downstream signaling outputs. Here, we find that the insulin receptor substrate (IRS)−1 modulates how long ligand-activated IGF-IR remains at the cell surface before undergoing endocytosis in mammalian cells. IRS-1 interacts with the clathrin adaptor complex AP2. IRS-1, but not an AP2-binding-deficient mutant, delays AP2-mediated IGF-IR endocytosis after the ligand stimulation. Mechanistically, IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; for this reason, IGF-IR avoids rapid endocytosis and prolongs its activity on the cell surface. Accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt activation and augments FoxO-mediated transcription. Our study establishes a new role for IRS-1 as an endocytic regulator of IGF-IR that ensures sustained IGF bioactivity, independent of its classic role as an adaptor in IGF-IR signaling. PMID:29661273

An RNA-binding protein, Qki5, regulates embryonic neural stem cells through pre-mRNA processing in cell adhesion signaling.

PubMed

Hayakawa-Yano, Yoshika; Suyama, Satoshi; Nogami, Masahiro; Yugami, Masato; Koya, Ikuko; Furukawa, Takako; Zhou, Li; Abe, Manabu; Sakimura, Kenji; Takebayashi, Hirohide; Nakanishi, Atsushi; Okano, Hideyuki; Yano, Masato

2017-09-15

Cell type-specific transcriptomes are enabled by the action of multiple regulators, which are frequently expressed within restricted tissue regions. In the present study, we identify one such regulator, Quaking 5 (Qki5), as an RNA-binding protein (RNABP) that is expressed in early embryonic neural stem cells and subsequently down-regulated during neurogenesis. mRNA sequencing analysis in neural stem cell culture indicates that Qki proteins play supporting roles in the neural stem cell transcriptome and various forms of mRNA processing that may result from regionally restricted expression and subcellular localization. Also, our in utero electroporation gain-of-function study suggests that the nuclear-type Qki isoform Qki5 supports the neural stem cell state. We next performed in vivo transcriptome-wide protein-RNA interaction mapping to search for direct targets of Qki5 and elucidate how Qki5 regulates neural stem cell function. Combined with our transcriptome analysis, this mapping analysis yielded a bona fide map of Qki5-RNA interaction at single-nucleotide resolution, the identification of 892 Qki5 direct target genes, and an accurate Qki5-dependent alternative splicing rule in the developing brain. Last, our target gene list provides the first compelling evidence that Qki5 is associated with specific biological events; namely, cell-cell adhesion. This prediction was confirmed by histological analysis of mice in which Qki proteins were genetically ablated, which revealed disruption of the apical surface of the lateral wall in the developing brain. These data collectively indicate that Qki5 regulates communication between neural stem cells by mediating numerous RNA processing events and suggest new links between splicing regulation and neural stem cell states. © 2017 Hayakawa-Yano et al.; Published by Cold Spring Harbor Laboratory Press.

Cell surface GRP78 facilitates hepatoma cells proliferation and migration by activating IGF-IR.

PubMed

Yin, Yancun; Chen, Chen; Chen, Jinliang; Zhan, Renhui; Zhang, Qiang; Xu, Xiaoyan; Li, Defang; Li, Minjing

2017-07-01

The 78kDa glucose regulated protein (GRP78) is a multifunctional chaperone that is involved in a variety of cellular processes. Insulin like growth factor I receptor (IGF-IR) often aberrant expresses in many types of tumor cells. The IGF-IR signaling plays key roles in carcinogenesis and maintenance of the malignant phenotype. The crosstalk between GRP78 and IGF-IR molecules has not well been illuminated. Here, we demonstrated a reciprocal regulation of GRP78 expression and IGF-IR pathway activation. IGF-I induced GRP78 expression in hepatoma cells. IGF-IR knockdown or IGF-IR inhibitor repressed GRP78 expression. Both phosphatidylinositol 3-kianase (PI3K) and mitogen-activated protein kinase (MAPK) pathways involved in IGF-I induction of GRP78 expression. Interestingly, treatment of hepatoma cells with IGF-I re-distributes GRP78 from endoplasmic reticulum (ER) to cell surface and promotes its physical interaction with IGF-IR. Also, GRP78 promotes IGF-IR phosphorylation and activation. Blocked of GRP78 by small interfering RNA or inhibition of GRP78 function by (-)-epigallocatechin gallate (EGCG) blocks IGF-I induced IGF-IR phosphorylation and its downstream signaling. Further, blocked cell surface GRP78 with antibody inhibits IGF-I stimulated cellular proliferation and migration. These data reveal an essential role for the molecular chaperone GRP78 in IGF-IR signaling and implicate the use of GRP78 inhibitors in blocking IGF-IR signaling in hepatoma cells. Copyright © 2017 Elsevier Inc. All rights reserved.

PKC and Rab13 mediate Ca2+ signal-regulated GLUT4 traffic.

PubMed

Deng, Bangli; Zhu, Xiaocui; Zhao, Yihe; Zhang, Da; Pannu, Alisha; Chen, Liming; Niu, Wenyan

2018-01-08

Exercise/muscle contraction increases cell surface glucose transporter 4 (GLUT4), leading to glucose uptake to regulate blood glucose level. Elevating cytosolic Ca 2+ mediates this effect, but the detailed mechanism is not clear yet. We used calcium ionophore ionomycin to raise intracellular cytosolic Ca 2+ level to explore the underlying mechanism. We showed that in L6 myoblast muscle cells stably expressing GLUT4myc, ionomycin increased cell surface GLUT4myc levels and the phosphorylation of AS160, TBC1D1. siPKCα and siPKCθ but not siPKCδ and siPKCε inhibited the ionomycin-increased cell surface GLUT4myc level. siPKCα, siPKCθ inhibited the phosphorylation of AS160 and TBC1D1 induced by ionomycin. siPKCα and siPKCθ prevented ionomycin-inhibited endocytosis of GLUT4myc. siPKCθ, but not siPKCα inhibited ionomycin-stimulated exocytosis of GLUT4myc. siRab13 but not siRab8a, siRab10 and siRab14 inhibited the exocytosis of GLUT4myc promoted by ionomycin. In summary, ionomycin-promoted exocytosis of GLUT4 is partly reversed by siPKCθ, whereas ionomycin-inhibited endocytosis of GLUT4 requires both siPKCα and siPKCθ. PKCα and PKCθ contribute to ionomycin-induced phosphorylation of AS160 and TBC1D1. Rab13 is required for ionomycin-regulated GLUT4 exocytosis. Copyright © 2017 Elsevier Inc. All rights reserved.

KSHV cell attachment sites revealed by ultra sensitive tyramide signal amplification (TSA) localize to membrane microdomains that are up-regulated on mitotic cells.

PubMed

Garrigues, H Jacques; Rubinchikova, Yelena E; Rose, Timothy M

2014-03-01

Cell surface structures initiating attachment of Kaposi's sarcoma-associated herpesvirus (KSHV) were characterized using purified hapten-labeled virions visualized by confocal microscopy with a sensitive fluorescent enhancement using tyramide signal amplification (TSA). KSHV attachment sites were present in specific cellular domains, including actin-based filopodia, lamellipodia, ruffled membranes, microvilli and intercellular junctions. Isolated microdomains were identified on the dorsal surface, which were heterogeneous in size with a variable distribution that depended on cellular confluence and cell cycle stage. KSHV binding domains ranged from scarce on interphase cells to dense and continuous on mitotic cells, and quantitation of bound virus revealed a significant increase on mitotic compared to interphase cells. KSHV also bound to a supranuclear domain that was distinct from microdomains in confluent and interphase cells. These results suggest that rearrangement of the cellular membrane during mitosis induces changes in cell surface receptors implicated in the initial attachment stage of KSHV entry. Copyright © 2014 Elsevier Inc. All rights reserved.

The neural cell adhesion molecule promotes FGFR-dependent phosphorylation and membrane targeting of the exocyst complex to induce exocytosis in growth cones.

PubMed

Chernyshova, Yana; Leshchyns'ka, Iryna; Hsu, Shu-Chan; Schachner, Melitta; Sytnyk, Vladimir

2011-03-09

The exocyst complex is an essential regulator of polarized exocytosis involved in morphogenesis of neurons. We show that this complex binds to the intracellular domain of the neural cell adhesion molecule (NCAM). NCAM promotes FGF receptor-mediated phosphorylation of two tyrosine residues in the sec8 subunit of the exocyst complex and is required for efficient recruitment of the exocyst complex to growth cones. NCAM at the surface of growth cones induces Ca(2+)-dependent vesicle exocytosis, which is blocked by an inhibitor of L-type voltage-dependent Ca(2+) channels and tetanus toxin. Preferential exocytosis in growth cones underlying neurite outgrowth is inhibited in NCAM-deficient neurons as well as in neurons transfected with phosphorylation-deficient sec8 and dominant-negative peptides derived from the intracellular domain of NCAM. Thus, we reveal a novel role for a cell adhesion molecule in that it regulates addition of the new membrane to the cell surface of growth cones in developing neurons.

Expression Analysis of the Transmembrane Mucin MUC20 in Human Corneal and Conjunctival Epithelia

PubMed Central

Woodward, Ashley M.; Argüeso, Pablo

2014-01-01

Purpose. Cell surface mucins are a group of highly O-glycosylated transmembrane glycoproteins responsible for the protection of epithelial cells on mucosal surfaces. The aim of this study was to investigate the localization and regulation of mucin 20 (MUC20) at the ocular surface. Methods. Localization of MUC20 in human corneal and conjunctival epithelia was evaluated by immunofluorescence microscopy. Immortalized corneal (HCLE) and conjunctival (HCjE) cell lines were grown at different stages of differentiation and subjected to quantitative PCR and Western blot analyses. Cell surface proteins on apical cell membranes were biotinylated and isolated by neutravidin chromatography. Results. The MUC20 was detected throughout the entire human ocular surface epithelia, predominantly in cell membranes within intermediate cell layers. In conjunctiva, MUC20 also was observed in the cytoplasm of apical cells within the stratified squamous epithelium, but not in goblet cells. Quantitative PCR and immunoblotting demonstrated expression of MUC20 in HCLE and HCjE cells. Induction of differentiation with serum-containing medium resulted in upregulation of MUC20 mRNA and protein. Biotin labeling of the surface of stratified cultures revealed low levels of MUC20 protein on apical glycocalyces. Further, MUC20 was not detected in the cell culture media or in human tears, suggesting that the extracellular domain of MUC20 is not released from the ocular surface as described previously for other cell surface mucins. Conclusions. Our results indicate that MUC20 is a novel transmembrane mucin expressed by the human corneal and conjunctival epithelia, and suggest that differential expression of MUC20 during differentiation has a role in maintaining ocular surface homeostasis. PMID:25168902

The behavior of MC3T3-E1 cells on chitosan/poly-L-lysine composite films: effect of nanotopography, surface chemistry, and wettability.

PubMed

Zheng, Zhenhuan; Zhang, Ling; Kong, Lijun; Wang, Aijun; Gong, Yandao; Zhang, Xiufang

2009-05-01

In the present work, a series of composite films were produced from chitosan/poly-L-lysine blend solutions. The surface topography, chemistry, and wettability of composite films were characterized by atomic force microscopy (AFM), attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy, and contact angle assay, respectively. For all composite films, blending with poly-L-lysine induced changes in surface chemistry and wettability. Interestingly, it was also found that increasing poly-L-lysine weight fraction in blend solutions could result in different nanoscaled surface topographic features, which displayed particle-, granule-, or fiber-dominant morphologies. MC3T3-E1 osteoblast-like cells were cultured on all composite films to evaluate the effects of surface nanotopography, chemistry, and wettability on cell behavior. The observations indicated that MC3T3-E1 cell behavior was affected by surface topography, chemistry, and wettability simultaneously and that cells showed strong responses to surface topography. On fiber-dominant surface, cells fully spread with obvious cytoskeleton organization and exhibited significantly higher level of adhesion and proliferation compared with particle- or granule-dominant surfaces. Furthermore, fiber-dominant surface also induced greater expression of mature osteogenic marker osteocalcin and higher mineralization based on RT-PCR and von Kossa staining. The results suggest that topographic modification of chitosan substratum at the nanoscale may be exploited in regulating cell behavior for its applications in tissue engineering.

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB

PubMed Central

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D.; Garner, Ethan C.; Walker, Suzanne

2014-01-01

Summary The bacterial actin homolog MreB, which is critical for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids, but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis. PMID:25402772

Lipid-linked cell wall precursors regulate membrane association of bacterial actin MreB.

PubMed

Schirner, Kathrin; Eun, Ye-Jin; Dion, Mike; Luo, Yun; Helmann, John D; Garner, Ethan C; Walker, Suzanne

2015-01-01

The bacterial actin homolog MreB, which is crucial for rod shape determination, forms filaments that rotate around the cell width on the inner surface of the cytoplasmic membrane. What determines filament association with the membranes or with other cell wall elongation proteins is not known. Using specific chemical and genetic perturbations while following MreB filament motion, we find that MreB membrane association is an actively regulated process that depends on the presence of lipid-linked peptidoglycan precursors. When precursors are depleted, MreB filaments disassemble into the cytoplasm, and peptidoglycan synthesis becomes disorganized. In cells that lack wall teichoic acids but continue to make peptidoglycan, dynamic MreB filaments are observed, although their presence is not sufficient to establish a rod shape. We propose that the cell regulates MreB filament association with the membrane, allowing rapid and reversible inactivation of cell wall enzyme complexes in response to the inhibition of cell wall synthesis.

Hyaluronan, CD44, and Emmprin Regulate Lactate Efflux and Membrane Localization of Monocarboxylate Transporters in Human Breast Carcinoma Cells

PubMed Central

Slomiany, Mark G.; Grass, G. Daniel; Robertson, Angela D.; Yang, Xiao Y.; Maria, Bernard L.; Beeson, Craig; Toole, Bryan P.

2013-01-01

Interactions of hyaluronan with CD44 in tumor cells play important cooperative roles in various aspects of malignancy and drug resistance. Emmprin (CD147; basigin)is a cell surface glycoprotein of the immunoglobulin superfamily that is highly up-regulated in malignant cancer cells and stimulates hyaluronan production, as well as several downstream signaling pathways. Emmprin also interacts with various monocarboxylate transporters (MCT). Malignant cancer cells use the glycolytic pathway and require MCTs to efflux lactate that results from glycolysis. Glycolysis and lactate secretion contribute to malignant cell behaviors and drug resistance in tumor cells. In the present study, we find that perturbation of endogenous hyaluronan, using small hyaluronan oligosaccharides, rapidly inhibits lactate efflux from breast carcinoma cells; down-regulation of emmprin, using emmprin small interfering RNA, also results in decreased efflux. In addition, we find that CD44 coimmunoprecipitates with MCT1, MCT4, and emmprin and colocalizes with these proteins at the plasma membrane. Moreover, after treatment of the cells with hyaluronan oligosaccharides, CD44, MCT1, and MCT4 become localized intracellularly whereas emmprin remains at the cell membrane. Together, these data indicate that constitutive interactions among hyaluronan, CD44, and emmprin contribute to regulation of MCT localization and function in the plasma membrane of breast carcinoma cells. PMID:19176383

MEAT SCIENCE AND MUSCLE BIOLOGY SYMPOSIUM

PubMed Central

Bi, P.; Kuang, S.

2012-01-01

Stem cell niche plays a critical role in regulating the behavior and function of adult stem cells that underlie tissue growth, maintenance, and regeneration. In the skeletal muscle, stem cells, called satellite cells, contribute to postnatal muscle growth and hypertrophy, and thus, meat production in agricultural animals. Satellite cells are located adjacent to mature muscle fibers underneath a sheath of basal lamina. Microenvironmental signals from extracellular matrix mediated by the basal lamina and from the host myofiber both impinge on satellite cells to regulate their activity. Furthermore, several types of muscle interstitial cells, including intramuscular preadipocytes and connective tissue fibroblasts, have recently been shown to interact with satellite cells and actively regulate the growth and regeneration of postnatal skeletal muscles. From this regard, interstitial adipogenic cells are not only important for marbling and meat quality, but also represent an additional cellular component of the satellite cell niche. At the molecular level, these interstitial cells may interact with satellite cells through cell surface ligands, such as delta-like 1 homolog (Dlk1) protein whose overexpression is thought to be responsible for muscle hypertrophy in callipyge sheep. In fact, extracellular Dlk1 protein has been shown to promote the myogenic differentiation of satellite cells. Understanding the cellular and molecular mechanisms within the stem cell niche that regulate satellite cell differentiation and maintain muscle homeostasis may lead to promising approaches to optimizing muscle growth and composition, thus improving meat production and quality. PMID:22100594

Wise retained in the endoplasmic reticulum inhibits Wnt signaling by reducing cell surface LRP6.

PubMed

Guidato, Sonia; Itasaki, Nobue

2007-10-15

The Wnt signaling pathway is tightly regulated by extracellular and intracellular modulators. Wise was isolated as a secreted protein capable of interacting with the Wnt co-receptor LRP6. Studies in Xenopus embryos revealed that Wise either enhances or inhibits the Wnt pathway depending on the cellular context. Here we show that the cellular localization of Wise has distinct effects on the Wnt pathway readout. While secreted Wise either synergizes or inhibits the Wnt signals depending on the partner ligand, ER-retained Wise consistently blocks the Wnt pathway. ER-retained Wise reduces LRP6 on the cell surface, making cells less susceptible to the Wnt signal. This study provides a cellular mechanism for the action of Wise and introduces the modulation of cellular susceptibility to Wnt signals as a novel mechanism of the regulation of the Wnt pathway.

Superficial physicochemical properties of polyurethane biomaterials as osteogenic regulators in human mesenchymal stem cells fates.

PubMed

Shahrousvand, Mohsen; Sadeghi, Gity Mir Mohamad; Shahrousvand, Ehsan; Ghollasi, Marzieh; Salimi, Ali

2017-08-01

All of the cells' interactions are done through their surfaces. Evaluation of surface physicochemical scaffolds along with other factors is important and determines the fate of stem cells. In this work, biodegradable and biocompatible polyester/polyether based polyurethanes (PUs) were synthesized by polycaprolactone diol (PCL) and poly (tetra methylene ether) glycol (PTMEG) as the soft segment. To assess better the impact of surface parameters such as stiffness and roughness effects on osteogenic differentiation of the human mesenchymal stem cell (hMSC), the dimension effect of substrates was eliminated and two-dimensional membranes were produced by synthesized polyurethane. Surface and bulk properties of prepared 2D membranes such as surface chemistry, roughness, stiffness and tensile behavior were evaluated by Attenuated total reflectance Fourier transform infrared (ATR-FTIR), atomic force microscopy (AFM) and tensile behavior. The prepared 2D PU films had suitable hydrophilicity, biodegradability, water absorption, surface roughness and bulk strength. The hMSCs showed greater osteogenesis expression in PU substrates with more roughness and stiffness than others. The results demonstrated that surface parameters along with other differentiation cues have a synergistic effect on stem cells fates. Copyright © 2017 Elsevier B.V. All rights reserved.

Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface protein assemblies

PubMed Central

Caiolfa, Valeria R.; Zamai, Moreno; Malengo, Gabriele; Andolfo, Annapaola; Madsen, Chris D.; Sutin, Jason; Digman, Michelle A.; Gratton, Enrico; Blasi, Francesco; Sidenius, Nicolai

2007-01-01

To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for dynamic studies of GPI-anchored molecules in live cells at steady state and in the absence of cross-linker/clustering agents. PMID:18056417

A Short Isoform of Human Cytomegalovirus US3 Functions as a Dominant Negative Inhibitor of the Full-Length Form

PubMed Central

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J.; Kang, Seongman; Ahn, Kwangseog

2006-01-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells. PMID:16699020

A short isoform of human cytomegalovirus US3 functions as a dominant negative inhibitor of the full-length form.

PubMed

Shin, Jinwook; Park, Boyoun; Lee, Sungwook; Kim, Youngkyun; Biegalke, Bonita J; Kang, Seongman; Ahn, Kwangseog

2006-06-01

Human cytomegalovirus encodes four unique short (US) region proteins, each of which is independently sufficient for causing the down-regulation of major histocompatibility complex (MHC) class I molecules on the cell surface. This down-regulation enables infected cells to evade recognition by cytotoxic T lymphocytes (CTLs) but makes them vulnerable to lysis by natural killer (NK) cells, which lyse those cells that lack MHC class I molecules. The 22-kDa US3 glycoprotein is able to down-regulate the surface expression of MHC class I molecules by dual mechanisms: direct endoplasmic reticulum retention by physical association and/or tapasin inhibition. The alternative splicing of the US3 gene generates two additional products, including 17-kDa and 3.5-kDa truncated isoforms; however, the functional significance of these isoforms during viral infection is unknown. Here, we describe a novel mode of self-regulation of US3 function that uses the endogenously produced truncated isoform. The truncated isoform itself neither binds to MHC class I molecules nor prevents the full-length US3 from interacting with MHC class I molecules. Instead, the truncated isoform associates with tapasin and competes with full-length US3 for binding to tapasin; thus, it suppresses the action of US3 that causes the disruption of the function of tapasin. Our results indicate that the truncated isoform of the US3 locus acts as a dominant negative regulator of full-length US3 activity. These data reflect the manner in which the virus has developed temporal survival strategies during viral infection against immune surveillance involving both CTLs and NK cells.

Characteristic Changes in Cell Surface Glycosylation Accompany Intestinal Epithelial Cell (IEC) Differentiation: High Mannose Structures Dominate the Cell Surface Glycome of Undifferentiated Enterocytes.

PubMed

Park, Dayoung; Brune, Kristin A; Mitra, Anupam; Marusina, Alina I; Maverakis, Emanual; Lebrilla, Carlito B

2015-11-01

Changes in cell surface glycosylation occur during the development and differentiation of cells and have been widely correlated with the progression of several diseases. Because of their structural diversity and sensitivity to intra- and extracellular conditions, glycans are an indispensable tool for analyzing cellular transformations. Glycans present on the surface of intestinal epithelial cells (IEC) mediate interactions with billions of native microorganisms, which continuously populate the mammalian gut. A distinct feature of IECs is that they differentiate as they migrate upwards from the crypt base to the villus tip. In this study, nano-LC/ESI QTOF MS profiling was used to characterize the changes in glycosylation that correspond to Caco-2 cell differentiation. As Caco-2 cells differentiate to form a brush border membrane, a decrease in high mannose type glycans and a concurrent increase in fucosylated and sialylated complex/hybrid type glycans were observed. At day 21, when cells appear to be completely differentiated, remodeling of the cell surface glycome ceases. Differential expression of glycans during IEC maturation appears to play a key functional role in regulating the membrane-associated hydrolases and contributes to the mucosal surface innate defense mechanisms. Developing methodologies to rapidly identify changes in IEC surface glycans may lead to a rapid screening approach for a variety of disease states affecting the GI tract. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

GABAB receptor cell surface export is controlled by an endoplasmic reticulum gatekeeper

PubMed Central

Doly, Stéphane; Shirvani, Hamasseh; Gäta, Gabriel; Meye, Frank; Emerit, Michel-Boris; Enslen, Hervé; Achour, Lamia; Pardo-Lopez, Liliana; Kwon, Yang Seung; Armand, Vincent; Gardette, Robert; Giros, Bruno; Gassmann, Martin; Bettler, Bernhard; Mameli, Manuel; Darmon, Michèle; Marullo, Stefano

2016-01-01

Summary Endoplasmic reticulum (ER) release and cell surface export of many G protein-coupled receptors (GPCRs), are tightly regulated. For GABAB receptors of GABA, the major mammalian inhibitory neurotransmitter, the ligand-binding GB1 subunit is maintained in the ER by unknown mechanisms in the absence of hetero-dimerization with the GB2 subunit. We report that GB1 retention is regulated by a specific gatekeeper, PRAF2. This ER resident transmembrane protein binds to GB1, preventing its progression in the biosynthetic pathway. GB1 release occurs upon competitive displacement from PRAF2 by GB2. PRAF2 concentration, relative to that of GB1 and GB2, tightly controls cell surface receptor density and controls GABAB function in neurons. Experimental perturbation of PRAF2 levels in vivo caused marked hyperactivity disorders in mice. These data reveal an unanticipated major impact of specific ER gate-keepers on GPCR function and identify PRAF2 as a new molecular target with therapeutic potential for psychiatric and neurological diseases involving GABAB function. PMID:26033241

Genetic dissection of early endosomal recycling highlights a TORC1-independent role for Rag GTPases

PubMed Central

2017-01-01

Endocytosed cell surface membrane proteins rely on recycling pathways for their return to the plasma membrane. Although endosome-to-plasma membrane recycling is critical for many cellular processes, much of the required machinery is unknown. We discovered that yeast has a recycling route from endosomes to the cell surface that functions efficiently after inactivation of the sec7-1 allele of Sec7, which controls transit through the Golgi. A genetic screen based on an engineered synthetic reporter that exclusively follows this pathway revealed that recycling was subject to metabolic control through the Rag GTPases Gtr1 and Gtr2, which work downstream of the exchange factor Vam6. Gtr1 and Gtr2 control the recycling pathway independently of TORC1 regulation through the Gtr1 interactor Ltv1. We further show that the early-endosome recycling route and its control though the Vam6>Gtr1/Gtr2>Ltv1 pathway plays a physiological role in regulating the abundance of amino acid transporters at the cell surface. PMID:28768685

Regulation of P2Y1 receptor traffic by sorting Nexin 1 is retromer independent.

PubMed

Nisar, Shaista; Kelly, Eamonn; Cullen, Pete J; Mundell, Stuart J

2010-04-01

The activity and traffic of G-protein coupled receptors (GPCRs) is tightly controlled. Recent work from our laboratory has shown that P2Y(1) and P2Y(12) responsiveness is rapidly and reversibly modulated in human platelets and that the underlying mechanism requires receptor trafficking as an essential part of this process. However, little is known about the molecular mechanisms underlying P2Y receptor traffic. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. These functions may in part be due to interactions of SNX1 with the mammalian retromer complex. In this study, we investigated the role of SNX1 in P2Y receptor trafficking. We show that P2Y(1) receptors recycle via a slow recycling pathway that is regulated by SNX1, whereas P2Y(12) receptors return to the cell surface via a rapid route that is SNX1 independent. SNX1 inhibition caused a dramatic increase in the rate of P2Y(1) receptor recycling, whereas inhibition of Vps26 and Vps35 known to be present in retromer had no effect, indicating that SNX1 regulation of P2Y(1) receptor recycling is retromer independent. In addition, inhibition of SNX4, 6 and 17 proteins did not affect P2Y(1) receptor recycling. SNX1 has also been implicated in GPCR degradation; however, we provide evidence that P2Y receptor degradation is SNX1 independent. These data describe a novel function of SNX1 in the regulation of P2Y(1) receptor recycling and suggest that SNX1 plays multiple roles in endocytic trafficking of GPCRs.

Estrogen-dependent regulation of sodium/hydrogen exchanger-3 (NHE3) expression via estrogen receptor β in proximal colon of pregnant mice.

PubMed

Choijookhuu, Narantsog; Sato, Yoko; Nishino, Tomoya; Endo, Daisuke; Hishikawa, Yoshitaka; Koji, Takehiko

2012-05-01

Although constipation is very common during pregnancy, the exact mechanism is unknown. We hypothesized that the involvement of estrogen receptor (ER) in the regulation of electrolyte transporter in the colon leads to constipation. In this study, the intestines of normal female ICR mouse and pregnant mice were examined for the expression of ERα and ERβ by immunohistochemistry and in situ hybridization. ERβ, but not ERα, was expressed in surface epithelial cells of the proximal, but not distal, colon on pregnancy days 10, 15, and 18, but not day 5, and the number of ERβ-positive cells increased significantly during pregnancy. Expression of NHE3, the gene that harbors estrogen response element, examined by immunohistochemistry and western blotting, was localized in the surface epithelial cells of the proximal colon and increased in parallel with ERβ expression. In ovariectomized mice, NHE3 expression was only marginal and was up-regulated after treatment with 17β-estradiol (E(2)), but not E(2) + ICI 182,780 (estrogen receptor antagonist). Moreover, knock-down of ERβ expression by electroporetically transfected siRNA resulted in a significant reduction of NHE3 expression. These results indicate that ERβ regulates the expression of NHE3 in the proximal colon of pregnant mice through estrogen action, suggesting the involvement of increased sodium absorption by up-regulated NHE3 in constipation during pregnancy.

The distribution of IL-13 receptor alpha1 expression on B cells, T cells and monocytes and its regulation by IL-13 and IL-4.

PubMed

Graber, P; Gretener, D; Herren, S; Aubry, J P; Elson, G; Poudrier, J; Lecoanet-Henchoz, S; Alouani, S; Losberger, C; Bonnefoy, J Y; Kosco-Vilbois, M H; Gauchat, J F

1998-12-01

To study the expression of IL-13 receptor alpha1 (IL-13Ralpha1), specific monoclonal antibodies (mAb) were generated. Surface expression of the IL-13Ralpha1 on B cells, monocytes and T cells was assessed by flow cytometry using these specific mAb. Among tonsillar B cells, the expression was the highest on the IgD+ CD38- B cell subpopulation which is believed to represent naive B cells. Expression was also detectable on a large fraction of the IgD-CD38- B cells but not on CD38+ B cells. Activation under conditions which promote B cell Ig class switching up-regulated the expression of the receptor. However, the same stimuli had an opposite effect for IL-13Ralpha1 expression levels on monocytes. While IL-13Ralpha1 mRNA was clearly detectable in T cell preparations, no surface expression was detected. However, permeabilization of the T cells showed a clear intracellular expression of the receptor. A soluble form of the receptor was immunoprecipitated from the supernatant of activated peripheral T cells, suggesting that T cell IL-13Ralpha1 might have functions unrelated to the capacity to form a type II IL-4/IL-13R with IL-4Ralpha.

The Tomato MIXTA-Like Transcription Factor Coordinates Fruit Epidermis Conical Cell Development and Cuticular Lipid Biosynthesis and Assembly1

PubMed Central

Lotan, Orfa; Alkan, Noam; Tsimbalist, Tatiana; Rechav, Katya; Fernandez-Moreno, Josefina-Patricia; Widemann, Emilie; Grausem, Bernard; Pinot, Franck; Costa, Fabrizio; Aharoni, Asaph

2015-01-01

The epidermis of aerial plant organs is the primary source of building blocks forming the outer surface cuticular layer. To examine the relationship between epidermal cell development and cuticle assembly in the context of fruit surface, we investigated the tomato (Solanum lycopersicum) MIXTA-like gene. MIXTA/MIXTA-like proteins, initially described in snapdragon (Antirrhinum majus) petals, are known regulators of epidermal cell differentiation. Fruit of transgenically silenced SlMIXTA-like tomato plants displayed defects in patterning of conical epidermal cells. They also showed altered postharvest water loss and resistance to pathogens. Transcriptome and cuticular lipids profiling coupled with comprehensive microscopy revealed significant modifications to cuticle assembly and suggested SlMIXTA-like to regulate cutin biosynthesis. Candidate genes likely acting downstream of SlMIXTA-like included cytochrome P450s (CYPs) of the CYP77A and CYP86A subfamilies, LONG-CHAIN ACYL-COA SYNTHETASE2, GLYCEROL-3-PHOSPHATE SN-2-ACYLTRANSFERASE4, and the ATP-BINDING CASSETTE11 cuticular lipids transporter. As part of a larger regulatory network of epidermal cell patterning and L1-layer identity, we found that SlMIXTA-like acts downstream of SlSHINE3 and possibly cooperates with homeodomain Leu zipper IV transcription factors. Hence, SlMIXTA-like is a positive regulator of both cuticle and conical epidermal cell formation in tomato fruit, acting as a mediator of the tight association between fruit cutin polymer formation, cuticle assembly, and epidermal cell patterning. PMID:26443676

VANGL2 interacts with integrin αv to regulate matrix metalloproteinase activity and cell adhesion to the extracellular matrix.

PubMed

Jessen, Tammy N; Jessen, Jason R

2017-12-15

Planar cell polarity (PCP) proteins are implicated in a variety of morphogenetic processes including embryonic cell migration and potentially cancer progression. During zebrafish gastrulation, the transmembrane protein Vang-like 2 (VANGL2) is required for PCP and directed cell migration. These cell behaviors occur in the context of a fibrillar extracellular matrix (ECM). While it is thought that interactions with the ECM regulate cell migration, it is unclear how PCP proteins such as VANGL2 influence these events. Using an in vitro cell culture model system, we previously showed that human VANGL2 negatively regulates membrane type-1 matrix metalloproteinase (MMP14) and activation of secreted matrix metalloproteinase 2 (MMP2). Here, we investigated the functional relationship between VANGL2, integrin αvβ3, and MMP2 activation. We provide evidence that VANGL2 regulates cell surface integrin αvβ3 expression and adhesion to fibronectin, laminin, and vitronectin. Inhibition of MMP14/MMP2 activity suppressed the cell adhesion defect in VANGL2 knockdown cells. Furthermore, our data show that MMP14 and integrin αv are required for increased proteolysis by VANGL2 knockdown cells. Lastly, we have identified integrin αvβ3 as a novel VANGL2 binding partner. Together, these findings begin to dissect the molecular underpinnings of how VANGL2 regulates MMP activity and cell adhesion to the ECM. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Function and regulation of primary cilia and intraflagellar transport proteins in the skeleton.

PubMed

Yuan, Xue; Serra, Rosa A; Yang, Shuying

2015-01-01

Primary cilia are microtubule-based organelles that project from the cell surface to enable transduction of various developmental signaling pathways. The process of intraflagellar transport (IFT) is crucial for the building and maintenance of primary cilia. Ciliary dysfunction has been found in a range of disorders called ciliopathies, some of which display severe skeletal dysplasias. In recent years, interest has grown in uncovering the function of primary cilia/IFT proteins in bone development, mechanotransduction, and cellular regulation. We summarize recent advances in understanding the function of cilia and IFT proteins in the regulation of cell differentiation in osteoblasts, osteocytes, chondrocytes, and mesenchymal stem cells (MSCs). We also discuss the mechanosensory function of cilia and IFT proteins in bone cells, cilia orientation, and other functions of cilia in chondrocytes. © 2014 New York Academy of Sciences.

Two novel WD40 domain–containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

PubMed Central

Shi, Yufeng; Stefan, Christopher J.; Rue, Sarah M.; Teis, David; Emr, Scott D.

2011-01-01

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway. PMID:21880895

Small-Molecule Sigma1 Modulator Induces Autophagic Degradation of PD-L1.

PubMed

Maher, Christina M; Thomas, Jeffrey D; Haas, Derick A; Longen, Charles G; Oyer, Halley M; Tong, Jane Y; Kim, Felix J

2018-02-01

Emerging evidence suggests that Sigma1 ( SIGMAR1 , also known as sigma-1 receptor) is a unique ligand-regulated integral membrane scaffolding protein that contributes to cellular protein and lipid homeostasis. Previously, we demonstrated that some small-molecule modulators of Sigma1 alter endoplasmic reticulum (ER)-associated protein homeostasis pathways in cancer cells, including the unfolded protein response and autophagy. Programmed death-ligand 1 (PD-L1) is a type I integral membrane glycoprotein that is cotranslationally inserted into the ER and is processed and transported through the secretory pathway. Once at the surface of cancer cells, PD-L1 acts as a T-cell inhibitory checkpoint molecule and suppresses antitumor immunity. Here, we demonstrate that in Sigma1-expressing triple-negative breast and androgen-independent prostate cancer cells, PD-L1 protein levels were suppressed by RNAi knockdown of Sigma1 and by small-molecule inhibition of Sigma1. Sigma1-mediated action was confirmed by pharmacologic competition between Sigma1-selective inhibitor and activator ligands. When administered alone, the Sigma1 inhibitor decreased cell surface PD-L1 expression and suppressed functional interaction of PD-1 and PD-L1 in a coculture of T cells and cancer cells. Conversely, the Sigma1 activator increased PD-L1 cell surface expression, demonstrating the ability to positively and negatively modulate Sigma1 associated PD-L1 processing. We discovered that the Sigma1 inhibitor induced degradation of PD-L1 via autophagy, by a mechanism distinct from bulk macroautophagy or general ER stress-associated autophagy. Finally, the Sigma1 inhibitor suppressed IFNγ-induced PD-L1. Our data demonstrate that small-molecule Sigma1 modulators can be used to regulate PD-L1 in cancer cells and trigger its degradation by selective autophagy. Implications: Sigma1 modulators sequester and eliminate PD-L1 by autophagy, thus preventing functional PD-L1 expression at the cell surface. This posits Sigma1 modulators as novel therapeutic agents in PD-L1/PD-1 blockade strategies that regulate the tumor immune microenvironment. Visual Overview: http://mcr.aacrjournals.org/content/molcanres/16/2/243/F1.large.jpg Mol Cancer Res; 16(2); 243-55. ©2017 AACR . ©2017 American Association for Cancer Research.

Association of Cell Surface Mucins with Galectin-3 Contributes to the Ocular Surface Epithelial Barrier*

PubMed Central

Argüeso, Pablo; Guzman-Aranguez, Ana; Mantelli, Flavio; Cao, Zhiyi; Ricciuto, Jessica; Panjwani, Noorjahan

2009-01-01

Maintenance of an intact mucosal barrier is critical to preventing damage to and infection of wet-surfaced epithelia. The mechanism of defense has been the subject of much investigation, and there is evidence now implicating O-glycosylated mucins on the epithelial cell surface. Here we investigate a new role for the carbohydrate-binding protein galectin-3 in stabilizing mucosal barriers through its interaction with mucins on the apical glycocalyx. Using the surface of the eye as a model system, we found that galectin-3 colocalized with two distinct membrane-associated mucins, MUC1 and MUC16, on the apical surface of epithelial cells and that both mucins bound to galectin-3 affinity columns in a galactose-dependent manner. Abrogation of the mucin-galectin interaction in four different mucosal epithelial cell types using competitive carbohydrate inhibitors of galectin binding, β-lactose and modified citrus pectin, resulted in decreased levels of galectin-3 on the cell surface with concomitant loss of barrier function, as indicated by increased permeability to rose bengal diagnostic dye. Similarly, down-regulation of mucin O-glycosylation using a stable tetracycline-inducible RNA interfering system to knockdown c1galt1 (T-synthase), a critical galactosyltransferase required for the synthesis of core 1 O-glycans, resulted in decreased cell surface O-glycosylation, reduced cell surface galectin-3, and increased epithelial permeability. Taken together, these results suggest that galectin-3 plays a key role in maintaining mucosal barrier function through carbohydrate-dependent interactions with cell surface mucins. PMID:19556244

Annexin A8 controls leukocyte recruitment to activated endothelial cells via cell surface delivery of CD63

NASA Astrophysics Data System (ADS)

Poeter, Michaela; Brandherm, Ines; Rossaint, Jan; Rosso, Gonzalo; Shahin, Victor; Skryabin, Boris V.; Zarbock, Alexander; Gerke, Volker; Rescher, Ursula

2014-04-01

To enable leukocyte adhesion to activated endothelium, the leukocyte receptor P-selectin is released from Weibel-Palade bodies (WPB) to the endothelial cell surface where it is stabilized by CD63. Here we report that loss of annexin A8 (anxA8) in human umbilical vein endothelial cells (HUVEC) strongly decreases cell surface presentation of CD63 and P-selectin, with a concomitant reduction in leukocyte rolling and adhesion. We confirm the compromised leukocyte adhesiveness in inflammatory-activated endothelial venules of anxA8-deficient mice. We find that WPB of anxA8-deficient HUVEC contain less CD63, and that this is caused by improper transport of CD63 from late multivesicular endosomes to WPB, with CD63 being retained in intraluminal vesicles. Consequently, reduced CD63 cell surface levels are seen following WPB exocytosis, resulting in enhanced P-selectin re-internalization. Our data support a model in which anxA8 affects leukocyte recruitment to activated endothelial cells by supplying WPB with sufficient amounts of the P-selectin regulator CD63.

The melanocortin receptor agonist NDP-MSH impairs the allostimulatory function of dendritic cells.

PubMed

Rennalls, La'Verne P; Seidl, Thomas; Larkin, James M G; Wellbrock, Claudia; Gore, Martin E; Eisen, Tim; Bruno, Ludovica

2010-04-01

As alpha-melanocyte-stimulating hormone (alpha-MSH) is released by immunocompetent cells and has potent immunosuppressive properties, it was determined whether human dendritic cells (DCs) express the receptor for this hormone. Reverse transcription-polymerase chain reaction detected messenger RNA specific for all of the known melanocortin receptors in DCs. Mixed lymphocyte reactions also revealed that treatment with [Nle(4), DPhe(7)]-alpha-MSH (NDP-MSH), a potent alpha-MSH analogue, significantly reduced the ability of DCs to stimulate allogeneic T cells. The expression of various cell surface adhesion, maturation and costimulatory molecules on DCs was also investigated. Although treatment with NDP-MSH did not alter the expression of CD83 and major histocompatibility complex class I and II, the surface expression of CD86 (B7.2), intercellular adhesion molecule (ICAM-1/CD54) and CD1a was reduced. In summary, our data indicate that NDP-MSH inhibits the functional activity of DCs, possibly by down-regulating antigen-presenting and adhesion molecules and that these events may be mediated via the extracellular signal-regulated kinase 1 and 2 pathway.

