Super-complexes of adhesion GPCRs and neural guidance receptors

NASA Astrophysics Data System (ADS)

Jackson, Verity A.; Mehmood, Shahid; Chavent, Matthieu; Roversi, Pietro; Carrasquero, Maria; Del Toro, Daniel; Seyit-Bremer, Goenuel; Ranaivoson, Fanomezana M.; Comoletti, Davide; Sansom, Mark S. P.; Robinson, Carol V.; Klein, Rüdiger; Seiradake, Elena

2016-04-01

Latrophilin adhesion-GPCRs (Lphn1-3 or ADGRL1-3) and Unc5 cell guidance receptors (Unc5A-D) interact with FLRT proteins (FLRT1-3), thereby promoting cell adhesion and repulsion, respectively. How the three proteins interact and function simultaneously is poorly understood. We show that Unc5D interacts with FLRT2 in cis, controlling cell adhesion in response to externally presented Lphn3. The ectodomains of the three proteins bind cooperatively. Crystal structures of the ternary complex formed by the extracellular domains reveal that Lphn3 dimerizes when bound to FLRT2:Unc5, resulting in a stoichiometry of 1:1:2 (FLRT2:Unc5D:Lphn3). This 1:1:2 complex further dimerizes to form a larger `super-complex' (2:2:4), using a previously undescribed binding motif in the Unc5D TSP1 domain. Molecular dynamics simulations, point-directed mutagenesis and mass spectrometry demonstrate the stability and molecular properties of these complexes. Our data exemplify how receptors increase their functional repertoire by forming different context-dependent higher-order complexes.

Guidance of Axons by Local Coupling of Retrograde Flow to Point Contact Adhesions.

PubMed

Nichol, Robert H; Hagen, Kate M; Lumbard, Derek C; Dent, Erik W; Gómez, Timothy M

2016-02-17

Growth cones interact with the extracellular matrix (ECM) through integrin receptors at adhesion sites termed point contacts. Point contact adhesions link ECM proteins to the actin cytoskeleton through numerous adaptor and signaling proteins. One presumed function of growth cone point contacts is to restrain or "clutch" myosin-II-based filamentous actin (F-actin) retrograde flow (RF) to promote leading edge membrane protrusion. In motile non-neuronal cells, myosin-II binds and exerts force upon actin filaments at the leading edge, where clutching forces occur. However, in growth cones, it is unclear whether similar F-actin-clutching forces affect axon outgrowth and guidance. Here, we show in Xenopus spinal neurons that RF is reduced in rapidly migrating growth cones on laminin (LN) compared with non-integrin-binding poly-d-lysine (PDL). Moreover, acute stimulation with LN accelerates axon outgrowth over a time course that correlates with point contact formation and reduced RF. These results suggest that RF is restricted by the assembly of point contacts, which we show occurs locally by two-channel imaging of RF and paxillin. Further, using micropatterns of PDL and LN, we demonstrate that individual growth cones have differential RF rates while interacting with two distinct substrata. Opposing effects on RF rates were also observed in growth cones treated with chemoattractive and chemorepulsive axon guidance cues that influence point contact adhesions. Finally, we show that RF is significantly attenuated in vivo, suggesting that it is restrained by molecular clutching forces within the spinal cord. Together, our results suggest that local clutching of RF can control axon guidance on ECM proteins downstream of axon guidance cues. Here, we correlate point contact adhesions directly with clutching of filamentous actin retrograde flow (RF), which our findings strongly suggest guides developing axons. Acute assembly of new point contact adhesions is temporally and

Fundamentals of protein and cell interactions in biomaterials.

PubMed

Aiyelabegan, Hammed Tanimowo; Sadroddiny, Esmaeil

2017-04-01

The extracellular matrix (ECM) is an active and complex microenvironment with outstanding biomechanical, biophysical, and biochemical characteristics, which can indirectly or directly controls cell adhesion, migration, proliferation, and differentiation, as well as partaking in regeneration and homeostasis of organs and tissues. The ECM has captivated a great deal of attention with the rapid progress of tissue engineering (TE) in the field of regenerative medicine (RM). Approaches to TE, RM and cancer therapy center on the necessity to deliver cell signals to direct cell proliferation and differentiation. These "external signals" are induced from cell-cell, and cell-ECM, interactions, as well as from physico-chemical, mechanical stimuli and growth factors. With the advent of new biomaterials such as casein, we gave a general insight into cell-ECM protein interactions in biomaterials and their applications in TE, RM and cancer therapy. An account of the main ECM molecules and cellular receptors with emphasis on integrins and its ligands was given, their effect on the induction of particular signal transduction pathways is also elucidated. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Single and collective cell migration: the mechanics of adhesions

PubMed Central

De Pascalis, Chiara; Etienne-Manneville, Sandrine

2017-01-01

Chemical and physical properties of the environment control cell proliferation, differentiation, or apoptosis in the long term. However, to be able to move and migrate through a complex three-dimensional environment, cells must quickly adapt in the short term to the physical properties of their surroundings. Interactions with the extracellular matrix (ECM) occur through focal adhesions or hemidesmosomes via the engagement of integrins with fibrillar ECM proteins. Cells also interact with their neighbors, and this involves various types of intercellular adhesive structures such as tight junctions, cadherin-based adherens junctions, and desmosomes. Mechanobiology studies have shown that cell–ECM and cell–cell adhesions participate in mechanosensing to transduce mechanical cues into biochemical signals and conversely are responsible for the transmission of intracellular forces to the extracellular environment. As they migrate, cells use these adhesive structures to probe their surroundings, adapt their mechanical properties, and exert the appropriate forces required for their movements. The focus of this review is to give an overview of recent developments showing the bidirectional relationship between the physical properties of the environment and the cell mechanical responses during single and collective cell migration. PMID:28684609

The DRF motif of CXCR6 as chemokine receptor adaptation to adhesion.

PubMed

Koenen, Andrea; Babendreyer, Aaron; Schumacher, Julian; Pasqualon, Tobias; Schwarz, Nicole; Seifert, Anke; Deupi, Xavier; Ludwig, Andreas; Dreymueller, Daniela

2017-01-01

The CXC-chemokine receptor 6 (CXCR6) is a class A GTP-binding protein-coupled receptor (GPCRs) that mediates adhesion of leukocytes by interacting with the transmembrane cell surface-expressed chemokine ligand 16 (CXCL16), and also regulates leukocyte migration by interacting with the soluble shed variant of CXCL16. In contrast to virtually all other chemokine receptors with chemotactic activity, CXCR6 carries a DRF motif instead of the typical DRY motif as a key element in receptor activation and G protein coupling. In this work, modeling analyses revealed that the phenylalanine F3.51 in CXCR6 might have impact on intramolecular interactions including hydrogen bonds by this possibly changing receptor function. Initial investigations with embryonic kidney HEK293 cells and further studies with monocytic THP-1 cells showed that mutation of DRF into DRY does not influence ligand binding, receptor internalization, receptor recycling, and protein kinase B (AKT) signaling. Adhesion was slightly decreased in a time-dependent manner. However, CXCL16-induced calcium signaling and migration were increased. Vice versa, when the DRY motif of the related receptor CX3CR1 was mutated into DRF the migratory response towards CX3CL1 was diminished, indicating that the presence of a DRF motif generally impairs chemotaxis in chemokine receptors. Transmembrane and soluble CXCL16 play divergent roles in homeostasis, inflammation, and cancer, which can be beneficial or detrimental. Therefore, the DRF motif of CXCR6 may display a receptor adaptation allowing adhesion and cell retention by transmembrane CXCL16 but reducing the chemotactic response to soluble CXCL16. This adaptation may avoid permanent or uncontrolled recruitment of inflammatory cells as well as cancer metastasis.

Genistein suppresses adhesion-induced protein tyrosine phosphorylation and invasion of B16-BL6 melanoma cells.

PubMed

Yan, C; Han, R

1998-07-03

Protein tyrosine phosphorylation occurs as one of the earlier events in cancer cell-extracellular matrix (ECM) interaction. With immunoblot analysis and immunofluorescence microscopy, genistein was found to suppress the tyrosine phosphorylation of proteins located at the cell periphery, including a 125 kDa protein, when B16-BL6 melanoma cells attached to and interacted with ECM. When accompanied by the suppression of adhesion-induced protein tyrosine phosphorylation, the invasive potential of B16-BL6 cells through reconstituted basement membrane was decreased significantly. However, neither adhesive capability nor cell growth was significantly affected by genistein. Therefore, the interruption of cancer cell-ECM interaction by suppression of protein tyrosine phosphorylation may contribute to invasion prevention of genistein.

Using self-assembled monolayers to pattern ECM proteins and cells on substrates.

PubMed

Ostuni, Emanuele; Whitesides, George M; Ingber, Donald E; Chen, Christopher S

2009-01-01

We present a method that uses microcontact printing of alkanethiols on gold to generate patterned substrates presenting "islands" of extracellular matrix (ECM) surrounded by nonadhesive regions such that single cells attach and spread only on the adhesive regions. We have used this micropatterning technology to demonstrate that mammalian cells can be switched between growth and apoptosis programs in the presence of saturating concentrations of growth factors by either promoting or preventing cell spreading (Science 276:1425-1428, 1997). From the perspective of fundamental cell biology, these results suggested that the local differentials in growth and viability that are critical for the formation of complex tissue patterns may be generated by local changes in cell-ECM interactions. In the context of cell culture technologies, such as bioreactors and cellular engineering applications, the regulation of cell function by cell shape indicates that the adhesive microenvironment around cells can be carefully optimized by patterning a substrate in addition to using soluble factors (Biotech. Prog. 14:356-363, 1998). Micropatterning technology is playing a central role both in our understanding how ECM and cell shape regulate cell physiology and in facilitating the development of cellular biosensor and tissue engineering applications (Science 264:696-698, 1994; J. Neurosci. Res. 13:213-20, 1985; Biotech. Bioeng. 43:792-800, 1994).

The DRF motif of CXCR6 as chemokine receptor adaptation to adhesion

PubMed Central

Koenen, Andrea; Babendreyer, Aaron; Schumacher, Julian; Pasqualon, Tobias; Schwarz, Nicole; Seifert, Anke; Deupi, Xavier

2017-01-01

The CXC-chemokine receptor 6 (CXCR6) is a class A GTP-binding protein-coupled receptor (GPCRs) that mediates adhesion of leukocytes by interacting with the transmembrane cell surface-expressed chemokine ligand 16 (CXCL16), and also regulates leukocyte migration by interacting with the soluble shed variant of CXCL16. In contrast to virtually all other chemokine receptors with chemotactic activity, CXCR6 carries a DRF motif instead of the typical DRY motif as a key element in receptor activation and G protein coupling. In this work, modeling analyses revealed that the phenylalanine F3.51 in CXCR6 might have impact on intramolecular interactions including hydrogen bonds by this possibly changing receptor function. Initial investigations with embryonic kidney HEK293 cells and further studies with monocytic THP-1 cells showed that mutation of DRF into DRY does not influence ligand binding, receptor internalization, receptor recycling, and protein kinase B (AKT) signaling. Adhesion was slightly decreased in a time-dependent manner. However, CXCL16-induced calcium signaling and migration were increased. Vice versa, when the DRY motif of the related receptor CX3CR1 was mutated into DRF the migratory response towards CX3CL1 was diminished, indicating that the presence of a DRF motif generally impairs chemotaxis in chemokine receptors. Transmembrane and soluble CXCL16 play divergent roles in homeostasis, inflammation, and cancer, which can be beneficial or detrimental. Therefore, the DRF motif of CXCR6 may display a receptor adaptation allowing adhesion and cell retention by transmembrane CXCL16 but reducing the chemotactic response to soluble CXCL16. This adaptation may avoid permanent or uncontrolled recruitment of inflammatory cells as well as cancer metastasis. PMID:28267793

Nucleation theory with delayed interactions: An application to the early stages of the receptor-mediated adhesion/fusion kinetics of lipid vesicles

NASA Astrophysics Data System (ADS)

Raudino, Antonio; Pannuzzo, Martina

2010-01-01

A semiquantitative theory aimed to describe the adhesion kinetics between soft objects, such as living cells or vesicles, has been developed. When rigid bodies are considered, the adhesion kinetics is successfully described by the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) picture, where the energy profile of two approaching bodies is given by a two asymmetrical potential wells separated by a barrier. The transition probability from the long-distance to the short-distance minimum defines the adhesion rate. Conversely, soft bodies might follow a different pathway to reach the short-distance minimum: thermally excited fluctuations give rise to local protrusions connecting the approaching bodies. These transient adhesion sites are stabilized by short-range adhesion forces (e.g., ligand-receptor interactions between membranes brought at contact distance), while they are destabilized both by repulsive forces and by the elastic deformation energy. Above a critical area of the contact site, the adhesion forces prevail: the contact site grows in size until the complete adhesion of the two bodies inside a short-distance minimum is attained. This nucleation mechanism has been developed in the framework of a nonequilibrium Fokker-Planck picture by considering both the adhesive patch growth and dissolution processes. In addition, we also investigated the effect of the ligand-receptor pairing kinetics at the adhesion site in the time course of the patch expansion. The ratio between the ligand-receptor pairing kinetics and the expansion rate of the adhesion site is of paramount relevance in determining the overall nucleation rate. The theory enables one to self-consistently include both thermodynamics (energy barrier height) and dynamic (viscosity) parameters, giving rise in some limiting cases to simple analytical formulas. The model could be employed to rationalize fusion kinetics between vesicles, provided the short-range adhesion transition is the rate

Nucleation theory with delayed interactions: an application to the early stages of the receptor-mediated adhesion/fusion kinetics of lipid vesicles.

PubMed

Raudino, Antonio; Pannuzzo, Martina

2010-01-28

A semiquantitative theory aimed to describe the adhesion kinetics between soft objects, such as living cells or vesicles, has been developed. When rigid bodies are considered, the adhesion kinetics is successfully described by the classical Derjaguin, Landau, Verwey, and Overbeek (DLVO) picture, where the energy profile of two approaching bodies is given by a two asymmetrical potential wells separated by a barrier. The transition probability from the long-distance to the short-distance minimum defines the adhesion rate. Conversely, soft bodies might follow a different pathway to reach the short-distance minimum: thermally excited fluctuations give rise to local protrusions connecting the approaching bodies. These transient adhesion sites are stabilized by short-range adhesion forces (e.g., ligand-receptor interactions between membranes brought at contact distance), while they are destabilized both by repulsive forces and by the elastic deformation energy. Above a critical area of the contact site, the adhesion forces prevail: the contact site grows in size until the complete adhesion of the two bodies inside a short-distance minimum is attained. This nucleation mechanism has been developed in the framework of a nonequilibrium Fokker-Planck picture by considering both the adhesive patch growth and dissolution processes. In addition, we also investigated the effect of the ligand-receptor pairing kinetics at the adhesion site in the time course of the patch expansion. The ratio between the ligand-receptor pairing kinetics and the expansion rate of the adhesion site is of paramount relevance in determining the overall nucleation rate. The theory enables one to self-consistently include both thermodynamics (energy barrier height) and dynamic (viscosity) parameters, giving rise in some limiting cases to simple analytical formulas. The model could be employed to rationalize fusion kinetics between vesicles, provided the short-range adhesion transition is the rate

Hyaluronan synthase 3 (HAS3) overexpression downregulates MV3 melanoma cell proliferation, migration and adhesion

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

Takabe, Piia, E-mail: piia.takabe@uef.fi; Bart, Geneviève; Ropponen, Antti

2015-09-10

Malignant skin melanoma is one of the most deadly human cancers. Extracellular matrix (ECM) influences the growth of malignant tumors by modulating tumor cells adhesion and migration. Hyaluronan is an essential component of the ECM, and its amount is altered in many tumors, suggesting an important role for hyaluronan in tumorigenesis. Nonetheless its role in melanomagenesis is not understood. In this study we produced a MV3 melanoma cell line with inducible expression of the hyaluronan synthase 3 (HAS3) and studied its effect on the behavior of the melanoma cells. HAS3 overexpression expanded the cell surface hyaluronan coat and decreased melanomamore » cell adhesion, migration and proliferation by cell cycle arrest at G1/G0. Melanoma cell migration was restored by removal of cell surface hyaluronan by Streptomyces hyaluronidase and by receptor blocking with hyaluronan oligosaccharides, while the effect on cell proliferation was receptor independent. Overexpression of HAS3 decreased ERK1/2 phosphorylation suggesting that inhibition of MAP-kinase signaling was responsible for these suppressive effects on the malignant phenotype of MV3 melanoma cells. - Highlights: • Inducible HAS3-MV3 melanoma cell line was generated using Lentiviral transduction. • HAS3 overexpression inhibits MV3 cell migration via hyaluronan–receptor interaction. • HAS3 overexpression decreases MV3 melanoma cell proliferation and adhesion. • ERK1/2 phosphorylation is downregulated by 50% in HAS3 overexpressing cells. • The results suggest that hyaluronan has anti-cancer like effects in melanoma.« less

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri.

PubMed

Matsuo, Yosuke; Miyoshi, Yukihiro; Okada, Sanae; Satoh, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion.

Theory and simulations of adhesion receptor dimerization on membrane surfaces.

PubMed

Wu, Yinghao; Honig, Barry; Ben-Shaul, Avinoam

2013-03-19

The equilibrium constants of trans and cis dimerization of membrane bound (2D) and freely moving (3D) adhesion receptors are expressed and compared using elementary statistical-thermodynamics. Both processes are mediated by the binding of extracellular subdomains whose range of motion in the 2D environment is reduced upon dimerization, defining a thin reaction shell where dimer formation and dissociation take place. We show that the ratio between the 2D and 3D equilibrium constants can be expressed as a product of individual factors describing, respectively, the spatial ranges of motions of the adhesive domains, and their rotational freedom within the reaction shell. The results predicted by the theory are compared to those obtained from a novel, to our knowledge, dynamical simulations methodology, whereby pairs of receptors perform realistic translational, internal, and rotational motions in 2D and 3D. We use cadherins as our model system. The theory and simulations explain how the strength of cis and trans interactions of adhesive receptors are affected both by their presence in the constrained intermembrane space and by the 2D environment of membrane surfaces. Our work provides fundamental insights as to the mechanism of lateral clustering of adhesion receptors after cell-cell contact and, more generally, to the formation of lateral microclusters of proteins on cell surfaces. Copyright © 2013 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Evaluation of cell binding activities of Leptospira ECM adhesins.

PubMed

Robbins, Gregory T; Hahn, Beth L; Evangelista, Karen V; Padmore, Lavinia; Aranda, Patrick S; Coburn, Jenifer

2015-04-01

Pathogenic spirochetes of the genus Leptospira are the causative agents of leptospirosis, a zoonotic infection that occurs globally. The bacteria colonize the renal proximal tubules of many animals and are shed in the urine. Contact with the urine, or with water contaminated with the urine of infected animals can cause infection of new host animals, including humans. Mechanisms of colonization of the proximal tubule and other tissues are not known, but specific interactions between bacterial adhesins and host substrates are likely to be critical in this process. Several extracellular matrix (ECM) adhesins have been previously identified, but more recently, it has been shown that Leptospira bind more efficiently to cells than ECM. In this work, recombinant forms of five putative Leptospira ECM adhesins, namely LipL32, Loa22, OmpL1, p31/LipL45, and LenA were evaluated for binding to cells as well as an expanded variety of ECM components. Reproducible and significant adhesin activity was demonstrated only for OmpL1, which bound to both mammalian cell lines tested and to glycosaminoglycans (GAGs). While determination of biologically significant bacterial adhesion activity will require generation of site-directed mutant strains, our results suggest that OmpL1 is a strong candidate for future evaluation regarding the roles of the adhesin activity of the protein during L. interrogans infection.

Evaluation of Cell Binding Activities of Leptospira ECM Adhesins

PubMed Central

Robbins, Gregory T.; Hahn, Beth L.; Evangelista, Karen V.; Padmore, Lavinia; Aranda, Patrick S.; Coburn, Jenifer

2015-01-01

Pathogenic spirochetes of the genus Leptospira are the causative agents of leptospirosis, a zoonotic infection that occurs globally. The bacteria colonize the renal proximal tubules of many animals and are shed in the urine. Contact with the urine, or with water contaminated with the urine of infected animals can cause infection of new host animals, including humans. Mechanisms of colonization of the proximal tubule and other tissues are not known, but specific interactions between bacterial adhesins and host substrates are likely to be critical in this process. Several extracellular matrix (ECM) adhesins have been previously identified, but more recently, it has been shown that Leptospira bind more efficiently to cells than ECM. In this work, recombinant forms of five putative Leptospira ECM adhesins, namely LipL32, Loa22, OmpL1, p31/LipL45, and LenA were evaluated for binding to cells as well as an expanded variety of ECM components. Reproducible and significant adhesin activity was demonstrated only for OmpL1, which bound to both mammalian cell lines tested and to glycosaminoglycans (GAGs). While determination of biologically significant bacterial adhesion activity will require generation of site-directed mutant strains, our results suggest that OmpL1 is a strong candidate for future evaluation regarding the roles of the adhesin activity of the protein during L. interrogans infection. PMID:25875373

Receptor-like Molecules on Human Intestinal Epithelial Cells Interact with an Adhesion Factor from Lactobacillus reuteri

PubMed Central

MATSUO, Yosuke; MIYOSHI, Yukihiro; OKADA, Sanae; SATOH, Eiichi

2012-01-01

A surface protein of Lactobacillus reuteri, mucus adhesion-promoting protein (MapA), is considered to be an adhesion factor. MapA is expressed in L. reuteri strains and adheres to piglet gastric mucus, collagen type I, and human intestinal epithelial cells such as Caco-2. The aim of this study was to identify molecules that mediate the attachment of MapA from L. reuteri to the intestinal epithelial cell surface by investigating the adhesion of MapA to receptor-like molecules on Caco-2 cells. MapA-binding receptor-like molecules were detected in Caco-2 cell lysates by 2D-PAGE. Two proteins, annexin A13 (ANXA13) and paralemmin (PALM), were identified by MALDI TOF-MS. The results of a pull-down assay showed that MapA bound directly to ANXA13 and PALM. Fluorescence microscopy studies confirmed that MapA binding to ANXA13 and PALM was colocalized on the Caco-2 cell membrane. To evaluate whether ANXA13 and PALM are important for MapA adhesion, ANXA13 and PALM knockdown cell lines were established. The adhesion of MapA to the abovementioned cell lines was reduced compared with that to wild-type Caco-2 cells. These knockdown experiments established the importance of these receptor-like molecules in MapA adhesion. PMID:24936355

Autophagy promotes degradation of internalized collagen and regulates distribution of focal adhesions to suppress cell adhesion

PubMed Central

Kawano, Shinichi; Esaki, Motohiro; Torisu, Kumiko; Matsuno, Yuichi; Kitazono, Takanari

2017-01-01

ABSTRACT Adhesion of cells to the extracellular matrix (ECM) via focal adhesions (FAs) is crucial for cell survival, migration, and differentiation. Although the regulation of FAs, including by integrins and the ECM, is important to cell behavior, how FAs are regulated is not well known. Autophagy is induced by both cell adhesion and cell detachment. Here, we showed that autophagosomes are located close to internalized collagen and paxillin, which is a well-known marker of FAs. Autophagy-deficient cells showed increased levels of internalized collagen compared with control cells. Moreover, paxillin exhibited a more peripheral distribution and the area of paxillin was increased, and adhesion-induced focal adhesion kinase signaling was impaired and adhesion was enhanced, in autophagy-deficient cells. These results suggest that autophagy suppressed cell adhesion by regulating internalized ECM and FAs. PMID:28970230

Local 3D matrix microenvironment regulates cell migration through spatiotemporal dynamics of contractility-dependent adhesions

NASA Astrophysics Data System (ADS)

Doyle, Andrew D.; Carvajal, Nicole; Jin, Albert; Matsumoto, Kazue; Yamada, Kenneth M.

2015-11-01

The physical properties of two-dimensional (2D) extracellular matrices (ECMs) modulate cell adhesion dynamics and motility, but little is known about the roles of local microenvironmental differences in three-dimensional (3D) ECMs. Here we generate 3D collagen gels of varying matrix microarchitectures to characterize their regulation of 3D adhesion dynamics and cell migration. ECMs containing bundled fibrils demonstrate enhanced local adhesion-scale stiffness and increased adhesion stability through balanced ECM/adhesion coupling, whereas highly pliable reticular matrices promote adhesion retraction. 3D adhesion dynamics are locally regulated by ECM rigidity together with integrin/ECM association and myosin II contractility. Unlike 2D migration, abrogating contractility stalls 3D migration regardless of ECM pore size. We find force is not required for clustering of activated integrins on 3D native collagen fibrils. We propose that efficient 3D migration requires local balancing of contractility with ECM stiffness to stabilize adhesions, which facilitates the detachment of activated integrins from ECM fibrils.

Distinct Effects of RGD-glycoproteins on Integrin-Mediated Adhesion and Osteogenic Differentiation of Human Mesenchymal Stem Cells

PubMed Central

Schwab, Elisabeth H.; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A.

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α5-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β3-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation. PMID:24324361

Distinct effects of RGD-glycoproteins on Integrin-mediated adhesion and osteogenic differentiation of human mesenchymal stem cells.

PubMed

Schwab, Elisabeth H; Halbig, Maria; Glenske, Kristina; Wagner, Alena-Svenja; Wenisch, Sabine; Cavalcanti-Adam, Elisabetta A

2013-01-01

The detailed interactions of mesenchymal stem cells (MSCs) with their extracellular matrix (ECM) and the resulting effects on MSC differentiation are still largely unknown. Integrins are the main mediators of cell-ECM interaction. In this study, we investigated the adhesion of human MSCs to fibronectin, vitronectin and osteopontin, three ECM glycoproteins which contain an integrin-binding sequence, the RGD motif. We then assayed MSCs for their osteogenic commitment in the presence of the different ECM proteins. As early as 2 hours after seeding, human MSCs displayed increased adhesion when plated on fibronectin, whereas no significant difference was observed when adhering either to vitronectin or osteopontin. Over a 10-day observation period, cell proliferation was increased when cells were cultured on fibronectin and osteopontin, albeit after 5 days in culture. The adhesive role of fibronectin was further confirmed by measurements of cell area, which was significantly increased on this type of substrate. However, integrin-mediated clusters, namely focal adhesions, were larger and more mature in MSCs adhering to vitronectin and osteopontin. Adhesion to fibronectin induced elevated expression of α₅-integrin, which was further upregulated under osteogenic conditions also for vitronectin and osteopontin. In contrast, during osteogenic differentiation the expression level of β₃-integrin was decreased in MSCs adhering to the different ECM proteins. When MSCs were cultured under osteogenic conditions, their commitment to the osteoblast lineage and their ability to form a mineralized matrix in vitro was increased in presence of fibronectin and osteopontin. Taken together these results indicate a distinct role of ECM proteins in regulating cell adhesion, lineage commitment and phenotype of MSCs, which is due to the modulation of the expression of specific integrin subunits during growth or osteogenic differentiation.

The Talin Head Domain Reinforces Integrin-Mediated Adhesion by Promoting Adhesion Complex Stability and Clustering

PubMed Central

Ellis, Stephanie J.; Lostchuck, Emily; Goult, Benjamin T.; Bouaouina, Mohamed; Fairchild, Michael J.; López-Ceballos, Pablo; Calderwood, David A.; Tanentzapf, Guy

2014-01-01

Talin serves an essential function during integrin-mediated adhesion in linking integrins to actin via the intracellular adhesion complex. In addition, the N-terminal head domain of talin regulates the affinity of integrins for their ECM-ligands, a process known as inside-out activation. We previously showed that in Drosophila, mutating the integrin binding site in the talin head domain resulted in weakened adhesion to the ECM. Intriguingly, subsequent studies showed that canonical inside-out activation of integrin might not take place in flies. Consistent with this, a mutation in talin that specifically blocks its ability to activate mammalian integrins does not significantly impinge on talin function during fly development. Here, we describe results suggesting that the talin head domain reinforces and stabilizes the integrin adhesion complex by promoting integrin clustering distinct from its ability to support inside-out activation. Specifically, we show that an allele of talin containing a mutation that disrupts intramolecular interactions within the talin head attenuates the assembly and reinforcement of the integrin adhesion complex. Importantly, we provide evidence that this mutation blocks integrin clustering in vivo. We propose that the talin head domain is essential for regulating integrin avidity in Drosophila and that this is crucial for integrin-mediated adhesion during animal development. PMID:25393120

Quantification of focal adhesion dynamics of cell movement based on cell-induced collagen matrix deformation using second-harmonic generation microscopy.

PubMed

Kang, Yong Guk; Jang, Hwanseok; Yang, Taeseok Daniel; Notbohm, Jacob; Choi, Youngwoon; Park, Yongdoo; Kim, Beop-Min

2018-06-01

Mechanical interactions of living cells with the surrounding environment via focal adhesion (FA) in three dimensions (3-D) play a key role in dynamic biological events, such as tissue regeneration, wound healing, and cancer invasion. Recently, several methods for observing 3-D cell-extracellular matrix (ECM) interactions have been reported, lacking solid and quantitative analysis on the dynamics of the physical interaction between the cell and the ECM. We measured the submicron displacements of ECM deformation in 3-D due to protrusion-retraction dynamics during cell migration, using second-harmonic generation without labeling the matrix structures. We then quantitatively analyzed the mechanical deformation between the ECM and the cells based on spatiotemporal volumetric correlations. The greatest deformations within the collagen matrix were found to occur at sites of colocalization of the FA site-related proteins vinculin and actin, which confirms that FA sites play a critical role in living cells within the ECM as a point for adhesion, traction, and migration. We believe that this modality can be used in studies of cell-ECM interaction during angiogenesis, wound healing, and metastasis. (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE).

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

PubMed

Jessen, Tammy N; Jessen, Jason R

2017-12-15

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

The FAK–Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin

PubMed Central

Swaminathan, Vinay; Fischer, R. S.; Waterman, Clare M.

2016-01-01

Cell migration is initiated in response to biochemical or physical cues in the environment that promote actin-mediated lamellipodial protrusion followed by the formation of nascent integrin adhesions (NAs) within the protrusion to drive leading edge advance. Although FAK is known to be required for cell migration through effects on focal adhesions, its role in NA formation and lamellipodial dynamics is unclear. Live-cell microscopy of FAK−/− cells with expression of phosphorylation deficient or a FERM-domain mutant deficient in Arp2/3 binding revealed a requirement for FAK in promoting the dense formation, transient stabilization, and timely turnover of NA within lamellipodia to couple actin-driven protrusion to adhesion and advance of the leading edge. Phosphorylation on Y397 of FAK promotes dense NA formation but is dispensable for transient NA stabilization and leading edge advance. In contrast, transient NA stabilization and advance of the cell edge requires FAK–Arp2/3 interaction, which promotes Arp2/3 localization to NA and reduces FAK activity. Haptosensing of extracellular matrix (ECM) concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading. Taken together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration. PMID:26842895

Binding constant of cell adhesion receptors and substrate-immobilized ligands depends on the distribution of ligands

NASA Astrophysics Data System (ADS)

Li, Long; Hu, Jinglei; Xu, Guangkui; Song, Fan

2018-01-01

Cell-cell adhesion and the adhesion of cells to tissues and extracellular matrix, which are pivotal for immune response, tissue development, and cell locomotion, depend sensitively on the binding constant of receptor and ligand molecules anchored on the apposing surfaces. An important question remains of whether the immobilization of ligands affects the affinity of binding with cell adhesion receptors. We have investigated the adhesion of multicomponent membranes to a flat substrate coated with immobile ligands using Monte Carlo simulations of a statistical mesoscopic model with biologically relevant parameters. We find that the binding of the adhesion receptors to ligands immobilized on the substrate is strongly affected by the ligand distribution. In the case of ligand clusters, the receptor-ligand binding constant can be significantly enhanced due to the less translational entropy loss of lipid-raft domains in the model cell membranes upon the formation of additional complexes. For ligands randomly or uniformly immobilized on the substrate, the binding constant is rather decreased since the receptors localized in lipid-raft domains have to pay an energetic penalty in order to bind ligands. Our findings help to understand why cell-substrate adhesion experiments for measuring the impact of lipid rafts on the receptor-ligand interactions led to contradictory results.

Cell-matrix adhesion characterization using multiple shear stress zones in single stepwise microchannel

NASA Astrophysics Data System (ADS)

Kim, Min-Ji; Doh, Il; Bae, Gab-Yong; Cha, Hyuk-Jin; Cho, Young-Ho

2014-08-01

This paper presents a cell chip capable to characterize cell-matrix adhesion by monitoring cell detachment rate. The proposed cell chip can supply multiple levels of shear stress in single stepwise microchannel. As epithelial-mesenchymal transition (EMT), one of hallmarks of cancer metastasis is closely associated to the interaction with extracelluar matrix (ECM), we took advantage of two lung cancer cell models with different adhesion properties to ECM depending their epithelial or mesenchymal properties, including the pair of lung cancer cells with (A549sh) or without E-cadherin expression (A549sh-Ecad), which would be optimal model to examine the alteration of adhesion properties after EMT induction. The cell-matrix adhesion resisting to shear stress appeared to be remarkably differed between lung cancer cells. The detachment rate of epithelial-like H358 and mesenchymal-like H460 cells was 53%-80% and 25%-66% in the shear stress range of 34-60 dyn/cm2, respectively. A549sh-Ecad cells exhibits lower detachment rate (5%-9%) compared to A549sh cells (14%-40%). By direct comparison of adhesion between A549sh and A549sh-Ecad, we demonstrated that A549shE-cad to mimic EMT were more favorable to the ECM attachment under the various levels of shear stress. The present method can be applied to quantitative analysis of tumor cell-ECM adhesion.

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins

PubMed Central

Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A.

2012-01-01

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis. PMID:22222499

Pathogenic Naegleria fowleri and non-pathogenic Naegleria lovaniensis exhibit differential adhesion to, and invasion of, extracellular matrix proteins.

PubMed

Jamerson, Melissa; da Rocha-Azevedo, Bruno; Cabral, Guy A; Marciano-Cabral, Francine

2012-03-01

Naegleria fowleri and Naegleria lovaniensis are closely related free-living amoebae found in the environment. N. fowleri causes primary amoebic meningoencephalitis (PAM), a rapidly fatal disease of the central nervous system, while N. lovaniensis is non-pathogenic. N. fowleri infection occurs when the amoebae access the nasal passages, attach to the nasal mucosa and its epithelial lining, and migrate to the brain. This process involves interaction with components of the host extracellular matrix (ECM). Since the ability to invade tissues can be a characteristic that distinguishes pathogenic from non-pathogenic amoebae, the objective of this study was to assess adhesion to, and invasion of, the ECM by these two related but distinct Naegleria species. N. fowleri exhibited a higher level of adhesion to the ECM components laminin-1, fibronectin and collagen I. Scanning electron microscopy revealed that N. fowleri attached on ECM substrata exhibited a spread-out appearance that included the presence of focal adhesion-like structures. Western immunoblotting revealed two integrin-like proteins for both species, but one of these, with a molecular mass of approximately 70 kDa, was detected at a higher level in N. fowleri. Confocal microscopy indicated that the integrin-like proteins co-localized to the focal adhesion-like structures. Furthermore, anti-integrin antibody decreased adhesion of N. fowleri to ECM components. Finally, N. fowleri disrupted 3D ECM scaffolds, while N. lovaniensis had a minimal effect. Collectively, these results indicate a distinction in adhesion to, and invasion of, ECM proteins between N. fowleri and N. lovaniensis.

Proteolytic and non-proteolytic regulation of collective cell invasion: tuning by ECM density and organization

PubMed Central

Kumar, Sandeep; Kapoor, Aastha; Desai, Sejal; Inamdar, Mandar M.; Sen, Shamik

2016-01-01

Cancer cells manoeuvre through extracellular matrices (ECMs) using different invasion modes, including single cell and collective cell invasion. These modes rely on MMP-driven ECM proteolysis to make space for cells to move. How cancer-associated alterations in ECM influence the mode of invasion remains unclear. Further, the sensitivity of the two invasion modes to MMP dynamics remains unexplored. In this paper, we address these open questions using a multiscale hybrid computational model combining ECM density-dependent MMP secretion, MMP diffusion, ECM degradation by MMP and active cell motility. Our results demonstrate that in randomly aligned matrices, collective cell invasion is more efficient than single cell invasion. Although increase in MMP secretion rate enhances invasiveness independent of cell–cell adhesion, sustenance of collective invasion in dense matrices requires high MMP secretion rates. However, matrix alignment can sustain both single cell and collective cell invasion even without ECM proteolysis. Similar to our in-silico observations, increase in ECM density and MMP inhibition reduced migration of MCF-7 cells embedded in sandwich gels. Together, our results indicate that apart from cell intrinsic factors (i.e., high cell–cell adhesion and MMP secretion rates), ECM density and organization represent two important extrinsic parameters that govern collective cell invasion and invasion plasticity. PMID:26832069

Nanolithographic control of the spatial organization of cellular adhesion receptors at the single-molecule level

PubMed Central

Schvartzman, Mark; Palma, Matteo; Sable, Julia; Abramson, Justin; Hu, Xian; Sheetz, Michael P.; Wind, Shalom J.

