Accumulation of cholesterol and increased demand for zinc in serum-deprived RPE cells

PubMed Central

Mishra, Sanghamitra; Peterson, Katherine; Yin, Lili; Berger, Alan; Fan, Jianguo

2016-01-01

Purpose Having observed that confluent ARPE-19 cells (derived from human RPE) survive well in high-glucose serum-free medium (SFM) without further feeding for several days, we investigated the expression profile of RPE cells under the same conditions. Methods Expression profiles were examined with microarray and quantitative PCR (qPCR) analyses, followed by western blot analysis of key regulated proteins. The effects of low-density lipoprotein (LDL) and zinc supplementation were examined with qPCR. Immunofluorescence was used to localize the LDL receptor and to examine LDL uptake. Cellular cholesterol levels were measured with filipin binding. Expression patterns in primary fetal RPE cells were compared using qPCR. Results Microarray analyses of gene expression in ARPE-19, confirmed with qPCR, showed upregulation of lipid and cholesterol biosynthesis pathways in SFM. At the protein level, the cholesterol synthesis control factor SRBEF2 was activated, and other key lipid synthesis proteins increased. Supplementation of SFM with LDL reversed the upregulation of lipid and cholesterol synthesis genes, but not of cholesterol transport genes. The LDL receptor relocated to the plasma membrane, and LDL uptake was activated by day 5–7 in SFM, suggesting increased demand for cholesterol. Confluent ARPE-19 cells in SFM accumulated intracellular cholesterol, compared with cells supplemented with serum, over 7 days. Over the same time course in SFM, the expression of metallothioneins decreased while the major zinc transporter was upregulated, consistent with a parallel increase in demand for zinc. Supplementation with zinc reversed expression changes for metallothionein genes, but not for other zinc-related genes. Similar patterns of regulation were also seen in primary fetal human RPE cells in SFM. Conclusions ARPE-19 cells respond to serum deprivation and starvation with upregulation of the lipid and cholesterol pathways, accumulation of intracellular cholesterol, and

Generation of Transplantable Retinal Pigmented Epithelial (RPE) Cells for Treatment of Age-Related Macular Degeneration (AMD).

PubMed

Surendran, Harshini; Rathod, Reena J; Pal, Rajarshi

2018-06-13

Age-related macular degeneration (AMD) is the foremost cause of blindness in people over the age of 60 worldwide. Clinically, this disease starts with distortion in central vision eventually leading to legal blindness. Vision loss has a significant impact on quality of life and incurs a substantial cost to the economy. Furthermore, AMD is a complex and progressive neurodegenerative disorder that triggers visual impairment due to the loss of retinal pigmented epithelium (RPE) and the light-sensitive photoreceptors that they support, protect and provide nutrition. Currently, there is no curative treatment for the most common form of this disease, i.e., dry AMD. A novel approach to treat AMD involves the transplantation of RPE cells derived from human induced pluripotent stem cells (iPSCs) in the outer retina. These iPSC-derived RPE cells not only show characteristics similar to native RPE but also could replace as well as regenerate damaged pathologic RPE and produce supportive growth factors and cytokines. Several clinical trials are being conducted taking advantage of a variety of cell- and tissue engineering-based approaches. Here, we present a simple, cost effective, and scalable cell-culture model for generation of purified RPE thus providing the foundation for developing an allogeneic cell therapy for AMD.

Overexpression of miR-183/-96/-182 triggers neuronal cell fate in Human Retinal Pigment Epithelial (hRPE) cells in culture.

PubMed

Davari, Maliheh; Soheili, Zahra-Soheila; Samiei, Shahram; Sharifi, Zohreh; Pirmardan, Ehsan Ranaei

2017-01-29

miR-183 cluster, composed of miR-183/-96/-182 genes, is highly expressed in the adult retina, particularly in photoreceptors. It involves in development, maturation and normal function of neuroretina. Ectopic overexpression of miR-183/-96/-182 genes was performed to assess reprogramming of hRPE cells. They were amplified from genomic DNA and cloned independently or in tandem configuration into pAAV.MCS vector. hRPE cells were then transfected with the recombinant constructs. Real-Time PCR was performed to measure the expression levels of miR-183/-96/-182 and that of several retina-specific neuronal genes such as OTX2, NRL, PDC and DCT. The transfected cells also were immunocytochemically examined for retina-specific neuronal markers, including Rhodopsin, red opsin, CRX, Thy1, CD73, recoverin and PKCα, to determine the cellular fate of the transfected hRPE cells. Data showed that upon miR-183/-96/-182 overexpression in hRPE cultures, the expression of neuronal genes including OTX2, NRL, PDC and DCT was also upregulated. Moreover, miR-183 cluster-treated hRPE cells were immunoreactive for neuronal markers such as Rhodopsin, red opsin, CRX and Thy1. Both transcriptional and translational upregulation of neuronal genes in miR-183 cluster-treated hRPE cells suggests that in vitro overexpression of miR-183 cluster could trigger reprogramming of hRPE cells to retinal neuron fate. Copyright © 2016 Elsevier Inc. All rights reserved.

Effect of SOCS1 overexpression on RPE cell activation by proinflammatory cytokines.

PubMed

Bazewicz, Magdalena; Draganova, Dafina; Makhoul, Maya; Chtarto, Abdel; Elmaleh, Valerie; Tenenbaum, Liliane; Caspers, Laure; Bruyns, Catherine; Willermain, François

2016-09-06

The purpose of this study was to investigate the in vitro effect of Suppressor Of Cytokine Signaling 1 (SOCS1) overexpression in retinal pigment epithelium (RPE) cells on their activation by pro-inflammatory cytokines IFNγ, TNFα and IL-17. Retinal pigment epithelium cells (ARPE-19) were stably transfected with the control plasmid pIRES2-AcGFP1 or the plasmid pSOCS1-IRES2-AcGFP1. They were stimulated by IFNγ (150ng/ml), TNFα (30ng/ml) or IL-17 (100ng/ml). The levels of SOCS1 mRNA were measured by real-time PCR. Signal Transducer and Activator of Transcription 1 (STAT1) phosphorylation and IκBα expression were analysed by western Blot (WB). IL-8 secretion was analysed by ELISA and expression of MHCII molecules and ICAM-1/CD54 by flow cytometry. Our data show that SOCS1 mRNA overexpression in RPE cells prevents IFNγ-induced SOCS1 mRNA increase and IFNγ-mediated STAT1 phosphorylation. Moreover, SOCS1 overexpression in RPE cells inhibits IFNγ-induced decrease of IL-8 secretion and prevents IFNγ-induced MHC II and ICAM1/CD54 upregulation. However, SOCS1 overexpression does not affect TNFα-induced IκBα degradation nor block TNFα-induced or IL-17-induced IL-8 secretion. On the contrary, IL-17-induced secretion is increased by SOCS1 overexpression. In conclusion, SOCS1 overexpression in RPE cells inhibits some IFNγ-mediated responses that lead to uveitis development. This notion raises the possibility that SOCS1 overexpression could be a novel target for treating non-infectious uveitis. However, some proinflammatory effects of TNFα and IL-17 stimulation on RPE are not blocked by SOCS1 overexpression. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Effects of Secreted Mast Cell Mediators on Retinal Pigment Epithelial Cells: Focus on Mast Cell Tryptase.

PubMed

Arai, Rei; Usui-Ouchi, Ayumi; Ito, Yosuke; Mashimo, Keitaro; Murakami, Akira; Ebihara, Nobuyuki

2017-01-01

Numerous mast cells are present in the choroid, but the effects of mast cell mediators on retinal pigment epithelial (RPE) cells are not well understood. We investigated the influence of mast cell mediators on RPE cells in vitro, focusing on tryptase. Expression of receptors was examined by the reverse transcription polymerase chain reaction. We also assessed production of interleukin 8 and vascular endothelial growth factor (VEGF) after RPE cells were stimulated with mast cell mediators by using an antibody array and enzyme-linked immunosorbent assay. Furthermore, we investigated the influence of tryptase on RPE cell migration and integrity by the scratch assay and the transepithelial resistance. RPE cells expressed protease-activated receptor 2 (PAR2), histamine receptor 1, tumor necrosis factor- α (TNF- α ) receptor 1, and CCR 1, 3, 4, 8, and 11. Tryptase, PAR2 agonists, histamine, and TNF- α all enhanced interleukin 8 production by RPE cells, while only tryptase enhanced VEGF production. Tryptase also enhanced expression of phosphorylated extracellular signal-regulated kinases 1/2, resulting in increased migration of RPE cells. However, tryptase did not alter epithelial integrity or the expression of zonula occludens-1 and junctional adhesion molecule-A by RPE cells. Mast cell mediators, especially tryptase, may influence RPE cell inflammation.

The influence of rAAV2-mediated SOX2 delivery into neonatal and adult human RPE cells; a comparative study.

PubMed

Ezati, Razie; Etemadzadeh, Azadeh; Soheili, Zahra-Soheila; Samiei, Shahram; Ranaei Pirmardan, Ehsan; Davari, Malihe; Najafabadi, Hoda Shams

2018-02-01

Cell replacement is a promising therapy for degenerative diseases like age-related macular degeneration (AMD). Since the human retina lacks regeneration capacity, much attention has been directed toward persuading for cells that can differentiate into retinal neurons. In this report, we have investigated reprogramming of the human RPE cells and concerned the effect of donor age on the cellular fate as a critical determinant in reprogramming competence. We evaluated the effect of SOX2 over-expression in human neonatal and adult RPE cells in cultures. The coding region of human SOX2 gene was cloned into adeno-associated virus (AAV2) and primary culture of human neonatal/adult RPE cells were infected by recombinant virus. De-differentiation of RPE to neural/retinal progenitor cells was investigated by quantitative real-time PCR and ICC for neural/retinal progenitor cells' markers. Gene expression analysis showed 80-fold and 12-fold over-expression for SOX2 gene in infected neonatal and adult hRPE cells, respectively. The fold of increase for Nestin in neonatal and adult hRPE cells was 3.8-fold and 2.5-fold, respectively. PAX6 expression was increased threefold and 2.5-fold in neonatal/adult treated cultures. Howbeit, we could not detect rhodopsin, and CHX10 expression in neonatal hRPE cultures and expression of rhodopsin in adult hRPE cells. Results showed SOX2 induced human neonatal/adult RPE cells to de-differentiate toward retinal progenitor cells. However, the increased number of PAX6, CHX10, Thy1, and rhodopsin positive cells in adult hRPE treated cultures clearly indicated the considerable generation of neuro-retinal terminally differentiated cells. © 2017 Wiley Periodicals, Inc.

Evaluation of RPE65, CRALBP, VEGF, CD68, and tyrosinase gene expression in human retinal pigment epithelial cells cultured on amniotic membrane.

PubMed

Akrami, Hassan; Soheili, Zahra-Soheila; Sadeghizadeh, Majid; Khalooghi, Keynoush; Ahmadieh, Hamid; Kanavi, Mojgan Rezaie; Samiei, Shahram; Pakravesh, Jalil

2011-06-01

The retinal pigment epithelium (RPE) plays a key role in the maintenance of the normal functions of the retina. Tissue engineering using amniotic membrane as a substrate to culture RPE cells may provide a promising new strategy to replace damaged RPE. We established a method of culturing RPE cells over the amniotic membrane as a support for their growth and transplantation. The transcription of specific genes involved in cellular function of native RPE, including RPE65, CRALBP, VEGF, CD68, and tyrosinase, were then measured using quantitative real-time PCR. Data showed a considerable increase in transcription of RPE65, CD68, and VEGF in RPE cells cultured on amniotic membrane. The amounts of CRALBP and tyrosinase transcripts were not affected. This may simply indicate that amniotic membrane restricted dedifferentiation of RPE cells in culture. The results suggest that amniotic membrane may be considered as an elective biological substrate for RPE cell culture.

The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

PubMed Central

Tsapara, Anna; Luthert, Phillip; Greenwood, John; Hill, Caroline S.

2010-01-01

Maintenance of the epithelial phenotype is crucial for tissue homeostasis. In the retina, dedifferentiation and loss of integrity of the retinal pigment epithelium (RPE) leads to retinal dysfunction and fibrosis. Transforming growth factor (TGF)-β critically contributes to RPE dedifferentiation and induces various responses, including increased Rho signaling, up-regulation of α-smooth muscle actin (SMA), and cell migration and dedifferentiation. Cellular TGF-β responses are stimulated by different signal transduction pathways: some are Smad dependent and others Smad independent. Alterations in Rho signaling are crucial to both types of TGF-β signaling, but how TGF-β-stimulates Rho signaling is poorly understood. Here, we show that primary RPE cells up-regulated GEF-H1 in response to TGF-β. GEF-H1 was the only detectable Rho exchange factor increased by TGF-β1 in a genome-wide expression analysis. GEF-H1 induction was Smad4-dependant and led to Rho activation. GEF-H1 inhibition counteracted α-SMA up-regulation and cell migration. In patients with retinal detachments and fibrosis, migratory RPE cells exhibited increased GEF-H1 expression, indicating that induction occurs in diseased RPE in vivo. Our data indicate that GEF-H1 is a target and functional effector of TGF-β by orchestrating Rho signaling to regulate gene expression and cell migration, suggesting that it represents a new marker and possible therapeutic target for degenerative and fibrotic diseases. PMID:20089843

AAV delivery of GRP78/BiP promotes adaptation of human RPE cell to ER stress.

PubMed

Ghaderi, Shima; Ahmadian, Shahin; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Samiei, Shahram; Kheitan, Samira; Pirmardan, Ehsan R

2018-02-01

Adeno associated virus (AAV)-mediated gene delivery of GRP78 (78 kDa glucose-regulated protein) attenuates the condition of endoplasmic reticulum (ER) stress and prevents apoptotic loss of photoreceptors in Retinitis pigmentosa (RP) rats. In the current study we overexpressed Grp78 with the help of AAV-2 in primary human retinal pigmented epithelium (hRPE) cell cultures and examined its effect on cell response to ER stress. The purpose of this work was studying potential stimulating effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress, as an in vitro model for RPE degeneration. To investigate the effect of Grp78 overexpression on unfolded protein response (UPR) markers under ER stress, hRPE primary cultures were transduced by recombinant virus rAAV/Grp78, and treated with ER stressor drug, tunicamycin. Expression changes of four UPR markers including GRP78, PERK, ATF6α, and GADD153/CHOP, were assessed by real-time PCR and western blotting. We found that GRP78 has a great contribution in modulation of UPR markers to favor adaptive response in ER-stressed hRPE cells. In fact, GRP78 overexpression affected adaptation and apoptotic phases of early UPR, through enhancement of two master regulators/ER stress sensors (PERK and ATF6α) and down-regulation of a key pro-apoptotic cascade activator (GADD153/CHOP). Together these findings demonstrate the promoting effect of GRP78 on adaptation/pro-survival of hRPE cells under ER stress. This protein with anti-apoptotic actions in the early UPR and important role in cell fate regulation, can be recruited as a useful candidate for future investigations of RPE degenerative diseases. © 2017 Wiley Periodicals, Inc.

Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye.

PubMed

Gao, Jiangyuan; Cui, Jing Z; To, Eleanor; Cao, Sijia; Matsubara, Joanne A

2018-01-12

Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), the primary cell type that is afflicted in AMD, undergoes programmed cell death in the late stages of the disease. However, the exact mechanisms for RPE degeneration in AMD are still unresolved. The prevailing theories consider that each cell death pathway works independently and without regulation of each other. Building upon our previous work in which we induced a short burst of inflammasome activity in vivo, we now investigate the effects of prolonged inflammasome activity on RPE cell death mechanisms in rats. Long-Evans rats received three intravitreal injections of amyloid beta (Aβ), once every 4 days, and were sacrificed at day 14. The vitreous samples were collected to assess the levels of secreted cytokines. The inflammasome activity was evaluated by both immunohistochemistry and western blot. The types of RPE cell death mechanisms were determined using specific cell death markers and morphological characterizations. We found robust inflammasome activation evident by enhanced caspase-1 immunoreactivity, augmented NF-κB nuclear translocalization, increased IL-1β vitreal secretion, and IL-18 protein levels. Moreover, we observed elevated proteolytic cleavage of caspase-3 and gasdermin D, markers for apoptosis and pyroptosis, respectively, in RPE-choroid tissues. There was also a significant reduction in the anti-apoptotic factor, X-linked inhibitor of apoptosis protein, consistent with the overall changes of RPE cells. Morphological analysis showed phenotypic characteristics of pyroptosis including RPE cell swelling. Our data suggest that two cell death pathways, pyroptosis and apoptosis, were activated in RPE cells after exposure to prolonged inflammasome activation, induced by a drusen component, Aβ. The involvement of two distinct cell death pathways in RPE sheds light on the potential interplay between

Epithelial phenotype and the RPE: is the answer blowing in the Wnt?

PubMed

Burke, Janice M

2008-11-01

Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell-cell adhesion and melanization are linked by a common signaling pathway: the Wnt/beta-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of

Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells.

PubMed

Bagheri, Abouzar; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Samiei, Shahram; Sheibani, Nader; Astaneh, Shamila Darvishalipour; Kanavi, Mozhgan Rezaei; Mohammadian, Azam

2015-01-01

Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells. Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) -1 and -2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT-PCR, and zymography. Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures. Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells.

Competitive quenching: a possible novel approach in protecting RPE cells from damage during PDT.

PubMed

Weinberger, Dov; Ron, Yonina; Lusky, Moshe; Gaaton, Dan; Orenstein, Arie; Blank, Michael; Mandel, Mathilda; Livnat, Tamar; Barliya, Tilda; Lavie, Gad

2005-04-01

The purpose of this study is to demonstrate feasibility of using our novel concept, termed competitive quenching, for protecting the choroidal extravascular compartment and retinal pigment epithelium (RPE) from verteporfin (VP)-induced phototoxicity using hypericin. Furthermore, we aim to achieve partitioning of the quencher, hypericin, in the extravascular space and VP within the microvascular compartment of the chorio-retinal complex in vivo. We protect RPE cells from damage inflicted by photoactivated VP by introducing hypericin into these cells prior to photosensitization to quench the photosensitizing activity of VP. Cell protection levels were measured by MTT and Hemacolor viability assays. Wavelength range used for VP photoexcitation (700 +/- 40 nm) excludes the absorption range of hypericin, preventing the latter from photoactivation. Pharmacokinetic conditions, in which hypericin spreads throughout the choroidal and retinal extravascular space while VP is confined to the vasculature, are delineated using double-fluorescence imaging. Cell viability increased 3- to 5-fold when 10-20 microM hypericin were present in RPE cells during photosensitization with 0.1-0.5 microM VP. VP fluorescence intensity was unchanged by the presence of hypericin in the cells. Hypericin administered intravenously to rats was confined to the choroidal vasculature after 15 min to 2 hr. Subsequently, hypericin partitioned to the choroidal and retinal extravascular space. VP administered at this time was confined to the microvasculature. RPE and choroid may potentially be protected by compartmentalizing hypericin to the extravascular compartment while VP administered shortly before photosensitization is confined to the microvasculature. Adverse photodynamic therapy (PDT) damage to choroidal tissues adjacent to neovasculature targeted for photoablation have the potential of being prevented by competitive quenching with hypericin.

Simultaneous application of bevacizumab and anti-CTGF antibody effectively suppresses proangiogenic and profibrotic factors in human RPE cells

PubMed Central

Bagheri, Abouzar; Ahmadieh, Hamid; Samiei, Shahram; Sheibani, Nader; Astaneh, Shamila Darvishalipour; Kanavi, Mozhgan Rezaei; Mohammadian, Azam

2015-01-01

Purpose Retinal pigment epithelial (RPE) cells play key roles in the development of choroidal neovascularization and subsequent fibrosis. We investigated the impact of bevacizumab, antihuman vascular endothelial growth factor (VEGF) antibody, and anticonnective tissue growth factor (anti-CTGF) neutralizing antibody, individually or in combination, on proangiogenic and profibrotic properties of RPE cells. Methods Primary cultures of human RPE cells were incubated with different concentrations of bevacizumab (0.25, 0.5, and 0.8 mg/ml) and/or anti-CTGF (10 μg/ml), and cell proliferation and apoptosis were determined. Expression and activity of proangiogenic and profibrotic genes including matrix metalloproteinases (MMP)-2 and 9, VEGFA, CTGF, vascular endothelial growth factor receptor-1 (VEGFR-1), cathepsin D, tissue inhibitor of metalloproteinases (TIMP) −1 and −2, and alpha smooth muscle actin (α-SMA) were assessed with slot blot, real-time RT–PCR, and zymography. Results Bevacizumab alone inhibited proliferation of RPE cells while anti-CTGF or bevacizumab and anti-CTGF combined had no inhibitory effect in this regard. Bevacizumab increased MMP-2, MMP-9, and cathepsin D but decreased VEGFA and VEGFR-1 expression. The CTGF level was increased by using 0.25 mg/ml bevacizumab but decreased at the 0.8 mg/ml concentration of bevacizumab. Treatment with anti-CTGF antibody decreased MMP-2 expression whereas combined treatment with bevacizumab and anti-CTGF resulted in decreased expression of MMP-2, TIMP-1, cathepsin D, VEGFA, CTGF, and α-SMA in the treated cultures. Conclusions Treatment of RPE cells with the combination of bevacizumab and anti-CTGF could effectively suppress the proangiogenic and profibrotic activity of RPE cells. PMID:25883524

Epithelial phenotype and the RPE: Is the answer blowing in the Wnt?

PubMed Central

Burke, Janice M.

2008-01-01

Cells of the human retinal pigment epithelium (RPE) have a regular epithelial cell shape within the tissue in situ, but for reasons that remain elusive the RPE shows an incomplete and variable ability to re-develop an epithelial phenotype after propagation in vitro. In other epithelial cell cultures, formation of an adherens junction (AJ) composed of E-cadherin plays an important early inductive role in epithelial morphogenesis, but E-cadherin is largely absent from the RPE. In this review, the contribution of cadherins, both minor (E-cadherin) and major (N-cadherin), to RPE phenotype development is discussed. Emphasis is placed on the importance for future studies of actin cytoskeletal remodeling during assembly of the AJ, which in epithelial cells results in an actin organization that is characteristically zonular. Other markers of RPE phenotype that are used to gauge the maturation state of RPE cultures including tissue-specific protein expression, protein polarity, and pigmentation are described. An argument is made that RPE epithelial phenotype, cadherin-based cell–cell adhesion and melanization are linked by a common signaling pathway: the Wnt/β-catenin pathway. Analyzing this pathway and its intersecting signaling networks is suggested as a useful framework for dissecting the steps in RPE morphogenesis. Also discussed is the effect of aging on RPE phenotype. Preliminary evidence is provided to suggest that light-induced sub-lethal oxidative stress to cultured ARPE-19 cells impairs organelle motility. Organelle translocation, which is mediated by stress-susceptible cytoskeletal scaffolds, is an essential process in cell phenotype development and retention. The observation of impaired organelle motility therefore raises the possibility that low levels of stress, which are believed to accompany RPE aging, may produce subtle disruptions of cell phenotype. Over time these would be expected to diminish the support functions performed by the RPE on behalf of

Exogenous NAD+ decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy

PubMed Central

Zhu, Ying; Zhao, Ke-ke; Tong, Yao; Zhou, Ya-li; Wang, Yi-xiao; Zhao, Pei-quan; Wang, Zhao-yang

2016-01-01

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD+) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD+ administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD+ administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD+ administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD+ against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD+ administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD+ administration might be potential value for the treatment of AMD. PMID:27240523

Exogenous NAD(+) decreases oxidative stress and protects H2O2-treated RPE cells against necrotic death through the up-regulation of autophagy.

PubMed

Zhu, Ying; Zhao, Ke-Ke; Tong, Yao; Zhou, Ya-Li; Wang, Yi-Xiao; Zhao, Pei-Quan; Wang, Zhao-Yang

2016-05-31

Increased oxidative stress, which can lead to the retinal pigment epithelium (RPE) cell death by inducing ATP depletion and DNA repair, is believed to be a prominent pathology in age-related macular degeneration (AMD). In the present study, we showed that and 0.1 mM nicotinamide adenine dinucleotide (NAD(+)) administration significantly blocked RPE cell death induced by 300 μM H2O2. Further investigation showed that H2O2 resulted in increased intracellular ROS level, activation of PARP-1 and subsequently necrotic death of RPE cells. Exogenous NAD(+) administration significantly decreased intracellular and intranuclear ROS levels in H2O2-treated RPE cells. In addition, NAD(+) administration to H2O2-treated RPE cells inhibited the activation of PARP-1 and protected the RPE cells against necrotic death. Moreover, exogenous NAD(+) administration up-regulated autophagy in the H2O2-treated RPE cells. Inhibition of autophagy by LY294002 blocked the decrease of intracellular and intranuclear ROS level. Besides, inhibition of autophagy by LY294002 abolished the protection of exogenous NAD(+) against H2O2-induced cell necrotic death. Taken together, our findings indicate that that exogenous NAD(+) administration suppresses H2O2-induced oxidative stress and protects RPE cells against PARP-1 mediated necrotic death through the up-regulation of autophagy. The results suggest that exogenous NAD(+) administration might be potential value for the treatment of AMD.

Biological Modulation of Mouse RPE Cells in Response to Subthreshold Diode Micropulse Laser Treatment.

PubMed

Li, Zhouyue; Song, Yanping; Chen, Xiao; Chen, Zhongshan; Ding, Qin

2015-11-01

Many clinical trials have demonstrated the effectiveness of subthreshold phototherapy with no visible damage in retinal vascular diseases, such as diabetic retinopathy. We aimed primarily to investigate the effect of subthreshold diode micropulse laser (SDM) treatment on mouse retinal pigmented epithelium (RPE) cells. The expression of angiogenesis-modulating cytokines in response to SDM was also explored. The least toxic laser dose was selected by measuring cell viability with MTT assay and 5 % duty cycle (DC) was chosen for use in further experiments. RPE cells were treated with laser-induced radiation ranging from 0 to 400 mW for 24 h. The apoptotic rate of RPE cells was assessed by flow cytometry. Expressions of vascular endothelial growth factor A (VEGF-A), transforming growth factor beta (TGF-β), basic fibroblast growth factor (bFGF), and pigment epithelium-derived factor (PEDF) were determined by Western Blotting and real-time PCR, respectively. After 24 h of laser irradiation, cell viability was reduced dose dependently and the effect was significant compared to the controls (P < 0.05). In addition, laser treatment with intensities of 100 and 200 mW with DC of 5 % produced no significant effect on cell viability and apoptosis as compared with the control group (P > 0.05). The protein and mRNA expressions of angiogenic stimulators (VEGF-A, TGF-β, and bFGF) were significantly down-regulated (P < 0.05), whereas those of the angiogenic inhibitor (PEDF) were up-regulated (P < 0.05). No significant difference was found between the cells treated with different intensities of laser radiation (P > 0.05). Our results showed that SDM treatment of the RPE cells suppressed the expression of choroid neovasculization-promoting cytokines and up-regulated the angiogenic inhibitor, PEDF without damaging the cells. Further investigation is needed to understand the mechanism and to optimize the use of SDM as a novel method of treatment for retinal vascular

Live-Cell Imaging of Phagosome Motility in Primary Mouse RPE Cells.

PubMed

Hazim, Roni; Jiang, Mei; Esteve-Rudd, Julian; Diemer, Tanja; Lopes, Vanda S; Williams, David S

2016-01-01

The retinal pigment epithelium (RPE) is a post-mitotic epithelial monolayer situated between the light-sensitive photoreceptors and the choriocapillaris. Given its vital functions for healthy vision, the RPE is a primary target for insults that result in blinding diseases, including age-related macular degeneration (AMD). One such function is the phagocytosis and digestion of shed photoreceptor outer segments. In the present study, we examined the process of trafficking of outer segment disk membranes in live cultures of primary mouse RPE, using high speed spinning disk confocal microscopy. This approach has enabled us to track phagosomes, and determine parameters of their motility, which are important for their efficient degradation.

Role of macrophage migration inhibitory factor (MIF) in the effects of oxidative stress on human retinal pigment epithelial cells.

PubMed

Ko, Ji-Ae; Sotani, Yasuyuki; Ibrahim, Diah Gemala; Kiuchi, Yoshiaki

2017-10-01

Proliferative vitreoretinopathy (PVR) is the major cause of treatment failure in individuals who undergo surgery for retinal detachment. The epithelial-mesenchymal transition (EMT) in retinal pigment epithelium (RPE) cells contributes to the pathogenesis of PVR. Oxidative stress is thought to play a role in the progression of retinal diseases including PVR. We have now examined the effects of oxidative stress on the EMT and related processes in the human RPE cell line. We found that H 2 O 2 induced the contraction of RPE cells in a three-dimensional collagen gel. Analysis of a cytokine array revealed that H 2 O 2 specifically increased the release of macrophage migration inhibitory factor (MIF) from RPE cells. Reverse transcription-polymerase chain reaction and immunoblot analyses showed that H 2 O 2 increased the expression of MIF in RPE cells. Immunoblot and immunofluorescence analyses revealed that H 2 O 2 upregulated the expression of α-SMA and vimentin and downregulated that of ZO-1 and N-cadherin. Consistent with these observations, the transepithelial electrical resistance of cell was reduced by exposure to H 2 O 2 . The effects of oxidative stress on EMT-related and junctional protein expression as well as on transepithelial electrical resistance were inhibited by antibodies to MIF, but they were not mimicked by treatment with recombinant MIF. Finally, analysis with a profiling array for mitogen-activated protein kinase signalling revealed that H 2 O 2 specifically induced the phosphorylation of p38 mitogen-activated protein kinase. Our results thus suggest that MIF may play a role in induction of the EMT and related processes by oxidative stress in RPE cells and that it might thereby contribute to the pathogenesis of PVR. Proliferative vitreoretinopathy is a major complication of rhegmatogenous retinal detachment, and both oxidative stress and induction of the EMT in RPE cells are thought to contribute to the pathogenesis of this condition. We have now

Drusen in patient-derived hiPSC-RPE models of macular dystrophies

PubMed Central

Galloway, Chad A.; Dalvi, Sonal; Hung, Sandy S. C.; MacDonald, Leslie A.; Latchney, Lisa R.; Wong, Raymond C. B.; Guymer, Robyn H.; Williams, David S.; Chung, Mina M.; Gamm, David M.; Pébay, Alice; Hewitt, Alex W.; Singh, Ruchira

2017-01-01

Age-related macular degeneration (AMD) and related macular dystrophies (MDs) are a major cause of vision loss. However, the mechanisms underlying their progression remain ill-defined. This is partly due to the lack of disease models recapitulating the human pathology. Furthermore, in vivo studies have yielded limited understanding of the role of specific cell types in the eye vs. systemic influences (e.g., serum) on the disease pathology. Here, we use human induced pluripotent stem cell-retinal pigment epithelium (hiPSC-RPE) derived from patients with three dominant MDs, Sorsby’s fundus dystrophy (SFD), Doyne honeycomb retinal dystrophy/malattia Leventinese (DHRD), and autosomal dominant radial drusen (ADRD), and demonstrate that dysfunction of RPE cells alone is sufficient for the initiation of sub-RPE lipoproteinaceous deposit (drusen) formation and extracellular matrix (ECM) alteration in these diseases. Consistent with clinical studies, sub-RPE basal deposits were present beneath both control (unaffected) and patient hiPSC-RPE cells. Importantly basal deposits in patient hiPSC-RPE cultures were more abundant and displayed a lipid- and protein-rich “drusen-like” composition. Furthermore, increased accumulation of COL4 was observed in ECM isolated from control vs. patient hiPSC-RPE cultures. Interestingly, RPE-specific up-regulation in the expression of several complement genes was also seen in patient hiPSC-RPE cultures of all three MDs (SFD, DHRD, and ADRD). Finally, although serum exposure was not necessary for drusen formation, COL4 accumulation in ECM, and complement pathway gene alteration, it impacted the composition of drusen-like deposits in patient hiPSC-RPE cultures. Together, the drusen model(s) of MDs described here provide fundamental insights into the unique biology of maculopathies affecting the RPE–ECM interface. PMID:28878022

Mouse Retinal Pigmented Epithelial Cell Lines retain their phenotypic characteristics after transfection with Human Papilloma Virus: A new tool to further the study of RPE biology

PubMed Central

Catanuto, Paola; Espinosa-Heidmann, Diego; Pereira-Simon, Simone; Sanchez, Patricia; Salas, Pedro; Hernandez, Eleut; Cousins, Scott W.; Elliot, Sharon J.

2009-01-01

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 16 month old (estrogen receptor knockout) ERKOα and ERKOβ mice and their C57Bl/6 wild type littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER) α and ERβ protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology. PMID:19013153

Quercetin-3-O-α-l-arabinopyranoside protects against retinal cell death via blue light-induced damage in human RPE cells and Balb-c mice.

PubMed

Kim, Jun; Jin, Hong Lan; Jang, Dae Sik; Jeong, Kwang Won; Choung, Se-Young

2018-04-25

Age-related macular degeneration (AMD) is among the increasing number of diseases causing irreversible blindness in the elderly. Dry AMD is characterized by the accumulation of lipofuscin in retinal pigment epithelium (RPE) cells. N-Retinylidene-N-retinylethanolamine (A2E), a component of lipofuscin, is oxidized to oxo-A2E under blue light illumination, leading to retinal cell death. The aim of this study was to investigate the protective effect and mechanism of quercetin-3-O-α-l-arabinopyranoside (QA) against blue light (BL)-induced damage in both RPE cells and mice models. Treatment by QA inhibited A2E uptake in RPE cells, as determined by a decrease in fluorescence intensity. QA also protected A2E-laden RPE cells against BL-induced apoptosis. QA inhibited C3 complement activation and poly (ADP-ribose) polymerase (PARP) cleavage, as determined by western blotting. QA showed an inhibitory effect on AP1 and NF-kB activity as estimated in a reporter gene assay. In addition, QA activated the gene expression of aryl hydrocarbon receptor target genes (CYP1A1, CYP1B1) in TCDD-treated RPE cells. In the mice model, oral administration of QA protected against retinal degeneration induced by BL exposure as determined by histological analyses (thickness of retinal layers and immunostaining for caspase-3). In addition, QA inhibited apoptosis and inflammation via inhibition of NF-kB p65 translocation, C3 activation, and PARP cleavage. Collectively, these results revealed the protective mechanism of QA against BL-induced retinal damage both in vitro and in vivo.

Loss of Hfe Leads to Progression of Tumor Phenotype in Primary Retinal Pigment Epithelial Cells

PubMed Central

Gnana-Prakasam, Jaya P.; Veeranan-Karmegam, Rajalakshmi; Coothankandaswamy, Veena; Reddy, Sushma K.; Martin, Pamela M.; Thangaraju, Muthusamy; Smith, Sylvia B.; Ganapathy, Vadivel

2013-01-01

Purpose. Hemochromatosis is a disorder of iron overload arising mostly from mutations in HFE. HFE is expressed in retinal pigment epithelium (RPE), and Hfe−/− mice develop age-related iron accumulation and retinal degeneration associated with RPE hyperproliferation. Here, the mechanism underlying the hyperproliferative phenotype in RPE was investigated. Methods. Cellular senescence was monitored by β-galactosidase activity. Gene expression was monitored by real-time PCR. Survivin was analyzed by Western blot and immunofluorescence. Migration and invasion were monitored using appropriate kits. Glucose transporters (GLUTs) were monitored by 3-O-methyl-D-glucose uptake. Histone deacetylases (HDACs) were studied by monitoring catalytic activity and acetylation status of histones H3/H4. Results. Hfe−/− RPE cells exhibited slower senescence rate and higher survivin expression than wild type cells. Hfe−/− cells migrated faster and showed greater glucose uptake and increased expression of GLUTs. The expression of HDACs and DNA methyltransferase (DNMTs) also was increased. Similarly, RPE cells from hemojuvelin (Hjv)-knockout mice, another model of hemochromatosis, also had increased expression of GLUTs, HDACs, and DNMTs. The expression of Slc5a8 was decreased in Hfe−/− RPE cells, but treatment with a DNA methylation inhibitor restored the transporter expression, indicating involvement of DNA methylation in the silencing of Slc5a8 in Hfe−/− cells. Conclusions. RPE cells from iron-overloaded mice exhibit several features of tumor cells: decreased senescence, enhanced migration, increased glucose uptake, and elevated levels of HDACs and DNMTs. These features are seen in Hfe−/− RPE cells as well as in Hjv−/− RPE cells, providing a molecular basis for the hyperproliferative phenotype of Hfe−/− and Hjv−/− RPE cells. PMID:23169885

Cultured Human Retinal Pigment Epithelial (hRPE) Sheets: A Search for Suitable Storage Conditions.

PubMed

Khan, Ayyad Z; Utheim, Tor P; Reppe, Sjur; Sandvik, Leiv; Lyberg, Torstein; Roald, Borghild B-H; Ibrahim, Ibrahim B; Eidet, Jon R

2018-04-01

The advancement of human retinal pigment epithelial cell (hRPE) replacement therapy is partly dependent on optimization of cell culture, cell preservation, and storage medium. This study was undertaken to search for a suitable storage temperature and storage medium for hRPE. hRPE monolayer sheets were cultured under standard conditions at 37°C and then randomized for storage at six temperatures (4, 16, 20, 24, 28, and 37°C) for 7 days. After revealing a suitable storage temperature, hRPE sheets were subsequently stored with and without the silk protein sericin added to the storage medium. Live/dead assay, light microscopy, pH, and phenotypic expression of various proteins were used to assess cell cultures stored at different temperatures. After 7 days of storage, hRPE morphology was best preserved at 4°C. Addition of sericin to the storage medium maintained the characteristic morphology of the preserved cells, and improved pigmentation and levels of pigmentation-related proteins in the cultured hRPE sheets following a 7-day storage period at 4°C.

Dynamics and detection of laser induced microbubbles in the retinal pigment epithelium (RPE)

NASA Astrophysics Data System (ADS)

Fritz, Andreas; Ptaszynski, Lars; Stoehr, Hardo; Brinkmann, Ralf

2007-07-01

Selective Retina Treatment (SRT) is a new method to treat eye diseases associated with disorders of the RPE. Selective RPE cell damage is achieved by applying a train of 1.7 μs laser pulses at 527 nm. The treatment of retinal diseases as e.g. diabetic maculopathy (DMP), is currently investigated within clinical studies, however 200 ns pulse durations are under investigation. Transient micro bubbles in the retinal pigment epithelium (RPE) are expected to be the origin of cell damage due to irradiation with laser pulses shorter than 50 μs. The bubbles emerge at the strongly absorbing RPE melanosomes. Cell membrane disruption caused by the transient associated volume increase is expected to be the origin of the angiographically observed RPE leakage. We investigate micro bubble formation and dynamics in porcine RPE using pulse durations of 150 ns. A laser interferometry system at 830 nm with the aim of an online dosimetry control for SRT was developed. Bubble formation was detected interferometrically and by fast flash photography. A correlation to cell damage observed with a vitality stain is found. A bubble detection algorithm is presented.

Monomethylfumarate Induces γ-Globin Expression and Fetal Hemoglobin Production in Cultured Human Retinal Pigment Epithelial (RPE) and Erythroid Cells, and in Intact Retina

PubMed Central

Promsote, Wanwisa; Makala, Levi; Li, Biaoru; Smith, Sylvia B.; Singh, Nagendra; Ganapathy, Vadivel; Pace, Betty S.; Martin, Pamela M.

2014-01-01

Purpose. Sickle retinopathy (SR) is a major cause of vision loss in sickle cell disease (SCD). There are no strategies to prevent SR and treatments are extremely limited. The present study evaluated (1) the retinal pigment epithelial (RPE) cell as a hemoglobin producer and novel cellular target for fetal hemoglobin (HbF) induction, and (2) monomethylfumarate (MMF) as an HbF-inducing therapy and abrogator of oxidative stress and inflammation in SCD retina. Methods. Human globin gene expression was evaluated by RT–quantitative (q)PCR in the human RPE cell line ARPE-19 and in primary RPE cells isolated from Townes humanized SCD mice. γ-Globin promoter activity was monitored in KU812 stable dual luciferase reporter expressing cells treated with 0 to 1000 μM dimethylfumarate, MMF, or hydroxyurea (HU; positive control) by dual luciferase assay. Reverse transcriptase–qPCR, fluorescence-activated cell sorting (FACS), immunofluorescence, and Western blot techniques were used to evaluate γ-globin expression and HbF production in primary human erythroid progenitors, ARPE-19, and normal hemoglobin producing (HbAA) and homozygous βs mutation (HbSS) RPE that were treated similarly, and in MMF-injected (1000 μM) HbAA and HbSS retinas. Dihydroethidium labeling and nuclear factor (erythroid-derived 2)-like 2 (Nrf2), IL-1β, and VEGF expression were also analyzed. Results. Retinal pigment epithelial cells express globin genes and synthesize adult and fetal hemoglobin MMF stimulated γ-globin expression and HbF production in cultured RPE and erythroid cells, and in HbSS mouse retina where it also reduced oxidative stress and inflammation. Conclusions. The production of hemoglobin by RPE suggests the potential involvement of this cell type in the etiology of SR. Monomethylfumarate influences multiple parameters consistent with improved retinal health in SCD and may therefore be of therapeutic potential in SR treatment. PMID:24825111

Cell Migration

PubMed Central

Trepat, Xavier; Chen, Zaozao; Jacobson, Ken

2015-01-01

Cell migration is fundamental to establishing and maintaining the proper organization of multicellular organisms. Morphogenesis can be viewed as a consequence, in part, of cell locomotion, from large-scale migrations of epithelial sheets during gastrulation, to the movement of individual cells during development of the nervous system. In an adult organism, cell migration is essential for proper immune response, wound repair, and tissue homeostasis, while aberrant cell migration is found in various pathologies. Indeed, as our knowledge of migration increases, we can look forward to, for example, abating the spread of highly malignant cancer cells, retarding the invasion of white cells in the inflammatory process, or enhancing the healing of wounds. This article is organized in two main sections. The first section is devoted to the single-cell migrating in isolation such as occurs when leukocytes migrate during the immune response or when fibroblasts squeeze through connective tissue. The second section is devoted to cells collectively migrating as part of multicellular clusters or sheets. This second type of migration is prevalent in development, wound healing, and in some forms of cancer metastasis. PMID:23720251

Two dietary polyphenols, fisetin and luteolin, reduce inflammation but augment DNA damage-induced toxicity in human RPE cells.

PubMed

Hytti, Maria; Szabó, Dora; Piippo, Niina; Korhonen, Eveliina; Honkakoski, Paavo; Kaarniranta, Kai; Petrovski, Goran; Kauppinen, Anu

2017-04-01

Plant-derived polyphenols are known to possess anti-inflammatory and antioxidant effects. In recent years, several studies have investigated their potential benefits for treating chronic diseases associated with prolonged inflammation and excessive oxidative stress, such as age-related macular degeneration (AMD). Previously, two polyphenols, fisetin and luteolin, have been reported to increase the survival of retinal pigment epithelial (RPE) cells suffering from oxidative stress as well as decreasing inflammation but the benefits of polyphenol therapy seem to depend on the model system used. Our aim was to analyze the effects of fisetin and luteolin on inflammation and cellular viability in a model of nonoxidative DNA damage-induced cell death in human RPE (hRPE) cells. Pretreatment of ARPE-19 or primary hRPE cells with the polyphenols augmented etoposide-induced cell death as measured by the lactate dehydrogenase and 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide assays. However, the treatment was able to reduce the release of two proinflammatory cytokines, IL-6 and IL-8, which were determined by enzyme-linked Immunosorbent assay. Analyses of caspase 3 activity, p53 acetylation and SIRT1 protein levels revealed the apoptotic nature of etoposide-evoked cell death and that fisetin and luteolin augmented the etoposide-induced acetylation of p53 and decreased SIRT1 levels. Taken together, our findings suggest that the cytoprotective effects of fisetin and luteolin depend on the stressor they need to combat, whereas their anti-inflammatory potential is sustained over a variety of model systems. Careful consideration of disease pathways will be necessary before fisetin or luteolin can be recommended as therapeutic agents for inflammatory diseases in general and specifically AMD. Copyright © 2017 Elsevier Inc. All rights reserved.