Natural killer cells: In health and disease.

PubMed

Mandal, Arundhati; Viswanathan, Chandra

2015-06-01

Natural killer (NK) cells constitute our bodies' frontline defense system, guarding against tumors and launching attacks against infections. The activities of NK cells are regulated by the interaction of various receptors expressed on their surfaces with cell surface ligands. While the role of NK cells in controlling tumor activity is relatively clear, the fact that they are also linked to various other disease conditions is now being highlighted. Here, we present an overview of the role of NK cells during normal body state as well as under diseased state. We discuss the possible utilization of these powerful cells as immunotherapeutic agents in combating diseases such as asthma, autoimmune diseases, and HIV-AIDS. This review also outlines current challenges in NK cell therapy. Copyright © 2015. Published by Elsevier B.V.

Global transcriptomic analysis of model human cell lines exposed to surface-modified gold nanoparticles: the effect of surface chemistry

NASA Astrophysics Data System (ADS)

Grzincic, E. M.; Yang, J. A.; Drnevich, J.; Falagan-Lotsch, P.; Murphy, C. J.

2015-01-01

Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14 000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected.Gold nanoparticles (Au NPs) are attractive for biomedical applications not only for their remarkable physical properties, but also for the ease of which their surface chemistry can be manipulated. Many applications involve functionalization of the Au NP surface in order to improve biocompatibility, attach targeting ligands or carry drugs. However, changes in cells exposed to Au NPs of different surface chemistries have been observed, and little is known about how Au NPs and their surface coatings may impact cellular gene expression. The gene expression of two model human cell lines, human dermal fibroblasts (HDF) and prostate cancer cells (PC3) was interrogated by microarray analysis of over 14 000 human genes. The cell lines were exposed to four differently functionalized Au NPs: citrate, poly(allylamine hydrochloride) (PAH), and lipid coatings combined with alkanethiols or PAH. Gene functional annotation categories and weighted gene correlation network analysis were used in order to connect gene expression changes to common cellular functions and to elucidate expression patterns between Au NP samples. Coated Au NPs affect genes implicated in proliferation, angiogenesis, and metabolism in HDF cells, and inflammation, angiogenesis, proliferation apoptosis regulation, survival and invasion in PC3 cells. Subtle changes in surface chemistry, such as the initial net charge, lability of the ligand, and underlying layers greatly influence the degree of expression change and the type of cellular pathway affected. Electronic supplementary information (ESI) available: UV-Vis spectra of Au NPs, the most significantly changed genes of HDF cells after Au NP incubation under GO accession number GO:0007049 ``cell cycle'', detailed information about the primer/probe sets used for RT-PCR validation of results. See DOI: 10.1039/c4nr05166a

Histone deacetylase 6 controls Notch3 trafficking and degradation in T-cell acute lymphoblastic leukemia cells.

PubMed

Pinazza, Marica; Ghisi, Margherita; Minuzzo, Sonia; Agnusdei, Valentina; Fossati, Gianluca; Ciminale, Vincenzo; Pezzè, Laura; Ciribilli, Yari; Pilotto, Giorgia; Venturoli, Carolina; Amadori, Alberto; Indraccolo, Stefano

2018-04-12

Several studies have revealed that endosomal sorting controls the steady-state levels of Notch at the cell surface in normal cells and prevents its inappropriate activation in the absence of ligands. However, whether this highly dynamic physiologic process can be exploited to counteract dysregulated Notch signaling in cancer cells remains unknown. T-ALL is a malignancy characterized by aberrant Notch signaling, sustained by activating mutations in Notch1 as well as overexpression of Notch3, a Notch paralog physiologically subjected to lysosome-dependent degradation in human cancer cells. Here we show that treatment with the pan-HDAC inhibitor Trichostatin A (TSA) strongly decreases Notch3 full-length protein levels in T-ALL cell lines and primary human T-ALL cells xenografted in mice without substantially reducing NOTCH3 mRNA levels. Moreover, TSA markedly reduced the levels of Notch target genes, including pTα, CR2, and DTX-1, and induced apoptosis of T-ALL cells. We further observed that Notch3 was post-translationally regulated following TSA treatment, with reduced Notch3 surface levels and increased accumulation of Notch3 protein in the lysosomal compartment. Surface Notch3 levels were rescued by inhibition of dynein with ciliobrevin D. Pharmacologic studies with HDAC1, 6, and 8-specific inhibitors disclosed that these effects were largely due to inhibition of HDAC6 in T-ALL cells. HDAC6 silencing by specific shRNA was followed by reduced Notch3 expression and increased apoptosis of T-ALL cells. Finally, HDAC6 silencing impaired leukemia outgrowth in mice, associated with reduction of Notch3 full-length protein in vivo. These results connect HDAC6 activity to regulation of total and surface Notch3 levels and suggest HDAC6 as a potential novel therapeutic target to lower Notch signaling in T-ALL and other Notch3-addicted tumors.

An investigation into the effect of surface roughness of stainless steel on human umbilical vein endothelial cell gene expression.

PubMed

McLucas, E; Moran, M T; Rochev, Y; Carroll, W M; Smith, T J

2006-01-01

The surface properties of vascular devices dictate the initial postimplantation reactions that occur and thus the efficacy of the implantation procedure. Over the last number of years, a number of different stent designs have emerged and stents are generally polished to a mirror finish during the manufacturing procedure. This study sought to investigate the effect of stainless steel surface roughness on endothelial cell gene expression using an appropriate cell culture in vitro assay system. Stainless steel discs were roughened by shot blasting or polished by mechanical polishing. The surface roughness of the treated and untreated discs was determined by atomic force microscopy (AFM). Cells were seeded on collagen type 1 gels and left to attach for 24 h. Stainless steel discs of varying roughness were then placed in contact with the cells and incubated for 24 h. RNA extractions and quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was then performed to determine the expression levels of candidate genes in the treated cells compared to suitable control cells. E-selectin and vascular cellular adhesion molecule (VCAM-1) were found to be significantly up-regulated in cells incubated with polished and roughened samples, indicating endothelial cell activation and inflammation. This study indicates that the surface roughness of stainless steel is an important surface property in the development of vascular stents.

The role of extracellular matrix metalloproteinase inducer (EMMPRIN) in the regulation of bovine endometrial cell functions.

PubMed

Mishra, Birendra; Kizaki, Keiichiro; Sato, Takashi; Ito, Akira; Hashizume, Kazuyoshi

2012-06-01

Extracellular matrix metalloproteinase inducer (EMMPRIN) is a cell surface glycoprotein that stimulates the production of several matrix metalloproteinases (MMPs) for tissue remodeling. Previously, we detected EMMPRIN in the bovine endometrium, and it is mainly expressed in the luminal and glandular epithelium whereas MMPs are expressed in the underlying stroma. From this expression pattern, we hypothesized that EMMPRIN may regulate stromal MMPs in endometrial cell functions. To test this hypothesis, a coculture of epithelial and stromal cells was performed using a transwell system. In the coculture, epithelial cells were cultured on the insert membrane and stromal cell on the surface of well plates. Expression of stromal MMP-2 and MMP-14 was significantly higher in coculture with epithelial cell. Further, with the addition of anti-EMMPRIN antibody into the epithelial cell compartment, the expression of stromal EMMPRIN and MMP-2 and MMP-14 was decreased. To identify the active site of EMMPRIN for the augmentation of MMPs, EMMPRIN synthetic peptides that correspond to the extracellular loop domain-I (EM1, EM2, EM3, and EM4) were added into the epithelial cell compartment, and only EM2 at a higher dose interfered with EMMPRIN-mediated expression of MMP-14. Next, we examined the effects of progesterone and/or estrogen on the expression of EMMPRIN, MMP-2, and MMP-14. Progesterone (300 nM) significantly stimulated the expression of EMMPRIN but had no effects on any of the MMPs. These results suggest that EMMPRIN derived from epithelial cells regulates MMPs in the endometrium under progesterone-rich conditions and may thereby modulate bovine endometrial cell functions during gestation.

Implant Surface Design Regulates Mesenchymal Stem Cell Differentiation and Maturation

PubMed Central

Boyan, B.D.; Cheng, A.; Olivares-Navarrete, R.; Schwartz, Z.

2016-01-01

Changes in dental implant materials, structural design, and surface properties can all affect biological response. While bulk properties are important for mechanical stability of the implant, surface design ultimately contributes to osseointegration. This article reviews the surface parameters of dental implant materials that contribute to improved cell response and osseointegration. In particular, we focus on how surface design affects mesenchymal cell response and differentiation into the osteoblast lineage. Surface roughness has been largely studied at the microscale, but recent studies have highlighted the importance of hierarchical micron/submicron/nanosurface roughness, as well as surface roughness in combination with surface wettability. Integrins are transmembrane receptors that recognize changes in the surface and mediate downstream signaling pathways. Specifically, the noncanonical Wnt5a pathway has been implicated in osteoblastic differentiation of cells on titanium implant surfaces. However, much remains to be elucidated. Only recently have studies been conducted on the differences in biological response to implants based on sex, age, and clinical factors; these all point toward differences that advocate for patient-specific implant design. Finally, challenges in implant surface characterization must be addressed to optimize and compare data across studies. An understanding of both the science and the biology of the materials is crucial for developing novel dental implant materials and surface modifications for improved osseointegration. PMID:26927483

[Membrane model of the regulation of proliferation: the theory and interpretation of an experiment].

PubMed

Volkov, E I

1983-04-01

The role of cell surface physical organization in the cell cycle regulation is analyzed within the framework of the earlier proposed theory (Chernavskii et al., 1982). Two models of cell surface are considered: hard-frame fluid-mosaic model (latticemosaic) and the fluid-mosaic one. The former deals with normal cells. The existence of integral carcasse or "frame" which is formed by the essential part of cross-linked membrane components and may have at least two different conformational states is hypothesized. The second model describes membranes of tumour cells. With the latter theory any mitogen (excluding the restoration of nutrient depletion) reduces the mechanical tensile strength of the frame and stimulates the general structural rearrangement of the plasma membrane. There are only two conformational transitions during the cell cycle which serve as signals for the beginning of S and M phases. If the values of tensile strength are great enough and therefore the conformational transitions are impossible, the cells pass into the resting (prereplicative--G01, or premitotical--G02) state. Three types of experiments are interpreted in the proposed theory: a) on differences in the action of growth factors on normal and tumour cell cycle, b) on the necessary condition for mitogenicity of lectins, c) on the stimulation of proliferation by mechanical deformation of cells.

Cell Cycle Progression of Human Cells Cultured in Rotating Bioreactor

NASA Technical Reports Server (NTRS)

Parks, Kelsey

2009-01-01

Space flight has been shown to alter the astronauts immune systems. Because immune performance is complex and reflects the influence of multiple organ systems within the host, scientists sought to understand the potential impact of microgravity alone on the cellular mechanisms critical to immunity. Lymphocytes and their differentiated immature form, lymphoblasts, play an important and integral role in the body's defense system. T cells, one of the three major types of lymphocytes, play a central role in cell-mediated immunity. They can be distinguished from other lymphocyte types, such as B cells and natural killer cells by the presence of a special receptor on their cell surface called T cell receptors. Reported studies have shown that spaceflight can affect the expression of cell surface markers. Cell surface markers play an important role in the ability of cells to interact and to pass signals between different cells of the same phenotype and cells of different phenotypes. Recent evidence suggests that cell-cycle regulators are essential for T-cell function. To trigger an effective immune response, lymphocytes must proliferate. The objective of this project is to investigate the changes in growth of human cells cultured in rotating bioreactors and to measure the growth rate and the cell cycle distribution for different human cell types. Human lymphocytes and lymphoblasts will be cultured in a bioreactor to simulate aspects of microgravity. The bioreactor is a cylindrical culture vessel that incorporates the aspects of clinostatic rotation of a solid fluid body around a horizontal axis at a constant speed, and compensates gravity by rotation and places cells within the fluid body into a sustained free-fall. Cell cycle progression and cell proliferation of the lymphocytes will be measured for a number of days. In addition, RNA from the cells will be isolated for expression of genes related in cell cycle regulations.

Optical regulation of protein adsorption and cell adhesion by photoresponsive GaN nanowires.

PubMed

Li, Jingying; Han, Qiusen; Zhang, Ying; Zhang, Wei; Dong, Mingdong; Besenbacher, Flemming; Yang, Rong; Wang, Chen

2013-10-09

Interfacing nanowires with living cells is attracting more and more interest due to the potential applications, such as cell culture engineering and drug delivery. We report on the feasibility of using photoresponsive semiconductor gallium nitride (GaN) nanowires (NWs) for regulating the behaviors of biomolecules and cells at the nano/biointerface. The GaN NWs have been fabricated by a facile chemical vapor deposition method. The superhydrophobicity to superhydrophilicity transition of the NWs is achieved by UV illumination. Bovine serum albumin adsorption could be modulated by photoresponsive GaN NWs. Tunable cell detachment and adhesion are also observed. The mechanism of the NW surface responsible for modulating both of protein adsorption and cell adhesion is discussed. These observations of the modulation effects on protein adsorption and cell adhesion by GaN NWs could provide a novel approach toward the regulation of the behaviors of biomolecules and cells at the nano/biointerface, which may be of considerable importance in the development of high-performance semiconductor nanowire-based biomedical devices for cell culture engineering, bioseparation, and diagnostics.

Study on the regulation of focal adesions and cortical actin by matrix nanotopography in 3D environment

NASA Astrophysics Data System (ADS)

Han, Jingjing; Lin, Keng-Hui; Chew, Lock Yue

2017-11-01

Matrix nanotopography plays an important role in regulating cell behaviors by providing spatial as well as mechanical cues for cells to sense. It has been proposed that nanoscale topography is possible to modulate the tensions which direct the formation of cytoskeleton and the organization of the membrane receptor within the cell, which in turn regulate intracellular mechanical and biochemical signaling. With current studies on this topic being performed mainly in 2D platforms, the question on how nanotopography can influence cell bahaviors in 3D environments has yet to be addressed. In this paper, we explored this question by placing cells in 3D hollow spherical polydimethylsiloxane scaffolds. After culturing rat embryonic fibroblast cells in two kinds of scaffold, one with smooth surface and the other with numerous nano-spikes, we observed that cells in the smooth scaffold have more anchoring sites and more focal adhesions than in the etched scaffold. Moreover, we found the presence of correlation between cortical actin, the important component for supporting cell attachment, and local cell geometry.

CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling

PubMed Central

Müller, Jennifer; Obermeier, Ingrid; Wöhner, Miriam; Brandl, Carolin; Mrotzek, Sarah; Angermüller, Sieglinde; Maity, Palash C.; Reth, Michael; Nitschke, Lars

2013-01-01

A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca2+ signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca2+ signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca2+ responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity. PMID:23836650

CD22 ligand-binding and signaling domains reciprocally regulate B-cell Ca2+ signaling.

PubMed

Müller, Jennifer; Obermeier, Ingrid; Wöhner, Miriam; Brandl, Carolin; Mrotzek, Sarah; Angermüller, Sieglinde; Maity, Palash C; Reth, Michael; Nitschke, Lars

2013-07-23

A high proportion of human B cells carry B-cell receptors (BCRs) that are autoreactive. Inhibitory receptors such as CD22 can downmodulate autoreactive BCR responses. With its extracellular domain, CD22 binds to sialic acids in α2,6 linkages in cis, on the surface of the same B cell or in trans, on other cells. Sialic acids are self ligands, as they are abundant in vertebrates, but are usually not expressed by pathogens. We show that cis-ligand binding of CD22 is crucial for the regulation of B-cell Ca(2+) signaling by controlling the CD22 association to the BCR. Mice with a mutated CD22 ligand-binding domain of CD22 showed strongly reduced Ca(2+) signaling. In contrast, mice with mutated CD22 immunoreceptor tyrosine-based inhibition motifs have increased B-cell Ca(2+) responses, increased B-cell turnover, and impaired survival of the B cells. Thus, the CD22 ligand-binding domain has a crucial function in regulating BCR signaling, which is relevant for controlling autoimmunity.

Adaptor protein containing PH domain, PTB domain and leucine zipper (APPL1) regulates the protein level of EGFR by modulating its trafficking

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jae-Rin; Hahn, Hwa-Sun; Kim, Young-Hoon

2011-11-11

Highlights: Black-Right-Pointing-Pointer APPL1 regulates the protein level of EGFR in response to EGF stimulation. Black-Right-Pointing-Pointer Depletion of APPL1 accelerates the movement of EGF/EGFR from the cell surface to the perinuclear region in response to EGF. Black-Right-Pointing-Pointer Knockdown of APPL1 enhances the activity of Rab5. -- Abstract: The EGFR-mediated signaling pathway regulates multiple biological processes such as cell proliferation, survival and differentiation. Previously APPL1 (adaptor protein containing PH domain, PTB domain and leucine zipper 1) has been reported to function as a downstream effector of EGF-initiated signaling. Here we demonstrate that APPL1 regulates EGFR protein levels in response to EGF stimulation.more » Overexpression of APPL1 enhances EGFR stabilization while APPL1 depletion by siRNA reduces EGFR protein levels. APPL1 depletion accelerates EGFR internalization and movement of EGF/EGFR from cell surface to the perinuclear region in response to EGF treatment. Conversely, overexpression of APPL1 decelerates EGFR internalization and translocation of EGF/EGFR to the perinuclear region. Furthermore, APPL1 depletion enhances the activity of Rab5 which is involved in internalization and trafficking of EGFR and inhibition of Rab5 in APPL1-depleted cells restored EGFR levels. Consistently, APPL1 depletion reduced activation of Akt, the downstream signaling effector of EGFR and this is restored by inhibition of Rab5. These findings suggest that APPL1 is required for EGFR signaling by regulation of EGFR stabilities through inhibition of Rab5.« less

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells

PubMed Central

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté

2017-01-01

ABSTRACT Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation. PMID:28089989

RhoA regulates actin network dynamics during apical surface emergence in multiciliated epithelial cells.

PubMed

Sedzinski, Jakub; Hannezo, Edouard; Tu, Fan; Biro, Maté; Wallingford, John B

2017-01-15

Homeostatic replacement of epithelial cells from basal precursors is a multistep process involving progenitor cell specification, radial intercalation and, finally, apical surface emergence. Recent data demonstrate that actin-based pushing under the control of the formin protein Fmn1 drives apical emergence in nascent multiciliated epithelial cells (MCCs), but little else is known about this actin network or the control of Fmn1. Here, we explore the role of the small GTPase RhoA in MCC apical emergence. Disruption of RhoA function reduced the rate of apical surface expansion and decreased the final size of the apical domain. Analysis of cell shapes suggests that RhoA alters the balance of forces exerted on the MCC apical surface. Finally, quantitative time-lapse imaging and fluorescence recovery after photobleaching studies argue that RhoA works in concert with Fmn1 to control assembly of the specialized apical actin network in MCCs. These data provide new molecular insights into epithelial apical surface assembly and could also shed light on mechanisms of apical lumen formation. © 2017. Published by The Company of Biologists Ltd.

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

PubMed Central

2012-01-01

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

PubMed

Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

2012-03-02

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

Hypoxia-inducible factor regulates alphavbeta3 integrin cell surface expression.

PubMed

Cowden Dahl, Karen D; Robertson, Sarah E; Weaver, Valerie M; Simon, M Celeste

2005-04-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of alpha and beta aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt(-/-) and Hifalpha(-/-) TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin alphavbeta3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O(2)). Culturing B16F0 melanoma cells at 1.5% O(2) resulted in increased alphavbeta3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O(2) tension influence placental invasion and tumor migration by increasing cell surface expression of alphavbeta3 integrin.

Hypoxia-inducible Factor Regulates αvβ3 Integrin Cell Surface Expression

PubMed Central

Cowden Dahl, Karen D.; Robertson, Sarah E.; Weaver, Valerie M.; Simon, M. Celeste

2005-01-01

Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and β aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvβ3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvβ3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvβ3 integrin. PMID:15689487

Barrier role of actin filaments in regulated mucin secretion from airway goblet cells.

PubMed

Ehre, Camille; Rossi, Andrea H; Abdullah, Lubna H; De Pestel, Kathleen; Hill, Sandra; Olsen, John C; Davis, C William

2005-01-01

Airway goblet cells secrete mucin onto mucosal surfaces under the regulation of an apical, phospholipase C/G(q)-coupled P2Y(2) receptor. We tested whether cortical actin filaments negatively regulate exocytosis in goblet cells by forming a barrier between secretory granules and plasma membrane docking sites as postulated for other secretory cells. Immunostaining of human lung tissues and SPOC1 cells (an epithelial, mucin-secreting cell line) revealed an apical distribution of beta- and gamma-actin in ciliated and goblet cells. In goblet cells, actin appeared as a prominent subplasmalemmal sheet lying between granules and the apical membrane, and it disappeared from SPOC1 cells activated by purinergic agonist. Disruption of actin filaments with latrunculin A stimulated SPOC1 cell mucin secretion under basal and agonist-activated conditions, whereas stabilization with jasplakinolide or overexpression of beta- or gamma-actin conjugated to yellow fluorescent protein (YFP) inhibited secretion. Myristoylated alanine-rich C kinase substrate, a PKC-activated actin-plasma membrane tethering protein, was phosphorylated after agonist stimulation, suggesting a translocation to the cytosol. Scinderin (or adseverin), a Ca(2+)-activated actin filament severing and capping protein was cloned from human airway and SPOC1 cells, and synthetic peptides corresponding to its actin-binding domains inhibited mucin secretion. We conclude that actin filaments negatively regulate mucin secretion basally in airway goblet cells and are dynamically remodeled in agonist-stimulated cells to promote exocytosis.

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix.

PubMed

Williams, B Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-07-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells.

Early Hematopoietic Zinc Finger Protein Prevents Tumor Cell Recognition by Natural Killer Cells1

PubMed Central

La Rocca, Rosanna; Fulciniti, Mariateresa; Lakshmikanth, Tadepally; Mesuraca, Maria; Ali, Talib Hassan; Mazzei, Valerio; Amodio, Nicola; Catalano, Lucio; Rotoli, Bruno; Ouerfelli, Ouathek; Grieco, Michele; Gulletta, Elio; Bond, Heather M.; Morrone, Giovanni; Ferrone, Soldano; Carbone, Ennio

2009-01-01

Early hematopoietic zinc finger/zinc finger protein 521 (EHZF/ZNF521) is a novel zinc finger protein expressed in hematopoietic stem and progenitor cells and is down-regulated during their differentiation. Its transcript is also abundant in some hematopoietic malignancies. Analysis of the changes in the antigenic profile of cells transfected with EHZF cDNA revealed up-regulation of HLA class I cell surface expression. This phenotypic change was associated with an increased level of HLA class I H chain, in absence of detectable changes in the expression of other Ag-processing machinery components. Enhanced resistance of target cells to NK cell-mediated cytotoxicity was induced by enforced expression of EHZF in the cervical carcinoma cell line HeLa and in the B lymphoblastoid cell line IM9. Preincubation of transfected cells with HLA class I Ag-specific mAb restored target cell susceptibility to NK cell-mediated lysis, indicating a specific role for HLA class I Ag up-regulation in the NK resistance induced by EHZF. A potential clinical significance of these findings is further suggested by the inverse correlation between EHZF and MHC class I expression levels, and autologous NK susceptibility of freshly explanted multiple myeloma cells. PMID:19342626

Regulation of osteogenesis by micro/nano hierarchical titanium surfaces through a Rock-Wnt5a feedback loop.

PubMed

Yu, Yonglin; Shen, Xinkun; Liu, Junjie; Hu, Yan; Ran, Qichun; Mu, Caiyun; Cai, Kaiyong

2018-05-28

Titanium substrates with micro/nano hierarchical features could positively mediate the osteogenesis of a titanium implant; nevertheless, the underlying molecular mechanism needs to be further revealed. In this work, we fabricated a micro/nano hierarchically structured Ti (MNT) sample and attempted to evaluate its topography-mediated biological effects and potential molecular mechanisms in vitro. The results proved that MNT could not only affect cell morphology and osteogenic differentiation, but also regulate ROCK activity cell biological functions of osteoblasts involved in ROCK activation, β-catenin accumulation, and high-Wnt5a expression in respect to topographical features. Moreover, blockade of ROCK activation resulted in significant inhibition of cell differentiation and Wnt5a expression. Furthermore, the anti-Wnt5a significantly down-regulated ROCK activity. In short, these results indicate the important role of ROCK-Wnt5a feedback loop in regulating cell differentiation by topographies. Copyright © 2018. Published by Elsevier B.V.

DAP12 impacts trafficking and surface stability of killer immunoglobulin-like receptors on natural killer cells

PubMed Central

Mulrooney, Tiernan J.; Posch, Phillip E.; Hurley, Carolyn Katovich

2013-01-01

KIR aid in the regulation of NK cell activity. In this study, the effect of the interaction between the KIR2DS and their adapter, DAP12, was investigated beyond the previously defined signaling function. Flow cytometry analysis showed enhanced KIR2DS surface expression on NKL cells when cotransfected with DAP12. Conversely, KIR2DS4 surface expression on primary cells was decreased when the cells were treated with DAP12-specific siRNA. Treatment of the KIR2DS and DAP12-transfected cells with CHX or BFA repressed KIR2DS surface expression, revealing a role for DAP12 in trafficking newly synthesized KIR to the cell surface. Immunoprecipitation of DAP12 revealed an interaction of DAP12 with an immature isoform of KIR2DS, indicating that the interaction likely initiates within the ER. An internalization assay demonstrated a significant impact of DAP12 on KIR2DS surface stability. Confocal microscopy showed that internalized KIR2DS molecules are recruited to lysosomal compartments independent of DAP12 expression. Our results suggest that in vivo conditions that adversely affect DAP12 expression will indirectly reduce surface expression and stability of KIR2DS. These effects could significantly impact ligand recognition and strength of signaling through KIR2DS molecules. PMID:23715743

Cancer Cell Glycocalyx Mediates Mechanostransduction and Flow-Regulated Invasion

PubMed Central

Qazi, Henry; Palomino, Rocio; Shi, Zhong-Dong; Munn, Lance L.; Tarbell, John M.

2014-01-01

Mammalian cells are covered by a surface proteoglycan (glycocalyx) layer, and it is known that blood vessel-lining endothelial cells use the glycocalyx to sense and transduce the shearing forces of blood flow into intracellular signals. Tumor cells in vivo are exposed to forces from interstitial fluid flow that may affect metastatic potential but are not reproduced by most in vitro cell motility assays. We hypothesized that glycocalyx-mediated mechanotransduction of interstitial flow shear stress is an un-recognized factor that can significantly enhance metastatic cell motility and play a role in augmentation of invasion. Involvement of MMP levels, cell adhesion molecules (CD44, α3 integrin), and glycocalyx components (heparan sulfate and hyaluronan) were investigated in a cell/collagen gel suspension model designed to mimic the interstitial flow microenvironment. Physiologic levels of flow upregulated MMP levels and enhanced the motility of metastatic cells. Blocking the flow-enhanced expression of MMP actvity or adhesion molecules (CD44 and integrins) resulted in blocking the flow-enhanced migratory activity. The presence of a glycocalyx-like layer was verified around tumor cells, and the degradation of this layer by hyaluronidase and heparinase blocked the flow-regulated invasion. This study shows for the first time that interstitial flow enhancement of metastatic cell motility can be mediated by the cell surface glycocalyx – a potential target for therapeutics. PMID:24077103

Reconstructing human pancreatic differentiation by mapping specific cell populations during development

PubMed Central

Ramond, Cyrille; Glaser, Nicolas; Berthault, Claire; Ameri, Jacqueline; Kirkegaard, Jeannette Schlichting; Hansson, Mattias; Honoré, Christian; Semb, Henrik; Scharfmann, Raphaël

2017-01-01

Information remains scarce on human development compared to animal models. Here, we reconstructed human fetal pancreatic differentiation using cell surface markers. We demonstrate that at 7weeks of development, the glycoprotein 2 (GP2) marks a multipotent cell population that will differentiate into the acinar, ductal or endocrine lineages. Development towards the acinar lineage is paralleled by an increase in GP2 expression. Conversely, a subset of the GP2+ population undergoes endocrine differentiation by down-regulating GP2 and CD142 and turning on NEUROG3, a marker of endocrine differentiation. Endocrine maturation progresses by up-regulating SUSD2 and lowering ECAD levels. Finally, in vitro differentiation of pancreatic endocrine cells derived from human pluripotent stem cells mimics key in vivo events. Our work paves the way to extend our understanding of the origin of mature human pancreatic cell types and how such lineage decisions are regulated. DOI: http://dx.doi.org/10.7554/eLife.27564.001 PMID:28731406

Mechanical regulation of T-cell functions

PubMed Central

Chen, Wei; Zhu, Cheng

2013-01-01

Summary T cells are key players of the mammalian adaptive immune system. They experience different mechanical microenvironments during their life cycles, from the thymus, secondary lymph organs, and peripheral tissues that are free of externally applied force but display variable substrate rigidities, to the blood and lymphatic circulation systems where complicated hydrodynamic forces are present. Regardless of whether T cells are subject to external forces or generate their own internal forces, they response and adapt to different biomechanical cues to modulate their adhesion, migration, trafficking, and triggering of immune functions through mechanical regulation of various molecules that bear force. These include adhesive receptors, immunoreceptors, motor proteins, cytoskeletal proteins, and their associated molecules. Here we discuss the forces acting on various surface and cytoplasmic proteins of a T cell in different mechanical milieus. We review existing data on how force regulates protein conformational changes and interactions with counter molecules, including integrins, actin, and the T-cell receptor, and how each relates to T-cell functions. PMID:24117820

The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity

PubMed Central

Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T.; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

2015-01-01

ABSTRACT Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. PMID:25616895

The breast cancer antigen 5T4 interacts with Rab11, and is a target and regulator of Rab11 mediated trafficking.

PubMed

Harris, Janelle L; Dave, Keyur; Gorman, Jeffrey; Khanna, Kum Kum

2018-06-01

5T4 is a transmembrane glycoprotein with limited expression in normal adult tissues and expression in some solid tumours. It is unclear whether 5T4 is preferentially expressed by stem or differentiated cell types. Modes of 5T4 regulation are unknown despite its ongoing development as a cancer immunotherapy target. Our aims were to clarify the differentiation status of 5T4 expressing cells in breast cancer and to understand the mechanism underlying 5T4 membrane presentation. We analysed 5T4 expression in breast cancer cell populations by flow cytometery and found that 5T4 is highly expressed on differentiated cells, where it localizes to focal adhesions. Using immunoprecipitation and mass spectrometry, we identified interactions between 5T4 and the membrane trafficking proteins Rab11, Rab18 and ARF6. Mechanistically we found that Rab11 and Rab18 have oppositional roles in controlling expression and surface presentation of 5T4. 5T4 depletion stabilizes Rab11 protein expression with a consequent stimulation transferrin surface labelling, indicating that 5T4 represses endocytic activity. Successful immunotherapeutic targeting of 5T4 requires surface presentation and different immunotherapy strategies require surface presentation versus endocytosis. While breast cancer cells with high 5T4 surface expression and rapid cell surface turnover would be susceptible to antibody-drug conjugates that rely on intracellular release, 5T4 positive cells with lower expression or lower turnover may still be responsive to T-cell mediated approaches. We find that endocytosis of 5T4 is strongly Rab11 dependent and as such Rab11 activity could affect the success or failure of 5T4-targetted immunotherapy, particularly for antibody-drug conjugate approaches. In fact, 5T4 itself represses Rab11 expression. This newly uncovered relationship between Rab11 and 5T4 suggests that breast tumours with high 5T4 expression may not have efficient endocytic uptake of 5T4-targetted immunotherapeutics. This should be considered when selecting amongst the different types of immunotherapies. Copyright © 2018 Elsevier Ltd. All rights reserved.

Chemoresistance induces enhanced adhesion and transendothelial penetration of neuroblastoma cells by down-regulating NCAM surface expression

PubMed Central

Blaheta, Roman A; Daher, Frederick H; Michaelis, Martin; Hasenberg, Christoph; Weich, Eva M; Jonas, Dietger; Kotchetkov, Rouslan; Doerr, Hans Willhelm; Cinatl, Jindrich

2006-01-01

Background Drug resistance to chemotherapy is often associated with increased malignancy in neuroblastoma (NB). One explanation for the link between resistance and malignancy might be that resistance facilitates cancer progression and invasion. To investigate this hypothesis, adhesion, transendothelial penetration and NCAM (CD56) adhesion receptor expression of drug-resistant versus drug-sensitive NB tumor cells were evaluated. Methods Acquired drug resistance was mimicked by exposing parental UKF-NB-2, UKF-NB-3 or IMR-32 tumor cells to increasing concentrations of vincristine- (VCR) or doxorubicin (DOX) to establish the resistant tumor cell sublines UKF-NB-2VCR, UKF-NB-2DOX, UKF-NB-3VCR, UKF-NB-3DOX, IMR-32VCR and IMR-32DOX. Additionally, the malignant behaviour of UKF-NB-4, which already possessed the intrinsic multidrug resistance (MDR) phenotype, was analyzed. UKF-NB-4 exposed to VCR or DOX were designated UKF-NB-4VCR or UKF-NB-4DOX. Combined phase contrast – reflection interference contrast microscopy was used to separately evaluate NB cell adhesion and penetration. NCAM was analyzed by flow cytometry, western blot and RT-PCR. Results VCR and DOX resistant tumor sublines showed enhanced adhesion and penetration capacity, compared to their drug naïve controls. Strongest effects were seen with UKF-NB-2VCR, UKF-NB-3VCR and IMR-32DOX. DOX or VCR treatment also evoked increased invasive behaviour of UKF-NB-4. The process of accelerated tumor invasion was accompanied by decreased NCAM surface and protein expression, and down-regulation of NCAM coding mRNA. Transfection of UKF-NB-4VCR cells with NCAM cDNA led to a significant receptor up-regulation, paralleled by diminished adhesion to an endothelial cell monolayer. Conclusion It is concluded that NB cells resistant to anticancer drugs acquire increased invasive capacity relative to non-resistant parental cells, and that enhanced invasion is caused by strong down-regulation of NCAM adhesion receptors. PMID:17181871

Micropatterned Azopolymer Surfaces Modulate Cell Mechanics and Cytoskeleton Structure.

PubMed

Rianna, Carmela; Ventre, Maurizio; Cavalli, Silvia; Radmacher, Manfred; Netti, Paolo A

2015-09-30

Physical and chemical characteristics of materials are important regulators of cell behavior. In particular, cell elasticity is a fundamental parameter that reflects the state of a cell. Surface topography finely modulates cell fate and function via adhesion mediated signaling and cytoskeleton generated forces. However, how topographies alter cell mechanics is still unclear. In this work we have analyzed the mechanical properties of peripheral and nuclear regions of NIH-3T3 cells on azopolymer substrates with different topographic patterns. Micrometer scale patterns in the form of parallel ridges or square lattices of surface elevations were encoded on light responsive azopolymer films by means of contactless optical methods. Cell mechanics was investigated by atomic force microscopy (AFM). Cells and consequently the cell cytoskeleton were oriented along the linear patterns affecting cytoskeletal structures, e.g., formation of actin stress fibers. Our data demonstrate that topographic substrate patterns are recognized by cells and mechanical information is transferred by the cytoskeleton. Furthermore, cytoskeleton generated forces deform the nucleus, changing its morphology that appears to be related to different mechanical properties in the nuclear region.

High Throughput Screening for Compounds That Alter Muscle Cell Glycosylation Identifies New Role for N-Glycans in Regulating Sarcolemmal Protein Abundance and Laminin Binding*

PubMed Central

Cabrera, Paula V.; Pang, Mabel; Marshall, Jamie L.; Kung, Raymond; Nelson, Stanley F.; Stalnaker, Stephanie H.; Wells, Lance; Crosbie-Watson, Rachelle H.; Baum, Linda G.

2012-01-01

Duchenne muscular dystrophy is an X-linked disorder characterized by loss of dystrophin, a cytoskeletal protein that connects the actin cytoskeleton in skeletal muscle cells to extracellular matrix. Dystrophin binds to the cytoplasmic domain of the transmembrane glycoprotein β-dystroglycan (β-DG), which associates with cell surface α-dystroglycan (α-DG) that binds laminin in the extracellular matrix. β-DG can also associate with utrophin, and this differential association correlates with specific glycosylation changes on α-DG. Genetic modification of α-DG glycosylation can promote utrophin binding and rescue dystrophic phenotypes in mouse dystrophy models. We used high throughput screening with the plant lectin Wisteria floribunda agglutinin (WFA) to identify compounds that altered muscle cell surface glycosylation, with the goal of finding compounds that increase abundance of α-DG and associated sarcolemmal glycoproteins, increase utrophin usage, and increase laminin binding. We identified one compound, lobeline, from the Prestwick library of Food and Drug Administration-approved compounds that fulfilled these criteria, increasing WFA binding to C2C12 cells and to primary muscle cells from wild type and mdx mice. WFA binding and enhancement by lobeline required complex N-glycans but not O-mannose glycans that bind laminin. However, inhibiting complex N-glycan processing reduced laminin binding to muscle cell glycoproteins, although O-mannosylation was intact. Glycan analysis demonstrated a general increase in N-glycans on lobeline-treated cells rather than specific alterations in cell surface glycosylation, consistent with increased abundance of multiple sarcolemmal glycoproteins. This demonstrates the feasibility of high throughput screening with plant lectins to identify compounds that alter muscle cell glycosylation and identifies a novel role for N-glycans in regulating muscle cell function. PMID:22570487

The herpes simplex virus receptor nectin-1 is down-regulated after trans-interaction with glycoprotein D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Stiles, Katie M.; Center for Oral Health Research, School of Dental Medicine University of Pennsylvania, Philadelphia, PA 19104; Milne, Richard S.B.

2008-03-30

During herpes simplex virus (HSV) entry, membrane fusion occurs either on the cell surface or after virus endocytosis. In both cases, binding of glycoprotein D (gD) to a receptor such as nectin-1 or HVEM is required. In this study, we co-cultured cells expressing gD with nectin-1 expressing cells to investigate the effects of gD on nectin-1 at cell contacts. After overnight co-cultures with gD expressing cells, there was a down-regulation of nectin-1 in B78H1-C10, SY5Y, A431 and HeLa cells, which HSV enters by endocytosis. In contrast, on Vero cells, which HSV enters at the plasma membrane, nectin-1 was not down-regulated.more » Further analysis of B78H1-derived cells showed that nectin-1 down-regulation corresponds to the ability of gD to bind nectin-1 and is achieved by internalization and low-pH-dependent degradation of nectin-1. Moreover, gD is necessary for virion internalization in B78H1 cells expressing nectin-1. These data suggest that the determinants of gD-mediated internalization of nectin-1 may direct HSV to an endocytic pathway during entry.« less

Zeta potential control for electrophoresis cells

NASA Technical Reports Server (NTRS)

Fogal, G. L.

1973-01-01

Zeta potential arises from fact that ions tend to be adsorbed on surface of cell walls. This potential interfaces with electric field sensed by migrating particles and degrades resolution of separation. By regulating sign and magnitude of applied potential induced charge can be used to increase or decrease effective wall zeta potential.

Vascular endothelial growth factor A (VEGF-A) decreases expression and secretion of pleiotrophin in a VEGF receptor-independent manner.

PubMed

Poimenidi, Evangelia; Theodoropoulou, Christina; Koutsioumpa, Marina; Skondra, Lamprini; Droggiti, Eirini; van den Broek, Marloes; Koolwijk, Pieter; Papadimitriou, Evangelia

2016-05-01

Vascular endothelial growth factor A (VEGF-A) is a key molecule in angiogenesis acting through VEGF receptors (VEGFRs), ανβ3 integrin, receptor protein tyrosine phosphatase beta/zeta (RPTPβ/ζ) and cell surface nucleolin (NCL). Pleiotrophin (PTN) stimulates endothelial cell migration and limits the angiogenic effects of VEGF-A165 to the levels of its own effect, possibly acting as a VEGF-A165 modifier. Since PTN and VEGF-A165 share receptors and actions on endothelial cells, in the present work we studied whether and how VEGF-A165 affects PTN expression or secretion. VEGF-A165 decreased PTN mRNA and protein levels acting at the transcriptional level. Bevacizumab, a selective VEGFR2 tyrosine kinase inhibitor and down-regulation of VEGFR2 expression by siRNA did not affect this decrease, suggesting that it is VEGFR-independent. VEGF-A121 also decreased PTN mRNA and protein levels, suggesting that heparin binding of VEGF-A165 is not involved. Blockage of cell surface NCL, lack of expression or mutation of β3 integrin and down-regulation of RPTPβ/ζ abolished the inhibitory effect of VEGF-A165 on PTN expression and secretion. Down-regulation of endogenous PTN in endothelial cells enhanced VEGF-A165-induced increase in migration and tube formation on matrigel. Collectively, these data suggest that VEGF-A down-regulates PTN expression and secretion through the RPTPβ/ζ-ανβ3-NCL axis to enhance its own effect on cell migration and further highlight the role of RPTPβ/ζ in VEGF-A actions. Copyright © 2016 Elsevier Inc. All rights reserved.