2011-01-01

The ability to control the placement of individual molecules promises to enable a wide range of applications and is a key challenge in nanoscience and nanotechnology. Many biological interactions, in particular, are sensitive to the precise geometric arrangement of proteins. We have developed a technique which combines molecular-scale nanolithography with site-selective biochemistry to create biomimetic arrays of individual protein binding sites. The binding sites can be arranged in heterogeneous patterns of virtually any possible geometry with a nearly unlimited number of degrees of freedom. We have used these arrays to explore how the geometric organization of the extracellular matrix (ECM) binding ligand RGD (Arg-Gly-Asp) affects cell adhesion and spreading. Systematic variation of spacing, density and cluster size of individual integrin binding sites was used to elicit different cell behavior. Cell spreading assays on arrays of different geometric arrangements revealed a dramatic increase in spreading efficiency when at least 4 liganded sites were spaced within 60 nm or less, with no dependence on global density. This points to the existence of a minimal matrix adhesion unit for fibronectin defined in space and stoichiometry. Developing an understanding of the ECM geometries that activate specific cellular functional complexes is a critical step toward controlling cell behavior. Potential practical applications range from new therapeutic treatments to the rational design of tissue scaffolds that can optimize healing without scarring. More broadly, spatial control at the single-molecule level can elucidate factors controlling individual molecular interactions and can enable synthesis of new systems based on molecular-scale architectures. PMID:21319842

The integrin expression profile modulates orientation and dynamics of force transmission at cell-matrix adhesions.

PubMed

Balcioglu, Hayri E; van Hoorn, Hedde; Donato, Dominique M; Schmidt, Thomas; Danen, Erik H J

2015-04-01

Integrin adhesion receptors connect the extracellular matrix (ECM) to the cytoskeleton and serve as bidirectional mechanotransducers. During development, angiogenesis, wound healing and cancer progression, the relative abundance of fibronectin receptors, including integrins α5β1 and αvβ3, changes, thus altering the integrin composition of cell-matrix adhesions. Here, we show that enhanced αvβ3 expression can fully compensate for loss of α5β1 and other β1 integrins to support outside-in and inside-out force transmission. α5β1 and αvβ3 each mediate actin cytoskeletal remodeling in response to stiffening or cyclic stretching of the ECM. Likewise, α5β1 and αvβ3 support cellular traction forces of comparable magnitudes and similarly increase these forces in response to ECM stiffening. However, cells using αvβ3 respond to lower stiffness ranges, reorganize their actin cytoskeleton more substantially in response to stretch, and show more randomly oriented traction forces. Centripetal traction force orientation requires long stress fibers that are formed through the action of Rho kinase (ROCK) and myosin II, and that are supported by α5β1. Thus, altering the relative abundance of fibronectin-binding integrins in cell-matrix adhesions affects the spatiotemporal organization of force transmission. © 2015. Published by The Company of Biologists Ltd.

Further Support for ECM Control of Receptor Trafficking and Signaling.

PubMed

Clegg, Lindsay; Mac Gabhann, Feilim

2017-01-01

Recently, Sack et al. (2016) presented an interesting, novel data set in Journal of Cellular Physiology examining the effect of substrate stiffness on VEGF processing and signaling. The data represent a clear contribution to the field. However, the authors' conclusion that "extracellular matrix binding is essential for VEGF internalization" conflicts with other knowledge in the field, and is not supported by their data. Instead, their data demonstrate the effect of heparin addition and changing ECM stiffness on both VEGF binding to fibronectin and VEGF binding to endothelial receptors. This is consistent with other work showing that matrix binding reduces VEGF-VEGFR internalization, shifting downstream signaling. J. Cell. Physiol. 232: 36-37, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Integrin binding and mechanical tension induce movement of mRNA and ribosomes to focal adhesions

NASA Technical Reports Server (NTRS)

Chicurel, M. E.; Singer, R. H.; Meyer, C. J.; Ingber, D. E.

1998-01-01

The extracellular matrix (ECM) activates signalling pathways that control cell behaviour by binding to cell-surface integrin receptors and inducing the formation of focal adhesion complexes (FACs). In addition to clustered integrins, FACs contain proteins that mechanically couple the integrins to the cytoskeleton and to immobilized signal-transducing molecules. Cell adhesion to the ECM also induces a rapid increase in the translation of preexisting messenger RNAs. Gene expression can be controlled locally by targeting mRNAs to specialized cytoskeletal domains. Here we investigate whether cell binding to the ECM promotes formation of a cytoskeletal microcompartment specialized for translational control at the site of integrin binding. High-resolution in situ hybridization revealed that mRNA and ribosomes rapidly and specifically localized to FACs that form when cells bind to ECM-coated microbeads. Relocation of these protein synthesis components to the FAC depended on the ability of integrins to mechanically couple the ECM to the contractile cytoskeleton and on associated tension-moulding of the actin lattice. Our results suggest a new type of gene regulation by integrins and by mechanical stress which may involve translation of mRNAs into proteins near the sites of signal reception.

Glucocorticoid-induced tumor necrosis factor receptor family-related ligand triggering upregulates vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 and promotes leukocyte adhesion.

PubMed

Lacal, Pedro Miguel; Petrillo, Maria Grazia; Ruffini, Federica; Muzi, Alessia; Bianchini, Rodolfo; Ronchetti, Simona; Migliorati, Graziella; Riccardi, Carlo; Graziani, Grazia; Nocentini, Giuseppe

2013-10-01

The interaction of glucocorticoid-induced tumor necrosis factor receptor-family related (GITR) protein with its ligand (GITRL) modulates different functions, including immune/inflammatory response. These effects are consequent to intracellular signals activated by both GITR and GITRL. Previous results have suggested that lack of GITR expression in GITR(-/-) mice decreases the number of leukocytes within inflamed tissues. We performed experiments to analyze whether the GITRL/GITR system modulates leukocyte adhesion and extravasation. For that purpose, we first evaluated the capability of murine splenocytes to adhere to endothelial cells (EC). Our results indicated that adhesion of GITR(-/-) splenocytes to EC was reduced as compared with wild-type cells, suggesting that GITR plays a role in adhesion and that this effect may be due to GITRL-GITR interaction. Moreover, adhesion was increased when EC were pretreated with an agonist GITR-Fc fusion protein, thus indicating that triggering of GITRL plays a role in adhesion by EC regulation. In a human in vitro model, the adhesion to human EC of HL-60 cells differentiated toward the monocytic lineage was increased by EC pretreatment with agonist GITR-Fc. Conversely, antagonistic anti-GITR and anti-GITRL Ab decreased adhesion, thus further indicating that GITRL triggering increases the EC capability to support leukocyte adhesion. EC treatment with GITR-Fc favored extravasation, as demonstrated by a transmigration assay. Notably, GITRL triggering increased intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression and anti-ICAM-1 and anti-VCAM-1 Abs reversed GITR-Fc effects. Our study demonstrates that GITRL triggering in EC increases leukocyte adhesion and transmigration, suggesting new anti-inflammatory therapeutic approaches based on inhibition of GITRL-GITR interaction.

Influence of surfaces modified with biomimetic extracellular matrices on adhesion and proliferation of mesenchymal stem cells and osteosarcoma cells.

PubMed

Cai, Rong; Kawazoe, Naoki; Chen, Guoping

2015-02-01

Preparation of surfaces modified with biomimetic extracellular matrices (ECMs) is important for investigation of the interaction between ECMs and cells. In the present study, surfaces modified with ECMs from normal somatic cells, stem cells and tumor cells were prepared by cell culture method. The ECMs derived from bone marrow-derived mesenchymal stem cells (MSCs), dermal fibroblasts (FBs), osteoblasts (OBs) and MG63 osteosarcoma cells were deposited on the surfaces of cell-culture polystyrene plates (TCPS). The ECMs from different cell types had different compositions. The effects of the ECM-deposited surfaces on the adhesion, spreading and proliferation of MSCs and MG63 human osteosarcoma cells were dependent on the type of both ECMs and cells. The surfaces deposited with ECMs from MSCs, FBs and OBs promoted cell adhesion more strongly than surfaces deposited with ECMs from MG63 cells and TCPS. Compared to TCPS, the ECM-deposited surfaces promoted proliferation of MSCs while they inhibited the proliferation of MG63 cells. Copyright © 2014 Elsevier B.V. All rights reserved.

A Novel Nectin-mediated Cell Adhesion Apparatus That Is Implicated in Prolactin Receptor Signaling for Mammary Gland Development*

PubMed Central

Kitayama, Midori; Mizutani, Kiyohito; Maruoka, Masahiro; Mandai, Kenji; Sakakibara, Shotaro; Ueda, Yuki; Komori, Takahide; Shimono, Yohei; Takai, Yoshimi

2016-01-01

Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development. PMID:26757815

A Nasal Epithelial Receptor for Staphylococcus aureus WTA Governs Adhesion to Epithelial Cells and Modulates Nasal Colonization

PubMed Central

Faulstich, Manuela; Grau, Timo; Severin, Yannik; Unger, Clemens; Hoffmann, Wolfgang H.; Rudel, Thomas; Autenrieth, Ingo B.; Weidenmaier, Christopher

2014-01-01

Nasal colonization is a major risk factor for S. aureus infections. The mechanisms responsible for colonization are still not well understood and involve several factors on the host and the bacterial side. One key factor is the cell wall teichoic acid (WTA) of S. aureus, which governs direct interactions with nasal epithelial surfaces. We report here the first receptor for the cell wall glycopolymer WTA on nasal epithelial cells. In several assay systems this type F-scavenger receptor, termed SREC-I, bound WTA in a charge dependent manner and mediated adhesion to nasal epithelial cells in vitro. The impact of WTA and SREC-I interaction on epithelial adhesion was especially pronounced under shear stress, which resembles the conditions found in the nasal cavity. Most importantly, we demonstrate here a key role of the WTA-receptor interaction in a cotton rat model of nasal colonization. When we inhibited WTA mediated adhesion with a SREC-I antibody, nasal colonization in the animal model was strongly reduced at the early onset of colonization. More importantly, colonization stayed low over an extended period of 6 days. Therefore we propose targeting of this glycopolymer-receptor interaction as a novel strategy to prevent or control S. aureus nasal colonization. PMID:24788600

Receptor-mediated membrane adhesion of lipid-polymer hybrid (LPH) nanoparticles studied by dissipative particle dynamics simulations

NASA Astrophysics Data System (ADS)

Li, Zhenlong; Gorfe, Alemayehu A.

2014-12-01

Lipid-polymer hybrid (LPH) nanoparticles represent a novel class of targeted drug delivery platforms that combine the advantages of liposomes and biodegradable polymeric nanoparticles. However, the molecular details of the interaction between LPHs and their target cell membranes remain poorly understood. We have investigated the receptor-mediated membrane adhesion process of a ligand-tethered LPH nanoparticle using extensive dissipative particle dynamics (DPD) simulations. We found that the spontaneous adhesion process follows a first-order kinetics characterized by two distinct stages: a rapid nanoparticle-membrane engagement, followed by a slow growth in the number of ligand-receptor pairs coupled with structural re-organization of both the nanoparticle and the membrane. The number of ligand-receptor pairs increases with the dynamic segregation of ligands and receptors toward the adhesion zone causing an out-of-plane deformation of the membrane. Moreover, the fluidity of the lipid shell allows for strong nanoparticle-membrane interactions to occur even when the ligand density is low. The LPH-membrane avidity is enhanced by the increased stability of each receptor-ligand pair due to the geometric confinement and the cooperative effect arising from multiple binding events. Thus, our results reveal the unique advantages of LPH nanoparticles as active cell-targeting nanocarriers and provide some general principles governing nanoparticle-cell interactions that may aid future design of LPHs with improved affinity and specificity for a given target of interest.

Neuropeptides, via specific receptors, regulate T cell adhesion to fibronectin.

PubMed

Levite, M; Cahalon, L; Hershkoviz, R; Steinman, L; Lider, O

1998-01-15

The ability of T cells to adhere to and interact with components of the blood vessel walls and the extracellular matrix is essential for their extravasation and migration into inflamed sites. We have found that the beta1 integrin-mediated adhesion of resting human T cells to fibronectin, a major glycoprotein component of the extracellular matrix, is induced by physiologic concentrations of three neuropeptides: calcitonin gene-related protein (CGRP), neuropeptide Y, and somatostatin; each acts via its own specific receptor on the T cell membrane. In contrast, substance P (SP), which coexists with CGRP in the majority of peripheral endings of sensory nerves, including those innervating the lymphoid organs, blocks T cell adhesion to fibronectin when induced by CGRP, neuropeptide Y, somatostatin, macrophage inflammatory protein-1beta, and PMA. Inhibition of T cell adhesion was obtained both by the intact SP peptide and by its 1-4 N-terminal and its 4-11, 5-11, and 6-11 C-terminal fragments, used at similar nanomolar concentrations. The inhibitory effects of the parent SP peptide and its fragments were abrogated by an SP NK-1 receptor antagonist, suggesting they all act through the same SP NK-1 receptor. These findings suggest that neuropeptides, by activating their specific T cell-expressed receptors, can provide the T cells with both positive (proadhesive) and negative (antiadhesive) signals and thereby regulate their function. Thus, neuropeptides may influence diverse physiologic processes involving integrins, including leukocyte-mediated migration and inflammation.

Direct Force Measurements of Receptor-Ligand Interactions on Living Cells

NASA Astrophysics Data System (ADS)

Eibl, Robert H.

The characterization of cell adhesion between two living cells at the level of single receptor-ligand bonds is an experimental challenge. This chapter describes how the extremely sensitive method of atomic force microscopy (AFM) based force spectroscopy can be applied to living cells in order to probe for cell-to-cell or cell-to-substrate interactions mediated by single pairs of adhesion receptors. In addition, it is outlined how single-molecule AFM force spectroscopy can be used to detect physiologic changes of an adhesion receptor in a living cell. This force spectroscopy allows us to detect in living cells rapidly changing, chemokine SDF-1 triggered activation states of single VLA-4 receptors. This recently developed AFM application will allow for the detailed investigation of the integrin-chemokine crosstalk of integrin activation mechanisms and on how other adhesion receptors are modulated in health and disease. As adhesion molecules, living cells and even bacteria can be studied by single-molecule AFM force spectroscopy, this method is set to become a powerful tool that can not only be used in biophysics, but in cell biology as well as in immunology and cancer research.

Monitoring of integrin-mediated adhesion of human ovarian cancer cells to model protein surfaces by quartz crystal resonators: evaluation in the impedance analysis mode.

PubMed

Li, Jing; Thielemann, Christiane; Reuning, Ute; Johannsmann, Diethelm

2005-01-15

The quartz crystal microbalance (QCM) was used to monitor specific, integrin-mediated adhesion of human ovarian cancer cells to distinct extracellular matrix (ECM) proteins immobilized on gold-coated quartz crystal resonators. The QCM was operated in the impedance analysis mode, where frequency shift as well as bandwidth are accessible on a broad range of overtones. The increase in bandwidth caused by covering the quartz resonator with cells was reproducible and largely independent of overtone order, whereas the frequency shift displayed some variability. Thus the bandwidth proved to be the more robust parameter for sensing cell adhesive events. The bandwidth increased in proportion to the number of seeded cells to the quartz crystal as long as the number was below 150,000 cells/ml. Comparing the resonance parameters on different harmonics, one finds that viscoelastic modeling with homogeneous layer systems cannot reproduce the results: lateral heterogeneity has to be taken into account. The differences in adhesive strength of human ovarian cancer cells towards selected ECM proteins monitored by QCM was in good agreement with data obtained by conventional cell adhesion assays. Strong cell adhesion was observed to the ECM proteins vitronectin (VN) and fibronectin (FN), while only weak attachment occurred on laminin. In order to prove specific, integrin alphavbeta3-mediated cell adhesion to its ligands FN and VN, the cyclic integrin alphavbeta3-directed peptide c(RGDfV) was used as competitor and significantly reversed cell adhesion. Since integrin interaction with ECM proteins is dependent on the presence of bivalent cations, cell detachment was also seen after treatment of cell monolayers with the chelator ethylene-dinitro-tetra-acetic acid (EDTA). The QCM technique is a reliable method to monitor cell adsorption to ECM-pretreated surfaces in real time. It may be an alternative tool for screening specific and selective antagonists of integrin/ECM interaction.

A viscoelastic-stochastic model of the effects of cytoskeleton remodelling on cell adhesion.

PubMed

Li, Long; Zhang, Wenyan; Wang, Jizeng

2016-10-01

Cells can adapt their mechanical properties through cytoskeleton remodelling in response to external stimuli when the cells adhere to the extracellular matrix (ECM). Many studies have investigated the effects of cell and ECM elasticity on cell adhesion. However, experiments determined that cells are viscoelastic and exhibiting stress relaxation, and the mechanism behind the effect of cellular viscoelasticity on the cell adhesion behaviour remains unclear. Therefore, we propose a theoretical model of a cluster of ligand-receptor bonds between two dissimilar viscoelastic media subjected to an applied tensile load. In this model, the distribution of interfacial traction is assumed to follow classical continuum viscoelastic equations, whereas the rupture and rebinding of individual molecular bonds are governed by stochastic equations. On the basis of this model, we determined that viscosity can significantly increase the lifetime, stability and dynamic strength of the adhesion cluster of molecular bonds, because deformation relaxation attributed to the viscoelastic property can increase the rebinding probability of each open bond and reduce the stress concentration in the adhesion area.

A viscoelastic–stochastic model of the effects of cytoskeleton remodelling on cell adhesion

PubMed Central

Li, Long; Zhang, Wenyan

2016-01-01

Cells can adapt their mechanical properties through cytoskeleton remodelling in response to external stimuli when the cells adhere to the extracellular matrix (ECM). Many studies have investigated the effects of cell and ECM elasticity on cell adhesion. However, experiments determined that cells are viscoelastic and exhibiting stress relaxation, and the mechanism behind the effect of cellular viscoelasticity on the cell adhesion behaviour remains unclear. Therefore, we propose a theoretical model of a cluster of ligand–receptor bonds between two dissimilar viscoelastic media subjected to an applied tensile load. In this model, the distribution of interfacial traction is assumed to follow classical continuum viscoelastic equations, whereas the rupture and rebinding of individual molecular bonds are governed by stochastic equations. On the basis of this model, we determined that viscosity can significantly increase the lifetime, stability and dynamic strength of the adhesion cluster of molecular bonds, because deformation relaxation attributed to the viscoelastic property can increase the rebinding probability of each open bond and reduce the stress concentration in the adhesion area. PMID:27853571

ECM remodeling and its plasticity

NASA Astrophysics Data System (ADS)

Feng, Jingchen; Jones, Christopher A. R.; Cibula, Matthew; Mao, Xiaoming; Sander, Leonard M.; Levine, Herbert; Sun, Bo

The mechanical interactions between cells and Extracellular Matrix (ECM) are of great importance in many cellular processes. These interactions are reciprocal, i.e. contracting cells pull and reorganize the surrounding matrix, while the remodeled matrix feeds back to regulate cell activities. Recent experiments show in collagen gels with densely distributed cells, aligned fiber bundles are formed in the direction between neighboring cells. Fibers flow into the center region between contracting cell pairs in this process, which causes the concentration of fibers in the fiber bundles to become significantly enhanced. Using an extended lattice-based model, we show that viscoelasticity plays an essential role in ECM remodeling and contributes to the enhanced concentration in fiber bundles. We further characterize ECM plasticity within our model and verify our results with rheometer experiments.

Retinoids induce integrin-independent lymphocyte adhesion through RAR-α nuclear receptor activity

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

Whelan, Jarrett T.; Wang, Lei; Chen, Jianming

2014-11-28

Highlights: • Transcription and translation are required for retinoid-induced lymphocyte adhesion. • RAR activation is sufficient to induced lymphocyte cell adhesion. • Vitamin D derivatives inhibit RAR-prompted lymphocyte adhesion. • Adhesion occurs through a novel binding site within ADAM disintegrin domains. • RARα is a key nuclear receptor for retinoid-dependent lymphocyte cell adhesion. - Abstract: Oxidative metabolites of vitamin A, in particular all-trans-retinoic acid (atRA), have emerged as key factors in immunity by specifying the localization of immune cells to the gut. Although it is appreciated that isomers of retinoic acid activate the retinoic acid receptor (RAR) and retinoid Xmore » receptor (RXR) family of nuclear receptors to elicit cellular changes, the molecular details of retinoic acid action remain poorly defined in immune processes. Here we employ a battery of agonists and antagonists to delineate the specific nuclear receptors utilized by retinoids to evoke lymphocyte cell adhesion to ADAM (adisintegrin and metalloprotease) protein family members. We report that RAR agonism is sufficient to promote immune cell adhesion in both immortal and primary immune cells. Interestingly, adhesion occurs independent of integrin function, and mutant studies demonstrate that atRA-induced adhesion to ADAM members required a distinct binding interface(s) as compared to integrin recognition. Anti-inflammatory corticosteroids as well as 1,25-(OH){sub 2}D{sub 3}, a vitamin D metabolite that prompts immune cell trafficking to the skin, potently inhibited the observed adhesion. Finally, our data establish that induced adhesion was specifically attributable to the RAR-α receptor isotype. The current study provides novel molecular resolution as to which nuclear receptors transduce retinoid exposure into immune cell adhesion.« less

Fabrication of type I collagen microcarrier using a microfluidic 3D T-junction device and its application for the quantitative analysis of cell-ECM interactions.

PubMed

Yoon, Junghyo; Kim, Jaehoon; Jeong, Hyo Eun; Sudo, Ryo; Park, Myung-Jin; Chung, Seok

2016-08-26

We presented a new quantitative analysis for cell and extracellular matrix (ECM) interactions, using cell-coated ECM hydrogel microbeads (hydrobeads) made of type I collagen. The hydrobeads can carry cells as three-dimensional spheroidal forms with an ECM inside, facilitating a direct interaction between the cells and ECM. The cells on hydrobeads do not have a hypoxic core, which opens the possibility for using as a cell microcarrier for bottom-up tissue reconstitution. This technique can utilize various types of cells, even MDA-MB-231 cells, which have weak cell-cell interactions and do not form spheroids in conventional spheroid culture methods. Morphological indices of the cell-coated hydrobead visually present cell-ECM interactions in a quantitative manner.

Activation of EGF Receptor Kinase by L1-mediated Homophilic Cell Interactions

PubMed Central

Islam, Rafique; Kristiansen, Lars V.; Romani, Susana; Garcia-Alonso, Luis; Hortsch, Michael

2004-01-01

Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo. PMID:14718570

Activation of EGF receptor kinase by L1-mediated homophilic cell interactions.

PubMed

Islam, Rafique; Kristiansen, Lars V; Romani, Susana; Garcia-Alonso, Luis; Hortsch, Michael

2004-04-01

Neural cell adhesion molecules (CAMs) are important players during neurogenesis and neurite outgrowth as well as axonal fasciculation and pathfinding. Some of these developmental processes entail the activation of cellular signaling cascades. Pharmacological and genetic evidence indicates that the neurite outgrowth-promoting activity of L1-type CAMs is at least in part mediated by the stimulation of neuronal receptor tyrosine kinases (RTKs), especially FGF and EGF receptors. It has long been suspected that neural CAMs might physically interact with RTKs, but their activation by specific cell adhesion events has not been directly demonstrated. Here we report that gain-of-function conditions of the Drosophila L1-type CAM Neuroglian result in profound sensory axon pathfinding defects in the developing Drosophila wing. This phenotype can be suppressed by decreasing the normal gene dosage of the Drosophila EGF receptor gene. Furthermore, in Drosophila S2 cells, cell adhesion mediated by human L1-CAM results in the specific activation of human EGF tyrosine kinase at cell contact sites and EGF receptors engage in a physical interaction with L1-CAM molecules. Thus L1-type CAMs are able to promote the adhesion-dependent activation of EGF receptor signaling in vitro and in vivo.

Modeling extracellular matrix (ECM) alterations in ovarian cancer by multiphoton excited fabrication of stromal models (Conference Presentation)

NASA Astrophysics Data System (ADS)

Campagnola, Paul J.; Ajeti, Visar; Lara, Jorge; Eliceiri, Kevin W.; Patankar, Mansh

2016-04-01

A profound remodeling of the extracellular matrix (ECM) occurs in human ovarian cancer but it unknown how this affects tumor growth, where this understanding could lead to better diagnostics and therapeutic approaches. We investigate the role of these ECM alterations by using multiphoton excited (MPE) polymerization to fabricate biomimetic models to investigate operative cell-matrix interactions in invasion/metastasis. First, we create nano/microstructured gradients mimicking the basal lamina to study adhesion/migration dynamics of ovarian cancer cells of differing metastatic potential. We find a strong haptotactic response that depends on both contact guidance and ECM binding cues. While we found enhanced migration for more invasive cells, the specifics of alignment and directed migration also depend on cell polarity. We further use MPE fabrication to create collagen scaffolds with complex, 3D submicron morphology. The stromal scaffold designs are derived directly from "blueprints" based on SHG images of normal, high risk, and malignant ovarian tissues. The models are seeded with different cancer cell lines and this allows decoupling of the roles of cell characteristics (metastatic potential) and ECM structure and composition (normal vs cancer) on adhesion/migration dynamics. We found the malignant stroma structure promotes enhanced migration and proliferation and also cytoskeletal alignment. Creating synthetic models based on fibers patterns further allows decoupling the topographic roles of the fibers themselves vs their alignment within the tissue. These models cannot be synthesized by other conventional fabrication methods and we suggest the MPE image-based fabrication method will enable a variety of studies in cancer biology.

A combinatorial extracellular matrix platform identifies cell-extracellular matrix interactions that correlate with metastasis

PubMed Central

Reticker-Flynn, Nathan E.; Braga Malta, David F.; Winslow, Monte M.; Lamar, John M.; Xu, Mary J.; Underhill, Gregory H.; Hynes, Richard O.; Jacks, Tyler E.; Bhatia, Sangeeta N.

2013-01-01

Extracellular matrix interactions play essential roles in normal physiology and many pathological processes. While the importance of ECM interactions in metastasis is well documented, systematic approaches to identify their roles in distinct stages of tumorigenesis have not been described. Here we report a novel screening platform capable of measuring phenotypic responses to combinations of ECM molecules. Using a genetic mouse model of lung adenocarcinoma, we measure the ECM-dependent adhesion of tumor-derived cells. Hierarchical clustering of the adhesion profiles differentiates metastatic cell lines from primary tumor lines. Furthermore, we uncovered that metastatic cells selectively associate with fibronectin when in combination with galectin-3, galectin-8, or laminin. We show that these molecules correlate with human disease and that their interactions are mediated in part by α3β1 integrin. Thus, our platform allowed us to interrogate interactions between metastatic cells and their microenvironments, and identified ECM and integrin interactions that could serve as therapeutic targets. PMID:23047680

The FAK-Arp2/3 interaction promotes leading edge advance and haptosensing by coupling nascent adhesions to lamellipodia actin.

PubMed

Swaminathan, Vinay; Fischer, R S; Waterman, Clare M

2016-04-01

Cell migration is initiated in response to biochemical or physical cues in the environment that promote actin-mediated lamellipodial protrusion followed by the formation of nascent integrin adhesions (NAs) within the protrusion to drive leading edge advance. Although FAK is known to be required for cell migration through effects on focal adhesions, its role in NA formation and lamellipodial dynamics is unclear. Live-cell microscopy of FAK(-/-)cells with expression of phosphorylation deficient or a FERM-domain mutant deficient in Arp2/3 binding revealed a requirement for FAK in promoting the dense formation, transient stabilization, and timely turnover of NA within lamellipodia to couple actin-driven protrusion to adhesion and advance of the leading edge. Phosphorylation on Y397 of FAK promotes dense NA formation but is dispensable for transient NA stabilization and leading edge advance. In contrast, transient NA stabilization and advance of the cell edge requires FAK-Arp2/3 interaction, which promotes Arp2/3 localization to NA and reduces FAK activity. Haptosensing of extracellular matrix (ECM) concentration during migration requires the interaction between FAK and Arp2/3, whereas FAK phosphorylation modulates mechanosensing of ECM stiffness during spreading. Taken together, our results show that mechanistically separable functions of FAK in NA are required for cells to distinguish distinct properties of their environment during migration. © 2016 Swaminathan et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Priming Endothelial Cells With a Melanoma-Derived Extracellular Matrix Triggers the Activation of αvβ3/VEGFR2 Axis.

PubMed

Helal-Neto, Edward; Brandão-Costa, Renata M; Saldanha-Gama, Roberta; Ribeiro-Pereira, Cristiane; Midlej, Victor; Benchimol, Marlene; Morandi, Verônica; Barja-Fidalgo, Christina

2016-11-01

The unique composition of tumor-produced extracellular matrix (ECM) can be a determining factor in changing the profile of endothelial cells in the tumor microenvironment. As the main receptor for ECM proteins, integrins can activate a series of signaling pathways related to cell adhesion, migration, and differentiation of endothelial cells that interact with ECM proteins. We studied the direct impact of the decellularized ECM produced by a highly metastatic human melanoma cell line (MV3) on the activation of endothelial cells and identified the intracellular signaling pathways associated with cell differentiation. Our data show that compared to the ECM derived from a human melanocyte cell line (NGM-ECM), ECM produced by a melanoma cell line (MV3-ECM) is considerably different in ultrastructural organization and composition and possesses a higher content of tenascin-C and laminin and a lower expression of fibronectin. When cultured directly on MV3-ECM, endothelial cells change morphology and show increased adhesion, migration, proliferation, and tubulogenesis. Interaction of endothelial cells with MV3-ECM induces the activation of integrin signaling, increasing FAK phosphorylation and its association with Src, which activates VEGFR2, potentiating the receptor response to VEGF. The blockage of αvβ3 integrin inhibited the FAK-Src association and VEGFR activation, thus reducing tubulogenesis. Together, our data suggest that the interaction of endothelial cells with the melanoma-ECM triggers integrin-dependent signaling, leading to Src pathway activation that may potentiate VEGFR2 activation and up-regulate angiogenesis. J. Cell. Physiol. 231: 2464-2473, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Delineating the roles of the GPIIb/IIIa and GP-Ib-IX-V platelet receptors in mediating platelet adhesion to adsorbed fibrinogen and albumin.

PubMed

Sivaraman, Balakrishnan; Latour, Robert A

2011-08-01

Platelet adhesion to adsorbed plasma proteins, such as fibrinogen (Fg), has been conventionally thought to be mediated by the GPIIb/IIIa receptor binding to Arg-Gly-Asp (RGD)-like motifs in the adsorbed protein. In previous studies, we showed that platelet adhesion response to adsorbed Fg and Alb was strongly influenced by the degree of adsorption-induced protein unfolding and that platelet adhesion was only partially blocked by soluble RGD, with RGD-blocked platelets adhering without activation. Based on these results, we hypothesized that in addition to the RGD-specific GPIIb/IIIa receptor, which mediates both adhesion and activation, a non-RGD-specific receptor set likely also plays a role in platelet adhesion (but not activation) to both Fg and albumin (Alb). To identify and elucidate the role of these receptors, in addition to GPIIb/IIIa, we also examined the GPIb-IX-V receptor complex, which has been shown to mediate platelet adhesion (but not activation) in studies by other groups. The platelet suspension was pretreated with either a GPIIb/IIIa-antagonist drug Aggrastat(®) or monoclonal antibodies 6B4 or 24G10 against GPIb-IX-V prior to adhesion on Fg- and Alb-coated OH- and CH(3)-functionalized alkanethiol self-assembled monolayer surfaces. The results revealed that GPIIb/IIIa is the primary receptor set involved in platelet adhesion to adsorbed Fg and Alb irrespective of their degree of adsorption-induced unfolding, while the GPIb-IX-V receptor complex plays an insignificant role. Overall, these studies provide novel insights into the molecular-level mechanisms mediating platelet interactions with adsorbed plasma proteins, thereby assisting the biomaterials field develop potent strategies for inhibiting platelet-protein interactions in the design of more hemocompatible cardiovascular biomaterials and effective anti-thrombotic therapies. Copyright © 2011 Elsevier Ltd. All rights reserved.

Adhesion molecules and receptors

USDA-ARS?s Scientific Manuscript database

Adhesion molecules are necessary for leukocyte trafficking and differentiation. They serve to initiate cell-cell interactions under conditions of shear, and they sustain the cell-cell and cell-matrix interactions needed for cellular locomotion. They also can serve directly as signaling molecules act...

Sliding tethered ligands add topological interactions to the toolbox of ligand–receptor design

PubMed Central

Bauer, Martin; Kékicheff, Patrick; Iss, Jean; Fajolles, Christophe; Charitat, Thierry; Daillant, Jean; Marques, Carlos M.

2015-01-01

Adhesion in the biological realm is mediated by specific lock-and-key interactions between ligand–receptor pairs. These complementary moieties are ubiquitously anchored to substrates by tethers that control the interaction range and the mobility of the ligands and receptors, thus tuning the kinetics and strength of the binding events. Here we add sliding anchoring to the toolbox of ligand–receptor design by developing a family of tethered ligands for which the spacer can slide at the anchoring point. Our results show that this additional sliding degree of freedom changes the nature of the adhesive contact by extending the spatial range over which binding may sustain a significant force. By introducing sliding tethered ligands with self-regulating length, this work paves the way for the development of versatile and reusable bio-adhesive substrates with potential applications for drug delivery and tissue engineering. PMID:26350224

EMMPRIN regulates β1 integrin-mediated adhesion through Kindlin-3 in human melanoma cells.

PubMed

Delyon, Julie; Khayati, Farah; Djaafri, Ibtissem; Podgorniak, Marie-Pierre; Sadoux, Aurélie; Setterblad, Niclas; Boutalbi, Zineb; Maouche, Kamel; Maskos, Uwe; Menashi, Suzanne; Lebbé, Céleste; Mourah, Samia

2015-06-01

EMMPRIN is known to promote tumor invasion through extracellular matrix (ECM) degradation. Here we report that EMMPRIN can regulate melanoma cell adhesion to the ECM through an interaction with β1 integrin involving kindlin-3. In this study, EMMPRIN knockdown in the human melanoma cell line M10 using siRNA decreased cell invasion and significantly increased cell adhesion and spreading. A morphological change from a round to a spread shape was observed associated with enhanced phalloidin-labelled actin staining. In situ proximity ligation assay and co-immunoprecipitation revealed that EMMPRIN silencing increased the interaction of β1 integrin with kindlin-3, a focal adhesion protein. This was associated with an increase in β1 integrin activation and a decrease in the phosphorylation of the downstream integrin kinase FAK. Moreover, the expression at both the transcript and protein level of kindlin-3 and of β1 integrin was inversely regulated by EMMPRIN. EMMPRIN did not regulate either talin expression or its interaction with β1 integrin. These results are consistent with our in vivo demonstration that EMMPRIN inhibition increased β1 integrin activation and its interaction with kindlin-3. To conclude, these findings reveal a new role of EMMPRIN in tumor cell migration through ß1 integrin/kindlin-3-mediated adhesion pathway. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Cell-cell and cell-extracellular matrix adhesions cooperate to organize actomyosin networks and maintain force transmission during dorsal closure.

PubMed

Goodwin, Katharine; Lostchuck, Emily E; Cramb, Kaitlyn M L; Zulueta-Coarasa, Teresa; Fernandez-Gonzalez, Rodrigo; Tanentzapf, Guy

2017-05-15

Tissue morphogenesis relies on the coordinated action of actin networks, cell-cell adhesions, and cell-extracellular matrix (ECM) adhesions. Such coordination can be achieved through cross-talk between cell-cell and cell-ECM adhesions. Drosophila dorsal closure (DC), a morphogenetic process in which an extraembryonic tissue called the amnioserosa contracts and ingresses to close a discontinuity in the dorsal epidermis of the embryo, requires both cell-cell and cell-ECM adhesions. However, whether the functions of these two types of adhesions are coordinated during DC is not known. Here we analyzed possible interdependence between cell-cell and cell-ECM adhesions during DC and its effect on the actomyosin network. We find that loss of cell-ECM adhesion results in aberrant distributions of cadherin-mediated adhesions and actin networks in the amnioserosa and subsequent disruption of myosin recruitment and dynamics. Moreover, loss of cell-cell adhesion caused up-regulation of cell-ECM adhesion, leading to reduced cell deformation and force transmission across amnioserosa cells. Our results show how interdependence between cell-cell and cell-ECM adhesions is important in regulating cell behaviors, force generation, and force transmission critical for tissue morphogenesis. © 2017 Goodwin, Lostchuck, et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

L1-CAM and N-CAM: From Adhesion Proteins to Pharmacological Targets.

PubMed

Colombo, Federico; Meldolesi, Jacopo

2015-11-01

L1 cell adhesion molecule (L1-CAM) and neural cell adhesion molecule (N-CAM), key members of the immunoglobulin-like CAM (Ig-CAM) family, were first recognized to play critical roles in surface interactions of neurons, by binding with each other and with extracellular matrix (ECM) proteins. Subsequently, adhesion was recognized to include signaling due to both activation of β-integrin, with the generation of intracellular cascades, and integration with the surface cytoskeleton. The importance of the two Ig-CAMs was revealed by their activation of the tyrosine kinase receptors of fibroblast growth factor (FGF), epidermal growth factor (EGF), and nerve growth factor (NGF). Based on these complex signaling properties, L1-CAM and N-CAM have become of great potential pharmacological interest in neurons and cancers. Treatment of neurodegenerative disorders and cognitive deficits of neurons is aimed to increase the cell Ig-CAM tone, possibly provided by synthetic/mimetic peptides. In cancer cells, where Ig-CAMs are often overexpressed, the proteins are employed for prognosis. The approaches to therapy are based on protein downregulation, antibodies, and adoptive immunotherapy. Copyright © 2015 Elsevier Ltd. All rights reserved.

Monocyte-endothelial adhesion in chronic rheumatoid arthritis. In situ detection of selectin and integrin-dependent interactions.