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells.

PubMed

Liu, Ji; Lu, Wennan; Guha, Sonia; Baltazar, Gabriel C; Coffey, Erin E; Laties, Alan M; Rubenstein, Ronald C; Reenstra, William W; Mitchell, Claire H

2012-07-15

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTR(inh)-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4(-/-) mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization.

Cystic fibrosis transmembrane conductance regulator contributes to reacidification of alkalinized lysosomes in RPE cells

PubMed Central

Liu, Ji; Lu, Wennan; Guha, Sonia; Baltazar, Gabriel C.; Coffey, Erin E.; Laties, Alan M.; Rubenstein, Ronald C.; Reenstra, William W.

2012-01-01

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in lysosomal acidification has been difficult to determine. We demonstrate here that CFTR contributes more to the reacidification of lysosomes from an elevated pH than to baseline pH maintenance. Lysosomal alkalinization is increasingly recognized as a factor in diseases of accumulation, and we previously showed that cAMP reacidified alkalinized lysosomes in retinal pigmented epithelial (RPE) cells. As the influx of anions to electrically balance proton accumulation may enhance lysosomal acidification, the contribution of the cAMP-activated anion channel CFTR to lysosomal reacidification was probed. The antagonist CFTRinh-172 had little effect on baseline levels of lysosomal pH in cultured human RPE cells but substantially reduced the reacidification of compromised lysosomes by cAMP. Likewise, CFTR activators had a bigger impact on cells whose lysosomes had been alkalinized. Knockdown of CFTR with small interfering RNA had a larger effect on alkalinized lysosomes than on baseline levels. Inhibition of CFTR in isolated lysosomes altered pH. While CFTR and Lamp1 were colocalized, treatment with cAMP did not increase targeting of CFTR to the lysosome. The inhibition of CFTR slowed lysosomal degradation of photoreceptor outer segments while activation of CFTR enhanced their clearance from compromised lysosomes. Activation of CFTR acidified RPE lysosomes from the ABCA4−/− mouse model of recessive Stargardt's disease, whose lysosomes are considerably alkalinized. In summary, CFTR contributes more to reducing lysosomal pH from alkalinized levels than to maintaining baseline pH. Treatment to activate CFTR may thus be of benefit in disorders of accumulation associated with lysosomal alkalinization. PMID:22572847

The Status of RPE65 Gene Therapy Trials: Safety and Efficacy

PubMed Central

Pierce, Eric A.; Bennett, Jean

2015-01-01

Several groups have reported the results of clinical trials of gene augmentation therapy for Leber congenital amaurosis (LCA) because of mutations in the RPE65 gene. These studies have used subretinal injection of adeno-associated virus (AAV) vectors to deliver the human RPE65 cDNA to the retinal pigment epithelial (RPE) cells of the treated eyes. In all of the studies reported to date, this approach has been shown to be both safe and effective. The successful clinical trials of gene augmentation therapy for retinal degeneration caused by mutations in the RPE65 gene sets the stage for broad application of gene therapy to treat retinal degenerative disorders. PMID:25635059

RPE65 gene therapy slows cone loss in Rpe65-deficient dogs.

PubMed

Mowat, F M; Breuwer, A R; Bartoe, J T; Annear, M J; Zhang, Z; Smith, A J; Bainbridge, J W B; Petersen-Jones, S M; Ali, R R

2013-05-01

Recent clinical trials of retinal pigment epithelium gene (RPE65) supplementation therapy in Leber congenital amaurosis type 2 patients have demonstrated improvements in rod and cone function, but it may be some years before the effects of therapy on photoreceptor survival become apparent. The Rpe65-deficient dog is a very useful pre-clinical model in which to test efficacy of therapies, because the dog has a retina with a high degree of similarity to that of humans. In this study, we evaluated the effect of RPE65 gene therapy on photoreceptor survival in order to predict the potential benefit and limitations of therapy in patients. We examined the retinas of Rpe65-deficient dogs after RPE65 gene therapy to evaluate the preservation of rods and cone photoreceptor subtypes. We found that gene therapy preserves both rods and cones. While the moderate loss of rods in the Rpe65-deficient dog retina is slowed by gene therapy, S-cones are lost extensively and gene therapy can prevent that loss, although only within the treated area. Although LM-cones are not lost extensively, cone opsin mislocalization indicates that they are stressed, and this can be partially reversed by gene therapy. Our results suggest that gene therapy may be able to slow cone degeneration in patients if intervention is sufficiently early and also that it is probably important to treat the macula in order to preserve central function.

Repressed SIRT1/PGC-1α pathway and mitochondrial disintegration in iPSC-derived RPE disease model of age-related macular degeneration.

PubMed

Golestaneh, Nady; Chu, Yi; Cheng, Shuk Kei; Cao, Hong; Poliakov, Eugenia; Berinstein, Daniel M

2016-12-20

Study of age related macular degeneration (AMD) has been hampered by lack of human models that represent the complexity of the disease. Here we have developed a human in vitro disease model of AMD to investigate the underlying AMD disease mechanisms. Generation of iPSCs from retinal pigment epithelium (RPE) of AMD donors, age-matched normal donors, skin fibroblasts of a dry AMD patient, and differentiation of iPSCs into RPE (AMD RPE-iPSC-RPE, normal RPE-iPSC-RPE and AMD Skin-iPSC-RPE, respectively). Immunostaining, cell viability assay and reactive oxygen species (ROS) production under oxidative stress conditions, electron microscopy (EM) imaging, ATP production and glycogen concentration assays, quantitative real time PCR, western blot, karyotyping. The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE present functional impairment and exhibit distinct disease phenotypes compared to RPE-iPSC-RPE generated from normal donors (Normal RPE-iPSC-RPE). The AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE show increased susceptibility to oxidative stress and produced higher levels of reactive oxygen species (ROS) under stress in accordance with recent reports. The susceptibility to oxidative stress-induced cell death in AMD RPE-iPSC-RPE and Skin-iPSC-RPE was consistent with inability of the AMD RPE-iPSC-RPE and Skin-iPSC-RPE to increase SOD2 expression under oxidative stress. Phenotypic analysis revealed disintegrated mitochondria, accumulation of autophagosomes and lipid droplets in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE. Mitochondrial activity was significantly lower in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE compared to normal cells and glycogen concentration was significantly increased in the diseased cells. Furthermore, Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), a regulator of mitochondrial biogenesis and function was repressed, and lower expression levels of NAD-dependent deacetylase sirtuin1 (SIRT1) were found in AMD RPE-iPSC-RPE and AMD Skin-iPSC-RPE

Damage Thresholds for Exposure to NIR and Blue Lasers in an In Vitro RPE Cell System

DTIC Science & Technology

2006-07-01

damage , and to identify antioxidants capable of protecting these cells from laser-in- duced cell death. MATERIALS AND METHODS The human RPE cell...melanosomes in blue laser-induced damage in vitro, which confirms the view that melanin plays an important role in photochemical damage mechanisms in...community has only a validating role in the animal ED50 damage threshold data used by safety committees. Systems of in vitro analysis must be

Function of MYO7A in the Human RPE and the Validity of Shaker1 Mice as a Model for Usher Syndrome 1B

PubMed Central

Gibbs, Daniel; Diemer, Tanja; Khanobdee, Kornnika; Hu, Jane; Bok, Dean

2010-01-01

Purpose. To investigate the function of MYO7A in human RPE cells and to test the validity of using shaker1 RPE in preclinical studies on therapies for Usher syndrome 1B by comparing human and mouse cells. Methods. MYO7A was localized by immunofluorescence. Primary cultures of human and mouse RPE cells were used to measure melanosome motility and rod outer segment (ROS) phagocytosis and digestion. MYO7A was knocked down in the human RPE cells by RNAi to test for a mutant phenotype in melanosome motility. Results. The distribution of MYO7A in the RPE of human and mouse was found to be comparable, both in vivo and in primary cultures. Primary cultures of human RPE cells phagocytosed and digested ROSs with kinetics comparable to that of primary cultures of mouse RPE cells. Melanosome motility was also comparable, and, after RNAi knockdown, consisted of longer-range fast movements characteristic of melanosomes in shaker1 RPE. Conclusions. The localization and function of MYO7A in human RPE cells is comparable to that in mouse RPE cells. Although shaker1 retinas do not undergo degeneration, correction of mutant phenotypes in the shaker1 RPE represents a valid preclinical test for potential therapeutic treatments. PMID:19643958

A local complement response by RPE causes early-stage macular degeneration

PubMed Central

Fernandez-Godino, Rosario; Garland, Donita L.; Pierce, Eric A.

2015-01-01

Inherited and age-related macular degenerations (AMDs) are important causes of vision loss. An early hallmark of these disorders is the formation of sub-retinal pigment epithelium (RPE) basal deposits. A role for the complement system in MDs was suggested by genetic association studies, but direct functional connections between alterations in the complement system and the pathogenesis of MD remain to be defined. We used primary RPE cells from a mouse model of inherited MD due to a p.R345W mutation in EGF-containing fibulin-like extracellular matrix protein 1 (EFEMP1) to investigate the role of the RPE in early MD pathogenesis. Efemp1R345W RPE cells recapitulate the basal deposit formation observed in vivo by producing sub-RPE deposits in vitro. The deposits share features with basal deposits, and their formation was mediated by EFEMP1R345W or complement component 3a (C3a), but not by complement component 5a (C5a). Increased activation of complement appears to occur in response to an abnormal extracellular matrix (ECM), generated by the mutant EFEMP1R345W protein and reduced ECM turnover due to inhibition of matrix metalloproteinase 2 by EFEMP1R345W and C3a. Increased production of C3a also stimulated the release of cytokines such as interleukin (IL)-6 and IL-1B, which appear to have a role in deposit formation, albeit downstream of C3a. These studies provide the first direct indication that complement components produced locally by the RPE are involved in the formation of basal deposits. Furthermore, these results suggest that C3a generated by RPE is a potential therapeutic target for the treatment of EFEMP1-associated MD as well as AMD. PMID:26199322

Two Bioactive Molecular Weight Fractions of a Conditioned Medium Enhance RPE Cell Survival on Age-Related Macular Degeneration and Aged Bruch's Membrane

PubMed Central

Sugino, Ilene K.; Sun, Qian; Springer, Carola; Cheewatrakoolpong, Noounanong; Liu, Tong; Li, Hong; Zarbin, Marco A.

2016-01-01

Purpose To characterize molecular weight fractions of bovine corneal endothelial cell conditioned medium (CM) supporting retinal pigment epithelium (RPE) cell survival on aged and age-related macular degeneration (AMD) Bruch's membrane. Methods CM was subject to size separation using centrifugal filters. Retentate and filtrate fractions were tested for bioactivity by analyzing RPE survival on submacular Bruch's membrane of aged and AMD donor eyes and behavior on collagen I-coated tissue culture wells. Protein and peptide composition of active fractions was determined by mass spectrometry. Results Two bioactive fractions, 3-kDa filtrate and a 10-50–kDa fraction, were necessary for RPE survival on aged and AMD Bruch's membrane. The 3-kDa filtrate, but not the 10-50–kDa fraction, supported RPE growth on collagen 1‐coated tissue culture plates. Mass spectrometry of the 10-50–kDa fraction identified 175 extracellular proteins, including growth factors and extracellular matrix molecules. Transforming growth factor (TGF)β-2 was identified as unique to active CM. Peptides representing 29 unique proteins were identified in the 3-KDa filtrate. Conclusions These results indicate there is a minimum of two bioactive molecules in CM, one found in the 3-kDa filtrate and one in the 10-50–kDa fraction, and that bioactive molecules in both fractions must be present to ensure RPE survival on Bruch's membrane. Mass spectrometry analysis suggested proteins to test in future studies to identify proteins that may contribute to CM bioactivity. Translational Relevance Results of this study are the first steps in development of an adjunct to cell-based therapy to ensure cell transplant survival and functionality in AMD patients. PMID:26933521

Pulsewidth-dependent nature of laser-induced DNA damage in RPE cells

NASA Astrophysics Data System (ADS)

Hall, Rebecca M.; Glickman, Randolph D.; Rockwell, Benjamin A.; Kumar, Neeru; Noojin, Gary D.

2001-07-01

Ultrashort pulse laser radiation may produce cellular damage through unique mechanisms. Primary cultures of bovine retinal pigment epithelial (RPE) cells were exposed to the out put of a Ti:Sapphire laser producing 30 fs (mode-locked) pulses, 44 amplified fs pulses, or continuous wave exposures at 800 nm. Laser exposures at and below the damage threshold were studied. DNA damage was detected using single cell gel electrophoresis (comet assay). Unexposed (control) cells produced short tails with low tail moments. In contrast, all laser-exposed cells showed some degree of DNA fragmentation, but the size and shape of the resulting comets differed among the various modalities. CW-exposed cells produced generally light and relatively compact tails, suggesting fewer and larger DNA fragments, while mode-locked laser exposures (30 fs pulses) resulted in large and diffuse comets, indicating the DNA was fragmented into many very small pieces. Work is continuing to define the relationship of laser pulsewidth and intensity with the degree of DNA fragmentation. These results suggest that DNA damage may result from multiple mechanisms of laser-cell interaction, including multiphoton absorption.

Successful gene therapy in older Rpe65-deficient dogs following subretinal injection of an adeno-associated vector expressing RPE65.

PubMed

Annear, Matthew J; Mowat, Freya M; Bartoe, Joshua T; Querubin, Janice; Azam, Selina A; Basche, Mark; Curran, Paul G; Smith, Alexander J; Bainbridge, James W B; Ali, Robin R; Petersen-Jones, Simon M

2013-10-01

Young Rpe65-deficient dogs have been used as a model for human RPE65 Leber congenital amaurosis (RPE65-LCA) in proof-of-concept trials of recombinant adeno-associated virus (rAAV) gene therapy. However, there are relatively few reports of the outcome of rAAV gene therapy in Rpe65-deficient dogs older than 2 years of age. The purpose of this study was to investigate the success of this therapy in older Rpe65-deficient dogs. Thirteen eyes were treated in dogs between 2 and 6 years old. An rAAV2 vector expressing the human RPE65 cDNA driven by the human RPE65 promoter was delivered by subretinal injection. Twelve of the 13 eyes had improved retinal function as assessed by electroretinography, and all showed improvement in vision at low lighting intensities. Histologic examination of five of the eyes was performed but found no correlation between electroretinogram (ERG) rescue and numbers of remaining photoreceptors. We conclude that functional rescue is still possible in older dogs and that the use of older Rpe65-deficient dogs, rather than young Rpe65-deficient dogs that have very little loss of photoreceptors, more accurately models the situation when treating human RPE65-LCA patients.

Hyperglycaemia Exacerbates Choroidal Neovascularisation in Mice via the Oxidative Stress-Induced Activation of STAT3 Signalling in RPE Cells

PubMed Central

Wang, Yu-Sheng; Shi, Yuan-Yuan; Hou, Wei; Xu, Chun-Sheng; Wang, Hai-Yan; Ye, Zi; Yao, Li-Bo; Zhang, Jian

2012-01-01

Choroidal neovascularisation (CNV) that occurs as a result of age-related macular degeneration (AMD) causes severe vision loss among elderly patients. The relationship between diabetes and CNV remains controversial. However, oxidative stress plays a critical role in the pathogenesis of both AMD and diabetes. In the present study, we investigated the influence of diabetes on experimentally induced CNV and on the underlying molecular mechanisms of CNV. CNV was induced via photocoagulation in the ocular fundi of mice with streptozotocin-induced diabetes. The effect of diabetes on the severity of CNV was measured. An immunofluorescence technique was used to determine the levels of oxidative DNA damage by anti-8-hydroxy-2-deoxyguanosine (8-OHdG) antibody, the protein expression of phosphorylated signal transducer and activator of transcription 3 (p-STAT3) and vascular endothelial growth factor (VEGF), in mice with CNV. The production of reactive oxygen species (ROS) in retinal pigment epithelial (RPE) cells that had been cultured under high glucose was quantitated using the 2′,7′-dichlorofluorescein diacetate (DCFH-DA) method. p-STAT3 expression was examined using Western blot analysis. RT-PCR and ELISA processes were used to detect VEGF expression. Hyperglycaemia exacerbated the development of CNV in mice. Oxidative stress levels and the expression of p-STAT3 and VEGF were highly elevated both in mice and in cultured RPE cells. Treatment with the antioxidant compound N-acetyl-cysteine (NAC) rescued the severity of CNV in diabetic mice. NAC also inhibited the overexpression of p-STAT3 and VEGF in CNV and in RPE cells. The JAK-2/STAT3 pathway inhibitor AG490 blocked VEGF expression but had no effect on the production of ROS in vitro. These results suggest that hyperglycaemia promotes the development of CNV by inducing oxidative stress, which in turn activates STAT3 signalling in RPE cells. Antioxidant supplementation helped attenuate the development of CNV. Thus, our

Can mesenchymal cells undergo collective cell migration?

PubMed Central

Theveneau, Eric

2011-01-01

Cell migration is critical for proper development of the embryo and is also used by many cell types to perform their physiological function. For instance, cell migration is essential for immune cells to monitor the body and for epithelial cells to heal a wound whereas, in cancer cells, acquisition of migratory capabilities is a critical step toward malignancy. Migratory cells are often categorized into two groups: (1) mesenchymal cells, produced by an epithelium-to-mesenchyme transition, that undergo solitary migration and (2) epithelial-like cells which migrate collectively. However, on some occasions, mesenchymal cells may travel in large, dense groups and exhibit key features of collectively migrating cells such as coordination and cooperation. Here, using data published on neural crest cells, a highly invasive mesenchymal cell population that extensively migrate throughout the embryo, we explore the idea that mesenchymal cells, including cancer cells, might be able to undergo collective cell migration under certain conditions and discuss how they could do so. PMID:22274714

TNF-α mediates choroidal neovascularization by upregulating VEGF expression in RPE through ROS-dependent β-catenin activation.

PubMed

Wang, Haibo; Han, Xiaokun; Wittchen, Erika S; Hartnett, M Elizabeth

2016-01-01

Inflammation, oxidative stress, and angiogenesis have been proposed to interact in age-related macular degeneration. It has been postulated that external stimuli that cause oxidative stress can increase production of vascular endothelial growth factor (VEGF) in retinal pigment epithelial (RPE) cells. In this study, we tested the hypothesis that the inflammatory cytokine, tumor necrosis factor alpha (TNF-α), contributed to choroidal neovascularization (CNV) by upregulating VEGF in RPE through intracellular reactive oxygen species (ROS)-dependent signaling and sought to understand the mechanisms involved. In a murine laser-induced CNV model, 7 days after laser treatment and intravitreal neutralizing mouse TNF-α antibody or isotype immunoglobulin G (IgG) control, the following measurements were made: 1) TNF-α protein and VEGF protein in RPE/choroids with western blot, 2) CNV volume in RPE/choroidal flatmounts, and 3) semiquantification of oxidized phospholipids stained with E06 antibody within CNV with immunohistochemistry (IHC). In cultured human RPE cells treated with TNF-α or PBS control, 1) ROS generation was measured using the 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence assay, and 2) NOX4 protein and VEGF protein or mRNA were measured with western blot or quantitative real-time PCR in cells pretreated with apocynin or nicotinamide adenine dinucleotide phosphate-oxidase (NADPH) inhibitor, VAS 2870, or transfected with p22phox siRNA, and each was compared to its appropriate control. Western blots of phosphorylated p65 (p-p65), total p65 and β-actin, and quantitative real-time PCR of VEGF mRNA were measured in human RPE cells treated with TNF-α and pretreatment with the nuclear factor kappa B inhibitor, Bay 11-7082 or control. Western blots of β-catenin, VEGF, and p22phox and coimmunoprecipitation of β-catenin and T-cell transcriptional factor were performed in human RPE cells treated with TNF-α following pretreatment with �

Vitreous-induced cytoskeletal rearrangements via the Rac1 GTPase-dependent signaling pathway in human retinal pigment epithelial cells

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

Huang, Xionggao; Department of Ophthalmology, Hainan Medical College, Haikou; Wei, Yantao

2012-03-09

Highlights: Black-Right-Pointing-Pointer Vitreous induces morphological changes and cytoskeletal rearrangements in RPE cells. Black-Right-Pointing-Pointer Rac1 is activated in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition prevents morphological changes in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer Rac inhibition suppresses cytoskeletal rearrangements in vitreous-transformed RPE cells. Black-Right-Pointing-Pointer The vitreous-induced effects are mediated by a Rac1 GTPase/LIMK1/cofilin pathway. -- Abstract: Proliferative vitreoretinopathy (PVR) is mainly caused by retinal pigment epithelial (RPE) cell migration, invasion, proliferation and transformation into fibroblast-like cells that produce the extracellular matrix (ECM). The vitreous humor is known to play an important role in PVR. An epithelial-to-mesenchymal transdifferentiation (EMT) of human RPE cells inducedmore » by 25% vitreous treatment has been linked to stimulation of the mesenchymal phenotype, migration and invasion. Here, we characterized the effects of the vitreous on the cell morphology and cytoskeleton in human RPE cells. The signaling pathway that mediates these effects was investigated. Serum-starved RPE cells were incubated with 25% vitreous, and the morphological changes were examined by phase-contrast microscopy. Filamentous actin (F-actin) was examined by immunofluorescence and confocal microscopy. Protein phosphorylation of AKT, ERK1/2, Smad2/3, LIM kinase (LIMK) 1 and cofilin was analyzed by Western blot analysis. Vitreous treatment induced cytoskeletal rearrangements, activated Rac1 and enhanced the phosphorylation of AKT, ERK1/2 and Smad2/3. When the cells were treated with a Rac activation-specific inhibitor, the cytoskeletal rearrangements were prevented, and the phosphorylation of Smad2/3 was blocked. Vitreous treatment also enhanced the phosphorylation of LIMK1 and cofilin and the Rac inhibitor blocked this effect. We propose that

Live Imaging of LysoTracker-Labelled Phagolysosomes Tracks Diurnal Phagocytosis of Photoreceptor Outer Segment Fragments in Rat RPE Tissue Ex Vivo.

PubMed

Mao, Yingyu; Finnemann, Silvia C

2016-01-01

Renewal of rod photoreceptor outer segments in the mammalian eye involves synchronized diurnal shedding after light onset of spent distal outer segment fragments (POS) linked to swift clearance of shed POS from the subretinal space by the adjacent retinal pigment epithelium (RPE). Engulfed POS phagosomes in RPE cells mature to acidified phagolysosomes, which accomplish enzymatic degradation of POS macromolecules. Here, we used an acidophilic fluorophore LysoTracker to label acidic organelles in freshly dissected, live rat RPE tissue flat mounts. We observed that all RPE cells imaged contained numerous acidified POS phagolysosomes whose abundance per cell was dramatically increased 2 h after light onset as compared to either 1 h before or 4 h after light onset. Lack of organelles of similar diameter (of 1-2 μm) in phagocytosis-defective mutant RCS rat RPE confirmed that LysoTracker live imaging detected POS phagolysosomes. Lack of increase in lysosomal membrane protein LAMP-1 in RPE/choroid during the diurnal phagocytic burst suggests that formation of POS phagolysosomes in RPE in situ may not involve extra lysosome membrane biogenesis. Taken together, we report a new imaging approach that directly detects POS phagosome acidification and allows rapid tracking and quantification of POS phagocytosis by live RPE -tissue ex situ.

Fisetin inhibits epidermal growth factor-induced migration of ARPE-19 cells by suppression of AKT activation and Sp1-dependent MMP-9 expression.

PubMed

Lin, Hung-Yu; Chen, Yong-Syuan; Wang, Kai; Chien, Hsiang-Wen; Hsieh, Yi-Hsien; Yang, Shun-Fa

2017-01-01

Proliferative vitreoretinopathy (PVR) can result in abnormal migration of RPE cells. Fisetin is a naturally occurring compound that has been reported to have antitumor effects, but its effects on epidermal growth factor (EGF)-induced cell migration and the underlying mechanisms remain unclear. Effects of fisetin on EGF-induced cell viability and migration were examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and in vitro migration assays. Reverse transcription-PCR (RT-PCR) and immunoblotting were performed to evaluate matrix metallopeptidase-9 (MMP-9) expression and activation of specificity protein-1 (Sp1) and protein kinase B (AKT) in ARPE-19 cells treated with EGF and with or without fisetin. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to examine Sp1 transcription activity and MMP-9 binding activity. Fisetin did not affect ARPE-19 cell viability and significantly inhibited the EGF-induced migration capacity of ARPE-19 cells. Furthermore, fisetin exerted an antimigratory effect and suppressed MMP-9 mRNA and protein expression. Treatment with EGF induced phosphorylation of AKT and expression of MMP-9 and Sp1. Fisetin combined with LY294002 (an inhibitor of AKT) prevented the EGF-induced migration involved in downregulation of Sp1 and MMP-9 expression. Luciferase and ChIP assays suggested that fisetin remarkably decreased the EGF-induced transcription activity of MMP-9 and Sp1 and inhibited EGF-mediated Sp1 from directly binding to the MMP-9 promoter in ARPE-19 cells. Fisetin inhibited EGF-induced cell migration via modulation of AKT/Sp1-dependent MMP-9 transcriptional activity. Therefore, fisetin may be a potential agent in the treatment of migratory PVR diseases.

Voltage-dependent ion channels in the mouse RPE: comparison with Norrie disease mice.

PubMed

Wollmann, Guido; Lenzner, Steffen; Berger, Wolfgang; Rosenthal, Rita; Karl, Mike O; Strauss, Olaf

2006-03-01

We studied electrophysiological properties of cultured retinal pigment epithelial (RPE) cells from mouse and a mouse model for Norrie disease. Wild-type RPE cells revealed the expression of ion channels known from other species: delayed-rectifier K(+) channels composed of Kv1.3 subunits, inward rectifier K(+) channels, Ca(V)1.3 L-type Ca(2+) channels and outwardly rectifying Cl(-) channels. Expression pattern and the ion channel characteristics current density, blocker sensitivity, kinetics and voltage-dependence were compared in cells from wild-type and Norrie mice. Although no significant differences were observed, our study provides a base for future studies on ion channel function and dysfunction in transgenic mouse models.

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

PubMed Central

Riera, Marina; Fontrodona, Laura; Albert, Silvia; Ramirez, Diana Mora; Seriola, Anna; Salas, Anna; Muñoz, Yolanda; Ramos, David; Villegas-Perez, Maria Paz; Zapata, Miguel Angel; Raya, Angel; Ruberte, Jesus; Veiga, Anna; Garcia-Arumi, Jose

2016-01-01

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD. PMID:27006969

Collective cell migration in development

PubMed Central

Scarpa, Elena

2016-01-01

During embryonic development, tissues undergo major rearrangements that lead to germ layer positioning, patterning, and organ morphogenesis. Often these morphogenetic movements are accomplished by the coordinated and cooperative migration of the constituent cells, referred to as collective cell migration. The molecular and biomechanical mechanisms underlying collective migration of developing tissues have been investigated in a variety of models, including border cell migration, tracheal branching, blood vessel sprouting, and the migration of the lateral line primordium, neural crest cells, or head mesendoderm. Here we review recent advances in understanding collective migration in these developmental models, focusing on the interaction between cells and guidance cues presented by the microenvironment and on the role of cell–cell adhesion in mechanical and behavioral coupling of cells within the collective. PMID:26783298

Focal Adhesion-Independent Cell Migration.

PubMed

Paluch, Ewa K; Aspalter, Irene M; Sixt, Michael

2016-10-06

Cell migration is central to a multitude of physiological processes, including embryonic development, immune surveillance, and wound healing, and deregulated migration is key to cancer dissemination. Decades of investigations have uncovered many of the molecular and physical mechanisms underlying cell migration. Together with protrusion extension and cell body retraction, adhesion to the substrate via specific focal adhesion points has long been considered an essential step in cell migration. Although this is true for cells moving on two-dimensional substrates, recent studies have demonstrated that focal adhesions are not required for cells moving in three dimensions, in which confinement is sufficient to maintain a cell in contact with its substrate. Here, we review the investigations that have led to challenging the requirement of specific adhesions for migration, discuss the physical mechanisms proposed for cell body translocation during focal adhesion-independent migration, and highlight the remaining open questions for the future.

Fisetin inhibits epidermal growth factor–induced migration of ARPE-19 cells by suppression of AKT activation and Sp1-dependent MMP-9 expression

PubMed Central

Lin, Hung-Yu; Chen, Yong-Syuan; Wang, Kai; Chien, Hsiang-Wen

2017-01-01

Purpose Proliferative vitreoretinopathy (PVR) can result in abnormal migration of RPE cells. Fisetin is a naturally occurring compound that has been reported to have antitumor effects, but its effects on epidermal growth factor (EGF)–induced cell migration and the underlying mechanisms remain unclear. Methods Effects of fisetin on EGF-induced cell viability and migration were examined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and in vitro migration assays. Reverse transcription–PCR (RT–PCR) and immunoblotting were performed to evaluate matrix metallopeptidase-9 (MMP-9) expression and activation of specificity protein-1 (Sp1) and protein kinase B (AKT) in ARPE-19 cells treated with EGF and with or without fisetin. Luciferase and chromatin immunoprecipitation (ChIP) assays were performed to examine Sp1 transcription activity and MMP-9 binding activity. Results Fisetin did not affect ARPE-19 cell viability and significantly inhibited the EGF-induced migration capacity of ARPE-19 cells. Furthermore, fisetin exerted an antimigratory effect and suppressed MMP-9 mRNA and protein expression. Treatment with EGF induced phosphorylation of AKT and expression of MMP-9 and Sp1. Fisetin combined with LY294002 (an inhibitor of AKT) prevented the EGF-induced migration involved in downregulation of Sp1 and MMP-9 expression. Luciferase and ChIP assays suggested that fisetin remarkably decreased the EGF-induced transcription activity of MMP-9 and Sp1 and inhibited EGF-mediated Sp1 from directly binding to the MMP-9 promoter in ARPE-19 cells. Conclusions Fisetin inhibited EGF-induced cell migration via modulation of AKT/Sp1–dependent MMP-9 transcriptional activity. Therefore, fisetin may be a potential agent in the treatment of migratory PVR diseases. PMID:29296070

Histopathology and Functional Correlations in a Patient with a Mutation in RPE65, the Gene for Retinol Isomerase

PubMed Central

Rayborn, Mary E.; Li, Yong; Grossman, Gregory H.; Berson, Eliot L.; Hollyfield, Joe G.

2011-01-01

Purpose. Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51. Methods. Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors. Results. Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons. Conclusions. The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed. PMID:21931134

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq.

PubMed

Whitmore, S Scott; Wagner, Alex H; DeLuca, Adam P; Drack, Arlene V; Stone, Edwin M; Tucker, Budd A; Zeng, Shemin; Braun, Terry A; Mullins, Robert F; Scheetz, Todd E

2014-12-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

Transcriptomic analysis across nasal, temporal, and macular regions of human neural retina and RPE/choroid by RNA-Seq

PubMed Central

Whitmore, S. Scott; Wagner, Alex H.; DeLuca, Adam P.; Drack, Arlene V.; Stone, Edwin M.; Tucker, Budd A.; Zeng, Shemin; Braun, Terry A.; Mullins, Robert F.; Scheetz, Todd E.

2014-01-01

Proper spatial differentiation of retinal cell types is necessary for normal human vision. Many retinal diseases, such as Best disease and male germ cell associated kinase (MAK)-associated retinitis pigmentosa, preferentially affect distinct topographic regions of the retina. While much is known about the distribution of cell-types in the retina, the distribution of molecular components across the posterior pole of the eye has not been well-studied. To investigate regional difference in molecular composition of ocular tissues, we assessed differential gene expression across the temporal, macular, and nasal retina and retinal pigment epithelium (RPE)/choroid of human eyes using RNA-Seq. RNA from temporal, macular, and nasal retina and RPE/choroid from four human donor eyes was extracted, poly-A selected, fragmented, and sequenced as 100 bp read pairs. Digital read files were mapped to the human genome and analyzed for differential expression using the Tuxedo software suite. Retina and RPE/choroid samples were clearly distinguishable at the transcriptome level. Numerous transcription factors were differentially expressed between regions of the retina and RPE/choroid. Photoreceptor-specific genes were enriched in the peripheral samples, while ganglion cell and amacrine cell genes were enriched in the macula. Within the RPE/choroid, RPE-specific genes were upregulated at the periphery while endothelium associated genes were upregulated in the macula. Consistent with previous studies, BEST1 expression was lower in macular than extramacular regions. The MAK gene was expressed at lower levels in macula than in extramacular regions, but did not exhibit a significant difference between nasal and temporal retina. The regional molecular distinction is greatest between macula and periphery and decreases between different peripheral regions within a tissue. Datasets such as these can be used to prioritize candidate genes for possible involvement in retinal diseases with

Does the Timing of Measurement Alter Session-RPE in Boxers?

PubMed

Uchida, Marco C; Teixeira, Luis F M; Godoi, Vladmir J; Marchetti, Paulo H; Conte, Marcelo; Coutts, Aaron J; Bacurau, Reury F P

2014-01-01

The purpose of this study was to compare the influence of measuring the overall session rating of perceived exertion (session-RPE) at 10 vs. 30 minutes following exercise. Eight boxers completed three different standardized training sessions of different intensities (easy, moderate and hard) in a matchedpairs, randomized research design. Exercise intensity was assessed during each bout by measuring heart rate, blood lactate concentration and session-RPE. To assess the effect of measurement timing on session-RPE, RPE data were collected either 10 or 30 minutes post-exercise. There was no significant effect of measurement time on session-RPE values following easy (10 minutes: session-RPE = 1.3 ± 1.0 Arbitrary Unit (AU), %Heart Rate Reserve (HRR) = 49.5 ± 11.1, and ∆Blood lactate = -2.3 ± 16.3%; 30 minutes: session-RPE = 1.7 ± 1.0 AU, %HRR = 51.3 ± 10.8, and ∆Blood lactate = 0.7 ± 25.2%), moderate (10 minutes: session-RPE = 2.7 ± 1.6 AU, %HRR = 67.2 ± 10.8, and ∆Blood lactate = 2.2 ± 19%; 30 minutes: session-RPE = 2.5 ± 0.9 AU, %HRR = 67.2 ± 5.9, and ∆Blood lactate = 24.5 ± 17.1%) and hard (10 minutes: session-RPE = 5.7 ± 1.0 AU, %HRR = 88.1 ± 6.3, and ∆Blood lactate = 146.3 ± 87.9%; 30 minutes: session-RPE = 5.8 ± 1.9 AU, %HRR> = 83.3 ± 8.0, and ∆Blood lactate = 91.6 ± 39%) sessions. In conclusion, our findings suggest that session-RPE can be used in boxing training routines across a range of intensities and accurate measurements can be determined as early as 10 minutes after exercise. Key PointsIt is difficult to quantify and monitoring the external training load in martial arts (e.g. Aikido, Kung Fu, Judo) and physical combat sports (e.g. Boxing, Muay Thai), session RPE method appears to be a reliable method to quantifying training load in those sports.For many athletes it is impractical to wait 30 minutes after training session to provide a session-RPE. The present findings show that collecting ses-sion-RPE measures at 10 min

Insights into the pathogenesis of dominant retinitis pigmentosa associated with a D477G mutation in RPE65.

PubMed

Choi, Elliot H; Suh, Susie; Sander, Christopher L; Hernandez, Christian J Ortiz; Bulman, Elizabeth R; Khadka, Nimesh; Dong, Zhiqian; Shi, Wuxian; Palczewski, Krzysztof; Kiser, Philip D

2018-04-12

RPE65 is the essential trans-cis isomerase of the classical retinoid (visual) cycle. Mutations in RPE65 give rise to severe retinal dystrophies, most of which are associated with loss of protein function and recessive inheritance. The only known exception is a c.1430G>A (D477G) mutation that gives rise to dominant retinitis pigmentosa with delayed onset and choroidal and macular involvement. Position 477 is distant from functionally critical regions of RPE65. Hence, the mechanism of D477G pathogenicity remains unclear, although protein misfolding and aggregation mechanisms have been suggested. We characterized a D477G knock-in mouse model which exhibited mild age-dependent changes in retinal structure and function. Immunoblot analysis of protein extracts from the eyes of the knock-in mice demonstrated the presence of ubiquitinated RPE65 and reduced RPE65 expression. We observed an accumulation of retinyl esters in the knock-in mice as well as a delay in rhodopsin regeneration kinetics and diminished electroretinography responses, indicative of RPE65 functional impairment induced by the D477G mutation in vivo. However, a cell line expressing D477G RPE65 revealed protein expression levels, cellular localization, and retinoid isomerase activity comparable to cells expressing wild-type protein. Structural analysis of an RPE65 chimera suggested that the D477G mutation does not perturb protein folding or tertiary structure. Instead, the mutation generates an aggregation-prone surface that could induce cellular toxicity through abnormal complex formation as suggested by crystal packing analysis. These results indicate that a toxic gain-of-function induced by the D477G RPE65 substitution may play a role in the pathogenesis of this form of dominant retinitis pigmentosa.

Impaired Cargo Clearance in the Retinal Pigment Epithelium (RPE) Underlies Irreversible Blinding Diseases

PubMed Central

Keeling, Eloise; Lotery, Andrew J.

2018-01-01

Chronic degeneration of the Retinal Pigment Epithelium (RPE) is a precursor to pathological changes in the outer retina. The RPE monolayer, which lies beneath the neuroretina, daily internalises and digests large volumes of spent photoreceptor outer segments. Impaired cargo handling and processing in the endocytic/phagosome and autophagy pathways lead to the accumulation of lipofuscin and pyridinium bis-retinoid A2E aggregates and chemically modified compounds such as malondialdehyde and 4-hydroxynonenal within RPE. These contribute to increased proteolytic and oxidative stress, resulting in irreversible damage to post-mitotic RPE cells and development of blinding conditions such as age-related macular degeneration, Stargardt disease and choroideremia. Here, we review how impaired cargo handling in the RPE results in their dysfunction, discuss new findings from our laboratory and consider how newly discovered roles for lysosomes and the autophagy pathway could provide insights into retinopathies. Studies of these dynamic, molecular events have also been spurred on by recent advances in optics and imaging technology. Mechanisms underpinning lysosomal impairment in other degenerative conditions including storage disorders, α-synuclein pathologies and Alzheimer’s disease are also discussed. Collectively, these findings help transcend conventional understanding of these intracellular compartments as simple waste disposal bags to bring about a paradigm shift in the way lysosomes are perceived. PMID:29473871

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

Wang, Cheng-hu; Cao, Guo-Fan; Jiang, Qin, E-mail: Jqin710@vip.sina.com

Highlights: Black-Right-Pointing-Pointer TNF-{alpha} induces MMP-9 expression and secretion to promote RPE cell migration. Black-Right-Pointing-Pointer MAPK activation is not critical for TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer Akt and mTORC1 signaling mediate TNF-{alpha}-induced MMP-9 expression. Black-Right-Pointing-Pointer SIN1 knockdown showed no significant effect on MMP-9 expression by TNF-{alpha}. -- Abstract: Tumor necrosis factor-alpha (TNF-{alpha}) promotes in vitro retinal pigment epithelial (RPE) cell migration to initiate proliferative vitreoretinopathy (PVR). Here we report that TNF-{alpha} promotes human RPE cell migration by inducing matrix metallopeptidase 9 (MMP-9) expression. Inhibition of MMP-9 by its inhibitor or its neutralizing antibody inhibited TNF-{alpha}-induced in vitro RPE cell migration. Reversely, exogenously-addedmore » active MMP-9 promoted RPE cell migration. Suppression Akt/mTOR complex 1(mTORC1) activation by LY 294002 and rapamycin inhibited TNF-{alpha}-mediated MMP-9 expression. To introduce a constitutively active Akt (CA-Akt) in cultured RPE cells increased MMP-9 expression, and to block mTORC1 activation by rapamycin inhibited its effect. RNA interference (RNAi)-mediated silencing of SIN1, a key component of mTOR complex 2 (mTORC2), had no effect on MMP-9 expression or secretion. In conclusion, this study suggest that TNF-{alpha} promotes RPE cell migration by inducing MMP-9 expression through activation of Akt/ mTORC1, but not mTORC2 signaling.« less

Von Hippel-Lindau protein in the RPE is essential for normal ocular growth and vascular development.

PubMed

Lange, Clemens A K; Luhmann, Ulrich F O; Mowat, Freya M; Georgiadis, Anastasios; West, Emma L; Abrahams, Sabu; Sayed, Haroon; Powner, Michael B; Fruttiger, Marcus; Smith, Alexander J; Sowden, Jane C; Maxwell, Patrick H; Ali, Robin R; Bainbridge, James W B

2012-07-01

Molecular oxygen is essential for the development, growth and survival of multicellular organisms. Hypoxic microenvironments and oxygen gradients are generated physiologically during embryogenesis and organogenesis. In the eye, oxygen plays a crucial role in both physiological vascular development and common blinding diseases. The retinal pigment epithelium (RPE) is a monolayer of cells essential for normal ocular development and in the mature retina provides support for overlying photoreceptors and their vascular supply. Hypoxia at the level of the RPE is closely implicated in pathogenesis of age-related macular degeneration. Adaptive tissue responses to hypoxia are orchestrated by sophisticated oxygen sensing mechanisms. In particular, the von Hippel-Lindau tumour suppressor protein (pVhl) controls hypoxia-inducible transcription factor (HIF)-mediated adaptation. However, the role of Vhl/Hif1a in the RPE in the development of the eye and its vasculature is unknown. In this study we explored the function of Vhl and Hif1a in the developing RPE using a tissue-specific conditional-knockout approach. We found that deletion of Vhl in the RPE results in RPE apoptosis, aniridia and microphthalmia. Increased levels of Hif1a, Hif2a, Epo and Vegf are associated with a highly disorganised retinal vasculature, chorioretinal anastomoses and the persistence of embryonic vascular structures into adulthood. Additional inactivation of Hif1a in the RPE rescues the RPE morphology, aniridia, microphthalmia and anterior vasoproliferation, but does not rescue retinal vasoproliferation. These data demonstrate that Vhl-dependent regulation of Hif1a in the RPE is essential for normal RPE and iris development, ocular growth and vascular development in the anterior chamber, whereas Vhl-dependent regulation of other downstream pathways is crucial for normal development and maintenance of the retinal vasculature.

Action spectrum for photochemical retinal pigment epithelium (RPE) disruption in an in vivo monkey model

NASA Astrophysics Data System (ADS)

Zhang, Jie; Sabarinathan, Ranjani; Bubel, Tracy; Williams, David R.; Hunter, Jennifer J.

2016-03-01

Observations of RPE disruption and autofluorescence (AF) photobleaching at light levels below the ANSI photochemical maximum permissible exposure (MPE) (Morgan et al., 2008) indicates a demand to modify future light safety standards to protect the retina from harm. To establish safe light exposures, we measured the visible light action spectrum for RPE disruption in an in vivo monkey model with fluorescence adaptive optics retinal imaging. Using this high resolution imaging modality can provide insight into the consequences of light on a cellular level and allow for longitudinal monitoring of retinal changes. The threshold retinal radiant exposures (RRE) for RPE disruption were determined for 4 wavelengths (460, 488, 544, and 594 nm). The anaesthetized macaque retina was exposed to a uniform 0.5° × 0.5° field of view (FOV). Imaging within a 2° × 2° FOV was performed before, immediately after and at 2 week intervals for 10 weeks. At each wavelength, multiple RREs were tested with 4 repetitions each to determine the threshold for RPE disruption. For qualitative analysis, RPE disruption is defined as any detectable change from the pre exposure condition in the cell mosaic in the exposed region relative to the corresponding mosaic in the immediately surrounding area. We have tested several metrics to evaluate the RPE images obtained before and after exposure. The measured action spectrum for photochemical RPE disruption has a shallower slope than the current ANSI photochemical MPE for the same conditions and suggests that longer wavelength light is more hazardous than other measurements would suggest.