Tetraspanin CD9 modulates human lymphoma cellular proliferation via histone deacetylase activity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herr, Michael J.; Department of Medicine, The University of Tennessee Health Science Center, Memphis, TN 38163; Department of Molecular Sciences, The University of Tennessee Health Science Center, Memphis, TN 38163

2014-05-16

Highlights: • CD9 is differentially expressed in human Burkitt’s lymphoma cells. • We found that CD9 expression promotes these cells proliferation. • CD9 expression also increases HDAC activity. • HDAC inhibition decreased both cell proliferation and importantly CD9 expression. • CD9 may dictate HDAC efficacy and play a role in HDAC regulation. - Abstract: Non-Hodgkin Lymphoma (NHL) is a type of hematological malignancy that affects two percent of the overall population in the United States. Tetraspanin CD9 is a cell surface protein that has been thoroughly demonstrated to be a molecular facilitator of cellular phenotype. CD9 expression varies in twomore » human lymphoma cell lines, Raji and BJAB. In this report, we investigated the functional relationship between CD9 and cell proliferation regulated by histone deacetylase (HDAC) activity in these two cell lines. Introduction of CD9 expression in Raji cells resulted in significantly increased cell proliferation and HDAC activity compared to Mock transfected Raji cells. The increase in CD9–Raji cell proliferation was significantly inhibited by HDAC inhibitor (HDACi) treatment. Pretreatment of BJAB cells with HDAC inhibitors resulted in a significant decrease in endogenous CD9 mRNA and cell surface expression. BJAB cells also displayed decreased cell proliferation after HDACi treatment. These results suggest a significant relationship between CD9 expression and cell proliferation in human lymphoma cells that may be modulated by HDAC activity.« less

Leptin-mediated regulation of MT1-MMP localization is KIF1B dependent and enhances gastric cancer cell invasion.

PubMed

Dong, Zhaogang; Xu, Xiaofei; Du, Lutao; Yang, Yongmei; Cheng, Huanhuan; Zhang, Xin; Li, Zewu; Wang, Lili; Li, Juan; Liu, Hui; Qu, Xun; Wang, Chuanxin

2013-05-01

Leptin overexpression is closely correlated with gastric cancer (GC) invasion, but its exact effect and the underlying mechanism in tumorigenesis remain poorly understood. Membrane type 1-matrix metalloproteinase (MT1-MMP), a surface-anchored 'master switch' proteinase, is overexpressed and plays crucial roles in tumor invasion. Here, we characterized the influence of leptin on the generation and surface localization of MT1-MMP in GC and elucidated its molecular mechanisms. Our results revealed that leptin promoted GC cell invasion in vitro by upregulating MT1-MMP expression. Furthermore, cell surface biotinylation assay and flow cytometry demonstrated that the surface expression of MT1-MMP was also enhanced by leptin, and knockdown of kinesin family member 1B (KIF1B, a microtubule plus end-directed monomeric motor protein) by small interference RNA inhibited this process. Notably, coimmunoprecipitation analysis indicated that leptin enhanced the interaction of MT1-MMP with KIF1B in a time-dependent manner, which consequently contributed to GC cell invasion. Moreover, leptin increased MT1-MMP or KIF1B expression by the protein kinase B (AKT) pathway and extracellular signal-regulated kinase 1/2 partially participated in this process. However, only AKT was implicated in the leptin-mediated membrane localization of MT1-MMP. Immunohistochemistry analysis revealed that leptin, MT1-MMP and KIF1B are overexpressed in GC tissues, and they positively correlated with clinical stage and lymph node metastasis. These observations indicate that this regulatory network exists in vivo. Taken together, our findings suggest that leptin is an effective intracellular stimulator of MT1-MMP and that leptin-enhanced cell surface localization of MT1-MMP is dependent on KIF1B, which consequently plays a critical role in GC invasion.

High Cell Surface Expression of CD4 Allows Distinction of CD4+CD25+ Antigen-specific Effector T Cells from CD4+CD25+ Regulatory T Cells in Murine Experimental Autoimmune Encephalomyelitis

PubMed Central

Li, Jinzhu; Ridgway, William; Fathman, C. Garrison; Tse, Harley Y.; Shaw, Michael K.

2008-01-01

Analysis of T regulatory cells (Treg) and T effector cells (Teff) in experimental autoimmune encephalomyelitis is complicated by the fact that both cell types express CD4 and CD25. We demonstrate that encephalitogenic T cells, following antigen recognition, up regulate cell surface expression of CD4. The CD4high sub-population contains all of the antigen response as shown by proliferation and cytokine secretion, and only these cells are capable of transferring EAE to naive animals. On the other hand, a FACS separable CD25+ sub-population of cells displayed consistent levels of CD4 prior to and after antigen stimulation. These cells displayed characteristics of Treg, such as expressing high levels of the Foxp3 gene and the ability to suppress mitogenic T cell responses. PMID:17920698

Deletion of Rab GAP AS160 modifies glucose uptake and GLUT4 translocation in primary skeletal muscles and adipocytes and impairs glucose homeostasis.

PubMed

Lansey, Melissa N; Walker, Natalie N; Hargett, Stefan R; Stevens, Joseph R; Keller, Susanna R

2012-11-15

Tight control of glucose uptake in skeletal muscles and adipocytes is crucial to glucose homeostasis and is mediated by regulating glucose transporter GLUT4 subcellular distribution. In cultured cells, Rab GAP AS160 controls GLUT4 intracellular retention and release to the cell surface and consequently regulates glucose uptake into cells. To determine AS160 function in GLUT4 trafficking in primary skeletal muscles and adipocytes and investigate its role in glucose homeostasis, we characterized AS160 knockout (AS160(-/-)) mice. We observed increased and normal basal glucose uptake in isolated AS160(-/-) adipocytes and soleus, respectively, while insulin-stimulated glucose uptake was impaired and GLUT4 expression decreased in both. No such abnormalities were found in isolated AS160(-/-) extensor digitorum longus muscles. In plasma membranes isolated from AS160(-/-) adipose tissue and gastrocnemius/quadriceps, relative GLUT4 levels were increased under basal conditions and remained the same after insulin treatment. Concomitantly, relative levels of cell surface-exposed GLUT4, determined with a glucose transporter photoaffinity label, were increased in AS160(-/-) adipocytes and normal in AS160(-/-) soleus under basal conditions. Insulin augmented cell surface-exposed GLUT4 in both. These observations suggest that AS160 is essential for GLUT4 intracellular retention and regulation of glucose uptake in adipocytes and skeletal muscles in which it is normally expressed. In vivo studies revealed impaired insulin tolerance in the presence of normal (male) and impaired (female) glucose tolerance. Concurrently, insulin-elicited increases in glucose disposal were abolished in all AS160(-/-) skeletal muscles and liver but not in AS160(-/-) adipose tissues. This suggests AS160 as a target for differential manipulation of glucose homeostasis.

Multiple facets of sialomucin complex/MUC4, a membrane mucin and erbb2 ligand, in tumors and tissues (Y2K update).

PubMed

Carraway, K L; Price-Schiavi, S A; Komatsu, M; Idris, N; Perez, A; Li, P; Jepson, S; Zhu, X; Carvajal, M E; Carraway, C A

2000-01-01

Sialomucin complex (SMC, MUC4) is a high Mr glycoprotein heterodimer, composed of mucin (ASGP-1) and transmembrane (ASGP-2) subunits. ASGP-2 contains two EGF-like domains and acts as an intramembrane ligand for the receptor tyrosine kinase ErbB2. Transfection studies with SMC DNAs showed that SMC expression could markedly reduce both cell-cell and cell-matrix interactions in vitro and increase the growth of primary tumors and the formation of metastatic foci of human A375 melanoma cells as xenotransplants in nude mice, possibly through the ability to suppress apoptosis. SMC is expressed in most vulnerable epithelia as a protective agent, which is found in both membrane and soluble forms at luminal surfaces and secreted into fluids such as milk and tears. SMC appears to be constitutively expressed by most accessible epithelia, notable exceptions being the mammary gland and uterine luminal epithelium, in which it is tightly regulated during pregnancy. Down-regulation at the luminal uterine surface appears necessary for blastocyst implantation. TGF-b is a potent repressor of SMC expression in the mammary gland and uterus, though by different mechanisms. These combined results suggest that SMC has multiple functions in epithelia and is tightly regulated in those tissues where its special functions are required.

Allosteric Regulation of E-Cadherin Adhesion*

PubMed Central

Shashikanth, Nitesh; Petrova, Yuliya I.; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M.; Leckband, Deborah E.

2015-01-01

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120ctn, increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120ctn dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120ctn dephosphorylation. PMID:26175155

Activated release of membrane-anchored TGF-alpha in the absence of cytosol

PubMed Central

1993-01-01

The ectodomain of proTGF-alpha, a membrane-anchored growth factor, is converted into soluble TGF-alpha by a regulated cellular proteolytic system that recognizes proTGF-alpha via the C-terminal valine of its cytoplasmic tail. In order to define the biochemical components involved in proTGF-alpha cleavage, we have used cells permeabilized with streptolysin O (SLO) that have been extensively washed to remove cytosol. PMA, acting through a Ca(2+)-independent protein kinase C, activates cleavage as efficiently in permeabilized cells as it does in intact cells. ProTGF-alpha cleavage is also stimulated by GTP gamma S through a mechanism whose pharmacological properties suggest the involvement of a heterotrimeric G protein acting upstream of the PMA- sensitive Ca(2+)-independent protein kinase C. Activated proTGF-alpha cleavage is dependent on ATP hydrolysis, appears not to require vesicular traffic, and acts specifically on proTGF-alpha that has reached the cell surface. These results indicate that proTGF-alpha is cleaved from the cell surface by a regulated system whose signaling, recognition, and proteolytic components are retained in cells devoid of cytosol. PMID:8314849

Human leukocyte antigen E in human cytomegalovirus infection: friend or foe?

PubMed

Gong, Fang; Song, Shengli; Lv, Guozhong; Pan, Yuhong; Zhang, Dongqing; Jiang, Hong

2012-07-01

Human cytomegalovirus (HCMV) is a well-studied β-herpesvirus virus, which adopts a variety of strategies to evade immune surveillance. It has been reported that in HCMV-infected cells, classical major histocompatibility (MHC) class I molecules are down-regulated, but the MHC class Ib molecule human leukocyte antigen (HLA)-E is normally expressed or even overexpressed on the cell surface. HLA-E has been first described to interact with CD94/NKG2 receptors expressed mainly on the surface of natural killer (NK) cells, thus confining its role to the regulation of NK-cell function. The engagement of CD94/NKG2A with HLA-E, with a signal peptide of the HCMV glycoprotein UL40, usually induces inhibitory signals. However, HLA-E also serves as a ligand for the TCR expressed by αβCD8(+) T cells. Recognition of peptides presented by HLA-E may result in CD8(+) effector T-cell activation. These findings will help to understand more on both pathogenic and protective roles of HLA-E in HCMV infection. In this review, we discussed recent studies about the roles of HLA-E in HCMV infection.

Ion Transport by Pulmonary Epithelia

PubMed Central

Hollenhorst, Monika I.; Richter, Katrin; Fronius, Martin

2011-01-01

The lung surface of air-breathing vertebrates is formed by a continuous epithelium that is covered by a fluid layer. In the airways, this epithelium is largely pseudostratified consisting of diverse cell types such as ciliated cells, goblet cells, and undifferentiated basal cells, whereas the alveolar epithelium consists of alveolar type I and alveolar type II cells. Regulation and maintenance of the volume and viscosity of the fluid layer covering the epithelium is one of the most important functions of the epithelial barrier that forms the outer surface area of the lungs. Therefore, the epithelial cells are equipped with a wide variety of ion transport proteins, among which Na+, Cl−, and K+ channels have been identified to play a role in the regulation of the fluid layer. Malfunctions of pulmonary epithelial ion transport processes and, thus, impairment of the liquid balance in our lungs is associated with severe diseases, such as cystic fibrosis and pulmonary oedema. Due to the important role of pulmonary epithelial ion transport processes for proper lung function, the present paper summarizes the recent findings about composition, function, and ion transport properties of the airway epithelium as well as of the alveolar epithelium. PMID:22131798

Benzoxazole derivatives suppress lipopolysaccharide-induced mast cell activation.

PubMed

Cho, Kyung-Ah; Park, Minhwa; Kim, Yu-Hee; Choo, Hea-Young Park; Lee, Kyung Ho

2018-05-01

Mast cells are central regulators of allergic inflammation that function by releasing various proallergic inflammatory mediators, including histamine, eicosanoids and proinflammatory cytokines. Occasionally, bacterial infections may initiate or worsen allergic inflammation. A number of studies have indicated that activation of lipoxygenase in mast cells positive regulates allergic inflammatory responses by generating leukotrienes and proinflammatory cytokines. In the present study, the effects of benzoxazole derivatives on the lipopolysaccharide (LPS)‑induced expression of proinflammatory cytokines, production of histamine and surface expression of co‑stimulatory molecules on bone marrow-derived mast cells (BMMCs) were studied. The benzoxazole derivatives significantly reduced the expression of interleukin (IL)‑1β, IL‑6, IL‑13, tumor necrosis factor‑α, perilipin (PLIN) 2, and PLIN3 in BMMCs treated with LPS. Furthermore, histamine production was suppressed in BMMCs treated with LPS, or treated with phorbol-12-myristate-13-acetate/ionomycin. Benzoxazole derivatives marginally affected the surface expression of cluster of differentiation (CD)80 and CD86 on BMMCs in the presence of LPS, although LPS alone did not increase the expression of those proteins. Therefore, benzoxazole derivatives inhibited the secretion of proinflammatory cytokines in mast cells and may be potential candidate anti‑allergic agents to suppress mast cell activation.

Insulin regulates its own delivery to skeletal muscle by feed-forward actions on the vasculature

PubMed Central

Wang, Hong; Upchurch, Charles T.; Liu, Zhenqi

2011-01-01

Insulin, at physiological concentrations, regulates the volume of microvasculature perfused within skeletal and cardiac muscle. It can also, by relaxing the larger resistance vessels, increase total muscle blood flow. Both of these effects require endothelial cell nitric oxide generation and smooth muscle cell relaxation, and each could increase delivery of insulin and nutrients to muscle. The capillary microvasculature possesses the greatest endothelial surface area of the body. Yet, whether insulin acts on the capillary endothelial cell is not known. Here, we review insulin's actions at each of three levels of the arterial vasculature as well as recent data suggesting that insulin can regulate a vesicular transport system within the endothelial cell. This latter action, if it occurs at the capillary level, could enhance insulin delivery to muscle interstitium and thereby complement insulin's actions on arteriolar endothelium to increase insulin delivery. We also review work that suggests that this action of insulin on vesicle transport depends on endothelial cell nitric oxide generation and that insulin's ability to regulate this vesicular transport system is impaired by inflammatory cytokines that provoke insulin resistance. PMID:21610226

Loss of PTB or Negative Regulation of Notch mRNA Reveals Distinct Zones of Notch and Actin Protein Accumulation in Drosophila Embryo

PubMed Central

Wesley, Cedric S.; Guo, Heng; Chaudhry, Kanita A.; Thali, Markus J.; Yin, Jerry C.; Clason, Todd; Wesley, Umadevi V.

2011-01-01

Polypyrimidine Tract Binding (PTB) protein is a regulator of mRNA processing and translation. Genetic screens and studies of wing and bristle development during the post-embryonic stages of Drosophila suggest that it is a negative regulator of the Notch pathway. How PTB regulates the Notch pathway is unknown. Our studies of Drosophila embryogenesis indicate that (1) the Notch mRNA is a potential target of PTB, (2) PTB and Notch functions in the dorso-lateral regions of the Drosophila embryo are linked to actin regulation but not their functions in the ventral region, and (3) the actin-related Notch activity in the dorso-lateral regions might require a Notch activity at or near the cell surface that is different from the nuclear Notch activity involved in cell fate specification in the ventral region. These data raise the possibility that the Drosophila embryo is divided into zones of different PTB and Notch activities based on whether or not they are linked to actin regulation. They also provide clues to the almost forgotten role of Notch in cell adhesion and reveal a role for the Notch pathway in cell fusions. PMID:21750738

Three-dimensional manipulation of single cells using surface acoustic waves.

PubMed

Guo, Feng; Mao, Zhangming; Chen, Yuchao; Xie, Zhiwei; Lata, James P; Li, Peng; Ren, Liqiang; Liu, Jiayang; Yang, Jian; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2016-02-09

The ability of surface acoustic waves to trap and manipulate micrometer-scale particles and biological cells has led to many applications involving "acoustic tweezers" in biology, chemistry, engineering, and medicine. Here, we present 3D acoustic tweezers, which use surface acoustic waves to create 3D trapping nodes for the capture and manipulation of microparticles and cells along three mutually orthogonal axes. In this method, we use standing-wave phase shifts to move particles or cells in-plane, whereas the amplitude of acoustic vibrations is used to control particle motion along an orthogonal plane. We demonstrate, through controlled experiments guided by simulations, how acoustic vibrations result in micromanipulations in a microfluidic chamber by invoking physical principles that underlie the formation and regulation of complex, volumetric trapping nodes of particles and biological cells. We further show how 3D acoustic tweezers can be used to pick up, translate, and print single cells and cell assemblies to create 2D and 3D structures in a precise, noninvasive, label-free, and contact-free manner.

Isotropic actomyosin dynamics promote organization of the apical cell cortex in epithelial cells.

PubMed

Klingner, Christoph; Cherian, Anoop V; Fels, Johannes; Diesinger, Philipp M; Aufschnaiter, Roland; Maghelli, Nicola; Keil, Thomas; Beck, Gisela; Tolić-Nørrelykke, Iva M; Bathe, Mark; Wedlich-Soldner, Roland

2014-10-13

Although cortical actin plays an important role in cellular mechanics and morphogenesis, there is surprisingly little information on cortex organization at the apical surface of cells. In this paper, we characterize organization and dynamics of microvilli (MV) and a previously unappreciated actomyosin network at the apical surface of Madin-Darby canine kidney cells. In contrast to short and static MV in confluent cells, the apical surfaces of nonconfluent epithelial cells (ECs) form highly dynamic protrusions, which are often oriented along the plane of the membrane. These dynamic MV exhibit complex and spatially correlated reorganization, which is dependent on myosin II activity. Surprisingly, myosin II is organized into an extensive network of filaments spanning the entire apical membrane in nonconfluent ECs. Dynamic MV, myosin filaments, and their associated actin filaments form an interconnected, prestressed network. Interestingly, this network regulates lateral mobility of apical membrane probes such as integrins or epidermal growth factor receptors, suggesting that coordinated actomyosin dynamics contributes to apical cell membrane organization. © 2014 Klingner et al.

Characterization of atrial natriuretic peptide degradation by cell-surface peptidase activity on endothelial cells

NASA Technical Reports Server (NTRS)

Frost, S. J.; Whitson, P. A.

1993-01-01

Atrial natriuretic peptide (ANP) is a fluid-regulating peptide hormone that promotes vasorelaxation, natriuresis, and diuresis. The mechanisms for the release of ANP and for its clearance from the circulation play important roles in modulating its biological effects. Recently, we have reported that the cell surface of an endothelial cell line, CPA47, could degrade 125I-ANP in the presence of EDTA. In this study, we have characterized this degradation of 125I-ANP. The kinetics of ANP degradation by the surface of CPA47 cells were first order, with a Km of 320 +/- 60 nM and Vmax of 35 +/- 14 pmol of ANP degraded/10 min/10(5) cells at pH 7.4. ANP is degraded by the surface of CPA47 cells over a broad pH range from 7.0-8.5. Potato carboxypeptidase inhibitor and bestatin inhibited 125I-ANP degradation, suggesting that this degradative activity on the surface of CPA47 cells has exopeptidase characteristics. The selectivity of CPA47 cell-surface degradation of ANP was demonstrated when 125I-ANP degradation was inhibited in the presence of neuropeptide Y and angiotensin I and II but not bradykinin, bombesin, endothelin-1, or substance P. The C-terminal amino acids phe26 and tyr28 were deduced to be important for ANP interaction with the cell-surface peptidase(s) based on comparison of the IC50 of various ANP analogues and other natriuretic peptides for the inhibition of ANP degradation. These data suggest that a newly characterized divalent cation-independent exopeptidase(s) that selectively recognizes ANP and some other vasoactive peptides exists on the surface of endothelial cells.

Proteomic Analysis of Secretomes of Oncolytic Herpes Simplex Virus-Infected Squamous Cell Carcinoma Cells

PubMed Central

Tada, Shinya; Hamada, Masakazu; Yura, Yoshiaki

2018-01-01

Oncolytic herpes simplex virus type 1 (HSV-1) strain RH2 induced immunogenic cell death (ICD) with the release and surface exposure of damage-associated molecular patterns (DAMPs) in squamous cell carcinoma (SCC) SCCVII cells. The supernatants of RH2-infected SCCVII cells also exhibited antitumor ability by intratumoral administration in SCCVII tumor-bearing mice. The supernatants of RH2-infected cells and mock-infected cells were concentrated to produce Med24 and MedC for proteomic analyses. In Med24, the up- and down-regulated proteins were observed. Proteins including filamin, tubulin, t-complex protein 1 (TCP-1), and heat shock proteins (HSPs), were up-regulated, while extracellular matrix (ECM) proteins were markedly down-regulated. Viral proteins were detected in Med 24. These results indicate that HSV-1 RH2 infection increases the release of danger signal proteins and viral gene products, but decreases the release of ECM components. These changes may alter the tumor microenvironment (TME) and contribute to enhancement of anti-tumor immunity against SCC. PMID:29360750

MRCK-1 drives apical constriction in C. elegans by linking developmental patterning to force generation

PubMed Central

Marston, Daniel J.; Higgins, Christopher D.; Peters, Kimberly A.; Cupp, Timothy D.; Dickinson, Daniel J.; Pani, Ariel M.; Moore, Regan P.; Cox, Amanda H.; Kiehart, Daniel P.; Goldstein, Bob

2016-01-01

Summary Apical constriction is a change in cell shape that drives key morphogenetic events including gastrulation and neural tube formation. Apical force-producing actomyosin networks drive apical constriction by contracting while connected to cell-cell junctions. The mechanisms by which developmental patterning regulates these actomyosin networks and associated junctions with spatial precision are not fully understood. Here, we identify a myosin light chain kinase MRCK-1 as a key regulator of C. elegans gastrulation that integrates spatial and developmental patterning information. We show that MRCK-1 is required for activation of contractile actomyosin dynamics and elevated cortical tension in the apical cell cortex of endodermal precursor cells. MRCK-1 is apically localized by active Cdc42 at the external, cell-cell contact-free surfaces of apically constricting cells, downstream of cell fate determination mechanisms. We establish that the junctional components α-catenin, β-catenin, and cadherin become highly enriched at the apical junctions of apically-constricting cells, and that MRCK-1 and myosin activity are required in vivo for this enrichment. Taken together, our results define mechanisms that position a myosin activator to a specific cell surface where it both locally increases cortical tension and locally enriches junctional components to facilitate apical constriction. These results reveal crucial links that can tie spatial information to local force generation to drive morphogenesis. PMID:27451898

ICAM-1-expressing neutrophils exhibit enhanced effector functions in murine models of endotoxemia.

PubMed

Woodfin, Abigail; Beyrau, Martina; Voisin, Mathieu-Benoit; Ma, Bin; Whiteford, James R; Hordijk, Peter L; Hogg, Nancy; Nourshargh, Sussan

2016-02-18

Intracellular adhesion molecule-1 (ICAM-1) is a transmembrane glycoprotein expressed on the cell surface of numerous cell types such as endothelial and epithelial cells, vascular smooth muscle cells, and certain leukocyte subsets. With respect to the latter, ICAM-1 has been detected on neutrophils in several clinical and experimental settings, but little is known about the regulation of expression or function of neutrophil ICAM-1. In this study, we report on the de novo induction of ICAM-1 on the cell surface of murine neutrophils by lipopolysaccharide (LPS), tumor necrosis factor, and zymosan particles in vitro. The induction of neutrophil ICAM-1 was associated with enhanced phagocytosis of zymosan particles and reactive oxygen species (ROS) generation. Conversely, neutrophils from ICAM-1-deficient mice were defective in these effector functions. Mechanistically, ICAM-1-mediated intracellular signaling appeared to support neutrophil ROS generation and phagocytosis. In vivo, LPS-induced inflammation in the mouse cremaster muscle and peritoneal cavity led to ICAM-1 expression on intravascular and locally transmigrated neutrophils. The use of chimeric mice deficient in ICAM-1 on myeloid cells demonstrated that neutrophil ICAM-1 was not required for local neutrophil transmigration, but supported optimal intravascular and extravascular phagocytosis of zymosan particles. Collectively, the present results shed light on regulation of expression and function of ICAM-1 on neutrophils and identify it as an additional regulator of neutrophil effector responses in host defense. © 2016 by The American Society of Hematology.

T-Cell Surface Antigens and sCD30 as Biomarkers of the Risk of Rejection in Solid Organ Transplantation.

PubMed

Wieland, Eberhard; Shipkova, Maria

2016-04-01

T-cell activation is a characteristic of organ rejection. T cells, located in the draining lymph nodes of the transplant recipient, are faced with non-self-molecules presented by antigen presenting cells and become activated. Activated T cells are characterized by up-regulated surface antigens, such as costimulatory molecules, adhesion molecules, chemokine receptors, and major histocompatibility complex class II molecules. Surface antigen expression can be followed by flow cytometry using monoclonal antibodies in either cell function assays using donor-specific or nonspecific stimulation of isolated cells or whole blood and without stimulation on circulating lymphocytes. Molecules such as CD30 can be proteolytically cleaved off the surface of activated cells in vivo, and the determination of the soluble protein (sCD30) in serum or plasma is performed by immunoassays. As promising biomarkers for rejection and long-term transplant outcome, CD28 (costimulatory receptor for CD80 and CD86), CD154 (CD40 ligand), and sCD30 (tumor necrosis factor receptor superfamily, member 8) have been identified. Whereas cell function assays are time-consuming laboratory-developed tests which are difficult to standardize, commercial assays are frequently available for soluble proteins. Therefore, more data from clinical trials have been published for sCD30 compared with the surface antigens on activated T cells. This short review summarizes the association between selected surface antigens and immunosuppression, and rejection in solid organ transplantation.

Epithelial adhesion molecules and the regulation of intestinal homeostasis during neutrophil transepithelial migration

PubMed Central

Sumagin, Ronen; Parkos, Charles A

2014-01-01

Epithelial adhesion molecules play essential roles in regulating cellular function and maintaining mucosal tissue homeostasis. Some form epithelial junctional complexes to provide structural support for epithelial monolayers and act as a selectively permeable barrier separating luminal contents from the surrounding tissue. Others serve as docking structures for invading viruses and bacteria, while also regulating the immune response. They can either obstruct or serve as footholds for the immune cells recruited to mucosal surfaces. Currently, it is well appreciated that adhesion molecules collectively serve as environmental cue sensors and trigger signaling events to regulate epithelial function through their association with the cell cytoskeleton and various intracellular adapter proteins. Immune cells, particularly neutrophils (PMN) during transepithelial migration (TEM), can modulate adhesion molecule expression, conformation, and distribution, significantly impacting epithelial function and tissue homeostasis. This review discusses the roles of key intestinal epithelial adhesion molecules in regulating PMN trafficking and outlines the potential consequences on epithelial function. PMID:25838976

A central role for vesicle trafficking in epithelial neoplasia: Intracellular highways to carcinogenesis

PubMed Central

Goldenring, James R.

2014-01-01

Epithelial cell carcinogenesis involves the loss of polarity, alteration of polarized protein presentation, dynamic cell morphology changes, increased proliferation and increased cell motility and invasion. Elements of membrane vesicle trafficking underlie all of these processes. Specific membrane trafficking regulators, including Rab small GTPases, through the coordinated dynamics of intracellular trafficking along cytoskeletal pathways, determine cell surface presentation of proteins and overall function of both differentiated and neoplastic cells. While mutations in vesicle trafficking proteins may not be direct drivers of transformation, elements of the machinery of vesicle movement play critical roles in the phenotypes of neoplastic cells. Therefore, the regulators of membrane vesicle trafficking decisions are critical mediators of the full spectrum of cell physiologies driving cancer cell biology, including initial loss of polarity, invasion and metastasis. Targeting of these fundamental intracellular processes may provide important points for manipulation of cancer cell behaviour. PMID:24108097

Effects of space flight on surface marker expression

NASA Astrophysics Data System (ADS)

Sonnenfeld, G.

1999-01-01

Space flight has been shown to affect expression of several cell surface markers. These markers play important roles in regulation of immune responses, including CD4 and CD8. The studies have involved flight of experimental animals and humans followed by analysis of tissue samples (blood in humans, rats and monkeys, spleen, thymus, lymph nodes and bone marrow in rodents). The degree and direction of the changes induced by space flight have been determined by the conditions of the flight. Also, there may be compartmentalization of the response of surface markers to space flight, with differences in the response of cells isolated from blood and local immune tissue. The same type of compartmentalization was also observed with cell adhesion molecules (integrins). In this case, the expression of integrins from lymph node cells differed from that of splenocytes isolated from rats immediately after space flight. Cell culture studies have indicated that there may be an inhibition in conversion of a precursor cell line to cells exhibiting mature macrophage characteristics after space flight, however, these experiments were limited as a result of technical difficulties. In general, it is clear that space flight results in alterations of cell surface markers. The biological significance of these changes remains to be established.

Supramolecular regulation of bioorthogonal catalysis in cells using nanoparticle-embedded transition metal catalysts

NASA Astrophysics Data System (ADS)

Tonga, Gulen Yesilbag; Jeong, Youngdo; Duncan, Bradley; Mizuhara, Tsukasa; Mout, Rubul; Das, Riddha; Kim, Sung Tae; Yeh, Yi-Cheun; Yan, Bo; Hou, Singyuk; Rotello, Vincent M.

2015-07-01

Bioorthogonal catalysis broadens the functional possibilities of intracellular chemistry. Effective delivery and regulation of synthetic catalytic systems in cells are challenging due to the complex intracellular environment and catalyst instability. Here, we report the fabrication of protein-sized bioorthogonal nanozymes through the encapsulation of hydrophobic transition metal catalysts into the monolayer of water-soluble gold nanoparticles. The activity of these catalysts can be reversibly controlled by binding a supramolecular cucurbit[7]uril ‘gate-keeper’ onto the monolayer surface, providing a biomimetic control mechanism that mimics the allosteric regulation of enzymes. The potential of this gated nanozyme for use in imaging and therapeutic applications was demonstrated through triggered cleavage of allylcarbamates for pro-fluorophore activation and propargyl groups for prodrug activation inside living cells.

Notch Signaling Modulates MUC16 Biosynthesis in an In Vitro Model of Human Corneal and Conjunctival Epithelial Cell Differentiation

PubMed Central

Xiong, Linjie; Woodward, Ashley M.

2011-01-01

Purpose. Notch proteins are a family of transmembrane receptors that coordinate binary cell fate decisions and differentiation in wet-surfaced epithelia. We sought to determine whether Notch signaling contributes to maintaining mucosal homeostasis by modulating the biosynthesis of cell surface-associated mucins in an in vitro model of human corneal (HCLE) and conjunctival (HCjE) epithelial cell differentiation. Methods. HCLE and HCjE cells were grown at different stages of differentiation, representing nondifferentiated (preconfluent and confluent) and differentiated (stratified) epithelial cultures. Notch signaling was blocked with the γ-secretase inhibitor dibenzazepine (DBZ). The presence of Notch intracellular domains (Notch1 to Notch3) and mucin protein (MUC1, -4, -16) was evaluated by electrophoresis and Western blot analysis. Mucin gene expression was determined by TaqMan real-time polymerase chain reaction. Results. Here we demonstrate that Notch3 is highly expressed in undifferentiated and differentiated HCLE and HCjE cells, and that Notch1 and Notch2 biosynthesis is enhanced by induction of differentiation with serum-containing media. Inhibition of Notch signaling with DBZ impaired MUC16 biosynthesis in a concentration-dependent manner in undifferentiated cells at both preconfluent and confluent stages, but not in postmitotic stratified cells. In contrast to protein levels, the amount of MUC16 transcripts were not significantly reduced after DBZ treatment, suggesting that Notch regulates MUC16 posttranscriptionally. Immunoblots of DBZ-treated epithelial cells grown at different stages of differentiation revealed no differences in the levels of MUC1 and MUC4. Conclusions. These results indicate that MUC16 biosynthesis is posttranscriptionally regulated by Notch signaling at early stages of epithelial cell differentiation, and suggest that Notch activation contributes to maintaining a mucosal phenotype at the ocular surface. PMID:21508102

Extracellular Matrix and Redox Signaling in Cellular Responses to Stress.

PubMed

Roberts, David D

2017-10-20

Cells in multicellular organisms communicate extensively with neighboring cells and distant organs using a variety of secreted proteins and small molecules. Cells also reside in a structural extracellular matrix (ECM), and changes in its composition, mechanical properties, and post-translational modifications provide additional layers of communication. This Forum addresses emerging mechanisms by which redox signaling controls and is controlled by changes in the ECM, focusing on the roles of matricellular proteins. These proteins engage specific cell surface signaling receptors, integrins, and proteoglycans to regulate the biosynthesis and catabolism of redox signaling molecules and the activation of their signal transducers. These signaling pathways, in turn, regulate the composition of ECM and its function. Covalent post-translational modifications of ECM by redox molecules further regulate its structure and function. Recent studies of acute injuries and chronic disease have identified important pathophysiological roles for this cross-talk and new therapeutic opportunities. Antioxid. Redox Signal. 27, 771-773.

Regulation of Plasminogen Activation on Cell Surfaces and Fibrin.

PubMed

Urano, Tetsumei; Castellino, Francis J; Suzuki, Yuko

2018-05-20

The fibrinolytic system dissolves fibrin and maintains vascular patency. Recent advances in imaging analyses allowed visualization of the spatiotemporal regulatory mechanism of fibrinolysis, as well as its regulation by other plasma haemostasis cofactors. Vascular endothelial cells (VECs) retain tissue-type plasminogen activator (tPA) after secretion and maintain high plasminogen (plg) activation potential on their surfaces. As in plasma, the serpin, plasminogen activator inhibitor type 1 (PAI-1), regulates fibrinolytic potential via inhibition of the VEC surface-bound plg activator, tPA. Once fibrin is formed, plg activation by tPA is initiated and effectively amplified on the surface of fibrin, and fibrin is rapidly degraded. The specific binding of plg and tPA to lytic edges of partly degraded fibrin via newly generated C-terminal lysine residues, which amplifies fibrin digestion, is a central aspect of this pathophysiological mechanism. Thrombomodulin (TM) plays a role in the attenuation of the plg binding on fibrin and the associated fibrinolysis, which is reversed by a carboxypeptidase B inhibitor. This suggests that the plasma procarboxypeptidase B, thrombin activatable fibrinolysis inhibitor (TAFI), which is activated by thrombin bound to TM on VECs, is a critical aspect of the regulation of plg activation on VECs and subsequent fibrinolysis. Platelets also contain PAI-1, TAFI, TM and the fibrin crosslinking enzyme, Factor (F) XIIIa, and either secrete or expose these agents upon activation in order to regulate fibrinolysis. In this review, the native machinery of plg activation and fibrinolysis, as well as their spatiotemporal regulatory mechanisms, as revealed by imaging analyses, are discussed. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

A2A adenosine receptor ligand binding and signalling is allosterically modulated by adenosine deaminase.

PubMed

Gracia, Eduard; Pérez-Capote, Kamil; Moreno, Estefanía; Barkešová, Jana; Mallol, Josefa; Lluís, Carme; Franco, Rafael; Cortés, Antoni; Casadó, Vicent; Canela, Enric I

2011-05-01

A2ARs (adenosine A2A receptors) are highly enriched in the striatum, which is the main motor control CNS (central nervous system) area. BRET (bioluminescence resonance energy transfer) assays showed that A2AR homomers may act as cell-surface ADA (adenosine deaminase; EC 3.5.4.4)-binding proteins. ADA binding affected the quaternary structure of A2ARs present on the cell surface. ADA binding to adenosine A2ARs increased both agonist and antagonist affinity on ligand binding to striatal membranes where these proteins are co-expressed. ADA also increased receptor-mediated ERK1/2 (extracellular-signal-regulated kinase 1/2) phosphorylation. Collectively, the results of the present study show that ADA, apart from regulating the concentration of extracellular adenosine, may behave as an allosteric modulator that markedly enhances ligand affinity and receptor function. This powerful regulation may have implications for the physiology and pharmacology of neuronal A2ARs.

Reduction of CD147 surface expression on primary T cells leads to enhanced cell proliferation.

PubMed

Biegler, Brian; Kasinrerk, Watchara

2012-12-01

CD147 is a ubiquitously expressed membrane glycoprotein that has numerous functional associations in health and disease. However, the molecular mechanisms by which CD147 participates in these processes are unclear. Establishing physiologically relevant silencing of CD147 in primary T cells could provide clues essential for elucidating some aspects of CD147 biology. To date, achieving the knockdown of CD147 in primary T cells has remained elusive. Utilizing RNA interference and the Nucleofector transfection system, we were able to reduce the expression of CD147 in primary T cells. Comparison of basic functions, such as proliferation and CD25 expression, were then made between control populations and populations with reduced expression. Up-regulation of CD147 was found upon T-cell activation, indicating a role in T-cell responses. To better understand the possible importance of this up-regulation, we knocked down the expression of CD147 using RNA interference. When compared to control populations the CD147 knockdown populations exhibited increased proliferation. This alteration of cell proliferation, however, was not linked to a change in CD25 expression. We achieved reduction of CD147 surface expression in primary T cells by siRNA-mediated gene silencing. Our results point to CD147 having a possible negative regulatory role in T cell-mediated immune responses.

Mechanisms regulating cell membrane localization of the chemokine receptor CXCR4 in human hepatocarcinoma cells.

PubMed

Cepeda, Edgar B; Dediulia, Tatjana; Fernando, Joan; Bertran, Esther; Egea, Gustavo; Navarro, Estanislao; Fabregat, Isabel

2015-05-01

Hepatocellular carcinoma (HCC) cells with a mesenchymal phenotype show an asymmetric subcellular distribution of the chemokine receptor CXCR4, which is required for cell migration and invasion. In this work we examine the mechanisms that regulate the intracellular trafficking of CXCR4 in HCC cells. Results indicate that HCC cells present CXCR4 at the cell surface, but most of this protein is in endomembranes colocalizing with markers of the Golgi apparatus and recycling endosomes. The presence of high protein levels of CXCR4 present at the cell surface correlates with a mesenchymal-like phenotype and a high autocrine activation of the Transforming Growth Factor-beta (TGF-β) pathway. CXCR4 traffics along the Golgi/exocyst/plasma membrane pathway and requires EXOC4 (Sec8) component of the exocyst complex. HCC cells use distinct mechanisms for the CXCR4 internalization such as dynamin-dependent endocytosis and macropinocytosis. Regardless of the endocytic mechanisms, colocalization of CXCR4 and Rab11 is observed, which could be involved not only in receptor recycling but also in its post-Golgi transport. In summary, this work highlights membrane trafficking pathways whose pharmacological targeting could subsequently result in the inactivation of one of the main guiding mechanisms used by metastatic cells to colonize secondary organs and tissues. Copyright © 2015 Elsevier B.V. All rights reserved.

Synergistic responses of superficial chemistry and micro topography of titanium created by wire-type electric discharge machining.