PubMed Central

Grober, J S; Bowen, B L; Ebling, H; Athey, B; Thompson, C B; Fox, D A; Stoolman, L M

1993-01-01

Blood monocytes are the principal reservoir for tissue macrophages in rheumatoid synovitis. Receptor-mediated adhesive interactions between circulating cells and the synovial venules initiate recruitment. These interactions have been studied primarily in cultured endothelial cells. Thus the functional activities of specific adhesion receptors, such as the endothelial selectins and the leukocytic integrins, have not been evaluated directly in diseased tissues. We therefore examined monocyte-microvascular interactions in rheumatoid synovitis by modifying the Stamper-Woodruff frozen section binding assay initially developed to study lymphocyte homing. Specific binding of monocytes to venules lined by low or high endothelium occurred at concentrations as low as 5 x 10(5) cells/ml. mAbs specific for P-selectin (CD62, GMP-140/PADGEM) blocked adhesion by > 90% in all synovitis specimens examined. In contrast, P-selectin-mediated adhesion to the microvasculature was either lower or absent in frozen sections of normal foreskin and placenta. mAbs specific for E-selectin (ELAM-1) blocked 20-50% of monocyte attachment in several RA synovial specimens but had no effect in others. mAbs specific for LFA-1, Mo1/Mac 1, the integrin beta 2-chain, and L-selectin individually inhibited 30-40% of adhesion. An mAb specific for the integrin beta 1-chain inhibited the attachment of elutriated monocytes up to 20%. We conclude that P-selectin associated with the synovial microvasculature initiates shear-resistant adhesion of monocytes in the Stamper-Woodruff assay and stabilizes bonds formed by other selectins and the integrins. Thus the frozen section binding assay permits direct evaluation of leukocyte-microvascular adhesive interactions in inflamed tissues and suggests a prominent role for P-selectin in monocyte recruitment in vivo. Images PMID:7685772

Disruption of the Aspergillus fumigatus ECM33 homologue results in rapid conidial germination, antifungal resistance and hypervirulence.

PubMed

Romano, Jacob; Nimrod, Guy; Ben-Tal, Nir; Shadkchan, Yona; Baruch, Koti; Sharon, Haim; Osherov, Nir

2006-07-01

The ECM33/SPS2 family of glycosylphosphatidylinositol-anchored proteins plays an important role in maintaining fungal cell wall integrity and virulence. However, the precise molecular role of these proteins is unknown. In this work, AfuEcm33, the gene encoding the ECM33 homologue in the important pathogenic fungus Aspergillus fumigatus, has been cloned and its function analysed. It is shown that disruption of AfuEcm33 results in rapid conidial germination, increased cell-cell adhesion, resistance to the antifungal agent caspofungin and increased virulence in an immunocompromised mouse model for disseminated aspergillosis. These results suggest that the protein encoded by AfuEcm33 is involved in key aspects of cell wall morphogenesis and plays an important role in A. fumigatus virulence.

Reprogramming of the Ovarian Tumor Stroma by Activation of a Biomechanical ECM Switch

DTIC Science & Technology

2016-09-01

Denatured collagen was detec- ted with anticollagen antibody (1:1000). For integrin-blocking enzyme -linked immunosorbent assay, wells were coated with...migration on denatured collagen; it failed to reduce cell adhesion. Moreover a peptide antagonist of alpha 10 beta 1 may inhibit ovarian tumor growth in...stromal cell adhesion, migration and proliferation on distinct ECM substrates including native and denatured collagen. 4   D). As outlined in aim 2

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells.

PubMed

Massimi, Mara; Devirgiliis, Laura Conti

2007-02-01

In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy. A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.

Adhesive interactions with wood

Treesearch

Charles R. Frihart

2004-01-01

While the chemistry for the polymerization of wood adhesives has been studied systematically and extensively, the critical aspects of the interaction of adhesives with wood are less clearly understood. General theories of bond formation need to be modified to take into account the porosity of wood and the ability of chemicals to be absorbed into the cell wall....

Triflavin, an Arg‐Gly‐Asp‐containing Antiplatelet Peptide Inhibits Cell‐substratum Adhesion and Melanoma Cell‐induced Lung Colonization

PubMed Central

Sheu, Joen R.; Lin, Chao H.; Chung, Jih L.; Teng, Che M.

1992-01-01

Triflavin, an Arg‐Gly‐Asp (RGD) containing peptide purified from Trimeresurus flavoviridis snake venom, inhibits human platelet aggregation by blocking fibrinogen binding to fibrinogen receptors associated with glycoprotein Ilb/IIIa complex. In this study, we show that triflavin (1‐30 μg/mouse) inhibits B16‐F10 melanoma cell‐induced lung colonization in C57BL/6 mice in a dose‐dependent manner. In vitro, triflavin dose‐dependently inhibits adhesion of B16‐F10 melanoma cells to extracellular matrices (ECMs; i.e., fibronectin, fibrinogen, vitronectin, and collagen type I). Triflavin is approximately 600‐800 times more potent than GRGDS at inhibiting cell adhesion. In addition, triflavin dose‐dependently inhibits B16‐F10 cell‐induced platelet aggregation. These results imply that the inhibitory effect of triflavin on the adhesion of tumor cells to ECMs (e.g., fibronectin, vitronectin and collagen type I) and/or tumor cell‐induced platelet aggregation may be partially responsible for its antimetastatic activity in C57BL/6 mice. PMID:1399825

Cell adhesion controlled by adhesion G protein-coupled receptor GPR124/ADGRA2 is mediated by a protein complex comprising intersectins and Elmo-Dock.

PubMed

Hernández-Vásquez, Magda Nohemí; Adame-García, Sendi Rafael; Hamoud, Noumeira; Chidiac, Rony; Reyes-Cruz, Guadalupe; Gratton, Jean Philippe; Côté, Jean-François; Vázquez-Prado, José

2017-07-21

Developmental angiogenesis and the maintenance of the blood-brain barrier involve endothelial cell adhesion, which is linked to cytoskeletal dynamics. GPR124 (also known as TEM5/ADGRA2) is an adhesion G protein-coupled receptor family member that plays a pivotal role in brain angiogenesis and in ensuring a tight blood-brain barrier. However, the signaling properties of GPR124 remain poorly defined. Here, we show that ectopic expression of GPR124 promotes cell adhesion, additive to extracellular matrix-dependent effect, coupled with filopodia and lamellipodia formation and an enrichment of a pool of the G protein-coupled receptor at actin-rich cellular protrusions containing VASP, a filopodial marker. Accordingly, GPR124-expressing cells also displayed increased activation of both Rac and Cdc42 GTPases. Mechanistically, we uncover novel direct interactions between endogenous GPR124 and the Rho guanine nucleotide exchange factors Elmo/Dock and intersectin (ITSN). Small fragments of either Elmo or ITSN1 that bind GPR124 blocked GPR124-induced cell adhesion. In addition, Gβγ interacts with the C-terminal tail of GPR124 and promotes the formation of a GPR124-Elmo complex. Furthermore, GPR124 also promotes the activation of the Elmo-Dock complex, as measured by Elmo phosphorylation on a conserved C-terminal tyrosine residue. Interestingly, Elmo and ITSN1 also interact with each other independently of their GPR124-recognition regions. Moreover, endogenous phospho-Elmo and ITSN1 co-localize with GPR124 at lamellipodia of adhering endothelial cells, where GPR124 expression contributes to polarity acquisition during wound healing. Collectively, our results indicate that GPR124 promotes cell adhesion via Elmo-Dock and ITSN. This constitutes a previously unrecognized complex formed of atypical and conventional Rho guanine nucleotide exchange factors for Rac and Cdc42 that is putatively involved in GPR124-dependent angiogenic responses. © 2017 by The American Society for

The state diagram for cell adhesion under flow: leukocyte rolling and firm adhesion.

PubMed

Chang, K C; Tees, D F; Hammer, D A

2000-10-10

Leukocyte adhesion under flow in the microvasculature is mediated by binding between cell surface receptors and complementary ligands expressed on the surface of the endothelium. Leukocytes adhere to endothelium in a two-step mechanism: rolling (primarily mediated by selectins) followed by firm adhesion (primarily mediated by integrins). Using a computational method called "Adhesive Dynamics," we have simulated the adhesion of a cell to a surface in flow, and elucidated the relationship between receptor-ligand functional properties and the dynamics of adhesion. We express this relationship in a state diagram, a one-to-one map between the biophysical properties of adhesion molecules and various adhesive behaviors. Behaviors that are observed in simulations include firm adhesion, transient adhesion (rolling), and no adhesion. We varied the dissociative properties, association rate, bond elasticity, and shear rate and found that the unstressed dissociation rate, k(r)(o), and the bond interaction length, gamma, are the most important molecular properties controlling the dynamics of adhesion. Experimental k(r)(o) and gamma values from the literature for molecules that are known to mediate rolling adhesion fall within the rolling region of the state diagram. We explain why L-selectin-mediated rolling, which has faster k(r)(o) than other selectins, is accompanied by a smaller value for gamma. We also show how changes in association rate, shear rate, and bond elasticity alter the dynamics of adhesion. The state diagram (which must be mapped for each receptor-ligand system) presents a concise and comprehensive means of understanding the relationship between bond functional properties and the dynamics of adhesion mediated by receptor-ligand bonds.

Designing ECM-mimetic Materials Using Protein Engineering

PubMed Central

Cai, Lei; Heilshorn, Sarah C.

2014-01-01

The natural extracellular matrix (ECM), with its multitude of evolved cell-instructive and cell-responsive properties, provides inspiration and guidelines for the design of engineered biomaterials. One strategy to create ECM-mimetic materials is the modular design of protein-based engineered ECM (eECM) scaffolds. This modular design strategy involves combining multiple protein domains with different functionalities into a single, modular polymer sequence, resulting in a multifunctional matrix with independent tunability of the individual domain functions. These eECMs often enable decoupled control over multiple material properties for fundamental studies of cell-matrix interactions. In addition, since the eECMs are frequently composed entirely of bioresorbable amino acids, these matrices have immense clinical potential for a variety of regenerative medicine applications. This brief review demonstrates how fundamental knowledge gained from structure-function studies of native proteins can be exploited in the design of novel protein-engineered biomaterials. While the field of protein-engineered biomaterials has existed for over 20 years, the community is only now beginning to fully explore the diversity of functional peptide modules that can be incorporated into these materials. We have chosen to highlight recent examples that either (1) demonstrate exemplary use as matrices with cell-instructive and cell-responsive properties or (2) demonstrate outstanding creativity in terms of novel molecular-level design and macro-level functionality. PMID:24365704

Utilization of Glycosaminoglycans/Proteoglycans as Carriers for Targeted Therapy Delivery

PubMed Central

Misra, Suniti; Hascall, Vincent C.; Atanelishvili, Ilia; Moreno Rodriguez, Ricardo; Markwald, Roger R.; Ghatak, Shibnath

2015-01-01

The outcome of patients with cancer has improved significantly in the past decade with the incorporation of drugs targeting cell surface adhesive receptors, receptor tyrosine kinases, and modulation of several molecules of extracellular matrices (ECMs), the complex composite of collagens, glycoproteins, proteoglycans, and glycosaminoglycans that dictates tissue architecture. Cancer tissue invasive processes progress by various oncogenic strategies, including interfering with ECM molecules and their interactions with invasive cells. In this review, we describe how the ECM components, proteoglycans and glycosaminoglycans, influence tumor cell signaling. In particular this review describes how the glycosaminoglycan hyaluronan (HA) and its major receptor CD44 impact invasive behavior of tumor cells, and provides useful insight when designing new therapeutic strategies in the treatment of cancer. PMID:26448753

The non-receptor tyrosine kinase Lyn controls neutrophil adhesion by recruiting the CrkL–C3G complex and activating Rap1 at the leading edge

PubMed Central

He, Yuan; Kapoor, Ashish; Cook, Sara; Liu, Shubai; Xiang, Yang; Rao, Christopher V.; Kenis, Paul J. A.; Wang, Fei

2011-01-01

Establishing new adhesions at the extended leading edges of motile cells is essential for stable polarity and persistent motility. Despite recent identification of signaling pathways that mediate polarity and chemotaxis in neutrophils, little is known about molecular mechanisms governing cell–extracellular-matrix (ECM) adhesion in these highly polarized and rapidly migrating cells. Here, we describe a signaling pathway in neutrophils that is essential for localized integrin activation, leading edge attachment and persistent migration during chemotaxis. This pathway depends upon Gi-protein-mediated activation and leading edge recruitment of Lyn, a non-receptor tyrosine kinase belonging to the Src kinase family. We identified the small GTPase Rap1 as a major downstream effector of Lyn to regulate neutrophil adhesion during chemotaxis. Depletion of Lyn in neutrophil-like HL-60 cells prevented chemoattractant-induced Rap1 activation at the leading edge of the cell, whereas ectopic expression of Rap1 largely rescued the defects induced by Lyn depletion. Furthermore, Lyn controls spatial activation of Rap1 by recruiting the CrkL–C3G protein complex to the leading edge. Together, these results provide novel mechanistic insights into the poorly understood signaling network that controls leading edge adhesion during chemotaxis of neutrophils, and possibly other amoeboid cells. PMID:21628423

ECM Proteins Glycosylation and Relation to Diabetes

NASA Astrophysics Data System (ADS)

Pernodet, Nadine; Bloomberg, Ayla; Sood, Vandana; Slutsky, Lenny; Ge, Shouren; Clark, Richard; Rafailovich, Miriam

2004-03-01

The chemical modification and crosslinking of proteins by sugar glycosylation contribute to the aging of tissue proteins, and acceleration of this reaction during hyperglycemia is implicated in the pathogenesis of diabetic complications, such as disorder of the wound healing. Advanced glycation endproducts (AGEs) formation and protein crosslinking are irreversible processes that alter the structural and functional properties of proteins, lipid components and nucleic acids. And the mechanism, by which it happens, is not clear. Fibrinogen and fibronectin are plasma proteins, which play a major role in human wound healing. Fibrinogen converts to an insoluble fibrin "gel" following a cut, which eventually forms a clot to prevent blood loss, to direct cell adhesion and migration for forming scars. Fibronectin is a critical protein for cell adhesion and migration in wound healing. The effects of glucose on the binding of these plasma proteins from the extra cellular matrix (ECM) were followed at different concentrations by atomic force microscopy and lateral force modulation to measure the mechanical response of the samples. Glucose solutions (1, 2, and 3mg/mL) were incubated with the protein (100 mg/ml) and silicon (Si) substrates spun with sulfonated polystyrene (SPS) 28% for five days. Data showed that not only the organization of the protein on the surface was affected but also its mechanical properties. At 3 mg/mL glucose, Fn fibers were observed to be harder than those of the control, in good agreement with our hypothesis that glycosylation hardens tissues by crosslinking of proteins in the ECM and might cause fibers to break more easily.

Plant-derived micronutrients suppress monocyte adhesion to cultured human aortic endothelial cell layer by modulating its extracellular matrix composition.

PubMed

Ivanov, Vadim; Ivanova, Svetlana; Kalinovsky, Tatiana; Niedzwiecki, Aleksandra; Rath, Matthias

2008-07-01

Monocyte adhesion to endothelium plays an important role in atherosclerosis. We investigated the effects of micronutrients on monocyte-binding properties of extracellular matrix (ECM) produced by human aortic endothelial cells (AoEC). Confluent cultures of AoEC were exposed to ascorbic acid, quercetin, gotu kola extract (10% asiatic acid), green tea extract (40% epigallocatechin gallate), or a mixture of these micronutrients for 48 hours. AoEC-produced ECM was exposed by differential treatment. U937 monocyte adhesion was assayed by fluorescence. ECM composition was assayed immunochemically and with radiolabeled metabolic precursors. AoEC exposure to micronutrients reduced ECM capacity to bind monocytes in a dose-dependent manner. This effect was accompanied by profound changes in the ECM composition. Correlation analysis revealed that changes in monocyte adhesion to ECM had the strongest positive correlation with ECM content for laminin (CC = 0.9681, P < 0.01), followed by fibronectin, collagens type III, I, and IV, biglycan, heparan sulfate, and elastin. The strongest negative correlation was with chondroitin sulfate (CC = -0.9623, P < 0.01), followed by perlecan and versican. Individual micronutrients had diverse effects on ECM composition and binding properties, and their mixture was the most effective treatment. In conclusion, micronutrient-dependent reduction of monocyte adhesion to endothelium is partly mediated through specific modulation of ECM composition and properties.

International Union of Basic and Clinical Pharmacology. XCIV. Adhesion G Protein–Coupled Receptors

PubMed Central

Aust, Gabriela; Araç, Demet; Engel, Felix B.; Formstone, Caroline; Fredriksson, Robert; Hall, Randy A.; Harty, Breanne L.; Kirchhoff, Christiane; Knapp, Barbara; Krishnan, Arunkumar; Liebscher, Ines; Lin, Hsi-Hsien; Martinelli, David C.; Monk, Kelly R.; Peeters, Miriam C.; Piao, Xianhua; Prömel, Simone; Schöneberg, Torsten; Schwartz, Thue W.; Singer, Kathleen; Stacey, Martin; Ushkaryov, Yuri A.; Vallon, Mario; Wolfrum, Uwe; Wright, Mathew W.; Xu, Lei; Langenhan, Tobias

2015-01-01

The Adhesion family forms a large branch of the pharmacologically important superfamily of G protein–coupled receptors (GPCRs). As Adhesion GPCRs increasingly receive attention from a wide spectrum of biomedical fields, the Adhesion GPCR Consortium, together with the International Union of Basic and Clinical Pharmacology Committee on Receptor Nomenclature and Drug Classification, proposes a unified nomenclature for Adhesion GPCRs. The new names have ADGR as common dominator followed by a letter and a number to denote each subfamily and subtype, respectively. The new names, with old and alternative names within parentheses, are: ADGRA1 (GPR123), ADGRA2 (GPR124), ADGRA3 (GPR125), ADGRB1 (BAI1), ADGRB2 (BAI2), ADGRB3 (BAI3), ADGRC1 (CELSR1), ADGRC2 (CELSR2), ADGRC3 (CELSR3), ADGRD1 (GPR133), ADGRD2 (GPR144), ADGRE1 (EMR1, F4/80), ADGRE2 (EMR2), ADGRE3 (EMR3), ADGRE4 (EMR4), ADGRE5 (CD97), ADGRF1 (GPR110), ADGRF2 (GPR111), ADGRF3 (GPR113), ADGRF4 (GPR115), ADGRF5 (GPR116, Ig-Hepta), ADGRG1 (GPR56), ADGRG2 (GPR64, HE6), ADGRG3 (GPR97), ADGRG4 (GPR112), ADGRG5 (GPR114), ADGRG6 (GPR126), ADGRG7 (GPR128), ADGRL1 (latrophilin-1, CIRL-1, CL1), ADGRL2 (latrophilin-2, CIRL-2, CL2), ADGRL3 (latrophilin-3, CIRL-3, CL3), ADGRL4 (ELTD1, ETL), and ADGRV1 (VLGR1, GPR98). This review covers all major biologic aspects of Adhesion GPCRs, including evolutionary origins, interaction partners, signaling, expression, physiologic functions, and therapeutic potential. PMID:25713288

Mechanism of mast cell adhesion to human tenocytes in vitro.

PubMed

Behzad, Hayedeh; Tsai, Shu-Huei; Nassab, Paulina; Mousavizadeh, Rouhollah; McCormack, Robert G; Scott, Alex

2015-01-01

Mast cells and fibroblasts are two key players involved in many fibrotic and degenerative disorders. In the present study we examined the nature of binding interactions between human mast cells and tendon fibroblasts (tenocytes). In the mast cell-fibroblast co-culture model, mast cells were shown to spontaneously bind to tenocytes, in a process that was partially mediated by α5β1 integrin receptors. The same receptors on mast cells significantly mediated binding of these cells to tissue culture plates in the presence of tenocyte-conditioned media; the tenocyte-derived fibronectin in the media was shown to also play a major role in these binding activities. Upon binding to tenocytes or tissue culture plates, mast cells acquired an elongated phenotype, which was dependent on α5β1 integrin and tenocyte fibronectin. Additionally, tenocyte-derived fibronectin significantly enhanced mRNA expression of the adhesion molecule, THY1, by mast cells. Our data suggests that α5β1 integrin mediates binding of mast cells to human tenocyte and to tenocyte-derived ECM proteins, in particular fibronectin. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Intraplatelet reactive oxygen species (ROS) correlate with the shedding of adhesive receptors, microvesiculation and platelet adhesion to collagen during storage: Does endogenous ROS generation downregulate platelet adhesive function?

PubMed

Ghasemzadeh, Mehran; Hosseini, Ehteramolsadat; Roudsari, Zahra Oushyani; Zadkhak, Parvin

2018-03-01

Platelets storage lesion is mainly orchestrated by platelet activating signals during storage. Reactive oxygen species (ROS) are being considered as important signaling molecules modulating platelet function while their production has also been shown to be augmented by platelet activation. This study investigated to what extent endogenous ROS generation during platelet storage could be correlated with platelet receptor shedding, microvesiculation and adhesive function. 10 PRP-platelet concentrates were subjected to flow cytometry analysis to examine the generation of intraplatelet ROS on days 1, 5 and 7 after storage. In 5 day-stored platelets considering 40% of ROS generation as a cutoff point, samples were divided into two groups of those with higher or lower levels of ROS. The expression of adhesion receptors (GPVI, GPIbα), the amount of microparticles and phosphatidylserine exposure in each group were then examined by flow cytometry. Platelet receptor shedding and adhesion to collagen matrix were respectively measured by western blotting and microscopic assays. Our data showed lowered expression of GPIbα (p < 0.05) and GPVI in samples with ROS > 40% than those with ROS ≤ 40%, whereas receptors shedding and microvesiculation were (p < 0.05) elevated in platelets with higher levels of ROS. Functionally, we observed significantly (p < 0.05) lower levels of platelet adhesion to collagen matrix in samples with ROS generation more than 40%. Taken together, we showed correlations between intraplatelet ROS generation and either platelet receptors or microparticle shedding as well as platelet adhesive capacity to collagen. These findings suggest that augmented ROS generation during storage might be relevant to down-regulation of platelet adhesive function. Copyright © 2018 Elsevier Ltd. All rights reserved.

Adhesive interactions of biologically inspired soft condensed matter

NASA Astrophysics Data System (ADS)

Anderson, Travers Heath

Improving our fundamental understanding of the surface interactions between complex materials is needed to improve existing materials and products as well as develop new ones. The object of this research was to apply the measurements of fundamental surface interactions to real world problems facing chemical engineers and materials scientists. I focus on three systems of biologically inspired soft condensed matter, with an emphasis on the adhesive interactions between them. The formation of phospholipid bilayers of the neutral lipid, dimyristoyl-phosphatidylcholine (DMPC) on silica surfaces from vesicles in aqueous solutions was investigated. The process involves five stages: vesicle adhesion to the substrate surfaces, steric interactions with neighboring vesicles, rupture, spreading via hydrophobic fusion of bilayer edges, and ejection of excess lipid, trapped water and ions into the solution. The forces between DMPC bilayers and silica were measured in the Surface Forces Apparatus (SFA) in phosphate buffered saline. The adhesion energy was found to be much stronger than the expected adhesion predicted by van der Waals interactions, likely due to an attractive electrostatic interaction. The effects of non-adsorbing cationic polyelectrolytes on the interactions between supported cationic surfactant bilayers were studied using the SFA. Addition of polyelectrolyte has a number of effects on the interactions including the induction of a depletion-attraction and screening of the double-layer repulsion. Calculations are made that allow for the conversion of the adhesion energy measured in the SFA to the overall interaction energy between vesicles in solution, which determines the stability behavior of vesicle dispersions. Mussels use a variety of dihydroxyphenyl-alanine (DOPA) rich proteins specifically tailored to adhering to wet surfaces. The SFA was used to study the role of DOPA on the adhesive properties of these proteins to TiO 2 and mica using both real mussel

Neural cell adhesion molecule-deficient beta-cell tumorigenesis results in diminished extracellular matrix molecule expression and tumour cell-matrix adhesion.

PubMed

Håkansson, Joakim; Xian, Xiaojie; He, Liqun; Ståhlberg, Anders; Nelander, Sven; Samuelsson, Tore; Kubista, Mikael; Semb, Henrik

2005-01-01

To understand by which mechanism neural cell adhesion molecule (N-CAM) limits beta tumour cell disaggregation and dissemination, we searched for potential downstream genes of N-CAM during beta tumour cell progression by gene expression profiling. Here, we show that N-CAM-deficient beta-cell tumorigenesis is associated with changes in the expression of genes involved in cell-matrix adhesion and cytoskeletal dynamics, biological processes known to affect the invasive and metastatic behaviour of tumour cells. The extracellular matrix (ECM) molecules emerged as the primary target, i.e. N-CAM deficiency resulted in down-regulated mRNA expression of a broad range of ECM molecules. Consistent with this result, deficient deposition of major ECM stromal components, such as fibronectin, laminin 1 and collagen IV, was observed. Moreover, N-CAM-deficient tumour cells displayed defective matrix adhesion. These results offer a potential mechanism for tumour cell disaggregation during N-CAM-deficient beta tumour cell progression. Prospective consequences of these findings for the role of N-CAM in beta tumour cell dissemination are discussed.

Intercellular adhesion molecule-1 augments myoblast adhesion and fusion through homophilic trans-interactions.

PubMed

Pizza, Francis X; Martin, Ryan A; Springer, Evan M; Leffler, Maxwell S; Woelmer, Bryce R; Recker, Isaac J; Leaman, Douglas W

2017-07-11

The overall objective of the study was to identify mechanisms through which intercellular adhesion molecule-1 (ICAM-1) augments the adhesive and fusogenic properties of myogenic cells. Hypotheses were tested using cultured myoblasts and fibroblasts, which do not constitutively express ICAM-1, and myoblasts and fibroblasts forced to express full length ICAM-1 or a truncated form lacking the cytoplasmic domain of ICAM-1. ICAM-1 mediated myoblast adhesion and fusion were quantified using novel assays and cell mixing experiments. We report that ICAM-1 augments myoblast adhesion to myoblasts and myotubes through homophilic trans-interactions. Such adhesive interactions enhanced levels of active Rac in adherent and fusing myoblasts, as well as triggered lamellipodia, spreading, and fusion of myoblasts through the signaling function of the cytoplasmic domain of ICAM-1. Rac inhibition negated ICAM-1 mediated lamellipodia, spreading, and fusion of myoblasts. The fusogenic property of ICAM-1-ICAM-1 interactions was restricted to myogenic cells, as forced expression of ICAM-1 by fibroblasts did not augment their fusion to ICAM-1+ myoblasts/myotubes. We conclude that ICAM-1 augments myoblast adhesion and fusion through its ability to self-associate and initiate Rac-mediated remodeling of the actin cytoskeleton.

The structure of cell-matrix adhesions: the new frontier.

PubMed

Hanein, Dorit; Horwitz, Alan Rick

2012-02-01

Adhesions between the cell and the extracellular matrix (ECM) are mechanosensitive multi-protein assemblies that transmit force across the cell membrane and regulate biochemical signals in response to the chemical and mechanical environment. These combined functions in force transduction, signaling and mechanosensing contribute to cellular phenotypes that span development, homeostasis and disease. These adhesions form, mature and disassemble in response to actin organization and physical forces that originate from endogenous myosin activity or external forces by the extracellular matrix. Despite advances in our understanding of the protein composition, interactions and regulation, our understanding of matrix adhesion structure and organization, how forces affect this organization, and how these changes dictate specific signaling events is limited. Insights across multiple structural levels are acutely needed to elucidate adhesion structure and ultimately the molecular basis of signaling and mechanotransduction. Here we describe the challenges and recent advances and prospects for unraveling the structure of cell-matrix adhesions and their response to force. Copyright © 2011 Elsevier Ltd. All rights reserved.

Species specificity in cell-substrate interactions in medusae.

PubMed

Schmid, V; Bally, A

1988-10-01

A new system is described for the study of ECM-tissue interactions, using the ECM (called mesogloea) of various cnidarians and isolated striated muscle and endodermal tissue of jellyfish. The mesogloea consists mainly of water and collagen. It is present in all cnidarians and can be isolated without enzyme treatment. It can be used as a substrate to which cells and tissues adhere and on which they spread and migrate. Tissues of striated muscle and endoderm adhere and spread not only on mesogloea from regions they normally cover, but also from other regions of the animal. However, adhesion and spreading are highly species-specific. Species-specific adhesion is found throughout the whole mass of mesogloea even at regions where cells do not occur naturally. The cell adhesion factor can be extracted from the mesogloea so that the mesogloea no longer shows any cell adhesion properties. The extract consists mainly of a cysteine-containing collagen.

Syndecan-2 is upregulated in colorectal cancer cells through interactions with extracellular matrix produced by stromal fibroblasts.

PubMed

Vicente, Carolina Meloni; Ricci, Ritchelli; Nader, Helena Bonciani; Toma, Leny

2013-05-25

The extracellular matrix (ECM) influences the structure, viability and functions of cells and tissues. Recent evidence indicates that tumor cells and stromal cells interact through direct cell-cell contact, the production of ECM components and the secretion of growth factors. Syndecans are a family of transmembrane heparan sulfate proteoglycans that are involved in cell adhesion, motility, proliferation and differentiation. Syndecan-2 has been found to be highly expressed in colorectal cancer cell lines and appears to be critical for cancer cell behavior. We have examined the effect of stromal fibroblast-produced ECM on the production of proteoglycans by colorectal cancer cell lines. Our results showed that in a highly metastatic colorectal cancer cell line, HCT-116, syndecan-2 expression is enhanced by fibroblast ECM, while the expression of other syndecans decreased. Of the various components of the stromal ECM, fibronectin was the most important in stimulating the increase in syndecan-2 expression. The co-localization of syndecan-2 and fibronectin suggests that these two molecules are involved in the adhesion of HCT-116 cells to the ECM. Additionally, we demonstrated an increase in the expression of integrins alpha-2 and beta-1, in addition to an increase in the expression of phospho-FAK in the presence of fibroblast ECM. Furthermore, blocking syndecan-2 with a specific antibody resulted in a decrease in cell adhesion, migration, and organization of actin filaments. Overall, these results show that interactions between cancer cells and stromal ECM proteins induce significant changes in the behavior of cancer cells. In particular, a shift from the expression of anti-tumorigenic syndecans to the tumorigenic syndecan-2 may have implications in the migratory behavior of highly metastatic tumor cells.

Depressed immune surveillance against cancer: role of deficient T cell: extracellular matrix interactions.

PubMed

Górski, A; Castronovo, V; Stepień-Sopniewska, B; Grieb, P; Ryba, M; Mrowiec, T; Korczak-Kowalska, G; Wierzbicki, P; Matysiak, W; Dybowska, B

1994-07-01

Although T cells infiltrate malignant tumors, the local immune response is usually inefficient and tumors escape destruction. While extracellular matrix proteins strongly costimulate T cell responses in normal individuals, our studies indicate that peripheral blood T cells from cancer patients and tumor infiltrating cells respond poorly or are resistant to stimulative signals mediated by collagen I and IV and fibronectin. Moreover, the adhesive properties of cancer T cells are markedly depressed. Those functional deficiencies are paralleled by variable deficits in integrin and non-integrin T cell receptors for extracellular matrix. Immunotherapy with BCG causes a dramatic but transient increase in T cell: ECM interactions.

A role for the PDZ-binding domain of the coxsackie B virus and adenovirus receptor (CAR) in cell adhesion and growth.

PubMed

Excoffon, Katherine J D Ashbourne; Hruska-Hageman, Alesia; Klotz, Michael; Traver, Geri L; Zabner, Joseph

2004-09-01

The coxsackie and adenovirus receptor (CAR) plays a role in viral infection, maintenance of the junction adhesion complex in polarized epithelia, and modulation of cellular growth properties. As a viral receptor, the C-terminus appears to play no role indicating that the major function of CAR is to tether the virus to the cell. By contrast, the C-terminus is known to play a role in cellular localization and probably has a significant function in CAR-mediated adhesion and cell growth properties. We hypothesized that the CAR PDZ (PSD-95/Disc-large/ZO-1) binding motif interacts with PDZ-domain-containing proteins to modulate the cellular phenotype. CAR was modified by deleting the last four amino acids (CARDeltaGSIV) and evaluated for cell-cell adhesion in polarized primary human airway epithelia and growth characteristics in stably transfected L-cells. Although ablation of the CAR PDZ-binding motif did not affect adenoviral infection, it did have a significant effect both on cell-cell adhesion and on cell growth. Expression of CARDeltaGSIV failed to increase the transepithelial resistance in polarized epithelia to the same degree as wild-type CAR and failed to act as a growth modulator in L-cells. Furthermore, we provide evidence for three new CAR interacting partners, including MAGI-1b, PICK1 and PSD-95. CAR appears to interact with several distinct PDZ-domain-containing proteins and may exert its biological function through these interactions.

Ligand-induced adhesion to activated endothelium and to vascular cell adhesion molecule-1 in lymphocytes transfected with the N-formyl peptide receptor.

PubMed

Honda, S; Campbell, J J; Andrew, D P; Engelhardt, B; Butcher, B A; Warnock, R A; Ye, R D; Butcher, E C

1994-04-15

Binding of FMLP to the neutrophil N-formyl peptide receptor (FPR) transmits signals through pertussis toxin-sensitive G proteins triggering Ca2+ flux, superoxide production, granule exocytosis, and neutrophil aggregation and adhesion involving the beta 2 (CD18) integrins. Expression of the FPR in mouse fibroblasts or human kidney cells has been shown to confer an N-formyl peptide-inducible Ca2+ flux in transfectants. Here we demonstrate that the transfected receptor can also support ligand-induced alterations in cellular adhesion. We established stable transfectants of mouse L1-2 pre-B cells with cDNA for human FPR (L1-2 FPR cells). The transfectants bind N-formyl-Nle-Leu-Phe-Nle-Tyr-Lys-fluorescein with 1.4 x 10(5) sites per cell and a dissociation constant of 3.3 nM. Stimulation with FMLP induces a transient Ca2+ flux. FMLP also triggers adhesion of L1-2 FPR cells to TNF-alpha- or LPS-activated bEnd3 cells (mouse brain-derived endothelial cells) and to purified mouse VCAM-1. Binding is inhibited by Abs to VCAM-1 and to the alpha-chain of its lymphocyte receptor (the alpha 4 beta 1 integrin, VLA-4). Stimulation with FMLP does not induce a change in cell surface expression of alpha 4. Induced adhesion to VCAM-1 is rapid, detectable at the earliest times measurable (30 to 60 s after FMLP addition), and is inhibited by pertussis toxin. We conclude that FPR can mediate integrin activation not only in neutrophils but also in lymphocytes, and can trigger rapid adhesion via lymphocyte alpha 4 beta 1. The adhesion of lymphocytes is critical to their migration and targeting; our results suggest the possibility of manipulating adhesive responses through expression of chemoattractant receptors in lymphoid cells engineered for cellular therapy, allowing targeted adhesion and potentially migration in response to locally administered ligands.

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix

PubMed Central

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-01-01

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPBKKK−, a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of α4β1 and α4β7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB–GAG interaction in the chemokine-like activity of this protein. PMID:11867726

Interaction with glycosaminoglycans is required for cyclophilin B to trigger integrin-mediated adhesion of peripheral blood T lymphocytes to extracellular matrix.

PubMed

Allain, Fabrice; Vanpouille, Christophe; Carpentier, Mathieu; Slomianny, Marie-Christine; Durieux, Sandrine; Spik, Geneviève

2002-03-05

Cyclophilins A and B (CyPA and CyPB) are cyclosporin A-binding proteins that are involved in inflammatory events. We have reported that CyPB interacts with two types of cell-surface-binding sites. The first site corresponds to a functional receptor and requires interaction with the central core of CyPB. This region is highly conserved in cyclophilins, suggesting that CyPA and CyPB might share biological activities mediated by interaction with this receptor. The second site is identified with glycosaminoglycans (GAGs), the binding region located in the N terminus of CyPB. The difference in the N-terminal extensions of CyPA and CyPB suggests that a unique interaction with GAGs might account for selective activity of CyPB. To explore this hypothesis, we analyzed the lymphocyte responses triggered by CyPA, CyPB, and CyPB(KKK-), a mutant unable to interact with GAGs. The three ligands seemed capable enough to elicit calcium signal and chemotaxis by binding to the same signaling receptor. In contrast, only CyPB enhanced firm adhesion of T cells to the extracellular matrix. This activity depended on the interactions with GAGs and signaling receptor. CyPB-mediated adhesion required CD147 presumably because it was a costimulatory molecule and was related to an activation of alpha4beta1 and alpha4beta7 integrins. Finally, we showed that CyPB was capable mainly to enhance T cell adhesion of the CD4+CD45RO+ subset. The present data indicate that CyPB rather than CyPA is a proinflammatory factor for T lymphocytes and highlight the crucial role of CyPB-GAG interaction in the chemokine-like activity of this protein.

EMMPRIN (CD147) is a novel receptor for platelet GPVI and mediates platelet rolling via GPVI-EMMPRIN interaction.