Drosophila hemocyte migration: an in vivo assay for directional cell migration.

PubMed

Moreira, Carolina G A; Regan, Jennifer C; Zaidman-Rémy, Anna; Jacinto, Antonio; Prag, Soren

2011-01-01

This protocol describes an in vivo assay for random and directed hemocyte migration in Drosophila. Drosophila is becoming an increasingly powerful model system for in vivo cell migration analysis, combining unique genetic tools with translucency of the embryo and pupa, which allows direct imaging and traceability of different cell types. In the assay we present here, we make use of the hemocyte response to epithelium wounding to experimentally induce a transition from random to directed migration. Time-lapse confocal microscopy of hemocyte migration in untreated conditions provides a random cell migration assay that allows identification of molecular mechanisms involved in this complex process. Upon laser-induced wounding of the thorax epithelium, a rapid chemotactic response changes hemocyte migratory behavior into a directed migration toward the wound site. This protocol provides a direct comparison of cells during both types of migration in vivo, and combined with recently developed resources such as transgenic RNAi, is ideal for forward genetic screens.

Activated Retinal Pigment Epithelium, an Optical Coherence Tomography Biomarker for Progression in Age-Related Macular Degeneration

PubMed Central

Curcio, Christine A.; Zanzottera, Emma C.; Ach, Thomas; Balaratnasingam, Chandrakumar; Freund, K. Bailey

2017-01-01

Purpose To summarize and contextualize recent histology and clinical imaging publications on retinal pigment epithelium (RPE) fate in advanced age-related macular degeneration (AMD); to support RPE activation and migration as important precursors to atrophy, manifest as intraretinal hyperreflective foci in spectral-domain optical coherence tomography (SDOCT). Methods The Project MACULA online resource for AMD histopathology was surveyed systematically to form a catalog of 15 phenotypes of RPE and RPE-derived cells and layer thicknesses in advanced disease. Phenotypes were also sought in correlations with clinical longitudinal eye-tracked SDOCT and with ex vivo imaging–histopathology correlations in geographic atrophy (GA) and pigment epithelium detachments (PED). Results The morphology catalog suggested two main pathways of RPE fate: basolateral shedding of intracellular organelles (apparent apoptosis in situ) and activation with anterior migration. Acquired vitelliform lesions may represent a third pathway. Migrated cells are packed with RPE organelles and confirmed as hyperreflective on SDOCT. RPE layer thickening due to cellular dysmorphia and thick basal laminar deposit is observed near the border of GA. Drusenoid PED show a life cycle of slow growth and rapid collapse preceded by RPE layer disruption and anterior migration. Conclusions RPE activation and migration comprise an important precursor to atrophy that can be observed at the cellular level in vivo via validated SDOCT. Collapse of large drusen and drusenoid PED appears to occur when RPE death and migration prevent continued production of druse components. Data implicate excessive diffusion distance from choriocapillaris in RPE death as well as support a potential benefit in targeting drusen in GA. PMID:28785769

Regulation of Cell Migration in Breast Cancer

DTIC Science & Technology

2011-04-01

the wound healing, assay by scarring and Oris plate migration assay, transwell migration assay and live - cell imaging studies. Cell migration capacity...evaluated by the use of techniques that include the wound healing assay by scarring and Oris plate migration assay, transwell migration assay and live - cell imaging studies

Collective cell migration during inflammatory response

NASA Astrophysics Data System (ADS)

Wu, Di; Stroka, Kimberly; Aranda-Espinoza, Helim

2012-02-01

Wound scratch healing assays of endothelial cell monolayers is a simple model to study collective cell migration as a function of biological signals. A signal of particular interest is the immune response, which after initial wounding in vivo causes the release of various inflammatory factors such as tumor necrosis alpha (TNF-α). TNF-α is an innate inflammatory cytokine that can induce cell growth, cell necrosis, and change cell morphology. We studied the effects of TNF-α on collective cell migration using the wound healing assays and measured several migration metrics, such as rate of scratch closure, velocities of leading edge and bulk cells, closure index, and velocity correlation functions between migrating cells. We observed that TNF-α alters all migratory metrics as a function of the size of the scratch and TNF-α content. The changes observed in migration correlate with actin reorganization upon TNF-α exposure.

Cell migration in microengineered tumor environments.

PubMed

Um, Eujin; Oh, Jung Min; Granick, Steve; Cho, Yoon-Kyoung

2017-12-05

Recent advances in microengineered cell migration platforms are discussed critically with a focus on how cell migration is influenced by engineered tumor microenvironments, the medical relevance being to understand how tumor microenvironments may promote or suppress the progression of cancer. We first introduce key findings in cancer cell migration under the influence of the physical environment, which is systematically controlled by microengineering technology, followed by multi-cues of physico-chemical factors, which represent the complexity of the tumor environment. Recognizing that cancer cells constantly communicate not only with each other but also with tumor-associated cells such as vascular, fibroblast, and immune cells, and also with non-cellular components, it follows that cell motility in tumor microenvironments, especially metastasis via the invasion of cancer cells into the extracellular matrix and other tissues, is closely related to the malignancy of cancer-related mortality. Medical relevance of forefront research realized in microfabricated devices, such as single cell sorting based on the analysis of cell migration behavior, may assist personalized theragnostics based on the cell migration phenotype. Furthermore, we urge development of theory and numerical understanding of single or collective cell migration in microengineered platforms to gain new insights in cancer metastasis and in therapeutic strategies.

Gene Therapy Rescues Cone Structure and Function in the 3-Month-Old rd12 Mouse: A Model for Midcourse RPE65 Leber Congenital Amaurosis

PubMed Central

Li, Xia; Li, Wensheng; Dai, Xufeng; Kong, Fansheng; Zheng, Qinxiang; Zhou, Xiangtian; Lü, Fan; Chang, Bo; Rohrer, Bärbel; Hauswirth, William. W.; Qu, Jia; Pang, Ji-jing

2011-01-01

Purpose. RPE65 function is necessary in the retinal pigment epithelium (RPE) to generate chromophore for all opsins. Its absence results in vision loss and rapid cone degeneration. Recent Leber congenital amaurosis type 2 (LCA with RPE65 mutations) phase I clinical trials demonstrated restoration of vision on RPE65 gene transfer into RPE cells overlying cones. In the rd12 mouse, a naturally occurring model of RPE65-LCA early cone degeneration was observed; however, some peripheral M-cones remained. A prior study showed that AAV-mediated RPE65 expression can prevent early cone degeneration. The present study was conducted to test whether the remaining cones in older rd12 mice can be rescued. Methods. Subretinal treatment with the scAAV5-smCBA-hRPE65 vector was initiated at postnatal day (P)14 and P90. After 2 months, electroretinograms were recorded, and cone morphology was analyzed by using cone-specific peanut agglutinin and cone opsin–specific antibodies. Results. Cone degeneration started centrally and spread ventrally, with cells losing cone-opsin staining before that for the PNA-lectin–positive cone sheath. Gene therapy starting at P14 resulted in almost wild-type M- and S-cone function and morphology. Delaying gene-replacement rescued the remaining M-cones, and most important, more M-cone opsin–positive cells were identified than were present at the onset of gene therapy, suggesting that opsin expression could be reinitiated in cells with cone sheaths. Conclusions. The results support and extend those of the previous study that gene therapy can stop early cone degeneration, and, more important, they provide proof that delayed treatment can restore the function and morphology of the remaining cones. These results have important implications for the ongoing LCA2 clinical trials. PMID:21169527

Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1.

PubMed

Sangha, Susan; Varshney, Nishi; Fras, Mary; Smyth, Kim; Rosenegger, David; Parvez, Kashif; Sadamoto, Hisayo; Lukowiak, Ken

2004-01-01

The central pattern generator (CPG) that drives aerial respiratory behaviour in Lymnaea consists of 3 neurons. One of these, RPeD1--the cell that initiates activity in the circuit, plays an absolutely necessary role as a site for memory formation, memory reconsolidation, and extinction. Using an operant conditioning training procedure that results in a long-term non-declarative memory (LTM), we decrease the occurrence of aerial respiratory behaviour. Since snails can still breathe cutaneously learning this procedure is not harmful. Concomitant with behavioural memory are changes in the spiking activity of RPeD1. Going beyond neural correlates of memory we directly show that RPeD1 is a necessary site for LTM formation. Expanding on this finding we show that this neuron is also a necessary site for memory reconsolidation and 'Pavlovian' extinction. As far as we can determine, this is the first time a single neuron has been shown to be a necessary site for these different aspects memory. RPeD1 is thus a key neuron mediating different hierarchical aspects of memory. We are now in a position to determine the necessary neuronal, molecular and proteomic events in this neuron that are causal to memory formation, reconsolidation and extinction.

The newt (Cynops pyrrhogaster) RPE65 promoter: molecular cloning, characterization and functional analysis.

PubMed

Casco-Robles, Martin Miguel; Miura, Tomoya; Chiba, Chikafumi

2015-06-01

The adult newt has the ability to regenerate the neural retina following injury, a process achieved primarily by the retinal pigment epithelium (RPE). To deliver exogenous genes to the RPE for genetic manipulation of regenerative events, we isolated the newt RPE65 promoter region by genome walking. First, we cloned the 2.8 kb RPE65 promoter from the newt, Cynops pyrrhogaster. Sequence analysis revealed several conserved regulatory elements described previously in mouse and human RPE65 promoters. Second, having previously established an I-SceI-mediated transgenic protocol for the newt, we used it here to examine the -657 bp proximal promoter of RPE65. The promoter assay used with F0 transgenic newts confirmed transgene expression of mCherry fluorescent protein in the RPE. Using bioinformatic tools and the TRANSFAC database, we identified a 340 bp CpG island located between -635 and -296 bp in the promoter; this region contains response elements for the microphthalmia-associated transcription factor known as MITF (CACGTG, CATGTG), and E-boxes (CANNTG). Sex-determining region box 9 (or SOX9) response element previously reported in the regulation of RPE genes (including RPE65) was also identified in the newt RPE65 promoter. Third, we identified DNA motif boxes in the newt RPE65 promoter that are conserved among other vertebrates. The newt RPE65 promoter is an invaluable tool for site-specific delivery of exogenous genes or genetic manipulation systems for the study of retinal regeneration in this animal.

The Mechanics of Single Cell and Collective Migration of Tumor Cells

PubMed Central

Lintz, Marianne; Muñoz, Adam; Reinhart-King, Cynthia A.

2017-01-01

Metastasis is a dynamic process in which cancer cells navigate the tumor microenvironment, largely guided by external chemical and mechanical cues. Our current understanding of metastatic cell migration has relied primarily on studies of single cell migration, most of which have been performed using two-dimensional (2D) cell culture techniques and, more recently, using three-dimensional (3D) scaffolds. However, the current paradigm focused on single cell movements is shifting toward the idea that collective migration is likely one of the primary modes of migration during metastasis of many solid tumors. Not surprisingly, the mechanics of collective migration differ significantly from single cell movements. As such, techniques must be developed that enable in-depth analysis of collective migration, and those for examining single cell migration should be adopted and modified to study collective migration to allow for accurate comparison of the two. In this review, we will describe engineering approaches for studying metastatic migration, both single cell and collective, and how these approaches have yielded significant insight into the mechanics governing each process. PMID:27814431

Embryonic cell-cell adhesion: a key player in collective neural crest migration.

PubMed

Barriga, Elias H; Mayor, Roberto

2015-01-01

Cell migration is essential for morphogenesis, adult tissue remodeling, wound healing, and cancer cell migration. Cells can migrate as individuals or groups. When cells migrate in groups, cell-cell interactions are crucial in order to promote the coordinated behavior, essential for collective migration. Interestingly, recent evidence has shown that cell-cell interactions are also important for establishing and maintaining the directionality of these migratory events. We focus on neural crest cells, as they possess extraordinary migratory capabilities that allow them to migrate and colonize tissues all over the embryo. Neural crest cells undergo an epithelial-to-mesenchymal transition at the same time than perform directional collective migration. Cell-cell adhesion has been shown to be an important source of planar cell polarity and cell coordination during collective movement. We also review molecular mechanisms underlying cadherin turnover, showing how the modulation and dynamics of cell-cell adhesions are crucial in order to maintain tissue integrity and collective migration in vivo. We conclude that cell-cell adhesion during embryo development cannot be considered as simple passive resistance to force, but rather participates in signaling events that determine important cell behaviors required for cell migration. © 2015 Elsevier Inc. All rights reserved.

Multi-Cellular Logistics of Collective Cell Migration

PubMed Central

Yamao, Masataka; Naoki, Honda; Ishii, Shin

2011-01-01

During development, the formation of biological networks (such as organs and neuronal networks) is controlled by multicellular transportation phenomena based on cell migration. In multi-cellular systems, cellular locomotion is restricted by physical interactions with other cells in a crowded space, similar to passengers pushing others out of their way on a packed train. The motion of individual cells is intrinsically stochastic and may be viewed as a type of random walk. However, this walk takes place in a noisy environment because the cell interacts with its randomly moving neighbors. Despite this randomness and complexity, development is highly orchestrated and precisely regulated, following genetic (and even epigenetic) blueprints. Although individual cell migration has long been studied, the manner in which stochasticity affects multi-cellular transportation within the precisely controlled process of development remains largely unknown. To explore the general principles underlying multicellular migration, we focus on the migration of neural crest cells, which migrate collectively and form streams. We introduce a mechanical model of multi-cellular migration. Simulations based on the model show that the migration mode depends on the relative strengths of the noise from migratory and non-migratory cells. Strong noise from migratory cells and weak noise from surrounding cells causes “collective migration,” whereas strong noise from non-migratory cells causes “dispersive migration.” Moreover, our theoretical analyses reveal that migratory cells attract each other over long distances, even without direct mechanical contacts. This effective interaction depends on the stochasticity of the migratory and non-migratory cells. On the basis of these findings, we propose that stochastic behavior at the single-cell level works effectively and precisely to achieve collective migration in multi-cellular systems. PMID:22205934

Minimal Effects of VEGF and Anti-VEGF Drugs on the Permeability or Selectivity of RPE Tight Junctions

PubMed Central

Peng, Shaomin; Adelman, Ron A.

2010-01-01

Purpose. Bevacizumab and ranibizumab are currently used to treat age-related macular degeneration by neutralizing vascular endothelial growth factor (VEGF). In this study, the potential side effects on the outer blood–retinal barrier were examined. Methods. Human fetal RPE (hfRPE) cells were used because they are highly differentiated in culture. The claudin composition of RPE tight junctions was determined by RT-PCR, immunoblot analysis, and immunofluorescence. ELISA assays monitored the secretion and trafficking of VEGF and a fluid-phase marker, methylpolyethylene glycol (mPEG). Tight junction functions were assessed by the conductance of K+ and Na+ (derived from the transepithelial electrical resistance, TER) and the flux of NaCl and mPEG. Results. Claudin-3, claudin-10, and claudin-19 were detected in RPE tight junctions. VEGF was secreted in equal amounts across the apical and basolateral membranes, but the apical membrane was more active in endocytosing and degrading VEGF. Exogenous VEGF and mPEG crossed the RPE monolayer by transcytosis, predominantly in the apical-to-basal direction. RPE tight junctions were selective for K+, but did not discriminate between Na+ and Cl−. VEGF, bevacizumab, and ranibizumab had minimal effects on TER, permeation of mPEG, and selectivity for K+, Na+, and Cl−. They had minimal effects on the expression and distribution of the claudins. Conclusions. RPE has mechanisms for maintaining low concentrations of VEGF in the subretinal space that include endocytosis and degradation and fluid-phase transcytosis in the apical-to-basal direction. RPE tight junctions are selective for K+ over Na+ and Cl−. Permeability and selectivity of the junctions are not affected by VEGF, bevacizumab, or ranibizumab. PMID:20042644

Evaluation of RPE-Select: A Web-Based Respiratory Protective Equipment Selector Tool.

PubMed

Vaughan, Nick; Rajan-Sithamparanadarajah, Bob; Atkinson, Robert

2016-08-01

This article describes the evaluation of an open-access web-based respiratory protective equipment selector tool (RPE-Select, accessible at http://www.healthyworkinglives.com/rpe-selector). This tool is based on the principles of the COSHH-Essentials (C-E) control banding (CB) tool, which was developed for the exposure risk management of hazardous chemicals in the workplace by small and medium sized enterprises (SMEs) and general practice H&S professionals. RPE-Select can be used for identifying adequate and suitable RPE for dusts, fibres, mist (solvent, water, and oil based), sprays, volatile solids, fumes, gases, vapours, and actual or potential oxygen deficiency. It can be applied for substances and products with safety data sheets as well as for a large number of commonly encountered process-generated substances (PGS), such as poultry house dusts or welding fume. Potential international usability has been built-in by using the Hazard Statements developed for the Globally Harmonised System (GHS) and providing recommended RPE in picture form as well as with a written specification. Illustration helps to compensate for the variabilities in assigned protection factors across the world. RPE-Select uses easily understandable descriptions/explanations and an interactive stepwise flow for providing input/answers at each step. The output of the selection process is a report summarising the user input data and a selection of RPE, including types of filters where applicable, from which the user can select the appropriate one for each wearer. In addition, each report includes 'Dos' and 'Don'ts' for the recommended RPE. RPE-Select outcomes, based on up to 20 hypothetical use scenarios, were evaluated in comparison with other available RPE selection processes and tools, and by 32 independent users with a broad range of familiarities with industrial use scenarios in general and respiratory protection in particular. For scenarios involving substances having safety data sheets

Evaluation of RPE-Select: A Web-Based Respiratory Protective Equipment Selector Tool

PubMed Central

Vaughan, Nick; Rajan-Sithamparanadarajah, Bob; Atkinson, Robert

2016-01-01

This article describes the evaluation of an open-access web-based respiratory protective equipment selector tool (RPE-Select, accessible at http://www.healthyworkinglives.com/rpe-selector). This tool is based on the principles of the COSHH-Essentials (C-E) control banding (CB) tool, which was developed for the exposure risk management of hazardous chemicals in the workplace by small and medium sized enterprises (SMEs) and general practice H&S professionals. RPE-Select can be used for identifying adequate and suitable RPE for dusts, fibres, mist (solvent, water, and oil based), sprays, volatile solids, fumes, gases, vapours, and actual or potential oxygen deficiency. It can be applied for substances and products with safety data sheets as well as for a large number of commonly encountered process-generated substances (PGS), such as poultry house dusts or welding fume. Potential international usability has been built-in by using the Hazard Statements developed for the Globally Harmonised System (GHS) and providing recommended RPE in picture form as well as with a written specification. Illustration helps to compensate for the variabilities in assigned protection factors across the world. RPE-Select uses easily understandable descriptions/explanations and an interactive stepwise flow for providing input/answers at each step. The output of the selection process is a report summarising the user input data and a selection of RPE, including types of filters where applicable, from which the user can select the appropriate one for each wearer. In addition, each report includes ‘Dos’ and ‘Don’ts’ for the recommended RPE. RPE-Select outcomes, based on up to 20 hypothetical use scenarios, were evaluated in comparison with other available RPE selection processes and tools, and by 32 independent users with a broad range of familiarities with industrial use scenarios in general and respiratory protection in particular. For scenarios involving substances having safety

Probable Chemical Hypoxia Effects on Progress of CNV Through Induction of Promoter CpG Demethylation and Overexpression of IL17RC in Human RPE Cells.

PubMed

Alivand, Mohammad Reza; Sabouni, Farzaneh; Soheili, Zahra-Soheila

2016-09-01

To survey the changes of promoter CpG methylation status and mRNA expression of IL17RC (interleukin 17 receptor C) gene in retinal pigment epithelium (RPE) cells under chemical hypoxia condition for choroidal neovascularization (CNV) modeling in vitro. RPE cells were cultured in both untreated as a control group and treated by cobalt chloride media as a hypoxia group for various concentrations (100-150μM) and times (24-36 hrs.) To confirm chemical hypoxia condition, mRNA expression of HIF (Hypoxia Inducible Factor) -1α, -2α, and Vascular Endothelial Growth Factor (VEGF) was compared between two groups by Real-time PCR. Also, in normoxia and hypoxia conditions, IL17RC expression changes and promoter CpG methylation status were evaluated by Real-time PCR and methylation-specific PCR (MSP) techniques, respectively. Overexpression of HIF-1α, HIF-2α, and VEGF was significant in hypoxia versus normoxia conditions. Our data showed overexpression of IL17RC (2.1- to 6.3-fold) and decreasing of its promoter methylation in comparison with hypoxia and normoxia conditions. It was found that there are significant association between promoter methylation status and expression of IL17RC in chemical hypoxia condition. Therefore, methylation of IL17RC could play as a marker in CNV and degeneration of RPE cells in vitro. Additionally, HIF-α and methylation phenomena may be considered as critical targets for blocking in angiogenesis of age-related degeneration in future studies.

Features specific to retinal pigment epithelium cells derived from three-dimensional human embryonic stem cell cultures - a new donor for cell therapy.

PubMed

Wu, Wei; Zeng, Yuxiao; Li, Zhengya; Li, Qiyou; Xu, Haiwei; Yin, Zheng Qin

2016-04-19

Retinal pigment epithelium (RPE) transplantation is a particularly promising treatment of retinal degenerative diseases affecting RPE-photoreceptor complex. Embryonic stem cells (ESCs) provide an abundant donor source for RPE transplantation. Herein, we studied the time-course characteristics of RPE cells derived from three-dimensional human ESCs cultures (3D-RPE). We showed that 3D-RPE cells possessed morphology, ultrastructure, gene expression profile, and functions of authentic RPE. As differentiation proceeded, 3D-RPE cells could mature gradually with decreasing proliferation but increasing functions. Besides, 3D-RPE cells could form polarized monolayer with functional tight junction and gap junction. When grafted into the subretinal space of Royal College of Surgeons rats, 3D-RPE cells were safe and efficient to rescue retinal degeneration. This study showed that 3D-RPE cells were a new donor for cell therapy of retinal degenerative diseases.

Modelling collective cell migration of neural crest

PubMed Central

Szabó, András; Mayor, Roberto

2016-01-01

Collective cell migration has emerged in the recent decade as an important phenomenon in cell and developmental biology and can be defined as the coordinated and cooperative movement of groups of cells. Most studies concentrate on tightly connected epithelial tissues, even though collective migration does not require a constant physical contact. Movement of mesenchymal cells is more independent, making their emergent collective behaviour less intuitive and therefore lending importance to computational modelling. Here we focus on such modelling efforts that aim to understand the collective migration of neural crest cells, a mesenchymal embryonic population that migrates large distances as a group during early vertebrate development. By comparing different models of neural crest migration, we emphasize the similarity and complementary nature of these approaches and suggest a future direction for the field. The principles derived from neural crest modelling could aid understanding the collective migration of other mesenchymal cell types. PMID:27085004

Lung cells support osteosarcoma cell migration and survival.

PubMed

Yu, Shibing; Fourman, Mitchell Stephen; Mahjoub, Adel; Mandell, Jonathan Brendan; Crasto, Jared Anthony; Greco, Nicholas Giuseppe; Weiss, Kurt Richard

2017-01-25

Osteosarcoma (OS) is the most common primary bone tumor, with a propensity to metastasize to the lungs. Five-year survival for metastatic OS is below 30%, and has not improved for several decades despite the introduction of multi-agent chemotherapy. Understanding OS cell migration to the lungs requires an evaluation of the lung microenvironment. Here we utilized an in vitro lung cell and OS cell co-culture model to explore the interactions between OS and lung cells, hypothesizing that lung cells would promote OS cell migration and survival. The impact of a novel anti-OS chemotherapy on OS migration and survival in the lung microenvironment was also examined. Three human OS cell lines (SJSA-1, Saos-2, U-2) and two human lung cell lines (HULEC-5a, MRC-5) were cultured according to American Type Culture Collection recommendations. Human lung cell lines were cultured in growth medium for 72 h to create conditioned media. OS proliferation was evaluated in lung co-culture and conditioned media microenvironment, with a murine fibroblast cell line (NIH-3 T3) in fresh growth medium as controls. Migration and invasion were measured using a real-time cell analysis system. Real-time PCR was utilized to probe for Aldehyde Dehydrogenase (ALDH1) expression. Osteosarcoma cells were also transduced with a lentivirus encoding for GFP to permit morphologic analysis with fluorescence microscopy. The anti-OS efficacy of Disulfiram, an ALDH-inhibitor previously shown to inhibit OS cell proliferation and metastasis in vitro, was evaluated in each microenvironment. Lung-cell conditioned medium promoted osteosarcoma cell migration, with a significantly higher attractive effect on all three osteosarcoma cell lines compared to basic growth medium, 10% serum containing medium, and NIH-3 T3 conditioned medium (p <0.05). Lung cell conditioned medium induced cell morphologic changes, as demonstrated with GFP-labeled cells. OS cells cultured in lung cell conditioned medium had increased

Multiscale Cues Drive Collective Cell Migration

NASA Astrophysics Data System (ADS)

Nam, Ki-Hwan; Kim, Peter; Wood, David K.; Kwon, Sunghoon; Provenzano, Paolo P.; Kim, Deok-Ho

2016-07-01

To investigate complex biophysical relationships driving directed cell migration, we developed a biomimetic platform that allows perturbation of microscale geometric constraints with concomitant nanoscale contact guidance architectures. This permits us to elucidate the influence, and parse out the relative contribution, of multiscale features, and define how these physical inputs are jointly processed with oncogenic signaling. We demonstrate that collective cell migration is profoundly enhanced by the addition of contract guidance cues when not otherwise constrained. However, while nanoscale cues promoted migration in all cases, microscale directed migration cues are dominant as the geometric constraint narrows, a behavior that is well explained by stochastic diffusion anisotropy modeling. Further, oncogene activation (i.e. mutant PIK3CA) resulted in profoundly increased migration where extracellular multiscale directed migration cues and intrinsic signaling synergistically conspire to greatly outperform normal cells or any extracellular guidance cues in isolation.

Modelling collective cell migration of neural crest.

PubMed

Szabó, András; Mayor, Roberto

2016-10-01

Collective cell migration has emerged in the recent decade as an important phenomenon in cell and developmental biology and can be defined as the coordinated and cooperative movement of groups of cells. Most studies concentrate on tightly connected epithelial tissues, even though collective migration does not require a constant physical contact. Movement of mesenchymal cells is more independent, making their emergent collective behaviour less intuitive and therefore lending importance to computational modelling. Here we focus on such modelling efforts that aim to understand the collective migration of neural crest cells, a mesenchymal embryonic population that migrates large distances as a group during early vertebrate development. By comparing different models of neural crest migration, we emphasize the similarity and complementary nature of these approaches and suggest a future direction for the field. The principles derived from neural crest modelling could aid understanding the collective migration of other mesenchymal cell types. Copyright © 2016 Elsevier Ltd. All rights reserved.

Modeling keratinocyte wound healing dynamics: Cell-cell adhesion promotes sustained collective migration.

PubMed

Nardini, John T; Chapnick, Douglas A; Liu, Xuedong; Bortz, David M

2016-07-07

The in vitro migration of keratinocyte cell sheets displays behavioral and biochemical similarities to the in vivo wound healing response of keratinocytes in animal model systems. In both cases, ligand-dependent Epidermal Growth Factor Receptor (EGFR) activation is sufficient to elicit collective cell migration into the wound. Previous mathematical modeling studies of in vitro wound healing assays assume that physical connections between cells have a hindering effect on cell migration, but biological literature suggests a more complicated story. By combining mathematical modeling and experimental observations of collectively migrating sheets of keratinocytes, we investigate the role of cell-cell adhesion during in vitro keratinocyte wound healing assays. We develop and compare two nonlinear diffusion models of the wound healing process in which cell-cell adhesion either hinders or promotes migration. Both models can accurately fit the leading edge propagation of cell sheets during wound healing when using a time-dependent rate of cell-cell adhesion strength. The model that assumes a positive role of cell-cell adhesion on migration, however, is robust to changes in the leading edge definition and yields a qualitatively accurate density profile. Using RNAi for the critical adherens junction protein, α-catenin, we demonstrate that cell sheets with wild type cell-cell adhesion expression maintain migration into the wound longer than cell sheets with decreased cell-cell adhesion expression, which fails to exhibit collective migration. Our modeling and experimental data thus suggest that cell-cell adhesion promotes sustained migration as cells pull neighboring cells into the wound during wound healing. Copyright © 2016 Elsevier Ltd. All rights reserved.

Tumor cell migration in complex microenvironments

PubMed Central

Polacheck, William J.; Zervantonakis, Ioannis K.; Kamm, Roger D.

2012-01-01

Tumor cell migration is essential for invasion and dissemination from primary solid tumors and for the establishment of lethal secondary metastases at distant organs. In vivo and in vitro models enabled identification of different factors in the tumor microenvironment that regulate tumor progression and metastasis. However, the mechanisms by which tumor cells integrate these chemical and mechanical signals from multiple sources to navigate the complex microenvironment remain poorly understood. In this review, we discuss the factors that influence tumor cell migration with a focus on the migration of transformed carcinoma cells. We provide an overview of the experimental and computational methods that allow the investigation of tumor cell migration, and we highlight the benefits and shortcomings of the various assays. We emphasize that the chemical and mechanical stimulus paradigms are not independent and that crosstalk between them motivates the development of new assays capable of applying multiple, simultaneous stimuli and imaging the cellular migratory response in real-time. These next-generation assays will more closely mimic the in vivo microenvironment to provide new insights into tumor progression, inform techniques to control tumor cell migration, and render cancer more treatable. PMID:22926411

Yap is essential for retinal progenitor cell cycle progression and RPE cell fate acquisition in the developing mouse eye.

PubMed

Kim, Jin Young; Park, Raehee; Lee, Jin Hwan J; Shin, Jinyeon; Nickas, Jenna; Kim, Seonhee; Cho, Seo-Hee

2016-11-15

Yap functions as a transcriptional regulator by acting together with sequence-specific DNA binding factors and transcription cofactors to mediate cell proliferation in developing epithelial tissues and tumors. An upstream kinase cascade controls nuclear localization and function in response to partially identified exogenous signals, including cell-to-cell contact. Nevertheless, its role in CNS development is poorly understood. In order to investigate Yap function in developing CNS, we characterized the cellular outcomes after selective Yap gene ablation in developing ocular tissues. When Yap was lost, presumptive retinal pigment epithelium acquired anatomical and molecular characteristics resembling those of the retinal epithelium rather than of RPE, including loss of pigmentation, pseudostratified epithelial morphology and ectopic induction of markers for retinal progenitor cells, like Chx10, and neurons, like β-Tubulin III. In addition, developing retina showed signs of progressive degeneration, including laminar folding, thinning and cell loss, which resulted from multiple defects in cell proliferation and survival, and in junction integrity. Furthermore, Yap-deficient retinal progenitors displayed decreased S-phase cells and altered cell cycle progression. Altogether, our studies not only illustrate the canonical function of Yap in promoting the proliferation of progenitors, but also shed new light on its evolutionarily conserved, instructive role in regional specification, maintenance of junctional integrity and precise regulation of cell proliferation during neuroepithelial development. Copyright © 2016 Elsevier Inc. All rights reserved.

Quantitative Fundus Autofluorescence in Best Vitelliform Macular Dystrophy: RPE Lipofuscin is not Increased in Non-Lesion Areas of Retina.

PubMed

Sparrow, Janet R; Duncker, Tobias; Woods, Russell; Delori, François C

2016-01-01

Since the lipofuscin of retinal pigment epithelial (RPE) cells has been implicated in the pathogenesis of Best vitelliform macular dystrophy, we quantified fundus autofluorescence (quantitative fundus autofluorescence, qAF) as an indirect measure of RPE lipofuscin levels. Mean non-lesion qAF was found to be within normal limits for age. By spectral domain optical coherence tomography (SD-OCT) vitelliform lesions presented as fluid-filled subretinal detachments containing reflective material. We discuss photoreceptor outer segment debris as the source of the intense fluorescence of these lesions and loss of anion channel functioning as an explanation for the bullous photoreceptor-RPE detachment. Unexplained is the propensity of the disease for central retina.

Follow-the-leader cell migration requires biased cell-cell contact and local microenvironmental signals

NASA Astrophysics Data System (ADS)

Wynn, Michelle L.; Rupp, Paul; Trainor, Paul A.; Schnell, Santiago; Kulesa, Paul M.

2013-06-01

Directed cell migration often involves at least two types of cell motility that include multicellular streaming and chain migration. However, what is unclear is how cell contact dynamics and the distinct microenvironments through which cells travel influence the selection of one migratory mode or the other. The embryonic and highly invasive neural crest (NC) are an excellent model system to study this question since NC cells have been observed in vivo to display both of these types of cell motility. Here, we present data from tissue transplantation experiments in chick and in silico modeling that test our hypothesis that cell contact dynamics with each other and the microenvironment promote and sustain either multicellular stream or chain migration. We show that when premigratory cranial NC cells (at the pre-otic level) are transplanted into a more caudal region in the head (at the post-otic level), cells alter their characteristic stream behavior and migrate in chains. Similarly, post-otic NC cells migrate in streams after transplantation into the pre-otic hindbrain, suggesting that local microenvironmental signals dictate the mode of NC cell migration. Simulations of an agent-based model (ABM) that integrates the NC cell behavioral data predict that chain migration critically depends on the interplay of biased cell-cell contact and local microenvironment signals. Together, this integrated modeling and experimental approach suggests new experiments and offers a powerful tool to examine mechanisms that underlie complex cell migration patterns.

Nanotopography guides and directs cell migration in amoeboid and epithelial cells

NASA Astrophysics Data System (ADS)

Lee, Rachel; Das, Satarupa; Hourwitz, Matthew; Sun, Xiaoyu; Parent, Carole; Fourkas, John; Losert, Wolfgang

Cell migration plays a critical role in development, angiogenesis, immune response, wound healing, and cancer metastasis. In many cases, cells also move in the context of a matrix of collagen fibers, and the alignment of these fibers can both affect the migration phenotype and guide cells. Here we show that both fast and slow migrating cells - amoeboid HL-60 and epithelial MCF10A - are affected in similar ways by micro/nanostructures with dimensions similar to those of collagen fibers. Cell alignment enhances the efficiency of migration by increasing directional persistence.

Glial cell migration in the eye disc.

PubMed

Silies, Marion; Yuva, Yeliz; Engelen, Daniel; Aho, Annukka; Stork, Tobias; Klämbt, Christian

2007-11-28

Any complex nervous system is made out of two major cell types, neurons and glial cells. A hallmark of glial cells is their pronounced ability to migrate. En route to their final destinations, glial cells are generally guided by neuronal signals. Here we show that in the developing visual system of Drosophila glial cell migration is largely controlled by glial-glial interactions and occurs independently of axonal contact. Differentiation into wrapping glia is initiated close to the morphogenetic furrow. Using single cell labeling experiments we identified six distinct glial cell types in the eye disc. The migratory glial population is separated from the wrapping glial cells by the so-called carpet cells, extraordinary large glial cells, each covering a surface area of approximately 10,000 epithelial cells. Subsequent cell ablation experiments demonstrate that the carpet glia regulates glial migration in the eye disc epithelium and suggest a new model underlying glial migration and differentiation in the developing visual system.

Three gene-targeted mouse models of RNA splicing factor RP show late-onset RPE and retinal degeneration.

PubMed

Graziotto, John J; Farkas, Michael H; Bujakowska, Kinga; Deramaudt, Bertrand M; Zhang, Qi; Nandrot, Emeline F; Inglehearn, Chris F; Bhattacharya, Shomi S; Pierce, Eric A

2011-01-01

Mutations in genes that produce proteins involved in mRNA splicing, including pre-mRNA processing factors 3, 8, and 31 (PRPF3, 8, and 31), RP9, and SNRNP200 are common causes of the late-onset inherited blinding disorder retinitis pigmentosa (RP). It is not known how mutations in these ubiquitously expressed genes lead to retina-specific disease. To investigate the pathogenesis of the RNA splicing factor forms of RP, the authors generated and characterized the retinal phenotypes of Prpf3-T494M, Prpf8-H2309P knockin mice. The retinal ultrastructure of Prpf31-knockout mice was also investigated. The knockin mice have single codon alterations in their endogenous Prpf3 and Prpf8 genes that mimic the most common disease causing mutations in human PRPF3 and PRPF8. The Prpf31-knockout mice mimic the null alleles that result from the majority of mutations identified in PRPF31 patients. The retinal phenotypes of the gene targeted mice were evaluated by electroretinography (ERG), light, and electron microscopy. The RPE cells of heterozygous Prpf3(+/T494M) and Prpf8(+/H2309P) knockin mice exhibited loss of the basal infoldings and vacuolization, with accumulation of amorphous deposits between the RPE and Bruch[b]'s membrane at age two years. These changes were more severe in the homozygous mice, and were associated with decreased rod function in the Prpf3-T494M mice. Similar degenerative changes in the RPE were detected in Prpf31(±) mice at one year of age. The finding of similar degenerative changes in RPE cells of all three mouse models suggests that the RPE may be the primary cell type affected in the RNA splicing factor forms of RP. The relatively late-onset phenotype observed in these mice is consistent with the typical adult onset of disease in patients with RP.

Mechanical Coordination of Single-Cell and Collective-Cell Amoeboid Migration

NASA Astrophysics Data System (ADS)

Del Alamo, Juan Carlos

Amoeboid migration consists of the sequential repetition of pseudopod extensions and retractions driven by actin polymerization and actomyosin contraction, and requires cells to apply mechanical forces on their surroundings. We measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examine wild-type cells as well as mutants with defects in contractility, F-actin polymerization, internal F-actin crosslinking, and cortical integrity. We find that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. The 3D pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate, and we show that these pushing forces are relevant for cell invasion and migration in three-dimensional environments. We observe that cells migrate mainly by forming two stationary adhesion sites at the front and back of the cell, over which the cell body moves forward in a step-wise fashion. During this process, the traction forces at each adhesion site are switched off and subsequently their direction is reversed. The cell migration speed is found to be proportional to the rate at which cells are able regulate these forces to produce the cell shape changes needed for locomotion, which is increased when axial contractility overcomes the stabilizing effect of cortical tension. This spatiotemporal coordination is conserved in streams of multiple migratory cells connected head to tail, which also migrate by exerting traction forces on stationary sites. Furthermore, we observe that trailing cells reuse the adhesion sites of the leading cells. Finally, we provide evidence that the above modes of migration may be conserved in a range of other amoeboid-type moving cells such as neutrophils.

Quantitative Fundus Autofluorescence in Best Vitelliform Macular Dystrophy: RPE Lipofuscin is not Increased in Non-Lesion Areas of Retina

PubMed Central

Duncker, Tobias; Woods, Russell; Delori, François C.

2018-01-01

Since the lipofuscin of retinal pigment epithelial (RPE) cells has been implicated in the pathogenesis of Best vitelliform macular dystrophy, we quantified fundus autofluorescence (quantitative fundus autofluorescence, qAF) as an indirect measure of RPE lipofuscin levels. Mean non-lesion qAF was found to be within normal limits for age. By spectral domain optical coherence tomography (SD-OCT) vitelliform lesions presented as fluid-filled subretinal detachments containing reflective material. We discuss photoreceptor outer segment debris as the source of the intense fluorescence of these lesions and loss of anion channel functioning as an explanation for the bullous photoreceptor-RPE detachment. Unexplained is the propensity of the disease for central retina. PMID:26427423

Session-RPE Method for Training Load Monitoring: Validity, Ecological Usefulness, and Influencing Factors

PubMed Central

Haddad, Monoem; Stylianides, Georgios; Djaoui, Leo; Dellal, Alexandre; Chamari, Karim

2017-01-01

Purpose: The aim of this review is to (1) retrieve all data validating the Session-rating of perceived exertion (RPE)-method using various criteria, (2) highlight the rationale of this method and its ecological usefulness, and (3) describe factors that can alter RPE and users of this method should take into consideration. Method: Search engines such as SPORTDiscus, PubMed, and Google Scholar databases in the English language between 2001 and 2016 were consulted for the validity and usefulness of the session-RPE method. Studies were considered for further analysis when they used the session-RPE method proposed by Foster et al. in 2001. Participants were athletes of any gender, age, or level of competition. Studies using languages other than English were excluded in the analysis of the validity and reliability of the session-RPE method. Other studies were examined to explain the rationale of the session-RPE method and the origin of RPE. Results: A total of 950 studies cited the Foster et al. study that proposed the session RPE-method. 36 studies have examined the validity and reliability of this proposed method using the modified CR-10. Conclusion: These studies confirmed the validity and good reliability and internal consistency of session-RPE method in several sports and physical activities with men and women of different age categories (children, adolescents, and adults) among various expertise levels. This method could be used as “standing alone” method for training load (TL) monitoring purposes though some recommend to combine it with other physiological parameters as heart rate. PMID:29163016

Directional Collective Cell Migration Emerges as a Property of Cell Interactions

PubMed Central

Woods, Mae L.; Carmona-Fontaine, Carlos; Barnes, Chris P.; Couzin, Iain D.; Mayor, Roberto; Page, Karen M.

2014-01-01

Collective cell migration is a fundamental process, occurring during embryogenesis and cancer metastasis. Neural crest cells exhibit such coordinated migration, where aberrant motion can lead to fatality or dysfunction of the embryo. Migration involves at least two complementary mechanisms: contact inhibition of locomotion (a repulsive interaction corresponding to a directional change of migration upon contact with a reciprocating cell), and co-attraction (a mutual chemoattraction mechanism). Here, we develop and employ a parameterized discrete element model of neural crest cells, to investigate how these mechanisms contribute to long-range directional migration during development. Motion is characterized using a coherence parameter and the time taken to reach, collectively, a target location. The simulated cell group is shown to switch from a diffusive to a persistent state as the response-rate to co-attraction is increased. Furthermore, the model predicts that when co-attraction is inhibited, neural crest cells can migrate into restrictive regions. Indeed, inhibition of co-attraction in vivo and in vitro leads to cell invasion into restrictive areas, confirming the prediction of the model. This suggests that the interplay between the complementary mechanisms may contribute to guidance of the neural crest. We conclude that directional migration is a system property and does not require action of external chemoattractants. PMID:25181349

Plasma-mediated transfection of RPE

NASA Astrophysics Data System (ADS)

Palanker, D.; Chalberg, T.; Vankov, A.; Huie, P.; Molnar, F. E.; Butterwick, A.; Calos, M.; Marmor, M.; Blumenkranz, M. S.

2006-02-01

A major obstacle in applying gene therapy to clinical practice is the lack of efficient and safe gene delivery techniques. Viral delivery has encountered a number of serious problems including immunological reactions and malignancy. Non-viral delivery methods (liposomes, sonoporation and electroporation) have either low efficiency in-vivo or produce severe collateral damage to ocular tissues. We discovered that tensile stress greatly increases the susceptibility of cellular membranes to electroporation. For synchronous application of electric field and mechanical stress, both are generated by the electric discharge itself. A pressure wave is produced by rapid vaporization of the medium. To prevent termination of electric current by the vapor cavity it is ionized thus restoring its electric conductivity. For in-vivo experiments with rabbits a plasmid DNA was injected into the subretinal space, and RPE was treated trans-sclerally with an array of microelectodes placed outside the eye. Application of 250-300V and 100-200 μs biphasic pulses via a microelectrode array resulted in efficient transfection of RPE without visible damage to the retina. Gene expression was quantified and monitored using bioluminescence (luciferase) and fluorescence (GFP) imaging. Transfection efficiency of RPE with this new technique exceeded that of standard electroporation by a factor 10,000. Safe and effective non-viral DNA delivery to the mammalian retina may help to materialize the enormous potential of the ocular gene therapy. Future experiments will focus on continued characterization of the safety and efficacy of this method and evaluation of long-term transgene expression in the presence of phiC31 integrase.