PubMed

Kataoka, Yu; Tamaki, Yukimichi; Miyazaki, Takashi

2011-01-01

Wire-type electric discharge machining has been applied to the manufacture of endosseous titanium implants as this computer associated technique allows extremely accurate complex sample shaping with an optimal micro textured surface during the processing. Since the titanium oxide layer is sensitively altered by each processing, the authors hypothesized that this technique also up-regulates biological responses through the synergistic effects of the superficial chemistry and micro topography. To evaluate the respective in vitro cellular responses on the superficial chemistry and micro topography of titanium surface processed by wire-type electric discharge, we used titanium-coated epoxy resin replica of the surface. An oxide layer on the titanium surface processed by wire-type electric discharge activated the initial responses of osteoblastic cells through an integrin-mediated mechanism. Since the mRNA expression of ALP on those replicas was up-regulated compared to smooth titanium samples, the micro topography of a titanium surface processed by wire-type electric discharge promotes the osteogenic potential of cells. The synergistic response of the superficial chemistry and micro topography of titanium processed by wire-type electric discharge was demonstrated in this study.

Revealing the cell-material interface with nanometer resolution by FIB-SEM

PubMed Central

Santoro, Francesca; Zhao, Wenting; Joubert, Lydia-Marie; Duan, Liting; Schnitker, Jan; van de Burgt, Yoeri; Lou, Hsin-Ya; Liu, Bofei; Salleo, Alberto; Cui, Lifeng; Cui, Yi; Cui, Bianxiao

2018-01-01

The interface between cells and non-biological surfaces regulates cell attachment, chronic tissue responses, and ultimately the success of medical implants or biosensors. Clinical and laboratory studies show that topological features of the surface profoundly influences cellular responses, e.g. titanium surfaces with nano- and microtopographical structures enhance osteoblast attachment and host-implant integration as compare to smooth surface. To understand how cells and tissues respond to different topographical features, it is of critical importance to directly visualize the cell-materials interface at the relevant nanometer length scale. Here, we present a new method for in situ examination of the cell-to-material interface at any desired location, based on focused-ion beam milling and scanning electron microscopy imaging (FIB-SEM) to resolve the cell membrane-to-material interface with 10 nm resolution. By examining how cell membranes interact with topographical features such as nanoscale protrusions or invaginations, we discovered that the cell membrane readily deforms inward and wraps around protruding structures, but hardly deforms outward to contour invaginating structures. This asymmetric membrane response (inward vs. outward deformation) causes the cleft width between the cell membrane and the nanostructure surface to vary for more than an order of magnitude. Our results suggest that surface topology is a crucial consideration for the development of medical implants or biosensors whose performances are strongly influenced by the cell-to-material interface. We anticipate that the method can be used to explore the direct interaction of cells/tissue with medical devices such as metal implants in the future. PMID:28682058

Poly(dopamine) coating of 3D printed poly(lactic acid) scaffolds for bone tissue engineering.

PubMed

Kao, Chia-Tze; Lin, Chi-Chang; Chen, Yi-Wen; Yeh, Chia-Hung; Fang, Hsin-Yuan; Shie, Ming-You

2015-11-01

3D printing is a versatile technique to generate large quantities of a wide variety of shapes and sizes of polymer. The aim of this study is to develop functionalized 3D printed poly(lactic acid) (PLA) scaffolds and use a mussel-inspired surface coating to regulate cell adhesion, proliferation and differentiation of human adipose-derived stem cells (hADSCs). We prepared PLA 3D scaffolds coated with polydopamine (PDA). The chemical composition and surface properties of PDA/PLA were characterized by XPS. PDA/PLA modulated hADSCs' responses in several ways. Firstly, adhesion and proliferation, and cell cycle of hADSCs cultured on PDA/PLA were significantly enhanced relative to those on PLA. In addition, the collagen I secreted from cells was increased and promoted cell attachment and cell cycle progression were depended on the PDA content. In osteogenesis assay, the ALP activity and osteocalcin of hADSCs cultured on PDA/PLA were significantly higher than seen in those cultured on pure PLA scaffolds. Moreover, hADSCs cultured on PDA/PLA showed up-regulation of the ang-1 and vWF proteins associated with angiogenic differentiation. Our results demonstrate that the bio-inspired coating synthetic PLA polymer can be used as a simple technique to render the surfaces of synthetic scaffolds active, thus enabling them to direct the specific responses of hADSCs. Copyright © 2015 Elsevier B.V. All rights reserved.

CD47: A Master Regulator of Stemness | Center for Cancer Research

Cancer.gov

Identifying the pathways cells use to regulate proliferation, differentiation, and survival are essential for designing new treatments to stimulate organ and tissue repair following injury and for diseases as diverse as cancer and diabetes. The thrombospondin-1 receptor CD47 seems to limit cell survival and regeneration after stress. At the same time, CD47 levels are increased on the surface of cancer cells, which show enhanced proliferation and survival. To understand this apparent paradox, David Roberts, Ph.D., in CCR’s Laboratory of Pathology and his colleagues decided to investigate CD47’s mechanism of action using CD47- and thrombospondin-1-null mice.

LAPTM5 promotes lysosomal degradation of intracellular CD3ζ but not of cell surface CD3ζ.

PubMed

Kawai, Yohei; Ouchida, Rika; Yamasaki, Sho; Dragone, Leonard; Tsubata, Takeshi; Wang, Ji-Yang

2014-07-01

The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.

Primary Cilia: Highly Sophisticated Biological Sensors

PubMed Central

Abou Alaiwi, Wissam A.; Lo, Shao T.; Nauli, Surya M.

2009-01-01

Primary cilia, thin hair-like structures protruding from the apical surface of most mammalian cells, have gained the attention of many researchers over the past decade. Primary cilia are microtubule-filled sensory organelles that are enclosed within the ciliary membrane. They originate at the cell surface from the mother centriole that becomes the mature basal body. In this review, we will discuss recent literatures on the roles of cilia as sophisticated sensory organelles. With particular emphasis on vascular endothelia and renal epithelia, the mechanosensory role of cilia in sensing fluid shear stress will be discussed. Also highlighted is the ciliary involvement in cell cycle regulation, development, cell signaling and cancer. Finally, primary cilia-related disorders will be briefly described. PMID:22423203

ITEL Experiment Module and its Flight on MASER9

NASA Astrophysics Data System (ADS)

Löth, K.; Schneider, H.; Larsson, B.; Jansson, O.; Houltz, Y.

2002-01-01

The ITEL (Interfacial Turbulence in Evaporating Liquid) module is built under contract from the European Space Agency (ESA) and is scheduled to fly onboard a Sounding Rocket (MASER 9) in March 2002. The project is conducted by Swedish Space Corporation (SSC) with Lambda-X as a subcontractor responsible for the optical system. The Principle Investigator is Pierre Colinet from Université Libre de Bruxelles (ULB). The experiment in ITEL on Maser 9 is part of a research program, which will make use of the International Space Station. The purpose of the flight on Maser 9 is to observe the cellular convection (Marangoni-Bénard instability) which arise when the surface tension varies with temperature yielding thermocapillary instabilities. During the 6 minutes of microgravity of the ITEL experiment, a highly volatile liquid layer (ethyl alcohol) will be evaporated, and the convection phenomena generated by the evaporation process will be visualized. Due to the cooling by latent heat consumption at the level of the evaporating free surface, a temperature gradient is induced perpendicularly to it. The flight experiment module contains one experiment cell, including a gas system for regulation of nitrogen flow over the evaporating surface and an injection unit that is used for injection of liquid into the cell both initially and during surface regulation. The experiment cell is equipped with pressure and flow sensors as well as thermocouples both inside the liquid and at different positions in the cell. Two optical diagnostic systems have been developed around the experiment cell. An interferometric optical tomograph measures the 3-dimensional distribution of temperature in the evaporating liquid and a Schlieren system visualizes the temperature gradients inside the liquid together with the liquid surface deformation. A PC/104 based electronic system is used for management and control of the experiment. The electronic system handles measurements, housekeeping, image capture system, surface and pressure regulation as well as storage of data. The images are stored onboard on three DV tape recorders. At flight, video images as well as data is sent to ground and the experiment can be controlled via telecommands. In this presentation we will focus on the technical parts of the experiment, the overall module and the preliminary technical results obtained from the flight, including reconstructions of 3-dimensional temperature distributions.

Protein profiling of single epidermal cell types from Arabidopsis thaliana using surface-enhanced laser desorption and ionization technology.

PubMed

Ebert, Berit; Melle, Christian; Lieckfeldt, Elke; Zöller, Daniela; von Eggeling, Ferdinand; Fisahn, Joachim

2008-08-25

Here, we describe a novel approach for investigating differential protein expression within three epidermal cell types. In particular, 3000 single pavement, basal, and trichome cells from leaves of Arabidopsis thaliana were harvested by glass micro-capillaries. Subsequently, these single cell samples were joined to form pools of 100 individual cells and analyzed using the ProteinChip technology; SELDI: surface-enhanced laser desorption and ionization. As a result, numerous protein signals that were differentially expressed in the three epidermal cell types could be detected. One of these proteins was characterized by tryptical digestion and subsequent identification via tandem quadrupole-time of flight (Q-TOF) mass spectrometry. Down regulation of this sequenced small subunit precursor of ribulose-1,5 bisphosphate carboxylase(C) oxygenase(O) (RuBisCo) in trichome and basal cells indicates the sink status of these cell types that are located on the surface of A. thaliana source leaves. Based on the obtained protein profiles, we suggest a close functional relationship between basal and trichome cells at the protein level.

POSH regulates Hippo signaling through ubiquitin-mediated expanded degradation.

PubMed

Ma, Xianjue; Guo, Xiaowei; Richardson, Helena E; Xu, Tian; Xue, Lei

2018-02-27

The Hippo signaling pathway is a master regulator of organ growth, tissue homeostasis, and tumorigenesis. The activity of the Hippo pathway is controlled by various upstream components, including Expanded (Ex), but the precise molecular mechanism of how Ex is regulated remains poorly understood. Here we identify Plenty of SH3s (POSH), an E3 ubiquitin ligase, as a key component of Hippo signaling in Drosophila POSH overexpression synergizes with loss of Kibra to induce overgrowth and up-regulation of Hippo pathway target genes. Furthermore, knockdown of POSH impedes dextran sulfate sodium-induced Yorkie-dependent intestinal stem cell renewal, suggesting a physiological role of POSH in modulating Hippo signaling. Mechanistically, POSH binds to the C-terminal of Ex and is essential for the Crumbs-induced ubiquitination and degradation of Ex. Our findings establish POSH as a crucial regulator that integrates the signal from the cell surface to negatively regulate Ex-mediated Hippo activation in Drosophila .

Monocyte:T cell interaction regulates human T cell activation through a CD28/CD46 crosstalk

PubMed Central

Charron, Lauren; Doctrinal, Axelle; Choileain, Siobhan Ni; Astier, Anne L.

2015-01-01

T cell activation requires engagement of the T cell receptor and of at least one costimulatory molecule. The key role of CD28 in inducing T cell activation has been reported several decades ago and the molecular mechanisms involved well described. The complement regulator CD46 also acts as a costimulatory molecule for T cells but, in contrast to CD28, has the ability to drive T cell differentiation from producing some IFNγ to secreting some potent anti-inflammatory IL-10, acquiring a so-called Type I regulatory phenotype (Tr1). Proteolytic cleavage of CD46 occurs upon costimulation and is important for T cell activation and IL-10 production. The observation that CD46 cleavage was reduced when PBMC were costimulated compared to purified naive T cells led us to hypothesize that interactions between different cell types within the PBMC were able to modulate the CD46 pathway. We show that CD46 downregulation is also reduced when CD4+ T cells are co-cultured with autologous monocytes. Indeed, monocyte:T cell co-cultures impaired CD46–mediated T cell differentiation and coactivation, by reducing downregulation of surface CD46, lowering induction of the early activation marker CD69, as well as reducing the levels of IL-10 secretion. Blocking of CD86 could partly restore CD69 expression and cytokine secretion, demonstrating that the CD28-CD86 pathway regulates CD46 activation. Direct concomitant ligation of CD28 and CD46 on CD4+ T cells also modulated CD46 expression and regulated cytokine production. These data identify a crosstalk between two main costimulatory pathways and provide novel insights into the regulation of human T cell activation. PMID:25787182

There and Back Again: Development and Regeneration of the Zebrafish Lateral Line System

PubMed Central

Thomas, Eric D.; Cruz, Ivan A.; Hailey, Dale W.; Raible, David W.

2014-01-01

The zebrafish lateral line is a sensory system used to detect changes in water flow. It is comprised of clusters of mechanosensory hair cells called neuromasts. The lateral line is initially established by a migratory group of cells, called a primordium, that deposits neuromasts at stereotyped locations along the surface of the fish. Wnt, FGF, and Notch signaling are all important regulators of various aspects of lateral line development, from primordium migration to hair cell specification. As zebrafish age, the organization of the lateral line becomes more complex in order to accommodate the fish’s increased size. This expansion is regulated by many of the same factors involved in the initial development. Furthermore, unlike mammalian hair cells, lateral line hair cells have the capacity to regenerate after damage. New hair cells arise from the proliferation and differentiation of surrounding support cells, and the molecular and cellular pathways regulating this are beginning to be elucidated. All in all, the zebrafish lateral line has proven to be an excellent model in which to study a diverse array of processes, including collective cell migration, cell polarity, cell fate, and regeneration. PMID:25330982

The planar cell polarity protein VANGL2 coordinates remodeling of the extracellular matrix

PubMed Central

Williams, B. Blairanne; Mundell, Nathan; Dunlap, Julie; Jessen, Jason

2012-01-01

Understanding how planar cell polarity (PCP) is established, maintained, and coordinated in migrating cell populations is an important area of research with implications for both embryonic morphogenesis and tumor cell invasion. We recently reported that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell surface level of membrane type-1 matrix metalloproteinase (MMP14 or MT1-MMP). Here, we further discuss these findings in terms of extracellular matrix (ECM) remodeling, cell migration, and zebrafish gastrulation. We also demonstrate that VANGL2 function impacts the focal degradation of ECM by human cancer cells including the formation or stability of invadopodia. Together, our findings implicate MMP14 as a downstream effector of VANGL2 signaling and suggest a model whereby the regulation of pericellular proteolysis is a fundamental aspect of PCP in migrating cells. PMID:23060953

Cigarette Smoke–Induced CXCR3 Receptor Up-Regulation Mediates Endothelial Apoptosis

PubMed Central

Green, Linden A.; Petrusca, Daniela; Rajashekhar, Gangaraju; Gianaris, Tom; Schweitzer, Kelly S.; Wang, Liang; Justice, Matthew J.; Petrache, Irina

2012-01-01

Endothelial monocyte–activating polypeptide II (EMAP II) and interferon-inducible protein (IP)–10 are proinflammatory mediators, which in addition to their chemokine activities, selectively induce apoptosis in endothelial cells and are up-regulated in the lungs of cigarette smoke–exposed humans. Previously, we showed that EMAP II is an essential mediator of cigarette smoke–induced lung emphysema in mice linking endothelial cell apoptosis with inflammation. Here we addressed the role of the CXCR3 receptor in EMAP II–induced and IP-10–induced apoptosis in endothelial cells and its regulation by cigarette smoke. We found that both neutralizing antibodies and small inhibitory RNA to CXCR3 abrogated EMAP II–induced and IP-10–induced endothelial caspase-3 activation and DNA fragmentation. CXCR3 receptor surface expression in human lung microvascular endothelial cells and in lung tissue endothelium was up-regulated by exposure to cigarette smoke. In tissue culture conditions, EMAP II–induced and IP-10–induced apoptosis was enhanced by preincubation with cigarette smoke extract. Interestingly, serum starvation also induced CXCR3 up-regulation and enhanced EMAP II–induced endothelial apoptosis. Signal transduction via p38 mitogen-activated protein kinase activation was essential for CXCR3-induced cell death, but not for CXCR3 receptor up-regulation by cigarette smoke. In turn, protein nitration was required for CXCR3 receptor up-regulation by cigarette smoke and consequently for subsequent CXCR3-induced cell death. In conclusion, the concerted up-regulation of proinflammatory EMAP II, IP-10, and CXCR3 by cigarette smoke could sustain a cascade of cell death that may promote the alveolar tissue loss noted in human emphysema. PMID:22936405

Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes

PubMed Central

Lacanna, Egidio; Bigosch, Colette; Kaever, Volkhard; Boehm, Alex

2016-01-01

ABSTRACT DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli. Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface. IMPORTANCE Bacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment. E. coli possesses a plethora of c-di-GMP-producing diguanylate cyclases, including DgcZ. Our study expands the knowledge on the role of DgcZ in regulation of surface attachment and suggests that it interconnects surface sensing and adhesion via multiple routes. PMID:27402625

Evidence for Escherichia coli Diguanylate Cyclase DgcZ Interlinking Surface Sensing and Adhesion via Multiple Regulatory Routes.

PubMed

Lacanna, Egidio; Bigosch, Colette; Kaever, Volkhard; Boehm, Alex; Becker, Anke

2016-09-15

DgcZ is the main cyclic dimeric GMP (c-di-GMP)-producing diguanylate cyclase (DGC) controlling biosynthesis of the exopolysaccharide poly-β-1,6-N-acetylglucosamine (poly-GlcNAc or PGA), which is essential for surface attachment of Escherichia coli Although the complex regulation of DgcZ has previously been investigated, its primary role and the physiological conditions under which the protein is active are not fully understood. Transcription of dgcZ is regulated by the two-component system CpxAR activated by the lipoprotein NlpE in response to surface sensing. Here, we show that the negative effect of a cpxR mutation and the positive effect of nlpE overexpression on biofilm formation both depend on DgcZ. Coimmunoprecipitation data suggest several potential interaction partners of DgcZ. Interaction with FrdB, a subunit of the fumarate reductase complex (FRD) involved in anaerobic respiration and in control of flagellum assembly, was further supported by a bacterial-two-hybrid assay. Furthermore, the FRD complex was required for the increase in DgcZ-mediated biofilm formation upon induction of oxidative stress by addition of paraquat. A DgcZ-mVENUS fusion protein was found to localize at one bacterial cell pole in response to alkaline pH and carbon starvation. Based on our data and previous knowledge, an integrative role of DgcZ in regulation of surface attachment is proposed. We speculate that both DgcZ-stimulated PGA biosynthesis and interaction of DgcZ with the FRD complex contribute to impeding bacterial escape from the surface. Bacterial cells can grow by clonal expansion to surface-associated biofilms that are ubiquitous in the environment but also constitute a pervasive problem related to bacterial infections. Cyclic dimeric GMP (c-di-GMP) is a widespread bacterial second messenger involved in regulation of motility and biofilm formation, and plays a primary role in bacterial surface attachment. E. coli possesses a plethora of c-di-GMP-producing diguanylate cyclases, including DgcZ. Our study expands the knowledge on the role of DgcZ in regulation of surface attachment and suggests that it interconnects surface sensing and adhesion via multiple routes. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Recruitment of EB1, a Master Regulator of Microtubule Dynamics, to the Surface of the Theileria annulata Schizont

PubMed Central

Woods, Kerry L.; Theiler, Romina; Mühlemann, Marcus; Segiser, Adrian; Huber, Sandra; Ansari, Hifzur R.; Pain, Arnab; Dobbelaere, Dirk A. E.

2013-01-01

The apicomplexan parasite Theileria annulata transforms infected host cells, inducing uncontrolled proliferation and clonal expansion of the parasitized cell population. Shortly after sporozoite entry into the target cell, the surrounding host cell membrane is dissolved and an array of host cell microtubules (MTs) surrounds the parasite, which develops into the transforming schizont. The latter does not egress to invade and transform other cells. Instead, it remains tethered to host cell MTs and, during mitosis and cytokinesis, engages the cell's astral and central spindle MTs to secure its distribution between the two daughter cells. The molecular mechanism by which the schizont recruits and stabilizes host cell MTs is not known. MT minus ends are mostly anchored in the MT organizing center, while the plus ends explore the cellular space, switching constantly between phases of growth and shrinkage (called dynamic instability). Assuming the plus ends of growing MTs provide the first point of contact with the parasite, we focused on the complex protein machinery associated with these structures. We now report how the schizont recruits end-binding protein 1 (EB1), a central component of the MT plus end protein interaction network and key regulator of host cell MT dynamics. Using a range of in vitro experiments, we demonstrate that T. annulata p104, a polymorphic antigen expressed on the schizont surface, functions as a genuine EB1-binding protein and can recruit EB1 in the absence of any other parasite proteins. Binding strictly depends on a consensus SxIP motif located in a highly disordered C-terminal region of p104. We further show that parasite interaction with host cell EB1 is cell cycle regulated. This is the first description of a pathogen-encoded protein to interact with EB1 via a bona-fide SxIP motif. Our findings provide important new insight into the mode of interaction between Theileria and the host cell cytoskeleton. PMID:23675298

Surface Expression of Hsp25 and Hsp72 Differentially Regulates Tumor Growth and Metastasis

PubMed Central

Bausero, María A.; Page, Diana T.; Osinaga, Eduardo; Asea, Alexzander

2006-01-01

The expression of unique surface structures on tumors that allow for recognition and activation of host immunocompetent cells plays an important role in determining tumor growth and/or metastasis. Recent studies have identified an important role for heat shock proteins (Hsp) in antitumor surveillance; however, the exact role of Hsp expressed on the surface of tumors has not been fully addressed. In this study, we show that 4T1 mammary adenocarcinoma cells sorted for high Hsp25 surface expression (Hsp25high) grow significantly faster than cells sorted for intermediate Hsp25 surface expression (Hsp25intermediate) or wild-type 4T1 cells implanted into the abdominal breast gland of female BALB/c mice (p < 0.05). In addition, histological examination of lung tissues revealed that Hsp25high 4T1 cells metastasized to the lungs more aggressively than either Hsp25intermediate or wild-type 4T1 cells (p < 0.05). Exposure of 4T1 cells to nonlethal heat shock (43°C, 30 min) induced the surface expression of Hsp72 and a concomitant reduction in Hsp25 surface expression. The growth and metastastic potential of Hsp72+ 4T1 cells was significantly less than that of Hsp25high, Hsp25intermediate or wild-type 4T1 cells (p < 0.05). Taken together, these studies identify an important role for expression of Hsp25 and Hsp72 during tumor growth and metastatic spread which might be helpful in the design of antimetastatic therapies. PMID:15627887

Surface expression of Hsp25 and Hsp72 differentially regulates tumor growth and metastasis.

PubMed

Bausero, María A; Page, Diana T; Osinaga, Eduardo; Asea, Alexzander

2004-01-01

The expression of unique surface structures on tumors that allow for recognition and activation of host immunocompetent cells plays an important role in determining tumor growth and/or metastasis. Recent studies have identified an important role for heat shock proteins (Hsp) in antitumor surveillance; however, the exact role of Hsp expressed on the surface of tumors has not been fully addressed. In this study, we show that 4T1 mammary adenocarcinoma cells sorted for high Hsp25 surface expression (Hsp25(high)) grow significantly faster than cells sorted for intermediate Hsp25 surface expression (Hsp25(intermediate)) or wild-type 4T1 cells implanted into the abdominal breast gland of female BALB/c mice (p < 0.05). In addition, histological examination of lung tissues revealed that Hsp25(high) 4T1 cells metastasized to the lungs more aggressively than either Hsp25(intermediate) or wild-type 4T1 cells (p < 0.05). Exposure of 4T1 cells to nonlethal heat shock (43 degrees C, 30 min) induced the surface expression of Hsp72 and a concomitant reduction in Hsp25 surface expression. The growth and metastastic potential of Hsp72(+) 4T1 cells was significantly less than that of Hsp25(high), Hsp25(intermediate) or wild-type 4T1 cells (p < 0.05). Taken together, these studies identify an important role for expression of Hsp25 and Hsp72 during tumor growth and metastatic spread which might be helpful in the design of antimetastatic therapies. Copyright 2004 S. Karger AG, Basel.

Multiscale Model of Swarming Bacteria

NASA Astrophysics Data System (ADS)

Alber, Mark

2011-03-01

Many bacteria can rapidly traverse surfaces from which they are extracting nutrient for growth. They generate flat, spreading colonies, called swarms because they resemble swarms of insects. In the beginning of the talk, swarms of the M. xanthus will be described in detail. Individual M. xanthus cells are elongated; they always move in the direction of their long axis; and they are in constant motion, repeatedly touching each other. As a cell glides, the slime capsule of a cell interacts with the bare agar surface, non-oriented slime which arises from the surface contact with the slime capsule, or oriented slime trails. Remarkably, cells regularly reverse their gliding directions. In this talk a detailed cell- and behavior-based computational model of M. xanthus swarming will be used to demonstrate that reversals of gliding direction and cell bending are essential for swarming and that specific reversal frequencies result in optimal swarming rate of the whole population. This suggests that the circuit regulating reversals evolved to its current sensitivity under selection for growth achieved by swarming.

Opening up of plasmalemma type-1 VDAC to form apoptotic "find me signal" pathways is essential in early apoptosis - evidence from the pathogenesis of cystic fibrosis resulting from failure of apoptotic cell clearance followed by sterile inflammation.

PubMed

Thinnes, Friedrich P

2014-04-01

Cell membrane-standing type-1 VDAC is involved in cell volume regulation and thus apoptosis. The channel has been shown to figure as a pathway for osmolytes of varying classes, ATP included. An early event in apoptotic cell death is the release of "find me signals" by cells that enter the apoptotic process. ATP is one of those signals. Apoptotic cells this way attract phagocytes for an immunologically silent cell clearance. Thus, whenever apoptosis fails by a blockade of plasmalemma type-1 VDAC processes of sterile inflammation must be assumed for cell elimination. This is evident from a close look on the pathogenetic process of cystic fibrosis (CF). However, in normal airway epithelia two different anion channels cooperate to guarantee an appropriate volume of airway surface liquid (ASL) necessary for surface clearing: the cystic fibrosis conductance regulator (CFTR) and the outwardly rectifying chloride channel (ORCC) complex also called "alternate chloride channel" and under the control of the CFTR. There are arguments, that type-1 VDAC forms the channel part of the ORCC complex, and it has been shown that CFTR and type-1 VDAC co-localize in the apical membranes of human surface respiratory epithelium. In cystic fibrosis, the central cAMP-dependent regulation of ion and water transport via functional CFTR is lost. Here, CFTR molecules do not reach the apical membranes of airway epithelia anymore or work in an insufficient way, respectively. In addition, type-1 VDAC is no longer available to work as a "find me signal" pathway. In consequence, clearing away of apoptotic cells is blocked. There are experimental data on the channel characteristics of type-1 VDAC under the anion channel blocker DIDS (4,4-diisothiocyanato-stilbenedisulphonic acid) that argue in favor of this hypothesis. Together, type-1 VDAC should be kept as a "find me signal" pathway, which may give way to several classes of such signals. Copyright © 2014 Elsevier Inc. All rights reserved.

Cellular and Molecular Biology of Airway Mucins

PubMed Central

Lillehoj, Erik P.; Kato, Kosuke; Lu, Wenju; Kim, Kwang C.

2017-01-01

Airway mucus constitutes a thin layer of airway surface liquid with component macromolecules that covers the luminal surface of the respiratory tract. The major function of mucus is to protect the lungs through mucociliary clearance of inhaled foreign particles and noxious chemicals. Mucus is comprised of water, ions, mucin glycoproteins, and a variety of other macromolecules, some of which possess anti-microbial, anti-protease, and anti-oxidant activities. Mucins comprise the major protein component of mucus and exist as secreted and cell-associated glycoproteins. Secreted, gel-forming mucins are mainly responsible for the viscoelastic property of mucus, which is crucial for effective mucociliary clearance. Cell-associated mucins shield the epithelial surface from pathogens through their extracellular domains and regulate intracellular signaling through their cytoplasmic regions. However, neither the exact structures of mucin glycoproteins, nor the manner through which their expression is regulated, are completely understood. This chapter reviews what is currently known about the cellular and molecular properties of airway mucins. PMID:23445810

ADAM10 controls collagen signaling and cell migration on collagen by shedding the ectodomain of discoidin domain receptor 1 (DDR1)

PubMed Central

Shitomi, Yasuyuki; Thøgersen, Ida B.; Ito, Noriko; Leitinger, Birgit; Enghild, Jan J.; Itoh, Yoshifumi

2015-01-01

Discoidin domain receptor 1 (DDR1) is a receptor tyrosine kinase that binds and transmits signals from various collagens in epithelial cells. However, how DDR1–dependent signaling is regulated has not been understood. Here we report that collagen binding induces ADAM10-dependent ectodomain shedding of DDR1. DDR1 shedding is not a result of an activation of its signaling pathway, since DDR1 mutants defective in signaling were shed in an efficient manner. DDR1 and ADAM10 were found to be in a complex on the cell surface, but shedding did not occur unless collagen bound to DDR1. Using a shedding-resistant DDR1 mutant, we found that ADAM10-dependent DDR1 shedding regulates the half-life of collagen-induced phosphorylation of the receptor. Our data also revealed that ADAM10 plays an important role in regulating DDR1-mediated cell adhesion to achieve efficient cell migration on collagen matrices. PMID:25540428

The hypertonic environment differentially regulates wild-type CFTR and TNR-CFTR chloride channels.

PubMed

Lassance-Soares, Roberta M; Cheng, Jie; Krasnov, Kristina; Cebotaru, Liudmila; Cutting, Garry R; Souza-Menezes, Jackson; Morales, Marcelo M; Guggino, William B

2010-01-01

This study tested the hypotheses that the hypertonic environment of the renal medulla regulates the expression of cystic fibrosis transmembrane conductance regulator protein (CFTR) and its natural splice variant, TNR-CFTR. To accomplish this, Madin-Darby canine kidney (MDCK) stable cell lines expressing TNR-CFTR or CFTR were used. The cells were treated with hypertonic medium made with either NaCl or urea or sucrose (480 mOsm/kg or 560 mOsm/kg) to mimic the tonicity of the renal medulla environment. Western blot data showed that CFTR and TNR-CFTR total cell protein is increased by hypertonic medium, but using the surface biotinylation technique, only CFTR was found to be increased in cell plasma membrane. Confocal microscopy showed TNR-CFTR localization primarily at the endoplasmic reticulum and plasma membrane. In conclusion, CFTR and TNR-CFTR have different patterns of distribution in MDCK cells and they are modulated by a hypertonic environment, suggesting their physiological importance in renal medulla. Copyright © 2010 S. Karger AG, Basel.

Volume regulation and shape bifurcation in the cell nucleus

PubMed Central

Kim, Dong-Hwee; Li, Bo; Si, Fangwei; Phillip, Jude M.; Wirtz, Denis; Sun, Sean X.

2015-01-01

ABSTRACT Alterations in nuclear morphology are closely associated with essential cell functions, such as cell motility and polarization, and correlate with a wide range of human diseases, including cancer, muscular dystrophy, dilated cardiomyopathy and progeria. However, the mechanics and forces that shape the nucleus are not well understood. Here, we demonstrate that when an adherent cell is detached from its substratum, the nucleus undergoes a large volumetric reduction accompanied by a morphological transition from an almost smooth to a heavily folded surface. We develop a mathematical model that systematically analyzes the evolution of nuclear shape and volume. The analysis suggests that the pressure difference across the nuclear envelope, which is influenced by changes in cell volume and regulated by microtubules and actin filaments, is a major factor determining nuclear morphology. Our results show that physical and chemical properties of the extracellular microenvironment directly influence nuclear morphology and suggest that there is a direct link between the environment and gene regulation. PMID:26243474

Volume regulation and shape bifurcation in the cell nucleus.

PubMed

Kim, Dong-Hwee; Li, Bo; Si, Fangwei; Phillip, Jude M; Wirtz, Denis; Sun, Sean X

2015-09-15

Alterations in nuclear morphology are closely associated with essential cell functions, such as cell motility and polarization, and correlate with a wide range of human diseases, including cancer, muscular dystrophy, dilated cardiomyopathy and progeria. However, the mechanics and forces that shape the nucleus are not well understood. Here, we demonstrate that when an adherent cell is detached from its substratum, the nucleus undergoes a large volumetric reduction accompanied by a morphological transition from an almost smooth to a heavily folded surface. We develop a mathematical model that systematically analyzes the evolution of nuclear shape and volume. The analysis suggests that the pressure difference across the nuclear envelope, which is influenced by changes in cell volume and regulated by microtubules and actin filaments, is a major factor determining nuclear morphology. Our results show that physical and chemical properties of the extracellular microenvironment directly influence nuclear morphology and suggest that there is a direct link between the environment and gene regulation. © 2015. Published by The Company of Biologists Ltd.

RACK1 interacts with filamin-A to regulate plasma membrane levels of the cystic fibrosis transmembrane conductance regulator

PubMed Central

Smith, Laura; Litman, Paul; Kohli, Ekta; Amick, Joseph; Page, Richard C.; Misra, Saurav

2013-01-01

Mutations in cystic fibrosis transmembrane regulator (CFTR), a chloride channel in the apical membranes of secretory epithelial cells, underlie the fatal genetic disorder cystic fibrosis. Certain CFTR mutations, including the common mutation ΔF508-CFTR, result in greatly decreased levels of active CFTR at the apical membrane. Direct interactions between CFTR and the cytoskeletal adaptors filamin-A (FlnA) and Na+/H+ exchanger regulatory factor 1 (NHERF1) stabilize the expression and localization of CFTR at the plasma membrane. The scaffold protein receptor for activated C kinase 1 (RACK1) also stabilizes CFTR surface expression; however, RACK1 does not interact directly with CFTR and its mechanism of action is unknown. In the present study, we report that RACK1 interacts directly with FlnA in vitro and in a Calu-3 airway epithelial cell line. We mapped the interaction between RACK1 and FlnA to the WD4 and WD6 repeats of RACK1 and to a segment of the large rod domain of FlnA, consisting of immunoglobulin-like repeats 8–15. Disruption of the RACK1-FlnA interaction causes a reduction in CFTR surface levels. Our results suggest that a novel RACK1-FlnA interaction is an important regulator of CFTR surface localization. PMID:23636454

PU.1 regulates TCR expression by modulating GATA-3 activity

PubMed Central

Chang, Hua-Chen; Han, Ling; Jabeen, Rukhsana; Carotta, Sebastian; Nutt, Stephen L.; Kaplan, Mark H.

2009-01-01

The Ets transcription factor PU.1 is a master regulator for the development of multiple lineages during hematopoiesis. The expression pattern of PU.1 is dynamically regulated during early T lineage development in the thymus. We previously revealed that PU.1 delineates heterogeneity of effector Th2 populations. In this study, we further define the function of PU.1 on the Th2 phenotype using mice that specifically lack PU.1 in T cells using an lck-Cre transgene with a conditional Sfpi1 allele (Sfpi1lck-/-). While deletion of PU.1 by the lck-Cre transgene does not affect T cell development, Sfpi1lck-/- T cells have a lower activation threshold than wild type T cells. When TCR engagement is limiting, Sfpi1lck-/- T cells cultured in Th2 polarizing conditions secrete higher levels of Th2 cytokines and have greater cytokine homogeneity than wild type cells. We show that PU.1 modulates the levels of TCR expression in CD4+ T cells by regulating the DNA-binding activity of GATA-3 and limiting GATA-3 regulation of TCR gene expression. GATA-3 dependent regulation of TCR expression is also observed in Th1 and Th2 cells. In CD4+ T cells, PU.1 expression segregates into subpopulations of cells that have lower levels of surface TCR, suggesting that PU.1 contributes to the heterogeneity of TCR expression. Thus, we have identified a mechanism whereby increased GATA-3 function in the absence of the antagonizing activity of PU.1 leads to increased TCR expression, a reduced activation threshold and increased homogeneity in Th2 populations. PMID:19801513

Molecular analysis of human papillomavirus virus-like particle activated Langerhans cells in vitro.

PubMed

Woodham, Andrew W; Raff, Adam B; Da Silva, Diane M; Kast, W Martin

2015-01-01

Langerhans cells (LC) are the resident antigen-presenting cells in human epithelium, and are therefore responsible for initiating immune responses against human papillomaviruses (HPV) entering the epithelial and mucosal layers in vivo. Upon proper pathogenic stimulation, LC become activated causing an internal signaling cascade that results in the up-regulation of co-stimulatory molecules and the release of inflammatory cytokines. Activated LC then migrate to lymph nodes where they interact with antigen-specific T cells and initiate an adaptive T-cell response. However, HPV manipulates LC in a suppressive manner that alters these normal maturation responses. Here, in vitro LC activation assays for the detection of phosphorylated signaling intermediates, the up-regulation of activation-associated surface markers, and the release of inflammatory cytokines in response to HPV particles are described.

The role of a cell surface inhibitor in early signal transduction associated with the regulation of cell division and differentiation

NASA Technical Reports Server (NTRS)

Johnson, T. C.; Enebo, D. J.; Moos, P. J.; Fattaey, H. K.; Spooner, B. S. (Principal Investigator)

1992-01-01

Serum stimulation of quiescent human fibroblast cultures resulted in a hyperphosphorylation of the nuclear retinoblastoma gene susceptibility product (RB). However, serum stimulation in the presence of 9 x 10(-8) M of a purified bovine sialoglycopeptide (SGP) cell surface inhibitor abrogated the hyperphosphorylation of the RB protein and the subsequent progression of cells through the mitotic cycle. The experimental results suggest that the SGP mediated its cell cycle arrest at a site in the cell cycle that was at the time of RB phosphorylation or somewhat upstream of the modification of this regulatory protein of cell division. Both cells serum-deprived and serum stimulated in the presence of the SGP displayed only a hypophosphorylated RB protein, consistent with the SGP-mediated cell cycle arrest point being near the G1/S interface.

A bio-recognition device developed onto nano-crystals of carbonate apatite for cell-targeted gene delivery.

PubMed

Chowdhury, E H; Akaike, Toshihiro

2005-05-20

The DNA delivery to mammalian cells is an essential tool for analyzing gene structure, regulation, and function. The approach holds great promise for the further development of gene therapy techniques and DNA vaccination strategies to treat and control diseases. Here, we report on the establishment of a cell-specific gene delivery and expression system by physical adsorption of a cell-recognition molecule on the nano-crystal surface of carbonate apatite. As a model, DNA/nano-particles were successfully coated with asialofetuin to facilitate uptake by hepatocyte-derived cell lines through the asialoglycoprotein receptor (ASGPr) and albumin to prevent non-specific interactions of the particles with cell-surface. The resulting composite particles with dual surface properties could accelerate DNA uptake and enhance expression to a notable extent. Nano-particles coated with transferrin in the same manner dramatically enhanced transgene expression in the corresponding receptor-bearing cells and thus our newly developed strategy represents a universal phenomenon for anchoring a bio-recognition macromolecule on the apatite crystal surface for targeted gene delivery, having immediate applications in basic research laboratories and great promise for gene therapy. (c) 2005 Wiley Periodicals, Inc.

The role of surface charge density in cationic liposome-promoted dendritic cell maturation and vaccine-induced immune responses

NASA Astrophysics Data System (ADS)

Ma, Yifan; Zhuang, Yan; Xie, Xiaofang; Wang, Ce; Wang, Fei; Zhou, Dongmei; Zeng, Jianqiang; Cai, Lintao

2011-05-01

Cationic liposomes have emerged as a novel adjuvant and antigen delivery system to enhance vaccine efficacy. However, the role of surface charge density in cationic liposome-regulated immune responses has not yet been elucidated. In the present study, we prepared a series of DOTAP/DOPC cationic liposomes with different surface densities by incorporating varying amounts of DOPC (a neutral lipid) into DOTAP (a cationic lipid). The results showed that DOTAP/DOPC cationic liposome-regulated immune responses relied on the surface charge density, and might occur through ROS signaling. The liposomes with a relatively high charge density, such as DOTAP/DOPC 5 : 0 and 4 : 1 liposomes, potently enhanced dendritic cell maturation, ROS generaion, antigen uptake, as well as the production of OVA-specific IgG2a and IFN-γ. In contrast, low-charge liposomes, such as DOTAP/DOPC 1 : 4 liposome, failed to promote immune responses even at high concentrations, confirming that the immunoregulatory effect of cationic liposomes is mostly attributable to their surface charge density. Moreover, the DOTAP/DOPC 1 : 4 liposome suppressed anti-OVA antibody responses in vivo. Overall, maintaining an appropriate surface charge is crucial for optimizing the adjuvant effect of cationic liposomes and enhancing the efficacy of liposome-based vaccines.

Mammalian target of rapamycin signalling modulates amino acid uptake by regulating transporter cell surface abundance in primary human trophoblast cells.