PubMed

Seizer, Peter; Borst, Oliver; Langer, Harald F; Bültmann, Andreas; Münch, Götz; Herouy, Yared; Stellos, Konstantinos; Krämer, Björn; Bigalke, Boris; Büchele, Berthold; Bachem, Max G; Vestweber, Dietmar; Simmet, Thomas; Gawaz, Meinrad; May, Andreas E

2009-04-01

The Extracellular Matrix Metalloproteinase Inducer (EMMPRIN, CD147, basigin) is an immunoglobulin-like receptor expressed in various cell types. During cellular interactions homotypic EMMPRIN-EMMPRIN interactions are known to induce the synthesis of matrix metalloproteinases. Recently, we have identified EMMPRIN as a novel receptor on platelets. To our knowledge EMMPRIN has not been shown to serve as adhesion receptor, yet. Here we characterise platelet glycoprotein VI (GPVI) as a novel adhesion receptor for EMMPRIN. Human platelets were prestimulated with ADP and perfused over immobilised recombinant EMMPRIN-Fc or Fc-fragments under arterial shear conditions. ADP-stimulated platelets showed significantly enhanced rolling (but not enhanced firm adhesion) on immobilised EMMPRIN-Fc compared to Fc. Pretreatment of platelets with blocking mAbs anti-EMMPRIN or anti-GPVI leads to a significant reduction of rolling platelets on immobilised EMMPRIN-Fc, whereas pretreatment with blocking mAbs anti-p-selectin, anti-alpha4-integrin or anti-GPIIb/IIIa complex (20 microg/ml each) had no effect. Consistently, chinese hamster ovary (CHO) cells stably transfected with GPVI showed enhanced rolling (but not adhesion) on immobilised EMMPRIN-Fc in comparison to non-transfected CHO cells. Similarly, CHO cells stably transfected with EMMPRIN showed enhanced rolling on immobilised GPVI-Fc (or EMMPRIN-Fc) compared to non transfected CHO-cells. Finally, specific binding of EMMPRIN to GPVI was demonstrated by a modified ELISA and surface plasmon resonance technology with a dissociation constant of 88 nM. Platelet GPVI is a novel receptor for EMMPRIN and can mediate platelet rolling via GPVI-EMMPRIN interaction.

The glycocalyx promotes cooperative binding and clustering of adhesion receptors.

PubMed

Xu, Guang-Kui; Qian, Jin; Hu, Jinglei

2016-05-18

Cell adhesion plays a pivotal role in various biological processes, e.g., immune responses, cancer metastasis, and stem cell differentiation. The adhesion behaviors depend subtly on the binding kinetics of receptors and ligands restricted at the cell-substrate interfaces. Although much effort has been directed toward investigating the kinetics of adhesion molecules, the role of the glycocalyx, anchored on cell surfaces as an exterior layer, is still unclear. In this paper, we propose a theoretical approach to study the collective binding kinetics of a few and a large number of binders in the presence of the glycocalyx, representing the cases of initial and mature adhesions of cells, respectively. The analytical results are validated by finding good agreement with our Monte Carlo simulations. In the force loading case, the on-rate and affinity increase as more bonds form, whereas this cooperative effect is not observed in the displacement loading case. The increased thickness and stiffness of the glycocalyx tend to decrease the affinity for a few bonds, while they have less influence on the affinity for a large number of bonds. Moreover, for a flexible membrane with thermally-excited shape fluctuations, the glycocalyx is exhibited to promote the formation of bond clusters, mainly due to the cooperative binding of binders. This study helps to understand the cooperative kinetics of adhesion receptors under physiologically relevant loading conditions and sheds light on the novel role of the glycocalyx in cell adhesion.

Synergistic effect of two cell recognition systems: glycosphingolipid-glycosphingolipid interaction and integrin receptor interaction with pericellular matrix protein.

PubMed

Kojima, N; Hakomori, S

1991-12-01

GM3-expressing cells adhere, spread and migrate on plastic plates coated with Gg3, LacCer and Gb4, but not with other glycosphingolipids (GSLs). Thus, cell adhesion, spreading and migration through GSL-GSL interaction occur in an analogous fashion to the interaction of cells with adhesive matrix proteins [AP, e.g. fibronectin (FN), laminin (LN)] through their integrin receptors. In this study, the adhesion of two GM3-expressing cell lines (B16 melanoma and HEL299 fibroblast) on plastic plates co-coated with GSL plus AP is compared with adhesion on plates coated with GSL (Gg3 or LacCer) alone, or coated with AP alone. Results show that: (i) cell adhesion on GSL-coated plates takes place earlier in the incubation period than that on AP-coated plates; (ii) cell adhesion, as well as spreading, was greatly enhanced (in terms of strength and rapidity) on plates co-coated with GSL plus AP; (iii) repulsion (negative adhesion) of cells was observed on plates co-coated with AP plus N-acetyl-GM3 (NAcGM3) and was presumably based on repulsive NAcGM3-NAcGM3 interaction; (iv) GM3-dependent cell adhesion on GSL-coated plates, as well as synergistic promotion of cell adhesion (based on the GSL-GSL and AP-integrin systems), was suppressed by incubation of cells with anti-GM3 monoclonal antibody DH2 or sialidase. Synergistic adhesion of cells on GSL/AP co-coated plates was less inhibited by incubation with peptide sequences RGDS or YIGSR than was adhesion on plates coated with AP alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Computational modeling of three-dimensional ECM-rigidity sensing to guide directed cell migration.

PubMed

Kim, Min-Cheol; Silberberg, Yaron R; Abeyaratne, Rohan; Kamm, Roger D; Asada, H Harry

2018-01-16

Filopodia have a key role in sensing both chemical and mechanical cues in surrounding extracellular matrix (ECM). However, quantitative understanding is still missing in the filopodial mechanosensing of local ECM stiffness, resulting from dynamic interactions between filopodia and the surrounding 3D ECM fibers. Here we present a method for characterizing the stiffness of ECM that is sensed by filopodia based on the theory of elasticity and discrete ECM fiber. We have applied this method to a filopodial mechanosensing model for predicting directed cell migration toward stiffer ECM. This model provides us with a distribution of force and displacement as well as their time rate of changes near the tip of a filopodium when it is bound to the surrounding ECM fibers. Aggregating these effects in each local region of 3D ECM, we express the local ECM stiffness sensed by the cell and explain polarity in the cellular durotaxis mechanism.

Activation of cannabinoid CB2 receptor ameliorates atherosclerosis associated with suppression of adhesion molecules.

PubMed

Zhao, Yan; Yuan, Zuyi; Liu, Yan; Xue, Jiahong; Tian, Yuling; Liu, Weimin; Zhang, Weiping; Shen, Yan; Xu, Wei; Liang, Xiao; Chen, Tao

2010-03-01

Adhesion molecules have been implicated in the development and progression of atherosclerosis. Cannabinoids have been reported to modulate the migration and adhesion molecules expression of various cell types. Here we examined the effects of WIN55212-2, a cannabinoid receptor 1 (CB1-R)/cannabinoid receptor 2 (CB2-R) agonist on the development of atherosclerotic lesions in apolipoprotein E-deficient (ApoE-/-) mice, which are vulnerable because of their high plasma cholesterol and triacylglycerol levels, focusing on the expression of endothelial adhesion molecules. In the aorta of ApoE-/- mice, WIN55212-2 significantly reduced aortic root plaque area. The mechanism for this seemed to be reduced infiltration of macrophages into the atherosclerotic plaque which was also associated with reduced expression of vascular cellular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and P-selectin in the aorta. In vitro studies revealed reduced cell adhesion of a monocytic cell line (U937) to human umbilical vein endothelial cells after incubation with WIN55212-2. The reduction in macrophage adhesion also correlated with significant reductions in the expression of VCAM-1, ICAM-1, and P-selectin, indicating that reduced infiltration of macrophages in atherosclerotic plaques may occur as a result of the direct effect of WIN55212-2 on adhesion molecules in macrophages and endothelial cells. In conclusion, WIN55212-2 seems to have direct anti-atherosclerotic effects in an animal model of atherosclerosis. These effects were at least partly due to effects on the expression of VCAM-1, ICAM-1, and P-selectin, which led to reduced macrophage adhesion and infiltration. Furthermore, the protective effects completely blocked by the highly selective CB2 receptor antagonist AM630 suggest that these beneficial effects of WIN55212-2 may be mediated through the CB2 receptor.

Involvement of the orphan nuclear estrogen receptor-related receptor α in osteoclast adhesion and transmigration

PubMed Central

Bonnelye, Edith; Saltel, Frédéric; Chabadel, Anne; Zirngibl, Ralph A; Aubin, Jane E; Jurdic, Pierre

2010-01-01

The orphan nuclear receptor, estrogen receptor-related receptor α (ERRα) is expressed in osteoblasts and osteoclasts (OCs) and has been proposed to be a modulator of estrogen signaling. To determine the role of ERRα in OC biology, we knocked down ERRα activity by transient transfection of an siRNA directed against ERRα in the RAW264.7 monocyte–macrophage cell line that differentiates into OCs in the presence of receptor activator of nuclear factor κB-ligands and macrophage colony-stimulating factor. In parallel, stable RAW cell lines expressing a dominant-negative form of ERRα and green fluorescent protein (RAW-GFP-ERRαΔAF2) were used. Expression of OC markers was assessed by real-time PCR, and adhesion and transmigration tests were performed. Actin cytoskeletal organization was visualized using confocal microscopy. We found that RAW264.7 cells expressing siRNA directed against ERRα and RAW-GFP-ERRαΔAF2 OCs displayed abnormal spreading, and decreased osteopontin and β3 integrin subunit expression compared with the corresponding control cells. Decreased adhesion and the absence of podosome belts concomitant with abnormal localization of c-src were also observed in RAW-GFP-ERRαΔAF2-derived OCs. In addition, RAW-GFP-ERRαΔAF2-derived OCs failed to transmigrate through osteoblast cell layers. Our data show that the impairment of ERRα function does not alter OC precursor proliferation and differentiation but does alter the adhesion/spreading and migration capacities of mature OCs. PMID:20841427

Trabecular meshwork ECM remodeling in glaucoma: could RAS be a target?

PubMed

Agarwal, Puneet; Agarwal, Renu

2018-06-14

Disturbances of extracellular matrix (ECM) homeostasis in trabecular meshwork (TM) cause increased aqueous outflow resistance leading to elevated intraocular pressure (IOP) in glaucomatous eyes. Therefore, restoration of ECM homeostasis is a rational approach to prevent disease progression. Since renin-angiotensin system (RAS) inhibition positively alters ECM homeostasis in cardiovascular pathologies involving pressure and volume overload, it is likely that RAS inhibitors reduce IOP primarily by restoring ECM homeostasis. Areas covered: Current evidence showing the presence of RAS components in ocular tissue and its role in regulating aqueous humor dynamics is briefly summarized. The role of RAS in ECM remodeling is discussed both in terms of its effects on ECM synthesis and its breakdown. The mechanisms of ECM remodeling involving interactions of RAS with transforming growth factor-β, Wnt/β-catenin signaling, bone morphogenic proteins, connective tissue growth factor, and matrix metalloproteinases in ocular tissue are discussed. Expert opinion: Current literature strongly indicates a significant role of RAS in ECM remodeling in TM of hypertensive eyes. Hence, IOP-lowering effect of RAS inhibitors may primarily be attributed to restoration of ECM homeostasis in aqueous outflow pathways rather than its vascular effects. However, the mechanistic targets for RAS inhibitors have much wider distribution and consequences, which remain relatively unexplored in TM.

Fibrous nonlinear elasticity enables positive mechanical feedback between cells and ECMs

PubMed Central

Hall, Matthew S.; Alisafaei, Farid; Ban, Ehsan; Feng, Xinzeng; Hui, Chung-Yuen; Shenoy, Vivek B.; Wu, Mingming

2016-01-01

In native states, animal cells of many types are supported by a fibrous network that forms the main structural component of the ECM. Mechanical interactions between cells and the 3D ECM critically regulate cell function, including growth and migration. However, the physical mechanism that governs the cell interaction with fibrous 3D ECM is still not known. In this article, we present single-cell traction force measurements using breast tumor cells embedded within 3D collagen matrices. We recreate the breast tumor mechanical environment by controlling the microstructure and density of type I collagen matrices. Our results reveal a positive mechanical feedback loop: cells pulling on collagen locally align and stiffen the matrix, and stiffer matrices, in return, promote greater cell force generation and a stiffer cell body. Furthermore, cell force transmission distance increases with the degree of strain-induced fiber alignment and stiffening of the collagen matrices. These findings highlight the importance of the nonlinear elasticity of fibrous matrices in regulating cell–ECM interactions within a 3D context, and the cell force regulation principle that we uncover may contribute to the rapid mechanical tissue stiffening occurring in many diseases, including cancer and fibrosis. PMID:27872289

Anisotropic forces from spatially constrained focal adhesions mediate contact guidance directed cell migration

PubMed Central

Ray, Arja; Lee, Oscar; Win, Zaw; Edwards, Rachel M.; Alford, Patrick W.; Kim, Deok-Ho; Provenzano, Paolo P.

2017-01-01

Directed migration by contact guidance is a poorly understood yet vital phenomenon, particularly for carcinoma cell invasion on aligned collagen fibres. We demonstrate that for single cells, aligned architectures providing contact guidance cues induce constrained focal adhesion maturation and associated F-actin alignment, consequently orchestrating anisotropic traction stresses that drive cell orientation and directional migration. Consistent with this understanding, relaxing spatial constraints to adhesion maturation either through reduction in substrate alignment density or reduction in adhesion size diminishes the contact guidance response. While such interactions allow single mesenchymal-like cells to spontaneously ‘sense' and follow topographic alignment, intercellular interactions within epithelial clusters temper anisotropic cell–substratum forces, resulting in substantially lower directional response. Overall, these results point to the control of contact guidance by a balance of cell–substratum and cell–cell interactions, modulated by cell phenotype-specific cytoskeletal arrangements. Thus, our findings elucidate how phenotypically diverse cells perceive ECM alignment at the molecular level. PMID:28401884

α5β1 Integrin-Fibronectin Interactions Specify Liquid to Solid Phase Transition of 3D Cellular Aggregates

PubMed Central

Caicedo-Carvajal, Carlos E.; Shinbrot, Troy; Foty, Ramsey A.

2010-01-01

Background Tissue organization during embryonic development and wound healing depends on the ability of cells on the one hand to exchange adhesive bonds during active rearrangement and on the other to become fixed in place as tissue homeostasis is reached. Cells achieve these contradictory tasks by regulating either cell-cell adhesive bonds, mediated by cadherins, or cell-extracellular matrix (ECM) connections, regulated by integrins. Integrin α5β1 and soluble fibronectin (sFN) are key players in cell-ECM force generation and in ECM polymerization. Here, we explore the interplay between integrin α5β1 and sFN and its influence on tissue mechanical properties and cell sorting behavior. Methodology/Principal Findings We generated a series of cell lines varying in α5β1 receptor density. We then systematically explored the effects of different sFN concentrations on aggregate biomechanical properties using tissue surface tensiometry. We found previously unreported complex behaviors including the observation that interactions between fibronectin and integrin α5β1 generates biphasic tissue cohesion profiles. Specifically, we show that at constant sFn concentration, aggregate cohesion increases linearly as α5β1 receptor density is increased from low to moderate levels, producing a transition from viscoelastic-liquid to pseudo viscoelastic-solid behavior. However, further increase in receptor density causes an abrupt drop in tissue cohesion and a transition back to viscoelastic-liquid properties. We propose that this may be due to depletion of sFn below a critical value in the aggregate microenvironment at high α5β1 levels. We also show that differential expression of α5β1 integrin can promote phase-separation between cells. Conclusions/Significance The interplay between α5-integrin and sFn contributes significantly to tissue cohesion and, depending on their level of expression, can mediate a shift from liquid to elastic behavior. This interplay represents a

A disease-associated mutation in the adhesion GPCR BAI2 (ADGRB2) increases receptor signaling activity.

PubMed

Purcell, Ryan H; Toro, Camilo; Gahl, William A; Hall, Randy A

2017-12-01

Mutations in G protein-coupled receptors (GPCRs) that increase constitutive signaling activity can cause human disease. A de novo C-terminal mutation (R1465W) in the adhesion GPCR BAI2 (also known as ADGRB2) was identified in a patient suffering from progressive spastic paraparesis and other neurological symptoms. In vitro studies revealed that this mutation strongly increases the constitutive signaling activity of an N-terminally cleaved form of BAI2, which represents the activated form of the receptor. Further studies dissecting the mechanism(s) underling this effect revealed that wild-type BAI2 primarily couples to Gα z , with the R1465W mutation conferring increased coupling to Gα i . The R1465W mutation also increases the total and surface expression of BAI2. The mutation has no effect on receptor binding to β-arrestins, but does perturb binding to the endocytic protein endophilin A1, identified here as a novel interacting partner for BAI2. These studies provide new insights into the signaling capabilities of the adhesion GPCR BAI2/ADGRB2 and shed light on how an apparent gain-of-function mutation to the receptor's C-terminus may lead to human disease. © 2017 Wiley Periodicals, Inc.

Molecular mechanisms of mechanotransduction in integrin-mediated cell-matrix adhesion

PubMed Central

Li, Zhenhai; Lee, Hyunjung; Zhu, Cheng

2016-01-01

Cell-matrix adhesion complexes are multi-protein structures linking the extracellular matrix (ECM) to the cytoskeleton. They are essential to both cell motility and function by bidirectionally sensing and transmitting mechanical and biochemical stimulations. Several types of cell-matrix adhesions have been identified and they share many key molecular components, such as integrins and actin-integrin linkers. Mechanochemical coupling between ECM molecules and the actin cytoskeleton has been observed from the single cell to the single molecule level and from immune cells to neuronal cells. However, the mechanisms underlying force regulation of integrin-mediated mechanotransduction still need to be elucidated. In this review article, we focus on integrin-mediated adhesions and discuss force regulation of cell-matrix adhesions and key adaptor molecules, three different force-dependent behaviors, and molecular mechanisms for mechanochemical coupling in force regulation. PMID:27720950

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

Extracellular matrix biomimicry for the creation of investigational and therapeutic devices.

PubMed

Pellowe, Amanda S; Gonzalez, Anjelica L

2016-01-01

The extracellular matrix (ECM) is a web of fibrous proteins that serves as a scaffold for tissues and organs, and is important for maintaining homeostasis and facilitating cellular adhesion. Integrin transmembrane receptors are the primary adhesion molecules that anchor cells to the ECM, thus integrating cells with their microenvironments. Integrins play a critical role in facilitating cell-matrix interactions and promoting signal transduction, both from the cell to the ECM and vice versa, ultimately mediating cell behavior. For this reason, many advanced biomaterials employ biomimicry by replicating the form and function of fibrous ECM proteins. The ECM also acts as a reservoir for small molecules and growth factors, wherein fibrous proteins directly bind and present these bioactive moieties that facilitate cell activity. Therefore biomimicry can be enhanced by incorporating small molecules into ECM-like substrates. Biomimetic ECM materials have served as invaluable research tools for studying interactions between cells and the surrounding ECM, revealing that cell-matrix signaling is driven by mechanical forces, integrin engagement, and small molecules. Mimicking pathological ECMs has also elucidated disease specific cell behaviors. For example, biomimetic tumor microenvironments have been used to induce metastatic cell behaviors, and have thereby shown promise for in vitro cancer drug testing and targeting. Further, ECM-like substrates have been successfully employed for autologous cell recolonization for tissue engineering and wound healing. As we continue to learn more about the mechanical and biochemical characteristics of the ECM, these properties can be harnessed to develop new biomaterials, biomedical devices, and therapeutics. © 2015 Wiley Periodicals, Inc.

Dynamic adhesion of eryptotic erythrocytes to immobilized platelets via platelet phosphatidylserine receptors.

PubMed

Walker, Britta; Towhid, Syeda T; Schmid, Evi; Hoffmann, Sascha M; Abed, Majed; Münzer, Patrick; Vogel, Sebastian; Neis, Felix; Brucker, Sara; Gawaz, Meinrad; Borst, Oliver; Lang, Florian

2014-02-01

Glucose depletion of erythrocytes triggers suicidal erythrocyte death or eryptosis, which leads to cell membrane scrambling with phosphatidylserine exposure at the cell surface. Eryptotic erythrocytes adhere to endothelial cells by a mechanism involving phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 at the endothelial cell membrane. Nothing has hitherto been known about an interaction between eryptotic erythrocytes and platelets, the decisive cells in primary hemostasis and major players in thrombotic vascular occlusion. The present study thus explored whether and how glucose-depleted erythrocytes adhere to platelets. To this end, adhesion of phosphatidylserine-exposing erythrocytes to platelets under flow conditions was examined in a flow chamber model at arterial shear rates. Platelets were immobilized on collagen and further stimulated with adenosine diphosphate (ADP, 10 μM) or thrombin (0.1 U/ml). As a result, a 48-h glucose depletion triggered phosphatidylserine translocation to the erythrocyte surface and augmented the adhesion of erythrocytes to immobilized platelets, an effect significantly increased upon platelet stimulation. Adherence of erythrocytes to platelets was blunted by coating of erythrocytic phosphatidylserine with annexin V or by neutralization of platelet phosphatidylserine receptors CXCL16 and CD36 with respective antibodies. In conclusion, glucose-depleted erythrocytes adhere to platelets. The adhesive properties of platelets are augmented by platelet activation. Erythrocyte adhesion to immobilized platelets requires phosphatidylserine at the erythrocyte surface and CXCL16 as well as CD36 expression on platelets. Thus platelet-mediated erythrocyte adhesion may foster thromboocclusive complications in diseases with stimulated phosphatidylserine exposure of erythrocytes.

Integrin expression and glycosylation patterns regulate cell-matrix adhesion and alter with breast cancer progression.

PubMed

Singh, Chandrajeet; Shyanti, Ritis K; Singh, Virendra; Kale, Raosaheb K; Mishra, Jai P N; Singh, Rana P

2018-05-05

Integrins are the major cell adhesion glycoproteins involved in cell-extracellular matrix (ECM) interaction and metastasis. Further, glycosylation on integrin is necessary for its proper folding and functionality. Herein, differential expression of integrins viz., αvβ3 and αvβ6 was examined in MDA-MB-231, MDA-MB-468 and MCF-10A cells, which signify three different stages of breast cancer development from highly metastatic to non-tumorigenic stage. The expression of αvβ3 and αvβ6 integrins at mRNA and protein levels was observed in all three cell lines and the results displayed a distinct pattern of expression. Highly metastatic cells showed enhanced expression of αvβ3 than moderate metastatic and non-tumorigenic cells. The scenario was reversed in case of αvβ6 integrin, which was strongly expressed in moderate metastatic and non-tumorigenic cells. N-glycosylation of αvβ3 and αvβ6 integrins is required for the attachment of cells to ECM proteins like fibronectin. The cell adhesion properties were found to be different in these cancer cells with respect to the type of integrins expressed. The results testify that αvβ3 integrin in highly metastatic cells, αvβ6 integrin in both moderate metastatic and non-tumorigenic cells play an important role in cell adhesion. The investigation typify that N-glycosylation on integrins is also necessary for cell-ECM interaction. Further, glycosylation inhibition by Swainsonine is found to be more detrimental to invasive property of moderate metastatic cells. Conclusively, types of integrins expressed as well as their N-glycosylation pattern alter during the course of breast cancer progression. Copyright © 2018 Elsevier Inc. All rights reserved.

A Review of Cell Adhesion Studies for Biomedical and Biological Applications.

PubMed

Khalili, Amelia Ahmad; Ahmad, Mohd Ridzuan

2015-08-05

Cell adhesion is essential in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. The mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. The essential function of cell adhesion has created tremendous interests in developing methods for measuring and studying cell adhesion properties. The study of cell adhesion could be categorized into cell adhesion attachment and detachment events. The study of cell adhesion has been widely explored via both events for many important purposes in cellular biology, biomedical, and engineering fields. Cell adhesion attachment and detachment events could be further grouped into the cell population and single cell approach. Various techniques to measure cell adhesion have been applied to many fields of study in order to gain understanding of cell signaling pathways, biomaterial studies for implantable sensors, artificial bone and tooth replacement, the development of tissue-on-a-chip and organ-on-a-chip in tissue engineering, the effects of biochemical treatments and environmental stimuli to the cell adhesion, the potential of drug treatments, cancer metastasis study, and the determination of the adhesion properties of normal and cancerous cells. This review discussed the overview of the available methods to study cell adhesion through attachment and detachment events.

The endothelin B receptor plays a crucial role in the adhesion of neutrophils to the endothelium in sickle cell disease

PubMed Central

Koehl, Bérengère; Nivoit, Pierre; El Nemer, Wassim; Lenoir, Olivia; Hermand, Patricia; Pereira, Catia; Brousse, Valentine; Guyonnet, Léa; Ghinatti, Giulia; Benkerrou, Malika; Colin, Yves; Le Van Kim, Caroline; Tharaux, Pierre-Louis

2017-01-01

Although the primary origin of sickle cell disease is a hemoglobin disorder, many types of cells contribute considerably to the pathophysiology of the disease. The adhesion of neutrophils to activated endothelium is critical in the pathophysiology of sickle cell disease and targeting neutrophils and their interactions with endothelium represents an important opportunity for the development of new therapeutics. We focused on endothelin-1, a mediator involved in neutrophil activation and recruitment in tissues, and investigated the involvement of the endothelin receptors in the interaction of neutrophils with endothelial cells. We used fluorescence intravital microscopy analyses of the microcirculation in sickle mice and quantitative microfluidic fluorescence microscopy of human blood. Both experiments on the mouse model and patients indicate that blocking endothelin receptors, particularly ETB receptor, strongly influences neutrophil recruitment under inflammatory conditions in sickle cell disease. We show that human neutrophils have functional ETB receptors with calcium signaling capability, leading to increased adhesion to the endothelium through effects on both endothelial cells and neutrophils. Intact ETB function was found to be required for tumor necrosis factor α-dependent upregulation of CD11b on neutrophils. Furthermore, we confirmed that human neutrophils synthesize endothelin-1, which may be involved in autocrine and paracrine pathophysiological actions. Thus, the endothelin-ETB axis should be considered as a cytokine-like potent pro-inflammatory pathway in sickle cell disease. Blockade of endothelin receptors, including ETB, may provide major benefits for preventing or treating vaso-occlusive crises in sickle cell patients. PMID:28385784

The p+ 6He interaction from Ec.m.=0.5 to 25 MeV

NASA Astrophysics Data System (ADS)

Mackintosh, R. S.

2004-09-01

The p- 6He potential has been determined by inverting the S-matrix, calculated from single-channel RGM without absorption, over the energy range Ec.m.=0.5,1.0,…,25 MeV. Energy-dependent IP inversion was used, but with energy-independent spin-orbit terms. A potential with odd- and even-parity components reproduces Slj( E) from l=0 to l=4 over the whole energy range. The central components of the potential have a smooth, predominantly linear, energy dependence that is consistent with global phenomenology. We compare the similar neutron- 6Li (IAS) interaction. Various features in the potentials deserve explanation. The procedure employed here could extract dynamic polarization potentials for the p- 6He interaction from Slj( E) from multichannel RGM calculations, should these become available.

Tumor necrosis factor α (TNF-α) receptor-II is required for TNF-α–induced leukocyte-endothelial interaction in vivo

PubMed Central

Chandrasekharan, Unni M.; Siemionow, Maria; Unsal, Murat; Yang, Lin; Poptic, Earl; Bohn, Justin; Ozer, Kagan; Zhou, Zhongmin; Howe, Philip H.; Penn, Marc

2007-01-01

Tumor necrosis factor-α (TNF-α) binds to 2 distinct cell-surface receptors: TNF-α receptor-I (TNFR-I: p55) and TNF-α receptor-II (TNFR-II: p75). TNF-α induces leukocyte adhesion molecules on endothelial cells (ECs), which mediate 3 defined steps of the inflammatory response; namely, leukocyte rolling, firm adhesion, and transmigration. In this study, we have investigated the role of p75 in TNF-α–induced leukocyte adhesion molecules using cultured ECs derived from wild-type (WT), p75-null (p75−/−), or p55-null (p55−/−) mice. We observed that p75 was essential for TNF-α–induced E-selectin, vascular cell adhesion molecule 1 (VCAM-1), and intercellular adhesion molecule 1 (ICAM-1) expression. We also investigated the putative role of p75 in inflammation in vivo using an intravital microscopic approach with a mouse cremaster muscle model. TNF-α–stimulated leukocyte rolling, firm adhesion to ECs, and transmigration were dramatically reduced in p75−/− mice. Transplanted WT cremaster in p75−/− mice showed a robust leukocyte rolling and firm adhesion upon TNF-α activation, suggesting that the impairment in EC-leukocyte interaction in p75−/− mice is due to EC dysfunction. These results demonstrate, for the first time, that endothelial p75 is essential for TNF-α–induced leukocyte–endothelial-cell interaction. Our findings may contribute to the identification of novel p75-targeted therapeutic approaches for inflammatory diseases. PMID:17068152

Human mesenchymal stem cells target adhesion molecules and receptors involved in T cell extravasation.

PubMed

Benvenuto, Federica; Voci, Adriana; Carminati, Enrico; Gualandi, Francesca; Mancardi, Gianluigi; Uccelli, Antonio; Vergani, Laura

2015-12-10

Systemic delivery of bone marrow-derived mesenchymal stem cells (MSC) seems to be of benefit in the treatment of multiple sclerosis (MS), an autoimmune disease of the central nervous system (CNS) sustained by migration of T cells across the brain blood barrier (BBB) and subsequent induction of inflammatory lesions into CNS. MSC have been found to modulate several effector functions of T cells. In this study, we investigated the effects of MSC on adhesion molecules and receptors on T cell surface that sustain their transendothelial migration. We used different co-culture methods combined with real-time PCR and flow cytometry to evaluate the expression both at the mRNA and at the plasma-membrane level of α4 integrin, β2 integrin, ICAM-1 and CXCR3. In parallel, we assessed if MSC are able to modulate expression of adhesion molecules on the endothelial cells that interact with T cells during their transendothelial migration. Our in vitro analyses revealed that MSC: (i) inhibit proliferation and activation of both peripheral blood mononuclear cells (PBMC) and CD3(+)-selected lymphocytes through the release of soluble factors; (ii) exert suppressive effects on those surface molecules highly expressed by activated lymphocytes and involved in transendothelial migration; (iii) inhibit CXCL10-driven chemotaxis of CD3(+) cells; (iv) down-regulated expression of adhesion molecules on endothelial cells. Taken together, these data demonstrate that the immunosuppressive effect of MSC does not exclusively depends on their anti-proliferative activity on T cells, but also on the impairment of leukocyte migratory potential through the inhibition of the adhesion molecules and receptors that are responsible for T cell trafficking across BBB. This could suggest a new mechanism through which MSC modulate T cell responses.

UV-killed Staphylococcus aureus enhances adhesion and differentiation of osteoblasts on bone-associated biomaterials.

PubMed

Somayaji, Shankari N; Huet, Yvette M; Gruber, Helen E; Hudson, Michael C

2010-11-01

Titanium alloys (Ti) are the preferred material for orthopedic applications. However, very often, these metallic implants loosen over a long period and mandate revision surgery. For implant success, osteoblasts must adhere to the implant surface and deposit a mineralized extracellular matrix (ECM). Here, we utilized UV-killed Staphylococcus aureus as a novel osteoconductive coating for Ti surfaces. S. aureus expresses surface adhesins capable of binding to bone and biomaterials directly. Furthermore, interaction of S. aureus with osteoblasts activates growth factor-related pathways that potentiate osteogenesis. Although UV-killed S. aureus cells retain their bone-adhesive ability, they do not stimulate significant immune modulator expression. All of the abovementioned properties were utilized for a novel implant coating so as to promote osteoblast recruitment and subsequent cell functions on the bone-implant interface. In this study, osteoblast adhesion, proliferation, and mineralized ECM synthesis were measured on Ti surfaces coated with fibronectin with and without UV-killed bacteria. Osteoblast adhesion was enhanced on Ti alloy surfaces coated with bacteria compared to uncoated surfaces, while cell proliferation was sustained comparably on both surfaces. Osteoblast markers such as collagen, osteocalcin, alkaline phosphatase activity, and mineralized nodule formation were increased on Ti alloy coated with bacteria compared to uncoated surfaces.

Single Cell Force Spectroscopy for Quantification of Cellular Adhesion on Surfaces

NASA Astrophysics Data System (ADS)

Christenson, Wayne B.

Cell adhesion is an important aspect of many biological processes. The atomic force microscope (AFM) has made it possible to quantify the forces involved in cellular adhesion using a technique called single cell force spectroscopy (SCFS). AFM based SCFS offers versatile control over experimental conditions for probing directly the interaction between specific cell types and specific proteins, surfaces, or other cells. Transmembrane integrins are the primary proteins involved in cellular adhesion to the extra cellular matix (ECM). One of the chief integrins involved in the adhesion of leukocyte cells is alpha Mbeta2 (Mac-1). The experiments in this dissertation quantify the adhesion of Mac-1 expressing human embryonic kidney (HEK Mac-1), platelets, and neutrophils cells on substrates with different concentrations of fibrinogen and on fibrin gels and multi-layered fibrinogen coated fibrin gels. It was shown that multi-layered fibrinogen reduces the adhesion force of these cells considerably. A novel method was developed as part of this research combining total internal reflection microscopy (TIRFM) with SCFS allowing for optical microscopy of HEK Mac-1 cells interacting with bovine serum albumin (BSA) coated glass after interacting with multi-layered fibrinogen. HEK Mac-1 cells are able to remove fibrinogen molecules from the multi-layered fibrinogen matrix. An analysis methodology for quantifying the kinetic parameters of integrin-ligand interactions from SCFS experiments is proposed, and the kinetic parameters of the Mac-1 fibrinogen bond are quantified. Additional SCFS experiments quantify the adhesion of macrophages and HEK Mac-1 cells on functionalized glass surfaces and normal glass surfaces. Both cell types show highest adhesion on a novel functionalized glass surface that was prepared to induce macrophage fusion. These experiments demonstrate the versatility of AFM based SCFS, and how it can be applied to address many questions in cellular biology offering

Adhesion properties of Lactobacillus rhamnosus mucus-binding factor to mucin and extracellular matrix proteins.

PubMed

Nishiyama, Keita; Nakamata, Koichi; Ueno, Shintaro; Terao, Akari; Aryantini, Ni Putu Desy; Sujaya, I Nengah; Fukuda, Kenji; Urashima, Tadasu; Yamamoto, Yuji; Mukai, Takao

2015-01-01

We previously described potential probiotic Lactobacillus rhamnosus strains, isolated from fermented mare milk produced in Sumbawa Island, Indonesia, which showed high adhesion to porcine colonic mucin (PCM) and extracellular matrix (ECM) proteins. Recently, mucus-binding factor (MBF) was found in the GG strain of L. rhamnosus as a mucin-binding protein. In this study, we assessed the ability of recombinant MBF protein from the FSMM22 strain, one of the isolates of L. rhamnosus from fermented Sumbawa mare milk, to adhere to PCM and ECM proteins by overlay dot blot and Biacore assays. MBF bound to PCM, laminin, collagen IV, and fibronectin with submicromolar dissociation constants. Adhesion of the FSMM22 mbf mutant strain to PCM and ECM proteins was significantly less than that of the wild-type strain. Collectively, these results suggested that MBF contribute to L. rhamnosus host colonization via mucin and ECM protein binding.

Receptor type I and type II binding regions and the peptidyl-prolyl isomerase site of cyclophilin B are required for enhancement of T-lymphocyte adhesion to fibronectin.

PubMed

Carpentier, Mathieu; Allain, Fabrice; Slomianny, Marie-Christine; Durieux, Sandrine; Vanpouille, Christophe; Haendler, Bernard; Spik, Geneviève

2002-04-23

Cyclophilin B (CyPB), a cyclosporin A (CsA) binding protein, interacts with two types of binding sites at the surface of T-lymphocytes. The type I sites correspond to functional receptors involved in endocytosis and the type II sites to sulfated glycosaminoglycans (GAGs). Mutational analysis of CyPB has revealed that W128, which is part of the CsA-binding pocket, is implicated in the binding to the functional type I receptors and that two amino acid clusters located in the N-terminus ensure the binding to GAGs. The peptidyl-prolyl isomerase activity of CyPB is not required for receptor binding. We have recently demonstrated that CyPB enhances adhesion of peripheral blood T-lymphocytes to fibronectin, a component of the extracellular matrix. We intended to identify additional amino acids involved in the binding of CyPB to its functional type I receptor and to determine regions responsible for the stimulation of peripheral blood T-lymphocyte adhesion. We determined that residues R76, G77, K132, D155, and D158 of the calcineurin (CN) interacting region were implicated in the recognition of type I receptor but not of GAGs. We also found that two different changes in the N-terminal extension that abated binding to GAGs prevented adhesion of peripheral blood T-lymphocytes to coated CyPB, whereas abbrogation of the PPIase activity had no effect. On the other hand, the adhesion of peripheral blood T-lymphocytes to coated fibronectin was not stimulated by CyPB mutants devoid of either type I receptor or GAGs binding activity or by mutants of the PPIase site. Altogether, the results demonstrate that different regions of CyPB are involved in peripheral blood T-lymphocyte activation and imply a novel important physiological function for peptidyl-prolyl isomerase activity.

Integrin traffic - the update.

PubMed

De Franceschi, Nicola; Hamidi, Hellyeh; Alanko, Jonna; Sahgal, Pranshu; Ivaska, Johanna

2015-03-01

Integrins are a family of transmembrane cell surface molecules that constitute the principal adhesion receptors for the extracellular matrix (ECM) and are indispensable for the existence of multicellular organisms. In vertebrates, 24 different integrin heterodimers exist with differing substrate specificity and tissue expression. Integrin-extracellular-ligand interaction provides a physical anchor for the cell and triggers a vast array of intracellular signalling events that determine cell fate. Dynamic remodelling of adhesions, through rapid endocytic and exocytic trafficking of integrin receptors, is an important mechanism employed by cells to regulate integrin-ECM interactions, and thus cellular signalling, during processes such as cell migration, invasion and cytokinesis. The initial concept of integrin traffic as a means to translocate adhesion receptors within the cell has now been expanded with the growing appreciation that traffic is intimately linked to the cell signalling apparatus. Furthermore, endosomal pathways are emerging as crucial regulators of integrin stability and expression in cells. Thus, integrin traffic is relevant in a number of pathological conditions, especially in cancer. Nearly a decade ago we wrote a Commentary in Journal of Cell Science entitled 'Integrin traffic'. With the advances in the field, we felt it would be appropriate to provide the growing number of researchers interested in integrin traffic with an update. © 2015. Published by The Company of Biologists Ltd.