Quantitative Analysis of Cell Migration Using Optical Flow

PubMed Central

Boric, Katica; Orio, Patricio; Viéville, Thierry; Whitlock, Kathleen

2013-01-01

Neural crest cells exhibit dramatic migration behaviors as they populate their distant targets. Using a line of zebrafish expressing green fluorescent protein (sox10:EGFP) in neural crest cells we developed an assay to analyze and quantify cell migration as a population, and use it here to characterize in detail the subtle defects in cell migration caused by ethanol exposure during early development. The challenge was to quantify changes in the in vivo migration of all Sox10:EGFP expressing cells in the visual field of time-lapse movies. To perform this analysis we used an Optical Flow algorithm for motion detection and combined the analysis with a fit to an affine transformation. Through this analysis we detected and quantified significant differences in the cell migrations of Sox10:EGFP positive cranial neural crest populations in ethanol treated versus untreated embryos. Specifically, treatment affected migration by increasing the left-right asymmetry of the migrating cells and by altering the direction of cell movements. Thus, by applying this novel computational analysis, we were able to quantify the movements of populations of cells, allowing us to detect subtle changes in cell behaviors. Because cranial neural crest cells contribute to the formation of the frontal mass these subtle differences may underlie commonly observed facial asymmetries in normal human populations. PMID:23936049

Light Activated Cell Migration in Synthetic Extracellular Matrices

PubMed Central

Guo, Qiongyu; Wang, Xiaobo; Tibbitt, Mark W.; Anseth, Kristi S.; Montell, Denise J.; Elisseeff, Jennifer H.

2012-01-01

Synthetic extracellular matrices provide a framework in which cells can be exposed to defined physical and biological cues. However no method exists to manipulate single cells within these matrices. It is desirable to develop such methods in order to understand fundamental principles of cell migration and define conditions that support or inhibit cell movement within these matrices. Here, we present a strategy for manipulating individual mammalian stem cells in defined synthetic hydrogels through selective optical activation of Rac, which is an intracellular signaling protein that plays a key role in cell migration. Photoactivated cell migration in synthetic hydrogels depended on mechanical and biological cues in the biomaterial. Real-time hydrogel photodegradation was employed to create geometrically defined channels and spaces in which cells could be photoactivated to migrate. Cell migration speed was significantly higher in the photo-etched channels and cells could easily change direction of movement compared to the bulk hydrogels. PMID:22889487

Deconstructing (and reconstructing) cell migration.

PubMed

Maheshwari, G; Lauffenburger, D A

1998-12-01

An overriding objective in cell biology is to be able to relate properties of particular molecular components to cell behavioral functions and even physiology. In the "traditional" mode of molecular cell biology, this objective has been tackled on a molecule-by-molecule basis, and in the "future" mode sometimes termed "functional genomics," it might be attacked in a high-throughput, parallel manner. Regardless of the manner of approach, the relationship between molecular-level properties and cell-level function is exceedingly difficult to elucidate because of the large number of relevant components involved, their high degree of interconnectedness, and the inescapable fact that they operate as physico-chemical entities-according to the laws of kinetics and mechanics-in space and time within the cell. Cell migration is a prominent representative example of such a cell behavioral function that requires increased understanding for both scientific and technological advance. This article presents a framework, derived from an engineering perspective regarding complex systems, intended to aid in developing improved understanding of how properties of molecular components influence the function of cell migration. That is, cell population migration behavior can be deconstructed as follows: first in terms of a mathematical model comprising cell population parameters (random motility, chemotaxis/haptotaxis, and chemokinesis/haptokinesis coefficients), which in turn depend on characteristics of individual cell paths that can be analyzed in terms of a mathematical model comprising individual cell parameters (translocation speed, directional persistence time, chemotactic/haptotactic index), which in turn depend on cell-level physical processes underlying motility (membrane extension and retraction, cell/substratum adhesion, cell contractile force, front-vs.-rear asymmetry), which in turn depend on molecular-level properties of the plethora of components involved in governance and

Fine Tuning Cell Migration by a Disintegrin and Metalloproteinases

PubMed Central

Theodorou, K.

2017-01-01

Cell migration is an instrumental process involved in organ development, tissue homeostasis, and various physiological processes and also in numerous pathologies. Both basic cell migration and migration towards chemotactic stimulus consist of changes in cell polarity and cytoskeletal rearrangement, cell detachment from, invasion through, and reattachment to their neighboring cells, and numerous interactions with the extracellular matrix. The different steps of immune cell, tissue cell, or cancer cell migration are tightly coordinated in time and place by growth factors, cytokines/chemokines, adhesion molecules, and receptors for these ligands. This review describes how a disintegrin and metalloproteinases interfere with several steps of cell migration, either by proteolytic cleavage of such molecules or by functions independent of proteolytic activity. PMID:28260841

Long-Term Live Cell Imaging of Cell Migration: Effects of Pathogenic Fungi on Human Epithelial Cell Migration.

PubMed

Wöllert, Torsten; Langford, George M

2016-01-01

Long-term live cell imaging was used in this study to determine the responses of human epithelial cells to pathogenic biofilms formed by Candida albicans. Epithelial cells of the skin represent the front line of defense against invasive pathogens such as C. albicans but under certain circumstances, especially when the host's immune system is compromised, the skin barrier is breached. The mechanisms by which the fungal pathogen penetrates the skin and invade the deeper layers are not fully understood. In this study we used keratinocytes grown in culture as an in vitro model system to determine changes in host cell migration and the actin cytoskeleton in response to virulence factors produced by biofilms of pathogenic C. albicans. It is clear that changes in epithelial cell migration are part of the response to virulence factors secreted by biofilms of C. albicans and the actin cytoskeleton is the downstream effector that mediates cell migration. Our goal is to understand the mechanism by which virulence factors hijack the signaling pathways of the actin cytoskeleton to alter cell migration and thereby invade host tissues. To understand the dynamic changes of the actin cytoskeleton during infection, we used long-term live cell imaging to obtain spatial and temporal information of actin filament dynamics and to identify signal transduction pathways that regulate the actin cytoskeleton and its associated proteins. Long-term live cell imaging was achieved using a high resolution, multi-mode epifluorescence microscope equipped with specialized light sources, high-speed cameras with high sensitivity detectors, and specific biocompatible fluorescent markers. In addition to the multi-mode epifluorescence microscope, a spinning disk confocal long-term live cell imaging system (Olympus CV1000) equipped with a stage incubator to create a stable in vitro environment for long-term real-time and time-lapse microscopy was used. Detailed descriptions of these two long-term live

Translocation of the retinal pigment epithelium and formation of sub-retinal pigment epithelium deposit induced by subretinal deposit

PubMed Central

Zhao, Lian; Wang, Zhenfang; Liu, Yun; Song, Ying; Li, Yiwen; Laties, Alan M.

2007-01-01

Purpose A cardinal pathological feature of age-related macular degeneration (AMD) is the deposition of extracellular material between the retinal pigment epithelium (RPE) and Bruch's membrane, pathologically described as sub-RPE deposits. Both the presence and local organization of these deposits contribute to the clinical manifestations of AMD, including localized deposits clinically recognized as drusen. The biogenesis of sub-RPE deposits remains elusive. This work explores the pathological processes of sub-RPE deposit formation. Methods Matrigel was injected to the subretinal space of rats to create an amorphous deposit. Tissue sections were examined by light or confocal microscopy. Results In the presence of the subretinal deposit of Matrigel, RPE cells leave Bruch's membrane to migrate toward photoreceptors and then form a new layer between the deposit and photoreceptors, resulting in RPE translocation. The new RPE layer displaces the deposit to the sub-RPE location and therefore it becomes a sub-RPE deposit. The RPE mobilization requires the presence of photoreceptors. Bruch's membrane devoid of RPE attachment becomes vulnerable to invasion by new blood vessels from the choroid. Conclusions Our work supports a novel model of sub-RPE deposit formation in which excessive material first accumulates in the subretinal space, disrupting the physical contact between RPE cells and photoreceptors. To restore the contact, RPE cells migrate toward photoreceptors and form a new layer. The subretinal material is consequently displaced to the sub-RPE location and becomes sub-RPE deposit. Our data also provide evidence that the presence of sub-RPE deposit is sufficient to induce choroidal neovascularization to penetrate Bruch's membrane. PMID:17615538

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

Untangling cell tracks: Quantifying cell migration by time lapse image data analysis.

PubMed

Svensson, Carl-Magnus; Medyukhina, Anna; Belyaev, Ivan; Al-Zaben, Naim; Figge, Marc Thilo

2018-03-01

Automated microscopy has given researchers access to great amounts of live cell imaging data from in vitro and in vivo experiments. Much focus has been put on extracting cell tracks from such data using a plethora of segmentation and tracking algorithms, but further analysis is normally required to draw biologically relevant conclusions. Such relevant conclusions may be whether the migration is directed or not, whether the population has homogeneous or heterogeneous migration patterns. This review focuses on the analysis of cell migration data that are extracted from time lapse images. We discuss a range of measures and models used to analyze cell tracks independent of the biological system or the way the tracks were obtained. For single-cell migration, we focus on measures and models giving examples of biological systems where they have been applied, for example, migration of bacteria, fibroblasts, and immune cells. For collective migration, we describe the model systems wound healing, neural crest migration, and Drosophila gastrulation and discuss methods for cell migration within these systems. We also discuss the role of the extracellular matrix and subsequent differences between track analysis in vitro and in vivo. Besides methods and measures, we are putting special focus on the need for openly available data and code, as well as a lack of common vocabulary in cell track analysis. © 2017 International Society for Advancement of Cytometry. © 2017 International Society for Advancement of Cytometry.

Low Doses of Curcuma longa Modulates Cell Migration and Cell-Cell Adhesion.

PubMed

de Campos, Paloma Santos; Matte, Bibiana Franzen; Diel, Leonardo Francisco; Jesus, Luciano Henrique; Bernardi, Lisiane; Alves, Alessandro Menna; Rados, Pantelis Varvaki; Lamers, Marcelo Lazzaron

2017-09-01

Cell invasion and metastasis are involved in clinical failures in cancer treatment, and both events require the acquisition of a migratory behavior by tumor cells. Curcumin is a promising natural product with anti-proliferative activity, but its effects on cell migration are still unclear. We evaluated the effects of curcumin on the proliferation, apoptosis, migration, and cell-cell adhesion of keratinocyte, oral squamous cell carcinoma (OSCC), and fibroblast cell lines, as well as in a xenograft model of OSCC. Curcumin (2 μM) decreased cell proliferation in cell lines with mesenchymal characteristics, while cell death was detected only at 50 μM. We observed that highly migratory cells showed a decrease on migration speed and directionality when treated with 2 or 5 μM of curcumin (50% and 40%, respectively, p < 0.05). Using spheroids, we observed that curcumin dose dependently decreased cell-cell adhesion, especially on tumor-derived spheroids. Also, in a xenograft model with patient-derived OSCC cells, the administration of curcumin decreased tumor growth and aggressiveness when compared with untreated tumors, indicating the potential antitumor effect in oral cancer. These results suggest that lower doses of curcumin can influence several steps involved in tumorigenesis, including migration properties, suggesting a possible use in cancer therapy. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Impairment of the ubiquitin-proteasome pathway in RPE alters the expression of inflammation related genes

USDA-ARS?s Scientific Manuscript database

The ubiquitin-proteasome pathway (UPP) plays an important role in regulating gene expression. Retinal pigment epithelial cells (RPE) are a major source of ocular inflammatory cytokines. In this work we determined the relationship between impairment of the UPP and expression of inflammation-related f...

Gradient biomaterials and their influences on cell migration

PubMed Central

Wu, Jindan; Mao, Zhengwei; Tan, Huaping; Han, Lulu; Ren, Tanchen; Gao, Changyou

2012-01-01

Cell migration participates in a variety of physiological and pathological processes such as embryonic development, cancer metastasis, blood vessel formation and remoulding, tissue regeneration, immune surveillance and inflammation. The cells specifically migrate to destiny sites induced by the gradually varying concentration (gradient) of soluble signal factors and the ligands bound with the extracellular matrix in the body during a wound healing process. Therefore, regulation of the cell migration behaviours is of paramount importance in regenerative medicine. One important way is to create a microenvironment that mimics the in vivo cellular and tissue complexity by incorporating physical, chemical and biological signal gradients into engineered biomaterials. In this review, the gradients existing in vivo and their influences on cell migration are briefly described. Recent developments in the fabrication of gradient biomaterials for controlling cellular behaviours, especially the cell migration, are summarized, highlighting the importance of the intrinsic driving mechanism for tissue regeneration and the design principle of complicated and advanced tissue regenerative materials. The potential uses of the gradient biomaterials in regenerative medicine are introduced. The current and future trends in gradient biomaterials and programmed cell migration in terms of the long-term goals of tissue regeneration are prospected. PMID:23741610

Texture sensing of cytoskeletal dynamics in cell migration

NASA Astrophysics Data System (ADS)

Das, Satarupa; Lee, Rachel; Hourwitz, Matthew J.; Sun, Xiaoyu; Parent, Carole; Fourkas, John T.; Losert, Wolfgang

Migrating cells can be directed towards a target by gradients in properties such as chemical concentration or mechanical properties of the surrounding microenvironment. In previous studies we have shown that micro/nanotopographical features on scales comparable to those of natural collagen fibers can guide fast migrating amoeboid cells by aligning actin polymerization waves to such nanostructures. We find that actin microfilaments and microtubules are aligned along the nanoridge topographies, modulating overall cell polarity and directional migration in epithelial cells. This work shows that topographic features on a biologically relevant length scale can modulate migration outcomes by affecting the texture sensing property of the cytoskeleton.

3D cancer cell migration in a confined matrix

NASA Astrophysics Data System (ADS)

Alobaidi, Amani; Sun, Bo

Cancer cell migration is widely studied in 2D motion, which does not mimic the invasion processes in vivo. More recently, 3D cell migration studies have been performed. The ability of cancer cells to migrate within the extracellular matrix depends on the physical and biochemical features of the extracellular matrix. We present a model of cell motility in confined matrix geometry. The aim of the study is to study cancer migration in collagen matrix, as a soft tissue, to investigate their motility within the confined and surrounding collagen environment. Different collagen concentrations have been used to show the ability of these cancer cells to move through such a complex structure by measuring Cancer cell migration velocity as well as the displacement. Graduate student physics department.

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.

Plectin deficiency in liver cancer cells promotes cell migration and sensitivity to sorafenib treatment.

PubMed

Cheng, Chiung-Chi; Chao, Wei-Ting; Liao, Chen-Chun; Tseng, Yu-Hui; Lai, Yen-Chang Clark; Lai, Yih-Shyong; Hsu, Yung-Hsiang; Liu, Yi-Hsiang

2018-01-02

Plectin involved in activation of kinases in cell signaling pathway and plays important role in cell morphology and migration. Plectin knockdown promotes cell migration by activating focal adhesion kinase and Rac1-GTPase activity in liver cells. Sorafenib is a multi-targeting tyrosine kinase inhibitor that improves patient survival on hepatocellular carcinoma. The aim of this study is to investigate the correlation between the expression of plectin and cell migration as well as the sensitivity of hepatoma cell lines exposing to sorafenib. Hepatoma cell lines PLC/PRF/5 and HepG2 were used to examine the level of plectin expression and cell migration in comparison with Chang liver cell line. In addition, sensitivity of the 3 cell lines to sorafenib treatment was also measured. Expression of plectin was lower in PLC/PRF/5 and HepG2 hepatoma cells than that of Chang liver cells whereas HepG2 and PLC/PRF/5 cells exhibit higher rate of cell migration in trans-well migration assay. Immunohistofluorecent staining on E-cadherin revealed the highest rate of collective cell migration in HepG2 cells and the lowest was found in Chang liver cells. Likewise, HepG2 cell line was most sensitive to sorafenib treatment and Chang liver cells exhibited the least sensitivity. The drug sensitivity to sorafenib treatment showed inverse correlation with the expression of plectin. We suggest that plectin deficiency and increased E-cadherin in hepatoma cells were associated with higher rates of cell motility, collective cell migration as well as higher drug sensitivity to sorafenib treatment.

Directional Cell Migration in Response to Repeated Substratum Stretching

NASA Astrophysics Data System (ADS)

Okimura, Chika; Iwadate, Yoshiaki

2017-10-01

Crawling migration plays an essential role in a variety of biological phenomena, including development, wound healing, and immune system function. Migration properties such as anterior-posterior polarity, directionality, and velocity are regulated not only by the reception of a chemoattractant but also by sensing mechanical inputs from the external environment. In this review, we describe the mechanical response of migrating cells, particularly under repeated stretching of the elastic substratum, highlighting the fact that there appear to be two independent mechanosensing systems that generate the polarity needed for migration. Cells that have no stress fibers, such as Dictyostelium cells and neutrophil-like differentiated HL-60 cells, migrate perpendicular to the stretching direction via myosin II localization. Cells that do possess stress fibers, however, such as fish keratocytes, migrate parallel to the stretching via a stress-fiber-dependent process.

Motile membrane protrusions regulate cell-cell adhesion and migration of olfactory ensheathing glia.

PubMed

Windus, Louisa C E; Claxton, Christina; Allen, Chelsea L; Key, Brian; St John, James A

2007-12-01

Olfactory ensheathing cells (OECs) are candidates for therapeutic approaches for neural regeneration due to their ability to assist axon regrowth in central nervous system lesion models. However, little is understood about the processes and mechanisms underlying migration of these cells. We report here that novel lamellipodial protrusions, termed lamellipodial waves, are integral to OEC migration. Time-lapse imaging of migrating OECs revealed that these highly dynamic waves progress along the shaft of the cells and are crucial for mediating cell-cell adhesion. Without these waves, cell-cell adhesion does not occur and migrational rates decline. The activity of waves is modulated by both glial cell line-derived neurotrophic factor and inhibitors of the JNK and SRC kinases. Furthermore, the activity of lamellipodial waves can be modulated by Mek1, independently of leading edge activity. The ability to selectively regulate cell migration via lamellipodial waves has implications for manipulating the migratory behavior of OECs during neural repair. (c) 2007 Wiley-Liss, Inc.

Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling

PubMed Central

Norris, Megan L; Pauli, Andrea; Gagnon, James A; Lord, Nathan D; Rogers, Katherine W; Mosimann, Christian; Zon, Leonard I

2017-01-01

Toddler/Apela/Elabela is a conserved secreted peptide that regulates mesendoderm development during zebrafish gastrulation. Two non-exclusive models have been proposed to explain Toddler function. The ‘specification model’ postulates that Toddler signaling enhances Nodal signaling to properly specify endoderm, whereas the ‘migration model’ posits that Toddler signaling regulates mesendodermal cell migration downstream of Nodal signaling. Here, we test key predictions of both models. We find that in toddler mutants Nodal signaling is initially normal and increasing endoderm specification does not rescue mesendodermal cell migration. Mesodermal cell migration defects in toddler mutants result from a decrease in animal pole-directed migration and are independent of endoderm. Conversely, endodermal cell migration defects are dependent on a Cxcr4a-regulated tether of the endoderm to mesoderm. These results suggest that Toddler signaling regulates mesodermal cell migration downstream of Nodal signaling and indirectly affects endodermal cell migration via Cxcr4a-signaling. PMID:29117894

Gaussian Curvature Directs Stress Fiber Orientation and Cell Migration.

PubMed

Bade, Nathan D; Xu, Tina; Kamien, Randall D; Assoian, Richard K; Stebe, Kathleen J

2018-03-27

We show that substrates with nonzero Gaussian curvature influence the organization of stress fibers and direct the migration of cells. To study the role of Gaussian curvature, we developed a sphere-with-skirt surface in which a positive Gaussian curvature spherical cap is seamlessly surrounded by a negative Gaussian curvature draping skirt, both with principal radii similar to cell-length scales. We find significant reconfiguration of two subpopulations of stress fibers when fibroblasts are exposed to these curvatures. Apical stress fibers in cells on skirts align in the radial direction and avoid bending by forming chords across the concave gap, whereas basal stress fibers bend along the convex direction. Cell migration is also strongly influenced by the Gaussian curvature. Real-time imaging shows that cells migrating on skirts repolarize to establish a leading edge in the azimuthal direction. Thereafter, they migrate in that direction. This behavior is notably different from migration on planar surfaces, in which cells typically migrate in the same direction as the apical stress fiber orientation. Thus, this platform reveals that nonzero Gaussian curvature not only affects the positioning of cells and alignment of stress fiber subpopulations but also directs migration in a manner fundamentally distinct from that of migration on planar surfaces. Copyright © 2018 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Novel Epigenetic Controlling of Hypoxia Pathway Related to Overexpression and Promoter Hypomethylation of TET1 and TET2 in RPE Cells.

PubMed

Alivand, Mohammad Reza; Soheili, Zahra-Soheila; Pornour, Majid; Solali, Saeed; Sabouni, Farzaneh

2017-10-01

CpG methylation of DNA takes part in a specific epigenetic memory that plays crucial roles in the differentiation and abnormality of the cells. The methylation pattern aberration of genomes is affected in three ways, namely DNA methyltransferase (DNMT), ten-eleven translocation (TET), and methyl-binding domain (MBD) proteins. Of these, TET enzymes have recently been demonstrated to be master modifier enzymes in the DNA methylation process. Additionally, recent studies emphasize that not only epigenetic phenomena play a role in controlling hypoxia pathway, but the hypoxia condition also triggers hypomethylation of genomes that may help with the expression of hypoxia pathway genes. In this study, we suggested that TET1 and TET2 could play a role in the demethylation of genomes under chemical hypoxia conditions. Herein, the evaluating methylation status and mRNA expression of mentioned genes were utilized through real-time PCR and methylation-specific PCR (MSP), respectively. Our results showed that TET1 and TET2 genes were overexpressed (P < 0.05) under chemical hypoxia conditions in Retinal Pigment Epithelial (RPE) cells, whereas the promoter methylation status of them were hypomethylated in the same condition. Therefore, chemical hypoxia not only causes overexpression of TET1 and TET2 but also could gradually do promoter demethylation of same genes. This is the first study to show the relationship between epigenetics and the expression of mentioned genes related to hypoxia pathways. Furthermore, it seems that these associations in RPE cells are subjected to chemical hypoxia as a mechanism that could play a crucial role in methylation pattern changes of hypoxia-related diseases such as cancer and ischemia. J. Cell. Biochem. 118: 3193-3204, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Primordial Germ Cell Specification and Migration

PubMed Central

Marlow, Florence

2015-01-01

Primordial germ cells are the progenitor cells that give rise to the gametes. In some animals, the germline is induced by zygotic transcription factors, whereas in others, primordial germ cell specification occurs via inheritance of maternally provided gene products known as germ plasm. Once specified, the primordial germ cells of some animals must acquire motility and migrate to the gonad in order to survive. In all animals examined, perinuclear structures called germ granules form within germ cells. This review focuses on some of the recent studies, conducted by several groups using diverse systems, from invertebrates to vertebrates, which have provided mechanistic insight into the molecular regulation of germ cell specification and migration. PMID:26918157

Alginate as a cell culture substrate for growth and differentiation of human retinal pigment epithelial cells.

PubMed

Heidari, Razeih; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Davari, Maliheh; Nazemroaya, Fatemeh; Bagheri, Abouzar; Deezagi, Abdolkhalegh

2015-03-01

The purpose of this study was to evaluate retinal pigment epithelium (RPE) cells' behavior in alginate beads that establish 3D environment for cellular growth and mimic extracellular matrix versus the conventional 2D monolayer culture. RPE cells were encapsulated in alginate beads by dripping alginate cell suspension into CaCl2 solution. Beads were suspended in three different media including Dulbecco's modified Eagle's medium (DMEM)/F12 alone, DMEM/F12 supplemented with 10 % fetal bovine serum (FBS), and DMEM/F12 supplemented with 30 % human amniotic fluid (HAF). RPE cells were cultivated on polystyrene under the same conditions as controls. Cell phenotype, cell proliferation, cell death, and MTT assay, immunocytochemistry, and real-time RT-PCR were performed to evaluate the effect of alginate on RPE cells characteristics and integrity. RPE cells can survive and proliferate in alginate matrixes. Immunocytochemistry analysis exhibited Nestin, RPE65, and cytokeratin expressions in a reasonable number of cultured cells in alginate beads. Real-time PCR data demonstrated high levels of Nestin, CHX10, RPE65, and tyrosinase gene expressions in RPE cells immobilized in alginate when compared to 2D monolayer culture systems. The results suggest that alginate can be used as a reliable scaffold for maintenance of RPE cells' integrity and in vitro propagation of human retinal progenitor cells for cell replacement therapies in retinal diseases.

Silk Film Topography Directs Collective Epithelial Cell Migration

PubMed Central

Rosenblatt, Mark I.

2012-01-01

The following study provides new insight into how surface topography dictates directed collective epithelial cell sheet growth through the guidance of individual cell movement. Collective cell behavior of migrating human corneal limbal-epithelial cell sheets were studied on highly biocompatible flat and micro-patterned silk film surfaces. The silk film edge topography guided the migratory direction of individual cells making up the collective epithelial sheet, which resulted in a 75% increase in total culture elongation. This was due to a 3-fold decrease in cell sheet migration rate efficiency for movement perpendicular to the topography edge. Individual cell migration direction is preferred in the parallel approach to the edge topography where localization of cytoskeletal proteins to the topography’s edge region is reduced, which results in the directed growth of the collective epithelial sheet. Findings indicate customized biomaterial surfaces may be created to direct both the migration rate and direction of tissue epithelialization. PMID:23185573

Altered Expression of Retinal Molecular Markers in the Canine RPE65 Model of Leber Congenital Amaurosis

PubMed Central

Hernández, Maria; Pearce-Kelling, Susan E.; Rodriguez, F. David; Aguirre, Gustavo D.; Vecino, Elena

2010-01-01

Purpose. Leber congenital amaurosis (LCA) is a group of childhood-onset retinal diseases characterized by severe visual impairment or blindness. One form is caused by mutations in the RPE65 gene, which encodes the retinal pigment epithelium (RPE) isomerase. In this study, the retinal structure and expression of molecular markers for different retinal cell types were characterized, and differences between control and RPE65 mutant dogs during the temporal evolution of the disease were analyzed. Methods. Retinas from normal and mutant dogs of different ages were examined by immunofluorescence with a panel of 16 different antibodies. Results. Cones and rods were preserved in the mutant retinas, and the number of cones was normal. However, there was altered expression of cone arrestin and delocalization of rod opsin. The ON bipolar cells showed sprouting of the dendritic arbors toward the outer nuclear layer (ONL) and retraction of their axons in the inner nuclear layer (INL). A decreased expression of GABA, and an increased expression of intermediate filament glial markers was also found in the mutant retinas. These changes were more evident in the adult than the young mutant retinas. Conclusions. The structure of the retina is well preserved in the mutant retina, but several molecular changes take place in photoreceptors and in bipolar and amacrine cells. Some of these changes are structural, whereas others reflect a change in localization of the examined proteins. This study provides new information that can be applied to the interpretation of outcomes of retinal gene therapy in animal models and humans. PMID:20671290

Effects of TNF-alpha on Endothelial Cell Collective Migration

NASA Astrophysics Data System (ADS)

Chen, Desu; Wu, Di; Helim Aranda-Espinoza, Jose; Losert, Wolfgang

2013-03-01

Tumor necrosis factor (TNF-alpha) is a small cell-signaling protein usually released by monocytes and macrophages during an inflammatory response. Previous work had shown the effects of TNF-alpha on single cell morphology, migration, and biomechanical properties. However, the effect on collective migrations remains unexplored. In this work, we have created scratches on monolayers of human umbilical endothelial cells (HUVECs) treated with 25ng/mL TNF-alpha on glass substrates. The wound healing like processes were imaged with phase contrast microscopy. Quantitative analysis of the collective migration of cells treated with TNF-alpha indicates that these cells maintain their persistent motion and alignment better than untreated cells. In addition, the collective migration was characterized by measuring the amount of non-affine deformations of the wound healing monolayer. We found a lower mean non-affinity and narrower distribution of non-affinities upon TNF-alpha stimulation. These results suggest that TNF-alpha introduces a higher degree of organized cell collective migration.

Low-level stretching accelerates cell migration into a gap.

PubMed

Toume, Samer; Gefen, Amit; Weihs, Daphne

2017-08-01

We observed that radially stretching cell monolayers at a low level (3%) increases the rate at which they migrate to close a gap formed by in vitro injury. Wound healing has been shown to accelerate in vivo when deformations are topically applied, for example, by negative pressure wound therapy. However, the direct effect of deformations on cell migration during gap closure is still unknown. Thus, we have evaluated the effect of radially applied, sustained (static) tensile strain on the kinematics of en mass cell migration. Monolayers of murine fibroblasts were cultured on stretchable, linear-elastic substrates that were subjected to different tensile strains, using a custom-designed three-dimensionally printed stretching apparatus. Immediately following stretching, the monolayer was 'wounded' at its centre, and cell migration during gap closure was monitored and quantitatively evaluated. We observed a significant increase in normalised migration rates and a reduction of gap closure time with 3% stretching, relative to unstretched controls or 6% stretch. Interestingly, the initial gap area was linearly correlated with the maximum migration rate, especially when stretching was applied. Therefore, small deformations applied to cell monolayers during gap closure enhance en mass cell migration associated with wound healing and can be used to fine-tune treatment protocols. © 2016 Medicalhelplines.com Inc and John Wiley & Sons Ltd.

Autoimmune retinopathy with RPE hypersensitivity and 'negative ERG' in X-linked hyper-IgM syndrome.

PubMed

Schuster, Andreas; Apfelstedt-Sylla, Eckart; Pusch, Carsten M; Zrenner, Eberhart; Thirkill, Charles E

2005-01-01

To report the clinical, electrophysiological, and immunological features of a patient with X-linked hyper-IgM immunodeficiency syndrome type 1 (HIGM1) accompanied by a novel type of autoimmune retinopathy, including retinal pigment epithelium (RPE) hypersensitivity. Comprehensive ophthalmological examinations, electrophysiological function testing, and inquiries into the immunological status of a 13-year-old presenting with subacute loss of vision in association with a molecularly confirmed diagnosis of HIGM1 were performed. The patient was genotyped by a PCR-based sequence tag content mapping strategy to define the genetic defect within the causative X-HIM gene TNFSF5. Since conventional allogenic bone marrow transplantation has been reported to cure HIGM1, a peripheral blood stem-cell transplantation was performed. (1) The patient's reduced visual acuity included prolonged dark adaptation and visual field constriction. Electrophysiology revealed a 'negative ERG' indicating post-receptoral dysfunction. (2) Initial immunological examination of the patient's serum identified abnormal antibody activity with components of the photoreceptors and the inner nuclear layer. The patient later developed indications of RPE hypersensitivity. A massively reduced light-peak to dark-trough ratio of the EOG slow oscillations (L/D ratio) corresponded to impaired RPE-photoreceptor complex function. (3) Molecular genetic analyses revealed the patient to be nullizygous for the tumor necrosis factor ligand member 5 gene (TNFSF5; CD40LG). A large chromosomal deletion of approximately 27.6-32.3 kb in size was identified in Xq26. (4) The transplant with its associated immunomodulation appeared to worsen rather than improve the patient's condition. The fundus appearance and electrophysiological function testing revealed indications of atypical retinal degeneration. However, the clinical course and the serological findings were consistent with those of ocular autoimmunity involving both

S-Fms signalobody enhances myeloid cell growth and migration.

PubMed

Kawahara, Masahiro; Hitomi, Azusa; Nagamune, Teruyuki

2014-07-01

Since receptor tyrosine kinases (RTKs) control various cell fates in many types of cells, mimicry of RTK functions is promising for artificial control of cell fates. We have previously developed single-chain Fv (scFv)/receptor chimeras named signalobodies that can mimic receptor signaling in response to a specific antigen. While the RTK-based signalobodies enabled us to control cell growth and migration, further extension of applicability in another cell type would underlie the impact of the RTK-based signalobodies. In this study, we applied the scFv-c-Fms (S-Fms) signalobody in a murine myeloid progenitor cell line, FDC-P1. S-Fms transduced a fluorescein-conjugated BSA (BSA-FL)-dependent growth signal and activated downstream signaling molecules including MEK, ERK, Akt, and STAT3, which are major constituents of Ras/MAPK, PI3K/Akt, and JAK/STAT signaling pathways. In addition, S-Fms transduced a migration signal as demonstrated by the transwell-based migration assay. Direct real-time observation of the cells further confirmed that FDC/S-Fms cells underwent directional cell migration toward a positive gradient of BSA-FL. These results demonstrated the utility of the S-Fms signalobody for controlling growth and migration of myeloid cells. Further extension of our approach includes economical large-scale production of practically relevant blood cells as well as artificial control of cell migration for tissue regeneration and immune response. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Deterministic Migration-Based Separation of White Blood Cells.

PubMed

Kim, Byeongyeon; Choi, Young Joon; Seo, Hyekyung; Shin, Eui-Cheol; Choi, Sungyoung

2016-10-01

Functional and phenotypic analyses of peripheral white blood cells provide useful clinical information. However, separation of white blood cells from peripheral blood requires a time-consuming, inconvenient process and thus analyses of separated white blood cells are limited in clinical settings. To overcome this limitation, a microfluidic separation platform is developed to enable deterministic migration of white blood cells, directing the cells into designated positions according to a ridge pattern. The platform uses slant ridge structures on the channel top to induce the deterministic migration, which allows efficient and high-throughput separation of white blood cells from unprocessed whole blood. The extent of the deterministic migration under various rheological conditions is explored, enabling highly efficient migration of white blood cells in whole blood and achieving high-throughput separation of the cells (processing 1 mL of whole blood less than 7 min). In the separated cell population, the composition of lymphocyte subpopulations is well preserved, and T cells secrete cytokines without any functional impairment. On the basis of the results, this microfluidic platform is a promising tool for the rapid enrichment of white blood cells, and it is useful for functional and phenotypic analyses of peripheral white blood cells. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Microtubule release from the centrosome in migrating cells

PubMed Central

Abal, Miguel; Piel, Matthieu; Bouckson-Castaing, Veronique; Mogensen, Mette; Sibarita, Jean-Baptiste; Bornens, Michel

2002-01-01

In migrating cells, force production relies essentially on a polarized actomyosin system, whereas the spatial regulation of actomyosin contraction and substrate contact turnover involves a complex cooperation between the microtubule (MT) and the actin filament networks (Goode, B.L., D.G. Drubin, and G. Barnes. 2000. Curr. Opin. Cell Biol., 12:63–71). Targeting and capture of MT plus ends at the cell periphery has been described, but whether or not the minus ends of these MTs are anchored at the centrosome is not known. Here, we show that release of short MTs from the centrosome is frequent in migrating cells and that their transport toward the cell periphery is blocked when dynein activity is impaired. We further show that MT release, but not MT nucleation or polymerization dynamics, is abolished by overexpression of the centrosomal MT-anchoring protein ninein. In addition, a dramatic inhibition of cell migration was observed; but, contrary to cells treated by drugs inhibiting MT dynamics, polarized membrane ruffling activity was not affected in ninein overexpressing cells. We thus propose that the balance between MT minus-end capture and release from the centrosome is critical for efficient cell migration. PMID:12473683

Perceptually regulated training at RPE13 is pleasant and improves physical health.

PubMed

Parfitt, Gaynor; Evans, Harrison; Eston, Roger

2012-08-01

Despite endorsement by various health organizations, there is a lack of research on the effectiveness of perceptually regulated exercise training (PRET) as a method of exercise intensity prescription. The purpose of this study was to confirm the efficacy of an 8-wk PRET program clamped at RPE13 to improve aerobic fitness and cardiovascular health. The affective response to this method of exercise prescription was also assessed. Sedentary volunteers (age = 34.3 ± 13.0 yr, weight = 72.5 ± 13.7 kg, height = 1.7 ± 0.1 m) were randomly assigned to either a training (n = 16) or a control (n = 10) group. All participants completed a graded exercise test to determine aerobic capacity at baseline and after the intervention. Participants allocated to the training group performed 30 min of PRET at RPE13 on the Borg 6-20 RPE Scale on three occasions per week for 8 wk. Affective valence was measured using the Feeling Scale. The RPE-regulated training resulted in improvements (P < 0.01) in V˙O(2max), mean arterial pressure, total cholesterol, and body mass index in the training group across time. During training at RPE13, V˙O(2) increased (P < 0.01) from week 1 (19.2 ± 1.1 mL·kg·min) to week 8 (23.4 ± 1.1 mL·kg·min). On average, affect was positive and stable throughout training (3.4 ± 1.2). Affect measured at RPE13 in the baseline and postintervention graded exercise tests increased in the training group (3.1 ± .9 to 3.7 ± 1.1, P < 0.05), whereas it decreased in the control group (2.8 ± 1.1 to 2.6 ± 1). Sedentary individuals were able to use PRET at RPE13 to improve their cardiovascular health and fitness, and on average, the exercise intensities selected were perceived to feel pleasant.

Profound re-organization of cell surface proteome in equine retinal pigment epithelial cells in response to in vitro culturing.

PubMed

Szober, Christoph M; Hauck, Stefanie M; Euler, Kerstin N; Fröhlich, Kristina J H; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A

2012-10-31

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses' vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

Effects of the vegetable polyphenols epigallocatechin-3-gallate, luteolin, apigenin, myricetin, quercetin, and cyanidin in primary cultures of human retinal pigment epithelial cells

PubMed Central

Chen, Rui; Grosche, Antje; Reichenbach, Andreas; Wiedemann, Peter; Bringmann, Andreas; Kohen, Leon

2014-01-01

Purpose Vegetable polyphenols (bioflavonoids) have been suggested to represent promising drugs for treating cancer and retinal diseases. We compared the effects of various bioflavonoids (epigallocatechin-3-gallate [EGCG], luteolin, apigenin, myricetin, quercetin, and cyanidin) on the physiological properties and viability of cultured human retinal pigment epithelial (RPE) cells. Methods Human RPE cells were obtained from several donors within 48 h of death. Secretion of vascular endothelial growth factor (VEGF) was determined with enzyme-linked immunosorbent assay. Messenger ribonucleic acid levels were determined with real-time reverse transcription polymerase chain reaction. Cellular proliferation was investigated with a bromodeoxyuridine immunoassay, and chemotaxis was examined with a Boyden chamber assay. The number of viable cells was determined by Trypan Blue exclusion. Apoptosis and necrosis rates were determined with a DNA fragmentation enzyme-linked immunosorbent assay. The phosphorylation level of signaling proteins was revealed by western blotting. Results With the exception of EGCG, all flavonoids tested decreased dose-dependently the RPE cell proliferation, migration, and secretion of VEGF. EGCG inhibited the secretion of VEGF evoked by CoCl2-induced hypoxia. The gene expression of VEGF was reduced by myricetin at low concentrations and elevated at higher concentrations. Luteolin, apigenin, myricetin, and quercetin induced significant decreases in the cell viability at higher concentration, by triggering cellular necrosis. Cyanidin reduced the rate of RPE cell necrosis. Myricetin caused caspase-3 independent RPE cell necrosis mediated by free radical generation and activation of calpain and phospholipase A2. The myricetin- and quercetin-induced RPE cell necrosis was partially inhibited by necrostatin-1, a blocker of programmed necrosis. Most flavonoids tested diminished the phosphorylation levels of extracellular signal-regulated kinases 1/2 and Akt

Collective cell migration without proliferation: density determines cell velocity and wave velocity

NASA Astrophysics Data System (ADS)

Tlili, Sham; Gauquelin, Estelle; Li, Brigitte; Cardoso, Olivier; Ladoux, Benoît; Delanoë-Ayari, Hélène; Graner, François

2018-05-01

Collective cell migration contributes to embryogenesis, wound healing and tumour metastasis. Cell monolayer migration experiments help in understanding what determines the movement of cells far from the leading edge. Inhibiting cell proliferation limits cell density increase and prevents jamming; we observe long-duration migration and quantify space-time characteristics of the velocity profile over large length scales and time scales. Velocity waves propagate backwards and their frequency depends only on cell density at the moving front. Both cell average velocity and wave velocity increase linearly with the cell effective radius regardless of the distance to the front. Inhibiting lamellipodia decreases cell velocity while waves either disappear or have a lower frequency. Our model combines conservation laws, monolayer mechanical properties and a phenomenological coupling between strain and polarity: advancing cells pull on their followers, which then become polarized. With reasonable values of parameters, this model agrees with several of our experimental observations. Together, our experiments and model disantangle the respective contributions of active velocity and of proliferation in monolayer migration, explain how cells maintain their polarity far from the moving front, and highlight the importance of strain-polarity coupling and density in long-range information propagation.

Force transmission in migrating cells

PubMed Central

Sauser, Roger; Ambrosi, Davide; Meister, Jean-Jacques; Verkhovsky, Alexander B.

2010-01-01

During cell migration, forces generated by the actin cytoskeleton are transmitted through adhesion complexes to the substrate. To investigate the mechanism of force generation and transmission, we analyzed the relationship between actin network velocity and traction forces at the substrate in a model system of persistently migrating fish epidermal keratocytes. Front and lateral sides of the cell exhibited much stronger coupling between actin motion and traction forces than the trailing cell body. Further analysis of the traction–velocity relationship suggested that the force transmission mechanisms were different in different cell regions: at the front, traction was generated by a gripping of the actin network to the substrate, whereas at the sides and back, it was produced by the network’s slipping over the substrate. Treatment with inhibitors of the actin–myosin system demonstrated that the cell body translocation could be powered by either of the two different processes, actomyosin contraction or actin assembly, with the former associated with significantly larger traction forces than the latter. PMID:20100912

Cancer Cell Migration in 3D

NASA Astrophysics Data System (ADS)

Wirtz, Denis

2014-03-01

Two-dimensional (2D) in vitro culture systems have for a number of years provided a controlled and versatile environment for mechanistic studies of cell adhesion, polarization, and migration, three interrelated cell functions critical to cancer metastasis. However, the organization and functions of focal adhesion proteins, protrusion machinery, and microtubule-based polarization in cells embedded in physiologically more relevant 3D extracellular matrices is qualitatively different from their organization and functions on conventional 2D planar substrates. This talk will describe the implications of the dependence of focal adhesion protein-based cell migration on micro-environmental dimensionality (1D vs. 2D vs.. 3D), how cell micromechanics plays a critical role in promoting local cell invasion, and associated validation in mouse models. We will discuss the implications of this work in cancer metastasis.

Robotic Patterning a Superhydrophobic Surface for Collective Cell Migration Screening.

PubMed

Pang, Yonggang; Yang, Jing; Hui, Zhixin; Grottkau, Brian E

2018-04-01

Collective cell migration, in which cells migrate as a group, is fundamental in many biological and pathological processes. There is increasing interest in studying the collective cell migration in high throughput. Cell scratching, insertion blocker, and gel-dissolving techniques are some methodologies used previously. However, these methods have the drawbacks of cell damage, substrate surface alteration, limitation in medium exchange, and solvent interference. The superhydrophobic surface, on which the water contact angle is greater than 150 degrees, has been recently utilized to generate patterned arrays. Independent cell culture areas can be generated on a substrate that functions the same as a conventional multiple well plate. However, so far there has been no report on superhydrophobic patterning for the study of cell migration. In this study, we report on the successful development of a robotically patterned superhydrophobic array for studying collective cell migration in high throughput. The array was developed on a rectangular single-well cell culture plate consisting of hydrophilic flat microwells separated by the superhydrophobic surface. The manufacturing process is robotic and includes patterning discrete protective masks to the substrate using 3D printing, robotic spray coating of silica nanoparticles, robotic mask removal, robotic mini silicone blocker patterning, automatic cell seeding, and liquid handling. Compared with a standard 96-well plate, our system increases the throughput by 2.25-fold and generates a cell-free area in each well non-destructively. Our system also demonstrates higher efficiency than conventional way of liquid handling using microwell plates, and shorter processing time than manual operating in migration assays. The superhydrophobic surface had no negative impact on cell viability. Using our system, we studied the collective migration of human umbilical vein endothelial cells and cancer cells using assays of endpoint

Quantitative analysis of random migration of cells using time-lapse video microscopy.