PubMed

Rosario, Fredrick J; Kanai, Yoshikatsu; Powell, Theresa L; Jansson, Thomas

2013-02-01

Abnormal fetal growth increases the risk for perinatal complications and predisposes for the development of obesity, diabetes and cardiovascular disease later in life. Emerging evidence suggests that changes in placental amino acid transport directly contribute to altered fetal growth. However, the molecular mechanisms regulating placental amino acid transport are largely unknown. Here we combined small interfering (si) RNA-mediated silencing approaches with protein expression/localization and functional studies in cultured primary human trophoblast cells to test the hypothesis that mammalian target of rapamycin complex 1 (mTORC1) and 2 (mTORC2) regulate amino acid transporters by post-translational mechanisms. Silencing raptor (inhibits mTORC1) or rictor (inhibits mTORC2) markedly decreased basal System A and System L amino acid transport activity but had no effect on growth factor-stimulated amino acid uptake. Simultaneous inhibition of mTORC1 and 2 completely inhibited both basal and growth factor-stimulated amino acid transport activity. In contrast, mTOR inhibition had no effect on serotonin transport. mTORC1 or mTORC2 silencing markedly decreased the plasma membrane expression of specific System A (SNAT2, SLC38A2) and System L (LAT1, SLC7A5) transporter isoforms without affecting global protein expression. In conclusion, mTORC1 and mTORC2 regulate human trophoblast amino acid transporters by modulating the cell surface abundance of specific transporter isoforms. This is the first report showing regulation of amino acid transport by mTORC2. Because placental mTOR activity and amino acid transport are decreased in human intrauterine growth restriction our data are consistent with the possibility that dysregulation of placental mTOR plays an important role in the development of abnormal fetal growth.

The NOTCH Ligand JAG1 Regulates GDNF Expression in Sertoli Cells

PubMed Central

Garcia, Thomas X.; Parekh, Parag; Gandhi, Pooja; Sinha, Krishna

2017-01-01

In the seminiferous epithelium of the testis, Sertoli cells are key niche cells directing proliferation and differentiation of spermatogonial stem cells (SSCs) into spermatozoa. Sertoli cells produce glial cell line-derived neurotrophic factor (GDNF), which is essential for SSC self-renewal and progenitor expansion. While the role of GDNF in the testis stem cell niche is established, little is known about how this factor is regulated. Our previous studies on NOTCH activity in Sertoli cells demonstrated a role of this pathway in limiting stem/progenitor cell numbers, thus ultimately downregulating sperm cell output. In this study we demonstrate through a double-mutant mouse model that NOTCH signaling in Sertoli cells functions solely through the canonical pathway. Further, we demonstrate through Dual luciferase assay and chromatin immunoprecipitation quantitative polymerase chain reaction (ChIP-qPCR) analysis that the NOTCH targets HES1 and HEY1, which are transcriptional repressors, directly downregulate GDNF expression by binding to the Gdnf promoter, thus antagonizing the effects of FSH/cAMP. Finally, we demonstrate that testicular stem/progenitors cells are activating NOTCH signaling in Sertoli cells in vivo and in vitro through the NOTCH ligand JAG1 at their surface, indicating that these cells may ensure their own homeostasis through negative feedback regulation. PMID:28051360

Molecular targets and signaling pathways regulated by nuclear translocation of syndecan-1.

PubMed

Szatmári, Tünde; Mundt, Filip; Kumar-Singh, Ashish; Möbus, Lena; Ötvös, Rita; Hjerpe, Anders; Dobra, Katalin

2017-12-08

The cell-surface heparan sulfate proteoglycan syndecan-1 is important for tumor cell proliferation, migration, and cell cycle regulation in a broad spectrum of malignancies. Syndecan-1, however, also translocates to the cell nucleus, where it might regulate various molecular functions. We used a fibrosarcoma model to dissect the functions of syndecan-1 related to the nucleus and separate them from functions related to the cell-surface. Nuclear translocation of syndecan-1 hampered the proliferation of fibrosarcoma cells compared to the mutant lacking nuclear localization signal. The growth inhibitory effect of nuclear syndecan-1 was accompanied by significant accumulation of cells in the G0/G1 phase, which indicated a possible G1/S phase arrest. We implemented multiple, unsupervised global transcriptome and proteome profiling approaches and combined them with functional assays to disclose the molecular mechanisms that governed nuclear translocation and its related functions. We identified genes and pathways related to the nuclear compartment with network enrichment analysis of the transcriptome and proteome. The TGF-β pathway was activated by nuclear syndecan-1, and three genes were significantly altered with the deletion of nuclear localization signal: EGR-1 (early growth response 1), NEK11 (never-in-mitosis gene a-related kinase 11), and DOCK8 (dedicator of cytokinesis 8). These candidate genes were coupled to growth and cell-cycle regulation. Nuclear translocation of syndecan-1 influenced the activity of several other transcription factors, including E2F, NFκβ, and OCT-1. The transcripts and proteins affected by syndecan-1 showed a striking overlap in their corresponding biological processes. These processes were dominated by protein phosphorylation and post-translation modifications, indicative of alterations in intracellular signaling. In addition, we identified molecules involved in the known functions of syndecan-1, including extracellular matrix organization and transmembrane transport. Collectively, abrogation of nuclear translocation of syndecan-1 resulted in a set of changes clustering in distinct patterns, which highlighted the functional importance of nuclear syndecan-1 in hampering cell proliferation and the cell cycle. This study emphasizes the importance of the localization of syndecan-1 when considering its effects on tumor cell fate.

Role of ER Export Signals in Controlling Surface Potassium Channel Numbers

NASA Astrophysics Data System (ADS)

Ma, Dzwokai; Zerangue, Noa; Lin, Yu-Fung; Collins, Anthony; Yu, Mei; Jan, Yuh Nung; Yeh Jan, Lily

2001-01-01

Little is known about the identity of endoplasmic reticulum (ER) export signals and how they are used to regulate the number of proteins on the cell surface. Here, we describe two ER export signals that profoundly altered the steady-state distribution of potassium channels and were required for channel localization to the plasma membrane. When transferred to other potassium channels or a G protein-coupled receptor, these ER export signals increased the number of functional proteins on the cell surface. Thus, ER export of membrane proteins is not necessarily limited by folding or assembly, but may be under the control of specific export signals.

The Plasma Membrane Sialidase NEU3 Regulates the Malignancy of Renal Carcinoma Cells by Controlling β1 Integrin Internalization and Recycling*

PubMed Central

Tringali, Cristina; Lupo, Barbara; Silvestri, Ilaria; Papini, Nadia; Anastasia, Luigi; Tettamanti, Guido; Venerando, Bruno

2012-01-01

The human plasma membrane sialidase NEU3 is a key enzyme in the catabolism of membrane gangliosides, is crucial in the regulation of cell surface processes, and has been demonstrated to be significantly up-regulated in renal cell carcinomas (RCCs). In this report, we show that NEU3 regulates β1 integrin trafficking in RCC cells by controlling β1 integrin recycling to the plasma membrane and controlling activation of the epidermal growth factor receptor (EGFR) and focal adhesion kinase (FAK)/protein kinase B (AKT) signaling. NEU3 silencing in RCC cells increased the membrane ganglioside content, in particular the GD1a content, and changed the expression of key regulators of the integrin recycling pathway. In addition, NEU3 silencing up-regulated the Ras-related protein RAB25, which directs internalized integrins to lysosomes, and down-regulated the chloride intracellular channel protein 3 (CLIC3), which induces the recycling of internalized integrins to the plasma membrane. In this manner, NEU3 silencing enhanced the caveolar endocytosis of β1 integrin, blocked its recycling and reduced its levels at the plasma membrane, and, consequently, inhibited EGFR and FAK/AKT. These events had the following effects on the behavior of RCC cells: they (a) decreased drug resistance mediated by the block of autophagy and the induction of apoptosis; (b) decreased metastatic potential mediated by down-regulation of the metalloproteinases MMP1 and MMP7; and (c) decreased adhesion to collagen and fibronectin. Therefore, our data identify NEU3 as a key regulator of the β1 integrin-recycling pathway and FAK/AKT signaling and demonstrate its crucial role in RCC malignancy. PMID:23139422

Plant cell surface receptor-mediated signaling - a common theme amid diversity.

PubMed

He, Yunxia; Zhou, Jinggeng; Shan, Libo; Meng, Xiangzong

2018-01-29

Sessile plants employ a diverse array of plasma membrane-bound receptors to perceive endogenous and exogenous signals for regulation of plant growth, development and immunity. These cell surface receptors include receptor-like kinases (RLKs) and receptor-like proteins (RLPs) that harbor different extracellular domains for perception of distinct ligands. Several RLK and RLP signaling pathways converge at the somatic embryogenesis receptor kinases (SERKs), which function as shared co-receptors. A repertoire of receptor-like cytoplasmic kinases (RLCKs) associate with the receptor complexes to relay intracellular signaling. Downstream of the receptor complexes, mitogen-activated protein kinase (MAPK) cascades are among the key signaling modules at which the signals converge, and these cascades regulate diverse cellular and physiological responses through phosphorylation of different downstream substrates. In this Review, we summarize the emerging common theme that underlies cell surface receptor-mediated signaling pathways in Arabidopsis thaliana : the dynamic association of RLKs and RLPs with specific co-receptors and RLCKs for signal transduction. We further discuss how signaling specificities are maintained through modules at which signals converge, with a focus on SERK-mediated receptor signaling. © 2018. Published by The Company of Biologists Ltd.

Mitochondrial F1Fo-ATP synthase translocates to cell surface in hepatocytes and has high activity in tumor-like acidic and hypoxic environment.

PubMed

Ma, Zhan; Cao, Manlin; Liu, Yiwen; He, Yiqing; Wang, Yingzhi; Yang, Cuixia; Wang, Wenjuan; Du, Yan; Zhou, Muqing; Gao, Feng

2010-08-01

F1Fo-ATP synthase was originally thought to exclusively locate in the inner membrane of the mitochondria. However, recent studies prove the existence of ectopic F1Fo-ATP synthase on the outside of the cell membrane. Ectopic ATP synthase was proposed as a marker for tumor target therapy. Nevertheless, the protein transport mechanism of the ectopic ATP synthase is still unclear. The specificity of the ectopic ATP synthase, with regard to tumors, is questioned because of its widespread expression. In the current study, we constructed green fluorescent protein-ATP5B fusion protein and introduced it into HepG2 cells to study the localization of the ATP synthase. The expression of ATP5B was analyzed in six cell lines with different 'malignancies'. These cells were cultured in both normal and tumor-like acidic and hypoxic conditions. The results suggested that the ectopic expression of ATP synthase is a consequence of translocation from the mitochondria. The expression and catalytic activity of ectopic ATP synthase were similar on the surface of malignant cells as on the surface of less malignant cells. Interestingly, the expression of ectopic ATP synthase was not up-regulated in tumor-like acidic and hypoxic microenvironments. However, the catalytic activity of ectopic ATP synthase was up-regulated in tumor-like microenvironments. Therefore, the specificity of ectopic ATP synthase for tumor target therapy relies on the high level of catalytic activity that is observed in acidic and hypoxic microenvironments in tumor tissues.

Cutting Edge: Active TGF-β1 Released from GARP/TGF-β1 Complexes on the Surface of Stimulated Human B Lymphocytes Increases Class-Switch Recombination and Production of IgA.

PubMed

Dedobbeleer, Olivier; Stockis, Julie; van der Woning, Bas; Coulie, Pierre G; Lucas, Sophie

2017-07-15

Production of active TGF-β is regulated at a posttranslational level and implies release of the mature cytokine dimer from the inactive, latent TGF-β precursor. There are several cell-type specific mechanisms of TGF-β activation. We identified a new mechanism operating on the surface of human regulatory T cells and involving membrane protein GARP, which binds latent TGF-β1. The paracrine activity of regulatory T cell-derived TGF-β1 contributes to immunosuppression and can be inhibited with anti-GARP Abs. Whether other immune cell types use surface GARP to activate latent TGF-β1 was not known. We show in this study that stimulated, human B lymphocytes produce active TGF-β1 from surface GARP/latent TGF-β1 complexes with isotype switching to IgA production. Copyright © 2017 by The American Association of Immunologists, Inc.

Fine Tuning of Tissues' Viscosity and Surface Tension through Contractility Suggests a New Role for α-Catenin

PubMed Central

Stirbat, Tomita Vasilica; Mgharbel, Abbas; Bodennec, Selena; Ferri, Karine; Mertani, Hichem C.; Rieu, Jean-Paul; Delanoë-Ayari, Hélène

2013-01-01

What governs tissue organization and movement? If molecular and genetic approaches are able to give some answers on these issues, more and more works are now giving a real importance to mechanics as a key component eventually triggering further signaling events. We chose embryonic cell aggregates as model systems for tissue organization and movement in order to investigate the origin of some mechanical constraints arising from cells organization. Steinberg et al. proposed a long time ago an analogy between liquids and tissues and showed that indeed tissues possess a measurable tissue surface tension and viscosity. We question here the molecular origin of these parameters and give a quantitative measurement of adhesion versus contractility in the framework of the differential interfacial tension hypothesis. Accompanying surface tension measurements by angle measurements (at vertexes of cell-cell contacts) at the cell/medium interface, we are able to extract the full parameters of this model: cortical tensions and adhesion energy. We show that a tunable surface tension and viscosity can be achieved easily through the control of cell-cell contractility compared to cell-medium one. Moreover we show that -catenin is crucial for this regulation to occur: these molecules appear as a catalyser for the remodeling of the actin cytoskeleton underneath cell-cell contact, enabling a differential contractility between the cell-medium and cell-cell interface to take place. PMID:23390488

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria

PubMed Central

Maria, Zahra; Campolo, Allison R.; Lacombe, Veronique A.

2015-01-01

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes. PMID:26720696

Diabetes Alters the Expression and Translocation of the Insulin-Sensitive Glucose Transporters 4 and 8 in the Atria.

PubMed

Maria, Zahra; Campolo, Allison R; Lacombe, Veronique A

2015-01-01

Although diabetes has been identified as a major risk factor for atrial fibrillation, little is known about glucose metabolism in the healthy and diabetic atria. Glucose transport into the cell, the rate-limiting step of glucose utilization, is regulated by the Glucose Transporters (GLUTs). Although GLUT4 is the major isoform in the heart, GLUT8 has recently emerged as a novel cardiac isoform. We hypothesized that GLUT-4 and -8 translocation to the atrial cell surface will be regulated by insulin and impaired during insulin-dependent diabetes. GLUT protein content was measured by Western blotting in healthy cardiac myocytes and type 1 (streptozotocin-induced, T1Dx) diabetic rodents. Active cell surface GLUT content was measured using a biotinylated photolabeled assay in the perfused heart. In the healthy atria, insulin stimulation increased both GLUT-4 and -8 translocation to the cell surface (by 100% and 240%, respectively, P<0.05). Upon insulin stimulation, we reported an increase in Akt (Th308 and s473 sites) and AS160 phosphorylation, which was positively (P<0.05) correlated with GLUT4 protein content in the healthy atria. During diabetes, active cell surface GLUT-4 and -8 content was downregulated in the atria (by 70% and 90%, respectively, P<0.05). Akt and AS160 phosphorylation was not impaired in the diabetic atria, suggesting the presence of an intact insulin signaling pathway. This was confirmed by the rescued translocation of GLUT-4 and -8 to the atrial cell surface upon insulin stimulation in the atria of type 1 diabetic subjects. In conclusion, our data suggest that: 1) both GLUT-4 and -8 are insulin-sensitive in the healthy atria through an Akt/AS160 dependent pathway; 2) GLUT-4 and -8 trafficking is impaired in the diabetic atria and rescued by insulin treatment. Alterations in atrial glucose transport may induce perturbations in energy production, which may provide a metabolic substrate for atrial fibrillation during diabetes.

Lactate signalling regulates fungal β-glucan masking and immune evasion

PubMed Central

Ballou, Elizabeth R.; Avelar, Gabriela M.; Childers, Delma S.; Mackie, Joanna; Bain, Judith M.; Wagener, Jeanette; Kastora, Stavroula L.; Panea, Mirela D.; Hardison, Sarah E.; Walker, Louise A.; Erwig, Lars P.; Munro, Carol A.; Gow, Neil A.R.; Brown, Gordon D.; MacCallum, Donna M.; Brown, Alistair J.P.

2017-01-01

Summary Paragraph As they proliferate, fungi expose antigens at their cell surface that are potent stimulators of the innate immune response, and yet the commensal fungus Candida albicans is able to colonize immuno-competent individuals. We show that C. albicans may evade immune detection by presenting a moving immunological target. We report that the exposure of β-glucan, a key Pathogen Associated Molecular Pattern (PAMP) located at the cell surface of C. albicans and other pathogenic Candida species, is modulated in response to changes in carbon source. Exposure to lactate induces β-glucan masking in C. albicans via a signaling pathway that has recruited an evolutionarily conserved receptor (Gpr1) and transcriptional factor (Crz1) from other well-characterized pathways. In response to lactate, these regulators control the expression of cell wall related genes that contribute to β-glucan masking. This represents the first description of active PAMP masking by a Candida species, a process that reduces the visibility of the fungus to the immune system. PMID:27941860

Lipoxin A4 Counter-regulates Histamine-stimulated Glycoconjugate Secretion in Conjunctival Goblet Cells.

PubMed

Hodges, Robin R; Li, Dayu; Shatos, Marie A; Serhan, Charles N; Dartt, Darlene A

2016-11-08

Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A 4 (LXA 4 ), are produced during inflammation returning the tissue to homeostasis and are also produced in non-inflamed tissues. The purpose of this study was to determine the actions of LXA 4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca 2+ ] ([Ca 2+ ] i ) and on histamine-stimulated responses. LXA 4 increased mucin secretion and [Ca 2+ ] i , and activated ERK1/2 in human goblet cells. Addition of LXA 4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA 4 responses. LXA 4 inhibited histamine-stimulated increases in mucin secretion, [Ca 2+ ] i , and ERK1/2 activation through activation of β-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA 4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases.

Lipoxin A4 Counter-regulates Histamine-stimulated Glycoconjugate Secretion in Conjunctival Goblet Cells

PubMed Central

Hodges, Robin R.; Li, Dayu; Shatos, Marie A.; Serhan, Charles N.; Dartt, Darlene A.

2016-01-01

Conjunctival goblet cells synthesize and secrete mucins which play an important role in protecting the ocular surface. Pro-resolution mediators, such as lipoxin A4 (LXA4), are produced during inflammation returning the tissue to homeostasis and are also produced in non-inflamed tissues. The purpose of this study was to determine the actions of LXA4 on cultured human conjunctival goblet cell mucin secretion and increase in intracellular [Ca2+] ([Ca2+]i) and on histamine-stimulated responses. LXA4 increased mucin secretion and [Ca2+]i, and activated ERK1/2 in human goblet cells. Addition of LXA4 before resolvin D1 (RvD1) decreased RvD1 responses though RvD1 did not block LXA4 responses. LXA4 inhibited histamine-stimulated increases in mucin secretion, [Ca2+]i, and ERK1/2 activation through activation of β-adrenergic receptor kinase 1. We conclude that conjunctival goblet cells respond to LXA4 through the ALX/FPR2 receptor to maintain homeostasis of the ocular surface and regulate histamine responses and could provide a new therapeutic approach for allergic conjunctivitis and dry eye diseases. PMID:27824117

CXCR4-SDF-1 signalling, locomotion, chemotaxis and adhesion.

PubMed

Kucia, Magda; Jankowski, Kacper; Reca, Ryan; Wysoczynski, Marcin; Bandura, Laura; Allendorf, Daniel J; Zhang, Jin; Ratajczak, Janina; Ratajczak, Mariusz Z

2004-03-01

Chemokines, small pro-inflammatory chemoattractant cytokines, that bind to specific G-protein-coupled seven-span transmembrane receptors present on plasma membranes of target cells are the major regulators of cell trafficking. In addition some chemokines have been reported to modulate cell survival and growth. Moreover, compelling evidence is accumulating that cancer cells may employ several mechanisms involving chemokine-chemokine receptor axes during their metastasis that also regulate the trafficking of normal cells. Of all the chemokines, stromal-derived factor-1 (SDF-1), an alpha-chemokine that binds to G-protein-coupled CXCR4, plays an important and unique role in the regulation of stem/progenitor cell trafficking. First, SDF-1 regulates the trafficking of CXCR4+ haemato/lymphopoietic cells, their homing/retention in major haemato/lymphopoietic organs and accumulation of CXCR4+ immune cells in tissues affected by inflammation. Second, CXCR4 plays an essential role in the trafficking of other tissue/organ specific stem/progenitor cells expressing CXCR4 on their surface, e.g., during embryo/organogenesis and tissue/organ regeneration. Third, since CXCR4 is expressed on several tumour cells, these CXCR4 positive tumour cells may metastasize to the organs that secrete/express SDF-1 (e.g., bones, lymph nodes, lung and liver). SDF-1 exerts pleiotropic effects regulating processes essential to tumour metastasis such as locomotion of malignant cells, their chemoattraction and adhesion, as well as plays an important role in tumour vascularization. This implies that new therapeutic strategies aimed at blocking the SDF-1-CXCR4 axis could have important applications in the clinic by modulating the trafficking of haemato/lymphopoietic cells and inhibiting the metastatic behaviour of tumour cells as well. In this review, we focus on a role of the SDF-1-CXCR4 axis in regulating the metastatic behaviour of tumour cells and discuss the molecular mechanisms that are essential to this process.

Biofunctionalization of a titanium surface with a nano-sawtooth structure regulates the behavior of rat bone marrow mesenchymal stem cells

PubMed Central

Zhang, Wenjie; Li, Zihui; Liu, Yan; Ye, Dongxia; Li, Jinhua; Xu, Lianyi; Wei, Bin; Zhang, Xiuli; Liu, Xuanyong; Jiang, Xinquan

2012-01-01

Background: The topography of an implant surface can serve as a powerful signaling cue for attached cells and can enhance the quality of osseointegration. A series of improved implant surfaces functionalized with nanoscale structures have been fabricated using various methods. Methods: In this study, using an H2O2 process, we fabricated two size-controllable sawtooth-like nanostructures with different dimensions on a titanium surface. The effects of the two nano-sawtooth structures on rat bone marrow mesenchymal stem cells (BMMSCs) were evaluated without the addition of osteoinductive chemical factors. Results: These new surface modifications did not adversely affect cell viability, and rat BMMSCs demonstrated a greater increase in proliferation ability on the surfaces of the nano-sawtooth structures than on a control plate. Furthermore, upregulated expression of osteogenic-related genes and proteins indicated that the nano-sawtooth structures promote osteoblastic differentiation of rat BMMSCs. Importantly, the large nano-sawtooth structure resulted in the greatest cell responses, including increased adhesion, proliferation, and differentiation. Conclusion: The enhanced adhesion, proliferation, and osteogenic differentiation abilities of rat BMMSCs on the nano-sawtooth structures suggest the potential to induce improvements in bone-titanium integration in vivo. Our study reveals the key role played by the nano-sawtooth structures on a titanium surface for the fate of rat BMMSCs and provides insights into the study of stem cell-nanostructure relationships and the related design of improved biomedical implant surfaces. PMID:22927760

Local Inflammatory Cues Regulate Differentiation and Persistence of CD8+ Tissue-Resident Memory T Cells.

PubMed

Bergsbaken, Tessa; Bevan, Michael J; Fink, Pamela J

2017-04-04

Many pathogens initiate infection at mucosal surfaces, and tissue-resident memory T (Trm) cells play an important role in protective immunity, yet the tissue-specific signals that regulate Trm differentiation are poorly defined. During Yersinia infection, CD8 + T cell recruitment to areas of inflammation within the intestine is required for differentiation of the CD103 - CD69 + Trm subset. Intestinal proinflammatory microenvironments have elevated interferon (IFN)-β and interleukin-12 (IL-12), which regulated Trm markers, including CD103. Type I interferon-receptor- or IL-12-receptor-deficient T cells functioned similarly to wild-type (WT) cells during infection; however, the inability of T cells to respond to inflammation resulted in defective differentiation of CD103 - CD69 + Trm cells and reduced Trm persistence. Intestinal macrophages were the main producers of IFN-β and IL-12 during infection, and deletion of CCR2 + IL-12-producing cells reduced the size of the CD103 - Trm population. These data indicate that intestinal inflammation drives phenotypic diversity and abundance of Trm cells for optimal tissue-specific immunity. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

The Role of Titanium Surface Microtopography on Adhesion, Proliferation, Transformation, and Matrix Deposition of Corneal Cells.

PubMed

Zhou, Chengxin; Lei, Fengyang; Chodosh, James; Paschalis, Eleftherios I

2016-04-01

Titanium (Ti) is an excellent implantable biomaterial that can be further enhanced by surface topography optimization. Despite numerous data from orthopedics and dentistry, the effect of Ti surface topography on ocular cells is still poorly understood. In light of the recent adaptation of Ti in the Boston Keratoprosthesis artificial cornea, we attempted to perform an extended evaluation of the effect of Ti surface topography on corneal cell adhesion, proliferation, cytotoxicity, transformation, and matrix deposition. Different surface topographies were generated on medical grade Ti-6Al-4V-ELI (extra-low interstitial), with linearly increased roughness (polished to grit blasted). Biological response was evaluated in vitro using human corneal limbal epithelial (HCLE) cells, stromal fibroblasts (HCF), and endothelial cells (HCEnC). None of the Ti surface topographies caused cytotoxicity to any of the three corneal cell types. However, rough Ti surface inhibited HCLE and HCF cell adhesion and proliferation, while HCEnC proliferation was unaffected. Long-term experiments with HCF revealed that rough Ti surface with R(a) (the arithmetic average of the profile height from the mean line) ≥ 1.15 μm suppressed HCF focal adhesion kinase phosphorylation, changed fibroblast morphology, and caused less aligned and reduced deposition of collagen matrix as compared to smooth Ti (R(a) ≤ 0.08 μm). In the presence of transforming growth factor β1 (TGFβ1) stimulation, rough Ti inhibited alpha-smooth muscle actin (α-SMA) expression and collagen deposition, leading to decreased myofibroblast transformation and disorganization of the collagen fibrils as compared to smooth Ti. This study suggests that Ti surface topography regulates corneal cell behavior in a tissue-dependent manner that varies across the corneal strata. Contrary to the accepted paradigm, smooth surface topography can enhance cell adhesion and proliferation and increase matrix deposition by corneal cells.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells.

PubMed

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-06-21

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype.

Cellular prion protein controls stem cell-like properties of human glioblastoma tumor-initiating cells

PubMed Central

Corsaro, Alessandro; Bajetto, Adriana; Thellung, Stefano; Begani, Giulia; Villa, Valentina; Nizzari, Mario; Pattarozzi, Alessandra; Solari, Agnese; Gatti, Monica; Pagano, Aldo; Würth, Roberto; Daga, Antonio; Barbieri, Federica; Florio, Tullio

2016-01-01

Prion protein (PrPC) is a cell surface glycoprotein whose misfolding is responsible for prion diseases. Although its physiological role is not completely defined, several lines of evidence propose that PrPC is involved in self-renewal, pluripotency gene expression, proliferation and differentiation of neural stem cells. Moreover, PrPC regulates different biological functions in human tumors, including glioblastoma (GBM). We analyzed the role of PrPC in GBM cell pathogenicity focusing on tumor-initiating cells (TICs, or cancer stem cells, CSCs), the subpopulation responsible for development, progression and recurrence of most malignancies. Analyzing four GBM CSC-enriched cultures, we show that PrPC expression is directly correlated with the proliferation rate of the cells. To better define its role in CSC biology, we knocked-down PrPC expression in two of these GBM-derived CSC cultures by specific lentiviral-delivered shRNAs. We provide evidence that CSC proliferation rate, spherogenesis and in vivo tumorigenicity are significantly inhibited in PrPC down-regulated cells. Moreover, PrPC down-regulation caused loss of expression of the stemness and self-renewal markers (NANOG, Sox2) and the activation of differentiation pathways (i.e. increased GFAP expression). Our results suggest that PrPC controls the stemness properties of human GBM CSCs and that its down-regulation induces the acquisition of a more differentiated and less oncogenic phenotype. PMID:27229535

Directed Evolution to Engineer Monobody for FRET Biosensor Assembly and Imaging at Live-Cell Surface.

PubMed

Limsakul, Praopim; Peng, Qin; Wu, Yiqian; Allen, Molly E; Liang, Jing; Remacle, Albert G; Lopez, Tyler; Ge, Xin; Kay, Brian K; Zhao, Huimin; Strongin, Alex Y; Yang, Xiang-Lei; Lu, Shaoying; Wang, Yingxiao

2018-04-19

Monitoring enzymatic activities at the cell surface is challenging due to the poor efficiency of transport and membrane integration of fluorescence resonance energy transfer (FRET)-based biosensors. Therefore, we developed a hybrid biosensor with separate donor and acceptor that assemble in situ. The directed evolution and sequence-function analysis technologies were integrated to engineer a monobody variant (PEbody) that binds to R-phycoerythrin (R-PE) dye. PEbody was used for visualizing the dynamic formation/separation of intercellular junctions. We further fused PEbody with the enhanced CFP and an enzyme-specific peptide at the extracellular surface to create a hybrid FRET biosensor upon R-PE capture for monitoring membrane-type-1 matrix metalloproteinase (MT1-MMP) activities. This biosensor revealed asymmetric distribution of MT1-MMP activities, which were high and low at loose and stable cell-cell contacts, respectively. Therefore, directed evolution and rational design are promising tools to engineer molecular binders and hybrid FRET biosensors for monitoring molecular regulations at the surface of living cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Secreted CLCA1 modulates TMEM16A to activate Ca(2+)-dependent chloride currents in human cells.

PubMed

Sala-Rabanal, Monica; Yurtsever, Zeynep; Nichols, Colin G; Brett, Tom J

2015-03-17

Calcium-activated chloride channel regulator 1 (CLCA1) activates calcium-dependent chloride currents; neither the target, nor mechanism, is known. We demonstrate that secreted CLCA1 activates calcium-dependent chloride currents in HEK293T cells in a paracrine fashion, and endogenous TMEM16A/Anoctamin1 conducts the currents. Exposure to exogenous CLCA1 increases cell surface levels of TMEM16A and cellular binding experiments indicate CLCA1 engages TMEM16A on the surface of these cells. Altogether, our data suggest that CLCA1 stabilizes TMEM16A on the cell surface, thus increasing surface expression, which results in increased calcium-dependent chloride currents. Our results identify the first Cl(-) channel target of the CLCA family of proteins and establish CLCA1 as the first secreted direct modifier of TMEM16A activity, delineating a unique mechanism to increase currents. These results suggest cooperative roles for CLCA and TMEM16 proteins in influencing the physiology of multiple tissues, and the pathology of multiple diseases, including asthma, COPD, cystic fibrosis, and certain cancers.

Dendritic Cells: A Spot on Sialic Acid

PubMed Central

Crespo, Hélio J.; Lau, Joseph T. Y.; Videira, Paula A.

2013-01-01

Glycans decorating cell surface and secreted proteins and lipids occupy the juncture where critical host–host and host-pathogen interactions occur. The role of glycan epitopes in cell–cell and cell-pathogen adhesive events is already well-established, and cell surface glycan structures change rapidly in response to stimulus and inflammatory cues. Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how glycans and their changes contribute to the overall immune response remains poorly defined. Sialic acids are unique sugars that usually occupy the terminal position of the glycan chains and may be modified by external factors, such as pathogens, or upon specific physiological cellular events. At cell surface, sialic acid-modified structures form the key fundamental determinants for a number of receptors with known involvement in cellular adhesiveness and cell trafficking, such as the Selectins and the Siglec families of carbohydrate recognizing receptors. Dendritic cells (DCs) preside over the transition from innate to the adaptive immune repertoires, and no other cell has such relevant role in antigen screening, uptake, and its presentation to lymphocytes, ultimately triggering the adaptive immune response. Interestingly, sialic acid-modified structures are involved in all DC functions, such as antigen uptake, DC migration, and capacity to prime T cell responses. Sialic acid content changes along DC differentiation and activation and, while, not yet fully understood, these changes have important implications in DC functions. This review focuses on the developmental regulation of DC surface sialic acids and how manipulation of DC surface sialic acids can affect immune-critical DC functions by altering antigen endocytosis, pathogen and tumor cell recognition, cell recruitment, and capacity for T cell priming. The existing evidence points to a potential of DC surface sialylation as a therapeutic target to improve and diversify DC-based therapies. PMID:24409183

Brain-derived neurotrophic factor (BDNF) induces sustained intracellular Ca2+ elevation through the up-regulation of surface transient receptor potential 3 (TRPC3) channels in rodent microglia.

PubMed

Mizoguchi, Yoshito; Kato, Takahiro A; Seki, Yoshihiro; Ohgidani, Masahiro; Sagata, Noriaki; Horikawa, Hideki; Yamauchi, Yusuke; Sato-Kasai, Mina; Hayakawa, Kohei; Inoue, Ryuji; Kanba, Shigenobu; Monji, Akira

2014-06-27

Microglia are immune cells that release factors, including proinflammatory cytokines, nitric oxide (NO), and neurotrophins, following activation after disturbance in the brain. Elevation of intracellular Ca(2+) concentration ([Ca(2+)]i) is important for microglial functions such as the release of cytokines and NO from activated microglia. There is increasing evidence suggesting that pathophysiology of neuropsychiatric disorders is related to the inflammatory responses mediated by microglia. Brain-derived neurotrophic factor (BDNF) is a neurotrophin well known for its roles in the activation of microglia as well as in pathophysiology and/or treatment of neuropsychiatric disorders. In this study, we sought to examine the underlying mechanism of BDNF-induced sustained increase in [Ca(2+)]i in rodent microglial cells. We observed that canonical transient receptor potential 3 (TRPC3) channels contribute to the maintenance of BDNF-induced sustained intracellular Ca(2+) elevation. Immunocytochemical technique and flow cytometry also revealed that BDNF rapidly up-regulated the surface expression of TRPC3 channels in rodent microglial cells. In addition, pretreatment with BDNF suppressed the production of NO induced by tumor necrosis factor α (TNFα), which was prevented by co-adiministration of a selective TRPC3 inhibitor. These suggest that BDNF induces sustained intracellular Ca(2+) elevation through the up-regulation of surface TRPC3 channels and TRPC3 channels could be important for the BDNF-induced suppression of the NO production in activated microglia. We show that TRPC3 channels could also play important roles in microglial functions, which might be important for the regulation of inflammatory responses and may also be involved in the pathophysiology and/or the treatment of neuropsychiatric disorders. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Ubiquitination of basal VEGFR2 regulates signal transduction and endothelial function

PubMed Central

Smith, Gina A.; Fearnley, Gareth W.; Abdul-Zani, Izma; Wheatcroft, Stephen B.; Tomlinson, Darren C.; Harrison, Michael A.

2017-01-01

ABSTRACT Cell surface receptors can undergo recycling or proteolysis but the cellular decision-making events that sort between these pathways remain poorly defined. Vascular endothelial growth factor A (VEGF-A) and vascular endothelial growth factor receptor 2 (VEGFR2) regulate signal transduction and angiogenesis, but how signaling and proteolysis is regulated is not well understood. Here, we provide evidence that a pathway requiring the E1 ubiquitin-activating enzyme UBA1 controls basal VEGFR2 levels, hence metering plasma membrane receptor availability for the VEGF-A-regulated endothelial cell response. VEGFR2 undergoes VEGF-A-independent constitutive degradation via a UBA1-dependent ubiquitin-linked pathway. Depletion of UBA1 increased VEGFR2 recycling from endosome-to-plasma membrane and decreased proteolysis. Increased membrane receptor availability after UBA1 depletion elevated VEGF-A-stimulated activation of key signaling enzymes such as PLCγ1 and ERK1/2. Although UBA1 depletion caused an overall decrease in endothelial cell proliferation, surviving cells showed greater VEGF-A-stimulated responses such as cell migration and tubulogenesis. Our study now suggests that a ubiquitin-linked pathway regulates the balance between receptor recycling and degradation which in turn impacts on the intensity and duration of VEGF-A-stimulated signal transduction and the endothelial response. PMID:28798148

Control of proliferation and osteogenic differentiation of human dental-pulp-derived stem cells by distinct surface structures.

PubMed

Kolind, K; Kraft, D; Bøggild, T; Duch, M; Lovmand, J; Pedersen, F S; Bindslev, D A; Bünger, C E; Foss, M; Besenbacher, F

2014-02-01

The ability to control the behavior of stem cells provides crucial benefits, for example, in tissue engineering and toxicity/drug screening, which utilize the stem cell's capacity to engineer new tissues for regenerative purposes and the testing of new drugs in vitro. Recently, surface topography has been shown to influence stem cell differentiation; however, general trends are often difficult to establish due to differences in length scales, surface chemistries and detailed surface topographies. Here we apply a highly versatile screening approach to analyze the interplay of surface topographical parameters on cell attachment, morphology, proliferation and osteogenic differentiation of human mesenchymal dental-pulp-derived stem cells (DPSCs) cultured with and without osteogenic differentiation factors in the medium (ODM). Increasing the inter-pillar gap size from 1 to 6 μm for surfaces with small pillar sizes of 1 and 2 μm resulted in decreased proliferation and in more elongated cells with long pseudopodial protrusions. The same alterations of pillar topography, up to an inter-pillar gap size of 4 μm, also resulted in enhanced mineralization of DPSCs cultured without ODM, while no significant trend was observed for DPSCs cultured with ODM. Generally, cells cultured without ODM had a larger deposition of osteogenic markers on structured surfaces relative to the unstructured surfaces than what was found when culturing with ODM. We conclude that the topographical design of biomaterials can be optimized for the regulation of DPSC differentiation and speculate that the inclusion of ODM alters the ability of the cells to sense surface topographical cues. These results are essential in order to transfer the use of this highly proliferative, easily accessible stem cell into the clinic for use in cell therapy and regenerative medicine. Copyright © 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

T-cell receptor revision: friend or foe?

PubMed

Hale, J Scott; Fink, Pamela J

2010-04-01

T-cell receptor (TCR) revision is a process of tolerance induction by which peripheral T cells lose surface expression of an autoreactive TCR, reinduce expression of the recombinase machinery, rearrange genes encoding extrathymically generated TCRs for antigen, and express these new receptors on the cell surface. We discuss the evidence for this controversial tolerance mechanism below. Despite the apparent heresy of post-thymic gene rearrangement, we argue here that TCR revision follows the rules obeyed by maturing thymocytes undergoing gene recombination. Expression of the recombinase is carefully controlled both spatially and temporally, and may be initiated by loss of signals through surface TCRs. The resulting TCR repertoire is characterized by its diversity, self major histocompatibility complex restriction, self tolerance, and ability to mount productive immune responses specific for foreign antigens. Hence, TCR revision is a carefully regulated process of tolerance induction that can contribute to the protection of the individual against invading pathogens while preserving the integrity of self tissue.

Gene regulation by mechanical forces

NASA Technical Reports Server (NTRS)

Oluwole, B. O.; Du, W.; Mills, I.; Sumpio, B. E.

1997-01-01

Endothelial cells are subjected to various mechanical forces in vivo from the flow of blood across the luminal surface of the blood vessel. The purpose of this review was to examine the data available on how these mechanical forces, in particular cyclic strain, affect the expression and regulation of endothelial cell function. Studies from various investigators using models of cyclic strain in vitro have shown that various vasoactive mediators such as nitric oxide and prostacyclin are induced by the effect of mechanical deformation, and that the expression of these mediators may be regulated at the transcription level by mechanical forces. There also seems to be emerging evidence that endothelial cells may also act as mechanotransducers, whereby the transmission of external forces induces various cytoskeletal changes and second messenger cascades. Furthermore, it seems these forces may act on specific response elements of promoter genes.