Role for cell adhesion and glycosyl (HNK-1 and oligomannoside) recognition in the sharpening of the regenerating retinotectal projection in goldfish.

PubMed

Schmidt, J T; Schachner, M

1998-12-01

Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7-21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76-86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1-positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26 degrees versus 11-12 degrees in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7 degrees. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18 degrees. Thus

Contractile forces originating from Cancer Diskiod regulated by geometrical ECM properties

NASA Astrophysics Data System (ADS)

Alobaidi, Amani; Sun, Bo

Cancer cell migration in three-dimensional extracellular matrix is a major cause of death for cancer patients. Although extensive studies have enlightened detailed mechanism of single cell 3D invasion and cell-ECM interaction, 3D collective cancer invasion is still poorly understood. To capture collective cancer invasion with more realistic, we developed a novel 3D invasion assay, Diskiod In Geometrically Micropatterned ECM (DIGME). DIGME allows us to independently controlling the shape the shape of tumor organoids, microstructure and spatial heterogeneity of the extracellular matrix all at the same time. Here we study the affect of contractile forces originating from different geometrical cancer diskiods. We show that cancer invasion could be regulated by geometrical ECM properties.

A Review of Cell Adhesion Studies for Biomedical and Biological Applications

PubMed Central

Ahmad Khalili, Amelia; Ahmad, Mohd Ridzuan

2015-01-01

Cell adhesion is essential in cell communication and regulation, and is of fundamental importance in the development and maintenance of tissues. The mechanical interactions between a cell and its extracellular matrix (ECM) can influence and control cell behavior and function. The essential function of cell adhesion has created tremendous interests in developing methods for measuring and studying cell adhesion properties. The study of cell adhesion could be categorized into cell adhesion attachment and detachment events. The study of cell adhesion has been widely explored via both events for many important purposes in cellular biology, biomedical, and engineering fields. Cell adhesion attachment and detachment events could be further grouped into the cell population and single cell approach. Various techniques to measure cell adhesion have been applied to many fields of study in order to gain understanding of cell signaling pathways, biomaterial studies for implantable sensors, artificial bone and tooth replacement, the development of tissue-on-a-chip and organ-on-a-chip in tissue engineering, the effects of biochemical treatments and environmental stimuli to the cell adhesion, the potential of drug treatments, cancer metastasis study, and the determination of the adhesion properties of normal and cancerous cells. This review discussed the overview of the available methods to study cell adhesion through attachment and detachment events. PMID:26251901

A tumor growth model with deformable ECM

NASA Astrophysics Data System (ADS)

Sciumè, G.; Santagiuliana, R.; Ferrari, M.; Decuzzi, P.; Schrefler, B. A.

2014-12-01

Existing tumor growth models based on fluid analogy for the cells do not generally include the extracellular matrix (ECM), or if present, take it as rigid. The three-fluid model originally proposed by the authors and comprising tumor cells (TC), host cells (HC), interstitial fluid (IF) and an ECM, considered up to now only a rigid ECM in the applications. This limitation is here relaxed and the deformability of the ECM is investigated in detail. The ECM is modeled as a porous solid matrix with Green-elastic and elasto-visco-plastic material behavior within a large strain approach. Jauman and Truesdell objective stress measures are adopted together with the deformation rate tensor. Numerical results are first compared with those of a reference experiment of a multicellular tumor spheroid (MTS) growing in vitro, then three different tumor cases are studied: growth of an MTS in a decellularized ECM, growth of a spheroid in the presence of host cells and growth of a melanoma. The influence of the stiffness of the ECM is evidenced and comparison with the case of a rigid ECM is made. The processes in a deformable ECM are more rapid than in a rigid ECM and the obtained growth pattern differs. The reasons for this are due to the changes in porosity induced by the tumor growth. These changes are inhibited in a rigid ECM. This enhanced computational model emphasizes the importance of properly characterizing the biomechanical behavior of the malignant mass in all its components to correctly predict its temporal and spatial pattern evolution.

The Coxsackievirus and Adenovirus Receptor: a new adhesion protein in cochlear development.

PubMed

Excoffon, Katherine J D A; Avenarius, Matthew R; Hansen, Marlan R; Kimberling, William J; Najmabadi, Hossein; Smith, Richard J H; Zabner, Joseph

2006-05-01

The Coxsackievirus and Adenovirus Receptor (CAR) is an essential regulator of cell growth and adhesion during development. The gene for CAR, CXADR, is located within the genomic locus for Usher syndrome type 1E (USH1E). Based on this and a physical interaction with harmonin, the protein responsible for USH1C, we hypothesized that CAR may be involved in cochlear development and that mutations in CXADR may be responsible for USH1E. The expression of CAR in the cochlea was determined by PCR and immunofluorescence microscopy. We found that CAR expression is highly regulated during development. In neonatal mice, CAR is localized to the junctions of most cochlear cell types but is restricted to the supporting and strial cells in adult cochlea. A screen of two populations consisting of non-syndromic deaf and Usher 1 patients for mutations in CXADR revealed one haploid mutation (P356S). Cell surface expression, viral receptor activity, and localization of the mutant form of CAR were indistinguishable from wild-type CAR. Although we were unable to confirm a role for CAR in autosomal recessive, non-syndromic deafness, or Usher syndrome type 1, based on its regulation, localization, and molecular interactions, CAR remains an attractive candidate for genetic deafness.

Platelet receptor polymorphisms do not influence Staphylococcus aureus–platelet interactions or infective endocarditis

PubMed Central

Daga, Shruti; Shepherd, James G.; Callaghan, J. Garreth S.; Hung, Rachel K.Y.; Dawson, Dana K.; Padfield, Gareth J.; Hey, Shi Y.; Cartwright, Robyn A.; Newby, David E.; Fitzgerald, J. Ross

2011-01-01

Cardiac vegetations result from bacterium–platelet adherence, activation and aggregation, and are associated with increased morbidity and mortality in infective endocarditis. The GPIIb/IIIa and FcγRIIa platelet receptors play a central role in platelet adhesion, activation and aggregation induced by endocarditis pathogens such as Staphylococcus aureus, but the influence of known polymorphisms of these receptors on the pathogenesis of infective endocarditis is unknown. We determined the GPIIIa platelet antigen PlA1/A2 and FcγRIIa H131R genotype of healthy volunteers (n = 160) and patients with infective endocarditis (n = 40), and investigated the influence of these polymorphisms on clinical outcome in infective endocarditis and S. aureus–platelet interactions in vitro. Platelet receptor genotype did not correlate with development of infective endocarditis, vegetation characteristics on echocardiogram or the composite clinical end-point of embolism, heart failure, need for surgery or mortality (P > 0.05 for all), even though patients with the GPIIIa PlA1/A1 genotype had increased in vivo platelet activation (P = 0.001). Furthermore, neither GPIIIa PlA1/A2 nor FcγRIIa H131R genotype influenced S. aureus-induced platelet adhesion, activation or aggregation in vitro (P > 0.05). Taken together, our data suggest that the GPIIIa and FcγRIIa platelet receptor polymorphisms do not influence S. aureus–platelet interactions in vitro or the clinical course of infective endocarditis. PMID:21044892

Local alignment vectors reveal cancer cell-induced ECM fiber remodeling dynamics

PubMed Central

Lee, Byoungkoo; Konen, Jessica; Wilkinson, Scott; Marcus, Adam I.; Jiang, Yi

2017-01-01

Invasive cancer cells interact with the surrounding extracellular matrix (ECM), remodeling ECM fiber network structure by condensing, degrading, and aligning these fibers. We developed a novel local alignment vector analysis method to quantitatively measure collagen fiber alignment as a vector field using Circular Statistics. This method was applied to human non-small cell lung carcinoma (NSCLC) cell lines, embedded as spheroids in a collagen gel. Collagen remodeling was monitored using second harmonic generation imaging under normal conditions and when the LKB1-MARK1 pathway was disrupted through RNAi-based approaches. The results showed that inhibiting LKB1 or MARK1 in NSCLC increases the collagen fiber alignment and captures outward alignment vectors from the tumor spheroid, corresponding to high invasiveness of LKB1 mutant cancer cells. With time-lapse imaging of ECM micro-fiber morphology, the local alignment vector can measure the dynamic signature of invasive cancer cell activity and cell-migration-induced ECM and collagen remodeling and realigning dynamics. PMID:28045069

Dock mediates Scar- and WASp-dependent actin polymerization through interaction with cell adhesion molecules in founder cells and fusion-competent myoblasts.

PubMed

Kaipa, Balasankara Reddy; Shao, Huanjie; Schäfer, Gritt; Trinkewitz, Tatjana; Groth, Verena; Liu, Jianqi; Beck, Lothar; Bogdan, Sven; Abmayr, Susan M; Önel, Susanne-Filiz

2013-01-01

The formation of the larval body wall musculature of Drosophila depends on the asymmetric fusion of two myoblast types, founder cells (FCs) and fusion-competent myoblasts (FCMs). Recent studies have established an essential function of Arp2/3-based actin polymerization during myoblast fusion, formation of a dense actin focus at the site of fusion in FCMs, and a thin sheath of actin in FCs and/or growing muscles. The formation of these actin structures depends on recognition and adhesion of myoblasts that is mediated by cell surface receptors of the immunoglobulin superfamily. However, the connection of the cell surface receptors with Arp2/3-based actin polymerization is poorly understood. To date only the SH2-SH3 adaptor protein Crk has been suggested to link cell adhesion with Arp2/3-based actin polymerization in FCMs. Here, we propose that the SH2-SH3 adaptor protein Dock, like Crk, links cell adhesion with actin polymerization. We show that Dock is expressed in FCs and FCMs and colocalizes with the cell adhesion proteins Sns and Duf at cell-cell contact points. Biochemical data in this study indicate that different domains of Dock are involved in binding the cell adhesion molecules Duf, Rst, Sns and Hbs. We emphasize the importance of these interactions by quantifying the enhanced myoblast fusion defects in duf dock, sns dock and hbs dock double mutants. Additionally, we show that Dock interacts biochemically and genetically with Drosophila Scar, Vrp1 and WASp. Based on these data, we propose that Dock links cell adhesion in FCs and FCMs with either Scar- or Vrp1-WASp-dependent Arp2/3 activation.

The receptor for advanced glycation end-products (RAGE) is only present in mammals, and belongs to a family of cell adhesion molecules (CAMs).

PubMed

Sessa, Luca; Gatti, Elena; Zeni, Filippo; Antonelli, Antonella; Catucci, Alessandro; Koch, Michael; Pompilio, Giulio; Fritz, Günter; Raucci, Angela; Bianchi, Marco E

2014-01-01

The human receptor for advanced glycation endproducts (RAGE) is a multiligand cell surface protein belonging to the immunoglobulin superfamily, and is involved in inflammatory and immune responses. Most importantly, RAGE is considered a receptor for HMGB1 and several S100 proteins, which are Damage-Associated Molecular Pattern molecules (DAMPs) released during tissue damage. In this study we show that the Ager gene coding for RAGE first appeared in mammals, and is closely related to other genes coding for cell adhesion molecules (CAMs) such as ALCAM, BCAM and MCAM that appeared earlier during metazoan evolution. RAGE is expressed at very low levels in most cells, but when expressed at high levels, it mediates cell adhesion to extracellular matrix components and to other cells through homophilic interactions. Our results suggest that RAGE evolved from a family of CAMs, and might still act as an adhesion molecule, in particular in the lung where it is highly expressed or under pathological conditions characterized by an increase of its protein levels.

Effect of Loading History on Airway Smooth Muscle Cell-Matrix Adhesions.

PubMed

Irons, Linda; Owen, Markus R; O'Dea, Reuben D; Brook, Bindi S

2018-06-05

Integrin-mediated adhesions between airway smooth muscle (ASM) cells and the extracellular matrix (ECM) regulate how contractile forces generated within the cell are transmitted to its external environment. Environmental cues are known to influence the formation, size, and survival of cell-matrix adhesions, but it is not yet known how they are affected by dynamic fluctuations associated with tidal breathing in the intact airway. Here, we develop two closely related theoretical models to study adhesion dynamics in response to oscillatory loading of the ECM, representing the dynamic environment of ASM cells in vivo. Using a discrete stochastic-elastic model, we simulate individual integrin binding and rupture events and observe two stable regimes in which either bond formation or bond rupture dominate, depending on the amplitude of the oscillatory loading. These regimes have either a high or low fraction of persistent adhesions, which could affect the level of strain transmission between contracted ASM cells and the airway tissue. For intermediate loading, we observe a region of bistability and hysteresis due to shared loading between existing bonds; the level of adhesion depends on the loading history. These findings are replicated in a related continuum model, which we use to investigate the effect of perturbations mimicking deep inspirations (DIs). Because of the bistability, a DI applied to the high adhesion state could either induce a permanent switch to a lower adhesion state or allow a return of the system to the high adhesion state. Transitions between states are further influenced by the frequency of oscillations, cytoskeletal or ECM stiffnesses, and binding affinities, which modify the magnitudes of the stable adhesion states as well as the region of bistability. These findings could explain (in part) the transient bronchodilatory effect of a DI observed in asthmatics compared to a more sustained effect in normal subjects. Copyright © 2018 Biophysical

Extracellular matrix and its receptors in Drosophila neural development

PubMed Central

Broadie, Kendal; Baumgartner, Stefan; Prokop, Andreas

2011-01-01

Extracellular matrix (ECM) and matrix receptors are intimately involved in most biological processes. The ECM plays fundamental developmental and physiological roles in health and disease, including processes underlying the development, maintenance and regeneration of the nervous system. To understand the principles of ECM-mediated functions in the nervous system, genetic model organisms like Drosophila provide simple, malleable and powerful experimental platforms. This article provides an overview of ECM proteins and receptors in Drosophila. It then focuses on their roles during three progressive phases of neural development: 1) neural progenitor proliferation, 2) axonal growth and pathfinding and 3) synapse formation and function. Each section highlights known ECM and ECM-receptor components and recent studies done in mutant conditions to reveal their in vivo functions, all illustrating the enormous opportunities provided when merging work on the nervous system with systematic research into ECM-related gene functions. PMID:21688401

In vitro Flow Adhesion Assay for Analyzing Shear-resistant Adhesion of Metastatic Cancer Cells to Endothelial Cells.

PubMed

Kang, Shin-Ae; Bajana, Sandra; Tanaka, Takemi

2016-02-20

Hematogenous metastasis is a primary cause of mortality from metastatic cancer. The shear-resistant adhesion of circulating tumor cells to the vascular endothelial cell surface under blood flow is an essential step in cell extravasation and further tissue invasion. This is similar to a process exploited by leukocytes for adhesion to inflamed blood vessels (leukocyte mimicry). The shear resistant adhesion is mediated by high affinity interactions between endothelial adhesion molecules and their counter receptor ligand expressed on circulating cells. Thus, weak interaction results in a rapid detachment of circulating cells from endothelium. Despite the critical role of vascular adhesion of cancer cells in hematogenous metastasis, our knowledge regarding this process has been limited due to the difficulty of mimicking dynamic flow conditions in vitro . In order to gain better insight into the shear-resistant adhesion of cancer cells to the endothelium, we developed a protocol for measuring the shear resistant adhesion of circulating tumor cells to endothelial cells under physiologic flow conditions by adapting a well established flow adhesion assay for inflammatory cells. This technique is useful to evaluate 1) the shear resistant adhesion competency of cancer cells and 2) the endothelial adhesion molecules necessary to support cancer cell adhesion (Kang et al. , 2015).

Interaction of human platelets with laminin and identification of the 67 kDa laminin receptor on platelets.

PubMed Central

Tandon, N N; Holland, E A; Kralisz, U; Kleinman, H K; Robey, F A; Jamieson, G A

1991-01-01

A microtitre adhesion assay has been developed to define parameters affecting the adherence of washed platelets to laminin. Adherence was optimally supported by Mg2+ and was inhibited by Ca2+ and by anti-laminin Fab fragments, but significant adhesion (75-90% of control) was found both in heparinized plasma containing physiological levels of bivalent cations and in plasma anti-coagulated with EGTA. Adherence was unaffected by platelet activation with ADP but was decreased by 50% by treatment with alpha-thrombin (1 unit/ml, 5 min). Adherence was unaffected by monospecific polyclonal antibodies to glycoprotein (GP) Ib and GPIV, and was normal with platelets from two patients with Glanzmann's thrombasthaenia, indicating that GPIb, the GPIIb/IIIa complex and GPIV are not involved in platelet-laminin interaction. Affinity chromatography of Triton-solubilized membranes on laminin-Sepharose followed by elution with 0.2 M-glycine/HCl (pH 2.85) identified a major band with a molecular mass of 67 kDa in the reduced and of 53 kDa in the unreduced form. This protein gave a positive reaction on Western blotting with a monospecific polyclonal antibody raised against the high-affinity laminin receptor isolated from human breast carcinoma tissue. The adhesion of platelets to laminin was inhibited by two monoclonal IgM antibodies specific to the LR-1 domain of the 67 kDa receptor. The binding protein was surface-oriented, as shown by flow cytofluorimetry and by the fact that it could be iodinated in intact platelets, but it was not labelled by the periodate-borotritide procedure, suggesting that it did not contain terminal sialic acid. The laminin-derived peptides Tyr-Ile-Gly-Ser-Arg and Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg-NH2, which constitute a complementary binding domain in laminin for the 67 kDa receptor, themselves supported platelet adhesion, bound to the receptor and inhibited the adhesion of platelets to laminin. In addition, Fab fragments of anti

Targeting and transport: How microtubules control focal adhesion dynamics

PubMed Central

Stehbens, Samantha

2012-01-01

Directional cell migration requires force generation that relies on the coordinated remodeling of interactions with the extracellular matrix (ECM), which is mediated by integrin-based focal adhesions (FAs). Normal FA turnover requires dynamic microtubules, and three members of the diverse group of microtubule plus-end-tracking proteins are principally involved in mediating microtubule interactions with FAs. Microtubules also alter the assembly state of FAs by modulating Rho GTPase signaling, and recent evidence suggests that microtubule-mediated clathrin-dependent and -independent endocytosis regulates FA dynamics. In addition, FA-associated microtubules may provide a polarized microtubule track for localized secretion of matrix metalloproteases (MMPs). Thus, different aspects of the molecular mechanisms by which microtubules control FA turnover in migrating cells are beginning to emerge. PMID:22908306

Annexin A6 contributes to the invasiveness of breast carcinoma cells by influencing the organization and localization of functional focal adhesions

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

Sakwe, Amos M., E-mail: asakwe@mmc.edu; Koumangoye, Rainelli; Guillory, Bobby

2011-04-01

The interaction of annexin A6 (AnxA6) with membrane phospholipids and either specific extracellular matrix (ECM) components or F-actin suggests that it may influence cellular processes associated with rapid plasma membrane reorganization such as cell adhesion and motility. Here, we examined the putative roles of AnxA6 in adhesion-related cellular processes that contribute to breast cancer progression. We show that breast cancer cells secrete annexins via the exosomal pathway and that the secreted annexins are predominantly cell surface-associated. Depletion of AnxA6 in the invasive BT-549 breast cancer cells is accompanied by enhanced anchorage-independent cell growth but cell-cell cohesion, cell adhesion/spreading onto collagenmore » type IV or fetuin-A, cell motility and invasiveness were strongly inhibited. To explain the loss in adhesion/motility, we show that vinculin-based focal adhesions in the AnxA6-depleted BT-549 cells are elongated and randomly distributed. These focal contacts are also functionally defective because the activation of focal adhesion kinase and the phosphoinositide-3 kinase/Akt pathway were strongly inhibited while the MAP kinase pathway remained constitutively active. Compared with normal human breast tissues, reduced AnxA6 expression in breast carcinoma tissues correlates with enhanced cell proliferation. Together this suggests that reduced AnxA6 expression contributes to breast cancer progression by promoting the loss of functional cell-cell and/or cell-ECM contacts and anchorage-independent cell proliferation.« less

The Synaptic Cell Adhesion Molecule, SynCAM1, Mediates Astrocyte-to-Astrocyte and Astrocyte-to-GnRH Neuron Adhesiveness in the Mouse Hypothalamus

PubMed Central

Sandau, Ursula S.; Mungenast, Alison E.; McCarthy, Jack; Biederer, Thomas; Corfas, Gabriel

2011-01-01

We previously identified synaptic cell adhesion molecule 1 (SynCAM1) as a component of a genetic network involved in the hypothalamic control of female puberty. Although it is well established that SynCAM1 is a synaptic adhesion molecule, its contribution to hypothalamic function is unknown. Here we show that, in addition to the expected neuronal localization illustrated by its presence in GnRH neurons, SynCAM1 is expressed in hypothalamic astrocytes. Cell adhesion assays indicated that SynCAM is recognized by both GnRH neurons and astrocytes as an adhesive partner and promotes cell-cell adhesiveness via homophilic, extracellular domain-mediated interactions. Alternative splicing of the SynCAM1 primary mRNA transcript yields four mRNAs encoding membrane-spanning SynCAM1 isoforms. Variants 1 and 4 are predicted to be both N and O glycosylated. Hypothalamic astrocytes and GnRH-producing GT1-7 cells express mainly isoform 4 mRNA, and sequential N- and O-deglycosylation of proteins extracted from these cells yields progressively smaller SynCAM1 species, indicating that isoform 4 is the predominant SynCAM1 variant expressed in astrocytes and GT1-7 cells. Neither cell type expresses the products of two other SynCAM genes (SynCAM2 and SynCAM3), suggesting that SynCAM-mediated astrocyte-astrocyte and astrocyte-GnRH neuron adhesiveness is mostly mediated by SynCAM1 homophilic interactions. When erbB4 receptor function is disrupted in astrocytes, via transgenic expression of a dominant-negative erbB4 receptor form, SynCAM1-mediated adhesiveness is severely compromised. Conversely, SynCAM1 adhesive behavior is rapidly, but transiently, enhanced in astrocytes by ligand-dependent activation of erbB4 receptors, suggesting that erbB4-mediated events affecting SynCAM1 function contribute to regulate astrocyte adhesive communication. PMID:21486931

Decoy receptor 3 promotes cell adhesion and enhances endometriosis development.

PubMed

Tsai, Hsiao-Wen; Huang, Ming-Ting; Wang, Peng-Hui; Huang, Ben-Shian; Chen, Yi-Jen; Hsieh, Shie-Liang

2018-02-01

Endometriosis is a multifactorial inflammatory disease with persistent activation of the nuclear factor-κB (NF-κB) signalling pathway. Aberrant adhesion of endometrium is the essential step in the progression of endometriosis, but the molecular mechanism of ectopic growth of endometrium is still unclear. Decoy receptor 3 (DcR3)/TNFRSF6B, a pleiotropic immunomodulator regulated by oestrogen, is able to activate focal adhesion kinase to promote cell adhesion. We found that DcR3 is upregulated in human ectopic endometrial cells via activation of the Akt-NF-κB signalling pathway, and its expression level correlates positively with that of the adhesion molecules intercellular adhesion molecule 1 (ICAM-1) and homing cell adhesion molecule (HCAM; CD44). In a multivariate regression model, DcR3 expression level was the most significant parameter associated with endometriosis severity. Knockdown of DcR3 not only downregulated the expression of ICAM-1 and HCAM, but also reduced cell adhesion and migration. In vivo investigation further showed that DcR3 promoted the growth and spread of endometrium, whereas knockdown of DcR3 by lentivirus-delivered short hairpin RNA inhibited ectopic adhesion of endometrium and abrogated endometriosis progression. These observations are in support of DcR3 playing a critical role in the pathogenesis of endometriosis, and the inhibition of DcR3 expression being a promising approach for the treatment of endometriosis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Integration of extracellular matrix with chitosan adhesive film for sutureless tissue fixation.

PubMed

Lauto, Antonio

2009-07-01

Extracellular matrices (ECMs) are currently applied in reconstructive surgery to enhance wound healing and tissue remodelling. Sutures and staples are usually employed to stabilize ECM on tissue although they may damage the matrix structure. In this investigation, a novel biocompatible bandage was developed to fix ECM on tissue without sutures. An adhesive film, based on chitosan, was integrated with small intestine submucosa (SIS) in a single bandage strip. This bandage was bonded to sheep small intestine upon laser irradiation of the chitosan film (P = 0.12 W, Fluence = 46+/-1 J/cm(2)) to assess tissue adhesion strength. Thermocouples were used to estimate temperatures under SIS during laser irradiation. Bandage strips were also mechanically tested to evaluate their tensile strength before and after irradiation. The bandage successfully bonded to intestine achieving a shear stress of 9.6+/- 1.6 kPa (n = 15). During laser irradiation, the temperature increased modestly to 31+/-2 degrees C (n = 14) beneath the ECM portion of the bandage. The bandage withstood a tensile strength of 3,122+/-780 and 3,384+/-791 kPa, before and after laser irradiation respectively (n = 10, P = 0.47, t-test). The SIS-chitosan bandage bonded effectively to tissue without sutures and preserved the ECM structure avoiding irreversible thermal denaturation of imbedded bioactive proteins.

Molecular analysis of antigen-independent adhesion forces between T and B lymphocytes.

PubMed Central

Amblard, F; Auffray, C; Sekaly, R; Fischer, A

1994-01-01

The low-affinity interactions underlying antigen recognition by T-cell receptors (TCRs) are thought to involve antigen-independent adhesion mechanisms. Using a hydrodynamic approach, we found that antigen-independent adhesion occurred between human B cells and resting T cells in a transient and temperature-dependent fashion. The mean cell-cell adhesion force was 0.32 x 10(-9) N and was generated by similar contributions (0.16 x 10(-9) N) of the LFA-1- and CD2-dependent adhesion pathways. After T-cell stimulation with a phorbol ester, the force contributed by LFA-1 was drastically increased, while that of CD2 was unaffected. We propose that weak receptor-mediated adhesion initiates antigen-independent intercellular contacts required for antigen recognition by the TCR and is upregulated following TCR engagement. The method used permits adhesion forces between living cells to be resolved at the molecular level and should prove valuable for the rapid assessment of interaction forces between various types of cells and cell-sized particles. Images PMID:7909604

Cell adhesion during bullet motion in capillaries.

PubMed

Takeishi, Naoki; Imai, Yohsuke; Ishida, Shunichi; Omori, Toshihiro; Kamm, Roger D; Ishikawa, Takuji

2016-08-01

A numerical analysis is presented of cell adhesion in capillaries whose diameter is comparable to or smaller than that of the cell. In contrast to a large number of previous efforts on leukocyte and tumor cell rolling, much is still unknown about cell motion in capillaries. The solid and fluid mechanics of a cell in flow was coupled with a slip bond model of ligand-receptor interactions. When the size of a capillary was reduced, the cell always transitioned to "bullet-like" motion, with a consequent decrease in the velocity of the cell. A state diagram was obtained for various values of capillary diameter and receptor density. We found that bullet motion enables firm adhesion of a cell to the capillary wall even for a weak ligand-receptor binding. We also quantified effects of various parameters, including the dissociation rate constant, the spring constant, and the reactive compliance on the characteristics of cell motion. Our results suggest that even under the interaction between P-selectin glycoprotein ligand-1 (PSGL-1) and P-selectin, which is mainly responsible for leukocyte rolling, a cell is able to show firm adhesion in a small capillary. These findings may help in understanding such phenomena as leukocyte plugging and cancer metastasis. Copyright © 2016 the American Physiological Society.

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking

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

Rossier, Olivier; Giannone, Grégory; CNRS, Interdisciplinary Institute for Neuroscience, UMR 5297, F-33000 Bordeaux

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements andmore » interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells.« less

The journey of integrins and partners in a complex interactions landscape studied by super-resolution microscopy and single protein tracking.

PubMed

Rossier, Olivier; Giannone, Grégory

2016-04-10

Cells adjust their adhesive and cytoskeletal organizations according to changes in the biochemical and physical nature of their surroundings. In return, by adhering and generating forces on the extracellular matrix (ECM) cells organize their microenvironment. Integrin-dependent focal adhesions (FAs) are the converging zones integrating biochemical and biomechanical signals arising from the ECM and the actin cytoskeleton. Thus, integrin-mediated adhesion and mechanotransduction, the conversion of mechanical forces into biochemical signals, are involved in critical cellular functions such as migration, proliferation and differentiation, and their deregulation contributes to pathologies including cancer. A challenging problem is to decipher how stochastic protein movements and interactions lead to formation of dynamic architecture such as integrin-dependent adhesive structures. In this review, we will describe recent advances made possible by super-resolution microscopies and single molecule tracking approaches that provided new understanding on the organization and the dynamics of integrins and intracellular regulators at the nanoscale in living cells. Copyright © 2015. Published by Elsevier Inc.

Suppression of adhesion-induced protein tyrosine phosphorylation decreases invasive and metastatic potentials of B16-BL6 melanoma cells by protein tyrosine kinase inhibitor genistein.

PubMed

Yan, C; Han, R

1997-01-01

Protein tyrosine kinase (PTK) appears to be involved in the activation of signaling during cell attachment to and spreading on extracellular matrix (ECM) in the metastatic cascade. To verify the assumption that PTK inhibitors might impair ECM signaling and prevent cancer metastasis, the highly metastatic B16-BL6 mouse melanoma cells were exposed to the PTK inhibitor genistein for 3 days. The ability of the cells to invade through reconstituted basement membrane (Matrigel) and to establish experimental pulmonary metastatic foci in C57BL/6 mice decreased after genistein exposure. The genistein-treated cells were also prevented from attaching to Matrigel and spread extremely poorly on the ECM substratum. Immunoblot analysis showed that tyrosine phosphorylation of a 125-kD protein in response to cell spreading on Matrigel was suppressed in the genistein-treated cells. Adhesion-induced protein tyrosine phosphorylation represents the earlier and specific event in the activation of ECM signaling, so this result implied ECM signaling was impaired in the treated cells. With immunofluorescence microscopy, the adhesion-induced tyrosine phosphorylated proteins were located at the pericytoplasms of well-spread cells, but not at the periphery of poorly spread genistein-treated cells. Therefore, this paper suggests that genistein might impair ECM signaling and subsequently prevent cancer cells from spreading well and invading or establishing metastasis through the suppression of adhesion-induced protein tyrosine phosphorylation. PTKs and adhesion-induced protein tyrosine phosphorylation might play a role in the control of invasion and metastasis.

Diffusion and intermembrane distance: case study of avidin and E-cadherin mediated adhesion.

PubMed

Fenz, Susanne F; Merkel, Rudolf; Sengupta, Kheya

2009-01-20

We present a biomimetic model system for cell-cell adhesion consisting of a giant unilamellar vesicle (GUV) adhering via specific ligand-receptor interactions to a supported lipid bilayer (SLB). The modification of in-plane diffusion of tracer lipids and receptors in the SLB membrane due to adhesion to the GUV is reported. Adhesion was mediated by either biotin-neutravidin (an avidin analogue) or the extracellular domains of the cell adhesion molecule E-cadherin (Ecad). In the strong interaction (biotin-avidin) case, binding of soluble receptors to the SLB alone led to reduced diffusion of tracer lipids. From theoretical considerations, this could be attributed partially to introduction of obstacles and partially to viscous effects. Further specific binding of a GUV membrane caused additional slowing down of tracers (up to 15%) and immobilization of receptors, and led to accumulation of receptors in the adhesion zone until full coverage was achieved. The intermembrane distance was measured to be 7 nm from microinterferometry (RICM). We show that a crowding effect due to the accumulated receptors alone is not sufficient to account for the slowing downan additional friction from the membrane also plays a role. In the weak binding case (Ecad), the intermembrane distance was about 50 nm, corresponding to partial overlap of the Ecad domains. No significant change in diffusion of tracer lipids was observed upon either protein binding or subsequent vesicle binding. The former was probably due to very small effective size of the obstacles introduced into the bilayer by Ecad binding, whereas the latter was due to the fact that, with such high intermembrane distance, the resulting friction is negligible. We conclude that the effect of intermembrane adhesion on diffusion depends strongly on the choice of the receptors.

The interaction between LYVE-1 with hyaluronan on the cell surface may play a role in the diversity of adhesion to cancer cells.

PubMed

Du, Yan; Liu, Hua; He, Yiqing; Liu, Yiwen; Yang, Cuixia; Zhou, Muqing; Wang, Wenjuan; Cui, Lian; Hu, Jiajie; Gao, Feng

2013-01-01

Hyaluronan (HA), a simple disaccharide unit, can polymerize and is considered a primary component of the extracellular matrix, which has a wide range of biological functions. In recent years, HA was found on the surface of tumor cells. According to previous reports, differing HA content on the cell surface of tumor cells is closely related to lymph node metastases, but the mechanisms mediating this process remained unclear. This research intended to study the surface content of HA on tumor cells and analyze cell adhesive changes caused by the interaction between HA and its lymphatic endothelial receptor (LYVE-1). We screened and observed high HA content on HS-578T breast cells and low HA content on MCF-7 breast cells through particle exclusion, immunofluorescence and flow cytometry experiments. The expression of LYVE-1, the lymph-vessel specific HA receptor, was consistent with our previous report and enhanced the adhesion of HA(high)-HS-578T cells to COS-7(LYVE-1(+)) through HA in cell static adhesion and dynamic parallel plate flow chamber experiments. MCF-7 breast cells contain little HA on the surface; however, our results showed little adhesion difference between MCF-7 cells and COS-7(LYVE-1(+)) and COS-7(LYVE-1(-)) cells. Similar results were observed concerning the adhesion of HS-578T cells or MCF-7 cells to SVEC4-10 cells. Furthermore, we observed for the first time that the cell surface HA content of high transfer tumor cells was rich, and we visualized the cross-linking of HA cable structures, which may activate LYVE-1 on lymphatic endothelial cells, promoting tumor adhesion. In summary, high-low cell surface HA content of tumor cells through the interaction with LYVE-1 leads to adhesion differences.

Anti-LRP/LR Specific Antibody IgG1-iS18 Impedes Adhesion and Invasion of Liver Cancer Cells

PubMed Central

Chetty, Carryn; Khumalo, Thandokuhle; Da Costa Dias, Bianca; Reusch, Uwe; Knackmuss, Stefan; Little, Melvyn; Weiss, Stefan F. T.

2014-01-01

Two key events, namely adhesion and invasion, are pivotal to the occurrence of metastasis. Importantly, the 37 kDa/67 kDa laminin receptor (LRP/LR) has been implicated in enhancing these two events thus facilitating cancer progression. In the current study, the role of LRP/LR in the adhesion and invasion of liver cancer (HUH-7) and leukaemia (K562) cells was investigated. Flow cytometry revealed that the HUH-7 cells displayed significantly higher cell surface LRP/LR levels compared to the poorly-invasive breast cancer (MCF-7) control cells, whilst the K562 cells displayed significantly lower cell surface LRP/LR levels in comparison to the MCF-7 control cells. However, Western blotting and densitometric analysis revealed that all three tumorigenic cell lines did not differ significantly with regards to total LRP/LR levels. Furthermore, treatment of liver cancer cells with anti-LRP/LR specific antibody IgG1-iS18 (0.2 mg/ml) significantly reduced the adhesive potential of cells to laminin-1 and the invasive potential of cells through the ECM-like Matrigel, whilst leukaemia cells showed no significant differences in both instances. Additionally, Pearson's correlation coefficients suggested direct proportionality between cell surface LRP/LR levels and the adhesive and invasive potential of liver cancer and leukaemia cells. These findings suggest the potential use of anti-LRP/LR specific antibody IgG1-iS18 as an alternative therapeutic tool for metastatic liver cancer through impediment of the LRP/LR- laminin-1 interaction. PMID:24798101

Changes in E-cadherin rigidity sensing regulate cell adhesion.

PubMed

Collins, Caitlin; Denisin, Aleksandra K; Pruitt, Beth L; Nelson, W James

2017-07-18

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin-dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell-cell adhesion assay and live cell imaging of cell-cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell-cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell-cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell-cell adhesion.