PubMed

Jain, Prachi; Worthylake, Rebecca A; Alahari, Suresh K

2012-05-13

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion (1, 2). During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues (3) provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment. Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration (2). Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration. Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function (4), hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO(2) supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

Surface topography during neural stem cell differentiation regulates cell migration and cell morphology.

PubMed

Czeisler, Catherine; Short, Aaron; Nelson, Tyler; Gygli, Patrick; Ortiz, Cristina; Catacutan, Fay Patsy; Stocker, Ben; Cronin, James; Lannutti, John; Winter, Jessica; Otero, José Javier

2016-12-01

We sought to determine the contribution of scaffold topography to the migration and morphology of neural stem cells by mimicking anatomical features of scaffolds found in vivo. We mimicked two types of central nervous system scaffolds encountered by neural stem cells during development in vitro by constructing different diameter electrospun polycaprolactone (PCL) fiber mats, a substrate that we have shown to be topographically similar to brain scaffolds. We compared the effects of large fibers (made to mimic blood vessel topography) with those of small-diameter fibers (made to mimic radial glial process topography) on the migration and differentiation of neural stem cells. Neural stem cells showed differential migratory and morphological reactions with laminin in different topographical contexts. We demonstrate, for the first time, that neural stem cell biological responses to laminin are dependent on topographical context. Large-fiber topography without laminin prevented cell migration, which was partially reversed by treatment with rock inhibitor. Cell morphology complexity assayed by fractal dimension was inhibited in nocodazole- and cytochalasin-D-treated neural precursor cells in large-fiber topography, but was not changed in small-fiber topography with these inhibitors. These data indicate that cell morphology has different requirements on cytoskeletal proteins dependent on the topographical environment encountered by the cell. We propose that the physical structure of distinct scaffolds induces unique signaling cascades that regulate migration and morphology in embryonic neural precursor cells. J. Comp. Neurol. 524:3485-3502, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cell Migration in 1D and 2D Nanofiber Microenvironments.

PubMed

Estabridis, Horacio M; Jana, Aniket; Nain, Amrinder; Odde, David J

2018-03-01

Understanding how cells migrate in fibrous environments is important in wound healing, immune function, and cancer progression. A key question is how fiber orientation and network geometry influence cell movement. Here we describe a quantitative, modeling-based approach toward identifying the mechanisms by which cells migrate in fibrous geometries having well controlled orientation. Specifically, U251 glioblastoma cells were seeded onto non-electrospinning Spinneret based tunable engineering parameters fiber substrates that consist of networks of suspended 400 nm diameter nanofibers. Cells were classified based on the local fiber geometry and cell migration dynamics observed by light microscopy. Cells were found in three distinct geometries: adhering two a single fiber, adhering to two parallel fibers, and adhering to a network of orthogonal fibers. Cells adhering to a single fiber or two parallel fibers can only move in one dimension along the fiber axis, whereas cells on a network of orthogonal fibers can move in two dimensions. We found that cells move faster and more persistently in 1D geometries than in 2D, with cell migration being faster on parallel fibers than on single fibers. To explain these behaviors mechanistically, we simulated cell migration in the three different geometries using a motor-clutch based model for cell traction forces. Using nearly identical parameter sets for each of the three cases, we found that the simulated cells naturally replicated the reduced migration in 2D relative to 1D geometries. In addition, the modestly faster 1D migration on parallel fibers relative to single fibers was captured using a correspondingly modest increase in the number of clutches to reflect increased surface area of adhesion on parallel fibers. Overall, the integrated modeling and experimental analysis shows that cell migration in response to varying fibrous geometries can be explained by a simple mechanical readout of geometry via a motor-clutch mechanism.

Physical confinement alters tumor cell adhesion and migration phenotypes

PubMed Central

Balzer, Eric M.; Tong, Ziqiu; Paul, Colin D.; Hung, Wei-Chien; Stroka, Kimberly M.; Boggs, Amanda E.; Martin, Stuart S.; Konstantopoulos, Konstantinos

2012-01-01

Cell migration on planar surfaces is driven by cycles of actin protrusion, integrin-mediated adhesion, and myosin-mediated contraction; however, this mechanism may not accurately describe movement in 3-dimensional (3D) space. By subjecting cells to restrictive 3D environments, we demonstrate that physical confinement constitutes a biophysical stimulus that alters cell morphology and suppresses mesenchymal motility in human breast carcinoma (MDA-MB-231). Dorsoventral polarity, stress fibers, and focal adhesions are markedly attenuated by confinement. Inhibitors of myosin, Rho/ROCK, or β1-integrins do not impair migration through 3-μm-wide channels (confinement), even though these treatments repress motility in 50-μm-wide channels (unconfined migration) by ≥50%. Strikingly, confined migration persists even when F-actin is disrupted, but depends largely on microtubule (MT) dynamics. Interfering with MT polymerization/depolymerization causes confined cells to undergo frequent directional changes, thereby reducing the average net displacement by ≥80% relative to vehicle controls. Live-cell EB1-GFP imaging reveals that confinement redirects MT polymerization toward the leading edge, where MTs continuously impact during advancement of the cell front. These results demonstrate that physical confinement can induce cytoskeletal alterations that reduce the dependence of migrating cells on adhesion-contraction force coupling. This mechanism may explain why integrins can exhibit reduced or altered function during migration in 3D environments.—Balzer, E. M., Tong, Z., Paul, C. D., Hung, W.-C., Stroka, K. M., Boggs, A. E., Martin, S. S., Konstantopoulos, K. Physical confinement alters tumor cell adhesion and migration phenotypes. PMID:22707566

Collisions of deformable cells lead to collective migration

NASA Astrophysics Data System (ADS)

Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

2015-03-01

Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility - acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.

Collisions of deformable cells lead to collective migration

DOE PAGES

Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

2015-03-17

Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility – acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignmentmore » of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.« less

Collisions of deformable cells lead to collective migration

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

Löber, Jakob; Ziebert, Falko; Aranson, Igor S.

Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility – acto-myosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignmentmore » of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility.« less

Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi

PubMed Central

Parker, Aimee; Maclaren, Oliver J.; Fletcher, Alexander G.; Muraro, Daniele; Kreuzaler, Peter A.; Byrne, Helen M.; Maini, Philip K.; Watson, Alastair J. M.; Pin, Carmen

2017-01-01

The functional integrity of the intestinal epithelial barrier relies on tight coordination of cell proliferation and migration, with failure to regulate these processes resulting in disease. It is not known whether cell proliferation is sufficient to drive epithelial cell migration during homoeostatic turnover of the epithelium. Nor is it known precisely how villus cell migration is affected when proliferation is perturbed. Some reports suggest that proliferation and migration may not be related while other studies support a direct relationship. We used established cell-tracking methods based on thymine analog cell labeling and developed tailored mathematical models to quantify cell proliferation and migration under normal conditions and when proliferation is reduced and when it is temporarily halted. We found that epithelial cell migration velocities along the villi are coupled to cell proliferation rates within the crypts in all conditions. Furthermore, halting and resuming proliferation results in the synchronized response of cell migration on the villi. We conclude that cell proliferation within the crypt is the primary force that drives cell migration along the villus. This methodology can be applied to interrogate intestinal epithelial dynamics and characterize situations in which processes involved in cell turnover become uncoupled, including pharmacological treatments and disease models.—Parker, A., Maclaren, O. J., Fletcher, A. G., Muraro, D., Kreuzaler, P. A., Byrne, H. M., Maini, P. K., Watson, A. J. M., Pin, C. Cell proliferation within small intestinal crypts is the principal driving force for cell migration on villi. PMID:27811059

Role of medullary progenitor cells in epithelial cell migration and proliferation

PubMed Central

Chen, Dong; Chen, Zhiyong; Zhang, Yuning; Park, Chanyoung; Al-Omari, Ahmed

2014-01-01

This study is aimed at characterizing medullary interstitial progenitor cells and to examine their capacity to induce tubular epithelial cell migration and proliferation. We have isolated a progenitor cell side population from a primary medullary interstitial cell line. We show that the medullary progenitor cells (MPCs) express CD24, CD44, CXCR7, CXCR4, nestin, and PAX7. MPCs are CD34 negative, which indicates that they are not bone marrow-derived stem cells. MPCs survive >50 passages, and when grown in epithelial differentiation medium develop phenotypic characteristics of epithelial cells. Inner medulla collecting duct (IMCD3) cells treated with conditioned medium from MPCs show significantly accelerated cell proliferation and migration. Conditioned medium from PGE2-treated MPCs induce tubule formation in IMCD3 cells grown in 3D Matrigel. Moreover, most of the MPCs express the pericyte marker PDGFR-b. Our study shows that the medullary interstitium harbors a side population of progenitor cells that can differentiate to epithelial cells and can stimulate tubular epithelial cell migration and proliferation. The findings of this study suggest that medullary pericyte/progenitor cells may play a critical role in collecting duct cell injury repair. PMID:24808539

A simple non-perturbing cell migration assay insensitive to proliferation effects.

PubMed

Glenn, Honor L; Messner, Jacob; Meldrum, Deirdre R

2016-08-18

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells.

A simple non-perturbing cell migration assay insensitive to proliferation effects

PubMed Central

Glenn, Honor L.; Messner, Jacob; Meldrum, Deirdre R.

2016-01-01

Migration is a fundamental cellular behavior that plays an indispensable role in development and homeostasis, but can also contribute to pathology such as cancer metastasis. Due to its relevance to many aspects of human health, the ability to accurately measure cell migration is of broad interest, and numerous approaches have been developed. One of the most commonly employed approaches, because of its simplicity and throughput, is the exclusion zone assay in which cells are allowed to migrate into an initially cell-free region. A major drawback of this assay is that it relies on simply counting cells in the exclusion zone and therefore cannot distinguish the effects of proliferation from migration. We report here a simple modification to the exclusion zone migration assay that exclusively measures cell migration and is not affected by proliferation. This approach makes use of a lineage-tracing vital stain that is retained through cell generations and effectively reads out migration relative to the original, parental cell population. This modification is simple, robust, non-perturbing, and inexpensive. We validate the method in a panel of cell lines under conditions that inhibit or promote migration and demonstrate its use in normal and cancer cell lines as well as primary cells. PMID:27535324

Fibronectin in cell adhesion and migration via N-glycosylation

PubMed Central

Hsiao, Cheng-Te; Cheng, Hung-Wei; Huang, Chi-Ming; Li, Hao-Ru; Ou, Meng-Hsin; Huang, Jie-Rong; Khoo, Kay-Hooi; Yu, Helen Wenshin; Chen, Yin-Quan; Wang, Yang-Kao; Chiou, Arthur; Kuo, Jean-Cheng

2017-01-01

Directed cell migration is an important step in effective wound healing and requires the dynamic control of the formation of cell-extracellular matrix interactions. Plasma fibronectin is an extracellular matrix glycoprotein present in blood plasma that plays crucial roles in modulating cellular adhesion and migration and thereby helping to mediate all steps of wound healing. In order to seek safe sources of plasma fibronectin for its practical use in wound dressing, we isolated fibronectin from human (homo) and porcine plasma and demonstrated that both have a similar ability as a suitable substrate for the stimulation of cell adhesion and for directing cell migration. In addition, we also defined the N-glycosylation sites and N-glycans present on homo and porcine plasma fibronectin. These N-glycosylation modifications of the plasma fibronectin synergistically support the integrin-mediated signals to bring about mediating cellular adhesion and directed cell migration. This study not only determines the important function of N-glycans in both homo and porcine plasma fibronectin-mediated cell adhesion and directed cell migration, but also reveals the potential applications of porcine plasma fibronectin if it was applied as a material for clinical wound healing and tissue repair. PMID:29050309

Role of the p55-gamma subunit of PI3K in ALK-induced cell migration: RNAi-based selection of cell migration regulators.

PubMed

Seo, Minchul; Kim, Jong-Heon; Suk, Kyoungho

2017-05-04

Recently, unbiased functional genetic selection identified novel cell migration-regulating genes. This RNAi-based functional selection was performed using 63,996 pooled lentiviral shRNAs targeting 21,332 mouse genes. After five rounds of selection using cells with accelerated or impaired migration, shRNAs were retrieved and identified by half-hairpin barcode sequencing using cells with the selected phenotypes. This selection process led to the identification of 29 novel cell migration regulators. One of these candidates, anaplastic lymphoma kinase (ALK), was further investigated. Subsequent studies revealed that ALK promoted cell migration through the PI3K-AKT pathway via the p55γ regulatory subunit of PI3K, rather than more commonly used p85 subunit. Western blot and immunohistochemistry studies using mouse brain tissues revealed similar temporal expression patterns of ALK, phospho-p55γ, and phospho-AKT during different stages of development. These data support an important role for the p55γ subunit of PI3K in ALK-induced cell migration during brain development.

Wavelength dependence of intracellular nitric oxide levels in hTERT-RPE cells in vitro

NASA Astrophysics Data System (ADS)

Pope, Nathaniel J.; Powell, Samantha M.; Wigle, Jeffrey C.

2018-02-01

Photobiomodulation (PBM) refers to the beneficial effects of low doses of light whether mediating therapeutic effects for pathophysiological processes, or stimulating resistance to physiological challenges. While much is known about beneficial outcomes, our understanding of the molecular mechanisms underlying these observed effects is still limited. It has been hypothesized that increases in ATP stimulate downstream signaling through transcription factors. However, it is also known that PBM can induce elevated levels of nitric oxide (NO) in cells, which is thought to occur by release of NO bound to cytochrome-c oxidase (COX). NO is a powerful signaling molecule involved in a host of biological responses; however, the mechanisms of NO production and the role of NO in the PBM response have received little attention. Utilizing human retinal pigmented epithelium cells (RPE) in vitro, coupled with a multi-laser exposure set-up, we have begun to systematically investigate the mechanism of NO production and function in the PBM response. Our data indicates that while NO levels are elevated following single exposures to 447, 532, 635 or 808 nm, the strength of the response is wavelength-dependent, and the response can be modulated by sequential exposures to two different wavelengths. Additionally, this wavelength-dependent rise in NO is independent of the function of nitric oxide synthase, and highly dependent on the source of electrons feeding the electron transport chain of the light-exposed cells. In sum, these results provide a roadmap for interrogating the molecular mechanisms of PBM, and provide novel tools and methods for dissecting NO signaling networks.

Contacting co-culture of human retinal microvascular endothelial cells alters barrier function of human embryonic stem cell derived retinal pigment epithelial cells.

PubMed

Skottman, H; Muranen, J; Lähdekorpi, H; Pajula, E; Mäkelä, K; Koivusalo, L; Koistinen, A; Uusitalo, H; Kaarniranta, K; Juuti-Uusitalo, K

2017-10-01

Here we evaluated the effects of human retinal microvascular endothelial cells (hREC) on mature human embryonic stem cell (hESC) derived retinal pigment epithelial (RPE) cells. The hESC-RPE cells (Regea08/017, Regea08/023 or Regea11/013) and hREC (ACBRI 181) were co-cultured on opposite sides of transparent membranes for up to six weeks. Thereafter barrier function, small molecule permeability, localization of RPE and endothelial cell marker proteins, cellular fine structure, and growth factor secretion of were evaluated. After co-culture, the RPE specific CRALBP and endothelial cell specific von Willebrand factor were appropriately localized. In addition, the general morphology, pigmentation, and fine structure of hESC-RPE cells were unaffected. Co-culture increased the barrier function of hESC-RPE cells, detected both with TEER measurements and cumulative permeability of FD4 - although the differences varied among the cell lines. Co-culturing significantly altered VEGF and PEDF secretion, but again the differences were cell line specific. The results of this study showed that co-culture with hREC affects hESC-RPE functionality. In addition, co-culture revealed drastic cell line specific differences, most notably in growth factor secretion. This model has the potential to be used as an in vitro outer blood-retinal barrier model for drug permeability testing. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Collective cell migration of thyroid carcinoma cells: a beneficial ability to override unfavourable substrates.

PubMed

Lobastova, Liudmila; Kraus, Dominik; Glassmann, Alexander; Khan, Dilaware; Steinhäuser, Christian; Wolff, Christina; Veit, Nadine; Winter, Jochen; Probstmeier, Rainer

2017-02-01

Tumor cell invasion and metastasis are life threatening events. Invasive tumor cells tend to migrate as collective sheets. In the present in vitro study we aimed to (i) assess whether collective tumor cells gain benefits in their migratory potential compared to single cells and (ii) to identify its putative underlying molecular mechanisms. The migratory potential of single and collective carcinoma cells was assessed using video time lapse microscopy and cell migration assays in the absence and presence of seven potential gap junction inhibitors or the Rac1 inhibitor Z62954982. The perturbation of gap junctions was assessed using a dye diffusion assay. In addition, LDH-based cytotoxicity and RT-PCR-based expression analyses were performed. Whereas single breast, cervix and thyroid carcinoma cells were virtually immobile on unfavourable plastic surfaces, we found that they gained pronounced migratory capacities as collectives under comparable conditions. Thyroid carcinoma cells, that were studied in more detail, were found to express specific subsets of connexins and to form active gap junctions as revealed by dye diffusion analysis. Although all potential gap junction blockers suppressed intercellular dye diffusion in at least one of the cell lines tested, only two of them were found to inhibit collective cell migration and none of them to inhibit single cell migration. In the presence of the Rac1 inhibitor Z62954982 collective migration, but not single cell migration, was found to be reduced up to 20 %. Our data indicate that collective migration enables tumor cells to cross otherwise unfavourable substrate areas. This capacity seems to be independent of intercellular communication via gap junctions, whereas Rac1-dependent intracellular signalling seems to be essential.

Validity and reliability of the session-RPE method for quantifying training load in karate athletes.

PubMed

Tabben, M; Tourny, C; Haddad, M; Chaabane, H; Chamari, K; Coquart, J B

2015-04-24

To test the construct validity and reliability of the session rating of perceived exertion (sRPE) method by examining the relationship between RPE and physiological parameters (heart rate: HR and blood lactate concentration: [La --] ) and the correlations between sRPE and two HR--based methods for quantifying internal training load (Banister's method and Edwards's method) during karate training camp. Eighteen elite karate athletes: ten men (age: 24.2 ± 2.3 y, body mass: 71.2 ± 9.0 kg, body fat: 8.2 ± 1.3% and height: 178 ± 7 cm) and eight women (age: 22.6 ± 1.2 y, body mass: 59.8 ± 8.4 kg, body fat: 20.2 ± 4.4%, height: 169 ± 4 cm) were included in the study. During training camp, subjects participated in eight karate--training sessions including three training modes (4 tactical--technical, 2 technical--development, and 2 randori training), during which RPE, HR, and [La -- ] were recorded. Significant correlations were found between RPE and physiological parameters (percentage of maximal HR: r = 0.75, 95% CI = 0.64--0.86; [La --] : r = 0.62, 95% CI = 0.49--0.75; P < 0.001). Moreover, individual sRPE was significantly correlated with two HR--based methods for quantifying internal training load ( r = 0.65--0.95; P < 0.001). The sRPE method showed the high reliability of the same intensity across training sessions (Cronbach's α = 0.81, 95% CI = 0.61--0.92). This study demonstrates that the sRPE method is valid for quantifying internal training load and intensity in karate.

Balancing Cell Migration with Matrix Degradation Enhances Gene Delivery to Cells Cultured Three-Dimensionally Within Hydrogels

PubMed Central

Shepard, Jaclyn A.; Huang, Alyssa; Shikanova, Ariella; Shea, Lonnie D.

2010-01-01

In regenerative medicine, hydrogels are employed to fill defects and support the infiltration of cells that can ultimately regenerate tissue. Gene delivery within hydrogels targeting infiltrating cells has the potential to promote tissue formation, but the delivery efficiency of nonviral vectors within hydrogels is low hindering their applicability in tissue regeneration. To improve their functionality, we have conducted a mechanistic study to investigate the contribution of cell migration and matrix degradation on gene delivery. In this report, lipoplexes were entrapped within hydrogels based on poly(ethylene glycol) (PEG) crosslinked with peptides containing matrix metalloproteinase degradable sequences. The mesh size of these hydrogels is substantially less than the size of the entrapped lipoplexes, which can function to retain vectors. Cell migration and transfection were simultaneously measured within hydrogels with varying density of cell adhesion sites (Arg-Gly-Asp peptides) and solids content. Increasing RGD density increased expression levels up to 100-fold, while greater solids content sustained expression levels for 16 days. Increasing RGD density and decreasing solids content increased cell migration, which indicates expression levels increase with increased cell migration. Initially exposing cells to vector resulted in transient expression that declined after 2 days, verifying the requirement of migration to sustain expression. Transfected cells were predominantly located within the population of migrating cells for hydrogels that supported cell migration. Although the small mesh size retained at least 70% of the lipoplexes in the absence of cells after 32 days, the presence of cells decreased retention to 10% after 16 days. These results indicate that vectors retained within hydrogels contact migrating cells, and that persistent cell migration can maintain elevated expression levels. Thus matrix degradation and cell migration are fundamental design

Session-RPE for quantifying load of different youth taekwondo training sessions.

PubMed

Lupo, Corrado; Capranica, Laura; Cortis, Cristina; Guidotti, Flavia; Bianco, Antonino; Tessitore, Antonio

2017-03-01

The session rating of perceived exertion (session-RPE) proved to be a valuable method to quantify the internal training load (ITL) in taekwondo. However, no study validated this method in youth taekwondo athletes performing different training sessions. Thus this study aimed at evaluating the reliability of the session-RPE to monitor the ITL of prepubescent taekwondo athletes during pre-competitive (PC) and competitive (C) training sessions. Five female (age: 12.0±0.7 y; height: 1.54±0.08 m; body mass: 48.8±7.3 kg) and four male (age: 12.0±0.8 yrs; height: 1.55±0.07 m; body mass: 47.3±5.3 kg) taekwondo athletes were monitored during 100 individual sessions (PC: N.=33; C: N.=67). The Edwards' HR method was used as reference measure of ITL; the CR-10 RPE scale was administered at 1- and 30-minutes from the end of each session. No difference for gender emerged. The ITLs of C (Edwards: 228±40 arbitrary units, AU) resulted higher than that of PC (192±26 AU; P=0.04). Although all training typologies and data collections achieved significant correlations between Edwards' and session-RPE methods, a large relationship (r =0.71, P<0.001) emerged only for PC sessions evaluated at 30 minutes of the recovery phases. Findings support coaches of prepubescent taekwondo athletes to successfully use session-RPE to monitor the ITL of different training typologies. However, PC training evaluated at 30 minutes of the recovery phase represents the best condition for a highly reliable ITL perception.

Activation of a C. elegans Antennapedia homologue in migrating cells controls their direction of migration.

PubMed

Salser, S J; Kenyon, C

1992-01-16

Anterior-posterior patterning in insects, vertebrates and nematodes involves members of conserved Antennapedia-class homeobox gene clusters (HOM-C) that are thought to give specific body regions their identities. The effects of these genes on region-specific body structures have been described extensively, particularly in Drosophila, but little is known about how HOM-C genes affect the behaviours of cells that migrate into their domains of function. In Caenorhabditis elegans, the Antennapedia-like HOM-C gene mab-5 not only specifies postembryonic fates of cells in a posterior body region, but also influences the migration of mesodermal and neural cells that move through this region. Here we show that as one neuroblast migrates into this posterior region, it switches on mab-5 gene expression; mab-5 then acts as a developmental switch to control the migratory behaviour of the neuroblast descendants. HOM-C genes can therefore not only direct region-specific patterns of cell division and differentiation, but can also act within migrating cells to programme region-specific migratory behaviour.

Steering cell migration by alternating blebs and actin-rich protrusions.

PubMed

Diz-Muñoz, Alba; Romanczuk, Pawel; Yu, Weimiao; Bergert, Martin; Ivanovitch, Kenzo; Salbreux, Guillaume; Heisenberg, Carl-Philipp; Paluch, Ewa K

2016-09-02

High directional persistence is often assumed to enhance the efficiency of chemotactic migration. Yet, cells in vivo usually display meandering trajectories with relatively low directional persistence, and the control and function of directional persistence during cell migration in three-dimensional environments are poorly understood. Here, we use mesendoderm progenitors migrating during zebrafish gastrulation as a model system to investigate the control of directional persistence during migration in vivo. We show that progenitor cells alternate persistent run phases with tumble phases that result in cell reorientation. Runs are characterized by the formation of directed actin-rich protrusions and tumbles by enhanced blebbing. Increasing the proportion of actin-rich protrusions or blebs leads to longer or shorter run phases, respectively. Importantly, both reducing and increasing run phases result in larger spatial dispersion of the cells, indicative of reduced migration precision. A physical model quantitatively recapitulating the migratory behavior of mesendoderm progenitors indicates that the ratio of tumbling to run times, and thus the specific degree of directional persistence of migration, are critical for optimizing migration precision. Together, our experiments and model provide mechanistic insight into the control of migration directionality for cells moving in three-dimensional environments that combine different protrusion types, whereby the proportion of blebs to actin-rich protrusions determines the directional persistence and precision of movement by regulating the ratio of tumbling to run times.

Using Single-Protein Tracking to Study Cell Migration.

PubMed

Orré, Thomas; Mehidi, Amine; Massou, Sophie; Rossier, Olivier; Giannone, Grégory

2018-01-01

To get a complete understanding of cell migration, it is critical to study its orchestration at the molecular level. Since the recent developments in single-molecule imaging, it is now possible to study molecular phenomena at the single-molecule level inside living cells. In this chapter, we describe how such approaches have been and can be used to decipher molecular mechanisms involved in cell migration.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PubMed

Wistow, Graeme; Bernstein, Steven L; Wyatt, M Keith; Fariss, Robert N; Behal, Amita; Touchman, Jeffrey W; Bouffard, Gerald; Smith, Don; Peterson, Katherine

2002-06-15

The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Automated Tracking of Cell Migration with Rapid Data Analysis.

PubMed

DuChez, Brian J

2017-09-01

Cell migration is essential for many biological processes including development, wound healing, and metastasis. However, studying cell migration often requires the time-consuming and labor-intensive task of manually tracking cells. To accelerate the task of obtaining coordinate positions of migrating cells, we have developed a graphical user interface (GUI) capable of automating the tracking of fluorescently labeled nuclei. This GUI provides an intuitive user interface that makes automated tracking accessible to researchers with no image-processing experience or familiarity with particle-tracking approaches. Using this GUI, users can interactively determine a minimum of four parameters to identify fluorescently labeled cells and automate acquisition of cell trajectories. Additional features allow for batch processing of numerous time-lapse images, curation of unwanted tracks, and subsequent statistical analysis of tracked cells. Statistical outputs allow users to evaluate migratory phenotypes, including cell speed, distance, displacement, and persistence, as well as measures of directional movement, such as forward migration index (FMI) and angular displacement. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

Nucleus and nucleus-cytoskeleton connections in 3D cell migration

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

Liu, Lingling, E-mail: liulingling2012@163.com; Luo, Qing, E-mail: qing.luo@cqu.edu.cn; Sun, Jinghui, E-mail: sunjhemail@163.com

Cell migration plays an important role in many physiological and pathological settings, ranging from embryonic development to cancer metastasis. Currently, accumulating data suggest that cells migrating in three-dimensional (3D) environments show well-defined differences compared to their well-established two-dimensional (2D) counterparts. During 3D migration, the cell body and nucleus must deform to allow cellular passage through the available spaces, and the deformability of the relatively rigid nucleus may constitute a limiting step. Here, we highlight the key evidence regarding the role of the nuclear mechanics in 3D migration, including the molecular components that govern the stiffness of the nucleus and reviewmore » how the nuclear dynamics are connected to and controlled by cytoskeleton-based migration machinery. Intriguingly, nuclear movement must be coordinated with the cytoskeletal dynamics at the leading and trailing edges, which in turn impact the cytoplasmic dynamics that affect the migration efficiency. Thus, we suggest that alterations in the nuclear structure may facilitate cellular reorganizations that are necessary for efficient migration. - Graphical abstract: Schematic representations of a cell migrating on a 2D substrate and a cell migrating in a 3D extracellular matrix environment. (A) Nucleus-cytoskeleton connections are essential to 3D migration. Mechanical signals are transduced by integrins at the cell surface and channeled to cytoskeletal proteins, which generates prestress. The nucleus-cytoskeleton connections can either act as a stable skeleton to anchor the nuclei or provide active force to move the nuclei. The LINC complex is responsible for the nucleo-cytoskeletal coupling. Nesprins connect the cytoskeletal proteins to the inner nuclear membrane proteins SUN1 and SUN2. The SUN proteins connect to the lamins that form the lamina, which attaches to the chromatin. This physical connectivity transmits the mechanical signals from

Neuronal cell migration in C. elegans: regulation of Hox gene expression and cell position.

PubMed

Harris, J; Honigberg, L; Robinson, N; Kenyon, C

1996-10-01

In C. elegans, the Hox gene mab-5, which specifies the fates of cells in the posterior body region, has been shown to direct the migrations of certain cells within its domain of function. mab-5 expression switches on in the neuroblast QL as it migrates into the posterior body region. mab-5 activity is then required for the descendants of QL to migrate to posterior rather than anterior positions. What information activates Hox gene expression during this cell migration? How are these cells subsequently guided to their final positions? We address these questions by describing four genes, egl-20, mig-14, mig-1 and lin-17, that are required to activate expression of mab-5 during migration of the QL neuroblast. We find that two of these genes, egl-20 and mig-14, also act in a mab-5-independent way to determine the final stopping points of the migrating Q descendants. The Q descendants do not migrate toward any obvious physical targets in wild-type or mutant animals. Therefore, these genes appear to be part of a system that positions the migrating Q descendants along the anteroposterior axis.

The construct validity of session RPE during an intensive camp in young male Karate athletes.

PubMed

Padulo, Johnny; Chaabène, Helmi; Tabben, Montassar; Haddad, Monoem; Gevat, Cecilia; Vando, Stefano; Maurino, Lucio; Chaouachi, Anis; Chamari, Karim

2014-04-01

the aim of this study was to assess the validity of the session rating of perceived exertion (RPE) method and two objective HR-based methods for quantifying karate's training load (TL) in young Karatekas. eleven athletes (age 12.50±1.84 years) participated in this study. The training period/camp was performed on 5 consecutive days with two training session (s) per-day (d). Construct validity of RPE method in young Karate athletes, was studied by correlation analysis between RPE session's training load and both Edwards and Banister's training impulse score' method. significant relationship was found between inter-day (n-11 × d-5 × s-2 = 110) sessions RPE and Edwards (r values from 0.84 to 0.92 p < 0.001) and Banister's (r values from 0.84 to 0.97 p < 0.001), respectively. this study showed that session-RPE can be considered a valid method for quantifying karate's training load in young karate athletes.

Rho GTPases and Regulation of Cell Migration and Polarization in Human Corneal Epithelial Cells

PubMed Central

Hou, Aihua; Toh, Li Xian; Gan, Kah Hui; Lee, Khee Jin Ryan; Manser, Edward; Tong, Louis

2013-01-01

Purpose Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. Methods Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. Results Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. Conclusion Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells. PMID:24130842

Cerium migration during PEM fuel cell assembly and operation

DOE PAGES

Baker, Andrew M.; Torraco, Dennis; Judge, Elizabeth J.; ...

2015-09-14

Cerium migration between PEM fuel cell components is influenced by potential-driven mobility, ionic diffusion, and gradients in water content. These factors were investigated in ex situ experiments and in operating fuel cells. Potential-induced migration was measured ex situ in hydrated window cells. Cerium-containing MEAs were also fabricated and tested under ASTs. MEA disassembly and subsequent XRF analysis were used to observe rapid cerium migration during cell assembly and operation. During MEA hot pressing, humidification, and low RH operation at OCV, ionic diffusion causes uniform migration from the membrane into the catalyst layers. During high RH operation at OCV, in-plane ceriummore » gradients arise due to variations in water content. These gradients may diminish the scavenging efficacy of cerium by reducing its proximity to generated radicals.« less

Cancer cell motility: lessons from migration in confined spaces

PubMed Central

Paul, Colin D.; Mistriotis, Panagiotis; Konstantopoulos, Konstantinos

2017-01-01

Time-lapse, deep-tissue imaging made possible by advances in intravital microscopy has demonstrated the importance of tumour cell migration through confining tracks in vivo. These tracks may either be endogenous features of tissues or be created by tumour or tumour-associated cells. Importantly, migration mechanisms through confining microenvironments are not predicted by 2D migration assays. Engineered in vitro models have been used to delineate the mechanisms of cell motility through confining spaces encountered in vivo. Understanding cancer cell locomotion through physiologically relevant confining tracks could be useful in developing therapeutic strategies to combat metastasis. PMID:27909339

SOX15 regulates proliferation and migration of endometrial cancer cells.

PubMed

Rui, Xiaohui; Xu, Yun; Jiang, Xiping; Guo, Caixia; Jiang, Jingting

2017-10-31

The study aimed to investigate the effects of Sry-like high mobility group box 15 ( SOX15 ) on proliferation and migration of endometrial cancer (EC) cells. Immunohistochemistry (IHC) was applied to determine the expression of SOX15 in EC tissues and adjacent tissues. We used cell transfection method to construct the HEC-1-A and Ishikawa cell lines with stable overexpression and low expression SOX15 Reverse-transcription quantitative real-time PCR (RT-qPCR) and Western blot were performed to examine expression of SOX15 mRNA and SOX15 protein, respectively. By conducting a series of cell proliferation assay and migration assay, we analyzed the influence of SOX15 overexpression or low expression on EC cell proliferation and migration. The expression of SOX15 mRNA and protein in EC tissues was significantly lower than that in adjacent tissues. After lentivirus-transfecting SOX15 , the expression level of SOX15 mRNA and protein was significantly increased in cells of SOX15 group, and decreased in sh- SOX15 group. Overexpression of SOX15 could suppress cell proliferation, while down-regulation of SOX15 increased cell proliferation. Flow cytometry results indicated that overexpression of SOX15 induced the ratio of cell-cycle arrest in G 1 stage. In addition, Transwell migration assay results showed that SOX15 overexpression significantly inhibited cell migration, and also down-regulation of SOX15 promoted the migration. As a whole, SOX15 could regulate the proliferation and migration of EC cells and up- regulation of SOX15 could be valuable for EC treatment. © 2017 The Author(s).

Alignment of cell division axes in directed epithelial cell migration

NASA Astrophysics Data System (ADS)

Marel, Anna-Kristina; Podewitz, Nils; Zorn, Matthias; Oskar Rädler, Joachim; Elgeti, Jens

2014-11-01

Cell division is an essential dynamic event in tissue remodeling during wound healing, cancer and embryogenesis. In collective migration, tensile stresses affect cell shape and polarity, hence, the orientation of the cell division axis is expected to depend on cellular flow patterns. Here, we study the degree of orientation of cell division axes in migrating and resting epithelial cell sheets. We use microstructured channels to create a defined scenario of directed cell invasion and compare this situation to resting but proliferating cell monolayers. In experiments, we find a strong alignment of the axis due to directed flow while resting sheets show very weak global order, but local flow gradients still correlate strongly with the cell division axis. We compare experimental results with a previously published mesoscopic particle based simulation model. Most of the observed effects are reproduced by the simulations.

The Golgi in Cell Migration: Regulation by Signal Transduction and Its Implications for Cancer Cell Metastasis

PubMed Central

Millarte, Valentina; Farhan, Hesso

2012-01-01

Migration and invasion are fundamental features of metastatic cancer cells. The Golgi apparatus, an organelle involved in posttranslational modification and sorting of proteins, is widely accepted to regulate directional cell migration. In addition, mounting evidence suggests that the Golgi is a hub for different signaling pathways. In this paper we will give an overview on how polarized secretion and microtubule nucleation at the Golgi regulate directional cell migration. We will review different signaling pathways that signal to and from the Golgi. Finally, we will discuss how these signaling pathways regulate the role of the Golgi in cell migration and invasion. We propose that by identifying regulators of the Golgi, we might be able to uncover unappreciated modulators of cell migration. Uncovering the regulatory network that orchestrates cell migration is of fundamental importance for the development of new therapeutic strategies against cancer cell metastasis. PMID:22623902

3D printing of biomimetic microstructures for cancer cell migration.

PubMed

Huang, Tina Qing; Qu, Xin; Liu, Justin; Chen, Shaochen

2014-02-01

To understand the physical behavior and migration of cancer cells, a 3D in vitro micro-chip in hydrogel was created using 3D projection printing. The micro-chip has a honeycomb branched structure, aiming to mimic 3D vascular morphology to test, monitor, and analyze differences in the behavior of cancer cells (i.e. HeLa) vs. non-cancerous cell lines (i.e. 10 T1/2). The 3D Projection Printing system can fabricate complex structures in seconds from user-created designs. The fabricated microstructures have three different channel widths of 25, 45, and 120 microns wide to reflect a range of blood vessel diameters. HeLa and 10 T1/2 cells seeded within the micro-chip were then analyzed for morphology and cell migration speed. 10 T1/2 cells exhibited greater changes in morphology due to channel size width than HeLa cells; however, channel width had a limited effect on 10 T1/2 cell migration while HeLa cancer cell migration increased as channel width decreased. This physiologically relevant 3D cancer tissue model has the potential to be a powerful tool for future drug discoveries and cancer migration studies.

Engineered Models of Confined Cell Migration

PubMed Central

Paul, Colin D.; Hung, Wei-Chien; Wirtz, Denis; Konstantopoulos, Konstantinos

2017-01-01

Cells in the body are physically confined by neighboring cells, tissues, and the extracellular matrix. Although physical confinement modulates intracellular signaling and the underlying mechanisms of cell migration, it is difficult to study in vivo. Furthermore, traditional two-dimensional cell migration assays do not recapitulate the complex topographies found in the body. Therefore, a number of experimental in vitro models that confine and impose forces on cells in well-defined microenvironments have been engineered. We describe the design and use of microfluidic microchannel devices, grooved substrates, micropatterned lines, vertical confinement devices, patterned hydrogels, and micropipette aspiration assays for studying cell responses to confinement. Use of these devices has enabled the delineation of changes in cytoskeletal reorganization, cell–substrate adhesions, intracellular signaling, nuclear shape, and gene expression that result from physical confinement. These assays and the physiologically relevant signaling pathways that have been elucidated are beginning to have a translational and clinical impact. PMID:27420571

Overexpression of Selenoprotein SelK in BGC-823 Cells Inhibits Cell Adhesion and Migration.

PubMed

Ben, S B; Peng, B; Wang, G C; Li, C; Gu, H F; Jiang, H; Meng, X L; Lee, B J; Chen, C L

2015-10-01

Effects of human selenoprotein SelK on the adhesion and migration ability of human gastric cancer BGC-823 cells using Matrigel adhesion and transwell migration assays, respectively, were investigated in this study. The Matrigel adhesion ability of BGC-823 cells that overexpressed SelK declined extremely significantly (p < 0.01) compared with that of the cells not expressing the protein. The migration ability of BGC-823 cells that overexpressed SelK also declined extremely significantly (p < 0.01). On the other hand, the Matrigel adhesion ability and migration ability of the cells that overexpressed C-terminally truncated SelK did not decline significantly. The Matrigel adhesion ability and migration ability of human embryonic kidney HEK-293 cells that overexpressed SelK did not show significant change (p > 0.05) with the cells that overexpressed the C-terminally truncated protein. In addition to the effect on Matrigel adhesion and migration, the overexpression of SelK also caused a loss in cell viability (as measured by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) colorimetric assay) and induced apoptosis as shown by confocal microscopy and flow cytometry. The cytosolic free Ca2+ level of these cells was significantly increased as detected by flow cytometry. But the overexpression of SelK in HEK-293 cells caused neither significant loss in cell viability nor apoptosis induction. Only the elevation of cytosolic free Ca2+ level in these cells was significant. Taken together, the results suggest that the overexpression of SelK can inhibit human cancer cell Matrigel adhesion and migration and cause both the loss in cell viability and induction of apoptosis. The release of intracellular Ca2+ from the endoplasmic reticulum might be a mechanism whereby the protein exerted its impact. Furthermore, only the full-length protein, but not C-terminally truncated form, was capable of producing such impact. The embryonic cells were not influenced by the

Guidance signalling regulates leading edge behaviour during collective cell migration of cardiac cells in Drosophila.

PubMed

Raza, Qanber; Jacobs, J Roger

2016-11-15

Collective cell migration is the coordinated movement of cells, which organize tissues during morphogenesis, repair and some cancers. The motile cell membrane of the advancing front in collective cell migration is termed the Leading Edge. The embryonic development of the vertebrate and Drosophila hearts are both characterized by the coordinated medial migration of a bilateral cluster of mesodermal cells. In Drosophila, the cardioblasts form cohesive bilateral rows that migrate collectively as a unit towards the dorsal midline to form the dorsal vessel. We have characterized the collective cell migration of cardioblasts as an in vivo quantitative model to study the behaviour of the Leading Edge. We investigated whether guidance signalling through Slit and Netrin pathways plays a role in cell migration during heart development. Through time-lapse imaging and quantitative assessment of migratory behaviour of the cardioblasts in loss-of-function mutants, we demonstrate that both Slit and Netrin mediated signals are autonomously and concomitantly required to maximize migration velocity, filopodial and lamellipodial activities. Additionally, we show that another Slit and Netrin receptor, Dscam1, the role of which during heart development was previously unknown, is required for both normal migration of cardioblasts and luminal expansion. Leading edge behaviour analysis revealed a dosage dependent genetic interaction between Slit and Netrin receptors suggesting that downstream signalling through these receptors converge on a common output that increases leading edge activity of the cardioblasts. Finally, we found that guidance signalling maintains the balance between epithelial and mesenchymal characteristics of the migrating cardioblasts. Copyright © 2016 Elsevier Inc. All rights reserved.

Cell Migration in Tissues: Explant Culture and Live Imaging.

PubMed

Staneva, Ralitza; Barbazan, Jorge; Simon, Anthony; Vignjevic, Danijela Matic; Krndija, Denis

2018-01-01

Cell migration is a process that ensures correct cell localization and function in development and homeostasis. In disease such as cancer, cells acquire an upregulated migratory capacity that leads to their dissemination throughout the body. Live imaging of cell migration allows for better understanding of cell behaviors in development, adult tissue homeostasis and disease. We have optimized live imaging procedures to track cell migration in adult murine tissue explants derived from: (1) healthy gut; (2) primary intestinal carcinoma; and (3) the liver, a common metastatic site. To track epithelial cell migration in the gut, we generated an inducible fluorescent reporter mouse, enabling us to visualize and track individual cells in unperturbed gut epithelium. To image intratumoral cancer cells, we use a spontaneous intestinal cancer model based on the activation of Notch1 and deletion of p53 in the mouse intestinal epithelium, which gives rise to aggressive carcinoma. Interaction of cancer cells with a metastatic niche, the mouse liver, is addressed using a liver colonization model. In summary, we describe a method for long-term 3D imaging of tissue explants by two-photon excitation microscopy. Explant culturing and imaging can help understand dynamic behavior of cells in homeostasis and disease, and would be applicable to various tissues.

ProBDNF inhibits collective migration and chemotaxis of rat Schwann cells.