Submembraneous microtubule cytoskeleton: regulation of microtubule assembly by heterotrimeric G proteins

PubMed Central

Roychowdhury, Sukla; Rasenick, Mark. M

2009-01-01

Heterotrimeric G proteins participate in signal transduction by transferring signals from cell surface receptors to intracellular effector molecules. G proteins also interact with microtubules and participate in microtubule-dependent centrosome/chromosome movement during cell division, as well as neuronal differentiation. In recent years, significant progress has been made in our understanding of the biochemical/functional interactions between G protein subunits (α and βγ) and microtubules, and the molecular details emerging from these studies suggest that α and βγ subunits of G proteins interact with tubulin/microtubules to regulate assembly/dynamics of microtubules, providing a novel mechanism for hormone or neurotransmitter induced rapid remodeling of cytoskeleton, regulation of mitotic spindle for centrosome/chromosome movements in cell division, and neuronal differentiation where structural plasticity mediated by microtubules is important for appropriate synaptic connections and signal transmission. PMID:18754776

Dual Effects of Cell Free Supernatants from Lactobacillus acidophilus and Lactobacillus rhamnosus GG in Regulation of MMP-9 by Up-Regulating TIMP-1 and Down-Regulating CD147 in PMA- Differentiated THP-1 Cells

PubMed Central

Maghsood, Faezeh; Mirshafiey, Abbas; Farahani, Mohadese M.; Modarressi, Mohammad Hossein; Jafari, Parvaneh; Motevaseli, Elahe

2018-01-01

Objective Recent studies have reported dysregulated expression of matrix metalloproteinases (MMPs), especially MMP-2, MMP-9, tissue inhibitor of metalloproteinase-1, -2 (TIMP-1, TIMP-2), and extracellular matrix metalloproteinase inducer (EMMPRIN/CD147) in activated macrophages of patients with inflammatory diseases. Therefore, MMP-2, MMP-9, and their regulators may represent a new target for treatment of inflammatory diseases. Probiotics, which are comprised of lactic acid bacteria, have the potential to modulate inflammatory responses. In this experimental study, we investigated the anti-inflammatory effects of cell-free supernatants (CFS) from Lactobacillus acidophilus (L. acidophilus) and L. rhamnosus GG (LGG) in phorbol myristate acetate (PMA)-differentiated THP-1 cells. Materials and Methods In this experimental study, PMA-differentiated THP-1 cells were treated with CFS from L. acidophilus, LGG and uninoculated bacterial growth media (as a control). The expression of MMP-2, MMP-9, TIMP-1, and TIMP-2 mRNAs were determined using real-time quantitative reverse transcription polymerase chain reaction (RT- PCR). The levels of cellular surface expression of CD147 were assessed by flow cytometry, and the gelatinolytic activity of MMP-2 and MMP-9 were determined by zymography. Results Our results showed that CFS from both L. acidophilus and LGG significantly inhibited the gene expression of MMP-9 (P=0.0011 and P=0.0005, respectively), increased the expression of TIMP-1 (P<0.0001), decreased the cell surface expression of CD147 (P=0.0307 and P=0.0054, respectively), and inhibited the gelatinolytic activity of MMP-9 (P=0.0003 and P<0.0001, respectively) in PMA-differentiated THP-1 cells. Although, MMP-2 expression and activity and TIMP-2 expression remained unchanged. Conclusion Our results indicate that CFS from L. acidophilus and LGG possess anti-inflammatory properties and can modulate the inflammatory response. PMID:29105390

Constrained Adherable Area of Nanotopographic Surfaces Promotes Cell Migration through the Regulation of Focal Adhesion via Focal Adhesion Kinase/Rac1 Activation.

PubMed

Lim, Jiwon; Choi, Andrew; Kim, Hyung Woo; Yoon, Hyungjun; Park, Sang Min; Tsai, Chia-Hung Dylan; Kaneko, Makoto; Kim, Dong Sung

2018-05-02

Cell migration is crucial in physiological and pathological processes such as embryonic development and wound healing; such migration is strongly guided by the surrounding nanostructured extracellular matrix. Previous studies have extensively studied the cell migration on anisotropic nanotopographic surfaces; however, only a few studies have reported cell migration on isotropic nanotopographic surfaces. We herein, for the first time, propose a novel concept of adherable area on cell migration using isotropic nanopore surfaces with sufficient nanopore depth by adopting a high aspect ratio. As the pore size of the nanopore surface was controlled to 200, 300, and 400 nm in a fixed center-to-center distance of 480 nm, it produced 86, 68, and 36% of adherable area, respectively, on the fabricated surface. A meticulous investigation of the cell migration in response to changes in the constrained adherable area of the nanotopographic surface showed 1.4-, 1.5-, and 1.6-fold increase in migration speeds and a 1.4-, 2-, and 2.5-fold decrease in the number of focal adhesions as the adherable area was decreased to 86, 68, and 36%, respectively. Furthermore, a strong activation of FAK/Rac1 signaling was observed to be involved in the promoted cell migration. These results suggest that the reduced adherable area promotes cell migration through decreasing the FA formation, which in turn upregulates FAK/Rac1 activation. The findings in this study can be utilized to control the cell migration behaviors, which is a powerful tool in the research fields involving cell migration such as promoting wound healing and tissue repair.

Cell Density Control of Staphylococcal Virulence Mediated by an Octapeptide Pheromone

NASA Astrophysics Data System (ADS)

Ji, Guangyong; Beavis, Ronald C.; Novick, Richard P.

1995-12-01

Some bacterial pathogens elaborate and secrete virulence factors in response to environmental signals, others in response to a specific host product, and still others in response to no discernible cue. In this study, we have demonstrated that the synthesis of Staphylococcus aureus virulence factors is controlled by a density-sensing system that utilizes an octapeptide produced by the organism itself. The octapeptide activates expression of the agr locus, a global regulator of the virulence response. This response involves the reciprocal regulation of genes encoding surface proteins and those encoding secreted virulence factors. As cells enter the postexponential phase, surface protein genes are repressed by agr and secretory protein genes are subsequently activated. The intracellular agr effector is a regulatory RNA, RNAIII, whose transcription is activated by an agr-encoded signal transduction system for which the octapeptide is the ligand.

R-Spondin1 regulates Wnt signaling by inhibiting internalization of LRP6

PubMed Central

Binnerts, Minke E.; Kim, Kyung-Ah; Bright, Jessica M.; Patel, Sejal M.; Tran, Karolyn; Zhou, Mei; Leung, John M.; Liu, Yi; Lomas, Woodrow E.; Dixon, Melissa; Hazell, Sophie A.; Wagle, Marie; Nie, Wen-Sheng; Tomasevic, Nenad; Williams, Jason; Zhan, Xiaoming; Levy, Michael D.; Funk, Walter D.; Abo, Arie

2007-01-01

The R-Spondin (RSpo) family of secreted proteins act as potent activators of the Wnt/β-catenin signaling pathway. We have previously shown that RSpo proteins can induce proliferative effects on the gastrointestinal epithelium in mice. Here we provide a mechanism whereby RSpo1 regulates cellular responsiveness to Wnt ligands by modulating the cell-surface levels of the coreceptor LRP6. We show that RSpo1 activity critically depends on the presence of canonical Wnt ligands and LRP6. Although RSpo1 does not directly activate LRP6, it interferes with DKK1/Kremen-mediated internalization of LRP6 through an interaction with Kremen, resulting in increased LRP6 levels on the cell surface. Our results support a model in which RSpo1 relieves the inhibition DKK1 imposes on the Wnt pathway. PMID:17804805

The Dentin Sialoprotein (DSP) Domain Regulates Dental Mesenchymal Cell Differentiation through a Novel Surface Receptor

PubMed Central

Wan, Chunyan; Yuan, Guohua; Luo, Daoshu; Zhang, Lu; Lin, Heng; Liu, Huan; Chen, Lei; Yang, Guobin; Chen, Shuo; Chen, Zhi

2016-01-01

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP aa183-219 stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP aa183-219 in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop. PMID:27430624

The Dentin Sialoprotein (DSP) Domain Regulates Dental Mesenchymal Cell Differentiation through a Novel Surface Receptor.

PubMed

Wan, Chunyan; Yuan, Guohua; Luo, Daoshu; Zhang, Lu; Lin, Heng; Liu, Huan; Chen, Lei; Yang, Guobin; Chen, Shuo; Chen, Zhi

2016-07-19

Dentin sialophosphoprotein (DSPP) is a dentin extracellular matrix protein that is processed into dentin sialoprotein (DSP), dentin glycoprotein (DGP) and dentin phosphoprotein (DPP). DSP is mainly expressed in odontoblasts. We hypothesized that DSP interacts with cell surface receptors and subsequently activates intracellular signaling. Using DSP as bait for screening a protein library, we demonstrate that DSP acts as a ligand and binds to integrin β6. The 36 amino acid residues of DSP are sufficient to bind to integrin β6. This peptide promoted cell attachment, migration, differentiation and mineralization of dental mesenchymal cells. In addition, DSP (aa183-219) stimulated phosphorylation of ERK1/2 and P38 kinases. This activation was inhibited by an anti-integrin β6 antibody and siRNA. Furthermore, we demonstrate that this DSP fragment induces SMAD1/5/8 phosphorylation and nuclear translocation via ERK1/2 and P38 signaling. SMAD1/5/8 binds to SMAD binding elements (SBEs) in the DSPP gene promoter. SBE mutations result in a decrease in DSPP transcriptional activity. Endogenous DSPP expression was up-regulated by DSP (aa183-219) in dental mesenchymal cells. The data in the current study demonstrate for the first time that this DSP domain acts as a ligand in a RGD-independent manner and is involved in intracellular signaling via interacting with integrin β6. The DSP domain regulates DSPP expression and odontoblast homeostasis via a positive feedback loop.

Ick Ciliary Kinase Is Essential for Planar Cell Polarity Formation in Inner Ear Hair Cells and Hearing Function.

PubMed

Okamoto, Shio; Chaya, Taro; Omori, Yoshihiro; Kuwahara, Ryusuke; Kubo, Shun; Sakaguchi, Hirofumi; Furukawa, Takahisa

2017-02-22

Cellular asymmetries play crucial roles in development and organ function. The planar cell polarity (PCP) signaling pathway is involved in the establishment of cellular asymmetry within the plane of a cell sheet. Inner ear sensory hair cells (HCs), which have several rows of staircase-like stereocilia and one kinocilium located at the vertex of the stereocilia protruding from the apical surface of each HC, exhibit a typical form of PCP. Although connections between cilia and PCP signaling in vertebrate development have been reported, their precise nature is not well understood. During inner ear development, several ciliary proteins are known to play a role in PCP formation. In the current study, we investigated a functional role for intestinal cell kinase (Ick), which regulates intraflagellar transport (IFT) at the tip of cilia, in the mouse inner ear. A lack of Ick in the developing inner ear resulted in PCP defects in the cochlea, including misorientation or misshaping of stereocilia and aberrant localization of the kinocilium and basal body in the apical and middle turns, leading to auditory dysfunction. We also observed abnormal ciliary localization of Ift88 in both HCs and supporting cells. Together, our results show that Ick ciliary kinase is essential for PCP formation in inner ear HCs, suggesting that ciliary transport regulation is important for PCP signaling. SIGNIFICANCE STATEMENT The cochlea in the inner ear is the hearing organ. Planar cell polarity (PCP) in hair cells (HCs) in the cochlea is essential for mechanotransduction and refers to the asymmetric structure consisting of stereociliary bundles and the kinocilium on the apical surface of the cell body. We reported previously that a ciliary kinase, Ick, regulates intraflagellar transport (IFT). Here, we found that loss of Ick leads to abnormal localization of the IFT component in kinocilia, PCP defects in HCs, and hearing dysfunction. Our study defines the association of ciliary transport regulation with PCP formation in HCs and hearing function. Copyright © 2017 the authors 0270-6474/17/372073-13$15.00/0.

Capacitance measurements of regulated exocytosis in mouse taste cells.

PubMed

Vandenbeuch, Aurelie; Zorec, Robert; Kinnamon, Sue C

2010-11-03

Exocytosis, consisting of the merger of vesicle and plasma membrane, is a common mechanism used by different types of nucleated cells to release their vesicular contents. Taste cells possess vesicles containing various neurotransmitters to communicate with adjacent taste cells and afferent nerve fibers. However, whether these vesicles engage in exocytosis on a stimulus is not known. Since vesicle membrane merger with the plasma membrane is reflected in plasma membrane area fluctuations, we measured membrane capacitance (C(m)), a parameter linearly related to membrane surface area. To investigate whether taste cells undergo regulated exocytosis, we used the compensated tight-seal whole-cell recording technique to monitor depolarization-induced changes in C(m) in the different types of taste cells. To identify taste cell types, mice expressing green fluorescent protein from the TRPM5 promoter or from the GAD67 promoter were used to discriminate type II and type III taste cells, respectively. Moreover, the cell types were also identified by monitoring their voltage-current properties. The results demonstrate that only type III taste cells show significant depolarization-induced increases in C(m), which were correlated to the voltage-activated calcium currents. The results suggest that type III, but neither type II nor type I cells exhibit depolarization-induced regulated exocytosis to release transmitter and activate gustatory afferent nerve fibers.

Reversible effects of sphingomyelin degradation on cholesterol distribution and metabolism in fibroblasts and transformed neuroblastoma cells.

PubMed Central

Pörn, M I; Slotte, J P

1990-01-01

Plasma-membrane sphingomyelin appears to be one of the major determinants of the preferential allocation of cell cholesterol into the plasma-membrane compartment, since removal of sphingomyelin leads to a dramatic redistribution of cholesterol within the cell [Slotte & Bierman (1988) Biochem. J. 250, 653-658]. In the present study we examined the long-term effects of sphingomyelin degradation on cholesterol redistribution in cells and determined the reversibility of the process. In a human lung fibroblast-cell line, removal of 80% of the sphingomyelin led to a rapid and transient up-regulation (3-fold) of acyl-CoA:cholesterol acyltransferase (ACAT) activity, and also, within 30 h, to the translocation of about 50% of the cell non-esterified cholesterol from a cholesterol oxidase-susceptible compartment (i.e. the cell surface) to oxidase-resistant compartments. At 49 h after the initial sphingomyelin degradation, the cell sphingomyelin level was back to 45% of the control level, and the direction of cell cholesterol flow was toward the cell surface, although the original distribution was not achieved. In a transformed neuroblastoma cell line (SH-SY5Y), the depletion of sphingomyelin led to a similarly rapid and transient up-regulation of ACAT activity, and to the translocation of about 25% of cell-surface cholesterol into internal membranes (within 3 h). The flow of cholesterol back to the cholesterol oxidase-susceptible pool was rapid, and a pretreatment cholesterol distribution was reached within 20-49 h. Also, the resynthesis of sphingomyelin was faster in SH-SY5Y neuroblastoma cells and reached control levels within 24 h. The findings of the present study show that the cellular redistribution of cholesterol, as induced by sphingomyelin degradation, is reversible and suggest that the normalization of cellular cholesterol distribution is linked to the re-synthesis of sphingomyelin. PMID:2222406

Development and functional evaluation of biomimetic silicone surfaces with hierarchical micro/nano-topographical features demonstrates favourable in vitro foreign body response of breast-derived fibroblasts.

PubMed

Kyle, Daniel J T; Oikonomou, Antonios; Hill, Ernie; Bayat, Ardeshir

2015-06-01

Reproducing extracellular matrix topographical cues, such as those present within acellular dermal matrix (ADM), in synthetic implant surfaces, may augment cellular responses, independent of surface chemistry. This could lead to enhanced implant integration and performance while reducing complications. In this work, the hierarchical micro and nanoscale features of ADM were accurately and reproducibly replicated in polydimethylsiloxane (PDMS), using an innovative maskless 3D grayscale fabrication process not previously reported. Human breast derived fibroblasts (n=5) were cultured on PDMS surfaces and compared to commercially available smooth and textured silicone implant surfaces, for up to one week. Cell attachment, proliferation and cytotoxicity, in addition to immunofluorescence staining, SEM imaging, qRT-PCR and cytokine array were performed. ADM PDMS surfaces promoted cell adhesion, proliferation and survival (p=<0.05), in addition to increased focal contact formation and spread fibroblast morphology when compared to commercially available implant surfaces. PCNA, vinculin and collagen 1 were up-regulated in fibroblasts on biomimetic surfaces while IL8, TNFα, TGFβ1 and HSP60 were down-regulated (p=<0.05). A reduced inflammatory cytokine response was also observed (p=<0.05). This study represents a novel approach to the development of functionalised biomimetic prosthetic implant surfaces which were demonstrated to significantly attenuate the acute in vitro foreign body reaction to silicone. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.

The Coxsackievirus and Adenovirus Receptor (CAR) Undergoes Ectodomain Shedding and Regulated Intramembrane Proteolysis (RIP)

PubMed Central

Houri, Nadia; Huang, Kuo-Cheng; Nalbantoglu, Josephine

2013-01-01

The Coxsackievirus and Adenovirus Receptor (CAR) is a cell adhesion molecule originally characterized as a virus receptor but subsequently shown to be involved in physiological processes such as neuronal and heart development, epithelial tight junction integrity, and tumour suppression. Proteolysis of cell adhesion molecules and a wide variety of other cell surface proteins serves as a mechanism for protein turnover and, in some cases, cell signaling. Metalloproteases such as A Disintegrin and Metalloprotease (ADAM) family members cleave cell surface receptors to release their substrates’ ectodomains, while the presenilin/ɣ-secretase complex mediates regulated intramembrane proteolysis (RIP), releasing intracellular domain fragments from the plasma membrane. In the case of some substrates such as Notch and amyloid precursor protein (APP), the released intracellular domains enter the nucleus to modulate gene expression. We report that CAR ectodomain is constitutively shed from glioma cells and developing neurons, and is also shed when cells are treated with the phorbol ester phorbol 12-myristate 13-acetate (PMA) and the calcium ionophore ionomycin. We identified ADAM10 as a sheddase of CAR using assays involving shRNA knockdown and rescue, overexpression of wild-type ADAM10 and inhibition of ADAM10 activity by addition of its prodomain. In vitro peptide cleavage, mass spectrometry and mutagenesis revealed the amino acids M224 to L227 of CAR as the site of ADAM10-mediated ectodomain cleavage. CAR also undergoes RIP by the presenilin/γ-secretase complex, and the intracellular domain of CAR enters the nucleus. Ectodomain shedding is a prerequisite for RIP of CAR. Thus, CAR belongs to the increasing list of cell surface molecules that undergo ectodomain shedding and that are substrates for ɣ-secretase-mediated RIP. PMID:24015300

Drosophila Big bang regulates the apical cytocortex and wing growth through junctional tension.

PubMed

Tsoumpekos, Giorgos; Nemetschke, Linda; Knust, Elisabeth

2018-03-05

Growth of epithelial tissues is regulated by a plethora of components, including signaling and scaffolding proteins, but also by junctional tension, mediated by the actomyosin cytoskeleton. However, how these players are spatially organized and functionally coordinated is not well understood. Here, we identify the Drosophila melanogaster scaffolding protein Big bang as a novel regulator of growth in epithelial cells of the wing disc by ensuring proper junctional tension. Loss of big bang results in the reduction of the regulatory light chain of nonmuscle myosin, Spaghetti squash. This is associated with an increased apical cell surface, decreased junctional tension, and smaller wings. Strikingly, these phenotypic traits of big bang mutant discs can be rescued by expressing constitutively active Spaghetti squash. Big bang colocalizes with Spaghetti squash in the apical cytocortex and is found in the same protein complex. These results suggest that in epithelial cells of developing wings, the scaffolding protein Big bang controls apical cytocortex organization, which is important for regulating cell shape and tissue growth. © 2018 Tsoumpekos et al.

Microvillar cell surface as a natural defense system against xenobiotics: a new interpretation of multidrug resistance.

PubMed

Lange, K; Gartzke, J

2001-08-01

The phenomenon of multidrug resistance (MDR) is reinterpreted on the basis of the recently proposed concept of microvillar signaling. According to this notion, substrate and ion fluxes across the surface of differentiated cells occur via transporters and ion channels that reside in membrane domains at the tips of microvilli (MV). The flux rates are regulated by the actin-based cytoskeletal core structure of MV, acting as a diffusion barrier between the microvillar tip compartment and the cytoplasm. The expression of this diffusion barrier system is a novel aspect of cell differentiation and represents a functional component of the natural defense system of epithelial cells against environmental hazardous ions and lipophilic compounds. Because of the specific organization of epithelial Ca(2+) signaling and the secretion, lipophilic compounds associated with the plasma membrane are transferred from the basal to the apical cell surface by a lipid flow mechanism. Drug release from the apical pole occurs by either direct secretion from the cell surface or metabolization by the microvillar cytochrome P-450 system and efflux of the metabolites and conjugation products through the large multifunctional anion channels localized in apical MV. The natural microvillar defense system also provides a mechanistic basis of acquired MDR in tumor cells. The microvillar surface organization is lost in rapidly growing cells such as tumor or embryonic cells but is restored during exposure of tumor cells to cytotoxins by induction of a prolonged G(0)/G(1) resting phase.

Multiple roles for the actin cytoskeleton during regulated exocytosis

PubMed Central

Porat-Shliom, Natalie; Milberg, Oleg; Masedunskas, Andrius; Weigert, Roberto

2014-01-01

Regulated exocytosis is the main mechanism utilized by specialized secretory cells to deliver molecules to the cell surface by virtue of membranous containers (i.e. secretory vesicles). The process involves a series of highly coordinated and sequential steps, which include the biogenesis of the vesicles, their delivery to the cell periphery, their fusion with the plasma membrane and the release of their content into the extracellular space. Each of these steps is regulated by the actin cytoskeleton. In this review, we summarize the current knowledge regarding the involvement of actin and its associated molecules during each of the exocytic steps in vertebrates, and suggest that the overall role of the actin cytoskeleton during regulated exocytosis is linked to the architecture and the physiology of the secretory cells under examination. Specifically, in neurons, neuroendocrine, endocrine, and hematopoietic cells, which contain small secretory vesicles that undergo rapid exocytosis (on the order of milliseconds), the actin cytoskeleton plays a role in pre-fusion events, where it acts primarily as a functional barrier and facilitates docking. In exocrine and other secretory cells, which contain large secretory vesicles that undergo slow exocytosis (seconds to minutes), the actin cytoskeleton plays a role in post-fusion events, where it regulates the dynamics of the fusion pore, facilitates the integration of the vesicles into the plasma membrane, provides structural support, and promotes the expulsion of large cargo molecules. PMID:22986507

Lipid raft regulates the initial spreading of melanoma A375 cells by modulating β1 integrin clustering.

PubMed

Wang, Ruifei; Bi, Jiajia; Ampah, Khamal Kwesi; Zhang, Chunmei; Li, Ziyi; Jiao, Yang; Wang, Xiaoru; Ba, Xueqing; Zeng, Xianlu

2013-08-01

Cell adhesion and spreading require integrins-mediated cell-extracellular matrix interaction. Integrins function through binding to extracellular matrix and subsequent clustering to initiate focal adhesion formation and actin cytoskeleton rearrangement. Lipid raft, a liquid ordered plasma membrane microdomain, has been reported to play major roles in membrane motility by regulating cell surface receptor function. Here, we identified that lipid raft integrity was required for β1 integrin-mediated initial spreading of melanoma A375 cells on fibronectin. We found that lipid raft disruption with methyl-β-cyclodextrin led to the inability of focal adhesion formation and actin cytoskeleton rearrangement by preventing β1 integrin clustering. Furthermore, we explored the possible mechanism by which lipid raft regulates β1 integrin clustering and demonstrated that intact lipid raft could recruit and modify some adaptor proteins, such as talin, α-actinin, vinculin, paxillin and FAK. Lipid raft could regulate the location of these proteins in lipid raft fractions and facilitate their binding to β1 integrin, which may be crucial for β1 integrin clustering. We also showed that lipid raft disruption impaired A375 cell migration in both transwell and wound healing models. Together, these findings provide a new insight for the relationship between lipid raft and the regulation of integrins. Copyright © 2013 Elsevier Ltd. All rights reserved.

The Wnt receptor Frizzled-4 modulates ADAM13 metalloprotease activity.

PubMed

Abbruzzese, Genevieve; Gorny, Anne-Kathrin; Kaufmann, Lilian T; Cousin, Hélène; Kleino, Iivari; Steinbeisser, Herbert; Alfandari, Dominique

2015-03-15

Cranial neural crest (CNC) cells are a transient population of stem cells that originate at the border of the neural plate and the epidermis, and migrate ventrally to contribute to most of the facial structures including bones, cartilage, muscles and ganglia. ADAM13 is a cell surface metalloprotease that is essential for CNC cell migration. Here, we show in Xenopus laevis embryos that the Wnt receptor Fz4 binds to the cysteine-rich domain of ADAM13 and negatively regulates its proteolytic activity in vivo. Gain of Fz4 function inhibits CNC cell migration and can be rescued by gain of ADAM13 function. Loss of Fz4 function also inhibits CNC cell migration and induces a reduction of mature ADAM13, together with an increase in the ADAM13 cytoplasmic fragment that is known to translocate into the nucleus to regulate gene expression. We propose that Fz4 associates with ADAM13 during its transport to the plasma membrane to regulate its proteolytic activity. © 2015. Published by The Company of Biologists Ltd.

The Cystic Fibrosis Transmembrane Conductance Regulator Potentiator Ivacaftor Augments Mucociliary Clearance Abrogating Cystic Fibrosis Transmembrane Conductance Regulator Inhibition by Cigarette Smoke.

PubMed

Raju, S Vamsee; Lin, Vivian Y; Liu, Limbo; McNicholas, Carmel M; Karki, Suman; Sloane, Peter A; Tang, Liping; Jackson, Patricia L; Wang, Wei; Wilson, Landon; Macon, Kevin J; Mazur, Marina; Kappes, John C; DeLucas, Lawrence J; Barnes, Stephen; Kirk, Kevin; Tearney, Guillermo J; Rowe, Steven M

2017-01-01

Acquired cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction may contribute to chronic obstructive pulmonary disease pathogenesis and is a potential therapeutic target. We sought to determine the acute effects of cigarette smoke on ion transport and the mucociliary transport apparatus, their mechanistic basis, and whether deleterious effects could be reversed with the CFTR potentiator ivacaftor (VX-770). Primary human bronchial epithelial (HBE) cells and human bronchi were exposed to cigarette smoke extract (CSE) and/or ivacaftor. CFTR function and expression were measured in Ussing chambers and by surface biotinylation. CSE-derived acrolein modifications on CFTR were determined by mass spectroscopic analysis of purified protein, and the functional microanatomy of the airway epithelia was measured by 1-μm resolution optical coherence tomography. CSE reduced CFTR-dependent current in HBE cells (P < 0.05) and human bronchi (P < 0.05) within minutes of exposure. The mechanism involved CSE-induced reduction of CFTR gating, decreasing CFTR open-channel probability by approximately 75% immediately after exposure (P < 0.05), whereas surface CFTR expression was partially reduced with chronic exposure, but was stable acutely. CSE treatment of purified CFTR resulted in acrolein modifications on lysine and cysteine residues that likely disrupt CFTR gating. In primary HBE cells, CSE reduced airway surface liquid depth (P < 0.05) and ciliary beat frequency (P < 0.05) within 60 minutes that was restored by coadministration with ivacaftor (P < 0.005). Cigarette smoking transmits acute reductions in CFTR activity, adversely affecting the airway surface. These effects are reversible by a CFTR potentiator in vitro, representing a potential therapeutic strategy in patients with chronic obstructive pulmonary disease with chronic bronchitis.

Neuro-immune interactions at barrier surfaces

PubMed Central

Veiga-Fernandes, Henrique; Mucida, Daniel

2016-01-01

Multidirectional interactions between the nervous and immune systems have been documented in homeostasis and pathologies ranging from multiple sclerosis to autism, and from leukemia to acute and chronic inflammation. Recent studies have addressed this crosstalk using cell-specific targeting, novel sequencing, imaging and analytical tools, shedding light on unappreciated mechanisms of neuro-immune regulation. This review focuses on neuro-immune interactions at barrier surfaces, mostly the gut, but also including the skin and the airways, areas densely populated by neurons and immune cells that constantly sense and adapt to tissue-specific environmental challenges. PMID:27153494

Altered Calcium Dynamics in Cardiac Cells Grown on Silane-Modified Surfaces

PubMed Central

Ravenscroft-Chang, Melissa S.; Stohlman, Jayna; Molnar, Peter; Natarajan, Anupama; Canavan, Heather E.; Teliska, Maggie; Stancescu, Maria; Krauthamer, Victor; Hickman, J.J.

2013-01-01

Chemically defined surfaces were created using self-assembled monolayers (SAMs) of hydrophobic and hydrophilic silanes as models for implant coatings, and the morphology and physiology of cardiac myocytes plated on these surfaces were studied in vitro. We focused on changes in intracellular Ca2+ because of its essential role in regulating heart cell function. The SAM-modified coverslips were analyzed using X-ray Photoelectron Spectroscopy to verify composition. The morphology and physiology of the cardiac cells were examined using fluorescence microscopy and intracellular Ca2+ imaging. The imaging experiments used the fluorescent ratiometric dye fura-2, AM to establish both the resting Ca2+ concentration and the dynamic responses to electrical stimulation. A significant difference in excitation-induced Ca2+ changes on the different silanated surfaces was observed. However, no significant change was noted based on the morphological analysis. This result implies a difference in internal Ca2+ dynamics, and thus cardiac function, occurs when the composition of the surface is different, and this effect is independent of cellular morphology. This finding has implications for histological examination of tissues surrounding implants, the choice of materials that could be beneficial as implant coatings and understanding of cell-surface interactions in cardiac systems. PMID:19828193

REEPs Are Membrane Shaping Adapter Proteins That Modulate Specific G Protein-Coupled Receptor Trafficking by Affecting ER Cargo Capacity

PubMed Central

Ho, Vincent K.; Angelotti, Timothy

2013-01-01

Receptor expression enhancing proteins (REEPs) were identified by their ability to enhance cell surface expression of a subset of G protein-coupled receptors (GPCRs), specifically GPCRs that have proven difficult to express in heterologous cell systems. Further analysis revealed that they belong to the Yip (Ypt-interacting protein) family and that some REEP subtypes affect ER structure. Yip family comparisons have established other potential roles for REEPs, including regulation of ER-Golgi transport and processing/neuronal localization of cargo proteins. However, these other potential REEP functions and the mechanism by which they selectively enhance GPCR cell surface expression have not been clarified. By utilizing several REEP family members (REEP1, REEP2, and REEP6) and model GPCRs (α2A and α2C adrenergic receptors), we examined REEP regulation of GPCR plasma membrane expression, intracellular processing, and trafficking. Using a combination of immunolocalization and biochemical methods, we demonstrated that this REEP subset is localized primarily to ER, but not plasma membranes. Single cell analysis demonstrated that these REEPs do not specifically enhance surface expression of all GPCRs, but affect ER cargo capacity of specific GPCRs and thus their surface expression. REEP co-expression with α2 adrenergic receptors (ARs) revealed that this REEP subset interacts with and alter glycosidic processing of α2C, but not α2A ARs, demonstrating selective interaction with cargo proteins. Specifically, these REEPs enhanced expression of and interacted with minimally/non-glycosylated forms of α2C ARs. Most importantly, expression of a mutant REEP1 allele (hereditary spastic paraplegia SPG31) lacking the carboxyl terminus led to loss of this interaction. Thus specific REEP isoforms have additional intracellular functions besides altering ER structure, such as enhancing ER cargo capacity, regulating ER-Golgi processing, and interacting with select cargo proteins. Therefore, some REEPs can be further described as ER membrane shaping adapter proteins. PMID:24098485

Adhesion and Growth of Vascular Smooth Muscle Cells on Nanostructured and Biofunctionalized Polyethylene

PubMed Central

Novotna, Katarina; Bacakova, Marketa; Kasalkova, Nikola Slepickova; Slepicka, Petr; Lisa, Vera; Svorcik, Vaclav; Bacakova, Lucie

2013-01-01

Cell colonization of synthetic polymers can be regulated by physical and chemical modifications of the polymer surface. High-density and low-density polyethylene (HDPE and LDPE) were therefore activated with Ar+ plasma and grafted with fibronectin (Fn) or bovine serum albumin (BSA). The water drop contact angle usually decreased on the plasma-treated samples, due to the formation of oxidized groups, and this decrease was inversely related to the plasma exposure time (50–300 s). The presence of nitrogen and sulfur on the polymer surface, revealed by X-ray photoelectron spectroscopy (XPS), and also by immunofluorescence staining, showed that Fn and BSA were bound to this surface, particularly to HDPE. Plasma modification and grafting with Fn and BSA increased the nanoscale surface roughness of the polymer. This was mainly manifested on HDPE. Plasma treatment and grafting with Fn or BSA improved the adhesion and growth of vascular smooth muscle cells in a serum-supplemented medium. The final cell population densities on day 6 after seeding were on an average higher on LDPE than on HDPE. In a serum-free medium, BSA grafted to the polymer surface hampered cell adhesion. Thus, the cell behavior on polyethylene can be modulated by its type, intensity of plasma modification, grafting with biomolecules, and composition of the culture medium. PMID:28809234

A protein crosslinking assay for measuring cell surface expression of glutamate receptor subunits in the rodent brain after in vivo treatments

PubMed Central

Boudreau, Amy C.; Milovanovic, Mike; Conrad, Kelly L.; Nelson, Christopher; Ferrario, Carrie R.; Wolf, Marina E.

2012-01-01

Trafficking of neurotransmitter receptors between intracellular and cell surface compartments is important for regulating neurotransmission. We developed a method for determining if an in vivo treatment has altered receptor distribution in a particular region of rodent brain. After the treatment, brain slices are rapidly prepared from the region of interest. Then cell surface-expressed receptors are covalently crosslinked to nearby proteins using the membrane-impermeable, bifunctional crosslinker bis(sulfosuccinimidyl)suberate (BS3). This increases the apparent molecular weight of surface receptors, while intracellular receptors are not modified. Thus, surface and intracellular receptor pools can be separated and quantified using SDS-PAGE and immunoblotting. This method is particularly useful for analyzing AMPA receptor subunits, offering advantages in accuracy, efficiency and cost compared to biotinylation. A disadvantage is that some antibodies no longer recognize their target protein after crosslinking. We have used this method to quantify changes in receptor distribution after acute and chronic exposure to psychomotor stimulants. PMID:22470150

Mechanism of abnormal growth in astrocytes derived from a mouse model of GM2 gangliosidosis.

PubMed

Kawashima, Nagako; Tsuji, Daisuke; Okuda, Tetsuya; Itoh, Kohji; Nakayama, Ken-ichi

2009-11-01

Sandhoff disease is a progressive neurodegenerative disorder caused by mutations in the HEXB gene which encodes the beta-subunit of N-acetyl-beta-hexosaminidase A and B, resulting in the accumulation of the ganglioside GM2. We isolated astrocytes from the neonatal brain of Sandhoff disease model mice in which the N-acetyl-beta-hexosaminidase beta-subunit gene is genetically disrupted (ASD). Glycolipid profiles revealed that GM2/GA2 accumulated in the lysosomes and not on the cell surface of ASD astrocytes. In addition, GM3 was increased on the cell surface. We found remarkable differences in the cell proliferation of ASD astrocytes when compared with cells isolated from wild-type mice, with a faster growth rate of ASD cells. In addition, we observed increased extracellular, signal-regulated kinase (ERK) phosphorylation in ASD cells, but Akt phosphorylation was decreased. Furthermore, the phosphorylation of ERK in ASD cells was not dependent upon extracellular growth factors. Treatment of ASD astrocytes with recombinant N-acetyl-beta-hexosaminidase A resulted in a decrease of their growth rate and ERK phosphorylation. These results indicated that the up-regulation of ERK phosphorylation and the increase in proliferation of ASD astrocytes were dependent upon GM2/GA2 accumulation. These findings may represent a mechanism in linking the nerve cell death and reactive gliosis observed in Sandhoff disease.

Chronic lithium treatment up-regulates cell surface Na(V)1.7 sodium channels via inhibition of glycogen synthase kinase-3 in adrenal chromaffin cells: enhancement of Na(+) influx, Ca(2+) influx and catecholamine secretion after lithium withdrawal.

PubMed

Yanagita, Toshihiko; Maruta, Toyoaki; Nemoto, Takayuki; Uezono, Yasuhito; Matsuo, Kiyotaka; Satoh, Shinya; Yoshikawa, Norie; Kanai, Tasuku; Kobayashi, Hideyuki; Wada, Akihiko

2009-09-01

In cultured bovine adrenal chromaffin cells expressing Na(V)1.7 isoform of voltage-dependent Na(+) channels, we have previously reported that lithium chloride (LiCl) inhibits function of Na(+) channels independent of glycogen synthase kinase-3 (GSK-3) (Yanagita et al., 2007). Here, we further examined the effects of chronic lithium treatment on Na(+) channels. LiCl treatment (1-30 mM, > or = 12 h) increased cell surface [(3)H]saxitoxin ([(3)H]STX) binding by approximately 32% without altering the affinity of [(3)H]STX binding. This increase was prevented by cycloheximide and actinomycin D. SB216763 and SB415286 (GSK-3 inhibitors) also increased cell surface [(3)H]STX binding by approximately 31%. Simultaneous treatment with LiCl and SB216763 or SB415286 did not produce an increased effect on [(3)H]STX binding compared with either treatment alone. LiCl increased Na(+) channel alpha-subunit mRNA level by 32% at 24 h. LiCl accelerated alpha-subunit gene transcription by 35% without altering alpha-subunit mRNA stability. In LiCl-treated cells, LiCl inhibited veratridine-induced (22)Na(+) influx as in untreated cells. However, washout of LiCl after chronic treatment enhanced veratridine-induced (22)Na(+) influx, (45)Ca(2+) influx and catecholamine secretion by approximately 30%. Washout of LiCl after 24 h treatment shifted concentration-response curve of veratridine upon (22)Na(+) influx upward, without altering its EC(50) value. Ptychodiscus brevis toxin-3 allosterically enhanced veratridine-induced (22)Na(+) influx by two-fold in untreated and LiCl-treated cells. Whole-cell patch-clamp analysis indicated that I-V curve and steady-state inactivation/activation curves were comparable between untreated and LiCl-treated cells. Thus, GSK-3 inhibition by LiCl up-regulated cell surface Na(V)1.7 via acceleration of alpha-subunit gene transcription, enhancing veratridine-induced Na(+) influx, Ca(2+) influx and catecholamine secretion.

Development of T follicular helper cells and their role in disease and immune system.

PubMed

Eivazi, Sadegh; Bagheri, Salman; Hashemzadeh, Mohammad Sadegh; Ghalavand, Majdedin; Qamsari, Elmira Safaie; Dorostkar, Ruhollah; Yasemi, Maryam

2016-12-01

The T follicular helper cells (TFH) are a subset of CD4+ T cells specialized to regulate antibody responses. The production of these cells is associated with the dendritic cells (DCs) and B cells. TFH cells help B cells form germinal centers (GC) differentiate into memory and plasma cells (antibody-secreting cells) as humoral responses. In addition, there is strong evidence that TFH cells play a pivotal role in the development of long-lived humoral immunity. Molecular factors such as transcription factors, surface receptors, cytokine and micro RNAs are involved in the formation of TFH cells. Such TFH cells are diagnosed by transcription factor (BCL-6), surface marker expression (including CXCR5, PD-1, ICOS and CD40L) and a unique cytokine production pattern (such as IL-21 and IL-6). Memory TFH cells, accompanied by memory B cells, are known to be formed during antibody responses. It is now clear that the precise control of TFH cells is critically important for both inducing the optimal affinity maturation of antibody responses and preventing self-reactivity. Exclusive controls of TFH cell function and production are essential for human health. However, it is important to note that excessive activities may lead to autoimmune diseases, while reduced activity often results in immunodeficiency. It has also been shown that TFH cells are associated with cancers such as angioimmunoblastic T-cell lymphoma (AITL), follicular T-cell lymphoma (FTCL) and nonspecific Peripheral T-cell lymphomas (PTCLs). The biology of TFH cells, including their differentiation and transcriptional regulation will be described in the present review. Some of The developments of these cells in immunodeficiency diseases, autoimmunity and cancer will also be taken into account. Copyright © 2016. Published by Elsevier Masson SAS.

The virulence regulator PrfA promotes biofilm formation by Listeria monocytogenes.

PubMed

Lemon, Katherine P; Freitag, Nancy E; Kolter, Roberto

2010-08-01

Listeria monocytogenes is a food-borne facultative intracellular pathogen. It is widespread in the environment and has several distinct life-styles. The key transcriptional activator PrfA positively regulates L. monocytogenes virulence genes to mediate the transition from extracellular, flagellum-propelled cell to intracellular pathogen. Here we report the first evidence that PrfA also has a significant positive impact on extracellular biofilm formation. Mutants lacking prfA were defective in surface-adhered biofilm formation. The DeltaprfA mutant exhibited wild-type flagellar motility, and its biofilm defect occurred after initial surface adhesion. We also observed that mutations that led to the constitutive expression of PrfA-dependent virulence genes had a minimal impact on biofilm formation. Furthermore, biofilm development was enhanced in a mutant encoding a PrfA protein variant unable to fully transition from the extracellular form to the virulent, intracellular activity conformation. These results indicate that PrfA positively regulates biofilm formation and suggest that PrfA has a global role in modulating the life-style of L. monocytogenes. The requirement of PrfA for optimal biofilm formation may provide selective pressure to maintain this critical virulence regulator when L. monocytogenes is outside host cells in the environment.