Changes in E-cadherin rigidity sensing regulate cell adhesion

PubMed Central

Collins, Caitlin; Pruitt, Beth L.; Nelson, W. James

2017-01-01

Mechanical cues are sensed and transduced by cell adhesion complexes to regulate diverse cell behaviors. Extracellular matrix (ECM) rigidity sensing by integrin adhesions has been well studied, but rigidity sensing by cadherins during cell adhesion is largely unexplored. Using mechanically tunable polyacrylamide (PA) gels functionalized with the extracellular domain of E-cadherin (Ecad-Fc), we showed that E-cadherin–dependent epithelial cell adhesion was sensitive to changes in PA gel elastic modulus that produced striking differences in cell morphology, actin organization, and membrane dynamics. Traction force microscopy (TFM) revealed that cells produced the greatest tractions at the cell periphery, where distinct types of actin-based membrane protrusions formed. Cells responded to substrate rigidity by reorganizing the distribution and size of high-traction-stress regions at the cell periphery. Differences in adhesion and protrusion dynamics were mediated by balancing the activities of specific signaling molecules. Cell adhesion to a 30-kPa Ecad-Fc PA gel required Cdc42- and formin-dependent filopodia formation, whereas adhesion to a 60-kPa Ecad-Fc PA gel induced Arp2/3-dependent lamellipodial protrusions. A quantitative 3D cell–cell adhesion assay and live cell imaging of cell–cell contact formation revealed that inhibition of Cdc42, formin, and Arp2/3 activities blocked the initiation, but not the maintenance of established cell–cell adhesions. These results indicate that the same signaling molecules activated by E-cadherin rigidity sensing on PA gels contribute to actin organization and membrane dynamics during cell–cell adhesion. We hypothesize that a transition in the stiffness of E-cadherin homotypic interactions regulates actin and membrane dynamics during initial stages of cell–cell adhesion. PMID:28674019

Role of YAP/TAZ in cell-matrix adhesion-mediated signalling and mechanotransduction

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

Dupont, Sirio, E-mail: sirio.dupont@unipd.it

2016-04-10

Signalling from the extracellular matrix (ECM) is a fundamental cellular input that sustains proliferation, opposes cell death and regulates differentiation. Through integrins, cells perceive both the chemical composition and physical properties of the ECM. In particular, cell behaviour is profoundly influenced by the mechanical elasticity or stiffness of the ECM, which regulates the ability of cells to develop forces through their contractile actomyosin cytoskeleton and to mature focal adhesions. This mechanosensing ability affects fundamental cellular functions, such that alterations of ECM stiffness is nowadays considered not a simple consequence of pathology, but a causative input driving aberrant cell behaviours. Wemore » here discuss recent advances on how mechanical signals intersect nuclear transcription and in particular the activity of YAP/TAZ transcriptional coactivators, known downstream transducers of the Hippo pathway and important effectors of ECM mechanical cues.« less

Listeria monocytogenes uses Listeria adhesion protein (LAP) to promote bacterial transepithelial translocation and induces expression of LAP receptor Hsp60.

PubMed

Burkholder, Kristin M; Bhunia, Arun K

2010-12-01

Listeria monocytogenes interaction with the intestinal epithelium is a key step in the infection process. We demonstrated that Listeria adhesion protein (LAP) promotes adhesion to intestinal epithelial cells and facilitates extraintestinal dissemination in vivo. The LAP receptor is a stress response protein, Hsp60, but the precise role for the LAP-Hsp60 interaction during Listeria infection is unknown. Here we investigated the influence of physiological stressors and Listeria infection on host Hsp60 expression and LAP-mediated bacterial adhesion, invasion, and transepithelial translocation in an enterocyte-like Caco-2 cell model. Stressors such as heat (41°C), tumor necrosis factor alpha (TNF-α) (100 U), and L. monocytogenes infection (10(4) to 10(6) CFU/ml) significantly (P < 0.05) increased plasma membrane and intracellular Hsp60 levels in Caco-2 cells and consequently enhanced LAP-mediated L. monocytogenes adhesion but not invasion of Caco-2 cells. In transepithelial translocation experiments, the wild type (WT) exhibited 2.7-fold more translocation through Caco-2 monolayers than a lap mutant, suggesting that LAP is involved in transepithelial translocation, potentially via a paracellular route. Short hairpin RNA (shRNA) suppression of Hsp60 in Caco-2 cells reduced WT adhesion and translocation 4.5- and 3-fold, respectively, while adhesion remained unchanged for the lap mutant. Conversely, overexpression of Hsp60 in Caco-2 cells enhanced WT adhesion and transepithelial translocation, but not those of the lap mutant. Furthermore, initial infection with a low dosage (10(6) CFU/ml) of L. monocytogenes increased plasma membrane and intracellular expression of Hsp60 significantly, which rendered Caco-2 cells more susceptible to subsequent LAP-mediated adhesion and translocation. These data provide insight into the role of LAP as a virulence factor during intestinal epithelial infection and pose new questions regarding the dynamics between the host stress response

Concentration-dependent rheological properties of ECM hydrogel for intracerebral delivery to a stroke cavity

PubMed Central

Massensini, Andre R.; Ghuman, Harmanvir; Saldin, Lindsey T.; Medberry, Christopher J.; Keane, Timothy J.; Nicholls, Francesca J.; Velankar, Sachin S.; Badylak, Stephen F.; Modo, Michel

2015-01-01

Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar tissue formation in many anatomical sites. However, the optimal shape and form of ECM scaffold for each clinical application can vary markedly. ECM hydrogels have been shown to promote chemotaxis and differentiation of neuronal stem cells, but minimally invasive delivery of such scaffold materials to the central nervous system (CNS) would require an injectable form. These ECM materials can be manufactured to exist in fluid phase at room temperature, while forming hydrogels at body temperature in a concentration-dependent fashion. Implantation into the lesion cavity after a stroke could hence provide a means to support endogenous repair mechanisms. Herein, we characterize the rheological properties of an ECM hydrogel composed of urinary bladder matrix (UBM) that influence its delivery and in vivo interaction with host tissue. There was a notable concentration-dependence in viscosity, stiffness, and elasticity; all characteristics important for minimally invasive intracerebral delivery. An efficient MRI-guided injection with drainage of fluid from the cavity is described to assess in situ hydrogel formation and ECM retention at different concentrations (0, 1, 2, 3, 4, and 8 mg/mL). Only ECM concentrations >3 mg/mL gelled within the stroke cavity. Lower concentrations were not retained within the cavity, but extensive permeation of the liquid phase ECM into the peri-infarct area was evident. The concentration of ECM hydrogel is hence an important factor affecting gelation, host-biomaterial interface, as well intra-lesion distribution. PMID:26318805

Integration of planar cell polarity and ECM signaling in elongation of the vertebrate body plan.

PubMed

Skoglund, Paul; Keller, Ray

2010-10-01

The shaping of the vertebrate embryonic body plan depends heavily on the narrowing and lengthening (convergence and extension) of embryonic tissues by cell intercalation, a process by which cells actively crawl between one another along the axis of convergence to produce a narrower, longer array. We discuss recent evidence that the vertebrate non-canonical Wnt/Planar Cell Polarity (PCP) pathway, known to directly function in polarizing the movements of intercalating cells, is also involved in the localized assembly of extracellular matrix (ECM). These cell-ECM interactions, in turn, are necessary for expression of the oriented, polarized cell intercalation. The mechanism of PCP/ECM interactions, their molecular signaling, and their mechanical consequences for morphogenesis are discussed with the goal of identifying important unsolved issues. Copyright © 2010 Elsevier Ltd. All rights reserved.

In vitro study of histamine and histamine receptor ligands influence on the adhesion of purified human eosinophils to endothelium.

PubMed

Grosicki, Marek; Wójcik, Tomasz; Chlopicki, Stefan; Kieć-Kononowicz, Katarzyna

2016-04-15

It is a well-known fact that histamine is involved in eosinophil-dependent inflammatory responses including cellular chemotaxis and migration. Nevertheless, the relative role of histamine receptors in the mechanisms of eosinophils adhesion to endothelial cells is not known. Therefore the aim of presented study was to examine the effect of selective histamine receptors ligands on eosinophils adhesion to endothelium. For that purpose the highly purified human eosinophils have been isolated from the peripheral blood. The viability and functional integrity of isolated eosinophils have been validated in several tests. Histamine as well as 4-methylhistamine (selective H4 agonist) in concentration-dependent manner significantly increased number of eosinophils that adhere to endothelium. Among the selective histamine receptors antagonist or H1 inverse agonist only JNJ7777120 (histamine H4 antagonist) and thioperamide (dual histamine H3/H4 antagonist) had direct effect on eosinophils adhesion to endothelial cells. Antagonists of H1 (diphenhydramine, mepyramine) H2 (ranitidine and famotidine) and H3 (pitolisant) histamine receptors were ineffective. To the best of our knowledge, this is the first study to demonstrate that histamine receptor H4 plays a dominant role in histamine-induced eosinophils adhesion to endothelium. Copyright © 2016 Elsevier B.V. All rights reserved.

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs): Positive and negative regulators in tumor cell adhesion.

PubMed

Bourboulia, Dimitra; Stetler-Stevenson, William G

2010-06-01

Cells adhere to one another and/or to matrices that surround them. Regulation of cell-cell (intercellular) and cell-matrix adhesion is tightly controlled in normal cells, however, defects in cell adhesion are common in the majority of human cancers. Multilateral communication among tumor cells with the extracellular matrix (ECM) and neighbor cells is accomplished through adhesion molecules, ECM components, proteolytic enzymes and their endogenous inhibitors. There is sufficient evidence to suggest that reduced adherence is a tumor cell property engaged during tumor progression. Tumor cells acquire the ability to change shape, detach and easily move through spaces disorganizing the normal tissue architecture. This property is due to changes in expression levels of adhesion molecules and/or due to elevated levels of secreted proteolytic enzymes, including matrix metalloproteinases (MMPs). Among other roles, MMPs degrade the ECM and, therefore, prepare the path for tumor cells to migrate, invade and spread to distant secondary areas, where they form metastasis. Tissue inhibitors of metalloproteinases or TIMPs control MMP activities and, therefore, minimize matrix degradation. Both MMPs and TIMPs are involved in tissue remodeling and decisively regulate tumor cell progression including tumor angiogenesis. In this review, we describe and discuss data that support the important role of MMPs and TIMPs in cancer cell adhesion and tumor progression. Published by Elsevier Ltd.

Cytotoxic-T-lymphocyte antigen 4 receptor signaling for lymphocyte adhesion is mediated by C3G and Rap1.

PubMed

Kloog, Yoel; Mor, Adam

2014-03-01

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders.

Cytotoxic-T-Lymphocyte Antigen 4 Receptor Signaling for Lymphocyte Adhesion Is Mediated by C3G and Rap1

PubMed Central

Kloog, Yoel

2014-01-01

T-lymphocyte adhesion plays a critical role in both inflammatory and autoimmune responses. The small GTPase Rap1 is the key coordinator mediating T-cell adhesion to endothelial cells, antigen-presenting cells, and virus-infected cells. We describe a signaling pathway, downstream of the cytotoxic T-lymphocyte antigen 4 (CTLA-4) receptor, leading to Rap1-mediated adhesion. We identified a role for the Rap1 guanine nucleotide exchange factor C3G in the regulation of T-cell adhesion and showed that this factor is required for both T-cell receptor (TCR)-mediated and CTLA-4-mediated T-cell adhesion. Our data indicated that C3G translocates to the plasma membrane downstream of TCR signaling, where it regulates activation of Rap1. We also showed that CTLA-4 receptor signaling mediates tyrosine phosphorylation in the C3G protein, and that this is required for augmented activation of Rap1 and increased adhesion mediated by leukocyte function-associated antigen type 1 (LFA-1). Zap70 is required for C3G translocation to the plasma membrane, whereas the Src family member Hck facilitates C3G phosphorylation. These findings point to C3G and Hck as promising potential therapeutic targets for the treatment of T-cell-dependent autoimmune disorders. PMID:24396067

Loss of miR-203 regulates cell shape and matrix adhesion through ROBO1/Rac/FAK in response to stiffness

PubMed Central

Le, Lily Thao-Nhi; Cazares, Oscar; Mouw, Janna K.; Chatterjee, Sharmila; Macias, Hector; Moran, Angel; Ramos, Jillian; Keely, Patricia J.; Weaver, Valerie M.

2016-01-01

Breast tumor progression is accompanied by changes in the surrounding extracellular matrix (ECM) that increase stiffness of the microenvironment. Mammary epithelial cells engage regulatory pathways that permit dynamic responses to mechanical cues from the ECM. Here, we identify a SLIT2/ROBO1 signaling circuit as a key regulatory mechanism by which cells sense and respond to ECM stiffness to preserve tensional homeostasis. We observed that Robo1 ablation in the developing mammary gland compromised actin stress fiber assembly and inhibited cell contractility to perturb tissue morphogenesis, whereas SLIT2 treatment stimulated Rac and increased focal adhesion kinase activity to enhance cell tension by maintaining cell shape and matrix adhesion. Further investigation revealed that a stiff ECM increased Robo1 levels by down-regulating miR-203. Consistently, patients whose tumor expressed a low miR-203/high Robo1 expression pattern exhibited a better overall survival prognosis. These studies show that cells subjected to stiffened environments up-regulate Robo1 as a protective mechanism that maintains cell shape and facilitates ECM adherence. PMID:26975850

Adhesion of liposomes: a quartz crystal microbalance study

NASA Astrophysics Data System (ADS)

Lüthgens, Eike; Herrig, Alexander; Kastl, Katja; Steinem, Claudia; Reiss, Björn; Wegener, Joachim; Pignataro, Bruno; Janshoff, Andreas

2003-11-01

Three different systems are presented, exploring the adhesion of liposomes mediated by electrostatic and lipid-protein interactions as well as molecular recognition of ligand receptor pairs. Liposomes are frequently used to gain insight into the complicated processes involving adhesion and subsequent events such as fusion and fission mainly triggered by specific proteins. We combined liposome technology with the quartz crystal microbalance (QCM) technique as a powerful tool to study the hidden interface between the membrane and functionalized surface. Electrostatic attraction and molecular recognition were employed to bind liposomes to the functionalized quartz crystal. The QCM was used to distinguish between adsorption of vesicles and rupture due to strong adhesive forces. Intact vesicles display viscoelastic behaviour, while planar lipid bilayers as a result of vesicle rupture can be modelled by a thin rigid film. Furthermore, the adhesion of cells was modelled successfully by receptor bearing liposomes. Scanning force microscopy was used to confirm the results obtained by QCM measurements.

An Inducible Endothelial Cell Surface Glycoprotein Mediates Melanoma Adhesion

NASA Astrophysics Data System (ADS)

Rice, G. Edgar; Bevilacqua, Michael P.

1989-12-01

Hematogenous metastasis requires the arrest and extravasation of blood-borne tumor cells, possibly involving direct adhesive interactions with vascular endothelium. Cytokine activation of cultured human endothelium increases adhesion of melanoma and carcinoma cell lines. An inducible 110-kD endothelial cell surface glycoprotein, designated INCAM-110, appears to mediate adhesion of melanoma cells. In addition, an inducible endothelial receptor for neutrophils, ELAM-1, supports the adhesion of a human colon carcinoma cell line. Thus, activation of vascular endothelium in vivo that results in increased expression of INCAM-110 and ELAM-1 may promote tumor cell adhesion and affect the incidence and distribution of metastases.

Dopamine D2 heteroreceptor complexes and their receptor-receptor interactions in ventral striatum: novel targets for antipsychotic drugs.

PubMed

Fuxe, Kjell; Borroto-Escuela, Dasiel O; Tarakanov, Alexander O; Romero-Fernandez, Wilber; Ferraro, Luca; Tanganelli, Sergio; Perez-Alea, Mileidys; Di Palma, Michael; Agnati, Luigi F

2014-01-01

This review is focused on the D2 heteroreceptor complexes within the ventral striatum with their receptor-receptor interactions and relevance for the treatment of schizophrenia. A "guide-and-clasp" manner for receptor-receptor interactions is proposed where "adhesive guides" may be amino acid triplet homologies, which were determined for different kinds of D2 heteroreceptor complexes. The first putative D2 heteroreceptor complex to be discovered in relation to schizophrenia was the A2A-D2 heteroreceptor complex where antagonistic A2A-D2 receptor-receptor interactions were demonstrated after A2A agonist treatment in the ventral striatum. The A2A agonist CGS 21680 with atypical antipsychotic properties may at least in part act by increasing β-arrestin2 signaling over the D2 protomer in the A2A-D2 heteroreceptor complex in the ventral striatum. The antagonistic NTS1-D2 interactions in the NTS1-D2 heteroreceptor complex in the ventral striatum are proposed as one molecular mechanism for the potential antipsychotic effects of NT. Indications were obtained that the psychotic actions of the 5-HT2AR hallucinogens LSD and DOI can involve enhancement of D2R protomer signaling via a biased agonist action at the 5-HT2A protomer in the D2-5-HT2A heteroreceptor complex in the ventral striatum. Facilitatory allosteric D2likeR-OTR interactions in heteroreceptor complexes in nucleus accumbens may have a role in social and emotional behaviors. By blocking the D2 protomers of these heteroreceptor complexes, antipsychotics can fail to reduce the negative symptoms of schizophrenia. The discovery of different types of D2 heteroreceptor complexes gives an increased understanding of molecular mechanisms involved in causing schizophrenia and new strategies for its treatment and understanding the side effects of antipsychotics. © 2014 Elsevier B.V. All rights reserved.

Site-directed mutagenesis of the chemokine receptor CXCR6 suggests a novel paradigm for interactions with the ligand CXCL16.

PubMed

Petit, Sarah J; Chayen, Naomi E; Pease, James E

2008-08-01

Chemokine receptor CXCR6 mediates the chemotaxis and adhesion of leukocytes to soluble and membrane-anchored forms of CXCL16, and is an HIV-1 co-receptor. Here, we describe the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type (WT) CXCR6, an N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar single nucleotide polymorphism, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 N terminus and inhibition of post-translational modifications of this region were without effect on receptor function. Likewise, N-terminal extension of CXCL16 resulted in a protein with decent potency and efficacy in chemotaxis and not, as anticipated, a CXCR6 antagonist. D176N and E274Q CXCR6 mutants were unable to interact with soluble CXCL16, suggesting a critical role for D176 and E274 in ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the E274Q mutant could promote robust adhesion to membrane-anchored CXCL16, suggesting that soluble and membrane-bound forms of CXCL16 possess distinct conformations. Collectively, our data suggest a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of CXCR6.

Ephrin-A1/EphA4-mediated adhesion of monocytes to endothelial cells.

PubMed

Jellinghaus, Stefanie; Poitz, David M; Ende, Georg; Augstein, Antje; Weinert, Sönke; Stütz, Beryl; Braun-Dullaeus, Rüdiger C; Pasquale, Elena B; Strasser, Ruth H

2013-10-01

The Eph receptors represent the largest family of receptor tyrosine kinases. Both Eph receptors and their ephrin ligands are cell-surface proteins, and they typically mediate cell-to-cell communication by interacting at sites of intercellular contact. The major aim of the present study was to investigate the involvement of EphA4-ephrin-A1 interaction in monocyte adhesion to endothelial cells, as this process is a crucial step during the initiation and progression of the atherosclerotic plaque. Immunohistochemical analysis of human atherosclerotic plaques revealed expression of EphA4 receptor and ephrin-A1 ligand in major cell types within the plaque. Short-time stimulation of endothelial cells with the soluble ligand ephrin-A1 leads to a fourfold increase in adhesion of human monocytes to endothelial cells. In addition, ephrin-A1 further increases monocyte adhesion to already inflamed endothelial cells. EphrinA1 mediates its effect on monocyte adhesion via the activated receptor EphA4. This ephrinA1/EphA4 induced process involves the activation of the Rho signaling pathway and does not require active transcription. Rho activation downstream of EphA4 leads to increased polymerization of actin filaments in endothelial cells. This process was shown to be crucial for the proadhesive effect of ephrin-A1. The results of the present study show that ephrin-A1-induced EphA4 forward signaling promotes monocyte adhesion to endothelial cells via activation of RhoA and subsequent stress-fiber formation by a non-transcriptional mechanism. Copyright © 2013 Elsevier B.V. All rights reserved.

Receptor-receptor interactions within receptor mosaics. Impact on neuropsychopharmacology.

PubMed

Fuxe, K; Marcellino, D; Rivera, A; Diaz-Cabiale, Z; Filip, M; Gago, B; Roberts, D C S; Langel, U; Genedani, S; Ferraro, L; de la Calle, A; Narvaez, J; Tanganelli, S; Woods, A; Agnati, L F

2008-08-01

Future therapies for diseases associated with altered dopaminergic signaling, including Parkinson's disease, schizophrenia and drug addiction or drug dependence may substantially build on the existence of intramembrane receptor-receptor interactions within dopamine receptor containing receptor mosaics (RM; dimeric or high-order receptor oligomers) where it is believed that the dopamine D(2) receptor may operate as the 'hub receptor' within these complexes. The constitutive adenosine A(2A)/dopamine D(2) RM, located in the dorsal striato-pallidal GABA neurons, are of particular interest in view of the demonstrated antagonistic A(2A)/D(2) interaction within these heteromers; an interaction that led to the suggestion and later demonstration that A(2A) antagonists could be used as novel anti-Parkinsonian drugs. Based on the likely existence of A(2A)/D(2)/mGluR5 RM located both extrasynaptically on striato-pallidal GABA neurons and on cortico-striatal glutamate terminals, multiple receptor-receptor interactions within this RM involving synergism between A(2A)/mGluR5 to counteract D(2) signaling, has led to the proposal of using combined mGluR5 and A(2A) antagonists as a future anti-Parkinsonian treatment. Based on the same RM in the ventral striato-pallidal GABA pathways, novel strategies for the treatment of schizophrenia, building on the idea that A(2A) agonists and/or mGluR5 agonists will help reduce the increased dopaminergic signaling associated with this disease, have been suggested. Such treatment may ensure the proper glutamatergic drive from the mediodorsal thalamic nucleus to the prefrontal cortex, one which is believed to be reduced in schizophrenia due to a dominance of D(2)-like signaling in the ventral striatum. Recently, A(2A) receptors also have been shown to counteract the locomotor and sensitizing actions of cocaine and increases in A(2A) receptors have also been observed in the nucleus accumbens after extended cocaine self-administration, probably

Improved degree of conversion of model self-etching adhesives through their interaction with dentin

PubMed Central

Zhang, Ying; Wang, Yong

2011-01-01

Objective To investigate the correlation of the chemical interaction between model self-etching adhesives and dentin with the degree of conversion (DC) of the adhesives. Methods The model self-etching adhesives contained bis[2-methacryloyloxy)ethyl] phosphate (2MP) and 2-hydroxyethyl methacrylate (HEMA) with a mass ratio of 1/1, and 0-40% water contents, respectively. The adhesives were applied either onto the prepared dentin surface or unreactive substrates (such as glass slides), agitated for 15s, then light-cured for 40s. The DCs of the adhesives were determined using micro-Raman spectral and mapping analysis. Results The DCs of the adhesives cured on the dentin substrate were found to be significantly higher than those on the unreactive glass substrate. Moreover, the DCs of the adhesives displayed a decreasing trend as the distance from the dentin surface became greater. The chemical interaction of the acidic 2MP/HEMA adhesives with the mineral apatite in dentin was proposed to play a significant role for the observations. The chemical interaction could be validated by the spectral comparison in the phosphate regions of 1100 cm−1 and 960 cm−1 in the Raman spectra. The results also revealed a notable influence of water content on the DC of adhesives. The DCs of the adhesive at 10% water content exhibited the highest DC level for both substrates. Conclusions Interaction with dentin dramatically improved the degree of conversion of self-etching adhesives. Our ability to chemically characterize the a/d interface including in situ detection of the DC distribution is very important in understanding self-etching adhesive bonding under in vivo conditions. PMID:22024375

Improved degree of conversion of model self-etching adhesives through their interaction with dentine.

PubMed

Zhang, Ying; Wang, Yong

2012-01-01

To investigate the correlation of the chemical interaction between model self-etching adhesives and dentine with the degree of conversion (DC) of the adhesives. The model self-etching adhesives contained bis[2-methacryloyloxy)ethyl] phosphate (2MP) and 2-hydroxyethyl methacrylate (HEMA) with a mass ratio of 1/1, and 0-40% water contents, respectively. The adhesives were applied either onto the prepared dentine surface or unreactive substrates (such as glass slides), agitated for 15s, then light-cured for 40s. The DCs of the adhesives were determined using micro-Raman spectral and mapping analysis. The DCs of the adhesives cured on the dentine substrate were found to be significantly higher than those on the unreactive glass substrate. Moreover, the DCs of the adhesives displayed a decreasing trend as the distance from the dentine surface became greater. The chemical interaction of the acidic 2MP/HEMA adhesives with the mineral apatite in dentine was proposed to play a significant role for the observations. The chemical interaction could be validated by the spectral comparison in the phosphate regions of 1100 cm(-1) and 960 cm(-1) in the Raman spectra. The results also revealed a notable influence of water content on the DC of adhesives. The DCs of the adhesive at 10% water content exhibited the highest DC level for both substrates. Interaction with dentine dramatically improved the degree of conversion of self-etching adhesives. Our ability to chemically characterise the a/d interface including in situ detection of the DC distribution is very important in understanding self-etching adhesive bonding under in vivo conditions. Copyright © 2011 Elsevier Ltd. All rights reserved.

Regulation of epithelial and lymphocyte cell adhesion by adenosine deaminase-CD26 interaction.

PubMed Central

Ginés, Silvia; Mariño, Marta; Mallol, Josefa; Canela, Enric I; Morimoto, Chikao; Callebaut, Christian; Hovanessian, Ara; Casadó, Vicent; Lluis, Carmen; Franco, Rafael

2002-01-01

The extra-enzymic function of cell-surface adenosine deaminase (ADA), an enzyme mainly localized in the cytosol but also found on the cell surface of monocytes, B cells and T cells, has lately been the subject of numerous studies. Cell-surface ADA is able to transduce co-stimulatory signals in T cells via its interaction with CD26, an integral membrane protein that acts as ADA-binding protein. The aim of the present study was to explore whether ADA-CD26 interaction plays a role in the adhesion of lymphocyte cells to human epithelial cells. To meet this aim, different lymphocyte cell lines (Jurkat and CEM T) expressing endogenous, or overexpressing human, CD26 protein were tested in adhesion assays to monolayers of colon adenocarcinoma human epithelial cells, Caco-2, which express high levels of cell-surface ADA. Interestingly, the adhesion of Jurkat and CEM T cells to a monolayer of Caco-2 cells was greatly dependent on CD26. An increase by 50% in the cell-to-cell adhesion was found in cells containing higher levels of CD26. Incubation with an anti-CD26 antibody raised against the ADA-binding site or with exogenous ADA resulted in a significant reduction (50-70%) of T-cell adhesion to monolayers of epithelial cells. The role of ADA-CD26 interaction in the lymphocyte-epithelial cell adhesion appears to be mediated by CD26 molecules that are not interacting with endogenous ADA (ADA-free CD26), since SKW6.4 (B cells) that express more cell-surface ADA showed lower adhesion than T cells. Adhesion stimulated by CD26 and ADA is mediated by T cell lymphocyte function-associated antigen. A role for ADA-CD26 interaction in cell-to-cell adhesion was confirmed further in integrin activation assays. FACS analysis revealed a higher expression of activated integrins on T cell lines in the presence of increasing amounts of exogenous ADA. Taken together, these results suggest that the ADA-CD26 interaction on the cell surface has a role in lymphocyte-epithelial cell adhesion. PMID

Heterophilic binding of the adhesion molecules poliovirus receptor and immunoglobulin superfamily 4A in the interaction between mouse spermatogenic and Sertoli cells.

PubMed

Wakayama, Tomohiko; Sai, Yoshimichi; Ito, Akihiko; Kato, Yukio; Kurobo, Miho; Murakami, Yoshinori; Nakashima, Emi; Tsuji, Akira; Kitamura, Yukihiko; Iseki, Shoichi

2007-06-01

The cell adhesion protein immunoglobulin superfamily 4A (IGSF4A) is expressed on the surfaces of spermatogenic cells in the mouse testis. During spermatogenesis, IGSF4A is considered to bind to the surface of Sertoli cells in a heterophilic manner. To identify this unknown partner of IGSF4A, we generated rat monoclonal antibodies against the membrane proteins of mouse Sertoli cells grown in primary culture. Using these monoclonal antibodies, we isolated a clone that immunostained Sertoli cells and reacted with the product of immunoprecipitation of the homogenate of mouse testis with anti-IGSF4A antibody. Subsequently, to identify the Sertoli cell membrane protein that is recognized by this monoclonal antibody, we performed expression cloning of a cDNA library from the mouse testis. As a result, we identified poliovirus receptor (PVR), which is another IGSF-type cell adhesion molecule, as the binding partner of IGSF4A. The antibodies raised against PVR and IGSF4A immunoprecipitated both antigens in the homogenate of mouse testis. Immunoreactivity for PVR was present in Sertoli cells but not in spermatogenic cells at all stages of spermatogenesis. Overexpression of PVR in TM4, a mouse Sertoli cell line, increased more than three-fold its capacity to adhere to Tera-2, which is a human cell line that expresses IGSF4A. These findings suggest that the heterophilic binding of PVR to IGSF4A is responsible, at least in part, for the interaction between Sertoli and spermatogenic cells during mouse spermatogenesis.

Conformational Changes in the Capsid of a Calicivirus upon Interaction with Its Functional Receptor

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

Ossiboff, Robert J.; Zhou, Yi; Lightfoot, Patrick J.

2010-07-19

Nonenveloped viral capsids are metastable structures that undergo conformational changes during virus entry that lead to interactions of the capsid or capsid fragments with the cell membrane. For members of the Caliciviridae, neither the nature of these structural changes in the capsid nor the factor(s) responsible for inducing these changes is known. Feline functional adhesion molecule A (fJAM-A) mediates the attachment and infectious viral entry of feline calicivirus (FCV). Here, we show that the infectivity of some FCV isolates is neutralized following incubation with the soluble receptor at 37 C. We used this property to select mutants resistant to preincubationmore » with the soluble receptor. We isolated and sequenced 24 soluble receptor-resistant (srr) mutants and characterized the growth properties and receptor-binding activities of eight mutants. The location of the mutations within the capsid structure of FCV was mapped using a new 3.6-{angstrom} structure of native FCV. The srr mutations mapped to the surface of the P2 domain were buried at the protruding domain dimer interface or were present in inaccessible regions of the capsid protein. Coupled with data showing that both the parental FCV and the srr mutants underwent increases in hydrophobicity upon incubation with the soluble receptor at 37 C, these findings indicate that FCV likely undergoes conformational change upon interaction with its receptor. Changes in FCV capsid conformation following its interaction with fJAM-A may be important for subsequent interactions of the capsid with cellular membranes, membrane penetration, and genome delivery.« less

ECM and ECM-like materials - Biomaterials for applications in regenerative medicine and cancer therapy.

PubMed

Hinderer, Svenja; Layland, Shannon Lee; Schenke-Layland, Katja

2016-02-01

Regenerative strategies such as stem cell-based therapies and tissue engineering applications are being developed with the aim to replace, remodel, regenerate or support damaged tissues and organs. In addition to careful cell type selection, the design of appropriate three-dimensional (3D) scaffolds is essential for the generation of bio-inspired replacement tissues. Such scaffolds are usually made of degradable or non-degradable biomaterials and can serve as cell or drug carriers. The development of more effective and efficient drug carrier systems is also highly relevant for novel cancer treatment strategies. In this review, we provide a summary of current approaches that employ ECM and ECM-like materials, or ECM-synthetic polymer hybrids, as biomaterials in the field of regenerative medicine. We further discuss the utilization of such materials for cell and drug delivery, and highlight strategies for their use as vehicles for cancer therapy. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

Non-human Primate and Rat Cardiac Fibroblasts show similar Extracellular Matrix-related and Cellular Adhesion Gene Responses to Substance P

PubMed Central

Meléndez, Giselle C.; Manteufel, Edward J.; Dehlin, Heather M.; Register, Thomas C.; Levick, Scott P.

2015-01-01

Background The sensory nerve neuropeptide substance P (SP) regulates cardiac fibrosis in rodents under pressure overload conditions. Interestingly, SP induces transient increase expression of specific genes in isolated rat cardiac fibroblasts, without resultant changes in cell function. This suggests that SP ‘primes’ fibroblasts, but does not directly activate them. We investigated whether these unusual findings are specific to rodent fibroblasts or are translatable to a larger animal model more closely related to humans. Methods We compared the effects of SP on genes associated with extracellular matrix (ECM) regulation, cell-cell adhesion, cell-matrix adhesion and ECM in cardiac fibroblasts isolated from a non-human primate and Sprague-Dawley rats. Results We found that rodent and non-human primate cardiac fibroblasts showed similar ECM regulation and cell adhesion gene expression responses to SP. There were, however, large discrepancies in ECM genes which did not result in collagen or laminin synthesis in rat or non-human primate fibroblasts in response to SP. Conclusions This study further supports the notion that SP serves as a ‘primer’ for fibroblasts rather than initiating direct effects and suggests that rodent fibroblasts are a suitable model for studying gene and functional responses to SP in the absence of human or non-human primate fibroblasts. PMID:25550118

42 CFR 456.725 - Funding of ECM system.

Code of Federal Regulations, 2011 CFR

2011-10-01

... 42 Public Health 4 2011-10-01 2011-10-01 false Funding of ECM system. 456.725 Section 456.725... Claims Management System for Outpatient Drug Claims § 456.725 Funding of ECM system. (a) For funds....722, FFP is available at a matching rate of 90 percent. After fiscal year 1992, ECM subsystems are...

42 CFR 456.725 - Funding of ECM system.

Code of Federal Regulations, 2010 CFR

2010-10-01

... 42 Public Health 4 2010-10-01 2010-10-01 false Funding of ECM system. 456.725 Section 456.725... Claims Management System for Outpatient Drug Claims § 456.725 Funding of ECM system. (a) For funds....722, FFP is available at a matching rate of 90 percent. After fiscal year 1992, ECM subsystems are...

Cellular Mechanics of Primary Human Cervical Fibroblasts: Influence of Progesterone and a Pro-inflammatory Cytokine.

PubMed

Shukla, Vasudha; Barnhouse, Victoria; Ackerman, William E; Summerfield, Taryn L; Powell, Heather M; Leight, Jennifer L; Kniss, Douglas A; Ghadiali, Samir N

2018-01-01

The leading cause of neonatal mortality, pre-term birth, is often caused by pre-mature ripening/opening of the uterine cervix. Although cervical fibroblasts play an important role in modulating the cervix's extracellular matrix (ECM) and mechanical properties, it is not known how hormones, i.e., progesterone, and pro-inflammatory insults alter fibroblast mechanics, fibroblast-ECM interactions and the resulting changes in tissue mechanics. Here we investigate how progesterone and a pro-inflammatory cytokine, IL-1β, alter the biomechanical properties of human cervical fibroblasts and the fibroblast-ECM interactions that govern tissue-scale mechanics. Primary human fibroblasts were isolated from non-pregnant cervix and treated with estrogen/progesterone, IL-1β or both. The resulting changes in ECM gene expression, matrix remodeling, traction force generation, cell-ECM adhesion and tissue contractility were monitored. Results indicate that IL-1β induces a significant reduction in traction force and ECM adhesion independent of pre-treatment with progesterone. These cell level effects altered tissue-scale mechanics where IL-1β inhibited the contraction of a collagen gel over 6 days. Interestingly, progesterone treatment alone did not modulate traction forces or gel contraction but did result in a dramatic increase in cell-ECM adhesion. Therefore, the protective effect of progesterone may be due to altered adhesion dynamics as opposed to altered ECM remodeling.

Numerical analysis of cell adhesion in capillary flow

NASA Astrophysics Data System (ADS)

Takeishi, Naoki; Imai, Yohsuke; Ishida, Shunichi; Omori, Toshihiro; Kamm, Roger; Ishikawa, Takuji

2016-11-01

Numerical simulation of cell adhesion was performed for capillaries whose diameter is comparable to or smaller than that of the cell. Despite a lot of works about leukocyte and tumor cell rolling, cell motion in capillaries has remained unclear. The solid and fluid mechanics of a cell in flow was coupled with a slip bond model of ligand-receptor interactions. When the size of a capillary was reduced, the cell always transitioned to "bullet-like" motion, with a consequent decrease in the velocity of the cell. A state diagram is obtained for various values of capillary diameter and receptor density. According to our numerical results, bullet motion enables firm adhesion of a cell to the capillary wall even for a weak ligand-receptor binding. We also quantified effects of various parameters, including the dissociation rate constant, the spring constant, and the reactive compliance on the characteristics of cell motion. Our results suggest that even under the interaction between PSGL-1 and P-selectin, which is mainly responsible for leukocyte rolling, a cell is able to show firm adhesion in a small capillary. These findings may help in understanding such phenomena as leukocyte plugging and cancer metastasis. This research was supported by JSPS KAKENHI Grant Numbers 25000008, 26107703, 14J03967. We also acknowledge support from the Tohoku University Division for International Advanced Research and Education Organization.

LAR-RPTP Clustering Is Modulated by Competitive Binding between Synaptic Adhesion Partners and Heparan Sulfate

PubMed Central

Won, Seoung Youn; Kim, Cha Yeon; Kim, Doyoun; Ko, Jaewon; Um, Ji Won; Lee, Sung Bae; Buck, Matthias; Kim, Eunjoon; Heo, Won Do; Lee, Jie-Oh; Kim, Ho Min

2017-01-01

The leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that direct axonal growth and neuronal regeneration. LAR-RPTPs are also synaptic adhesion molecules that form trans-synaptic adhesion complexes by binding to various postsynaptic adhesion ligands, such as Slit- and Trk-like family of proteins (Slitrks), IL-1 receptor accessory protein-like 1 (IL1RAPL1), interleukin-1 receptor accessory protein (IL-1RAcP) and neurotrophin receptor tyrosine kinase C (TrkC), to regulate synaptogenesis. Here, we determined the crystal structure of the human LAR-RPTP/IL1RAPL1 complex and found that lateral interactions between neighboring LAR-RPTP/IL1RAPL1 complexes in crystal lattices are critical for the higher-order assembly and synaptogenic activity of these complexes. Moreover, we found that LAR-RPTP binding to the postsynaptic adhesion ligands, Slitrk3, IL1RAPL1 and IL-1RAcP, but not TrkC, induces reciprocal higher-order clustering of trans-synaptic adhesion complexes. Although LAR-RPTP clustering was induced by either HS or postsynaptic adhesion ligands, the dominant binding of HS to the LAR-RPTP was capable of dismantling pre-established LAR-RPTP-mediated trans-synaptic adhesion complexes. These findings collectively suggest that LAR-RPTP clustering for synaptogenesis is modulated by a complex synapse-organizing protein network. PMID:29081732

Wet-surface–enhanced ellipsometric contrast microscopy identifies slime as a major adhesion factor during bacterial surface motility

PubMed Central

Ducret, Adrien; Valignat, Marie-Pierre; Mouhamar, Fabrice; Mignot, Tâm; Theodoly, Olivier

2012-01-01

In biology, the extracellular matrix (ECM) promotes both cell adhesion and specific recognition, which is essential for central developmental processes in both eukaryotes and prokaryotes. However, live studies of the dynamic interactions between cells and the ECM, for example during motility, have been greatly impaired by imaging limitations: mostly the ability to observe the ECM at high resolution in absence of specific staining by live microscopy. To solve this problem, we developed a unique technique, wet-surface enhanced ellipsometry contrast (Wet-SEEC), which magnifies the contrast of transparent organic materials deposited on a substrate (called Wet-surf) with exquisite sensitivity. We show that Wet-SEEC allows both the observation of unprocessed nanofilms as low as 0.2 nm thick and their accurate 3D topographic reconstructions, directly by standard light microscopy. We next used Wet-SEEC to image slime secretion, a poorly defined property of many prokaryotic and eukaryotic organisms that move across solid surfaces in absence of obvious extracellular appendages (gliding). Using combined Wet-SEEC and fluorescent-staining experiments, we observed slime deposition by gliding Myxococcus xanthus cells at unprecedented resolution. Altogether, the results revealed that in this bacterium, slime associates preferentially with the outermost components of the motility machinery and promotes its adhesion to the substrate on the ventral side of the cell. Strikingly, analogous roles have been proposed for the extracellular proteoglycans of gliding diatoms and apicomplexa, suggesting that slime deposition is a general means for gliding organisms to adhere and move over surfaces. PMID:22665761

The changing world of G protein-coupled receptors: from monomers to dimers and receptor mosaics with allosteric receptor-receptor interactions.