PubMed

Ding, You-Quan; Li, Xuan-Yang; Xia, Guan-Nan; Ren, Hong-Yi; Zhou, Xin-Fu; Su, Bing-Yin; Qi, Jian-Guo

2016-10-01

Schwann cell migration, including collective migration and chemotaxis, is essential for the formation of coordinate interactions between Schwann cells and axons during peripheral nerve development and regeneration. Moreover, limited migration of Schwann cells imposed a serious obstacle on Schwann cell-astrocytes intermingling and spinal cord repair after Schwann cell transplantation into injured spinal cords. Recent studies have shown that mature brain-derived neurotrophic factor, a member of the neurotrophin family, inhibits Schwann cell migration. The precursor form of brain-derived neurotrophic factor, proBDNF, was expressed in the developing or degenerating peripheral nerves and the injured spinal cords. Since "the yin and yang of neurotrophin action" has been established as a common sense, proBDNF would be expected to promote Schwann cell migration. However, we found, in the present study, that exogenous proBDNF also inhibited in vitro collective migration and chemotaxis of RSC 96 cells, a spontaneously immortalized rat Schwann cell line. Moreover, proBDNF suppressed adhesion and spreading of those cells. At molecular level, proBDNF inhibits F-actin polymerization and focal adhesion dynamics in cultured RSC 96 cells. Therefore, our results suggested a special case against the classical opinion of "the yin and yang of neurotrophin action" and implied that proBDNF might modulate peripheral nerve development or regeneration and spinal cord repair through perturbing native or transplanted Schwann cell migration. Copyright © 2016 Elsevier Ltd. All rights reserved.

Bleb Expansion in Migrating Cells Depends on Supply of Membrane from Cell Surface Invaginations.

PubMed

Goudarzi, Mohammad; Tarbashevich, Katsiaryna; Mildner, Karina; Begemann, Isabell; Garcia, Jamie; Paksa, Azadeh; Reichman-Fried, Michal; Mahabaleshwar, Harsha; Blaser, Heiko; Hartwig, Johannes; Zeuschner, Dagmar; Galic, Milos; Bagnat, Michel; Betz, Timo; Raz, Erez

2017-12-04

Cell migration is essential for morphogenesis, organ formation, and homeostasis, with relevance for clinical conditions. The migration of primordial germ cells (PGCs) is a useful model for studying this process in the context of the developing embryo. Zebrafish PGC migration depends on the formation of cellular protrusions in form of blebs, a type of protrusion found in various cell types. Here we report on the mechanisms allowing the inflation of the membrane during bleb formation. We show that the rapid expansion of the protrusion depends on membrane invaginations that are localized preferentially at the cell front. The formation of these invaginations requires the function of Cdc42, and their unfolding allows bleb inflation and dynamic cell-shape changes performed by migrating cells. Inhibiting the formation and release of the invaginations strongly interfered with bleb formation, cell motility, and the ability of the cells to reach their target. Copyright © 2017 Elsevier Inc. All rights reserved.

HA-1077 inhibits cell migration/invasion of oral squamous cell carcinoma.

PubMed

Moreira Carboni, Simone de Sales Costa; Rodrigues Lima, Nathália Alves; Pinheiro, Nanci Mendes; Tavares-Murta, Beatriz Martins; Crema, Virgínia Oliveira

2015-10-01

Oral squamous cell carcinoma (OSCC) is the most malignant lesion occurring in the head and neck. The Rho-kinases (ROCKs), effectors of Rho proteins, are involved in actin cytoskeletal organization, cell migration, and maintenance cortex. The HA-1077 inhibits the ROCKs. This study aimed to evaluate the effect of treatment with HA-1077 on cell motility in SCC-4 cells, a cell line originating from human OSCC. F-actin of SCC-4 cells treated or not with HA-1077 (1, 50 and 100 μmol/l), and also HA-1077 50 μmol/l and/or inhibitors Y-27632 30 μmol/l was stained with rhodamine-conjugated phalloidin and analyzed by confocal microscopy. Approximately 1×10 cells/well, control and treated with HA-1077 (25, 50, and 100 μmol/l) were added to the migration plate assay. In addition, 1×10 cells/well, control and treated with HA-1077 50 μmol/l, were tested by invasion assays (plate coated with Matrigel). The inhibition of ROCKs with HA-1077 and/or Y-27632 leads to morphological changes, affecting the organization of the actin. The inhibitory effect of HA-1077 (P<0.0001) was dose dependent as the number of cells migrated at 100 μmol/l was statistically different: 25 μmol/l (P<0.0001) and 50 μmol/l (P<0.01). The number of cells treated with HA-1077 50 μmol/l decreased compared with control cells that invaded through Matrigel (P<0.0001). This study shows an inhibitory effect of HA-1077 on cell migration and invasion, suggesting that the use of HA-1077 can be a potential therapy for OSCC.

Mechanical guidance of collective cell migration and invasion

NASA Astrophysics Data System (ADS)

Trepat, Xavier

A broad range of biological processes such as morphogenesis, tissue regeneration, and cancer invasion depend on the collective migration of epithelial cells. Guidance of collective cell migration is commonly attributed to soluble or immobilized chemical gradients. I will present novel mechanisms of collective cellular guidance that are physical in origin rather than chemical. Firstly, I will focus on how the mechanical interaction between the tumor and its stroma guides cancer cell invasion. I will show that cancer associated fibroblasts exert a physical force on cancer cells that enables their collective invasion. In the second part of my talk I will focus on durotaxis, the ability of cells to follow gradients of extracellular matrix stiffness. Durotaxis is well established as a single cell phenomenon but whether it can direct the motion of cell collectives is unknown. I will show that durotaxis emerges in cell collectives even if isolated constituent cells are unable to durotax. Collective durotaxis applies to a broad variety of epithelial cell types and requires the action of myosin motors and the integrity of cell-cell junctions. Collective durotaxis is more efficient than any previous report of single cell durotaxis; it thus emerges as robust mechanism to direct collective cell migration in development and disease.eplace this text with your abstract.

The construct validity of session RPE during an intensive camp in young male Karate athletes

PubMed Central

Padulo, Johnny; Chaabène, Helmi; Tabben, Montassar; Haddad, Monoem; Gevat, Cecilia; Vando, Stefano; Maurino, Lucio; Chaouachi, Anis; Chamari, Karim

2014-01-01

Summary Background: the aim of this study was to assess the validity of the session rating of perceived exertion (RPE) method and two objective HR-based methods for quantifying karate’s training load (TL) in young Karatekas. Methods: eleven athletes (age 12.50±1.84 years) participated in this study. The training period/camp was performed on 5 consecutive days with two training session (s) per-day (d). Construct validity of RPE method in young Karate athletes, was studied by correlation analysis between RPE session’s training load and both Edwards and Banister’s training impulse score’ method. Results: significant relationship was found between inter-day (n-11 × d-5 × s-2 = 110) sessions RPE and Edwards (r values from 0.84 to 0.92 p < 0.001) and Banister’s (r values from 0.84 to 0.97 p < 0.001), respectively Conclusion: this study showed that session-RPE can be considered a valid method for quantifying karate’s training load in young karate athletes. PMID:25332921

Tissue stiffening coordinates morphogenesis by triggering collective cell migration in vivo.

PubMed

Barriga, Elias H; Franze, Kristian; Charras, Guillaume; Mayor, Roberto

2018-02-22

Collective cell migration is essential for morphogenesis, tissue remodelling and cancer invasion. In vivo, groups of cells move in an orchestrated way through tissues. This movement involves mechanical as well as molecular interactions between cells and their environment. While the role of molecular signals in collective cell migration is comparatively well understood, how tissue mechanics influence collective cell migration in vivo remains unknown. Here we investigated the importance of mechanical cues in the collective migration of the Xenopus laevis neural crest cells, an embryonic cell population whose migratory behaviour has been likened to cancer invasion. We found that, during morphogenesis, the head mesoderm underlying the cephalic neural crest stiffens. This stiffening initiates an epithelial-to-mesenchymal transition in neural crest cells and triggers their collective migration. To detect changes in their mechanical environment, neural crest cells use mechanosensation mediated by the integrin-vinculin-talin complex. By performing mechanical and molecular manipulations, we show that mesoderm stiffening is necessary and sufficient to trigger neural crest migration. Finally, we demonstrate that convergent extension of the mesoderm, which starts during gastrulation, leads to increased mesoderm stiffness by increasing the cell density underneath the neural crest. These results show that convergent extension of the mesoderm has a role as a mechanical coordinator of morphogenesis, and reveal a link between two apparently unconnected processes-gastrulation and neural crest migration-via changes in tissue mechanics. Overall, we demonstrate that changes in substrate stiffness can trigger collective cell migration by promoting epithelial-to-mesenchymal transition in vivo. More broadly, our results raise the idea that tissue mechanics combines with molecular effectors to coordinate morphogenesis.

The role of Exo70 in vascular smooth muscle cell migration.

PubMed

Ma, Wenqing; Wang, Yu; Yao, Xiaomeng; Xu, Zijian; An, Liguo; Yin, Miao

2016-01-01

As a key subunit of the exocyst complex, Exo70 has highly conserved sequence and is widely found in yeast, mammals, and plants. In yeast, Exo70 mediates the process of exocytosis and promotes anchoring and integration of vesicles with the plasma membrane. In mammalian cells, Exo70 is involved in maintaining cell morphology, cell migration, cell connection, mRNA splicing, and other physiological processes, as well as participating in exocytosis. However, Exo70's function in mammalian cells has yet to be fully recognized. In this paper, the expression of Exo70 and its role in cell migration were studied in a rat vascular smooth muscle cell line A7r5. Immunofluorescent analysis the expression of Exo70, α-actin, and tubulin in A7r5 cells showed a co-localization of Exo70 and α-actin, we treated the cells with cytochalasin B to depolymerize α-actin, in order to further confirm the co-localization of Exo70 and α-actin. We analyzed Exo70 co-localization with actin at the edge of migrating cells by wound-healing assay to establish whether Exo70 might play a role in cell migration. Next, we analyzed the migration and invasion ability of A7r5 cells before and after RNAi silencing through the wound healing assay and transwell assay. The mechanism of interaction between Exo70 and cytoskeleton can be clarified by the immunoprecipitation techniques and wound-healing assay. The results showed that Exo70 and α-actin were co-localized at the leading edge of migrating cells. The ability of A7r5 to undergo cell migration was decreased when Exo70 expression was silenced by RNAi. Reducing Exo70 expression in RNAi treated A7r5 cells significantly lowered the invasion and migration ability of these cells compared to the normal cells. These results indicate that Exo70 participates in the process of A7r5 cell migration. This research is importance for the study on the pathological process of vascular intimal hyperplasia, since it provides a new research direction for the treatment of

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement

PubMed Central

Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S.; Riahi, Reza; Wong, Pak Kin

2016-01-01

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters. PMID:26936382

Probing Leader Cells in Endothelial Collective Migration by Plasma Lithography Geometric Confinement.

PubMed

Yang, Yongliang; Jamilpour, Nima; Yao, Baoyin; Dean, Zachary S; Riahi, Reza; Wong, Pak Kin

2016-03-03

When blood vessels are injured, leader cells emerge in the endothelium to heal the wound and restore the vasculature integrity. The characteristics of leader cells during endothelial collective migration under diverse physiological conditions, however, are poorly understood. Here we investigate the regulation and function of endothelial leader cells by plasma lithography geometric confinement generated. Endothelial leader cells display an aggressive phenotype, connect to follower cells via peripheral actin cables and discontinuous adherens junctions, and lead migrating clusters near the leading edge. Time-lapse microscopy, immunostaining, and particle image velocimetry reveal that the density of leader cells and the speed of migrating clusters are tightly regulated in a wide range of geometric patterns. By challenging the cells with converging, diverging and competing patterns, we show that the density of leader cells correlates with the size and coherence of the migrating clusters. Collectively, our data provide evidence that leader cells control endothelial collective migration by regualting the migrating clusters.

Co-culture with Sertoli cells promotes proliferation and migration of umbilical cord mesenchymal stem cells

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

Zhang, Fenxi, E-mail: fxzhang0824@gmail.com; Hong, Yan; Liang, Wenmei

Highlights: Black-Right-Pointing-Pointer Co-culture of Sertoli cells (SCs) with human umbilical cord mesenchymal stem cells (UCMSCs). Black-Right-Pointing-Pointer Presence of SCs dramatically increased proliferation and migration of UCMSCs. Black-Right-Pointing-Pointer Presence of SCs stimulated expression of Mdm2, Akt, CDC2, Cyclin D, CXCR4, MAPKs. -- Abstract: Human umbilical cord mesenchymal stem cells (hUCMSCs) have been recently used in transplant therapy. The proliferation and migration of MSCs are the determinants of the efficiency of MSC transplant therapy. Sertoli cells are a kind of 'nurse' cells that support the development of sperm cells. Recent studies show that Sertoli cells promote proliferation of endothelial cells and neuralmore » stem cells in co-culture. We hypothesized that co-culture of UCMSCs with Sertoli cells may also promote proliferation and migration of UCMSCs. To examine this hypothesis, we isolated UCMSCs from human cords and Sertoli cells from mouse testes, and co-cultured them using a Transwell system. We found that UCMSCs exhibited strong proliferation ability and potential to differentiate to other cell lineages such as osteocytes and adipocytes. The presence of Sertoli cells in co-culture significantly enhanced the proliferation and migration potential of UCMSCs (P < 0.01). Moreover, these phenotypic changes were accompanied with upregulation of multiple genes involved in cell proliferation and migration including phospho-Akt, Mdm2, phospho-CDC2, Cyclin D1, Cyclin D3 as well as CXCR4, phospho-p44 MAPK and phospho-p38 MAPK. These findings indicate that Sertoli cells boost UCMSC proliferation and migration potential.« less

Impact of jamming on collective cell migration

NASA Astrophysics Data System (ADS)

Nnetu, Kenechukwu David; Knorr, Melanie; Pawlizak, Steve; Fuhs, Thomas; Zink, Mareike; KäS, Josef A.

2012-02-01

Multi-cellular migration plays an important role in physiological processes such as embryogenesis, cancer metastasis and tissue repair. During migration, single cells undergo cycles of extension, adhesion and retraction resulting in morphological changes. In a confluent monolayer, there are inter-cellular interactions and crowding, however, the impact of these interactions on the dynamics and elasticity of the monolayer at the multi-cellular and single cell level is not well understood. Here we study the dynamics of a confluent epithelial monolayer by simultaneously measuring cell motion at the multi-cellular and single cell level for various cell densities and tensile elasticity. At the multi-cellular level, the system exhibited spatial kinetic transitions from isotropic to anisotropic migration on long times and the velocity of the monolayer decreased with increasing cell density. Moreover, the dynamics was spatially and temporally heterogeneous. Interestingly, the dynamics was also heterogeneous in wound-healing assays and the correlation length was fitted by compressed exponential. On the single cell scale, we observed transient caging effects with increasing cage rearrangement times as the system age due to an increase in density. Also, the density dependent elastic modulus of the monolayer scaled as a weak power law. Together, these findings suggest that caging effects at the single cell level initiates a slow and heterogeneous dynamics at the multi-cellular level which is similar to the glassy dynamics of deformable colloidal systems.

Chemokine-Dependent pH Elevation at the Cell Front Sustains Polarity in Directionally Migrating Zebrafish Germ Cells.

PubMed

Tarbashevich, Katsiaryna; Reichman-Fried, Michal; Grimaldi, Cecilia; Raz, Erez

2015-04-20

Directional cell migration requires cell polarization with respect to the distribution of the guidance cue. Cell polarization often includes asymmetric distribution of response components as well as elements of the motility machinery. Importantly, the function and regulation of most of these molecules are known to be pH dependent. Intracellular pH gradients were shown to occur in certain cells migrating in vitro, but the functional relevance of such gradients for cell migration and for the response to directional cues, particularly in the intact organism, is currently unknown. In this study, we find that primordial germ cells migrating in the context of the developing embryo respond to the graded distribution of the chemokine Cxcl12 by establishing elevated intracellular pH at the cell front. We provide insight into the mechanisms by which a polar pH distribution contributes to efficient cell migration. Specifically, we show that Carbonic Anhydrase 15b, an enzyme controlling the pH in many cell types, including metastatic cancer cells, is expressed in migrating germ cells and is crucial for establishing and maintaining an asymmetric pH distribution within them. Reducing the level of the protein and thereby erasing the pH elevation at the cell front resulted in abnormal cell migration and impaired arrival at the target. The basis for the disrupted migration is found in the stringent requirement for pH conditions in the cell for regulating contractility, for the polarization of Rac1 activity, and hence for the formation of actin-rich structures at the leading edge of the migrating cells. Copyright © 2015 Elsevier Ltd. All rights reserved.

Platelets Inhibit Migration of Canine Osteosarcoma Cells.

PubMed

Bulla, S C; Badial, P R; Silva, R C; Lunsford, K; Bulla, C

2017-01-01

The interaction between platelets and tumour cells is important for tumour growth and metastasis. Thrombocytopenia or antiplatelet treatment negatively impact on cancer metastasis, demonstrating potentially important roles for platelets in tumour progression. To our knowledge, there is no information regarding the role of platelets in cancer progression in dogs. This study was designed to test whether canine platelets affected the migratory behaviour of three canine osteosarcoma cell lines and to give insights of molecular mechanisms. Intact platelets, platelet lysate and platelet releasate inhibited the migration of canine osteosarcoma cell lines. Addition of blood leucocytes to the platelet samples did not alter the inhibitory effect on migration. Platelet treatment also significantly downregulated the transcriptional levels of SNAI2 and TWIST1 genes. The interaction between canine platelets or molecules released during platelet activation and these tumour cell lines inhibits their migration, which suggests that canine platelets might antagonize metastasis of canine osteosarcoma. This effect is probably due to, at least in part, downregulation of genes related to epithelial-mesenchymal transition. Copyright © 2016. Published by Elsevier Ltd.

Flow and diffusion in channel-guided cell migration.

PubMed

Marel, Anna-Kristina; Zorn, Matthias; Klingner, Christoph; Wedlich-Söldner, Roland; Frey, Erwin; Rädler, Joachim O

2014-09-02

Collective migration of mechanically coupled cell layers is a notable feature of wound healing, embryonic development, and cancer progression. In confluent epithelial sheets, the dynamics have been found to be highly heterogeneous, exhibiting spontaneous formation of swirls, long-range correlations, and glass-like dynamic arrest as a function of cell density. In contrast, the flow-like properties of one-sided cell-sheet expansion in confining geometries are not well understood. Here, we studied the short- and long-term flow of Madin-Darby canine kidney (MDCK) cells as they moved through microchannels. Using single-cell tracking and particle image velocimetry (PIV), we found that a defined averaged stationary cell current emerged that exhibited a velocity gradient in the direction of migration and a plug-flow-like profile across the advancing sheet. The observed flow velocity can be decomposed into a constant term of directed cell migration and a diffusion-like contribution that increases with density gradient. The diffusive component is consistent with the cell-density profile and front propagation speed predicted by the Fisher-Kolmogorov equation. To connect diffusion-mediated transport to underlying cellular motility, we studied single-cell trajectories and occurrence of vorticity. We discovered that the directed large-scale cell flow altered fluctuations in cellular motion at short length scales: vorticity maps showed a reduced frequency of swirl formation in channel flow compared with resting sheets of equal cell density. Furthermore, under flow, single-cell trajectories showed persistent long-range, random-walk behavior superimposed on drift, whereas cells in resting tissue did not show significant displacements with respect to neighboring cells. Our work thus suggests that active cell migration manifests itself in an underlying, spatially uniform drift as well as in randomized bursts of short-range correlated motion that lead to a diffusion-mediated transport

Modeling collective cell migration in geometric confinement

NASA Astrophysics Data System (ADS)

Tarle, Victoria; Gauquelin, Estelle; Vedula, S. R. K.; D'Alessandro, Joseph; Lim, C. T.; Ladoux, Benoit; Gov, Nir S.

2017-06-01

Monolayer expansion has generated great interest as a model system to study collective cell migration. During such an expansion the culture front often develops ‘fingers’, which we have recently modeled using a proposed feedback between the curvature of the monolayer’s leading edge and the outward motility of the edge cells. We show that this model is able to explain the puzzling observed increase of collective cellular migration speed of a monolayer expanding into thin stripes, as well as describe the behavior within different confining geometries that were recently observed in experiments. These comparisons give support to the model and emphasize the role played by the edge cells and the edge shape during collective cell motion.

Modeling collective cell migration in geometric confinement.

PubMed

Tarle, Victoria; Gauquelin, Estelle; Vedula, S R K; D'Alessandro, Joseph; Lim, C T; Ladoux, Benoit; Gov, Nir S

2017-05-03

Monolayer expansion has generated great interest as a model system to study collective cell migration. During such an expansion the culture front often develops 'fingers', which we have recently modeled using a proposed feedback between the curvature of the monolayer's leading edge and the outward motility of the edge cells. We show that this model is able to explain the puzzling observed increase of collective cellular migration speed of a monolayer expanding into thin stripes, as well as describe the behavior within different confining geometries that were recently observed in experiments. These comparisons give support to the model and emphasize the role played by the edge cells and the edge shape during collective cell motion.

Tropomyosin isoform Tpm2.1 regulates collective and amoeboid cell migration and cell aggregation in breast epithelial cells.

PubMed

Shin, HyeRim; Kim, Dayoung; Helfman, David M

2017-11-10

Metastasis dissemination is the result of various processes including cell migration and cell aggregation. These processes involve alterations in the expression and organization of cytoskeletal and adhesion proteins in tumor cells. Alterations in actin filaments and their binding partners are known to be key players in metastasis. Downregulation of specific tropomyosin (Tpm) isoforms is a common characteristic of transformed cells. In this study, we examined the role of Tpm2.1 in non-transformed MCF10A breast epithelial cells in cell migration and cell aggregation, because this isoform is downregulated in primary and metastatic breast cancer as well as various breast cancer cell lines. Downregulation of Tpm2.1 using siRNA or shRNA resulted in retardation of collective cell migration but increase in single cell migration and invasion. Loss of Tpm2.1 is associated with enhanced actomyosin contractility and increased expression of E-cadherin and β-catenin. Furthermore, inhibition of Rho-associated kinase (ROCK) recovered collective cell migration in Tpm2.1-silenced cells. We also found that Tpm2.1-silenced cells formed more compacted spheroids and exhibited faster cell motility when spheroids were re-plated on 2D surfaces coated with fibronectin and collagen. When Tpm2.1 was downregulated, we observed a decrease in the level of AXL receptor tyrosine kinase, which may explain the increased levels of E-cadherin and β-catenin. These studies demonstrate that Tpm2.1 functions as an important regulator of cell migration and cell aggregation in breast epithelial cells. These findings suggest that downregulation of Tpm2.1 may play a critical role during tumor progression by facilitating the metastatic potential of tumor cells.

Migration-stimulating factor (MSF) is over-expressed in non-small cell lung cancer and promotes cell migration and invasion in A549 cells over-expressing MSF

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

Deng, Xuefeng, E-mail: dengxfdoctor@hotmail.com; Department of Cardio-thoracic Surgery, Affiliated Hospital of Academy of Military Medical Sciences; Ma, Qunfeng

Migration-stimulating factor (MSF), an oncofetal truncated isoform of fibronectin, is a potent stimulator of cell invasion. However, its distribution and motogenic role in non-small cell lung cancer (NSCLC) have never been identified. In this study, real-time PCR and immunohistochemical staining (IHC) were performed to detect MSF mRNA and protein levels in tumor tissues and matched adjacent tumor-free tissues. Furthermore, to examine the effect of MSF on invasiveness, MSF was upregulated in A549 cells. The invasiveness and viability of A549 cells were then determined using a transwell migration assay and the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) viability assays, respectively. The expression level ofmore » MSF in NSCLC tissue was markedly higher than in matched adjacent tumor-free tissue. Additionally, the level of MSF protein expression in stage III and IV NSCLC samples was higher than in stage I and II NSCLC samples. More importantly, we also demonstrated that migration and invasion of A549 cells increased substantially after upregulating MSF, although proliferation remained unchanged. Meanwhile, we found no correlation between increasing motility and invasiveness of MSF-overexpressing cells and expression levels and activities of matrix metalloprotease MMP-2 and MMP-9. Our current study shows that MSF plays a role in migration and invasion of A549 cells and suggests that MSF may be a potential biomarker of NSCLC progression. - Highlights: • MSF expression was upregulated in NSCLC and correlated with TNM stages. • MSF may be a new biomarker for NSCLC progression. • MSF promoted migration and invasion in A549 cells, independent of MMP-2/MMP-9 expression.« less

Collisions of deformable cells lead to collective migration

NASA Astrophysics Data System (ADS)

Aranson, Igor; Löber, Jakob; Ziebert, Falko

2015-03-01

Collective migration of eukaryotic cells plays a fundamental role in tissue growth, wound healing and immune response. The motion, arising spontaneously or in response to chemical and mechanical stimuli, is also important for understanding life-threatening pathologies, such as cancer and metastasis formation. We present a phase-field model to describe the movement of many self-organized, interacting cells. The model takes into account the main mechanisms of cell motility - actomyosin dynamics, as well as substrate-mediated and cell-cell adhesion. It predicts that collective cell migration emerges spontaneously as a result of inelastic collisions between neighboring cells: collisions lead to a mutual alignment of the cell velocities and to the formation of coherently-moving multi-cellular clusters. Small cell-to-cell adhesion, in turn, reduces the propensity for large-scale collective migration, while higher adhesion leads to the formation of moving bands. Our study provides valuable insight into biological processes associated with collective cell motility. J. L. acknowledges funding from the German Science Foundation (DFG) within the GRK 1558. F. Z. acknowledges funding from the German Science Foundation (DFG) via Project ZI 1232/2-1. I. S. A. was supported by the US Department of Energy (DOE), Office of.

Multidisciplinary approaches to understanding collective cell migration in developmental biology.

PubMed

Schumacher, Linus J; Kulesa, Paul M; McLennan, Rebecca; Baker, Ruth E; Maini, Philip K

2016-06-01

Mathematical models are becoming increasingly integrated with experimental efforts in the study of biological systems. Collective cell migration in developmental biology is a particularly fruitful application area for the development of theoretical models to predict the behaviour of complex multicellular systems with many interacting parts. In this context, mathematical models provide a tool to assess the consistency of experimental observations with testable mechanistic hypotheses. In this review, we showcase examples from recent years of multidisciplinary investigations of neural crest cell migration. The neural crest model system has been used to study how collective migration of cell populations is shaped by cell-cell interactions, cell-environmental interactions and heterogeneity between cells. The wide range of emergent behaviours exhibited by neural crest cells in different embryonal locations and in different organisms helps us chart out the spectrum of collective cell migration. At the same time, this diversity in migratory characteristics highlights the need to reconcile or unify the array of currently hypothesized mechanisms through the next generation of experimental data and generalized theoretical descriptions. © 2016 The Authors.

Interstitial flow influences direction of tumor cell migration through competing mechanisms

PubMed Central

Polacheck, William J.; Charest, Joseph L.; Kamm, Roger D.

2011-01-01

Interstitial flow is the convective transport of fluid through tissue extracellular matrix. This creeping fluid flow has been shown to affect the morphology and migration of cells such as fibroblasts, cancer cells, endothelial cells, and mesenchymal stem cells. A microfluidic cell culture system was designed to apply stable pressure gradients and fluid flow and allow direct visualization of transient responses of cells seeded in a 3D collagen type I scaffold. We used this system to examine the effects of interstitial flow on cancer cell morphology and migration and to extend previous studies showing that interstitial flow increases the metastatic potential of MDA-MB-435S melanoma cells [Shields J, et al. (2007) Cancer Cell 11:526–538]. Using a breast carcinoma line (MDA-MB-231) we also observed cell migration along streamlines in the presence of flow; however, we further demonstrated that the strength of the flow as well as the cell density determined directional bias of migration along the streamline. In particular, we found that cells either at high seeding density or with the CCR-7 receptor inhibited migration against, rather than with the flow. We provide further evidence that CCR7-dependent autologous chemotaxis is the mechanism that leads to migration with the flow, but also demonstrate a competing CCR7-independent mechanism that causes migration against the flow. Data from experiments investigating the effects of cell concentration, interstitial flow rate, receptor activity, and focal adhesion kinase phosphorylation support our hypothesis that the competing stimulus is integrin mediated. This mechanism may play an important role in development of metastatic disease. PMID:21690404

Fibrin glue inhibits migration of ocular surface epithelial cells

PubMed Central

Yeung, A M; Faraj, L A; McIntosh, O D; Dhillon, V K; Dua, H S

2016-01-01

Purpose Fibrin glue has been used successfully in numerous ophthalmic surgical procedures. Recently, fibrin glue has been used in limbal stem cell transplantation to reduce both operative time and to negate the need for sutures. The aim of this study was to determine the effects of fibrin glue on epithelial cell migration in vitro. Methods Corneoscleral rims were split to retain the epithelial layer, Bowman's layer, and anterior stroma. Rims were cut into eight equal-sized pieces and were placed directly on culture plates or affixed with fibrin glue. Rims were maintained in culture for 25 days and epithelial cell growth was monitored. Cells were photographed to measure area or growth and immunofluorescence staining of explants for fibrin was performed. Results Explants that were glued demonstrated significantly delayed epithelial cell growth and migration as compared with explants without glue. By day 16, all fibrin glue had dissolved and coincided with onset of cell growth from glued explants. Cell growth commenced between days 3 and 4 for control explants without glue and around days 14–16 for explants with fibrin glue. Conclusions Fibrin glue delays epithelial cell migration by acting as a physical barrier and can potentially interfere with explant-derived limbal epithelial cell migration on to the corneal surface. We propose that glue should be used to attach the conjunctival frill of the limbal explant but care should be taken to ensure that the glue does not wrap around the explant if used to secure the explant as well. Strategic use of glue, to attach the recessed conjunctiva, can be advantageous in delaying conjunctival cell migration and reducing the need for sequential sector conjunctival epitheliectomy. PMID:27367746

Intracellular pH gradients in migrating cells.

PubMed

Martin, Christine; Pedersen, Stine F; Schwab, Albrecht; Stock, Christian

2011-03-01

Cell polarization along the axis of movement is required for migration. The localization of proteins and regulators of the migratory machinery to either the cell front or its rear results in a spatial asymmetry enabling cells to simultaneously coordinate cell protrusion and retraction. Protons might function as such unevenly distributed regulators as they modulate the interaction of focal adhesion proteins and components of the cytoskeleton in vitro. However, an intracellular pH (pH(i)) gradient reflecting a spatial asymmetry of protons has not been shown so far. One major regulator of pH(i), the Na(+)/H(+) exchanger NHE1, is essential for cell migration and accumulates at the cell front. Here, we test the hypothesis that the uneven distribution of NHE1 activity creates a pH(i) gradient in migrating cells. Using the pH-sensitive fluorescent dye BCECF, pH(i) was measured in five cell lines (MV3, B16V, NIH3T3, MDCK-F1, EA.hy926) along the axis of movement. Differences in pH(i) between the front and the rear end (ΔpH(i) front-rear) were present in all cell lines, and inhibition of NHE1 either with HOE642 or by absence of extracellular Na(+) caused the pH(i) gradient to flatten or disappear. In conclusion, pH(i) gradients established by NHE1 activity exist along the axis of movement.

Retinal pigment epithelium culture;a potential source of retinal stem cells.

PubMed

Akrami, Hassan; Soheili, Zahra-Soheila; Khalooghi, Keynoush; Ahmadieh, Hamid; Rezaie-Kanavi, Mojgan; Samiei, Shahram; Davari, Malihe; Ghaderi, Shima; Sanie-Jahromi, Fatemeh

2009-07-01

To establish human retinal pigment epithelial (RPE) cell culture as a source for cell replacement therapy in ocular diseases. Human cadaver globes were used to isolate RPE cells. Each globe was cut into several pieces of a few millimeters in size. After removing the sclera and choroid, remaining tissues were washed in phosphate buffer saline and RPE cells were isolated using dispase enzyme solution and cultured in Dulbecco's Modified Eagle's Medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Primary cultures of RPE cells were established and spheroid colonies related to progenitor/stem cells developed in a number of cultures. The colonies included purely pigmented or mixed pigmented and non-pigmented cells. After multiple cellular passages, several types of photoreceptors and neural-like cells were detected morphologically. Cellular plasticity in RPE cell cultures revealed promising results in terms of generation of stem/progenitor cells from human RPE cells. Whether the spheroids and neural-like retinal cells were directly derived from retinal stem cells or offspring of trans-differentiating or de-differentiating RPE cells remains to be answered.

Time-lapse cinematography of the capillary tube cell migration inhibition test.

PubMed

Bray, M A

1980-01-01

The kinetics of human and guinea pig cell migration inhibition have been studied using time-lapse cinematography of cells migrating from capillary tubes. Guinea pig and human cells exhibit markedly different kinetics in the absence of inhibitors. Specific antigen causes a dose-related inhibition of migration for up to 60 h using guinea pig cells and a peak of inhibition after 18 h using the human leucocyte system. The timing of measurement of maximum activity more critical for the latter test. The kinetics of lymphokine generation have been examined and the migration inhibitory activity of the plant mitogen (PHA), a Kurloff cell product and a continuous cell line supernatant have been compared with the inhibitory profiles of lymphokine preparations and specific antigen.

Retinal pigment epithelial cell multinucleation in the aging eye - a mechanism to repair damage and maintain homoeostasis.

PubMed

Chen, Mei; Rajapakse, Dinusha; Fraczek, Monika; Luo, Chang; Forrester, John V; Xu, Heping

2016-06-01

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age-related retinal degenerative disorders particularly age-related macular degeneration. During aging RPE cells decline in number, suggesting an age-dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose-dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted 'postmitotic' status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long-standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age-related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells. © 2016 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Transplantation of reprogrammed embryonic stem cells improves visual function in a mouse model for retinitis pigmentosa.

PubMed

Wang, Nan-Kai; Tosi, Joaquin; Kasanuki, Jennifer Mie; Chou, Chai Lin; Kong, Jian; Parmalee, Nancy; Wert, Katherine J; Allikmets, Rando; Lai, Chi-Chun; Chien, Chung-Liang; Nagasaki, Takayuki; Lin, Chyuan-Sheng; Tsang, Stephen H

2010-04-27

To study whether C57BL/6J-Tyr/J (C2J) mouse embryonic stem (ES) cells can differentiate into retinal pigment epithelial (RPE) cells in vitro and then restore retinal function in a model for retinitis pigmentosa: Rpe65/Rpe65 C57BL6 mice. Yellow fluorescent protein (YFP)-labeled C2J ES cells were induced to differentiate into RPE-like structures on PA6 feeders. RPE-specific markers are expressed from differentiated cells in vitro. After differentiation, ES cell-derived RPE-like cells were transplanted into the subretinal space of postnatal day 5 Rpe65/Rpe65 mice. Live imaging of YFP-labeled C2J ES cells demonstrated survival of the graft. Electroretinograms (ERGs) were performed on transplanted mice to evaluate the functional outcome of transplantation. RPE-like cells derived from ES cells sequentially express multiple RPE-specific markers. After transplantation, YFP-labeled cells can be tracked with live imaging for as long as 7 months. Although more than half of the mice were complicated with retinal detachments or tumor development, one fourth of the mice showed increased electroretinogram responses in the transplanted eyes. Rpe65/Rpe65 mice transplanted with RPE-like cells showed significant visual recovery during a 7-month period, whereas those injected with saline, PA6 feeders, or undifferentiated ES cells showed no rescue. ES cells can differentiate, morphologically, and functionally, into RPE-like cells. Based on these findings, differentiated ES cells have the potential for the development of new therapeutic approaches for RPE-specific diseases such as certain forms of retinitis pigmentosa and macular degeneration. Nevertheless, stringent control of retinal detachment and teratoma development will be necessary before initiation of treatment trials.

Induced Retro-Differentiation of Human Retinal Pigment Epithelial Cells on PolyHEMA.

PubMed

Nazemroaya, Fatemeh; Soheili, Zahra-Soheila; Samiei, Shahram; Deezagi, Abdolkhalegh; Ahmadieh, Hamid; Davari, Malihe; Heidari, Razeih; Bagheri, Abouzar; Darvishalipour-Astaneh, Shamila

2017-10-01

Retinal pigment epithelium (RPE) cells represent a great potential to rescue degenerated cells of the damaged retina. Activation of the virtually plastic properties of RPE cells may aid in recovery of retinal degenerative disorders without the need for entire RPE sheet transplantation. Poly (2-hydroxyethyl methacrylate)(PolyHEMA) is one of the most important hydrogels in the biomaterials world. This hydrophobic polymer does not normally support attachment of mammalian cells. In the current study we investigated the effect of PolyHEMA as a cell culture substrate on the growth, differentiation, and plasticity of hRPE cells. hRPE cells were isolated from neonatal human globes and cultured on PolyHEMA and polystyrene substrates (as controls) in 24-well culture plates. DMEM/F12 was supplemented with 10% fetal bovine serum (FBS) and/or 30% human amniotic fluid (HAF) for cultured cells on polystyrene and PolyHEMA coated vessels. Morphology, rate of cell proliferation and cell death, MTT assay, immunocytochemistry and Real-Time RT-PCR were performed to investigate the effects of PolyHEMA on the growth and differentiation of cultured hRPE cells. Proliferation rate of the cells that had been cultured on PolyHEMA was reduced; PolyHEMA did not induce cell death in the hRPE cultures. hRPE cells cultured on PolyHEMA formed many giant spheroid colonies. The giant colonies were re-cultured and the presence of retinal progenitor markers and markers of hRPE cells were detected in cell cultures on PolyHEMA. PolyHEMA seems to be promising for both maintenance and de-differentiation of hRPE cells and expansion of the retinal progenitor cells from the cultures that are originated from hRPE cells. J. Cell. Biochem. 118: 3080-3089, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments

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

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells’ functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells’ (RPC) markers were studied using immunocytochemistry (ICC). Emergedmore » cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. - Highlights: • Isolation of a spontaneously generated retinal pigmented epithelium cell line is reported. • The cells express some of the retinal progenitor cell markers in addition to the RPE markers. • The aforesaid cell line is highly transfecable and considerably infectable by AAV2. • These results confirm the great RPE plasticity and its invaluable potential in research

Endogenous cannabinoid receptor ligand induces the migration of human natural killer cells.

PubMed

Kishimoto, Seishi; Muramatsu, Mayumi; Gokoh, Maiko; Oka, Saori; Waku, Keizo; Sugiura, Takayuki

2005-02-01

2-Arachidonoylglycerol is an endogenous ligand for the cannabinoid receptors (CB1 and CB2). Evidence is gradually accumulating which shows that 2-arachidonoylglycerol plays important physiological roles in several mammalian tissues and cells, yet the details remain ambiguous. In this study, we first examined the effects of 2-arachidonoylglycerol on the motility of human natural killer cells. We found that 2-arachidonoylglycerol induces the migration of KHYG-1 cells (a natural killer leukemia cell line) and human peripheral blood natural killer cells. The migration of natural killer cells induced by 2-arachidonoylglycerol was abolished by treating the cells with SR144528, a CB2 receptor antagonist, suggesting that the CB2 receptor is involved in the 2-arachidonoylglycerol-induced migration. In contrast to 2-arachidonoylglycerol, anandamide, another endogenous cannabinoid receptor ligand, did not induce the migration. Delta9-tetrahydrocannabinol, a major psychoactive constituent of marijuana, also failed to induce the migration; instead, the addition of delta9-tetrahydrocannabinol together with 2-arachidonoylglycerol abolished the migration induced by 2-arachidonoylglycerol. It is conceivable that the endogenous ligand for the cannabinoid receptor, that is, 2-arachidonoylglycerol, affects natural killer cell functions such as migration, thereby contributing to the host-defense mechanism against infectious viruses and tumor cells.

Small-molecule-directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells.

PubMed

Maruotti, Julien; Sripathi, Srinivas R; Bharti, Kapil; Fuller, John; Wahlin, Karl J; Ranganathan, Vinod; Sluch, Valentin M; Berlinicke, Cynthia A; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z; Bhutto, Imran; Lutty, Gerard A; Zack, Donald J

2015-09-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule-only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE.

Small-molecule–directed, efficient generation of retinal pigment epithelium from human pluripotent stem cells

PubMed Central

Maruotti, Julien; Sripathi, Srinivas R.; Bharti, Kapil; Fuller, John; Wahlin, Karl J.; Ranganathan, Vinod; Sluch, Valentin M.; Berlinicke, Cynthia A.; Davis, Janine; Kim, Catherine; Zhao, Lijun; Wan, Jun; Qian, Jiang; Corneo, Barbara; Temple, Sally; Dubey, Ramin; Olenyuk, Bogdan Z.; Bhutto, Imran; Lutty, Gerard A.; Zack, Donald J.

2015-01-01

Age-related macular degeneration (AMD) is associated with dysfunction and death of retinal pigment epithelial (RPE) cells. Cell-based approaches using RPE-like cells derived from human pluripotent stem cells (hPSCs) are being developed for AMD treatment. However, most efficient RPE differentiation protocols rely on complex, stepwise treatments and addition of growth factors, whereas small-molecule–only approaches developed to date display reduced yields. To identify new compounds that promote RPE differentiation, we developed and performed a high-throughput quantitative PCR screen complemented by a novel orthogonal human induced pluripotent stem cell (hiPSC)-based RPE reporter assay. Chetomin, an inhibitor of hypoxia-inducible factors, was found to strongly increase RPE differentiation; combination with nicotinamide resulted in conversion of over one-half of the differentiating cells into RPE. Single passage of the whole culture yielded a highly pure hPSC-RPE cell population that displayed many of the morphological, molecular, and functional characteristics of native RPE. PMID:26269569

Decorin inhibits cell migration through a process requiring its glycosaminoglycan side chain.