Investigating Evolutionary Conservation of Dendritic Cell Subset Identity and Functions

PubMed Central

Vu Manh, Thien-Phong; Bertho, Nicolas; Hosmalin, Anne; Schwartz-Cornil, Isabelle; Dalod, Marc

2015-01-01

Dendritic cells (DCs) were initially defined as mononuclear phagocytes with a dendritic morphology and an exquisite efficiency for naïve T-cell activation. DC encompass several subsets initially identified by their expression of specific cell surface molecules and later shown to excel in distinct functions and to develop under the instruction of different transcription factors or cytokines. Very few cell surface molecules are expressed in a specific manner on any immune cell type. Hence, to identify cell types, the sole use of a small number of cell surface markers in classical flow cytometry can be deceiving. Moreover, the markers currently used to define mononuclear phagocyte subsets vary depending on the tissue and animal species studied and even between laboratories. This has led to confusion in the definition of DC subset identity and in their attribution of specific functions. There is a strong need to identify a rigorous and consensus way to define mononuclear phagocyte subsets, with precise guidelines potentially applicable throughout tissues and species. We will discuss the advantages, drawbacks, and complementarities of different methodologies: cell surface phenotyping, ontogeny, functional characterization, and molecular profiling. We will advocate that gene expression profiling is a very rigorous, largely unbiased and accessible method to define the identity of mononuclear phagocyte subsets, which strengthens and refines surface phenotyping. It is uniquely powerful to yield new, experimentally testable, hypotheses on the ontogeny or functions of mononuclear phagocyte subsets, their molecular regulation, and their evolutionary conservation. We propose defining cell populations based on a combination of cell surface phenotyping, expression analysis of hallmark genes, and robust functional assays, in order to reach a consensus and integrate faster the huge but scattered knowledge accumulated by different laboratories on different cell types, organs, and species. PMID:26082777

Prolactin and growth hormone in fish osmoregulation

USGS Publications Warehouse

Sakamoto, T.; McCormick, S.D.

2006-01-01

Prolactin is an important regulator of multiple biological functions in vertebrates, and has been viewed as essential to ion uptake as well as reduction in ion and water permeability of osmoregulatory surfaces in freshwater and euryhaline fish. Prolactin-releasing peptide seems to stimulate prolactin expression in the pituitary and peripheral organs during freshwater adaptation. Growth hormone, a member of the same family of hormones as prolactin, promotes acclimation to seawater in several teleost fish, at least in part through the action of insulin-like growth factor I. In branchial epithelia, development and differentiation of the seawater-type chloride cell (and their underlying biochemistry) is regulated by GH, IGF-I, and cortisol, whereas the freshwater-type chloride cell is regulated by prolactin and cortisol. In the epithelia of gastrointestinal tract, prolactin induces cell proliferation during freshwater adaptation, whereas cortisol stimulates both cell proliferation and apoptosis. We propose that control of salinity acclimation in teleosts by prolactin and growth hormone primarily involves regulation of cell proliferation, apoptosis, and differentiation (the latter including upregulation of specific ion transporters), and that there is an important interaction of these hormones with corticosteroids. ?? 2005 Elsevier Inc. All rights reserved.

Heterogeneous expression and regulation of CD40 in human hepatocellular carcinoma.

PubMed

Holub, Margareta; Zakeri, Schaker M; Lichtenberger, Cornelia; Pammer, Johannes; Paolini, Pierre; Leifeld, Ludger; Rockenschaub, Susanne; Wolschek, Markus F; Steger, Günther; Willheim, Martin; Gangl, Alfred; Reinisch, Walter

2003-02-01

CD40, a member of the tumour necrosis factor receptor family, plays a major role in adaptive immune responses and contributes to cancer surveillance. Conflicting results have been reported recently on the expression and function of CD40 in carcinomas. The aim of the present study was to investigate the role of CD40 in human hepatoma. CD40 expression was examined in hepatomas and derived cell lines by immunohistochemistry, flow cytometry and reverse transcriptase polymerase chain reaction. We investigated in hepatoma cell lines the regulation of CD40 by pro-inflammatory cytokines and the effects of its ligation with soluble CD40L on the expression of co-stimulatory and pro-apoptotic cell-surface molecules and survival. CD40 was detected with a similar frequency of about 40% in hepatoma specimens and derived cell lines but not in normal hepatocytes. Tumour necrosis factor alpha and its combination with interferon gamma upregulated CD40 only in intrinsically positive cell lines. CD40 ligation had no effect on cell viability or surface expression of CD54, CD80, CD86 or CD95. CD40 is expressed variably in human hepatoma and enhanced by distinct pro-inflammatory cytokines. The lack of detectable effects of CD40 ligation does not support a major role of this molecule in hepatocellular carcinoma biology.

PrP(C) regulates epidermal growth factor receptor function and cell shape dynamics in Neuro2a cells.

PubMed

Llorens, Franc; Carulla, Patricia; Villa, Ana; Torres, Juan M; Fortes, Puri; Ferrer, Isidre; del Río, José A

2013-10-01

The prion protein (PrP) plays a key role in prion disease pathogenesis. Although the misfolded and pathologic variant of this protein (PrP(SC)) has been studied in depth, the physiological role of PrP(C) remains elusive and controversial. PrP(C) is a cell-surface glycoprotein involved in multiple cellular functions at the plasma membrane, where it interacts with a myriad of partners and regulates several intracellular signal transduction cascades. However, little is known about the gene expression changes modulated by PrP(C) in animals and in cellular models. In this article, we present PrP(C)-dependent gene expression signature in N2a cells and its implication in the most overrepresented functions: cell cycle, cell growth and proliferation, and maintenance of cell shape. PrP(C) over-expression enhances cell proliferation and cell cycle re-entrance after serum stimulation, while PrP(C) silencing slows down cell cycle progression. In addition, MAP kinase and protein kinase B (AKT) pathway activation are under the regulation of PrP(C) in asynchronous cells and following mitogenic stimulation. These effects are due in part to the modulation of epidermal growth factor receptor (EGFR) by PrP(C) in the plasma membrane, where the two proteins interact in a multimeric complex. We also describe how PrP(C) over-expression modulates filopodia formation by Rho GTPase regulation mainly in an AKT-Cdc42-N-WASP-dependent pathway. © 2013 International Society for Neurochemistry.

Modulation of tumor necrosis factor (TNF) receptor expression during monocytic differentiation by glucocorticoids.

PubMed

Goppelt-Struebe, M; Reiser, C O; Schneider, N; Grell, M

1996-10-01

Regulation of tumor necrosis factor receptors by glucocorticoids was investigated during phorbol ester-induced monocytic differentiation. As model system the human monocytic cell lines U937 and THP-1, which express both types of TNF receptors (TNF-R60 and TNF-R80), were differentiated with tetradecanoyl phorbol-13-acetate (TPA, 5 x 10(-9) M) in the presence or absence of dexamethasone (10(-9) - 10(-6) M). Expression of TNF receptors was determined at the mRNA level by Northern blot analysis and at the protein level by FACS analysis. During differentiation, TNF-R60 mRNA was down-regulated, whereas TNF-R80 mRNA levels were increased. Dexamethasone had no effect on TNF-R60 mRNA expression but attenuated TNF-R80 mRNA expression in both cell lines. Cell surface expression of TNF-R60 protein remained essentially unchanged during differentiation of THP-1 cells, whereas a rapid down-regulation of TNF-R80 was observed that was followed by a slow recovery. Surface expression of TNF-R80 was not affected by dexamethasone, whereas TNF-R60 expression was reduced by about 25%. These results indicate differential regulation of the two types of TNF receptors at the mRNA and protein level during monocytic differentiation. Glucocorticoids interfered with mRNA expression of TNF-R80 and protein expression of TNF-R60, but the rather limited effect leaves the question of its functional relevance open. In contrast to other cytokine systems, TNF receptors do not appear to be major targets of glucocorticoid action.

CD45RO enriches for activated, highly mutated human germinal center B cells

PubMed Central

Jackson, Stephen M.; Harp, Natessa; Patel, Darshna; Zhang, Jeffrey; Willson, Savannah; Kim, Yoon J.; Clanton, Christian

2007-01-01

To date, there is no consensus regarding the influence of different CD45 isoforms during peripheral B-cell development. Examining correlations between surface CD45RO expression and various physiologic processes ongoing during the germinal center (GC) reaction, we hypothesized that GC B cells, like T cells, that up-regulate surface RO should progressively acquire phenotypes commonly associated with activated, differentiating lymphocytes. GC B cells (IgD−CD38+) were subdivided into 3 surface CD45RO fractions: RO−, RO+/−, and RO+. We show here that the average number of mutations per IgVH transcript increased in direct correlation with surface RO levels. Conjunctional use of RO and CD69 further delineated low/moderately and highly mutated fractions. Activation-induced cytidine deaminase (AID) mRNA was slightly reduced among RO+ GC B cells, suggesting that higher mutation averages are unlikely due to elevated somatic mutation activity. Instead, RO+ GC B cells were negative for Annexin V, comprised mostly (93%) of CD77− centrocytes, and were enriched for CD69+ cells. Collectively, RO+ GC B cells occupy what seems to be a specialized niche comprised mostly of centrocytes that may be in transition between activation states. These findings are among the first to sort GC B cells into populations enriched for live mutated cells solely using a single extracellular marker. PMID:17644737

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development.

PubMed

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-04-07

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3varepsilon proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3zeta-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development.

Tonic ubiquitylation controls T-cell receptor:CD3 complex expression during T-cell development

PubMed Central

Wang, Haopeng; Holst, Jeff; Woo, Seng-Ryong; Guy, Cliff; Bettini, Matt; Wang, Yao; Shafer, Aaron; Naramura, Mayumi; Mingueneau, Michaël; Dragone, Leonard L; Hayes, Sandra M; Malissen, Bernard; Band, Hamid; Vignali, Dario A A

2010-01-01

Expression of the T-cell receptor (TCR):CD3 complex is tightly regulated during T-cell development. The mechanism and physiological role of this regulation are unclear. Here, we show that the TCR:CD3 complex is constitutively ubiquitylated in immature double positive (DP) thymocytes, but not mature single positive (SP) thymocytes or splenic T cells. This steady state, tonic CD3 monoubiquitylation is mediated by the CD3ɛ proline-rich sequence, Lck, c-Cbl, and SLAP, which collectively trigger the dynamin-dependent downmodulation, lysosomal sequestration and degradation of surface TCR:CD3 complexes. Blocking this tonic ubiquitylation by mutating all the lysines in the CD3 cytoplasmic tails significantly upregulates TCR levels on DP thymocytes. Mimicking monoubiquitylation by expression of a CD3ζ-monoubiquitin (monoUb) fusion molecule significantly reduces TCR levels on immature thymocytes. Moreover, modulating CD3 ubiquitylation alters immunological synapse (IS) formation and Erk phosphorylation, thereby shifting the signalling threshold for positive and negative selection, and regulatory T-cell development. Thus, tonic TCR:CD3 ubiquitylation results in precise regulation of TCR expression on immature T cells, which is required to maintain the fidelity of T-cell development. PMID:20150895

VANGL2 regulates membrane trafficking of MMP14 to control cell polarity and migration.

PubMed

Williams, B Blairanne; Cantrell, V Ashley; Mundell, Nathan A; Bennett, Andrea C; Quick, Rachel E; Jessen, Jason R

2012-05-01

Planar cell polarity (PCP) describes the polarized orientation of cells within the plane of a tissue. Unlike epithelial PCP, the mechanisms underlying PCP signaling in migrating cells remain undefined. Here, the establishment of PCP must be coordinated with dynamic changes in cell adhesion and extracellular matrix (ECM) organization. During gastrulation, the membrane type-1 matrix metalloproteinase (MT1-MMP or MMP14) is required for PCP and convergence and extension cell movements. We report that the PCP protein Vang-like 2 (VANGL2) regulates the endocytosis and cell-surface availability of MMP14 in manner that is dependent on focal adhesion kinase. We demonstrate that zebrafish trilobite/vangl2 mutant embryos exhibit increased Mmp14 activity and decreased ECM. Furthermore, in vivo knockdown of Mmp14 partially rescues the Vangl2 loss-of-function convergence and extension phenotype. This study identifies a mechanism linking VANGL2 with MMP14 trafficking and suggests that establishment of PCP in migrating gastrula cells requires regulated proteolytic degradation or remodeling of the ECM. Our findings implicate matrix metalloproteinases as downstream effectors of PCP and suggest a broadly applicable mechanism whereby VANGL2 affects diverse morphogenetic processes.

Regulation of the Min Cell Division Inhibition Complex by the Rcs Phosphorelay in Proteus mirabilis.

PubMed

Howery, Kristen E; Clemmer, Katy M; Şimşek, Emrah; Kim, Minsu; Rather, Philip N

2015-08-01

A key regulator of swarming in Proteus mirabilis is the Rcs phosphorelay, which represses flhDC, encoding the master flagellar regulator FlhD4C2. Mutants in rcsB, the response regulator in the Rcs phosphorelay, hyperswarm on solid agar and differentiate into swarmer cells in liquid, demonstrating that this system also influences the expression of genes central to differentiation. To gain a further understanding of RcsB-regulated genes involved in swarmer cell differentiation, transcriptome sequencing (RNA-Seq) was used to examine the RcsB regulon. Among the 133 genes identified, minC and minD, encoding cell division inhibitors, were identified as RcsB-activated genes. A third gene, minE, was shown to be part of an operon with minCD. To examine minCDE regulation, the min promoter was identified by 5' rapid amplification of cDNA ends (5'-RACE), and both transcriptional lacZ fusions and quantitative real-time reverse transcriptase (qRT) PCR were used to confirm that the minCDE operon was RcsB activated. Purified RcsB was capable of directly binding the minC promoter region. To determine the role of RcsB-mediated activation of minCDE in swarmer cell differentiation, a polar minC mutation was constructed. This mutant formed minicells during growth in liquid, produced shortened swarmer cells during differentiation, and exhibited decreased swarming motility. This work describes the regulation and role of the MinCDE cell division system in P. mirabilis swarming and swarmer cell elongation. Prior to this study, the mechanisms that inhibit cell division and allow swarmer cell elongation were unknown. In addition, this work outlines for the first time the RcsB regulon in P. mirabilis. Taken together, the data presented in this study begin to address how P. mirabilis elongates upon contact with a solid surface. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Cell surface marker profiling of human tracheal basal cells reveals distinct subpopulations, identifies MST1/MSP as a mitogenic signal, and identifies new biomarkers for lung squamous cell carcinomas.

PubMed

Van de Laar, Emily; Clifford, Monica; Hasenoeder, Stefan; Kim, Bo Ram; Wang, Dennis; Lee, Sharon; Paterson, Josh; Vu, Nancy M; Waddell, Thomas K; Keshavjee, Shaf; Tsao, Ming-Sound; Ailles, Laurie; Moghal, Nadeem

2014-12-31

The large airways of the lungs (trachea and bronchi) are lined with a pseudostratified mucociliary epithelium, which is maintained by stem cells/progenitors within the basal cell compartment. Alterations in basal cell behavior can contribute to large airway diseases including squamous cell carcinomas (SQCCs). Basal cells have traditionally been thought of as a uniform population defined by basolateral position, cuboidal cell shape, and expression of pan-basal cell lineage markers like KRT5 and TP63. While some evidence suggests that basal cells are not all functionally equivalent, few heterogeneously expressed markers have been identified to purify and study subpopulations. In addition, few signaling pathways have been identified that regulate their cell behavior. The goals of this work were to investigate tracheal basal cell diversity and to identify new signaling pathways that regulate basal cell behavior. We used flow cytometry (FACS) to profile cell surface marker expression at a single cell level in primary human tracheal basal cell cultures that maintain stem cell/progenitor activity. FACS results were validated with tissue staining, in silico comparisons with normal basal cell and lung cancer datasets, and an in vitro proliferation assay. We identified 105 surface markers, with 47 markers identifying potential subpopulations. These subpopulations generally fell into more (~ > 13%) or less abundant (~ < 6%) groups. Microarray gene expression profiling supported the heterogeneous expression of these markers in the total population, and immunostaining of large airway tissue suggested that some of these markers are relevant in vivo. 24 markers were enriched in lung SQCCs relative to adenocarcinomas, with four markers having prognostic significance in SQCCs. We also identified 33 signaling receptors, including the MST1R/RON growth factor receptor, whose ligand MST1/MSP was mitogenic for basal cells. This work provides the largest description to date of molecular diversity among human large airway basal cells. Furthermore, these markers can be used to further study basal cell function in repair and disease, and may aid in the classification and study of SQCCs.

The Pseudomonas aeruginosa ribbon-helix-helix DNA-binding protein AlgZ (AmrZ) controls twitching motility and biogenesis of type IV pili.

PubMed

Baynham, Patricia J; Ramsey, Deborah M; Gvozdyev, Borys V; Cordonnier, Ellen M; Wozniak, Daniel J

2006-01-01

Pseudomonas aeruginosa is an opportunistic pathogen that is commonly found in water and soil. In order to colonize surfaces with low water content, P. aeruginosa utilizes a flagellum-independent form of locomotion called twitching motility, which is dependent upon the extension and retraction of type IV pili. This study demonstrates that AlgZ, previously identified as a DNA-binding protein absolutely required for transcription of the alginate biosynthetic operon, is required for twitching motility. AlgZ may be required for the biogenesis or function of type IV pili in twitching motility. Transmission electron microscopy analysis of an algZ deletion in nonmucoid PAO1 failed to detect surface pili. To examine expression and localization of PilA (the major pilin subunit), whole-cell extracts and cell surface pilin preparations were analyzed by Western blotting. While the PilA levels present in whole-cell extracts were similar for wild-type P. aeruginosa and P. aeruginosa with the algZ deletion, the amount of PilA on the surface of the cells was drastically reduced in the algZ mutant. Analysis of algZ and algD mutants indicates that the DNA-binding activity of AlgZ is essential for the regulation of twitching motility and that this is independent of the role of AlgZ in alginate expression. These data show that AlgZ DNA-binding activity is required for twitching motility independently of its role in alginate production and that this involves the surface localization of type IV pili. Given this new role in twitching motility, we propose that algZ (PA3385) be designated amrZ (alginate and motility regulator Z).

Rebamipide suppresses PolyI:C-stimulated cytokine production in human conjunctival epithelial cells.

PubMed

Ueta, Mayumi; Sotozono, Chie; Yokoi, Norihiko; Kinoshita, Shigeru

2013-09-01

We previously documented that ocular surface epithelial cells could regulate ocular surface inflammation and suggested that, while Toll-like receptor 3 upregulates, EP3, one of the prostaglandin E2 receptors, downregulates ocular surface inflammation. Others reported that rebamipide, a gastroprotective drug, could not only increase the gastric mucus production, but also suppressed gastric mucosal inflammation and that it was dominantly distributed in mucosal tissues. The eyedrop form of rebamipide, approved in Japan for use in the treatment of dry eye diseases, upregulates mucin secretion and production, thereby suppressing superficial punctate keratopathy on the ocular surface of patients with this disease. In the current study, we investigated whether rebamipide has anti- inflammatory effects on the ocular surface. To examine the effects of rebamipide on polyI:C-induced cytokine expression by primary human conjunctival epithelial cells, we used enzyme-linked immunosorbent assay and quantitative reverse transcription-polymerase chain reaction assay. We studied the effects of rebamipide on ocular surface inflammation in our murine experimental allergic conjunctivitis (EAC) model. Rebamipide could suppress polyI:C-induced cytokine production and the expression of mRNAs for CXCL10, CXCL11, RANTES, MCP-1, and IL-6 in human conjunctival epithelial cells. In our EAC model, the topical administration of rebamipide suppressed conjunctival allergic eosinophil infiltration. The topical application of rebamipide on the ocular surface might suppress ocular surface inflammation by suppressing the production of cytokines by ocular surface epithelial cells.

Neurotrophin responsiveness of sympathetic neurons is regulated by rapid mobilization of the p75 receptor to the cell surface through TrkA activation of Arf6.

PubMed

Edward Hickman, F; Stanley, Emily M; Carter, Bruce D

2018-05-22

The p75 neurotrophin receptor (p75NTR) plays an integral role in patterning the sympathetic nervous system during development. Initially, p75NTR is expressed at low levels as sympathetic axons project toward their targets, which enables neurotrophin-3 (NT3) to activate TrkA receptors and promote growth. Upon reaching nerve growth factor (NGF) producing tissues, p75NTR is up regulated resulting in formation of TrkA-p75 complexes, which are high affinity binding sites selective for NGF, thereby blunting NT3 signaling. The level of p75NTR expressed on the neuron surface is instrumental in regulating trophic factor response; however, the mechanisms by which p75NTR expression is regulated are poorly understood. Here, we demonstrate a rapid, translation independent increase in surface expression of p75NTR in response to NGF in rat sympathetic neurons. p75NTR was mobilized to the neuron surface from GGA3-postitive vesicles through activation of the GTPase Arf6, which was stimulated by NGF, but not NT3 binding to TrkA. Arf6 activation required PI3 kinase activity and was prevented by an inhibitor of the cytohesin family of Arf6 GEFs. Overexpression of a constitutively active Arf6 mutant (Q67L) was sufficient to significantly increase surface expression of p75NTR even in the absence of NGF. Functionally, expression of active Arf6 markedly attenuated the ability of NT3 to promote neuronal survival and neurite outgrowth while the NGF response was unaltered. These data suggest that NGF activation of Arf6 through TrkA is critical for the increase in p75NTR surface expression that enables the switch in neurotrophin responsiveness during development in the sympathetic nervous system. SIGNIFICANCE STATEMENT p75NTR is instrumental in the regulation of neuronal survival and apoptosis during development and is also implicated as a contributor to aberrant neurodegeneration in numerous conditions. Therefore, a better understanding of the mechanisms that mediate p75NTR surface availability, may provide insight into how and why neurodegenerative processes manifest and reveal new therapeutic targets. Results from this study indicate a novel mechanism by which p75NTR can be rapidly shuttled to the cell surface from existing intracellular pools and explores a unique pathway by which NGF regulates the sympathetic innervation of target tissues, which has profound consequences for the function of these organs. Copyright © 2018 the authors.

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus.

PubMed

Senesi, Sonia; Ghelardi, Emilia; Celandroni, Francesco; Salvetti, Sara; Parisio, Eva; Galizzi, Alessandro

2004-02-01

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.

Three-dimensional manipulation of single cells using surface acoustic waves

PubMed Central

Guo, Feng; Mao, Zhangming; Chen, Yuchao; Xie, Zhiwei; Lata, James P.; Li, Peng; Ren, Liqiang; Liu, Jiayang; Yang, Jian; Dao, Ming; Suresh, Subra; Huang, Tony Jun

2016-01-01

The ability of surface acoustic waves to trap and manipulate micrometer-scale particles and biological cells has led to many applications involving “acoustic tweezers” in biology, chemistry, engineering, and medicine. Here, we present 3D acoustic tweezers, which use surface acoustic waves to create 3D trapping nodes for the capture and manipulation of microparticles and cells along three mutually orthogonal axes. In this method, we use standing-wave phase shifts to move particles or cells in-plane, whereas the amplitude of acoustic vibrations is used to control particle motion along an orthogonal plane. We demonstrate, through controlled experiments guided by simulations, how acoustic vibrations result in micromanipulations in a microfluidic chamber by invoking physical principles that underlie the formation and regulation of complex, volumetric trapping nodes of particles and biological cells. We further show how 3D acoustic tweezers can be used to pick up, translate, and print single cells and cell assemblies to create 2D and 3D structures in a precise, noninvasive, label-free, and contact-free manner. PMID:26811444

Characterizing Spatial Organization of Cell Surface Receptors in Human Breast Cancer with STORM

NASA Astrophysics Data System (ADS)

Lyall, Evan; Chapman, Matthew R.; Sohn, Lydia L.

2012-02-01

Regulation and control of complex biological functions are dependent upon spatial organization of biological structures at many different length scales. For instance Eph receptors and their ephrin ligands bind when opposing cells come into contact during development, resulting in spatial organizational changes on the nanometer scale that lead to changes on the macro scale, in a process known as organ morphogenesis. One technique able to probe this important spatial organization at both the nanometer and micrometer length scales, including at cell-cell junctions, is stochastic optical reconstruction microscopy (STORM). STORM is a technique that localizes individual fluorophores based on the centroids of their point spread functions and then reconstructs a composite image to produce super resolved structure. We have applied STORM to study spatial organization of the cell surface of human breast cancer cells, specifically the organization of tyrosine kinase receptors and chemokine receptors. A better characterization of spatial organization of breast cancer cell surface proteins is necessary to fully understand the tumorigenisis pathways in the most common malignancy in United States women.

The Human Cell Surfaceome of Breast Tumors

PubMed Central

da Cunha, Júlia Pinheiro Chagas; Galante, Pedro Alexandre Favoretto; de Souza, Jorge Estefano Santana; Pieprzyk, Martin; Carraro, Dirce Maria; Old, Lloyd J.; Camargo, Anamaria Aranha; de Souza, Sandro José

2013-01-01

Introduction. Cell surface proteins are ideal targets for cancer therapy and diagnosis. We have identified a set of more than 3700 genes that code for transmembrane proteins believed to be at human cell surface. Methods. We used a high-throuput qPCR system for the analysis of 573 cell surface protein-coding genes in 12 primary breast tumors, 8 breast cell lines, and 21 normal human tissues including breast. To better understand the role of these genes in breast tumors, we used a series of bioinformatics strategies to integrates different type, of the datasets, such as KEGG, protein-protein interaction databases, ONCOMINE, and data from, literature. Results. We found that at least 77 genes are overexpressed in breast primary tumors while at least 2 of them have also a restricted expression pattern in normal tissues. We found common signaling pathways that may be regulated in breast tumors through the overexpression of these cell surface protein-coding genes. Furthermore, a comparison was made between the genes found in this report and other genes associated with features clinically relevant for breast tumorigenesis. Conclusions. The expression profiling generated in this study, together with an integrative bioinformatics analysis, allowed us to identify putative targets for breast tumors. PMID:24195083

Plasmin-Cleaved β-2-Glycoprotein 1 Is an Inhibitor of Angiogenesis

PubMed Central

Sakai, Taro; Balasubramanian, Krishnakumar; Maiti, Sourindra; Halder, Jyotsna B.; Schroit, Alan J.

2007-01-01

β-2-Glycoprotein 1, an abundant plasma glycoprotein, binds anionic cell surfaces and functions as a regulator of thrombosis. Here, we show that cleavage of the kringle domain at Lys317/Thr318 switches its function to a regulator of angiogenesis. In vitro, the cleaved protein specifically inhibited the proliferation and migration of endothelial cells. The protein was without effect on preformed endothelial cell tubes. In vivo, the cleaved protein inhibited neovascularization into subcutaneously implanted Matrigel and Gelfoam sponge implants and the growth of orthotopically injected tumors. Collectively, these data indicate that plasmin-cleaved β-2-glycoprotein 1 is a potent antiangiogenic and antitumor molecule of potential therapeutic significance. PMID:17872974

Growth regulation in tip-growing cells that develop on the epidermis.

PubMed

Honkanen, Suvi; Dolan, Liam

2016-12-01

Plants develop tip-growing extensions-root hairs and rhizoids-that initiate as swellings on the outer surface of individual epidermal cells. A conserved genetic mechanism controls the earliest stages in the initiation of these swellings. The same mechanism controls the formation of multicellular structures that develop from swellings on epidermal cells in early diverging land plants. Details of the molecular events that regulate the positioning of the swellings involve sterols and phosphatidylinositol phosphates. The final length of root hairs is determined by the intensity of a pulse of transcription factor synthesis. Genes encoding similar transcription factors control root hair development in cereals and are potential targets for crop improvement. Copyright © 2016. Published by Elsevier Ltd.

Analysis of telomerase activity based on a spired DNA tetrahedron TS primer.

PubMed

Li, Yan; Wen, Yanli; Wang, Lele; Liang, Wen; Xu, Li; Ren, Shuzhen; Zou, Ziying; Zuo, Xiaolei; Fan, Chunhai; Huang, Qing; Liu, Gang; Jia, Nengqin

2015-05-15

The development of sensitive telomerase biosensors is hindered by the restricted accessibility of telomere strand (TS) primer and the limited enzyme reaction space, which is mainly confined by the vertical distance. In this work, we designed an electrochemical telomerase biosensor based on a spired DNA tetrahedron TS primer (STTS). By adding a rigid dsDNA spire onto the top of the DNA tetrahedron, we successfully regulated the distance between the TS primer and the surface, and thus greatly facilitated the telomerase elongation on surface. The signal-to-noise ratio was 2 times higher than TSP without the spire structure. The limit of detection was calculated to be lower than 10 HeLa cells, which is at least 2 magnitudes lower than other surface extension-based electrochemical telomerase sensors without amplification. The practicability of STTS sensor was also demonstrated by analysing various other cell lines including cancer cells, stem cells of high telomerase activity and somatic cells of low telomerase activity. Copyright © 2014 Elsevier B.V. All rights reserved.

Activation of p44/42 in Human Natural Killer Cells Decreases Cell-surface Protein Expression: Relationship to Tributyltin-induced alterations of protein expression

PubMed Central

Dudimah, Fred D.; Abraha, Abraham; Wang, Xiaofei; Whalen, Margaret M.

2010-01-01

Tributyltin (TBT) activates the mitogen activated protein kinase (MAPK), p44/42 in human natural killer (NK) cells. TBT also reduces NK cytotoxic function and decreases the expression of several NK-cell proteins. To understand the role that p44/42 activation plays in TBT-induced loss of NK cell function, we have investigated how selective activation of p44/42 by phorbol 12-myristate 13-acetate (PMA) affects NK cells. Previously we showed that PMA caused losses of lytic function similar to those seen with TBT exposures. Here we examined activation of p44/42 in the regulation of NK-cell protein expression and how this regulation may explain the protein expression changes seen with TBT exposures. NK cells exposed to PMA were examined for levels of cell-surface proteins, granzyme mRNA, and perforin mRNA expression. The expression of CD11a, CD16, CD18, and CD56 were reduced, perforin mRNA levels were unchanged and granzyme mRNA levels were increased. To verify that activation of p44/42 was responsible for the alterations seen in CD11a, CD16, CD18, and CD56 with PMA, NK cells were treated with the p44/42 pathway inhibitor (PD98059) prior to PMA exposures. In the presence of PD98059, PMA caused no decreases in the expression of the cell-surface proteins. Results of these studies indicate that the activation of p44/42 may lead to the loss of NK cell cytotoxic function by decreasing the expression of CD11a, CD16, CD18, and CD56. Further, activation of p44/42 appears to be at least in part responsible for the TBT-induced decreases in expression of CD16, CD18, and CD56. PMID:20883105

Baclofen mediates neuroprotection on hippocampal CA1 pyramidal cells through the regulation of autophagy under chronic cerebral hypoperfusion

PubMed Central

Liu, Li; Li, Chang-jun; Lu, Yun; Zong, Xian-gang; Luo, Chao; Sun, Jun; Guo, Lian-jun

2015-01-01

GABA receptors play an important role in ischemic brain injury. Studies have indicated that autophagy is closely related to neurodegenerative diseases. However, during chronic cerebral hypoperfusion, the changes of autophagy in the hippocampal CA1 area, the correlation between GABA receptors and autophagy, and their influences on hippocampal neuronal apoptosis have not been well established. Here, we found that chronic cerebral hypoperfusion resulted in rat hippocampal atrophy, neuronal apoptosis, enhancement and redistribution of autophagy, down-regulation of Bcl-2/Bax ratio, elevation of cleaved caspase-3 levels, reduction of surface expression of GABAA receptor α1 subunit and an increase in surface and mitochondrial expression of connexin 43 (CX43) and CX36. Chronic administration of GABAB receptors agonist baclofen significantly alleviated neuronal damage. Meanwhile, baclofen could up-regulate the ratio of Bcl-2/Bax and increase the activation of Akt, GSK-3β and ERK which suppressed cytodestructive autophagy. The study also provided evidence that baclofen could attenuate the decrease in surface expression of GABAA receptor α1 subunit, and down-regulate surface and mitochondrial expression of CX43 and CX36, which might enhance protective autophagy. The current findings suggested that, under chronic cerebral hypoperfusion, the effects of GABAB receptors activation on autophagy regulation could reverse neuronal damage. PMID:26412641

Isolating and Analyzing Cells of the Pancreas Mesenchyme by Flow Cytometry.

PubMed

Epshtein, Alona; Sakhneny, Lina; Landsman, Limor

2017-01-28

The pancreas is comprised of epithelial cells that are required for food digestion and blood glucose regulation. Cells of the pancreas microenvironment, including endothelial, neuronal, and mesenchymal cells were shown to regulate cell differentiation and proliferation in the embryonic pancreas. In the adult, the function and mass of insulin-producing cells were shown to depend on cells in their microenvironment, including pericyte, immune, endothelial, and neuronal cells. Lastly, changes in the pancreas microenvironment were shown to regulate pancreas tumorigenesis. However, the cues underlying these processes are not fully defined. Therefore, characterizing the different cell types that comprise the pancreas microenvironment and profiling their gene expression are crucial to delineate the tissue development and function under normal and diseased states. Here, we describe a method that allows for the isolation of mesenchymal cells from the pancreas of embryonic, neonatal, and adult mice. This method utilizes the enzymatic digestion of mouse pancreatic tissue and the subsequent fluorescence-activated cell sorting (FACS) or flow-cytometric analysis of labeled cells. Cells can be labeled by either immunostaining for surface markers or by the expression of fluorescent proteins. Cell isolation can facilitate the characterization of genes and proteins expressed in cells of the pancreas mesenchyme. This protocol was successful in isolating and culturing highly enriched mesenchymal cell populations from the embryonic, neonatal, and adult mouse pancreas.

Interplay of the modified nucleotide phosphoadenosine 5'-phosphosulfate (PAPS) with global regulatory proteins in Escherichia coli: modulation of cyclic AMP (cAMP)-dependent gene expression and interaction with the HupA regulatory protein.

PubMed

Longo, Francesca; Motta, Sara; Mauri, Pierluigi; Landini, Paolo; Rossi, Elio

2016-11-25

In the bacterium Escherichia coli, some intermediates of the sulfate assimilation and cysteine biosynthesis pathway can act as signal molecules and modulate gene expression. In addition to sensing and utilization of sulphur sources, these signaling mechanisms also impact more global cell processes, such as resistance to antimicrobial agents and biofilm formation. In a recent work, we have shown that inactivation of the cysH gene, encoding phosphoadenosine-phosphosulfate (PAPS) reductase, and the consequent increase in intracellular PAPS concentration, strongly affect production of several cell surface-associated structures, enhancing surface adhesion and cell aggregation. In order to identify the molecular mechanism relaying intracellular PAPS concentration to regulation of cell surface-associated structures, we looked for mutations able to suppress the effects of cysH inactivation. We found that mutations in the adenylate cyclase-encoding cyaA gene abolished the effects of PAPS accumulation; consistent with this result, cyclic AMP (cAMP)-dependent gene expression appears to be increased in the cysH mutant. Experiments aimed at the direct identification of proteins interacting with either CysC or CysH, i.e. the PAPS-related proteins APS kinase and PAPS reductase, allowed us to identify several regulators, namely, CspC, CspE, HNS and HupA. Protein-protein interaction between HupA and CysH was confirmed by a bacterial two hybrid system, and inactivation of the hupA gene enhanced the effects of the cysH mutation in terms of production of cell surface-associated factors. Our results indicate that PAPS can modulate different regulatory systems, providing evidence that this molecule acts as a global signal molecule in E. coli. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Comparative Transcriptome Analysis of Desulfovibrio Vulgaris Grown in Planktonic Culture and Mature Biofilm on a Steel Surface

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Weiwen; Culley, David E.; Nie, Lei

2007-08-01

The build-up of biofilms of sulphate -reducing bacteria (SRB) on metals surfaces may lead to severe corrosion of iron. To understand the processes at molecular level, in this study, a whole-genome oligonucleotide microarray was used to examine differential expression patterns between planktonic populations and mature biofilm of model SRB species Desulfovibrio vulgaris. Statistical analysis revealed that 472 genes were differentially expressed (1.5 fold or more with a p value less than 0.025) when comparing biofilm to planktonic cells. Among the differentially expressed genes were several that corresponded to biofilm formation genes identified in many aerobic bacterial biofilms (i.e., Pseudomonas speciesmore » and Escherichia coli), such as down-regulation of genes encoding flagellin, flagellar motor switch protein and chemotaxis proteins involved in cell motility and induction of genes encoding sugar transferase and glycogen synthase involved in exopolysaccharide biosynthesis. In addition, D. vulgaris biofilm-bound cells exhibited decreased transcription of genes involved in protein synthesis, energy metabolism and sulfate reduction, as well as genes involved in general stress responses. These findings were all consistent with early suggestion that the average physiology of biofilm cells were similar to planktonic cells of stationary phases. Most notably, up-regulation of large number of outer membrane proteins was observed in D. vulgaris biofilm. Although their function is still unknown, the higher expression of these genes in D. vulgaris biofilm could implicate important roles formation and maintenance of multi-cellular consortium on metal surface. The study provided insights into the metabolic networks associated with D. vulgaris biofilm formation and maintenance on an iron surface.« less

Ca2+ Binding to EF Hands 1 and 3 Is Essential for the Interaction of Apoptosis-Linked Gene-2 with Alix/AIP1 in Ocular Melanoma†

PubMed Central

Subramanian, Lalita; Polans, Arthur S.; Walker, Teresa M.; van Ginkel, Paul R.; Bhattacharya, Saswati; Dellaria, Julia M.; Crabb, John W.; Cox, Jos; Durussel, Isabelle; Palczewski, Krzysztof

2005-01-01

Apoptosis-linked gene-2 (ALG-2) encodes a 22 kDa Ca2+-binding protein of the penta EF-hand family that is required for programmed cell death in response to various apoptotic agents. Here, we demonstrate that ALG-2 mRNA and protein are down-regulated in human uveal melanoma cells compared to their progenitor cells, normal melanocytes. The down regulation of ALG-2 may provide melanoma cells with a selective advantage. ALG-2 and its putative target molecule, Alix/AIP1, are localized primarily in the cytoplasm of melanocytes and melanoma cells independent of the intracellular Ca2+ concentration or the activation of apoptosis. Cross-linking and analytical centrifugation studies support a single-species dimer conformation of ALG-2, also independent of Ca2+ concentration. However, binding of Ca2+ to both EF-1 and EF-3 is necessary for ALG-2 interaction with Alix/AIP1 as demonstrated using surface plasmon resonance spectroscopy. Mutations in EF-5 result in reduced target interaction without alteration in Ca2+ affinity. The addition of N-terminal ALG-2 peptides, residues 1–22 or residues 7–17, does not alter the interaction of ALG-2 or an N-terminal deletion mutant of ALG-2 with Alix/AIP1, as might be expected from a model derived from the crystal structure of ALG-2. Fluorescence studies of ALG-2 demonstrate that an increase in surface hydrophobicity is primarily due to Ca2+ binding to EF-3, while Ca2+ binding to EF-1 has little effect on surface exposure of hydrophobic residues. Together, these data indicate that gross surface hydrophobicity changes are insufficient for target recognition. PMID:15366927

Defining an optimal surface chemistry for pluripotent stem cell culture in 2D and 3D

NASA Astrophysics Data System (ADS)

Zonca, Michael R., Jr.

Surface chemistry is critical for growing pluripotent stem cells in an undifferentiated state. There is great potential to engineer the surface chemistry at the nanoscale level to regulate stem cell adhesion. However, the challenge is to identify the optimal surface chemistry of the substrata for ES cell attachment and maintenance. Using a high-throughput polymerization and screening platform, a chemically defined, synthetic polymer grafted coating that supports strong attachment and high expansion capacity of pluripotent stem cells has been discovered using mouse embryonic stem (ES) cells as a model system. This optimal substrate, N-[3-(Dimethylamino)propyl] methacrylamide (DMAPMA) that is grafted on 2D synthetic poly(ether sulfone) (PES) membrane, sustains the self-renewal of ES cells (up to 7 passages). DMAPMA supports cell attachment of ES cells through integrin beta1 in a RGD-independent manner and is similar to another recently reported polymer surface. Next, DMAPMA has been able to be transferred to 3D by grafting to synthetic, polymeric, PES fibrous matrices through both photo-induced and plasma-induced polymerization. These 3D modified fibers exhibited higher cell proliferation and greater expression of pluripotency markers of mouse ES cells than 2D PES membranes. Our results indicated that desirable surfaces in 2D can be scaled to 3D and that both surface chemistry and structural dimension strongly influence the growth and differentiation of pluripotent stem cells. Lastly, the feasibility of incorporating DMAPMA into a widely used natural polymer, alginate, has been tested. Novel adhesive alginate hydrogels have been successfully synthesized by either direct polymerization of DMAPMA and methacrylic acid blended with alginate, or photo-induced DMAPMA polymerization on alginate nanofibrous hydrogels. In particular, DMAPMA-coated alginate hydrogels support strong ES cell attachment, exhibiting a concentration dependency of DMAPMA. This research provides a new avenue for stem cell culture and maintenance using an optimal organic-based chemistry.

Allosteric Regulation of E-Cadherin Adhesion.