PubMed

Fuxe, Kjell; Marcellino, Daniel; Borroto-Escuela, Dasiel Oscar; Frankowska, Malgorzata; Ferraro, Luca; Guidolin, Diego; Ciruela, Francisco; Agnati, Luigi F

2010-10-01

Based on indications of direct physical interactions between neuropeptide and monoamine receptors in the early 1980s, the term receptor-receptor interactions was introduced and later on the term receptor heteromerization in the early 1990s. Allosteric mechanisms allow an integrative activity to emerge either intramolecularly in G protein-coupled receptor (GPCR) monomers or intermolecularly via receptor-receptor interactions in GPCR homodimers, heterodimers, and receptor mosaics. Stable heteromers of Class A receptors may be formed that involve strong high energy arginine-phosphate electrostatic interactions. These receptor-receptor interactions markedly increase the repertoire of GPCR recognition, signaling and trafficking in which the minimal signaling unit in the GPCR homomers appears to be one receptor and one G protein. GPCR homomers and GPCR assemblies are not isolated but also directly interact with other proteins to form horizontal molecular networks at the plasma membrane.

CD44-mediated activation of α5β1-integrin, cortactin and paxillin signaling underpins adhesion of basal-like breast cancer cells to endothelium and Fibronectin-enriched matrices

PubMed Central

McFarlane, Suzanne; McFarlane, Cheryl; Montgomery, Nicola; Hill, Ashleigh; Waugh, David J.J.

2015-01-01

CD44 expression is elevated in basal-like breast cancer (BLBC) tissue, and correlates with increased efficiency of distant metastasis in patients and experimental models. We sought to characterize mechanisms underpinning CD44-promoted adhesion of BLBC cells to vascular endothelial monolayers and extracellular matrix (ECM) substrates. Stimulation with hyaluronan (HA), the native ligand for CD44, increased expression and activation of β1-integrin receptors, and increased α5-integrin subunit expression. Adhesion assays confirmed that CD44-signalling potentiated BLBC cell adhesion to endothelium and Fibronectin in an α5B1-integrin-dependent mechanism. Co-immunoprecipitation experiments confirmed HA-promoted association of CD44 with talin and the β1-integrin chain in BLBC cells. Knockdown of talin inhibited CD44 complexing with β1-integrin and repressed HA-induced, CD44-mediated activation of β1-integrin receptors. Immunoblotting confirmed that HA induced rapid phosphorylation of cortactin and paxillin, through a CD44-dependent and β1-integrin-dependent mechanism. Knockdown of CD44, cortactin or paxillin independently attenuated the adhesion of BL-BCa cells to endothelial monolayers and Fibronectin. Accordingly, we conclude that CD44 induced, integrin-mediated signaling not only underpins efficient adhesion of BLBC cells to BMECs to facilitate extravasation but initiates their adhesion to Fibronectin, enabling penetrant cancer cells to adhere more efficiently to underlying Fibronectin-enriched matrix present within the metastatic niche. PMID:26447611

Cell adhesion molecules in context

PubMed Central

2011-01-01

Cell adhesion molecules (CAMs) are now known to mediate much more than adhesion between cells and between cells and the extracellular matrix. Work by many researchers has illuminated their roles in modulating activation of molecules such as receptor tyrosine kinases, with subsequent effects on cell survival, migration and process extension. CAMs are also known to serve as substrates for proteases that can create diffusible fragments capable of signaling independently from the CAM. The diversity of interactions is further modulated by membrane rafts, which can co-localize or separate potential signaling partners to affect the likelihood of a given signaling pathway being activated. Given the ever-growing number of known CAMs and the fact that their heterophilic binding in cis or in trans can affect their interactions with other molecules, including membrane-bound receptors, one would predict a wide range of effects attributable to a particular CAM in a particular cell at a particular stage of development. The function(s) of a given CAM must therefore be considered in the context of the history of the cell expressing it and the repertoire of molecules expressed both by that cell and its neighbors. PMID:20948304

Inverted formin 2 in focal adhesions promotes dorsal stress fiber and fibrillar adhesion formation to drive extracellular matrix assembly

PubMed Central

Skau, Colleen T.; Plotnikov, Sergey V.; Doyle, Andrew D.; Waterman, Clare M.

2015-01-01

Actin filaments and integrin-based focal adhesions (FAs) form integrated systems that mediate dynamic cell interactions with their environment or other cells during migration, the immune response, and tissue morphogenesis. How adhesion-associated actin structures obtain their functional specificity is unclear. Here we show that the formin-family actin nucleator, inverted formin 2 (INF2), localizes specifically to FAs and dorsal stress fibers (SFs) in fibroblasts. High-resolution fluorescence microscopy and manipulation of INF2 levels in cells indicate that INF2 plays a critical role at the SF–FA junction by promoting actin polymerization via free barbed end generation and centripetal elongation of an FA-associated actin bundle to form dorsal SF. INF2 assembles into FAs during maturation rather than during their initial generation, and once there, acts to promote rapid FA elongation and maturation into tensin-containing fibrillar FAs in the cell center. We show that INF2 is required for fibroblasts to organize fibronectin into matrix fibers and ultimately 3D matrices. Collectively our results indicate an important role for the formin INF2 in specifying the function of fibrillar FAs through its ability to generate dorsal SFs. Thus, dorsal SFs and fibrillar FAs form a specific class of integrated adhesion-associated actin structure in fibroblasts that mediates generation and remodeling of ECM. PMID:25918420

Suppression of lysyl-tRNA synthetase, KRS, causes incomplete epithelial-mesenchymal transition and ineffective cell‑extracellular matrix adhesion for migration.

PubMed

Nam, Seo Hee; Kang, Minkyung; Ryu, Jihye; Kim, Hye-Jin; Kim, Doyeun; Kim, Dae Gyu; Kwon, Nam Hoon; Kim, Sunghoon; Lee, Jung Weon

2016-04-01

The cell-adhesion properties of cancer cells can be targeted to block cancer metastasis. Although cytosolic lysyl-tRNA synthetase (KRS) functions in protein synthesis, KRS on the plasma membrane is involved in cancer metastasis. We hypothesized that KRS is involved in cell adhesion-related signal transduction for cellular migration. To test this hypothesis, colon cancer cells with modulated KRS protein levels were analyzed for cell-cell contact and cell-substrate adhesion properties and cellular behavior. Although KRS suppression decreased expression of cell-cell adhesion molecules, cells still formed colonies without being scattered, supporting an incomplete epithelial mesenchymal transition. Noteworthy, KRS-suppressed cells still exhibited focal adhesions on laminin, with Tyr397-phopshorylated focal adhesion kinase (FAK), but they lacked laminin-adhesion-mediated extracellular signal-regulated kinase (ERK) and paxillin activation. KRS, p67LR and integrin α6β1 were found to interact, presumably to activate ERK for paxillin expression and Tyr118 phosphorylation even without involvement of FAK, so that specific inhibition of ERK or KRS in parental HCT116 cells blocked cell-cell adhesion and cell-substrate properties for focal adhesion formation and signaling activity. Together, these results indicate that KRS can promote cell-cell and cell-ECM adhesion for migration.

2D and 3D Matrices to Study Linear Invadosome Formation and Activity.

PubMed

Di Martino, Julie; Henriet, Elodie; Ezzoukhry, Zakaria; Mondal, Chandrani; Bravo-Cordero, Jose Javier; Moreau, Violaine; Saltel, Frederic

2017-06-02

Cell adhesion, migration, and invasion are involved in many physiological and pathological processes. For example, during metastasis formation, tumor cells have to cross anatomical barriers to invade and migrate through the surrounding tissue in order to reach blood or lymphatic vessels. This requires the interaction between cells and the extracellular matrix (ECM). At the cellular level, many cells, including the majority of cancer cells, are able to form invadosomes, which are F-actin-based structures capable of degrading ECM. Invadosomes are protrusive actin structures that recruit and activate matrix metalloproteinases (MMPs). The molecular composition, density, organization, and stiffness of the ECM are crucial in regulating invadosome formation and activation. In vitro, a gelatin assay is the standard assay used to observe and quantify invadosome degradation activity. However, gelatin, which is denatured collagen I, is not a physiological matrix element. A novel assay using type I collagen fibrils was developed and used to demonstrate that this physiological matrix is a potent inducer of invadosomes. Invadosomes that form along the collagen fibrils are known as linear invadosomes due to their linear organization on the fibers. Moreover, molecular analysis of linear invadosomes showed that the discoidin domain receptor 1 (DDR1) is the receptor involved in their formation. These data clearly demonstrate the importance of using a physiologically relevant matrix in order to understand the complex interactions between cells and the ECM.

Cross-talk between estradiol receptor and EGFR/IGF-IR signaling pathways in estrogen-responsive breast cancers: focus on the role and impact of proteoglycans.

PubMed

Skandalis, Spyros S; Afratis, Nikolaos; Smirlaki, Gianna; Nikitovic, Dragana; Theocharis, Achilleas D; Tzanakakis, George N; Karamanos, Nikos K

2014-04-01

In hormone-dependent breast cancer, estrogen receptors are the principal signaling molecules that regulate several cell functions either by the genomic pathway acting directly as transcription factors in the nucleus or by the non-genomic pathway interacting with other receptors and their adjacent pathways like EGFR/IGFR. It is well established in literature that EGFR and IGFR signaling pathways promote cell proliferation and differentiation. Moreover, recent data indicate the cross-talk between ERs and EGFR/IGFR signaling pathways causing a transformation of cell functions as well as deregulation on normal expression pattern of matrix molecules. Specifically, proteoglycans, a major category of extracellular matrix (ECM) and cell surface macromolecules, are modified during malignancy and cause alterations in cancer cell signaling, affecting eventually functional cell properties such as proliferation, adhesion and migration. The on-going strategies to block only one of the above signaling effectors result cancer cells to overcome such inactivation using alternative signaling pathways. In this article, we therefore review the underlying mechanisms in respect to the role of ERs and the involvement of cross-talk between ERs, IGFR and EGFR in breast cancer cell properties and expression of extracellular secreted and cell bound proteoglycans involved in cancer progression. Understanding such signaling pathways may help to establish new potential pharmacological targets in terms of using ECM molecules to design novel anticancer therapies. © 2013. Published by Elsevier B.V. All rights reserved.

Sickle red cell-endothelium interactions.

PubMed

Kaul, Dhananjay K; Finnegan, Eileen; Barabino, Gilda A

2009-01-01

Periodic recurrence of painful vaso-occlusive crisis is the defining feature of sickle cell disease. Among multiple pathologies associated with this disease, sickle red cell-endothelium interaction has been implicated as a potential initiating mechanism in vaso-occlusive events. This review focuses on various interrelated mechanisms involved in human sickle red cell adhesion. We discuss in vitro and microcirculatory findings on sickle red cell adhesion, its potential role in vaso-occlusion, and the current understanding of receptor-ligand interactions involved in this pathological phenomenon. In addition, we discuss the contribution of other cellular interactions (leukocytes recruitment and leukocyte-red cell interaction) to vaso-occlusion, as observed in transgenic sickle mouse models. Emphasis is given to recently discovered adhesion molecules that play a predominant role in mediating human sickle red cell adhesion. Finally, we analyze various therapeutic approaches for inhibiting sickle red cell adhesion by targeting adhesion molecules and also consider therapeutic strategies that target stimuli involved in endothelial activation and initiation of adhesion.

Integrin-extracellular matrix interactions in connective tissue remodeling and osteoblast differentiation

NASA Technical Reports Server (NTRS)

Globus, R. K.; Moursi, A.; Zimmerman, D.; Lull, J.; Damsky, C.

1995-01-01

The differentiaton of bone cells is a complex multistep process. Bone is somewhat unusual in that it is very actively and continually remodeled in the adult and that maintenance of its mass in the mature organism is exquisitely sensitive to mechanical as well as chemical signals. Bone is also unique because it consists of a very large amount of extracellular matrix (ECM) that is mineralized. The integrin family of ECM receptors has been shown to play an important role in tissue morphogenesis in several systems. Our studies on the regulation of matrix remodeling enzymes by integrins in rabbit synovial fibroblasts show that two b1 integrin fibronectin (FN) receptor complexes (alpha 5 beta 1 and alpha 4 beta 1) cooperate in detecting subtle changes in the composition of the ECM. As a result of signal transduction by these integrins, the levels of mRNA and protein for several members of the metalloproteinase family are regulated in these cells. We have also used antibody and RGD peptide perturbation studies to determine the significance of cell/ECM interactions to normal osteogenesis. We found that interactions between the cell binding domain of FN and integrins are required for both normal morphogenesis and gene expression in cultured osteoblasts that differentiate to form bone-like tissue in culture. These data lead us to propose that beta 1 integrins play an important role in osteoblast differentiation as well as in bone remodeling.

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

PubMed

Eberwein, Philipp; Laird, Dougal; Schulz, Simon; Reinhard, Thomas; Steinberg, Thorsten; Tomakidi, Pascal

2015-10-01

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events. Copyright © 2015 Elsevier B.V. All rights reserved.

Interplay between Rolling and Firm Adhesion Elucidated with a Cell-Free System Engineered with Two Distinct Receptor-Ligand Pairs

PubMed Central

Eniola, A. Omolola; Willcox, P. Jeanene; Hammer, Daniel A.

2003-01-01

The firm arrest of leukocytes to the endothelium during inflammation is known to be mediated by endothelial intercellular adhesion molecules (ICAMs) binding to activated integrins displayed on leukocyte surface. Selectin-ligand interactions, which mediate rolling, are believed to be important for facilitating firm adhesion, either by activating integrins or by facilitating the transition to firm adhesion by making it easier for integrins to bind. Although leukocytes employ two distinct adhesion molecules that mediate different states of adhesion, the fundamental biophysical mechanisms by which two pairs of adhesion molecules facilitate cell adhesion is not well understood. In this work, we attempt to understand the interaction between two molecular systems using a cell-free system in which polystyrene microspheres functionalized with the selectin ligand, sialyl LewisX (sLeX), and an antibody against ICAM-1, aICAM-1, are perfused over P-selectin/ICAM-1 coated surfaces in a parallel plate flow chamber. Separately, sLeX/P-selectin interactions support rolling and aICAM-1/ICAM-1 interactions mediate firm adhesion. Our results show that sLeX/aICAM-1 microspheres will firmly adhere to P-selectin/ICAM-1 coated surfaces, and that the extent of firm adhesion of microspheres is dependent on wall shear stress within the flow chamber, sLeX/aICAM-1 microsphere site density, and P-selectin/ICAM-1 surface density ratio. We show that P-selectin's interaction with sLeX mechanistically facilitates firm adhesion mediated by antibody binding to ICAM-1: the extent of firm adhesion for the same concentration of aICAM-1/ICAM-1 interaction is greater when sLeX/P-selectin interactions are present. aICAM-1/ICAM-1 interactions also stabilize rolling by increasing pause times and decreasing average rolling velocities. Although aICAM-1 is a surrogate for β2-integrin, the kinetics of association between aICAM-1 and ICAM-1 is within a factor of 1.5 of activated integrin binding ICAM-1

Urokinase receptor and CXCR4 are regulated by common microRNAs in leukaemia cells

PubMed Central

Alfano, Daniela; Gorrasi, Anna; Li Santi, Anna; Ricci, Patrizia; Montuori, Nunzia; Selleri, Carmine; Ragno, Pia

2015-01-01

The urokinase-type plasminogen activator (uPA) receptor (uPAR) focuses uPA proteolytic activity on the cell membrane, promoting localized degradation of extracellular matrix (ECM), and binds vitronectin (VN), mediating cell adhesion to the ECM. uPAR-bound uPA and VN induce proteolysis-independent intracellular signalling, regulating cell adhesion, migration, survival and proliferation. uPAR cross-talks with CXCR4, the receptor for the stroma-derived factor 1 chemokine. CXCR4 is crucial in the trafficking of hematopoietic stem cells from/to the bone marrow, which involves also uPAR. Both uPAR and CXCR4 are expressed in acute myeloid leukaemia (AML), with a lower expression in undifferentiated and myeloid subsets, and higher expression in myelomonocytic and promyelocytic subsets. We hypothesized a microRNA (miR)-mediated co-regulation of uPAR and CXCR4 expression, which could allow their cross-talk at the cell surface. We identified three miRs, miR-146a, miR-335 and miR-622, regulating the expression of both uPAR and CXCR4 in AML cell lines. Indeed, these miRs directly target the 3′untranslated region of both uPAR- and CXCR4-mRNAs; accordingly, uPAR/CXCR4 expression is reduced by their overexpression in AML cells and increased by their specific inhibitors. Overexpression of all three miRs impairs migration, invasion and proliferation of myelomonocytic cells. Interestingly, we observed an inverse relationship between uPAR/CXCR4 expression and miR-146a and miR-335 levels in AML blasts, suggesting their possible role in the regulation of uPAR/CXCR4 expression also in vivo. PMID:26082201

Inhibitory Effects of North American Wild Rice on Monocyte Adhesion and Inflammatory Modulators in Low-Density Lipoprotein Receptor-Knockout Mice.

PubMed

Moghadasian, Mohammed H; Zhao, Ruozhi; Ghazawwi, Nora; Le, Khuong; Apea-Bah, Franklin B; Beta, Trust; Shen, Garry X

2017-10-18

The present study examined the effects of wild rice on monocyte adhesion, inflammatory and fibrinolytic mediators in low-density lipoprotein receptor-knockout (LDLr-KO) mice. Male LDLr-KO mice received a cholesterol (0.06%, w/w)-supplemented diet with or without white or wild rice (60%, w/w) for 20 weeks. White rice significantly increased monocyte adhesion and abundances of monocyte chemoattractant protein-1, tissue necrosis factor-α, intracellular cell adhesion molecule-1, plasminogen activator inhibitor-1, urokinase plasminogen activator (uPA), and uPA receptor in aortae and hearts of LDLr-KO mice compared to the control diet. Wild rice inhibited monocyte adhesion to the aorta, atherosclerosis, and abundances of the inflammatory and fibrinolytic regulators in the cardiovascular tissue of LDLr-KO mice compared to white rice. White or wild rice did not significantly alter the levels of cholesterol, triglycerides, or antioxidant enzymes in plasma. The anti-atherosclerotic effect of wild rice may result from its inhibition on monocyte adhesion and inflammatory modulators in LDLr-KO mice.

Effect of long-range repulsive Coulomb interactions on packing structure of adhesive particles.

PubMed

Chen, Sheng; Li, Shuiqing; Liu, Wenwei; Makse, Hernán A

2016-02-14

The packing of charged micron-sized particles is investigated using discrete element simulations based on adhesive contact dynamic model. The formation process and the final obtained structures of ballistic packings are studied to show the effect of interparticle Coulomb force. It is found that increasing the charge on particles causes a remarkable decrease of the packing volume fraction ϕ and the average coordination number 〈Z〉, indicating a looser and chainlike structure. Force-scaling analysis shows that the long-range Coulomb interaction changes packing structures through its influence on particle inertia before they are bonded into the force networks. Once contact networks are formed, the expansion effect caused by repulsive Coulomb forces are dominated by short-range adhesion. Based on abundant results from simulations, a dimensionless adhesion parameter Ad*, which combines the effects of the particle inertia, the short-range adhesion and the long-range Coulomb interaction, is proposed and successfully scales the packing results for micron-sized particles within the latest derived adhesive loose packing (ALP) regime. The structural properties of our packings follow well the recent theoretical prediction which is described by an ensemble approach based on a coarse-grained volume function, indicating some kind of universality in the low packing density regime of the phase diagram regardless of adhesion or particle charge. Based on the comprehensive consideration of the complicated inter-particle interactions, our findings provide insight into the roles of short-range adhesion and repulsive Coulomb force during packing formation and should be useful for further design of packings.

The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis.

PubMed

Ishikawa, Tomomi; Kitano, Hisataka; Mamiya, Atsushi; Kokubun, Shinichiro; Hidai, Chiaki

2016-07-01

Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner. © 2016 The Author(s).

Dual ECM Biomimetic Scaffolds for Dental Pulp Regenerative Applications

PubMed Central

Huang, Chun-Chieh; Narayanan, Raghuvaran; Warshawsky, Noah; Ravindran, Sriram

2018-01-01

Dental pulp is a highly vascularized and innervated tissue that provides sensitivity and vitality to the tooth. Chronic caries results in an infected pulp tissue prone to necrosis. Existing clinical treatments replace the living pulp tissue with a non-responsive resin filling resulting in loss of tooth vitality. Tissue engineering approaches to dental pulp tissue regeneration have been investigated to preserve tooth vitality and function. However, a critical criterion is the choice of growth factors that may promote mesenchymal stem cell differentiation and more importantly, vascularization. But, the problems associated with growth factor dosage, delivery, safety, immunological and ectopic complications affect their translatory potential severely. The purpose of this study is to develop, characterize and evaluate a biomimetic native extracellular matrix (ECM) derived dual ECM scaffold that consists of a pulp-specific ECM to promote MSC attachment, proliferation and differentiation and an endothelial ECM to promote migration of host endothelial cells and eventual vascularization in vivo. Our results show that the dual ECM scaffolds possess similar properties as a pulp-ECM scaffold to promote MSC attachment and odontogenic differentiation in vitro. Additionally, when implanted subcutaneously in a tooth root slice model in vivo, the dual ECM scaffolds promoted robust odontogenic differentiation of both dental pulp and bone marrow derived MSCs and also extensive vascularization when compared to respective controls. These scaffolds are mass producible for clinical use and hence have the potential to replace root canal therapy as a treatment for chronic dental caries. PMID:29887803

ECM-Based Biohybrid Materials for Engineering Compliant, Matrix-Dense Tissues

PubMed Central

Bracaglia, Laura G.; Fisher, John P.

2015-01-01

An ideal tissue engineering scaffold should not only promote, but take an active role in, constructive remodeling and formation of site appropriate tissue. ECM-derived proteins provide unmatched cellular recognition, and therefore influence cellular response towards predicted remodeling behaviors. Materials built with only these proteins, however, can degrade rapidly or begin too weak to substitute for compliant, matrix-dense tissues. The focus of this review is on biohybrid materials that incorporate polymer components with ECM-derived proteins, to produce a substrate with desired mechanical and degradation properties, as well as actively guide tissue remodeling. Materials are described through four fabrication methods: (1) polymer and ECM-protein fibers woven together, (2) polymer and ECM proteins combined in a bilayer, (3) cell-built ECM on polymer scaffold, and (4) ECM proteins and polymers combined in a single hydrogel. Scaffolds from each fabrication method can achieve characteristics suitable for different types of tissue. In vivo testing has shown progressive remodeling in injury models, and suggests ECM-based biohybrid materials promote a prohealing immune response over single component alternatives. The prohealing immune response is associated with lasting success and long term host maintenance of the implant. PMID:26227679

ß3 integrin modulates transforming growth factor beta induced (TGFBI) function and paclitaxel response in ovarian cancer cells.

PubMed

Tumbarello, David A; Temple, Jillian; Brenton, James D

2012-05-28

The extracellular matrix (ECM) has a key role in facilitating the progression of ovarian cancer and we have shown recently that the secreted ECM protein TGFBI modulates the response of ovarian cancer to paclitaxel-induced cell death. We have determined TGFBI signaling from the extracellular environment is preferential for the cell surface αvß3 integrin heterodimer, in contrast to periostin, a TGFBI paralogue, which signals primarily via a ß1 integrin-mediated pathway. We demonstrate that suppression of ß1 integrin expression, in ß3 integrin-expressing ovarian cancer cells, increases adhesion to rTGFBI. In addition, Syndecan-1 and -4 expression is dispensable for adhesion to rTGFBI and loss of Syndecan-1 cooperates with the loss of ß1 integrin to further enhance adhesion to rTGFBI. The RGD motif present in the carboxy-terminus of TGFBI is necessary, but not sufficient, for SKOV3 cell adhesion and is dispensable for adhesion of ovarian cancer cells lacking ß3 integrin expression. In contrast to TGFBI, the carboxy-terminus of periostin, lacking a RGD motif, is unable to support adhesion of ovarian cancer cells. Suppression of ß3 integrin in SKOV3 cells increases resistance to paclitaxel-induced cell death while suppression of ß1 integrin has no effect. Furthermore, suppression of TGFBI expression stimulates a paclitaxel resistant phenotype while suppression of fibronectin expression, which primarily signals through a ß1 integrin-mediated pathway, increases paclitaxel sensitivity. Therefore, different ECM components use distinct signaling mechanisms in ovarian cancer cells and in particular, TGFBI preferentially interacts through a ß3 integrin receptor mediated mechanism to regulate the response of cells to paclitaxel-induced cell death.

Distinct Fcγ receptors mediate the effect of Serum Amyloid P on neutrophil adhesion and fibrocyte differentiation

PubMed Central

Cox, Nehemiah; Pilling, Darrell; Gomer, Richard H.

2014-01-01

The plasma protein Serum Amyloid P (SAP) reduces neutrophil adhesion, inhibits the differentiation of monocytes into fibroblast-like cells called fibrocytes, and promotes phagocytosis of cell debris by macrophages. Together, these effects of SAP reduce key aspects of inflammation and fibrosis, and SAP injections improve lung function in pulmonary fibrosis patients. SAP functions are mediated in part by Fcγ receptors, but the contribution of each Fcγ receptor is not fully understood. We found that amino acids Q55 and E126 in human SAP affect human fibrocyte differentiation and SAP binding to FcγRI. E126, K130 and Q128 affect neutrophil adhesion and SAP affinity for FcγRIIa. Q128 also affects phagocytosis by macrophages and SAP affinity for FcγRI. All the identified functionally significant amino acids in SAP form a binding site that is distinct from the previously described SAP-FcγRIIa binding site. Blocking FcγRI with an IgG blocking antibody reduces the SAP effect on fibrocyte differentiation, and ligating FcγRIIa with antibodies reduces neutrophil adhesion. Together, these results suggest that SAP binds to FcγRI on monocytes to inhibit fibrocyte differentiation, and binds to FcγRIIa on neutrophils to reduce neutrophil adhesion. PMID:25024390

Analysis of molecular pathways in pancreatic ductal adenocarcinomas with a bioinformatics approach.

PubMed

Wang, Yan; Li, Yan

2015-01-01

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer death worldwide. Our study aimed to reveal molecular mechanisms. Microarray data of GSE15471 (including 39 matching pairs of pancreatic tumor tissues and patient-matched normal tissues) was downloaded from Gene Expression Omnibus (GEO) database. We identified differentially expressed genes (DEGs) in PDAC tissues compared with normal tissues by limma package in R language. Then GO and KEGG pathway enrichment analyses were conducted with online DAVID. In addition, principal component analysis was performed and a protein-protein interaction network was constructed to study relationships between the DEGs through database STRING. A total of 532 DEGs were identified in the 38 PDAC tissues compared with 33 normal tissues. The results of principal component analysis of the top 20 DEGs could differentiate the PDAC tissues from normal tissues directly. In the PPI network, 8 of the 20 DEGs were all key genes of the collagen family. Additionally, FN1 (fibronectin 1) was also a hub node in the network. The genes of the collagen family as well as FN1 were significantly enriched in complement and coagulation cascades, ECM-receptor interaction and focal adhesion pathways. Our results suggest that genes of collagen family and FN1 may play an important role in PDAC progression. Meanwhile, these DEGs and enriched pathways, such as complement and coagulation cascades, ECM-receptor interaction and focal adhesion may be important molecular mechanisms involved in the development and progression of PDAC.

Bio-active molecules modified surfaces enhanced mesenchymal stem cell adhesion and proliferation

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

Mobasseri, Rezvan; Center for Nanofibers & Nanotechnology, Department of Mechanical Engineering, National University of Singapore, 117576; Tian, Lingling

Surface modification of the substrate as a component of in vitro cell culture and tissue engineering, using bio-active molecules including extracellular matrix (ECM) proteins or peptides derived ECM proteins can modulate the surface properties and thereby induce the desired signaling pathways in cells. The aim of this study was to evaluate the behavior of human bone marrow mesenchymal stem cells (hBM-MSCs) on glass substrates modified with fibronectin (Fn), collagen (Coll), RGD peptides (RGD) and designed peptide (R-pept) as bio-active molecules. The glass coverslips were coated with fibronectin, collagen, RGD peptide and R-peptide. Bone marrow mesenchymal stem cells were cultured on differentmore » substrates and the adhesion behavior in early incubation times was investigated using scanning electron microscopy (SEM) and confocal microscopy. The MTT assay was performed to evaluate the effect of different bio-active molecules on MSCs proliferation rate during 24 and 72 h. Formation of filopodia and focal adhesion (FA) complexes, two steps of cell adhesion process, were observed in MSCs cultured on bio-active molecules modified coverslips, specifically in Fn coated and R-pept coated groups. SEM image showed well adhesion pattern for MSCs cultured on Fn and R-pept after 2 h incubation, while the shape of cells cultured on Coll and RGD substrates indicated that they might experience stress condition in early hours of culture. Investigation of adhesion behavior, as well as proliferation pattern, suggests R-peptide as a promising bio-active molecule to be used for surface modification of substrate in supporting and inducing cell adhesion and proliferation. - Highlights: • Bioactive molecules modified surface is a strategy to design biomimicry scaffold. • Bi-functional Tat-derived peptide (R-pept) enhanced MSCs adhesion and proliferation. • R-pept showed similar influences to fibronectin on FA formation and attachment.« less

Surface control of blastospore attachment and ligand-mediated hyphae adhesion of Candida albicans.

PubMed

Varghese, Nisha; Yang, Sijie; Sejwal, Preeti; Luk, Yan-Yeung

2013-11-14

Adhesion on a surface via nonspecific attachment or multiple ligand-receptor interactions is a critical event for fungal infection by Candida albicans. Here, we find that the tri(ethylene glycol)- and d-mannitol-terminated monolayers do not resist the blastospore attachment, but prevent the hyphae adhesion of C. albicans. The hyphae adhesion can be facilitated by tripeptide sequences of arginine-glycine-aspartic acid (RGD) covalently decorated on a background of tri(ethylene glycol)-terminated monolayers. This adhesion mediated by selected ligands is sensitive to the scrambling of peptide sequences, and is inhibited by the presence of cyclic RGD peptides in the solution.

ECM1 and TMPRSS4 Are Diagnostic Markers of Malignant Thyroid Neoplasms and Improve the Accuracy of Fine Needle Aspiration Biopsy

PubMed Central

Kebebew, Electron; Peng, Miao; Reiff, Emily; Duh, Quan-Yang; Clark, Orlo H.; McMillan, Alex

2005-01-01

Objective: The objective of this study was to determine whether genes that regulate cellular invasion and metastasis are differentially expressed and could serve as diagnostic markers of malignant thyroid nodules. Summary and Background Data: Patients whose thyroid nodules have indeterminate or suspicious cytologic features on fine needle aspiration (FNA) biopsy require thyroidectomy because of a 20% to 30% risk of thyroid cancer. Cell invasion and metastasis is a hallmark of malignant phenotype; therefore, genes that regulate these processes might be differentially expressed and could serve as diagnostic markers of malignancy. Methods: Differentially expressed genes (2-fold higher or lower) in malignant versus benign thyroid neoplasms were identified by extracellular matrix and adhesion molecule cDNA array analysis and confirmed by real-time quantitative polymerase chain reaction (PCR). The area under the receiver operating characteristic (AUC) curve was calculated to determine diagnostic accuracy of gene expression level cutoffs established by logistic regression analysis. Results: By cDNA array analysis, ADAMTS8, ECM1, MMP8, PLAU, SELP, and TMPRSS4 were upregulated, and by quantitative PCR, ECM1, SELP, and TMPRSS4 mRNA expression was higher in malignant (n = 57) than in benign (n = 38) thyroid neoplasms (P< 0.002). ECM1 and TMPRSS4 mRNA expression levels were independent predictors of a malignant thyroid neoplasm (P < 0.003). The AUC was 0.956 for ECM1 and 0.926 for TMPRSS4. Combining both markers improved their diagnostic use (AUC 0.985; sensitivity, 91.7%; specificity, 89.8%; positive predictive value, 85.7%; negative predictive value, 82.8%). ECM1 and TMPRSS4 expression analysis improved the diagnostic accuracy of FNA biopsy in 35 of 38 indeterminate or suspicious results. The level of ECM1 mRNA expression was higher in TNM stage I differentiated thyroid cancers than in stage II and III tumors (P ≤ 0.031). Conclusions: ECM1 and TMPRSS4 are excellent

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release*

PubMed Central

Malenczyk, Katarzyna; Jazurek, Magdalena; Keimpema, Erik; Silvestri, Cristoforo; Janikiewicz, Justyna; Mackie, Ken; Di Marzo, Vincenzo; Redowicz, Maria J.; Harkany, Tibor; Dobrzyn, Agnieszka

2013-01-01

Endocannabinoid signaling has been implicated in modulating insulin release from β cells of the endocrine pancreas. β Cells express CB1 cannabinoid receptors (CB1Rs), and the enzymatic machinery regulating anandamide and 2-arachidonoylglycerol bioavailability. However, the molecular cascade coupling agonist-induced cannabinoid receptor activation to insulin release remains unknown. By combining molecular pharmacology and genetic tools in INS-1E cells and in vivo, we show that CB1R activation by endocannabinoids (anandamide and 2-arachidonoylglycerol) or synthetic agonists acutely or after prolonged exposure induces insulin hypersecretion. In doing so, CB1Rs recruit Akt/PKB and extracellular signal-regulated kinases 1/2 to phosphorylate focal adhesion kinase (FAK). FAK activation induces the formation of focal adhesion plaques, multimolecular platforms for second-phase insulin release. Inhibition of endocannabinoid synthesis or FAK activity precluded insulin release. We conclude that FAK downstream from CB1Rs mediates endocannabinoid-induced insulin release by allowing cytoskeletal reorganization that is required for the exocytosis of secretory vesicles. These findings suggest a mechanistic link between increased circulating and tissue endocannabinoid levels and hyperinsulinemia in type 2 diabetes. PMID:24089517

p38 Signaling and Receptor Recycling Events in a Microfluidic Endothelial Cell Adhesion Assay

PubMed Central

Vickers, Dwayne A. L.; Chory, Emma J.; Harless, Megan C.; Murthy, Shashi K.

2013-01-01

Adhesion-based microfluidic cell separation has proven to be very useful in applications ranging from cancer diagnostics to tissue engineering. This process involves functionalizing microchannel surfaces with a capture molecule. High specificity and purity capture can be achieved using this method. Despite these advances, little is known about the mechanisms that govern cell capture within these devices and their relationships to basic process parameters such as fluid shear stress and the presence of soluble factors. This work examines how the adhesion of human endothelial cells (ECs) is influenced by a soluble tetrapeptide, Arg-Glu-Asp-Val (REDV) and fluidic shear stress. The ability of these ECs to bind within microchannels coated with REDV is shown to be governed by shear- and soluble-factor mediated changes in p38 mitogen-activated protein kinase expression together with recycling of adhesion receptors from the endosome. PMID:23762436

Cell adhesion molecules, the extracellular matrix and oral squamous carcinoma.

PubMed

Lyons, A J; Jones, J

2007-08-01

Carcinomas are characterized by invasion of malignant cells into the underlying connective tissue and migration of malignant cells to form metastases at distant sites. These processes require alterations in cell-cell and cell-extracellular matrix interactions. As cell adhesion molecules play a role in cell-cell and cell-extracellular matrix adhesion and interactions they are involved in the process of tumour invasion and metastases. In epithelial tissues, receptors of the integrin family mediate adhesion to the adjacent matrix whereas cadherins largely mediate intercellular adhesion. These and other cell adhesion molecules such as intercellular adhesion molecule-1, CD44, dystroglycans and selectins, are involved and undergo changes in carcinomas, which provide possible targets for anti-cancer drug treatments. In the extracellular matrix that is associated with tumours, laminin 5, oncofetal fibronectin and tenascin C appear. The degree of expression of some of these moieties indicates prognosis in oral cancer and offer targets for antibody-directed radiotherapy. Metalloproteases which degrade the extracellular matrix are increased in carcinomas, and their activity is necessary for tumour angiogenesis and consequent invasion and metastases. Metalloprotease inhibitors have begun to produce decreases in mortality in clinical trials. This report provides a brief overview of our current understanding of cell adhesion molecules, the extracellular matrix, tumour invasion and metastasis.