PubMed

Merle, B; Durussel, L; Delmas, P D; Clézardin, P

1999-12-01

Several studies overwhelmingly support the notion that decorin (DCN) is involved in matrix assembly, and in the control of cell adhesion and proliferation. However, nothing is known about the role of DCN during cell migration. Cell migration is a tightly regulated process which requires both adhesion (at the leading edge of the cell) and de-adhesion (at the trailing edge of the cell) from the substratum. We have determined in this study the effect of DCN on MG-63 osteosarcoma cell migration and have analyzed whether its effect is mediated by the protein core and/or the glycosaminoglycan side chain. DCN impeded the migration-promoting effect of matrix molecules (fibronectin, collagen type I) known to interact with the proteoglycan. Conversely, DCN did not counteract the migration-promoting effect of fibrinogen lacking proteoglycan affinity. DCN bearing dermatan-sulfate chains (i.e., skin and cartilage DCN) was about 20-fold more effective in inhibiting cell migration than DCN bearing chondroitin-sulfate chains (i.e., bone DCN). In addition, chondroitinase AC-treatment of cartilage DCN (which specifically removes chondroitin-sulfate chains) did not attenuate the inhibitory effect of this proteoglycan, while cartilage DCN deprived of both chondroitin- and dermatan-sulfate chains failed to alter cell migration promoted by either fibronectin or its heparin- and cell-binding domains. These data assert that the dermatan-sulfate chains of DCN are responsible for a negative influence on cell migration. However, isolated glycosaminoglycans failed to alter cell migration promoted by fibronectin, indicating that strongly negatively charged glycosaminoglycans alone cannot account for the impaired cell motility seen with DCN. Overall, these results show that the inhibitory action of DCN is dependent of substratum binding, is differentially mediated by its glycosaminoglycan side chains (chondroitin-sulfate vs. dermatan-sulfate chains), and is independent of a steric hindrance

Stimulation of Cortical Myosin Phosphorylation by p114RhoGEF Drives Cell Migration and Tumor Cell Invasion

PubMed Central

Zihni, Ceniz; Harris, Andrew R.; Bailly, Maryse; Charras, Guillaume T.; Balda, Maria S.; Matter, Karl

2012-01-01

Actinomyosin activity is an important driver of cell locomotion and has been shown to promote collective cell migration of epithelial sheets as well as single cell migration and tumor cell invasion. However, the molecular mechanisms underlying activation of cortical myosin to stimulate single cell movement, and the relationship between the mechanisms that drive single cell locomotion and those that mediate collective cell migration of epithelial sheets are incompletely understood. Here, we demonstrate that p114RhoGEF, an activator of RhoA that associates with non-muscle myosin IIA, regulates collective cell migration of epithelial sheets and tumor cell invasion. Depletion of p114RhoGEF resulted in specific spatial inhibition of myosin activation at cell-cell contacts in migrating epithelial sheets and the cortex of migrating single cells, but only affected double and not single phosphorylation of myosin light chain. In agreement, overall elasticity and contractility of the cells, processes that rely on persistent and more constant forces, were not affected, suggesting that p114RhoGEF mediates process-specific myosin activation. Locomotion was p114RhoGEF-dependent on Matrigel, which favors more roundish cells and amoeboid-like actinomyosin-driven movement, but not on fibronectin, which stimulates flatter cells and lamellipodia-driven, mesenchymal-like migration. Accordingly, depletion of p114RhoGEF led to reduced RhoA, but increased Rac activity. Invasion of 3D matrices was p114RhoGEF-dependent under conditions that do not require metalloproteinase activity, supporting a role of p114RhoGEF in myosin-dependent, amoeboid-like locomotion. Our data demonstrate that p114RhoGEF drives cortical myosin activation by stimulating myosin light chain double phosphorylation and, thereby, collective cell migration of epithelial sheets and amoeboid-like motility of tumor cells. PMID:23185572

Androgen-Induced Cell Migration: Role of Androgen Receptor/Filamin A Association

PubMed Central

Castoria, Gabriella; D'Amato, Loredana; Ciociola, Alessandra; Giovannelli, Pia; Giraldi, Tiziana; Sepe, Leandra; Paolella, Giovanni; Barone, Maria Vittoria; Migliaccio, Antimo; Auricchio, Ferdinando

2011-01-01

Background Androgen receptor (AR) controls male morphogenesis, gametogenesis and prostate growth as well as development of prostate cancer. These findings support a role for AR in cell migration and invasiveness. However, the molecular mechanism involved in AR-mediated cell migration still remains elusive. Methodology/Principal Findings Mouse embryo NIH3T3 fibroblasts and highly metastatic human fibrosarcoma HT1080 cells harbor low levels of transcriptionally incompetent AR. We now report that, through extra nuclear action, AR triggers migration of both cell types upon stimulation with physiological concentrations of the androgen R1881. We analyzed the initial events leading to androgen-induced cell migration and observed that challenging NIH3T3 cells with 10 nM R1881 rapidly induces interaction of AR with filamin A (FlnA) at cytoskeleton. AR/FlnA complex recruits integrin beta 1, thus activating its dependent cascade. Silencing of AR, FlnA and integrin beta 1 shows that this ternary complex controls focal adhesion kinase (FAK), paxillin and Rac, thereby driving cell migration. FAK-null fibroblasts migrate poorly and Rac inhibition by EHT impairs motility of androgen-treated NIH3T3 cells. Interestingly, FAK and Rac activation by androgens are independent of each other. Findings in human fibrosarcoma HT1080 cells strengthen the role of Rac in androgen signaling. The Rac inhibitor significantly impairs androgen-induced migration in these cells. A mutant AR, deleted of the sequence interacting with FlnA, fails to mediate FAK activation and paxillin tyrosine phosphorylation in androgen-stimulated cells, further reinforcing the role of AR/FlnA interaction in androgen-mediated motility. Conclusions/Significance The present report, for the first time, indicates that the extra nuclear AR/FlnA/integrin beta 1 complex is the key by which androgen activates signaling leading to cell migration. Assembly of this ternary complex may control organ development and prostate cancer

Shape Analysis of the Peripapillary RPE Layer in Papilledema and Ischemic Optic Neuropathy

PubMed Central

Kupersmith, Mark J.; Rohlf, F. James

2011-01-01

Purpose. Geometric morphometrics (GM) was used to analyze the shape of the peripapillary retinal pigment epithelium–Bruch's membrane (RPE/BM) layer imaged on the SD-OCT 5-line raster in normal subjects and in patients with papilledema and ischemic optic neuropathy. Methods. Three groups of subjects were compared: 30 normals, 20 with anterior ischemic optic neuropathy (AION), and 25 with papilledema and intracranial hypertension. Twenty equidistant semilandmarks were digitized on OCT images of the RPE/BM layer spanning 2500 μm on each side of the neural canal opening (NCO). The data were analyzed using standard GM techniques, including a generalized least-squares Procrustes superimposition, principal component analysis, thin-plate spline (to visualize deformations), and permutation statistical analysis to evaluate differences in shape variables. Results. The RPE/BM layer in normals and AION have a characteristic V shape pointing away from the vitreous; the RPE/BM layer in papilledema has an inverted U shape, skewed nasally inward toward the vitreous. The differences were statistically significant. There was no significant difference in shapes between normals and AION. Pre- and posttreatment OCTs, in select cases of papilledema, showed that the inverted U-shaped RPE/BM moved posteriorly into a normal V shape as the papilledema resolved with weight loss or shunting. Conclusions. The shape difference in papilledema, absent in AION, cannot be explained by disc edema alone. The difference is a consequence of both the translaminar pressure gradient and the material properties of the peripapillary sclera. GM offers a novel way of statistically assessing shape differences of the peripapillary optic nerve head. PMID:21896851

Migration of guinea pig airway epithelial cells in response to bombesin analogues.

PubMed

Kim, J S; McKinnis, V S; White, S R

1997-03-01

Bombesin-like peptides within neuroepithelial cells elicit proliferation of normal and malignant airway epithelial cells. It is not clear that these peptides also elicit epithelial cell migration, a necessary component of airway repair after injury. We studied the effects of the bombesin analogues, gastrin releasing peptide (GRP) and neuromedin B (NMB), on guinea pig tracheal epithelial cell (GPTEC) migration. Primary GPTEC were allowed to migrate through 8-microm-pore gelatin-coated filters for 6 h in a chemotaxis chamber, after which the number of migrated cells per 10 high power fields (10 hpf) were counted. Both neuropeptides elicited migration of GPTEC: 24.8 +/- 4.5 cells for 10(-11) M NMB (P < 0.001 versus control, n = 4) and 16.8 +/- 1.2 cells for 10(-12) M GRP (P < 0.001 versus control, n = 8). Migration was attenuated substantially by a bombesin receptor antagonist. To investigate further the relationship of migration through a filter to the repair of a damaged epithelium, we studied the repair of epithelial cells by video microscopy. A 0.3- to 0.5-microm2 wound was created in a confluent monolayer of GPTEC, and wound closure was followed over 24 h. There was no significant acceleration in the rate of repair of GRP- or NMB-stimulated monolayers compared to control. These data demonstrate that GRP and NMB elicit migration of airway epithelial cells but may not play a significant role in the early repair of the airway epithelium in culture.

The lutheran/basal cell adhesion molecule promotes tumor cell migration by modulating integrin-mediated cell attachment to laminin-511 protein.

PubMed

Kikkawa, Yamato; Ogawa, Takaho; Sudo, Ryo; Yamada, Yuji; Katagiri, Fumihiko; Hozumi, Kentaro; Nomizu, Motoyoshi; Miner, Jeffrey H

2013-10-25

Cell-matrix interactions are critical for tumor cell migration. Lutheran (Lu), also known as basal cell adhesion molecule (B-CAM), competes with integrins for binding to laminin α5, a subunit of LM-511, a major component of basement membranes. Here we show that the preferential binding of Lu/B-CAM to laminin α5 promotes tumor cell migration. The attachment of Lu/B-CAM transfectants to LM-511 was slightly weaker than that of control cells, and this was because Lu/B-CAM disturbed integrin binding to laminin α5. Lu/B-CAM induced a spindle cell shape with pseudopods and promoted cell migration on LM-511. In addition, blocking with an anti-Lu/B-CAM antibody led to a flat cell shape and inhibited migration on LM-511, similar to the effects of an activating integrin β1 antibody. We conclude that tumor cell migration on LM-511 requires that Lu/B-CAM competitively modulates cell attachment through integrins. We suggest that this competitive interaction is involved in a balance between static and migratory cell behaviors.

Bursts of activity in collective cell migration

PubMed Central

Chepizhko, Oleksandr; Giampietro, Costanza; Mastrapasqua, Eleonora; Nourazar, Mehdi; Ascagni, Miriam; Sugni, Michela; Fascio, Umberto; Leggio, Livio; Malinverno, Chiara; Scita, Giorgio; Santucci, Stéphane; Alava, Mikko J.; Zapperi, Stefano; La Porta, Caterina A. M.

2016-01-01

Dense monolayers of living cells display intriguing relaxation dynamics, reminiscent of soft and glassy materials close to the jamming transition, and migrate collectively when space is available, as in wound healing or in cancer invasion. Here we show that collective cell migration occurs in bursts that are similar to those recorded in the propagation of cracks, fluid fronts in porous media, and ferromagnetic domain walls. In analogy with these systems, the distribution of activity bursts displays scaling laws that are universal in different cell types and for cells moving on different substrates. The main features of the invasion dynamics are quantitatively captured by a model of interacting active particles moving in a disordered landscape. Our results illustrate that collective motion of living cells is analogous to the corresponding dynamics in driven, but inanimate, systems. PMID:27681632

Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling.

PubMed

Sumitomo, M; Shen, R; Walburg, M; Dai, J; Geng, Y; Navarro, D; Boileau, G; Papandreou, C N; Giancotti, F G; Knudsen, B; Nanus, D M

2000-12-01

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). NEP substrates such as bombesin and endothelin-1 induce cell migration. We investigated the mechanisms of NEP regulation of cell migration in PC cells, including regulation of phosphorylation on tyrosine of focal adhesion kinase (FAK). Western analyses and cell migration assays revealed an inverse correlation between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression system inhibited bombesin- and endothelin-1-stimulated FAK phosphorylation and cell migration. This results from NEP-induced inhibition of neuropeptide-stimulated association of FAK with cSrc protein. Expression of a mutated catalytically inactive NEP protein also resulted in partial inhibition of FAK phosphorylation and cell migration. Coimmunoprecipitation experiments show that NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an NEP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K interaction, suggesting that NEP protein inhibits cell migration via a protein-protein interaction independent of its catalytic function. These experiments demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and PC cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated by NEP.

Neutral endopeptidase inhibits prostate cancer cell migration by blocking focal adhesion kinase signaling

PubMed Central

Sumitomo, Makoto; Shen, Ruoqian; Walburg, Marc; Dai, Jie; Geng, Yiping; Navarro, Daniel; Boileau, Guy; Papandreou, Christos N.; Giancotti, Filippo G.; Knudsen, Beatrice; Nanus, David M.

2000-01-01

Neutral endopeptidase 24.11 (NEP, CD10) is a cell-surface enzyme expressed by prostatic epithelial cells that cleaves and inactivates neuropeptides implicated in the growth of androgen-independent prostate cancer (PC). NEP substrates such as bombesin and endothelin-1 induce cell migration. We investigated the mechanisms of NEP regulation of cell migration in PC cells, including regulation of phosphorylation on tyrosine of focal adhesion kinase (FAK). Western analyses and cell migration assays revealed an inverse correlation between NEP expression and the levels of FAK phosphorylation and cell migration in PC cell lines. Constitutively expressed NEP, recombinant NEP, and induced NEP expression using a tetracycline-repressive expression system inhibited bombesin- and endothelin-1–stimulated FAK phosphorylation and cell migration. This results from NEP-induced inhibition of neuropeptide-stimulated association of FAK with cSrc protein. Expression of a mutated catalytically inactive NEP protein also resulted in partial inhibition of FAK phosphorylation and cell migration. Coimmunoprecipitation experiments show that NEP associates with tyrosine-phosphorylated Lyn kinase, which then binds the p85 subunit of phosphatidylinositol 3-kinase (PI3-K) resulting in an NEP-Lyn-PI3-K protein complex. This complex competitively blocks FAK-PI3-K interaction, suggesting that NEP protein inhibits cell migration via a protein-protein interaction independent of its catalytic function. These experiments demonstrate that NEP can inhibit FAK phosphorylation on tyrosine and PC cell migration through multiple pathways and suggest that cell migration which contributes to invasion and metastases in PC cells can be regulated by NEP. PMID:11104793

Cadherin-2 Is Required Cell Autonomously for Collective Migration of Facial Branchiomotor Neurons.

PubMed

Rebman, Jane K; Kirchoff, Kathryn E; Walsh, Gregory S

2016-01-01

Collective migration depends on cell-cell interactions between neighbors that contribute to their overall directionality, yet the mechanisms that control the coordinated migration of neurons remains to be elucidated. During hindbrain development, facial branchiomotor neurons (FBMNs) undergo a stereotypic tangential caudal migration from their place of birth in rhombomere (r)4 to their final location in r6/7. FBMNs engage in collective cell migration that depends on neuron-to-neuron interactions to facilitate caudal directionality. Here, we demonstrate that Cadherin-2-mediated neuron-to-neuron adhesion is necessary for directional and collective migration of FBMNs. We generated stable transgenic zebrafish expressing dominant-negative Cadherin-2 (Cdh2ΔEC) driven by the islet1 promoter. Cell-autonomous inactivation of Cadherin-2 function led to non-directional migration of FBMNs and a defect in caudal tangential migration. Additionally, mosaic analysis revealed that Cdh2ΔEC-expressing FBMNs are not influenced to migrate caudally by neighboring wild-type FBMNs due to a defect in collective cell migration. Taken together, our data suggest that Cadherin-2 plays an essential cell-autonomous role in mediating the collective migration of FBMNs.

Fast-crawling cell types migrate to avoid the direction of periodic substratum stretching

PubMed Central

Okimura, Chika; Ueda, Kazuki; Sakumura, Yuichi; Iwadate, Yoshiaki

2016-01-01

ABSTRACT To investigate the relationship between mechanical stimuli from substrata and related cell functions, one of the most useful techniques is the application of mechanical stimuli via periodic stretching of elastic substrata. In response to this stimulus, Dictyostelium discoideum cells migrate in a direction perpendicular to the stretching direction. The origins of directional migration, higher migration velocity in the direction perpendicular to the stretching direction or the higher probability of a switch of migration direction to perpendicular to the stretching direction, however, remain unknown. In this study, we applied periodic stretching stimuli to neutrophil-like differentiated HL-60 cells, which migrate perpendicular to the direction of stretch. Detailed analysis of the trajectories of HL-60 cells and Dictyostelium cells obtained in a previous study revealed that the higher probability of a switch of migration direction to that perpendicular to the direction of stretching was the main cause of such directional migration. This directional migration appears to be a strategy adopted by fast-crawling cells in which they do not migrate faster in the direction they want to go, but migrate to avoid a direction they do not want to go. PMID:26980079

A PDMS Device Coupled with Culture Dish for In Vitro Cell Migration Assay.

PubMed

Lv, Xiaoqing; Geng, Zhaoxin; Fan, Zhiyuan; Wang, Shicai; Pei, WeiHua; Chen, Hongda

2018-04-30

Cell migration and invasion are important factors during tumor progression and metastasis. Wound-healing assay and the Boyden chamber assay are efficient tools to investigate tumor development because both of them could be applied to measure cell migration rate. Therefore, a simple and integrated polydimethylsiloxane (PDMS) device was developed for cell migration assay, which could perform quantitative evaluation of cell migration behaviors, especially for the wound-healing assay. The integrated device was composed of three units, which included cell culture dish, PDMS chamber, and wound generation mold. The PDMS chamber was integrated with cell culture chamber and could perform six experiments under different conditions of stimuli simultaneously. To verify the function of this device, it was utilized to explore the tumor cell migration behaviors under different concentrations of fetal bovine serum (FBS) and transforming growth factor (TGF-β) at different time points. This device has the unique capability to create the "wound" area in parallel during cell migration assay and provides a simple and efficient platform for investigating cell migration assay in biomedical application.

Lamellipodin and the Scar/WAVE complex cooperate to promote cell migration in vivo

PubMed Central

Law, Ah-Lai; Vehlow, Anne; Kotini, Maria; Dodgson, Lauren; Soong, Daniel; Theveneau, Eric; Bodo, Cristian; Taylor, Eleanor; Navarro, Christel; Perera, Upamali; Michael, Magdalene; Dunn, Graham A.; Bennett, Daimark; Mayor, Roberto

2013-01-01

Cell migration is essential for development, but its deregulation causes metastasis. The Scar/WAVE complex is absolutely required for lamellipodia and is a key effector in cell migration, but its regulation in vivo is enigmatic. Lamellipodin (Lpd) controls lamellipodium formation through an unknown mechanism. Here, we report that Lpd directly binds active Rac, which regulates a direct interaction between Lpd and the Scar/WAVE complex via Abi. Consequently, Lpd controls lamellipodium size, cell migration speed, and persistence via Scar/WAVE in vitro. Moreover, Lpd knockout mice display defective pigmentation because fewer migrating neural crest-derived melanoblasts reach their target during development. Consistently, Lpd regulates mesenchymal neural crest cell migration cell autonomously in Xenopus laevis via the Scar/WAVE complex. Further, Lpd’s Drosophila melanogaster orthologue Pico binds Scar, and both regulate collective epithelial border cell migration. Pico also controls directed cell protrusions of border cell clusters in a Scar-dependent manner. Taken together, Lpd is an essential, evolutionary conserved regulator of the Scar/WAVE complex during cell migration in vivo. PMID:24247431

Notch1-Dll4 signaling and mechanical force regulate leader cell formation during collective cell migration

PubMed Central

Riahi, Reza; Sun, Jian; Wang, Shue; Long, Min; Zhang, Donna D.; Wong, Pak Kin

2015-01-01

At the onset of collective cell migration, a subset of cells within an initially homogenous population acquires a distinct “leader” phenotype with characteristic morphology and motility. However, the factors driving leader cell formation as well as the mechanisms regulating leader cell density during the migration process remain to be determined. Here, we use single cell gene expression analysis and computational modeling to show that leader cell identity is dynamically regulated by Dll4 signaling through both Notch1 and cellular stress in a migrating epithelium. Time-lapse microscopy reveals that Dll4 is induced in leader cells after the creation of the cell-free region and leader cells are regulated via Notch1-Dll4 lateral inhibition. Furthermore, mechanical stress inhibits Dll4 expression and leader cell formation in the monolayer. Collectively, our findings suggest that a reduction of mechanical force near the boundary promotes Notch1-Dll4 signaling to dynamically regulate the density of leader cells during collective cell migration. PMID:25766473

Characterization of a spontaneously generated murine retinal pigmented epithelium cell line; a model for in vitro experiments.

PubMed

Ranaei Pirmardan, Ehsan; Soheili, Zahra-Soheila; Samiei, Shahram; Ahmadieh, Hamid; Mowla, Seyed Javad; Ezzati, Razie; Naseri, Marzieh

2016-10-01

Retinal pigmented epithelium (RPE), the outermost layer of the retina, has a key role in maintaining retinal cells' functions. Severity of the culture of RPE cells has exerted many limitations to both in vitro and in vivo studies and its therapeutic applications. Therefore, establishment of RPE cell lines with high proliferative potential can considerably improve study of RPE cell biology. Here we report generation of a spontaneously immortalized murine RPE cell line in primary mouse RPE cell culture. Founded colonized cells were picked up and expression of RPE and retinal progenitor cells' (RPC) markers were studied using immunocytochemistry (ICC). Emerged cells cultured over 35 passages and population doubling times in different serum concentrations were calculated. We also investigated the ability of cells for becoming transfected by calcium-phosphate method and for becoming infected by adeno-associated virus serotype 2 (AAV2) using flow cytometry. Data showed that the cobblestone constituent cells expressed RPE65, cytokeratin and ZO1 and moreover several progenitor markers such as Pax6, Sox2, Nestin and Chx10. It revealed that, despite primary RPE cells, the newly emerged cells were easily transfectable and were highly infectable when compared with HEK293T cells. Our data indicated that the emerged mouse RPE cell line pretended RPC-like phenotype and also simultaneously expressed RPE markers. It would be a promising model for leading studies on RPE and RPC cells and substantially confirmed the great RPE plasticity and its invaluable potential in research studies. Copyright © 2016 Elsevier Inc. All rights reserved.

Impact of Tumor Cell Cytoskeleton Organization on Invasiveness and Migration: A Microchannel-Based Approach

PubMed Central

Rolli, Claudio G.; Seufferlein, Thomas; Kemkemer, Ralf; Spatz, Joachim P.

2010-01-01

Cell migration is a fundamental feature of the interaction of cells with their surrounding. The cell's stiffness and ability to deform itself are two major characteristics that rule migration behavior especially in three-dimensional tissue. We simulate this situation making use of a micro-fabricated migration chip to test the active invasive behavior of pancreatic cancer cells (Panc-1) into narrow channels. At a channel width of 7 µm cell migration through the channels was significantly impeded due to size exclusion. A striking increase in cell invasiveness was observed once the cells were treated with the bioactive lipid sphingosylphosphorylcholine (SPC) that leads to a reorganization of the cell's keratin network, an enhancement of the cell's deformability, and also an increase in the cell's migration speed on flat surfaces. The migration speed of the highly deformed cells inside the channels was three times higher than of cells on flat substrates but was not affected upon SPC treatment. Cells inside the channels migrated predominantly by smooth sliding while maintaining constant cell length. In contrast, cells on adhesion mediating narrow lines moved in a stepwise way, characterized by fluctuations in cell length. Taken together, with our migration chip we demonstrate that the dimensionality of the environment strongly affects the migration phenotype and we suggest that the spatial cytoskeletal keratin organization correlates with the tumor cell's invasive potential. PMID:20090950

Application of stem cell-derived retinal pigmented epithelium in retinal degenerative diseases: present and future.

PubMed

Luo, Mingyue; Chen, Youxin

2018-01-01

As a constituent of blood-retinal barrier and retinal outer segment (ROS) scavenger, retinal pigmented epithelium (RPE) is fundamental to normal function of retina. Malfunctioning of RPE contributes to the onset and advance of retinal degenerative diseases. Up to date, RPE replacement therapy is the only possible method to completely reverse retinal degeneration. Transplantation of human RPE stem cell-derived RPE (hRPESC-RPE) has shown some good results in animal models. With promising results in terms of safety and visual improvement, human embryonic stem cell-derived RPE (hESC-RPE) can be expected in clinical settings in the near future. Despite twists and turns, induced pluripotent stem cell-derived RPE (iPSC-RPE) is now being intensely investigated to overcome genetic and epigenetic instability. By far, only one patient has received iPSC-RPE transplant, which is a hallmark of iPSC technology development. During follow-up, no major complications such as immunogenicity or tumorigenesis have been observed. Future trials should keep focusing on the safety of stem cell-derived RPE (SC-RPE) especially in long period, and better understanding of the nature of stem cell and the molecular events in the process to generate SC-RPE is necessary to the prosperity of SC-RPE clinical application.

Evidence for tension-based regulation of Drosophila MAL and SRF during invasive cell migration.

PubMed

Somogyi, Kálmán; Rørth, Pernille

2004-07-01

Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. We report that this migration requires the activity of the transcriptional factor serum response factor (SRF) and its cofactor MAL-D and present evidence that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration.

Cell migration and antigen capture are antagonistic processes coupled by myosin II in dendritic cells

PubMed Central

Chabaud, Mélanie; Heuzé, Mélina L.; Bretou, Marine; Vargas, Pablo; Maiuri, Paolo; Solanes, Paola; Maurin, Mathieu; Terriac, Emmanuel; Le Berre, Maël; Lankar, Danielle; Piolot, Tristan; Adelstein, Robert S.; Zhang, Yingfan; Sixt, Michael; Jacobelli, Jordan; Bénichou, Olivier; Voituriez, Raphaël; Piel, Matthieu; Lennon-Duménil, Ana-Maria

2015-01-01

The immune response relies on the migration of leukocytes and on their ability to stop in precise anatomical locations to fulfil their task. How leukocyte migration and function are coordinated is unknown. Here we show that in immature dendritic cells, which patrol their environment by engulfing extracellular material, cell migration and antigen capture are antagonistic. This antagonism results from transient enrichment of myosin IIA at the cell front, which disrupts the back-to-front gradient of the motor protein, slowing down locomotion but promoting antigen capture. We further highlight that myosin IIA enrichment at the cell front requires the MHC class II-associated invariant chain (Ii). Thus, by controlling myosin IIA localization, Ii imposes on dendritic cells an intermittent antigen capture behaviour that might facilitate environment patrolling. We propose that the requirement for myosin II in both cell migration and specific cell functions may provide a general mechanism for their coordination in time and space. PMID:26109323

Slits Affect the Timely Migration of Neural Crest Cells via Robo Receptor

PubMed Central

Giovannone, Dion; Reyes, Michelle; Reyes, Rachel; Correa, Lisa; Martinez, Darwin; Ra, Hannah; Gomez, Gustavo; Kaiser, Josh; Ma, Le; Stein, Mary-Pat; de Bellard, Maria Elena

2013-01-01

SUMMARY Background Neural crest cells emerge by delamination from the dorsal neural tube and give rise to various components of the peripheral nervous system in vertebrate embryos. These cells change from non-motile into highly motile cells migrating to distant areas before further differentiation. Mechanisms controlling delamination and subsequent migration of neural crest cells are not fully understood. Slit2, a chemorepellant for axonal guidance that repels and stimulates motility of trunk neural crest cells away from the gut has recently been suggested to be a tumor suppressor molecule. The goal of this study was to further investigate the role of Slit2 in trunk neural crest cell migration by constitutive expression in neural crest cells. Results We found that Slit gain-of-function significantly impaired neural crest cell migration while Slit loss-of-function favored migration. In addition, we observed that the distribution of key cytoskeletal markers was disrupted in both gain and loss of function instances. Conclusions These findings suggest that Slit molecules might be involved in the processes that allow neural crest cells to begin migration and transitioning to a mesenchymal type. PMID:22689303

Fibronectin Modulates Cell Adhesion and Signaling to Promote Single Cell Migration of Highly Invasive Oral Squamous Cell Carcinoma

PubMed Central

Ramos, Grasieli de Oliveira; Bernardi, Lisiane; Lauxen, Isabel; Sant’Ana Filho, Manoel; Horwitz, Alan Rick; Lamers, Marcelo Lazzaron

2016-01-01

Cell migration is regulated by adhesion to the extracellular matrix (ECM) through integrins and activation of small RhoGTPases, such as RhoA and Rac1, resulting in changes to actomyosin organization. During invasion, epithelial-derived tumor cells switch from laminin-enriched basal membrane to collagen and fibronectin-enriched connective tissue. How this switch affects the tumor migration is still unclear. We tested the hypothesis that ECM dictates the invasiveness of Oral Squamous Cell Carcinoma (OSCC). We analyzed the migratory properties of two OSCC lines, a low invasive cell line with high e-cadherin levels (Linv/HE-cad) or a highly invasive cell line with low e-cadherin levels (Hinv/LE-cad), plated on different ECM components. Compared to laminin, fibronectin induced non-directional collective migration and decreased RhoA activity in Linv/HE-cad OSCC. For Hinv/LE-cad OSCC, fibronectin increased Rac1 activity and induced smaller adhesions, resulting in a fast single cell migration in both 2D and 3D environments. Consistent with these observations, human OSCC biopsies exhibited similar changes in cell-ECM adhesion distribution at the invasive front of the tumor, where cells encounter fibronectin. Our results indicate that ECM composition might induce a switch from collective to single cell migration according to tumor invasiveness due to changes in cell-ECM adhesion and the resulting signaling pathways that alter actomyosin organization. PMID:26978651

Modeling and predictions of biphasic mechanosensitive cell migration altered by cell-intrinsic properties and matrix confinement.

PubMed

Pathak, Amit

2018-04-12

Motile cells sense the stiffness of their extracellular matrix (ECM) through adhesions and respond by modulating the generated forces, which in turn lead to varying mechanosensitive migration phenotypes. Through modeling and experiments, cell migration speed is known to vary with matrix stiffness in a biphasic manner, with optimal motility at an intermediate stiffness. Here, we present a two-dimensional cell model defined by nodes and elements, integrated with subcellular modeling components corresponding to mechanotransductive adhesion formation, force generation, protrusions and node displacement. On 2D matrices, our calculations reproduce the classic biphasic dependence of migration speed on matrix stiffness and predict that cell types with higher force-generating ability do not slow down on very stiff matrices, thus disabling the biphasic response. We also predict that cell types defined by lower number of total receptors require stiffer matrices for optimal motility, which also limits the biphasic response. For a cell type with robust biphasic migration on 2D surface, simulations in channel-like confined environments of varying width and height predict faster migration in more confined matrices. Simulations performed in shallower channels predict that the biphasic mechanosensitive cell migration response is more robust on 2D micro-patterns as compared to the channel-like 3D confinement. Thus, variations in the dimensionality of matrix confinement alters the way migratory cells sense and respond to the matrix stiffness. Our calculations reveal new phenotypes of stiffness- and topography-sensitive cell migration that critically depend on both cell-intrinsic and matrix properties. These predictions may inform our understanding of various mechanosensitive modes of cell motility that could enable tumor invasion through topographically heterogeneous microenvironments. © 2018 IOP Publishing Ltd.

ALG2 regulates glioblastoma cell proliferation, migration and tumorigenicity

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

Zhang, Dunke; Wang, Feng; Pang, Yi

Apoptosis-linked gene-2 (ALG-2), also known as programmed cell death 6 (PDCD6), has recently been reported to be aberrantly expressed in various tumors and required for tumor cell viability. The aim of the present study was to investigate whether ALG-2 plays a crucial role in tumor cell proliferation, migration and tumorigenicity. In this study, we examined the expression of PDCD6 in glioblastoma cell lines and found that ALG-2 was generally expressed in glioblastoma cell lines. We also performed an analysis of an online database and found that high expression of ALG-2 was associated with poor prognosis (p = 0.039). We found that over-expressionmore » of ALG2 in glioblastoma could inhibit cell proliferation and, conversely, that down-regulation of ALG2 could promote cell proliferation. Further studies showed that over-expression of ALG2 inhibited the migration of tumor cells, whereas down-regulation of ALG2 promoted tumor cell migration. Finally, in vitro and in vivo studies showed that over-expression of ALG2 inhibited the tumorigenic ability of tumor cells, while down-regulation of ALG2 promoted tumor cell tumorigenic ability. In conclusion, ALG2 has a tumor suppressive role in glioblastoma and might be a potential target for the treatment of glioblastoma. - Highlights: • Low ALG2 expression is indicative of poor prognosis in glioblastoma patients. • ALG2 is required for cell proliferation in GBM cells. • ALG2 is involved in GBM cell migration. • ALG2 is involved in GBM cell self-renewal and tumorigenesis in vitro and in vivo.« less

A lateral signalling pathway coordinates shape volatility during cell migration

PubMed Central

Zhang, Liang; Luga, Valbona; Armitage, Sarah K.; Musiol, Martin; Won, Amy; Yip, Christopher M.; Plotnikov, Sergey V.; Wrana, Jeffrey L.

2016-01-01

Cell migration is fundamental for both physiological and pathological processes. Migrating cells usually display high dynamics in morphology, which is orchestrated by an integrative array of signalling pathways. Here we identify a novel pathway, we term lateral signalling, comprised of the planar cell polarity (PCP) protein Pk1 and the RhoGAPs, Arhgap21/23. We show that the Pk1–Arhgap21/23 complex inhibits RhoA, is localized on the non-protrusive lateral membrane cortex and its disruption leads to the disorganization of the actomyosin network and altered focal adhesion dynamics. Pk1-mediated lateral signalling confines protrusive activity and is regulated by Smurf2, an E3 ubiquitin ligase in the PCP pathway. Furthermore, we demonstrate that dynamic interplay between lateral and protrusive signalling generates cyclical fluctuations in cell shape that we quantify here as shape volatility, which strongly correlates with migration speed. These studies uncover a previously unrecognized lateral signalling pathway that coordinates shape volatility during productive cell migration. PMID:27226243

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin.

PubMed

Erdogan, Begum; Ao, Mingfang; White, Lauren M; Means, Anna L; Brewer, Bryson M; Yang, Lijie; Washington, M Kay; Shi, Chanjuan; Franco, Omar E; Weaver, Alissa M; Hayward, Simon W; Li, Deyu; Webb, Donna J

2017-11-06

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF-cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α-mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration. © 2017 Erdogan et al.

Cancer-associated fibroblasts promote directional cancer cell migration by aligning fibronectin

PubMed Central

Ao, Mingfang; White, Lauren M.; Means, Anna L.; Yang, Lijie; Washington, M. Kay; Franco, Omar E.; Li, Deyu; Webb, Donna J.

2017-01-01

Cancer-associated fibroblasts (CAFs) are major components of the carcinoma microenvironment that promote tumor progression. However, the mechanisms by which CAFs regulate cancer cell migration are poorly understood. In this study, we show that fibronectin (Fn) assembled by CAFs mediates CAF–cancer cell association and directional migration. Compared with normal fibroblasts, CAFs produce an Fn-rich extracellular matrix with anisotropic fiber orientation, which guides the cancer cells to migrate directionally. CAFs align the Fn matrix by increasing nonmuscle myosin II- and platelet-derived growth factor receptor α–mediated contractility and traction forces, which are transduced to Fn through α5β1 integrin. We further show that prostate cancer cells use αv integrin to migrate efficiently and directionally on CAF-derived matrices. We demonstrate that aligned Fn is a prominent feature of invasion sites in human prostatic and pancreatic carcinoma samples. Collectively, we present a new mechanism by which CAFs organize the Fn matrix and promote directional cancer cell migration. PMID:29021221

Retinal Pigment Epithelium Culture;a Potential Source of Retinal Stem Cells

PubMed Central

Akrami, Hassan; Soheili, Zahra-Soheila; Khalooghi, Keynoush; Ahmadieh, Hamid; Rezaie-Kanavi, Mojgan; Samiei, Shahram; Davari, Malihe; Ghaderi, Shima; Sanie-Jahromi, Fatemeh

2009-01-01

Purpose To establish human retinal pigment epithelial (RPE) cell culture as a source for cell replacement therapy in ocular diseases. Methods Human cadaver globes were used to isolate RPE cells. Each globe was cut into several pieces of a few millimeters in size. After removing the sclera and choroid, remaining tissues were washed in phosphate buffer saline and RPE cells were isolated using dispase enzyme solution and cultured in Dulbecco’s Modified Eagle’s Medium: Nutrient Mixture F-12 supplemented with 10% fetal calf serum. Results Primary cultures of RPE cells were established and spheroid colonies related to progenitor/stem cells developed in a number of cultures. The colonies included purely pigmented or mixed pigmented and non-pigmented cells. After multiple cellular passages, several types of photoreceptors and neural-like cells were detected morphologically. Conclusion Cellular plasticity in RPE cell cultures revealed promising results in terms of generation of stem/progenitor cells from human RPE cells. Whether the spheroids and neural-like retinal cells were directly derived from retinal stem cells or offspring of trans-differentiating or de-differentiating RPE cells remains to be answered. PMID:23198062

Cerium migration during PEM fuel cell accelerated stress testing

DOE PAGES

Baker, Andrew M.; Mukundan, Rangachary; Borup, Rodney L.; ...

2016-01-01

Cerium is a radical scavenger which improves polymer electrolyte membrane (PEM) fuel cell durability. During operation, however, cerium rapidly migrates in the PEM and into the catalyst layers (CLs). In this work, membrane electrode assemblies (MEAs) were subjected to accelerated stress tests (ASTs) under different humidity conditions. Cerium migration was characterized in the MEAs after ASTs using X-ray fluorescence. During fully humidified operation, water flux from cell inlet to outlet generated in-plane cerium gradients. Conversely, cerium profiles were flat during low humidity operation, where in-plane water flux was negligible, however, migration from the PEM into the CLs was enhanced. Humiditymore » cycling resulted in both in-plane cerium gradients due to water flux during the hydration component of the cycle, and significant migration into the CLs. Fluoride and cerium emissions into effluent cell waters were measured during ASTs and correlated, which signifies that ionomer degradation products serve as possible counter-ions for cerium emissions. Fluoride emission rates were also correlated to final PEM cerium contents, which indicates that PEM degradation and cerium migration are coupled. Lastly, it is proposed that cerium migrates from the PEM due to humidification conditions and degradation, and is subsequently stabilized in the CLs by carbon catalyst supports.« less

The role of backward cell migration in two-hit mutants' production in the stem cell niche.

PubMed

Bollas, Audrey; Shahriyari, Leili

2017-01-01

It has been discovered that there are two stem cell groups in the intestinal crypts: central stem cells (CeSCs), which are at the very bottom of the crypt, and border stem cells (BSCs), which are located between CeSCs and transit amplifying cells (TAs). Moreover, backward cell migration from BSCs to CeSCs has been observed. Recently, a bi-compartmental stochastic model, which includes CeSCs and BSCs, has been developed to investigate the probability of two-hit mutant production in the stem cell niche. In this project, we improve this stochastic model by adding the probability of backward cell migration to the model. The model suggests that the probability of two-hit mutant production increases when the frequency of backward cell migration increases. Furthermore, a small non-zero probability of backward cell migration leads to the largest range of optimal values for the frequency of symmetric divisions and the portion of divisions at each stem cell compartment in terms of delaying 2-hit mutant production. Moreover, the probability of two-hit mutant production is more sensitive to the probability of symmetric divisions than to the rate of backward cell migrations. The highest probability of two-hit mutant production corresponds to the case when all stem cell's divisions are asymmetric.

Emergent patterns of collective cell migration under tubular confinement.

PubMed

Xi, Wang; Sonam, Surabhi; Beng Saw, Thuan; Ladoux, Benoit; Teck Lim, Chwee

2017-11-15

Collective epithelial behaviors are essential for the development of lumens in organs. However, conventional assays of planar systems fail to replicate cell cohorts of tubular structures that advance in concerted ways on out-of-plane curved and confined surfaces, such as ductal elongation in vivo. Here, we mimic such coordinated tissue migration by forming lumens of epithelial cell sheets inside microtubes of 1-10 cell lengths in diameter. We show that these cell tubes reproduce the physiological apical-basal polarity, and have actin alignment, cell orientation, tissue organization, and migration modes that depend on the extent of tubular confinement and/or curvature. In contrast to flat constraint, the cell sheets in a highly constricted smaller microtube demonstrate slow motion with periodic relaxation, but fast overall movement in large microtubes. Altogether, our findings provide insights into the emerging migratory modes for epithelial migration and growth under tubular confinement, which are reminiscent of the in vivo scenario.

Carbon Ion Irradiation Inhibits Glioma Cell Migration Through Downregulation of Integrin Expression

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

Rieken, Stefan, E-mail: Stefan.Rieken@med.uni-heidelberg.de; Habermehl, Daniel; Wuerth, Lena

2012-05-01

Purpose: To investigate the effect of carbon ion irradiation on glioma cell migration. Methods and Materials: U87 and Ln229 glioma cells were irradiated with photons and carbon ions. Migration was analyzed 24 h after irradiation. Fluorescence-activated cell sorting analysis was performed in order to quantify surface expression of integrins. Results: Single photon doses of 2 Gy and 10 Gy enhanced {alpha}{sub {nu}}{beta}{sub 3} and {alpha}{sub {nu}}{beta}{sub 5} integrin expression and caused tumor cell hypermigration on both vitronectin (Vn) and fibronectin (Fn). Compared to integrin expression in unirradiated cells, carbon ion irradiation caused decreased integrin expression and inhibited cell migration onmore » both Vn and Fn. Conclusion: Photon radiotherapy (RT) enhances the risk of tumor cell migration and subsequently promotes locoregional spread via photon induction of integrin expression. In contrast to photon RT, carbon ion RT causes decreased integrin expression and suppresses glioma cell migration on both Vn and Fn, thus promising improved local control.« less

The Caenorhabditis elegans Q neuroblasts: A powerful system to study cell migration at single-cell resolution in vivo.

PubMed

Rella, Lorenzo; Fernandes Póvoa, Euclides E; Korswagen, Hendrik C

2016-04-01

During development, cell migration plays a central role in the formation of tissues and organs. Understanding the molecular mechanisms that drive and control these migrations is a key challenge in developmental biology that will provide important insights into disease processes, including cancer cell metastasis. In this article, we discuss the Caenorhabditis elegans Q neuroblasts and their descendants as a tool to study cell migration at single-cell resolution in vivo. The highly stereotypical migration of these cells provides a powerful system to study the dynamic cytoskeletal processes that drive migration as well as the evolutionarily conserved signaling pathways (including different Wnt signaling cascades) that guide the cells along their specific trajectories. Here, we provide an overview of what is currently known about Q neuroblast migration and highlight the live-cell imaging, genome editing, and quantitative gene expression techniques that have been developed to study this process. © 2016 Wiley Periodicals, Inc.

Migration of cells in a social context

PubMed Central

Vedel, Søren; Tay, Savaş; Johnston, Darius M.; Bruus, Henrik; Quake, Stephen R.

2013-01-01

In multicellular organisms and complex ecosystems, cells migrate in a social context. Whereas this is essential for the basic processes of life, the influence of neighboring cells on the individual remains poorly understood. Previous work on isolated cells has observed a stereotypical migratory behavior characterized by short-time directional persistence with long-time random movement. We discovered a much richer dynamic in the social context, with significant variations in directionality, displacement, and speed, which are all modulated by local cell density. We developed a mathematical model based on the experimentally identified “cellular traffic rules” and basic physics that revealed that these emergent behaviors are caused by the interplay of single-cell properties and intercellular interactions, the latter being dominated by a pseudopod formation bias mediated by secreted chemicals and pseudopod collapse following collisions. The model demonstrates how aspects of complex biology can be explained by simple rules of physics and constitutes a rapid test bed for future studies of collective migration of individual cells. PMID:23251032

Migration of cells in a social context.

PubMed

Vedel, Søren; Tay, Savaş; Johnston, Darius M; Bruus, Henrik; Quake, Stephen R

2013-01-02

In multicellular organisms and complex ecosystems, cells migrate in a social context. Whereas this is essential for the basic processes of life, the influence of neighboring cells on the individual remains poorly understood. Previous work on isolated cells has observed a stereotypical migratory behavior characterized by short-time directional persistence with long-time random movement. We discovered a much richer dynamic in the social context, with significant variations in directionality, displacement, and speed, which are all modulated by local cell density. We developed a mathematical model based on the experimentally identified "cellular traffic rules" and basic physics that revealed that these emergent behaviors are caused by the interplay of single-cell properties and intercellular interactions, the latter being dominated by a pseudopod formation bias mediated by secreted chemicals and pseudopod collapse following collisions. The model demonstrates how aspects of complex biology can be explained by simple rules of physics and constitutes a rapid test bed for future studies of collective migration of individual cells.

The mechanisms of substance P-mediated migration of bone marrow-derived mesenchymal stem cell-like ST2 cells.

PubMed

Dubon, Maria Jose; Park, Ki-Sook

2016-04-01

Substance P (SP) is known to induce the mobilization of bone marrow-derived mesenchymal stem cells (BM-MSCs) and thus participates in wound repair. However, the cellular and molecular mechanisms responsible for the SP-mediated migration of BM-MSCs were not fully understood. In the present study, we studied the molecular mechanisms that mediate the migration of the BM-derived MSC-like cell line ST2 in response to SP. Using a migration assay and western blot analysis, we noted that SP induced the chemotactic migration of ST2 cells through the intrinsic activation of extracellular signal-regulated kinases (ERKs) and protein kinase B (Akt), the phosphorylated expression levels of which were increased. We noted that Src is involved in the SP-mediated migration of ST2 cells and that focal adhesion kinase (FAK) was activated in the ST2 cells following SP treatment. Membrane ruffling increased in the ST2 cells after SP treatment, as was clearly demonstrated by immunocytochemical analysis. Importantly, using a blocking antibody against N-cadherin (GC-4), we studied cell migration and noted that SP mediated the migration of the ST2 cells through N-cadherin. The present study thus advanced our understanding of the mechanisms through which SP induces BM-MSC migration.

Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion.