PubMed

Shashikanth, Nitesh; Petrova, Yuliya I; Park, Seongjin; Chekan, Jillian; Maiden, Stephanie; Spano, Martha; Ha, Taekjip; Gumbiner, Barry M; Leckband, Deborah E

2015-08-28

Cadherins are transmembrane adhesion proteins that maintain intercellular cohesion in all tissues, and their rapid regulation is essential for organized tissue remodeling. Despite some evidence that cadherin adhesion might be allosterically regulated, testing of this has been hindered by the difficulty of quantifying altered E-cadherin binding affinity caused by perturbations outside the ectodomain binding site. Here, measured kinetics of cadherin-mediated intercellular adhesion demonstrated quantitatively that treatment with activating, anti-E-cadherin antibodies or the dephosphorylation of a cytoplasmic binding partner, p120(ctn), increased the homophilic binding affinity of E-cadherin. Results obtained with Colo 205 cells, which express inactive E-cadherin and do not aggregate, demonstrated that four treatments, which induced Colo 205 aggregation and p120(ctn) dephosphorylation, triggered quantitatively similar increases in E-cadherin affinity. Several processes can alter cell aggregation, but these results directly demonstrated the allosteric regulation of cell surface E-cadherin by p120(ctn) dephosphorylation. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

Force-activatable coating enables high-resolution cellular force imaging directly on regular cell culture surfaces.

PubMed

Sarkar, Anwesha; Zhao, Yuanchang; Wang, Yongliang; Wang, Xuefeng

2018-06-25

Integrin-transmitted cellular forces are crucial mechanical signals regulating a vast range of cell functions. Although various methods have been developed to visualize and quantify cellular forces at the cell-matrix interface, a method with high performance and low technical barrier is still in demand. Here we developed a force-activatable coating (FAC), which can be simply coated on regular cell culture apparatus' surfaces by physical adsorption, and turn these surfaces to force reporting platforms that enable cellular force mapping directly by fluorescence imaging. The FAC molecule consists of an adhesive domain for surface coating and a force-reporting domain which can be activated to fluoresce by integrin molecular tension. The tension threshold required for FAC activation is tunable in 10-60 piconewton (pN), allowing the selective imaging of cellular force contributed by integrin tension at different force levels. We tested the performance of two FACs with tension thresholds of 12 and 54 pN (nominal values), respectively, on both glass and polystyrene surfaces. Cellular forces were successfully mapped by fluorescence imaging on all the surfaces. FAC-coated surfaces also enable co-imaging of cellular forces and cell structures in both live cells and immunostained cells, therefore opening a new avenue for the study of the interplay of force and structure. We demonstrated the co-imaging of integrin tension and talin clustering in live cells, and concluded that talin clustering always occurs before the generation of integrin tension above 54 pN, reinforcing the notion that talin is an important adaptor protein for integrin tension transmission. Overall, FAC provides a highly convenient approach that is accessible to general biological laboratories for the study of cellular forces with high sensitivity and resolution, thus holding the potential to greatly boost the research of cell mechanobiology.

Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior

PubMed Central

Liberio, Michelle S.; Sadowski, Martin C.; Soekmadji, Carolina; Davis, Rohan A.; Nelson, Colleen C.

2014-01-01

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-l-lysine, poly-l-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-l-lysine and poly-l-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement. PMID:25375165

Retention in the Golgi apparatus and expression on the cell surface of Cfr/Esl-1/Glg-1/MG-160 are regulated by two distinct mechanisms.

PubMed

Miyaoka, Yuichiro; Kato, Hidenori; Ebato, Kazuki; Saito, Shigeru; Miyata, Naoko; Imamura, Toru; Miyajima, Atsushi

2011-11-15

Cfr (cysteine-rich fibroblast growth factor receptor) is an Fgf (fibroblast growth factor)-binding protein without a tyrosine kinase. We have shown previously that Cfr is involved in Fgf18 signalling via Fgf receptor 3c. However, as Cfr is also known as Glg (Golgi apparatus protein)-1 or MG-160 and occurs in the Golgi apparatus, it remains unknown how the distribution of Cfr is regulated. In the present study, we performed a mutagenic analysis of Cfr to show that two distinct regions contribute to its distribution and stability. First, the C-terminal region retains Cfr in the Golgi apparatus. Secondly, the Cfr repeats in the extracellular juxtamembrane region destabilizes Cfr passed through the Golgi apparatus. This destabilization does not depend on the cleavage and secretion of the extracellular domain of Cfr. Furthermore, we found that Cfr with a GPI (glycosylphosphatidylinositol) anchor was predominantly expressed on the cell surface in Ba/F3 cells and affected Fgf18 signalling in a similar manner to the full-length Cfr, indicating that the interaction of Cfr with Fgfs on the cell surface is important for its function in Fgf signalling. These results suggest that the expression of Cfr in the Golgi apparatus and on the plasma membrane is finely tuned through two distinct mechanisms for exhibiting different functions.

Urokinase receptor expression involves tyrosine phosphorylation of phosphoglycerate kinase.

PubMed

Shetty, Praveenkumar; Velusamy, Thirunavukkarasu; Bhandary, Yashodhar P; Liu, Ming C; Shetty, Sreerama

2010-02-01

The interaction of urokinase-type plasminogen activator (uPA) with its receptor, uPAR, plays a central role in several pathophysiological processes, including cancer. uPA induces its own cell surface receptor expression through stabilization of uPAR mRNA. The mechanism involves binding of a 51 nt uPAR mRNA coding sequence with phosphoglycerate kinase (PGK) to down regulate cell surface uPAR expression. Tyrosine phosphorylation of PGK mediated by uPA treatment enhances uPAR mRNA stabilization. In contrast, inhibition of tyrosine phosphorylation augments PGK binding to uPAR mRNA and attenuates uPA-induced uPAR expression. Mapping the specific peptide region of PGK indicated that its first quarter (amino acids 1-100) interacts with uPAR mRNA. To determine if uPAR expression by uPA is regulated through activation of tyrosine residues of PGK, we mutated the specific tyrosine residue and tested mutant PGK for its ability to interfere with uPAR expression. Inhibition of tyrosine phosphorylation by mutating Y76 residue abolished uPAR expression induced by uPA treatment. These findings collectively demonstrate that Y76 residue present in the first quarter of the PGK molecule is involved in lung epithelial cell surface uPAR expression. This region can effectively mimic the function of a whole PGK molecule in inhibiting tumor cell growth.

CD4 mimetics sensitize HIV-1-infected cells to ADCC.

PubMed

Richard, Jonathan; Veillette, Maxime; Brassard, Nathalie; Iyer, Shilpa S; Roger, Michel; Martin, Loïc; Pazgier, Marzena; Schön, Arne; Freire, Ernesto; Routy, Jean-Pierre; Smith, Amos B; Park, Jongwoo; Jones, David M; Courter, Joel R; Melillo, Bruno N; Kaufmann, Daniel E; Hahn, Beatrice H; Permar, Sallie R; Haynes, Barton F; Madani, Navid; Sodroski, Joseph G; Finzi, Andrés

2015-05-19

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.

CD4 mimetics sensitize HIV-1-infected cells to ADCC

PubMed Central

Richard, Jonathan; Veillette, Maxime; Brassard, Nathalie; Iyer, Shilpa S.; Roger, Michel; Martin, Loïc; Pazgier, Marzena; Schön, Arne; Freire, Ernesto; Routy, Jean-Pierre; Smith, Amos B.; Park, Jongwoo; Jones, David M.; Courter, Joel R.; Melillo, Bruno N.; Kaufmann, Daniel E.; Hahn, Beatrice H.; Permar, Sallie R.; Haynes, Barton F.; Madani, Navid; Sodroski, Joseph G.; Finzi, Andrés

2015-01-01

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection. PMID:25941367

Proteomic Definitions of Mesenchymal Stem Cells

PubMed Central

Maurer, Martin H.

2011-01-01

Mesenchymal stem cells (MSCs) are pluripotent cells isolated from the bone marrow and various other organs. They are able to proliferate and self-renew, as well as to give rise to progeny of at least the osteogenic, chondrogenic, and adipogenic lineages. Despite this functional definition, MSCs can also be defined by their expression of a distinct set of cell surface markers. In the current paper, studies investigating the proteome of human MSCs are reviewed with the aim to identify common protein markers of MSCs. The proteomic analysis of MSCs revealed a distinct set of proteins representing the basic molecular inventory, including proteins for (i) cell surface markers, (ii) the responsiveness to growth factors, (iii) the reuse of developmental signaling cascades in adult stem cells, (iv) the interaction with molecules of the extracellular matrix, (v) the expression of genes regulating transcription and translation, (vi) the control of the cell number, and (vii) the protection against cellular stress. PMID:21437194

Co-Requirement of PICK1 Binding and PKC Phosphorylation for Stable Surface Expression of the Metabotropic Glutamate Receptor mGluR7

PubMed Central

Suh, Young Ho; Pelkey, Kenneth A.; Lavezzari, Gabriela; Roche, Paul A.; Huganir, Richard L.; McBain, Chris J.; Roche, Katherine W.

2008-01-01

SUMMARY The presynaptic metabotropic glutamate receptor (mGluR) mGluR7 modulates excitatory neurotransmission by regulating neurotransmitter release, and plays a critical role in certain forms of synaptic plasticity. Although the dynamic regulation of mGluR7 surface expression governs a novel form of metaplasticity in the hippocampus, little is known about the molecular mechanisms regulating mGluR7 trafficking. We now show that mGluR7 surface expression is stabilized by both PKC phosphorylation and by receptor binding to the PDZ domain-containing protein PICK1. Phosphorylation of mGluR7 on serine 862 (S862) inhibits CaM binding thereby increasing mGluR7 surface expression and receptor binding to PICK1. Furthermore, in mice lacking PICK1, PKC-dependent increases in mGluR7 phosphorylation and surface expression are diminished, and mGluR7-dependent plasticity at mossy fiber-interneuron hippocampal synapses is impaired. These data support a model in which PICK1 binding and PKC phosphorylation act together to stabilize mGluR7 on the cell surface in vivo. PMID:18549785

Downregulation of GbpB, a Component of the VicRK Regulon, Affects Biofilm Formation and Cell Surface Characteristics of Streptococcus mutans▿ †

PubMed Central

Duque, Cristiane; Stipp, Rafael N.; Wang, Bing; Smith, Daniel J.; Höfling, José F.; Kuramitsu, Howard K.; Duncan, Margaret J.; Mattos-Graner, Renata O.

2011-01-01

The virulence of the dental caries pathogen Streptococcus mutans relies in part on the sucrose-dependent synthesis of and interaction with glucan, a major component of the extracellular matrix of tooth biofilms. However, the mechanisms by which secreted and/or cell-associated glucan-binding proteins (Gbps) produced by S. mutans participate in biofilm growth remain to be elucidated. In this study, we further investigate GbpB, an essential immunodominant protein with similarity to murein hydrolases. A conditional knockdown mutant that expressed gbpB antisense RNA under the control of a tetracycline-inducible promoter was constructed in strain UA159 (UACA2) and used to investigate the effects of GbpB depletion on biofilm formation and cell surface-associated characteristics. Additionally, regulation of gbpB by the two-component system VicRK was investigated, and phenotypic analysis of a vicK mutant (UAvicK) was performed. GbpB was directly regulated by VicR, and several phenotypic changes were comparable between UACA2 and UAvicK, although differences between these strains existed. It was established that GbpB depletion impaired initial phases of sucrose-dependent biofilm formation, while exogenous native GbpB partially restored the biofilm phenotype. Several cellular traits were significantly affected by GbpB depletion, including altered cell shape, decreased autolysis, increased cell hydrophobicity, and sensitivity to antibiotics and osmotic and oxidative stresses. These data provide the first experimental evidence for GbpB participation in sucrose-dependent biofilm formation and in cell surface properties. PMID:21078847

ADAM10 Regulates Notch Function in Intestinal Stem Cells of Mice

PubMed Central

Tsai, Yu-Hwai; VanDussen, Kelli L.; Sawey, Eric T.; Wade, Alex W.; Kasper, Chelsea; Rakshit, Sabita; Bhatt, Riha G.; Stoeck, Alex; Maillard, Ivan; Crawford, Howard C.; Samuelson, Linda C.; Dempsey, Peter J.

2014-01-01

BACKGROUND & AIMS ADAM10 is a cell surface sheddase that regulates physiological processes including Notch signaling. ADAM10 is expressed in all intestinal epithelial cell types but the requirement for ADAM10 signaling in crypt homeostasis is not well defined. METHODS We analyzed intestinal tissues from mice with constitutive (Vil-Cre;Adam10f/f mice) and conditional (Vil-CreER;Adam10f/f and Lgr5-CreER;Adam10f/f mice) deletion of ADAM10. We performed cell lineage tracing experiments in mice that expressed a gain-of-function allele of Notch in the intestine (Rosa26NICD) or mice with intestine-specific disruption of Notch (Rosa26DN-MAML), to examine the effects of ADAM10 deletion on cell fate specification and intestinal stem cell maintenance. RESULTS Loss of ADAM10 from developing and adult intestine caused lethality associated with altered intestinal morphology, reduced progenitor cell proliferation, and increased secretory cell differentiation. ADAM10 deletion led to the replacement of intestinal cell progenitors with 2 distinct, post-mitotic, secretory cell lineages: intermediate-like (Paneth/goblet) and enteroendocrine cells. Based on analysis of Rosa26NICD and Rosa26DN-MAML mice, we determined that ADAM10 controls these cell fate decisions by regulating Notch signaling. Cell lineage tracing experiments showed that ADAM10 is required for survival of Lgr5+ crypt-based columnar cells. Our findings indicate that Notch-activated stem cells have a competitive advantage for occupation of the stem cell niche. CONCLUSIONS ADAM10 acts in a cell autonomous manner within the intestinal crypt compartment to regulate Notch signaling. This process is required for progenitor cell lineage specification and crypt-based columnar cell maintenance. PMID:25038433

The yeast Arf-GAP Glo3p is required for the endocytic recycling of cell surface proteins.

PubMed

Kawada, Daiki; Kobayashi, Hiromu; Tomita, Tsuyoshi; Nakata, Eisuke; Nagano, Makoto; Siekhaus, Daria Elisabeth; Toshima, Junko Y; Toshima, Jiro

2015-01-01

Small GTP-binding proteins of the Ras superfamily play diverse roles in intracellular trafficking. Among them, the Rab, Arf, and Rho families function in successive steps of vesicle transport, in forming vesicles from donor membranes, directing vesicle trafficking toward target membranes and docking vesicles onto target membranes. These proteins act as molecular switches that are controlled by a cycle of GTP binding and hydrolysis regulated by guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). In this study we explored the role of GAPs in the regulation of the endocytic pathway using fluorescently labeled yeast mating pheromone α-factor. Among 25 non-essential GAP mutants, we found that deletion of the GLO3 gene, encoding Arf-GAP protein, caused defective internalization of fluorescently labeled α-factor. Quantitative analysis revealed that glo3Δ cells show defective α-factor binding to the cell surface. Interestingly, Ste2p, the α-factor receptor, was mis-localized from the plasma membrane to the vacuole in glo3Δ cells. Domain deletion mutants of Glo3p revealed that a GAP-independent function, as well as the GAP activity, of Glo3p is important for both α-factor binding and Ste2p localization at the cell surface. Additionally, we found that deletion of the GLO3 gene affects the size and number of Arf1p-residing Golgi compartments and causes a defect in transport from the TGN to the plasma membrane. Furthermore, we demonstrated that glo3Δ cells were defective in the late endosome-to-TGN transport pathway, but not in the early endosome-to-TGN transport pathway. These findings suggest novel roles for Arf-GAP Glo3p in endocytic recycling of cell surface proteins. Copyright © 2014 Elsevier B.V. All rights reserved.

Pim-1L Protects Cell Surface-Resident ABCA1 From Lysosomal Degradation in Hepatocytes and Thereby Regulates Plasma High-Density Lipoprotein Level.

PubMed

Katsube, Akira; Hayashi, Hisamitsu; Kusuhara, Hiroyuki

2016-12-01

ATP-binding cassette transporter A1 (ABCA1) exerts an atheroprotective action through the biogenesis of high-density lipoprotein in hepatocytes and prevents the formation of foam cells from macrophages. Controlling ABCA1 is a rational approach to improving atherosclerotic cardiovascular disease. Although much is known about the regulatory mechanism of ABCA1 synthesis, the molecular mechanism underpinning its degradation remains to be clearly described. ABCA1 possesses potential sites of phosphorylation by serine/threonine-protein kinase Pim-1 (Pim-1). Pim-1 depletion decreased the expression of cell surface-resident ABCA1 (csABCA1) and apolipoprotein A-I-mediated [ 3 H]cholesterol efflux in the human hepatoma cell line HepG2, but not in peritoneal macrophages from mice. In vitro kinase assay, immunoprecipitation, and immunocytochemistry suggested phosphorylation of csABCA1 by the long form of Pim-1 (Pim-1L). Cell surface biotinylation indicated that Pim-1L inhibited lysosomal degradation of csABCA1 involving the liver X receptor β, which interacts with csABCA1 and thereby protects it from ubiquitination and subsequent lysosomal degradation. Cell surface coimmunoprecipitation with COS-1 cells expressing extracellularly hemagglutinin-tagged ABCA1 showed that Pim-1L-mediated phosphorylation of csABCA1 facilitated the interaction between csABCA1 and liver X receptor β and thereby stabilized the csABCA1-Pim-1L complex. Mice deficient in Pim-1 kinase activity showed lower expression of ABCA1 in liver plasma membranes and lower plasma high-density lipoprotein levels than control mice. Pim-1L protects hepatic csABCA1 from lysosomal degradation by facilitating the physical interaction between csABCA1 and liver X receptor β and subsequent stabilization of the csABCA1-Pim-1L complex and thereby regulates the circulating level of high-density lipoprotein. Our findings may aid the development of high-density lipoprotein-targeted therapy. © 2016 American Heart Association, Inc.

CD24 tracks divergent pluripotent states in mouse and human cells

PubMed Central

Shakiba, Nika; White, Carl A.; Lipsitz, Yonatan Y.; Yachie-Kinoshita, Ayako; Tonge, Peter D; Hussein, Samer M. I.; Puri, Mira C.; Elbaz, Judith; Morrissey-Scoot, James; Li, Mira; Munoz, Javier; Benevento, Marco; Rogers, Ian M.; Hanna, Jacob H.; Heck, Albert J. R.; Wollscheid, Bernd; Nagy, Andras; Zandstra, Peter W

2015-01-01

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. PMID:26076835

In situ cell surface proteomics reveals differentially expressed membrane proteins in retinal pigment epithelial cells during autoimmune uveitis.

PubMed

Uhl, P B; Szober, C M; Amann, B; Alge-Priglinger, C; Ueffing, M; Hauck, S M; Deeg, C A

2014-09-23

Retinal pigment epithelium (RPE) builds the outer blood-retinal barrier of the eye and plays an important role in pathogenesis of the sight threatening disease equine recurrent uveitis (ERU). ERU is a spontaneous autoimmune mediated inflammatory disease characterised by the breakdown of the outer blood-retinal barrier and an influx of autoaggressive T-cells into the inner eye. Therefore, identification of molecular mechanisms contributing to changed function of blood-retinal barrier in ERU is important for the understanding of pathophysiology. Cell surface proteins of RPE collected from healthy horses and horses with ERU were captured by in situ biotinylation and analysed with high resolution mass spectrometry coupled to liquid chromatography (LC-MS/MS) to identify differentially expressed proteins. With label free differential proteomics, a total of 27 differently expressed cell surface proteins in diseased RPE could be detected. Significant down-regulation of three very interesting proteins, synaptotagmin 1, basigin and collectrin was verified and further characterised. We applied an innovative and successful method to detect changes in the plasma cell surface proteome of RPE cells in a spontaneous inflammatory eye disease, serving as a valuable model for human autoimmune uveitis. We were able to identify 27 differentially expressed plasma cell membrane proteins, including synaptotagmin 1, basigin and collectrin, which play important roles in cell adhesion, transport and cell communication. Copyright © 2014 Elsevier B.V. All rights reserved.

Effects of Structural Properties of Electrospun TiO2 Nano-fiber Meshes on their Osteogenic Potential

PubMed Central

Wang, Xiaokun; Gittens, Rolando A.; Song, Rosemary; Tannenbaum, Rina; Olivares-Navarrete, Rene; Schwartz, Zvi; Chen, Haifeng; Boyan, Barbara D.

2011-01-01

Ideal outcomes in the field of tissue engineering and regenerative medicine involve biomaterials that can enhance cell differentiation and production of local factors for natural tissue regeneration without the use of systemic drugs. Biomaterials typically used in tissue engineering applications include polymeric scaffolds that mimic the 3-D structural environment of the native tissue, but these are often functionalized with proteins or small peptides to improve their biological performance. For bone applications, titanium (Ti) implants, or more appropriately the titania (TiO2) passive oxide layer formed on their surface, have been shown to enhance osteoblast differentiation in vitro and to promote osseointegration in vivo. In this study we evaluated the effect on osteoblast differentiation of pure TiO2 nano-fiber meshes with different surface micro-roughness and nano-fiber diameters, prepared by the electrospinning method. MG63 cells were seeded on TiO2 meshes, and cell number, differentiation markers and local factor production were analyzed. The results showed that cells grew throughout the entire surfaces and with similar morphology in all groups. Cell number was sensitive to surface micro-roughness, whereas cell differentiation and local factor production was regulated by both surface roughness and nano-fiber diameter. These results indicate that scaffold structural cues alone can be used to drive cell differentiation and create an osteogenic environment without the use of exogenous factors. PMID:22075122

Heat shock protein 70 surface-positive tumor exosomes stimulate migratory and cytolytic activity of natural killer cells.

PubMed

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A; Multhoff, Gabriele

2005-06-15

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo-/Colo+) and colon (CX-/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo-/CX- derived exosomes were Hsp70 membrane positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes.

Heat Shock Protein 70 Surface-Positive Tumor Exosomes Stimulate Migratory and Cytolytic Activity of Natural Killer Cells

PubMed Central

Gastpar, Robert; Gehrmann, Mathias; Bausero, Maria A.; Asea, Alexzander; Gross, Catharina; Schroeder, Josef A.

2006-01-01

Detergent-soluble membrane vesicles are actively released by human pancreas (Colo−/Colo+) and colon (CX−/CX+) carcinoma sublines, differing in their capacity to present heat shock protein 70 (Hsp70)/Bag-4 on their plasma membranes. Floating properties, acetylcholine esterase activity, and protein composition characterized them as exosomes. An enrichment of Rab-4 documented their intracellular transport route from early endosomes to the plasma membrane. After solubilization, comparable amounts of cytosolic proteins, including tubulin, Hsp70, Hsc70, and Bag-4, but not ER-residing Grp94 and calnexin, were detectable in tumor-derived exosomes. However, with respect to the exosomal surface, only Colo+/CX+ but not Colo−/CX exosomes were Hsp70 membrane derived positive. Therefore, concomitant with an up-regulated cell surface density of activation markers, migration and Hsp70 reactivity of natural killer (NK) cells was stimulated selectively by Hsp70/Bag-4 surface-positive exosomes, but not by their negative counterparts and tumor cell lysates. Moreover, the exosome-mediated lytic activity of NK cells was blockable by Hsp70-specific antibody. As already shown for TKD stimulation, NK cells preincubated with Hsp70 surface-positive exosomes initiated apoptosis in tumors through granzyme B release. In summary, our data provide an explanation how Hsp70 reactivity in NK cells is induced by tumor-derived exosomes. PMID:15958569

Hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity in oral squamous cell carcinoma derived cells.

PubMed

Chaudhari, Pratik Rajeev; Charles, Silvania Emlit; D'Souza, Zinia Charlotte; Vaidya, Milind Murlidhar

2017-11-15

BPAG1e and Plectin are hemidesmosomal linker proteins which anchor intermediate filament proteins to the cell surface through β4 integrin. Recent reports indicate that these proteins play a role in various cellular processes apart from their known anchoring function. However, the available literature is inconsistent. Further, the previous study from our laboratory suggested that Keratin8/18 pair promotes cell motility and tumor progression by deregulating β4 integrin signaling in oral squamous cell carcinoma (OSCC) derived cells. Based on these findings, we hypothesized that linker proteins may have a role in neoplastic progression of OSCC. Downregulation of hemidesmosomal linker proteins in OSCC derived cells resulted in reduced cell migration accompanied by alterations in actin organization. Further, decreased MMP9 activity led to reduced cell invasion in linker proteins knockdown cells. Moreover, loss of these proteins resulted in reduced tumorigenic potential. SWATH analysis demonstrated upregulation of N-Myc downstream regulated gene 1 (NDRG1) in linker proteins downregulated cells as compared to vector control cells. Further, the defects in phenotype upon linker proteins ablation were rescued upon loss of NDRG1 in linker proteins knockdown background. These data together indicate that hemidesmosomal linker proteins regulate cell motility, invasion and tumorigenicity possibly through NDRG1 in OSCC derived cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Unsaturated fatty acids promote proliferation via ERK1/2 and Akt pathway in bovine mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yonezawa, Tomo; Haga, Satoshi; Kobayashi, Yosuke

2008-03-21

GPR40 has recently been identified as a G protein-coupled cell-surface receptor for long-chain fatty acids (LCFAs). The mRNA of the bovine ortholog of GPR40 (bGPR40) was detected by RT-PCR in cloned bovine mammary epithelial cells (bMEC) and in the bovine mammary gland at various stages of lactation. Oleate and linoleate caused an increase in intracellular Ca{sup 2+} concentrations in these cells, and significantly reduced forskolin-induced cAMP concentrations. Phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 and Akt kinase, which regulates cell proliferation and survival, was rapidly increased by oleate. Incubation with oleate and linoleate for 24 h significantly promoted cell proliferation.more » Moreover, in serum-free medium, oleate significantly stimulated cell proliferation during a 7-day culture. These results suggest that bGPR40 mediates LCFA signaling in mammary epithelial cells and thereby plays an important role in cell proliferation and survival.« less

Azospirillum brasilense Chemotaxis Depends on Two Signaling Pathways Regulating Distinct Motility Parameters

PubMed Central

Mukherjee, Tanmoy; Kumar, Dhivya; Burriss, Nathan; Xie, Zhihong

2016-01-01

ABSTRACT The genomes of most motile bacteria encode two or more chemotaxis (Che) systems, but their functions have been characterized in only a few model systems. Azospirillum brasilense is a motile soil alphaproteobacterium able to colonize the rhizosphere of cereals. In response to an attractant, motile A. brasilense cells transiently increase swimming speed and suppress reversals. The Che1 chemotaxis pathway was previously shown to regulate changes in the swimming speed, but it has a minor role in chemotaxis and root surface colonization. Here, we show that a second chemotaxis system, named Che4, regulates the probability of swimming reversals and is the major signaling pathway for chemotaxis and wheat root surface colonization. Experimental evidence indicates that Che1 and Che4 are functionally linked to coordinate changes in the swimming motility pattern in response to attractants. The effect of Che1 on swimming speed is shown to enhance the aerotactic response of A. brasilense in gradients, likely providing the cells with a competitive advantage in the rhizosphere. Together, the results illustrate a novel mechanism by which motile bacteria utilize two chemotaxis pathways regulating distinct motility parameters to alter movement in gradients and enhance the chemotactic advantage. IMPORTANCE Chemotaxis provides motile bacteria with a competitive advantage in the colonization of diverse niches and is a function enriched in rhizosphere bacterial communities, with most species possessing at least two chemotaxis systems. Here, we identify the mechanism by which cells may derive a significant chemotactic advantage using two chemotaxis pathways that ultimately regulate distinct motility parameters. PMID:27068592

Azospirillum brasilense Chemotaxis Depends on Two Signaling Pathways Regulating Distinct Motility Parameters.

PubMed

Mukherjee, Tanmoy; Kumar, Dhivya; Burriss, Nathan; Xie, Zhihong; Alexandre, Gladys

2016-06-15

The genomes of most motile bacteria encode two or more chemotaxis (Che) systems, but their functions have been characterized in only a few model systems. Azospirillum brasilense is a motile soil alphaproteobacterium able to colonize the rhizosphere of cereals. In response to an attractant, motile A. brasilense cells transiently increase swimming speed and suppress reversals. The Che1 chemotaxis pathway was previously shown to regulate changes in the swimming speed, but it has a minor role in chemotaxis and root surface colonization. Here, we show that a second chemotaxis system, named Che4, regulates the probability of swimming reversals and is the major signaling pathway for chemotaxis and wheat root surface colonization. Experimental evidence indicates that Che1 and Che4 are functionally linked to coordinate changes in the swimming motility pattern in response to attractants. The effect of Che1 on swimming speed is shown to enhance the aerotactic response of A. brasilense in gradients, likely providing the cells with a competitive advantage in the rhizosphere. Together, the results illustrate a novel mechanism by which motile bacteria utilize two chemotaxis pathways regulating distinct motility parameters to alter movement in gradients and enhance the chemotactic advantage. Chemotaxis provides motile bacteria with a competitive advantage in the colonization of diverse niches and is a function enriched in rhizosphere bacterial communities, with most species possessing at least two chemotaxis systems. Here, we identify the mechanism by which cells may derive a significant chemotactic advantage using two chemotaxis pathways that ultimately regulate distinct motility parameters. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Cell Type-Specific Regulation of Immunological Synapse Dynamics by B7 Ligand Recognition

PubMed Central

Brzostek, Joanna; Gascoigne, Nicholas R. J.; Rybakin, Vasily

2016-01-01

B7 proteins CD80 (B7-1) and CD86 (B7-2) are expressed on most antigen-presenting cells and provide critical co-stimulatory or inhibitory input to T cells via their T-cell-expressed receptors: CD28 and CTLA-4. CD28 is expressed on effector T cells and regulatory T cells (Tregs), and CD28-dependent signals are required for optimum activation of effector T cell functions. CD28 ligation on effector T cells leads to formation of distinct molecular patterns and induction of cytoskeletal rearrangements at the immunological synapse (IS). CD28 plays a critical role in recruitment of protein kinase C (PKC)-θ to the effector T cell IS. CTLA-4 is constitutively expressed on the surface of Tregs, but it is expressed on effector T cells only after activation. As CTLA-4 binds to B7 proteins with significantly higher affinity than CD28, B7 ligand recognition by cells expressing both receptors leads to displacement of CD28 and PKC-θ from the IS. In Tregs, B7 ligand recognition leads to recruitment of CTLA-4 and PKC-η to the IS. CTLA-4 plays a role in regulation of T effector and Treg IS stability and cell motility. Due to their important roles in regulating T-cell-mediated responses, B7 receptors are emerging as important drug targets in oncology. In this review, we present an integrated summary of current knowledge about the role of B7 family receptor–ligand interactions in the regulation of spatial and temporal IS dynamics in effector and Tregs. PMID:26870040

Laminar shear stress modulates endothelial luminal surface stiffness in a tissue-specific manner.

PubMed

Merna, Nick; Wong, Andrew K; Barahona, Victor; Llanos, Pierre; Kunar, Balvir; Palikuqi, Brisa; Ginsberg, Michael; Rafii, Shahin; Rabbany, Sina Y

2018-04-17

Endothelial cells form vascular beds in all organs and are exposed to a range of mechanical forces that regulate cellular phenotype. We sought to determine the role of endothelial luminal surface stiffness in tissue-specific mechanotransduction of laminar shear stress in microvascular mouse cells and the role of arachidonic acid in mediating this response. Microvascular mouse endothelial cells were subjected to laminar shear stress at 4 dynes/cm 2 for 12 hours in parallel plate flow chambers that enabled real-time optical microscopy and atomic force microscopy measurements of cell stiffness. Lung endothelial cells aligned parallel to flow, while cardiac endothelial cells did not. This rapid alignment was accompanied by increased cell stiffness. The addition of arachidonic acid to cardiac endothelial cells increased alignment and stiffness in response to shear stress. Inhibition of arachidonic acid in lung endothelial cells and embryonic stem cell-derived endothelial cells prevented cellular alignment and decreased cell stiffness. Our findings suggest that increased endothelial luminal surface stiffness in microvascular cells may facilitate mechanotransduction and alignment in response to laminar shear stress. Furthermore, the arachidonic acid pathway may mediate this tissue-specific process. An improved understanding of this response will aid in the treatment of organ-specific vascular disease. © 2018 John Wiley & Sons Ltd.

B lymphocytes confer immune tolerance via cell surface GARP-TGF-β complex.

PubMed

Wallace, Caroline H; Wu, Bill X; Salem, Mohammad; Ansa-Addo, Ephraim A; Metelli, Alessandra; Sun, Shaoli; Gilkeson, Gary; Shlomchik, Mark J; Liu, Bei; Li, Zihai

2018-04-05

GARP, a cell surface docking receptor for binding and activating latent TGF-β, is highly expressed by platelets and activated Tregs. While GARP is implicated in immune invasion in cancer, the roles of the GARP-TGF-β axis in systemic autoimmune diseases are unknown. Although B cells do not express GARP at baseline, we found that the GARP-TGF-β complex is induced on activated human and mouse B cells by ligands for multiple TLRs, including TLR4, TLR7, and TLR9. GARP overexpression on B cells inhibited their proliferation, induced IgA class-switching, and dampened T cell-independent antibody production. In contrast, B cell-specific deletion of GARP-encoding gene Lrrc32 in mice led to development of systemic autoimmune diseases spontaneously as well as worsening of pristane-induced lupus-like disease. Canonical TGF-β signaling more readily upregulates GARP in Peyer patch B cells than in splenic B cells. Furthermore, we demonstrated that B cells are required for the induction of oral tolerance of T cell-dependent antigens via GARP. Our studies reveal for the first time to our knowledge that cell surface GARP-TGF-β is an important checkpoint for regulating B cell peripheral tolerance, highlighting a mechanism of autoimmune disease pathogenesis.

Inhibition of Fas (CD95) expression and Fas-mediated apoptosis by oncogenic Ras.

PubMed

Fenton, R G; Hixon, J A; Wright, P W; Brooks, A D; Sayers, T J

1998-08-01

The ras oncogene plays an important role in the multistep progression to cancer by activation of signal transduction pathways that contribute to aberrant growth regulation. Although many of these effects are cell autonomous, the ras oncogene also regulates the expression of genes that alter host/tumor interactions. We now extend the mechanisms through which ras promotes tumor survival by demonstrating that oncogenic Ras inhibits expression of the fas gene and renders Ras-transformed cells resistant to Fas-induced apoptosis. A panel of Ras-transformed clones exhibited a marked inhibition in fas mRNA and Fas cell surface expression as compared with untransformed parental cell lines. Fas expression was induced by culture in the presence of IFN-gamma + tumor necrosis factor alpha; however, the maximal level attained in Ras transformants was approximately 10-fold below the level of untransformed cells. Whereas untransformed cells were sensitive to apoptotic death induced by cross-linking surface Fas (especially after cytokine treatment), Ras-transformed cells were very resistant to Fas-induced death even under the most stringent assay conditions. To demonstrate that this resistance was mediated by oncogenic Ras and not secondary genetic events, pools of Ras-transformed cells were generated using a highly efficient retroviral transduction technique. Transformed pools were assayed 6 days after infection and demonstrated a marked decrease in fas gene expression and Fas-mediated apoptosis. Oncogenic Ras did not promote general resistance to apoptosis, because ectopic expression of a fas cDNA in Ras-transformed cells restored sensitivity to Fas-induced apoptosis. These data indicate that oncogenic Ras inhibits basal levels of expression of the fas gene, and although cytokine signal transduction pathways are functional in these cells, the level of surface Fas expression remains below the threshold required for induction of apoptosis. These data identify a mechanism by which Ras-transformed cells may escape from host-mediated immune destruction.

Comparative analysis of cell wall surface glycan expression in Candida albicans and Saccharomyces cerevisiae yeasts by flow cytometry.

PubMed

Martínez-Esparza, M; Sarazin, A; Jouy, N; Poulain, D; Jouault, T

2006-07-31

The yeast Candida albicans is an opportunistic pathogen, part of the normal human microbial flora that causes infections in immunocompromised individuals with a high morbidity and mortality levels. Recognition of yeasts by host cells is based on components of the yeast cell wall, which are considered part of its virulence attributes. Cell wall glycans play an important role in the continuous interchange that regulates the balance between saprophytism and parasitism, and also between resistance and infection. Some of these molecular entities are expressed both by the pathogenic yeast C. albicans and by Saccharomyces cerevisiae, a related non-pathogenic yeast, involving similar molecular mechanisms and receptors for recognition. In this work we have exploited flow cytometry methods for probing surface glycans of the yeasts. We compared glycan expression by C. albicans and by S. cerevisiae, and studied the effect of culture conditions. Our results show that the expression levels of alpha- and beta-linked mannosides as well as beta-glucans can be successfully evaluated by flow cytometry methods using different antibodies independent of agglutination reactions. We also found that the surface expression pattern of beta-mannosides detected by monoclonal or polyclonal antibodies are differently modulated during the growth course. These data indicate that the yeast beta-mannosides exposed on mannoproteins and/or phospholipomannan are increased in stationary phase, whereas those linked to mannan are not affected by the yeast growth phase. The cytometric method described here represents a useful tool to investigate to what extent C. albicans is able to regulate its glycan surface expression and therefore modify its virulence properties.

JAM related proteins in mucosal homeostasis and inflammation

PubMed Central

Luissint, Anny-Claude; Nusrat, Asma; Parkos, Charles A.

2014-01-01

Mucosal surfaces are lined by epithelial cells that form a physical barrier protecting the body against external noxious substances and pathogens. At a molecular level, the mucosal barrier is regulated by tight junctions (TJs) that seal the paracellular space between adjacent epithelial cells. Transmembrane proteins within TJs include Junctional Adhesion Molecules (JAMs) that belong to the CTX (Cortical Thymocyte marker for Xenopus) family of proteins. JAM family encompasses three classical members (JAM-A, -B and –C) and related molecules including JAM4, JAM-Like protein (JAM-L), Coxsackie and Adenovirus Receptor (CAR), CAR-Like Membrane Protein (CLMP) and Endothelial cell-Selective Adhesion Molecule (ESAM). JAMs have multiple functions that include regulation of endothelial and epithelial paracellular permeability, leukocyte recruitment during inflammation, angiogenesis, cell migration and proliferation. In this review, we summarize the current knowledge regarding the roles of the JAM family members in the regulation of mucosal homeostasis and leukocyte trafficking with a particular emphasis on barrier function and its perturbation during pathological inflammation. PMID:24667924

Deletion of the COOH-Terminal Domain of CXC Chemokine Receptor 4 Leads to the Down-regulation of Cell-to-Cell Contact, Enhanced Motility and Proliferation in Breast Carcinoma Cells

PubMed Central

Ueda, Yukiko; Neel, Nicole F.; Schutyser, Evemie; Raman, Dayanidhi; Richmond, Ann

2009-01-01

The CXC chemokine receptor 4 (CXCR4) contributes to the metastasis of human breast cancer cells. The CXCR4 COOH-terminal domain (CTD) seems to play a major role in regulating receptor desensitization and down-regulation. We expressed either wild-type CXCR4 (CXCR4-WT) or CTD-truncated CXCR4 (CXCR4-ΔCTD) in MCF-7 human mammary carcinoma cells to determine whether the CTD is involved in CXCR4-modulated proliferation of mammary carcinoma cells. CXCR4-WT-transduced MCF-7 cells (MCF-7/CXCR4-WT cells) do not differ from vector-transduced MCF-7 control cells in morphology or growth rate. However, CXCR4-ΔCTD-transduced MCF-7 cells (MCF-7/CXCR4-ΔCTD cells) exhibit a higher growth rate and altered morphology, potentially indicating an epithelial-to-mesenchymal transition. Furthermore, extracellular signal-regulated kinase (ERK) activation and cell motility are increased in these cells. Ligand induces receptor association with β-arrestin for both CXCR4-WT and CXCR4-ΔCTD in these MCF-7 cells. Overexpressed CXCR4-WT localizes predominantly to the cell surface in unstimulated cells, whereas a significant portion of overexpressed CXCR4-ΔCTD resides intracellularly in recycling endosomes. Analysis with human oligomicroarray, Western blot, and immunohistochemistry showed that E-cadherin and Zonula occludens are down-regulated in MCF-7/CXCR4-ΔCTD cells. The array analysis also indicates that mesenchymal marker proteins and certain growth factor receptors are up-regulated in MCF-7/CXCR4-ΔCTD cells. These observations suggest that (a) the overexpression of CXCR4-ΔCTD leads to a gain-of-function of CXCR4-mediated signaling and (b) the CTD of CXCR4-WT may perform a feedback repressor function in this signaling pathway. These data will contribute to our understanding of how CXCR4-ΔCTD may promote progression of breast tumors to metastatic lesions. PMID:16740704