PubMed

Belo, Angelica; Cheng, Kunrong; Chahdi, Ahmed; Shant, Jasleen; Xie, Guofeng; Khurana, Sandeep; Raufman, Jean-Pierre

2011-05-01

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion.

Muscarinic receptor agonists stimulate human colon cancer cell migration and invasion

PubMed Central

Belo, Angelica; Cheng, Kunrong; Chahdi, Ahmed; Shant, Jasleen; Xie, Guofeng; Khurana, Sandeep

2011-01-01

Muscarinic receptors (CHRM) are overexpressed in colon cancer. To explore a role for muscarinic receptor signaling in colon cancer metastasis, we used human H508 and HT29 colon cancer cells that coexpress epidermal growth factor (ERBB) and CHRM3 receptors. In a wound closure model, following 8-h incubation of H508 cells with 100 μM ACh we observed a threefold increase in cell migration indistinguishable from the actions of epidermal growth factor (EGF). Atropine blocked the actions of ACh but not of EGF. In SNU-C4 colon cancer cells that express ERBB but not CHRM, EGF caused a threefold increase in migration; ACh had no effect. ACh-induced cell migration was attenuated by chemical inhibitors of ERBB1 activation, by anti-ERBB1 antibody, and by inhibitors of ERK and phosphatidylinositol 3-kinase (PI3K) signaling. Consistent with matrix metalloproteinase-7 (MMP7)-mediated release of an ERBB1 ligand, heparin binding epidermal growth factor-like growth factor (HBEGF), ACh-induced migration was inhibited by an MMP inhibitor and by anti-MMP7 and -HBEGF antibodies. ACh-induced cell migration was blocked by inhibiting RhoA and ROCK, key proteins that interact with the actin cytoskeleton. ACh-induced RhoA activation was attenuated by agents that inhibit ERBB1, ERK, and PI3K activation. Collectively, these findings indicate that ACh-induced cell migration is mediated by MMP7-mediated release of HBEGF, an ERBB ligand that activates ERBB1 and downstream ERK and PI3K signaling. In a cell invasion model, ACh-induced HT29 cell invasion was blocked by atropine. In concert with previous observations, these findings indicate that muscarinic receptor signaling plays a key role in colon cancer cell proliferation, survival, migration, and invasion. PMID:21273532

A pilgrim's progress: Seeking meaning in primordial germ cell migration.

PubMed

Cantú, Andrea V; Laird, Diana J

2017-10-01

Comparative studies of primordial germ cell (PGC) development across organisms in many phyla reveal surprising diversity in the route of migration, timing and underlying molecular mechanisms, suggesting that the process of migration itself is conserved. However, beyond the perfunctory transport of cellular precursors to their later arising home of the gonads, does PGC migration serve a function? Here we propose that the process of migration plays an additional role in quality control, by eliminating PGCs incapable of completing migration as well as through mechanisms that favor PGCs capable of responding appropriately to migration cues. Focusing on PGCs in mice, we explore evidence for a selective capacity of migration, considering the tandem regulation of proliferation and migration, cell-intrinsic and extrinsic control, the potential for tumors derived from failed PGC migrants, the potential mechanisms by which migratory PGCs vary in their cellular behaviors, and corresponding effects on development. We discuss the implications of a selective role of PGC migration for in vitro gametogenesis. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

The role of backward cell migration in two-hit mutants’ production in the stem cell niche

PubMed Central

Bollas, Audrey

2017-01-01

It has been discovered that there are two stem cell groups in the intestinal crypts: central stem cells (CeSCs), which are at the very bottom of the crypt, and border stem cells (BSCs), which are located between CeSCs and transit amplifying cells (TAs). Moreover, backward cell migration from BSCs to CeSCs has been observed. Recently, a bi-compartmental stochastic model, which includes CeSCs and BSCs, has been developed to investigate the probability of two-hit mutant production in the stem cell niche. In this project, we improve this stochastic model by adding the probability of backward cell migration to the model. The model suggests that the probability of two-hit mutant production increases when the frequency of backward cell migration increases. Furthermore, a small non-zero probability of backward cell migration leads to the largest range of optimal values for the frequency of symmetric divisions and the portion of divisions at each stem cell compartment in terms of delaying 2-hit mutant production. Moreover, the probability of two-hit mutant production is more sensitive to the probability of symmetric divisions than to the rate of backward cell migrations. The highest probability of two-hit mutant production corresponds to the case when all stem cell’s divisions are asymmetric. PMID:28931019

In vivo imaging of the retinal pigment epithelial cells

NASA Astrophysics Data System (ADS)

Morgan, Jessica Ijams Wolfing

The retinal pigment epithelial (RPE) cells form an important layer of the retina because they are responsible for providing metabolic support to the photoreceptors. Techniques to image the RPE layer include autofluorescence imaging with a scanning laser ophthalmoscope (SLO). However, previous studies were unable to resolve single RPE cells in vivo. This thesis describes the technique of combining autofluorescence, SLO, adaptive optics (AO), and dual-wavelength simultaneous imaging and registration to visualize the individual cells in the RPE mosaic in human and primate retina for the first time in vivo. After imaging the RPE mosaic non-invasively, the cell layer's structure and regularity were characterized using quantitative metrics of cell density, spacing, and nearest neighbor distances. The RPE mosaic was compared to the cone mosaic, and RPE imaging methods were confirmed using histology. The ability to image the RPE mosaic led to the discovery of a novel retinal change following light exposure; 568 nm exposures caused an immediate reduction in autofluorescence followed by either full recovery or permanent damage in the RPE layer. A safety study was conducted to determine the range of exposure irradiances that caused permanent damage or transient autofluorescence reductions. Additionally, the threshold exposure causing autofluorescence reduction was determined and reciprocity of radiant exposure was confirmed. Light exposures delivered by the AOSLO were not significantly different than those delivered by a uniform source. As all exposures tested were near or below the permissible light levels of safety standards, this thesis provides evidence that the current light safety standards need to be revised. Finally, with the retinal damage and autofluorescence reduction thresholds identified, the methods of RPE imaging were modified to allow successful imaging of the individual cells in the RPE mosaic while still ensuring retinal safety. This thesis has provided a

Envisioning migration: Mathematics in both experimental analysis and modeling of cell behavior

PubMed Central

Zhang, Elizabeth R.; Wu, Lani F.; Altschuler, Steven J.

2013-01-01

The complex nature of cell migration highlights the power and challenges of applying mathematics to biological studies. Mathematics may be used to create model equations that recapitulate migration, which can predict phenomena not easily uncovered by experiments or intuition alone. Alternatively, mathematics may be applied to interpreting complex data sets with better resolution—potentially empowering scientists to discern subtle patterns amid the noise and heterogeneity typical of migrating cells. Iteration between these two methods is necessary in order to reveal connections within the cell migration signaling network, as well as to understand the behavior that arises from those connections. Here, we review recent quantitative analysis and mathematical modeling approaches to the cell migration problem. PMID:23660413

HGF and c-Met Interaction Promotes Migration in Human Chondrosarcoma Cells

PubMed Central

Tsou, Hsi-Kai; Chen, Hsien-Te; Hung, Ya-Huey; Chang, Chia-Hao; Li, Te-Mao; Fong, Yi-Chin; Tang, Chih-Hsin

2013-01-01

Chondrosarcoma is a type of highly malignant tumor with a potent capacity for local invasion and causing distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Hepatocyte growth factor (HGF) has been demonstrated to stimulate cancer proliferation, migration, and metastasis. However, the effect of HGF on migration activity of human chondrosarcoma cells is not well known. Here, we found that human chondrosarcoma tissues demonstrated significant expression of HGF, which was higher than that in normal cartilage. We also found that HGF increased the migration and expression of matrix metalloproteinase (MMP)-2 in human chondrosarcoma cells. c-Met inhibitor and siRNA reduced HGF-increased cell migration and MMP-2 expression. HGF treatment resulted in activation of the phosphatidylinositol 3′-kinase (PI3K)/Akt/PKCδ/NF-κB pathway, and HGF-induced expression of MMP-2 and cell migration was inhibited by specific inhibitors or siRNA-knockdown of PI3K, Akt, PKCδ, and NF-κB cascades. Taken together, our results indicated that HGF enhances migration of chondrosarcoma cells by increasing MMP-2 expression through the c-Met receptor/PI3K/Akt/PKCδ/NF-κB signal transduction pathway. PMID:23320110

Method to study cell migration under uniaxial compression

PubMed Central

Srivastava, Nishit; Kay, Robert R.; Kabla, Alexandre J.

2017-01-01

The chemical, physical, and mechanical properties of the extracellular environment have a strong effect on cell migration. Aspects such as pore size or stiffness of the matrix influence the selection of the mechanism used by cells to propel themselves, including by pseudopods or blebbing. How a cell perceives its environment and how such a cue triggers a change in behavior are largely unknown, but mechanics is likely to be involved. Because mechanical conditions are often controlled by modifying the composition of the environment, separating chemical and physical contributions is difficult and requires multiple controls. Here we propose a simple method to impose a mechanical compression on individual cells without altering the composition of the matrix. Live imaging during compression provides accurate information about the cell's morphology and migratory phenotype. Using Dictyostelium as a model, we observe that a compression of the order of 500 Pa flattens the cells under gel by up to 50%. This uniaxial compression directly triggers a transition in the mode of migration from primarily pseudopodial to bleb driven in <30 s. This novel device is therefore capable of influencing cell migration in real time and offers a convenient approach with which to systematically study mechanotransduction in confined environments. PMID:28122819

Cell migration or cytokinesis and proliferation? – Revisiting the “go or grow” hypothesis in cancer cells in vitro

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

Garay, Tamás; Juhász, Éva; Molnár, Eszter

The mortality of patients with solid tumors is mostly due to metastasis that relies on the interplay between migration and proliferation. The “go or grow” hypothesis postulates that migration and proliferation spatiotemporally excludes each other. We evaluated this hypothesis on 35 cell lines (12 mesothelioma, 13 melanoma and 10 lung cancer) on both the individual cell and population levels. Following three-day-long videomicroscopy, migration, proliferation and cytokinesis-length were quantified. We found a significantly higher migration in mesothelioma cells compared to melanoma and lung cancer while tumor types did not differ in mean proliferation or duration of cytokinesis. Strikingly, we found inmore » melanoma and lung cancer a significant positive correlation between mean proliferation and migration. Furthermore, non-dividing melanoma and lung cancer cells displayed slower migration. In contrast, in mesothelioma there were no such correlations. Interestingly, negative correlation was found between cytokinesis-length and migration in melanoma. FAK activation was higher in melanoma cells with high motility. We demonstrate that the cancer cells studied do not defer proliferation for migration. Of note, tumor cells from various organ systems may differently regulate migration and proliferation. Furthermore, our data is in line with the observation of pathologists that highly proliferative tumors are often highly invasive. - Highlights: • We investigated the “go or grow” hypothesis in human cancer cells in vitro. • Proliferation and migration positively correlate in melanoma and lung cancer cells. • Duration of cytokinesis and migration shows inverse correlation. • Increased FAK activation is present in highly motile melanoma cells.« less

Cadherin-11 Mediates Contact Inhibition of Locomotion during Xenopus Neural Crest Cell Migration

PubMed Central

Becker, Sarah F. S.; Mayor, Roberto; Kashef, Jubin

2013-01-01

Collective cell migration is an essential feature both in embryonic development and cancer progression. The molecular mechanisms of these coordinated directional cell movements still need to be elucidated. The migration of cranial neural crest (CNC) cells during embryogenesis is an excellent model for collective cell migration in vivo. These highly motile and multipotent cells migrate directionally on defined routes throughout the embryo. Interestingly, local cell-cell interactions seem to be the key force for directionality. CNC cells can change their migration direction by a repulsive cell response called contact inhibition of locomotion (CIL). Cell protrusions collapse upon homotypic cell-cell contact and internal repolarization leads to formation of new protrusions toward cell-free regions. Wnt/PCP signaling was shown to mediate activation of small RhoGTPase RhoA and inhibition of cell protrusions at the contact side. However, the mechanism how a cell recognizes the contact is poorly understood. Here, we demonstrate that Xenopus cadherin-11 (Xcad-11) mediated cell-cell adhesion is necessary in CIL for directional and collective migration of CNC cells. Reduction of Xcad-11 adhesive function resulted in higher invasiveness of CNC due to loss of CIL. Additionally, transplantation analyses revealed that CNC migratory behaviour in vivo is non-directional and incomplete when Xcad-11 adhesive function is impaired. Blocking Wnt/PCP signaling led to similar results underlining the importance of Xcad-11 in the mechanism of CIL and directional migration of CNC. PMID:24392028

Looking into the future: Using induced pluripotent stem cells to build two and three dimensional ocular tissue for cell therapy and disease modeling.

PubMed

Song, Min Jae; Bharti, Kapil

2016-05-01

Retinal degenerative diseases are the leading cause of irreversible vision loss in developed countries. In many cases the diseases originate in the homeostatic unit in the back of the eye that contains the retina, retinal pigment epithelium (RPE) and the choriocapillaris. RPE is a central and a critical component of this homeostatic unit, maintaining photoreceptor function and survival on the apical side and choriocapillaris health on the basal side. In diseases like age-related macular degeneration (AMD), it is thought that RPE dysfunctions cause disease-initiating events and as the RPE degenerates photoreceptors begin to die and patients start loosing vision. Patient-specific induced pluripotent stem (iPS) cell-derived RPE provides direct access to a patient's genetics and allow the possibility of identifying the initiating events of RPE-associated degenerative diseases. Furthermore, iPS cell-derived RPE cells are being tested as a potential cell replacement in disease stages with RPE atrophy. In this article we summarize the recent progress in the field of iPS cell-derived RPE "disease modeling" and cell therapies and also discuss the possibilities of developing a model of the entire homeostatic unit to aid in studying disease processes in the future. This article is part of a Special Issue entitled SI: PSC and the brain. Published by Elsevier B.V.

Cell intrinsic modulation of Wnt signaling controls neuroblast migration in C. elegans.

PubMed

Mentink, Remco A; Middelkoop, Teije C; Rella, Lorenzo; Ji, Ni; Tang, Chung Yin; Betist, Marco C; van Oudenaarden, Alexander; Korswagen, Hendrik C

2014-10-27

Members of the Wnt family of secreted signaling proteins are key regulators of cell migration and axon guidance. In the nematode C. elegans, the migration of the QR neuroblast descendants requires multiple Wnt ligands and receptors. We found that the migration of the QR descendants is divided into three sequential phases that are each mediated by a distinct Wnt signaling mechanism. Importantly, the transition from the first to the second phase, which is the main determinant of the final position of the QR descendants along the anteroposterior body axis, is mediated through a cell-autonomous process in which the time-dependent expression of a Wnt receptor turns on the canonical Wnt/β-catenin signaling response that is required to terminate long-range anterior migration. Our results show that, in addition to direct guidance of cell migration by Wnt morphogenic gradients, cell migration can also be controlled indirectly through cell-intrinsic modulation of Wnt signaling responses.

Endothelial Cell Morphology and Migration are Altered by Changes in Gravitational Fields

NASA Technical Reports Server (NTRS)

Melhado, Caroline; Sanford, Gary; Harris-Hooker, Sandra

1997-01-01

Endothelial cell migration is important to vascular wall regeneration following injury or stress. However, the mechanism(s) governing this response is not well understood. The microgravity environment of space may complicate the response of these cells to injury. To date, there are no reports in this area. We examined how bovine aortic (BAEC) and pulmonary (BPEC) endothelial cells respond to denudation injury under hypergravity (HGrav) and simulated microgravity (MGrav), using image analysis. In 10% FBS, the migration of confluent BAEC and BPEC into the denuded area was not affected by HGrav or MGrav. However, in low FBS (0.5%), signficantly retarded migration under MGrav, and increased migration under HGrav was found. MGrav also decreased the migration of postconfluent BPEC while HGrav showed no difference. Both MGrav and HGrav strongly decreased the migration of postconfluent BAEC. Also, both cell lines showed significant morphological changes by scanning electron microscopy. These studies indicate that endothelial cell function is affected by changes in gravity.

Cytoglobin inhibits migration through PI3K/AKT/mTOR pathway in fibroblast cells.

PubMed

Demirci, Selami; Doğan, Ayşegül; Apdik, Hüseyin; Tuysuz, Emre Can; Gulluoglu, Sukru; Bayrak, Omer Faruk; Şahin, Fikrettin

2018-01-01

Cell proliferation and migration are crucial in many physiological processes including development, cancer, tissue repair, and wound healing. Cell migration is regulated by several signaling molecules. Identification of genes related to cell migration is required to understand molecular mechanism of non-healing chronic wounds which is a major concern in clinics. In the current study, the role of cytoglobin (CYGB) gene in fıbroblast cell migration and proliferation was described. L929 mouse fibroblast cells were transduced with lentiviral particles for CYGB and GFP, and analyzed for cell proliferation and migration ability. Fibroblast cells overexpressing CYGB displayed decreased cell proliferation, colony formation capacity, and cell migration. Phosphorylation levels of mTOR and two downstream effectors S6 and 4E-BP1 which take part in PI3K/AKT/mTOR signaling declined in CYGB-overexpressing cells. Microarray analysis indicated that CYGB overexpression leads to downregulation of cell proliferation, migration, and tumor growth associated genes in L929 cell line. This study demonstrated the role of CYGB in fibroblast cell motility and proliferation. CYGB could be a promising candidate for further studies as a potential target for diseases related to cell migration such as cancer and chronic wound treatment.

Directional cell migration in an extracellular pH gradient: a model study with an engineered cell line and primary microvascular endothelial cells.

PubMed

Paradise, Ranjani K; Whitfield, Matthew J; Lauffenburger, Douglas A; Van Vliet, Krystyn J

2013-02-15

Extracellular pH (pH(e)) gradients are characteristic of tumor and wound environments. Cell migration in these environments is critical to tumor progression and wound healing. While it has been shown previously that cell migration can be modulated in conditions of spatially invariant acidic pH(e) due to acid-induced activation of cell surface integrin receptors, the effects of pH(e) gradients on cell migration remain unknown. Here, we investigate cell migration in an extracellular pH(e) gradient, using both model α(v)β(3) CHO-B2 cells and primary microvascular endothelial cells. For both cell types, we find that the mean cell position shifts toward the acidic end of the gradient over time, and that cells preferentially polarize toward the acidic end of the gradient during migration. We further demonstrate that cell membrane protrusion stability and actin-integrin adhesion complex formation are increased in acidic pH(e), which could contribute to the preferential polarization toward acidic pH(e) that we observed for cells in pH(e) gradients. These results provide the first demonstration of preferential cell migration toward acid in a pH(e) gradient, with intriguing implications for directed cell migration in the tumor and wound healing environments. Copyright © 2012 Elsevier Inc. All rights reserved.

The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells.

PubMed

Shao, Genbao; Wang, Ranran; Sun, Aiqin; Wei, Jing; Peng, Ke; Dai, Qian; Yang, Wannian; Lin, Qiong

2018-02-19

EGFR-dependent cell migration plays an important role in lung cancer progression. Our previous study observed that the HECT E3 ubiquitin ligase NEDD4 is significantly correlated with tumor metastasis and required for migration and invasion signaling of EGFR in gastric cancer cells. However, how NEDD4 promotes the EGFR-dependent lung cancer cell migration is unknown. This study is to elucidate the mechanism by which NEDD4 mediates the EGFR lung cancer migration signaling. Lentiviral vector-loaded NEDD4 shRNA was used to deplete endogenous NEDD4 in lung cancer cell lines. Effects of the NEDD4 knockdown on the EGFR-dependent or independent lung cancer cell migration were determined using the wound-healing and transwell assays. Association of NEDD4 with activated EGFR was assayed by co-immunoprecipitation. Co-expression of NEDD4 with EGFR or PTEN was determined by immunohistochemical (IHC) staining in 63 lung adenocarcinoma tissue samples. Effects of NEDD4 ectopic expression or knockdown on PTEN ubiquitination and down-regulation, AKT activation and lysosomal secretion were examined using the GST-Uba pulldown assay, immunoblotting, immunofluorescent staining and a human cathepsin B ELISA assay respectively. The specific cathepsin B inhibitor CA-074Me was used for assessing the role of cathepsin B in lung cancer cell migration. Knockdown of NEDD4 significantly reduced EGF-stimulated cell migration in non-small cell lung carcinoma (NSCLC) cells. Co-immunoprecipitation assay found that NEDD4 is associated with EGFR complex upon EGF stimulation, and IHC staining indicates that NEDD4 is co-expressed with EGFR in lung adenocarcinoma tumor tissues, suggesting that NEDD4 might mediate lung cancer cell migration by interaction with the EGFR signaling complex. Interestingly, NEDD4 promotes the EGF-induced cathepsin B secretion, possibly through lysosomal exocytosis, as overexpression of the ligase-dead mutant of NEDD4 impedes lysosomal secretion, and knockdown of NEDD4

Subducted and melanotic cells in advanced age-related macular degeneration are derived from retinal pigment epithelium.

PubMed

Zanzottera, Emma C; Messinger, Jeffrey D; Ach, Thomas; Smith, R Theodore; Curcio, Christine A

2015-05-01

To describe, illustrate, and account for two cell types plausibly derived from RPE in geographic atrophy (GA) and choroidal neovascularization (CNV) of AMD, using melanosomes, lipofuscin, and basal laminar deposit (BLamD) as anatomical markers. Human donor eyes with GA (n = 13) or CNV (n = 39) were histologically processed, photodocumented, and analyzed for frequencies of occurrence. We defined RPE as cells containing spindle-shaped melanosomes and RPE lipofuscin, internal to basal lamina or BLamD, if present, or Bruch's membrane if not, and RPE-derived cells as those plausibly derived from RPE and not attached to basal lamina or BLamD. 'Subducted' cells contain RPE melanosomes and localize to the sub-RPE space, on Bruch's membrane. Credible transitional forms from RPE cells were seen. Grades of RPE overlying 'Subducted' cells were 'Atrophic with BLamD' (32.2% vs. 37.0% of 'Subducted,' for GA and CNV eyes, respectively), 'Dissociated' (22.0% vs. 21.7%), 'Nonuniform' (22.0% vs. 23.9%), and 'Sloughed' RPE (10.2% vs. 4.3%). Found exclusively in CNV scars, 'Melanotic' cells containing spherical melanosomes were adjacent to 'Entombed' RPE with spindle-shaped and spherical melanosomes. Of subretinal 'Melanotic' cells, 40.0% associated with 'Atrophy with BLamD,' 36.8% with 'Atrophy without BLamD,' and 20.6% with 'Entombed.' 'Dissociated' RPE within atrophic areas may be the source of 'Subducted' cells. 'Entombed' RPE within fibrovascular and fibrocellular scars may be the source of 'Melanotic' cells. An imaging correlate for 'Subducted' cells awaits discovery; 'Melanotic' cells appear gray-black in the CNV fundus. Results provide a basis for future molecular phenotyping studies.

The effects of acoustic vibration on fibroblast cell migration.

PubMed

Mohammed, Taybia; Murphy, Mark F; Lilley, Francis; Burton, David R; Bezombes, Frederic

2016-12-01

Cells are known to interact and respond to external mechanical cues and recent work has shown that application of mechanical stimulation, delivered via acoustic vibration, can be used to control complex cell behaviours. Fibroblast cells are known to respond to physical cues generated in the extracellular matrix and it is thought that such cues are important regulators of the wound healing process. Many conditions are associated with poor wound healing, so there is need for treatments/interventions, which can help accelerate the wound healing process. The primary aim of this research was to investigate the effects of mechanical stimulation upon the migratory and morphological properties of two different fibroblast cells namely; human lung fibroblast cells (LL24) and subcutaneous areolar/adipose mouse fibroblast cells (L929). Using a speaker-based system, the effects of mechanical stimulation (0-1600Hz for 5min) on the mean cell migration distance (μm) and actin organisation was investigated. The results show that 100Hz acoustic vibration enhanced cell migration for both cell lines whereas acoustic vibration above 100Hz was found to decrease cell migration in a frequency dependent manner. Mechanical stimulation was also found to promote changes to the morphology of both cell lines, particularly the formation of lamellipodia and filopodia. Overall lamellipodia was the most prominent actin structure displayed by the lung cell (LL24), whereas filopodia was the most prominent actin feature displayed by the fibroblast derived from subcutaneous areolar/adipose tissue. Mechanical stimulation at all the frequencies used here was found not to affect cell viability. These results suggest that low-frequency acoustic vibration may be used as a tool to manipulate the mechanosensitivity of cells to promote cell migration. Copyright © 2016 Elsevier B.V. All rights reserved.

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity

PubMed Central

Danson, Christopher M.; Pocha, Shirin M.; Bloomberg, Graham B.; Cory, Giles O.

2009-01-01

Summary The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration. PMID:18032787

Phosphorylation of WAVE2 by MAP kinases regulates persistent cell migration and polarity.

PubMed

Danson, Christopher M; Pocha, Shirin M; Bloomberg, Graham B; Cory, Giles O

2007-12-01

The WAVE family of proteins has long been implicated in the stimulus-dependent generation of lamellipodia at the leading edge of migrating cells, with WAVE2 in particular implicated in the formation of peripheral ruffles and chemotactic migration. However, the lack of direct visualisation of cell migration in WAVE2 mutants or knockdowns has made defining the mechanisms of WAVE2 regulation during cell migration difficult. We have characterised three MAP kinase phosphorylation sites within WAVE2 and analysed fibroblast behaviour in a scratch-wound model following introduction of transgenes encoding phospho-defective WAVE2. The cells exhibited an increase in migration speed, a decrease in the persistence of migration, and disruption of polarisation of the Golgi apparatus. All these effects could be mimicked by acute knockdown of endogenous WAVE2 expression with RNAi, indicating that phosphorylation of WAVE2 by MAP kinases regulates cell polarity during migration.

Human amniotic fluid promotes retinal pigmented epithelial cells' trans-differentiation into rod photoreceptors and retinal ganglion cells.

PubMed

Ghaderi, Shima; Soheili, Zahra-Soheila; Ahmadieh, Hamid; Davari, Maliheh; Jahromi, Fatemeh Sanie; Samie, Shahram; Rezaie-Kanavi, Mozhgan; Pakravesh, Jalil; Deezagi, Abdolkhalegh

2011-09-01

To evaluate the effect of human amniotic fluid (HAF) on retinal pigmented epithelial cells growth and trans-differentiation into retinal neurons, retinal pigmented epithelium (RPE) cells were isolated from neonatal human cadaver eye globes and cultured in Dulbecco's modified Eagle's medium-F12 supplemented with 10% fetal bovine serum (FBS). Confluent monolayer cultures were trypsinized and passaged using FBS-containing or HAF-containing media. Amniotic fluid samples were received from pregnant women in the first trimester of gestation. Cell proliferation and death enzyme-linked immunosorbent assays were performed to assess the effect of HAF on RPE cell growth. Trans-differentiation into rod photoreceptors and retinal ganglion cells was also studied using immunocytochemistry and real-time polymerase chain reaction techniques. Primary cultures of RPE cells were successfully established under FBS-containing or HAF-containing media leading to rapid cell growth and proliferation. When RPE cells were moved to in vitro culture system, they began to lose their differentiation markers such as pigmentation and RPE65 marker and trans-differentiated neural-like cells followed by spheroid colonies pertaining to stem/progenitor cells were morphologically detected. Immunocytochemistry (ICC) analysis of HAF-treated cultures showed a considerable expression of Rhodopsin gene (30% Rhodopsin-positive cells) indicating trans-differentiation of RPE cells to rod photoreceptors. Real-time polymerase chain reaction revealed an HAF-dose-dependant expression of Thy-1 gene (RGC marker) and significant promoting effect of HAF on RGCs generation. The data presented here suggest that HAF possesses invaluable stimulatory effect on RPE cells growth and trans-differentiation into retinal neurons. It can be regarded as a newly introduced enriched supplement in serum-free kinds of media used in neuro-retinal regeneration studies.

Live-cell imaging of migrating cells expressing fluorescently-tagged proteins in a three-dimensional matrix.

PubMed

Shih, Wenting; Yamada, Soichiro

2011-12-22

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network. By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and

Cell migration without a lamellipodium: translation of actin dynamics into cell movement mediated by tropomyosin.

PubMed

Gupton, Stephanie L; Anderson, Karen L; Kole, Thomas P; Fischer, Robert S; Ponti, Aaron; Hitchcock-DeGregori, Sarah E; Danuser, Gaudenz; Fowler, Velia M; Wirtz, Denis; Hanein, Dorit; Waterman-Storer, Clare M

2005-02-14

The actin cytoskeleton is locally regulated for functional specializations for cell motility. Using quantitative fluorescent speckle microscopy (qFSM) of migrating epithelial cells, we previously defined two distinct F-actin networks based on their F-actin-binding proteins and distinct patterns of F-actin turnover and movement. The lamellipodium consists of a treadmilling F-actin array with rapid polymerization-dependent retrograde flow and contains high concentrations of Arp2/3 and ADF/cofilin, whereas the lamella exhibits spatially random punctae of F-actin assembly and disassembly with slow myosin-mediated retrograde flow and contains myosin II and tropomyosin (TM). In this paper, we microinjected skeletal muscle alphaTM into epithelial cells, and using qFSM, electron microscopy, and immunolocalization show that this inhibits functional lamellipodium formation. Cells with inhibited lamellipodia exhibit persistent leading edge protrusion and rapid cell migration. Inhibition of endogenous long TM isoforms alters protrusion persistence. Thus, cells can migrate with inhibited lamellipodia, and we suggest that TM is a major regulator of F-actin functional specialization in migrating cells.

In vitro measurements of oxygen consumption rates in hTERT-RPE cells exposed to low levels of red light

NASA Astrophysics Data System (ADS)

Wigle, Jeffrey C.; Castellanos, Cherry C.

2016-03-01

Exposure to 2.88 J/cm2 of red light induces an adaptive response against a lethal pulse of 2.0 μm laser radiation in hTERT-RPE cells in vitro, but not in a knockdown mutant for vascular endothelial growth factor c (VEGF-C). The generally accepted initiation sequence for photobiomodulation is that absorption of red light by cytochome c oxidase (CCOX) of the electron transport chain increases the binding affinity of CCOX for O2 vs. nitric oxide (NO). This results in displacement of NO by O2 in the active site of CCOX, thereby increasing cellular respiration and intracellular ATP. We've previously reported that red-light exposure induces a small, but consistently reproducible, increase in NO levels in these cells. But the relative importance of NO and oxidative phosphorylation is unclear because little is known about the relative contributions of NO and ATP to the response. However, if NO dissociation from CCOX actually increases oxidative phosphorylation, one should see a corresponding increase in oxygen consumption. A Seahorse Extracellular Flux Analyzer was used to measure oxygen consumption rates (OCR) in normal and mutant cells as a proxy for oxidative phosphorylation. Both basal respiration and maximum respiration rates in normal cells are significantly higher than in the mutant. The normal cells have a significant amount of "excess capacity," whereas the VEGF-C(KD) have little or none. The OCR in exposed normal cells is lower than in unexposed cells when measured immediately after exposure. The exposures used for these experiments had no effect on the OCR in mutant cells.

Migrating Myeloid Cells Sense Temporal Dynamics of Chemoattractant Concentrations.

PubMed

Petrie Aronin, Caren E; Zhao, Yun M; Yoon, Justine S; Morgan, Nicole Y; Prüstel, Thorsten; Germain, Ronald N; Meier-Schellersheim, Martin

2017-11-21

Chemoattractant-mediated recruitment of hematopoietic cells to sites of pathogen growth or tissue damage is critical to host defense and organ homeostasis. Chemotaxis is typically considered to rely on spatial sensing, with cells following concentration gradients as long as these are present. Utilizing a microfluidic approach, we found that stable gradients of intermediate chemokines (CCL19 and CXCL12) failed to promote persistent directional migration of dendritic cells or neutrophils. Instead, rising chemokine concentrations were needed, implying that temporal sensing mechanisms controlled prolonged responses to these ligands. This behavior was found to depend on G-coupled receptor kinase-mediated negative regulation of receptor signaling and contrasted with responses to an end agonist chemoattractant (C5a), for which a stable gradient led to persistent migration. These findings identify temporal sensing as a key requirement for long-range myeloid cell migration to intermediate chemokines and provide insights into the mechanisms controlling immune cell motility in complex tissue environments. Published by Elsevier Inc.

Envisioning migration: mathematics in both experimental analysis and modeling of cell behavior.

PubMed

Zhang, Elizabeth R; Wu, Lani F; Altschuler, Steven J

2013-10-01

The complex nature of cell migration highlights the power and challenges of applying mathematics to biological studies. Mathematics may be used to create model equations that recapitulate migration, which can predict phenomena not easily uncovered by experiments or intuition alone. Alternatively, mathematics may be applied to interpreting complex data sets with better resolution--potentially empowering scientists to discern subtle patterns amid the noise and heterogeneity typical of migrating cells. Iteration between these two methods is necessary in order to reveal connections within the cell migration signaling network, as well as to understand the behavior that arises from those connections. Here, we review recent quantitative analysis and mathematical modeling approaches to the cell migration problem. Copyright © 2013 Elsevier Ltd. All rights reserved.

Session-RPE for quantifying the load of different youth basketball training sessions.

PubMed

Lupo, C; Tessitore, A; Gasperi, L; Gomez, Mar

2017-03-01

The aim of the study was to evaluate youth basketball training, verifying the reliability of the session-RPE method in relation to session duration (< and ≥ 80 minutes) and workout typology (reduced and high warm-up, conditioning, technical, tactical, game portions within a single session) categories. Six male youth basketball players (age, 16.5±0.5 years; height, 195.5±6.75 cm; body mass, 93.9±10.9 kg; and body mass index, 23.6±2.8 kg.m -2 ) were monitored (HR, type and duration of workouts) during 15 (66 individual) training sessions (80±26 minutes). Edwards' HR method was used as a reference measure of internal training load (ITL); the CR-10 RPE scale was administered 30 minutes after the end of each session. The results obtained showed that all comparisons between different session durations and workout portions revealed effects in term of Edwards' ITLs except for warm-up portions. Moderate to strong relationships between Edwards' and session- RPE methods emerged for all sessions (r = .85, P < .001), player's sessions (r range = .79 - .95, P < .001), session durations (< 80 minutes: r = .67, P < .001; ≥ 80 minutes: r = .75, P < .001), and workout portions (r range = .78 - .89, P range = .002 - < .001). The findings indicated that coaches of youth basketball players can successfully use session-RPE to monitor the ITL, regardless of session durations and workout portions.

Mast cell migration to Th2 stimulated airway smooth muscle from asthmatics

PubMed Central

Sutcliffe, A; Kaur, D; Page, S; Woodman, L; Armour, C L; Baraket, M; Bradding, P; Hughes, J M; Brightling, C E

2006-01-01

Background Mast cell microlocalisation within the airway smooth muscle (ASM) bundle is an important determinant of the asthmatic phenotype. We hypothesised that mast cells migrate towards ASM in response to ASM derived chemokines. Methods Primary ASM cultures from subjects with and without asthma were stimulated with interleukin (IL)‐1β, IL‐4, and IL‐13 alone and in combination. Mast cell chemotaxis towards these ASM supernatants was investigated, and the chemotaxins mediating migration by using specific blocking antibodies for stem cell factor (SCF) and the chemokine receptors CCR3, CXCR1, 3 and 4 as well as the Gi inhibitor pertussis toxin and the tyrosine kinase inhibitor genistein were defined. The concentrations of CCL11, CXCL8, CXCL10, TGF‐β, and SCF in the supernatants were measured and the effect of non‐asthmatic ASM supernatants on the mast cell chemotactic activity of asthmatic ASM was examined. Results Human lung mast cells and HMC‐1 cells migrated towards Th2 stimulated ASM from asthmatics but not non‐asthmatics. Mast cell migration was mediated through the combined activation of CCR3 and CXCR1. CCL11 and CXCL8 expression by ASM increased markedly after stimulation, but was similar in those with and without asthma. ASM supernatants from non‐asthmatics inhibited mast cell migration towards the asthmatic ASM supernatant. Conclusion Th2 stimulated ASM from asthmatics is chemotactic for mast cells. Non‐asthmatic ASM releases a mediator or mediators that inhibit mast cell migration towards stimulated asthmatic ASM. Specifically targeting mast cell migration into the ASM bundle may provide a novel treatment for asthma. PMID:16601090

G protein-coupled receptor kinase 2 positively regulates epithelial cell migration

PubMed Central

Penela, Petronila; Ribas, Catalina; Aymerich, Ivette; Eijkelkamp, Niels; Barreiro, Olga; Heijnen, Cobi J; Kavelaars, Annemieke; Sánchez-Madrid, Francisco; Mayor, Federico

2008-01-01

Cell migration requires integration of signals arising from both the extracellular matrix and messengers acting through G protein-coupled receptors (GPCRs). We find that increased levels of G protein-coupled receptor kinase 2 (GRK2), a key player in GPCR regulation, potentiate migration of epithelial cells towards fibronectin, whereas such process is decreased in embryonic fibroblasts from hemizygous GRK2 mice or upon knockdown of GRK2 expression. Interestingly, the GRK2 effect on fibronectin-mediated cell migration involves the paracrine/autocrine activation of a sphingosine-1-phosphate (S1P) Gi-coupled GPCR. GRK2 positively modulates the activity of the Rac/PAK/MEK/ERK pathway in response to adhesion and S1P by a mechanism involving the phosphorylation-dependent, dynamic interaction of GRK2 with GIT1, a key scaffolding protein in cell migration processes. Furthermore, decreased GRK2 levels in hemizygous mice result in delayed wound healing rate in vivo, consistent with a physiological role of GRK2 as a regulator of coordinated integrin and GPCR-directed epithelial cell migration. PMID:18369319

Impaired SIRT1 promotes the migration of vascular smooth muscle cell-derived foam cells.

PubMed

Zhang, Ming-Jie; Zhou, Yi; Chen, Lei; Wang, Xu; Pi, Yan; Long, Chun-Yan; Sun, Meng-Jiao; Chen, Xue; Gao, Chang-Yue; Li, Jing-Cheng; Zhang, Li-Li

2016-07-01

The formation of fat-laden foam cells, contributing to the fatty streaks of the plaques of atheroma, is the critical early process in atherosclerosis. The previous study demonstrated that vascular smooth muscle cells (VSMCs) contain a much larger burden of the excess cholesterol in comparison with monocyte-derived macrophages in human coronary atherosclerosis, as the main origin of foam cells. It is noteworthy that VSMC-derived foam cells are deposited in subintima but not media, where VSMCs normally deposit in. Therefore, migration from media to intima is an indispensable step for a VSMC to accrue neutral lipids and form foam cell. Whether this migration occurs paralleled with or prior to the formation of foam cell is still unclear. Herein, the present study was designed to test the VSMC migratory capability in the process of foam cell formation induced by oxidized low-density lipoprotein (oxLDL). In conclusion, we provide evidence that oxLDL induces the VSMC-derived foam cells formation with increased migration ability and MMP-9 expression, which were partly attributed to the impaired SIRT1 and enhanced nuclear factor-kappa B (NF-κB) activity. As activation of transient receptor potential vanilloid type 1 (TRPV1) has been reported to have anti-atherosclerotic effects, we investigated its role in oxLDL-treated VSMC migration. It is found that activating TRPV1 by capsaicin inhibits VSMC foam cell formation and the accompanied migration through rescuing the SIRT1 and suppressing NF-κB signaling. The present study provides evidence that SIRT1 may be a promising intervention target of atherosclerosis, and raises the prospect of TRPV1 in prevention and treatment of atherosclerosis.

Morphological and functional rescue in RCS rats after RPE cell line transplantation at a later stage of degeneration.

PubMed

Wang, Shaomei; Lu, Bin; Girman, Sergej; Holmes, Toby; Bischoff, Nicolas; Lund, Raymond D

2008-01-01

It is well documented that grafting of cells in the subretinal space of Royal College of Surgeons (RCS) rats limits deterioration of vision and loss of photoreceptors if performed early in postnatal life. What is unclear is whether cells introduced later, when photoreceptor degeneration is already advanced, can still be effective. This possibility was examined in the present study, using the human retinal pigment epithelial cell line, ARPE-19. Dystrophic RCS rats (postnatal day [P] 60) received subretinal injection of ARPE-19 cells (2 x 10(5)/3 microL/eye). Spatial frequency was measured by recording optomotor responses at P100 and P150, and luminance threshold responses were recorded from the superior colliculus at P150. Retinas were stained with cresyl violet, retinal cell-specific markers, and a human nuclear marker. Control animals were injected with medium alone. Animals comparably treated with grafts at P21 were available for comparison. All animals were treated with immunosuppression. Later grafts preserved both spatial frequency and threshold responses over the control and delayed photoreceptor degeneration. There were two to three layers of rescued photoreceptors even at P150, compared with a scattered single layer in sham and untreated control retinas. Retinal cell marker staining showed an orderly array of the inner retinal lamination. The morphology of the second-order neurons was better preserved around the grafted area than in regions distant from graft. Sham injection had little effect in rescuing the photoreceptors. RPE cell line transplants delivered later in the course of degeneration can preserve not only the photoreceptors and inner retinal lamination but also visual function in RCS rats. However, early intervention can achieve better rescue.

Epithelial-mesenchymal transition transcription factors control pluripotent adult stem cell migration in vivo in planarians

PubMed Central

Abnave, Prasad; Aboukhatwa, Ellen; Kosaka, Nobuyoshi; Thompson, James; Hill, Mark A.

2017-01-01

Migration of stem cells underpins the physiology of metazoan animals. For tissues to be maintained, stem cells and their progeny must migrate and differentiate in the correct positions. This need is even more acute after tissue damage by wounding or pathogenic infection. Inappropriate migration also underpins metastasis. Despite this, few mechanistic studies address stem cell migration during repair or homeostasis in adult tissues. Here, we present a shielded X-ray irradiation assay that allows us to follow stem cell migration in planarians. We demonstrate the use of this system to study the molecular control of stem cell migration and show that snail-1, snail-2 and zeb-1 EMT transcription factor homologs are necessary for cell migration to wound sites and for the establishment of migratory cell morphology. We also observed that stem cells undergo homeostatic migration to anterior regions that lack local stem cells, in the absence of injury, maintaining tissue homeostasis. This requires the polarity determinant notum. Our work establishes planarians as a suitable model for further in-depth study of the processes controlling stem cell migration in vivo. PMID:28893948

Reelin is involved in transforming growth factor-β1-induced cell migration in esophageal carcinoma cells.

PubMed

Yuan, Yi; Chen, Hongyan; Ma, Gang; Cao, Xiaofeng; Liu, Zhihua

2012-01-01

Reelin (RELN), which is a glycoprotein secreted by Cajal-Retzius cells of the developing cerebral cortex, plays an important role in neuronal migration, but its role in cell migration and cancer metastasis is largely unclear. Here, we showed that cell motility was significantly increased in KYSE-510 cells by TGF-β1 treatment. Moreover, TGF-β1 decreased RELN mRNA expression and overexpression of Reelin at least partly reversed TGF-β1-induced cell migration in KYSE-30 cells. Furthermore, this negative regulation of Reelin expression by TGF-β1 was through Snail, one transcription factor which was induced by TGF-β1 in KYSE-510 cells. RELN promoter activity was reduced in parallel with the induction of Snail after TGF-β1 treatment and Snail suppressed both RELN promoter activity and expression through binding to E-box sequences in the RELN promoter region in ESCC cells. Knockdown of RELN induced cell migration in KYSE-510 cells, together with the increase of mesenchymal markers expression. Taken together, Reelin is an essential negative regulator in the TGF-β1-induced cell migration process, and is suppressed by TGF-β pathway at the transcriptional level through Snail regulation. Therefore, the correlation of Reelin and TGF-β pathway was critical in cancer metastasis, and Reelin could be one potential anti-metastasis target in future clinical practice.

GROα overexpression drives cell migration and invasion in triple negative breast cancer cells.

PubMed