PH dependent adhesive peptides

DOEpatents

Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

2010-06-29

A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

Extracellular matrix structure.

PubMed

Theocharis, Achilleas D; Skandalis, Spyros S; Gialeli, Chrysostomi; Karamanos, Nikos K

2016-02-01

Extracellular matrix (ECM) is a non-cellular three-dimensional macromolecular network composed of collagens, proteoglycans/glycosaminoglycans, elastin, fibronectin, laminins, and several other glycoproteins. Matrix components bind each other as well as cell adhesion receptors forming a complex network into which cells reside in all tissues and organs. Cell surface receptors transduce signals into cells from ECM, which regulate diverse cellular functions, such as survival, growth, migration, and differentiation, and are vital for maintaining normal homeostasis. ECM is a highly dynamic structural network that continuously undergoes remodeling mediated by several matrix-degrading enzymes during normal and pathological conditions. Deregulation of ECM composition and structure is associated with the development and progression of several pathologic conditions. This article emphasizes in the complex ECM structure as to provide a better understanding of its dynamic structural and functional multipotency. Where relevant, the implication of the various families of ECM macromolecules in health and disease is also presented. Copyright © 2015 Elsevier B.V. All rights reserved.

42 CFR 456.725 - Funding of ECM system.

Code of Federal Regulations, 2014 CFR

2014-10-01

... 42 Public Health 4 2014-10-01 2014-10-01 false Funding of ECM system. 456.725 Section 456.725 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Claims Management System for Outpatient Drug Claims § 456.725 Funding of ECM system. (a) For funds...

42 CFR 456.725 - Funding of ECM system.

Code of Federal Regulations, 2012 CFR

2012-10-01

... 42 Public Health 4 2012-10-01 2012-10-01 false Funding of ECM system. 456.725 Section 456.725 Public Health CENTERS FOR MEDICARE & MEDICAID SERVICES, DEPARTMENT OF HEALTH AND HUMAN SERVICES... Claims Management System for Outpatient Drug Claims § 456.725 Funding of ECM system. (a) For funds...

Glycosylation-dependent galectin-receptor interactions promote Chlamydia trachomatis infection.

PubMed

Lujan, Agustin L; Croci, Diego O; Gambarte Tudela, Julián A; Losinno, Antonella D; Cagnoni, Alejandro J; Mariño, Karina V; Damiani, María T; Rabinovich, Gabriel A

2018-06-11

Chlamydia trachomatis ( Ct ) constitutes the most prevalent sexually transmitted bacterium worldwide. Chlamydial infections can lead to severe clinical sequelae including pelvic inflammatory disease, ectopic pregnancy, and tubal infertility. As an obligate intracellular pathogen, Ct has evolved multiple strategies to promote adhesion and invasion of host cells, including those involving both bacterial and host glycans. Here, we show that galectin-1 (Gal1), an endogenous lectin widely expressed in female and male genital tracts, promotes Ct infection. Through glycosylation-dependent mechanisms involving recognition of bacterial glycoproteins and N -glycosylated host cell receptors, Gal1 enhanced Ct attachment to cervical epithelial cells. Exposure to Gal1, mainly in its dimeric form, facilitated bacterial entry and increased the number of infected cells by favoring Ct - Ct and Ct -host cell interactions. These effects were substantiated in vivo in mice lacking Gal1 or complex β1-6-branched N -glycans. Thus, disrupting Gal1- N -glycan interactions may limit the severity of chlamydial infection by inhibiting bacterial invasion of host cells.

A simplified model for dynamics of cell rolling and cell-surface adhesion

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

Cimrák, Ivan, E-mail: ivan.cimrak@fri.uniza.sk

2015-03-10

We propose a three dimensional model for the adhesion and rolling of biological cells on surfaces. We study cells moving in shear flow above a wall to which they can adhere via specific receptor-ligand bonds based on receptors from selectin as well as integrin family. The computational fluid dynamics are governed by the lattice-Boltzmann method. The movement and the deformation of the cells is described by the immersed boundary method. Both methods are fully coupled by implementing a two-way fluid-structure interaction. The adhesion mechanism is modelled by adhesive bonds including stochastic rules for their creation and rupture. We explore amore » simplified model with dissociation rate independent of the length of the bonds. We demonstrate that this model is able to resemble the mesoscopic properties, such as velocity of rolling cells.« less

Tethered agonists: a new mechanism underlying adhesion G protein-coupled receptor activation.

PubMed

Schöneberg, Torsten; Liebscher, Ines; Luo, Rong; Monk, Kelly R; Piao, Xianhua

2015-06-01

The family of adhesion G protein-coupled receptors (aGPCRs) comprises 33 members in the human genome, which are subdivided into nine subclasses. Many aGPCRs undergo an autoproteolytic process via their GPCR Autoproteolysis-INducing (GAIN) domain during protein maturation to generate an N- and a C-terminal fragments, NTF and CTF, respectively. The NTF and CTF are non-covalently reassociated on the plasma membrane to form a single receptor unit. How aGPCRs are activated upon ligand binding remains one of the leading questions in the field of aGPCR research. Recent work from our labs and others shows that ligand binding can remove the NTF from the plasma membrane-bound CTF, exposing a tethered agonist which potently activates downstream signaling.

FERMT2 links cortical actin structures, plasma membrane tension and focal adhesion function to stabilize podocyte morphology.

PubMed

Yasuda-Yamahara, M; Rogg, M; Frimmel, J; Trachte, P; Helmstaedter, M; Schroder, P; Schiffer, M; Schell, C; Huber, T B

2018-01-11

Simplification and retraction of podocyte protrusions, generally termed as foot process effacement, is a uniform pathological pattern observed in the majority of glomerular disease, including focal segmental glomerulosclerosis. However, it is still incompletely understood how the interaction of cortical actin structures, actomyosin contractility and focal adhesions, is being orchestrated to control foot process morphology in health and disease. By uncovering the functional role of fermitin family member 2 (FERMT2 or kindlin-2) in podocytes, we provide now evidence, how cell-extracellular matrix (ECM) interactions modulate membrane tension and actomyosin contractility. A genetic modeling approach was applied by deleting FERMT2 in a set of in vivo systems as well as in CRISPR/Cas9 modified human podocytes. Loss of FERMT2 results in altered cortical actin composition, cell cortex destabilization associated with plasma membrane blebbing and a remodeling of focal adhesions. We further show that FERMT2 knockout podocytes have high levels of RhoA activation and concomitantly increased actomyosin contractility. Inhibition of actomyosin tension reverses the membrane blebbing phenotype. Thus, our findings establish a direct link between cell-matrix adhesions, cortical actin structures and plasma membrane tension allowing to better explain cell morphological changes in foot process effacement. Copyright © 2018 The Authors. Published by Elsevier B.V. All rights reserved.

Matrix stiffness modulates infection of endothelial cells by Listeria monocytogenes via expression of cell surface vimentin.

PubMed

Bastounis, Effie E; Yeh, Yi-Ting; Theriot, Julie A

2018-05-02

Extracellular matrix stiffness (ECM) is one of the many mechanical forces acting on mammalian adherent cells and an important determinant of cellular function. While the effect of ECM stiffness on many aspects of cellular behavior has been previously studied, how ECM stiffness might mediate susceptibility of host cells to infection by bacterial pathogens was hitherto unexplored. To address this open question, we manufactured hydrogels of varying physiologically-relevant stiffness and seeded human microvascular endothelial cells (HMEC-1) on them. We then infected HMEC-1 with the bacterial pathogen Listeria monocytogenes (Lm), and found that adhesion of Lm onto host cells increases monotonically with increasing matrix stiffness, an effect that requires the activity of focal adhesion kinase (FAK). We identified cell surface vimentin as a candidate surface receptor mediating stiffness-dependent adhesion of Lm to HMEC-1 and found that bacterial infection of these host cells is decreased when the amount of surface vimentin is reduced. Our results provide the first evidence that ECM stiffness can mediate the susceptibility of mammalian host cells to infection by a bacterial pathogen.

Role of cellular adhesions in tissue dynamics spectroscopy

NASA Astrophysics Data System (ADS)

Merrill, Daniel A.; An, Ran; Turek, John; Nolte, David

2014-02-01

Cellular adhesions play a critical role in cell behavior, and modified expression of cellular adhesion compounds has been linked to various cancers. We tested the role of cellular adhesions in drug response by studying three cellular culture models: three-dimensional tumor spheroids with well-developed cellular adhesions and extracellular matrix (ECM), dense three-dimensional cell pellets with moderate numbers of adhesions, and dilute three-dimensional cell suspensions in agarose having few adhesions. Our technique for measuring the drug response for the spheroids and cell pellets was biodynamic imaging (BDI), and for the suspensions was quasi-elastic light scattering (QELS). We tested several cytoskeletal chemotherapeutic drugs (nocodazole, cytochalasin-D, paclitaxel, and colchicine) on three cancer cell lines chosen from human colorectal adenocarcinoma (HT-29), human pancreatic carcinoma (MIA PaCa-2), and rat osteosarcoma (UMR-106) to exhibit differences in adhesion strength. Comparing tumor spheroid behavior to that of cell suspensions showed shifts in the spectral motion of the cancer tissues that match predictions based on different degrees of cell-cell contacts. The HT-29 cell line, which has the strongest adhesions in the spheroid model, exhibits anomalous behavior in some cases. These results highlight the importance of using three-dimensional tissue models in drug screening with cellular adhesions being a contributory factor in phenotypic differences between the drug responses of tissue and cells.

Mechanotransduction: all signals point to cytoskeleton, matrix, and integrins

NASA Technical Reports Server (NTRS)

Alenghat, Francis J.; Ingber, Donald E.

2002-01-01

Mechanical stresses modulate cell function by either activating or tuning signal transduction pathways. Mechanotransduction, the process by which cells convert mechanical stimuli into a chemical response, occurs both in cells specialized for sensing mechanical cues and in parenchymal cells whose primary function is not mechanosensory. However, common among the various responses to mechanical stress is the importance of direct or indirect connections between the internal cytoskeleton, the extracellular matrix (ECM), and traditional signal transducing molecules. In many instances, these elements converge at focal adhesions, sites of structural attachment between the cytoskeleton and ECM that are anchored by cell surface integrin receptors. Alenghat and Ingber discuss the accumulating evidence for the central role of cytoskeleton, ECM, and integrin-anchored focal adhesions in several mechanotransduction pathways.

Matrix MetalloProteinases (MMPs) andTissue Inhibitors of MetalloProteinases (TIMPs): positive and negative regulators intumor cell adhesion

PubMed Central

Bourboulia, Dimitra; Stetler-Stevenson, William G.

2010-01-01

Cells adhere to one another and/or to matrices that surround them. Regulation of cell-cell (intercellular) and cell-matrix adhesion is tightly controlled in normal cells, however, defects in cell adhesion are common in the majority of humancancers. Multilateral communication among tumor cells with the extracellular matrix (ECM) and neighbor cells is accomplished through adhesion molecules, ECM components, proteolytic enzymes and their endogenous inhibitors. There is sufficient evidence to suggest that reduced adherence is a tumor cell propertyengaged during tumor progression. Tumor cells acquire the ability to change shape, detach and easily move through spaces disorganizing the normal tissue architecture. This property is due to changes in expression levels of adhesion molecules and/or due to elevated levels of secreted proteolytic enzymes, including matrix metalloproteinases (MMPs). Among other roles, MMPsdegrade the ECMand, therefore, prepare the path for tumor cells to migrate, invade and spread to distant secondary areas, where they form metastasis. Tissue Inhibitors of Metalloproteinases or TIMPs control MMP activities and, therefore, minimize matrix degradation. Both MMPs and TIMPs are involved in tissue remodeling and decisively regulate tumor cell progression including tumor angiogenesis. In this review, we describe and discuss data that support the important role of MMPs and TIMPs in cancer cell adhesion and tumor progression. PMID:20470890

Impact of lipid rafts on the T -cell-receptor and peptide-major-histocompatibility-complex interactions under different measurement conditions

NASA Astrophysics Data System (ADS)

Li, Long; Xu, Guang-Kui; Song, Fan

2017-01-01

The interactions between T-cell receptor (TCR) and peptide-major-histocompatibility complex (pMHC), which enable T-cell development and initiate adaptive immune responses, have been intensively studied. However, a central issue of how lipid rafts affect the TCR-pMHC interactions remains unclear. Here, by using a statistical-mechanical membrane model, we show that the binding affinity of TCR and pMHC anchored on two apposing cell membranes is significantly enhanced because of the lipid raft-induced signaling protein aggregation. This finding may provide an alternative insight into the mechanism of T-cell activation triggered by very low densities of pMHC. In the case of cell-substrate adhesion, our results indicate that the loss of lateral mobility of the proteins on the solid substrate leads to the inhibitory effect of lipid rafts on TCR-pMHC interactions. Our findings help to understand why different experimental methods for measuring the impact of lipid rafts on the receptor-ligand interactions have led to contradictory conclusions.

Static Mechanical Loading Influences the Expression of Extracellular Matrix and Cell Adhesion Proteins in Vaginal Cells Derived From Premenopausal Women With Severe Pelvic Organ Prolapse.

PubMed

Kufaishi, Hala; Alarab, May; Drutz, Harold; Lye, Stephen; Shynlova, Oksana

2016-08-01

Primary human vaginal cells derived from women with severe pelvic organ prolapse (POP-HVCs) demonstrate altered cellular characteristics as compared to cells derived from asymptomatic women (control-HVCs). Using computer-controllable Flexcell stretch unit, we examined whether POP-HVCs react differently to mechanical loading as compared to control-HVCs by the expression of extracellular matrix (ECM) components, cell-ECM adhesion proteins, and ECM degrading and maturating enzymes. Vaginal tissue biopsies from premenopausal patients with Pelvic Organ Prolapse Quantification System stage ≥3 (n = 8) and asymptomatic controls (n = 7) were collected during vaginal hysterectomy or repair. Human vaginal cells were isolated by enzymatic digestion, seeded on collagen (COLI)-coated plates, and stretched (24 hours, 25% elongation). Total RNA was extracted, and 84 genes were screened using Human ECM and Adhesion Molecules polymerase chain reaction array; selected genes were verified by quantitative reverse transcription-polymerase chain reaction. Stretch-conditioned media (SCM) were collected and analyzed by protein array, immunoblotting, and zymography. In mechanically stretched control-HVCs, transcript levels of integrins (ITGA1, ITGA4, ITGAV, and ITGB1) and matrix metalloproteinases (MMPs) 2, 8, and 13 were downregulated (P < .05); in POP-HVCs, MMP1, MMP3, and MMP10, ADAMTS8 and 13, tissue inhibitor of metalloproteinases (TIMPs) 1 to 3, ITGA2, ITGA4, ITGA6, ITGB1, contactin (CNTN1), catenins (A1 and B1), and laminins (A3 and C1) were significantly upregulated, whereas COLs (1, 4, 5, 6, 11, and 12) and LOXL1 were downregulated. Human vaginal cells massively secrete MMPs and TIMPs proteins; MMP1, MMP8, MMP9 protein expression and MMP2 gelatinase activity were increased, whereas TIMP2 decreased in SCM from POP-HVCs compared to control-HVCs. Primary human vaginal cells derived from women with severe pelvic organ prolapse and control-HVCs react differentially to in vitro

An Analytical Model for Determining Two-Dimensional Receptor-Ligand Kinetics

PubMed Central

Cheung, Luthur Siu-Lun; Konstantopoulos, Konstantinos

2011-01-01

Cell-cell adhesive interactions play a pivotal role in major pathophysiological vascular processes, such as inflammation, infection, thrombosis, and cancer metastasis, and are regulated by hemodynamic forces generated by blood flow. Cell adhesion is mediated by the binding of receptors to ligands, which are both anchored on two-dimensional (2-D) membranes of apposing cells. Biophysical assays have been developed to determine the unstressed (no-force) 2-D affinity but fail to disclose its dependence on force. Here we develop an analytical model to estimate the 2-D kinetics of diverse receptor-ligand pairs as a function of force, including antibody-antigen, vascular selectin-ligand, and bacterial adhesin-ligand interactions. The model can account for multiple bond interactions necessary to mediate adhesion and resist detachment amid high hemodynamic forces. Using this model, we provide a generalized biophysical interpretation of the counterintuitive force-induced stabilization of cell rolling observed by a select subset of receptor-ligand pairs with specific intrinsic kinetic properties. This study enables us to understand how single-molecule and multibond biophysics modulate the macroscopic cell behavior in diverse pathophysiological processes. PMID:21575567

Interactions between Skeletal Muscle Myoblasts and their Extracellular Matrix Revealed by a Serum Free Culture System.

PubMed

Chaturvedi, Vishal; Dye, Danielle E; Kinnear, Beverley F; van Kuppevelt, Toin H; Grounds, Miranda D; Coombe, Deirdre R

2015-01-01

Decellularisation of skeletal muscle provides a system to study the interactions of myoblasts with muscle extracellular matrix (ECM). This study describes the efficient decellularisation of quadriceps muscle with the retention of matrix components and the use of this matrix for myoblast proliferation and differentiation under serum free culture conditions. Three decellularisation approaches were examined; the most effective was phospholipase A2 treatment, which removed cellular material while maximizing the retention of ECM components. Decellularised muscle matrices were then solubilized and used as substrates for C2C12 mouse myoblast serum free cultures. The muscle matrix supported myoblast proliferation and differentiation equally as well as collagen and fibronectin. Immunofluorescence analyses revealed that myoblasts seeded on muscle matrix and fibronectin differentiated to form long, well-aligned myotubes, while myoblasts seeded on collagen were less organized. qPCR analyses showed a time dependent increase in genes involved in skeletal muscle differentiation and suggested that muscle-derived matrix may stimulate an increased rate of differentiation compared to collagen and fibronectin. Decellularized whole muscle three-dimensional scaffolds also supported cell adhesion and spreading, with myoblasts aligning along specific tracts of matrix proteins within the scaffolds. Thus, under serum free conditions, intact acellular muscle matrices provided cues to direct myoblast adhesion and migration. In addition, myoblasts were shown to rapidly secrete and organise their own matrix glycoproteins to create a localized ECM microenvironment. This serum free culture system has revealed that the correct muscle ECM facilitates more rapid cell organisation and differentiation than single matrix glycoprotein substrates.

LGR5 receptor promotes cell-cell adhesion in stem cells and colon cancer cells via the IQGAP1-Rac1 pathway.

PubMed

Carmon, Kendra S; Gong, Xing; Yi, Jing; Wu, Ling; Thomas, Anthony; Moore, Catherine M; Masuho, Ikuo; Timson, David J; Martemyanov, Kirill A; Liu, Qingyun J

2017-09-08

Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/β-catenin signaling in vitro ; however, deletion of LGR5 in stem cells has little or no effect on Wnt/β-catenin signaling or cell proliferation in vivo Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell-cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell-cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell-cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1-Rac1 pathway to strengthen cell-cell adhesion in normal adult crypt stem cells and colon cancer cells. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host-Cell Interaction.

PubMed

Gil-Bona, Ana; Reales-Calderon, Jose A; Parra-Giraldo, Claudia M; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

2016-01-01

Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a "veil growth," never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain.

Epigenetic regulation of integrin-linked kinase expression depending on adhesion of gastric carcinoma cells.

PubMed

Kim, Yong-Bae; Lee, Sung-Yul; Ye, Sang-Kyu; Lee, Jung Weon

2007-02-01

Cell adhesion to the extracellular matrix (ECM) regulates gene expressions in diverse dynamic environments. However, the manner in which gene expressions are regulated by extracellular cues is largely unknown. In this study, suspended gastric carcinoma cells showed higher basal and transforming growth factor-beta1 (TGFbeta1)-mediated acetylations of histone 3 (H3) and Lys(9) of H3 and levels of integrin-linked kinase (ILK) mRNA and protein than did fibronectin-adherent cells did. Moreover, the insignificant acetylation and ILK expression in adherent cells were recovered by alterations of integrin signaling and actin organization, indicating a connection between cytoplasmic and nuclear changes. Higher acetylations in suspended cells were correlated with associations between Smad4, p300/CBP, and Lys(9)-acetylated H3. Meanwhile, adherent cells showed more associations between HDAC3, Ski, and MeCP2. Chromatin immunoprecipitations with anti-acetylated H3, Lys(9)-acetylated H3, or p300/CBP antibody resulted in more coprecipitated ILK promoter, correlated with enhanced ILK mRNA and protein levels, in suspended cells. Moreover, ILK expression inversely regulated cell adhesion to ECM proteins, and its overexpression enhanced cell growth in soft agar. These observations indicate that cell adhesion and/or its related molecular basis regulate epigenetic mechanisms leading to a loss of ILK transcription, which in turn regulates cell adhesion property in a feedback linkage.

Characterization of static adhesion of human platelets in plasma to protein surfaces in microplates.

PubMed

Eriksson, Andreas C; Whiss, Per A

2009-04-01

Platelet adhesion is a complex and important event for prevention of blood loss after vessel injury. This study investigated fundamental adhesive mechanisms occurring in an in-vitro assay developed for the measurement of static adhesion of human platelets in plasma. The aim was to gain methodological knowledge that could be used for interpretations of results from other studies using this specific assay. Involvement of adhesive receptors was investigated by the use of various antibodies as well as therapeutic drugs (abciximab, eptifibatide and tirofiban). Inhibitors of adenosine 5'-diphosphate receptors (cangrelor, MRS2179) and of thromboxane A(2) signalling (BM-531) were used to estimate the role of autocrine activation. Adhesion to collagen was found to be mainly mediated by alpha(2)beta(1) and to some extent by alpha(IIb)beta(3). Adhesion to fibrinogen was mediated by alpha(IIb)beta(3). In addition, adenosine 5'-diphosphate-induced adhesion to albumin was dependent on alpha(IIb)beta(3). Furthermore, experiments with cangrelor and BM-531 showed that the majority of the adhesive interactions tested were dependent on adenosine 5'-diphosphate or thromboxane A(2). We conclude that the mechanisms of adhesion measured by the static platelet adhesion assay are in accordance with the current knowledge regarding platelet activation and adhesion. Despite its simplicity, we suggest that this adhesion assay could be used as a screening device for the study of the influence of various surfaces and soluble substances on platelet adhesion.

Roles of specific membrane lipid domains in EGF receptor activation and cell adhesion molecule stabilization in a developing olfactory system.

PubMed

Gibson, Nicholas J; Tolbert, Leslie P; Oland, Lynne A

2009-09-29

Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-beta-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons.

Roles of Specific Membrane Lipid Domains in EGF Receptor Activation and Cell Adhesion Molecule Stabilization in a Developing Olfactory System

PubMed Central

Gibson, Nicholas J.; Tolbert, Leslie P.; Oland, Lynne A.

2009-01-01

Background Reciprocal interactions between glial cells and olfactory receptor neurons (ORNs) cause ORN axons entering the brain to sort, to fasciculate into bundles destined for specific glomeruli, and to form stable protoglomeruli in the developing olfactory system of an experimentally advantageous animal species, the moth Manduca sexta. Epidermal growth factor receptors (EGFRs) and the cell adhesion molecules (IgCAMs) neuroglian and fasciclin II are known to be important players in these processes. Methodology/Principal Findings We report in situ and cell-culture studies that suggest a role for glycosphingolipid-rich membrane subdomains in neuron-glia interactions. Disruption of these subdomains by the use of methyl-β-cyclodextrin results in loss of EGFR activation, depletion of fasciclin II in ORN axons, and loss of neuroglian stabilization in the membrane. At the cellular level, disruption leads to aberrant ORN axon trajectories, small antennal lobes, abnormal arrays of olfactory glomerul, and loss of normal glial cell migration. Conclusions/Significance We propose that glycosphingolipid-rich membrane subdomains (possible membrane rafts or platforms) are essential for IgCAM-mediated EGFR activation and for anchoring of neuroglian to the cytoskeleton, both required for normal extension and sorting of ORN axons. PMID:19787046

Nano-anisotropic surface coating based on drug immobilized pendant polymer to suppress macrophage adhesion response.

PubMed

Kaladhar, K; Renz, H; Sharma, C P

2015-04-01

Exploring drug molecules for material design, to harness concepts of nano-anisotropy and ligand-receptor interactions, are rather elusive. The aim of this study is to demonstrate the bottom-up design of a single-step and bio-interactive polymeric surface coating, based on drug based pendant polymer. This can be applied on to polystyrene (PS) substrates, to suppress macrophage adhesion and spreading. The drug molecule is used in this coating for two purposes. The first one is drug as a "pendant" group, to produce nano-anisotropic properties that can enable adhesion of the coatings to the substrate. The second purpose is to use the drug as a "ligand", to produce ligand-receptor interaction, between the bound ligand and receptors of albumin, to develop a self-albumin coat over the surface, by the preferential binding of albumin in biological environment, to reduce macrophage adhesion. Our in silico studies show that, diclofenac (DIC) is an ideal drug based "ligand" for albumin. This can also act as a "pendant" group with planar aryl groups. The combination of these two factors can help to harness, both nano-anisotropic properties and biological functions to the polymeric coating. Further, the drug, diclofenac (DIC) is immobilized to the polyvinyl alcohol (PVA), to develop the pendant polymer (PVA-DIC). The interaction of bound DIC with the albumin is a ligand-receptor based interaction, as per the studies by circular dichroism, differential scanning calorimetry, and SDS-PAGE. The non-polar π-π* interactions are regulating; the interactions between PVA bound DIC-DIC interactions, leading to "nano-anisotropic condensation" to form distinct "nano-anisotropic segments" inside the polymeric coating. This is evident from, the thermo-responsiveness and uniform size of nanoparticles, as well as regular roughness in the surface coating, with similar properties as that of nanoparticles. In addition, the hydrophobic DIC-polystyrene (PS) interactions, between the PVA

Stress relaxing hyaluronic acid-collagen hydrogels promote cell spreading, fiber remodeling, and focal adhesion formation in 3D cell culture.

PubMed

Lou, Junzhe; Stowers, Ryan; Nam, Sungmin; Xia, Yan; Chaudhuri, Ovijit

2018-02-01

The physical and architectural cues of the extracellular matrix (ECM) play a critical role in regulating important cellular functions such as spreading, migration, proliferation, and differentiation. Natural ECM is a complex viscoelastic scaffold composed of various distinct components that are often organized into a fibrillar microstructure. Hydrogels are frequently used as synthetic ECMs for 3D cell culture, but are typically elastic, due to covalent crosslinking, and non-fibrillar. Recent work has revealed the importance of stress relaxation in viscoelastic hydrogels in regulating biological processes such as spreading and differentiation, but these studies all utilize synthetic ECM hydrogels that are non-fibrillar. Key mechanotransduction events, such as focal adhesion formation, have only been observed in fibrillar networks in 3D culture to date. Here we present an interpenetrating network (IPN) hydrogel system based on HA crosslinked with dynamic covalent bonds and collagen I that captures the viscoelasticity and fibrillarity of ECM in tissues. The IPN hydrogels exhibit two distinct processes in stress relaxation, one from collagen and the other from HA crosslinking dynamics. Stress relaxation in the IPN hydrogels can be tuned by modulating HA crosslinker affinity, molecular weight of the HA, or HA concentration. Faster relaxation in the IPN hydrogels promotes cell spreading, fiber remodeling, and focal adhesion (FA) formation - behaviors often inhibited in other hydrogel-based materials in 3D culture. This study presents a new, broadly adaptable materials platform for mimicking key ECM features of viscoelasticity and fibrillarity in hydrogels for 3D cell culture and sheds light on how these mechanical and structural cues regulate cell behavior. Copyright © 2017 Elsevier Ltd. All rights reserved.

The coxsackievirus-adenovirus receptor reveals complex homophilic and heterophilic interactions on neural cells.

PubMed

Patzke, Christopher; Max, Klaas E A; Behlke, Joachim; Schreiber, Jadwiga; Schmidt, Hannes; Dorner, Armin A; Kröger, Stephan; Henning, Mechthild; Otto, Albrecht; Heinemann, Udo; Rathjen, Fritz G

2010-02-24

The coxsackievirus-adenovirus receptor (CAR) is a member of the Ig superfamily strongly expressed in the developing nervous system. Our histological investigations during development reveal an initial uniform distribution of CAR on all neural cells with a concentration on membranes that face the margins of the nervous system (e.g., the basal laminae and the ventricular side). At more advanced stages, CAR becomes downregulated and restricted to specific regions including areas rich in axonal and dendritic surfaces. To study the function of CAR on neural cells, we used the fiber knob of the adenovirus, extracellular CAR domains, blocking antibodies to CAR, as well as CAR-deficient neural cells. Blocking antibodies were found to inhibit neurite extension in retina organ and retinal explant cultures, whereas the application of the recombinant fiber knob of the adenovirus subtype Ad2 or extracellular CAR domains promoted neurite extension and adhesion to extracellular matrices. We observed a promiscuous interaction of CAR with extracellular matrix glycoproteins, which was deduced from analytical ultracentrifugation experiments, affinity chromatography, and adhesion assays. The membrane proximal Ig domain of CAR, termed D2, was found to bind to a fibronectin fragment, including the heparin-binding domain 2, which promotes neurite extension of wild type, but not of CAR-deficient neural cells. In contrast to heterophilic interactions, homophilic association of CAR involves both Ig domains, as was revealed by ultracentrifugation, chemical cross-linking, and adhesion studies. The results of these functional and binding studies are correlated to a U-shaped homodimer of the complete extracellular domains of CAR detected by x-ray crystallography.

The Molecular Architecture of Cell Adhesion: Dynamic Remodeling Revealed by Videonanoscopy.

PubMed

Sergé, Arnauld

2016-01-01

The plasma membrane delimits the cell, which is the basic unit of living organisms, and is also a privileged site for cell communication with the environment. Cell adhesion can occur through cell-cell and cell-matrix contacts. Adhesion proteins such as integrins and cadherins also constitute receptors for inside-out and outside-in signaling within proteolipidic platforms. Adhesion molecule targeting and stabilization relies on specific features such as preferential segregation by the sub-membrane cytoskeleton meshwork and within membrane proteolipidic microdomains. This review presents an overview of the recent insights brought by the latest developments in microscopy, to unravel the molecular remodeling occurring at cell contacts. The dynamic aspect of cell adhesion was recently highlighted by super-resolution videomicroscopy, also named videonanoscopy. By circumventing the diffraction limit of light, nanoscopy has allowed the monitoring of molecular localization and behavior at the single-molecule level, on fixed and living cells. Accessing molecular-resolution details such as quantitatively monitoring components entering and leaving cell contacts by lateral diffusion and reversible association has revealed an unexpected plasticity. Adhesion structures can be highly specialized, such as focal adhesion in motile cells, as well as immune and neuronal synapses. Spatiotemporal reorganization of adhesion molecules, receptors, and adaptors directly relates to structure/function modulation. Assembly of these supramolecular complexes is continuously balanced by dynamic events, remodeling adhesions on various timescales, notably by molecular conformation switches, lateral diffusion within the membrane and endo/exocytosis. Pathological alterations in cell adhesion are involved in cancer evolution, through cancer stem cell interaction with stromal niches, growth, extravasation, and metastasis.

The conveyor belt hypothesis for thymocyte migration: participation of adhesion and de-adhesion molecules.

PubMed

Villa-Verde, D M; Calado, T C; Ocampo, J S; Silva-Monteiro, E; Savino, W

1999-05-01

Thymocyte differentiation is the process by which bone marrow-derived precursors enter the thymus, proliferate, rearrange the genes and express the corresponding T cell receptors, and undergo positive and/or negative selection, ultimately yielding mature T cells that will represent the so-called T cell repertoire. This process occurs in the context of cell migration, whose cellular and molecular basis is still poorly understood. Kinetic studies favor the idea that these cells leave the organ in an ordered pattern, as if they were moving on a conveyor belt. We have recently proposed that extracellular matrix glycoproteins, such as fibronectin, laminin and type IV collagen, among others, produced by non-lymphoid cells both in the cortex and in the medulla, would constitute a macromolecular arrangement allowing differentiating thymocytes to migrate. Here we discuss the participation of both molecules with adhesive and de-adhesive properties in the intrathymic T cell migration. Functional experiments demonstrated that galectin-3, a soluble beta-galactoside-binding lectin secreted by thymic microenvironmental cells, is a likely candidate for de-adhesion proteins by decreasing thymocyte interaction with the thymic microenvironment.

Tumor necrosis factor alpha (TNF-alpha)-induced cell adhesion to human endothelial cells is under dominant control of one TNF receptor type, TNF-R55

PubMed Central

1993-01-01

Tumor necrosis factor alpha (TNF-alpha) is a pleiotropic cytokine triggering cell responses through two distinct membrane receptors. Stimulation of leukocyte adhesion to the endothelium is one of the many TNF-alpha activities and is explained by the upregulation of adhesion molecules on the endothelial cell surface. Human umbilical vein endothelial cells (HUVEC) were isolated, cultured, and demonstrated to express both TNF receptor types, TNF-R55 and TNF-R75. Cell adhesion to HUVEC was studied using the HL60, U937, and MOLT-4 cell lines. HUVEC were activated by either TNF-alpha, binding to both TNF-R55 and TNF- R75, and by receptor type-specific agonists, binding exclusively to TNF- R55 or to TNF-R75. The TNF-alpha-induced cell adhesion to HUVEC was found to be controlled almost exclusively by TNF-R55. This finding correlated with the exclusive activity of TNF-R55 in the TNF-alpha- dependent regulation of the expression of the intercellular adhesion molecule type 1 (ICAM-1), E-selectin, and vascular cell adhesion molecule type 1 (VCAM-1). The CD44 adhesion molecule in HUVEC was also found to be upregulated through TNF-R55. However, both TNF-R55 and TNF- R75 upregulate alpha 2 integrin expression in HUVEC. The predominant role of TNF-R55 in TNF-alpha-induced adhesion in HUVEC may correlate with its specific control of NF-kappa B activation, since kappa B elements are known to be present in ICAM-1, E-selectin, and VCAM-1 gene regulatory sequences. PMID:8386742

A fibronectin receptor on Candida albicans mediates adherence of the fungus to extracellular matrix

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

Klotz, S.A.; Smith, R.L.

1991-03-01

Binding of fibronectin, an extracellular matrix (ECM) protein, to Candida albicans was measured, and adherence of the fungus to immobilized ECM proteins, fibronectin, laminin, types I and IV collagen, and subendothelial ECM was studied. 125I-labeled fibronectin was inhibited from binding to the fungus by unlabeled human plasma fibronectin and by Arg-Gly-Asp (RGD), Gly-Arg-Gly-Glu-Ser-Pro (GRGESP), and Gly-Arg-Gly-Asp-Thr-Pro (GRGDTP), but binding was not inhibited by Gly-Arg-Gly-Asp-Ser-Pro. Soluble fibronectin, RGD, GRGESP, and GRGDTP also inhibited fungal adherence to the individual immobilized ECM proteins in a complex pattern, but only soluble fibronectin (10(-7) M) inhibited fungal adherence to subendothelial ECM. Thus, C. albicans possessesmore » at least one type of cell surface receptor for binding soluble fibronectin that can be inhibited with peptides. This receptor apparently is used to bind the fungus to immobilized ECM proteins and to subendothelial ECM and may play a role in the initiation of disseminated disease by bloodborne fungi by providing for adherence of the microorganisms to ECM proteins.« less

MMPs-Mediated ECM Remodeling

PubMed

Mali, Aniket V; Joshi, Asavari A; Hegde, Mahabaleshwar V; Kadam, Shivajirao S

2017-04-01

Background: To enhance their own survival, tumor cells can manipulate their microenvironment through remodeling of the extra cellular matrix (ECM). The urokinase-type plasminogen activator (uPA) system catalyzes plasmin production which further mediates activation of matrix metalloproteinases (MMPs) and plays an important role in breast cancer invasion and metastasis through ECM remodeling. This provides a potential target for therapeutic intervention of breast cancer treatment. Enterolactone (EL) is derived from dietary flax lignans in the human body and is known to have anti-breast cancer activity. We here investigated molecular and cellular mechanisms of EL action on the uPA-plasmin- MMPs system. Methods: MTT and trypan blue dye exclusion assays, anchorage-dependent clonogenic assays and wound healing assays were carried out to study effects on cell proliferation and viability, clonogenicity and migration capacity, respectively. Real-time PCR was employed to study gene expression and gelatin zymography was used to assess MMP-2 and MMP-9 activities. All data were statistically analysed and presented as mean ± SEM values. Results: All the findings collectively demonstrated anticancer and antimetastatic potential of EL with antiproliferative, antimigratory and anticlonogenic cellular mechanisms. EL was found to exhibit multiple control of plasmin activation by down-regulating uPA expression and also up-regulating its natural inhibitor, PAI-1, at the mRNA level. Further, EL was found to down-regulate expression of MMP-2 and MMP-9 genes, and up-regulate TIMP-1 and TIMP-2; natural inhibitors of MMP-2 and MMP-9, respectively. This may be as a consequence of inhibition of plasmin activation, resulting in robust control over migration and invasion of breast cancer cells during metastasis. Conclusions: EL suppresses proliferation, migration and metastasis of MDA-MB-231 breast cancer cells by inhibiting induced ECM remodeling by the ‘uPA-plasmin-MMPs system’. Creative

Role of surface layer collagen binding protein from indigenous Lactobacillus plantarum 91 in adhesion and its anti-adhesion potential against gut pathogen.

PubMed

Yadav, Ashok Kumar; Tyagi, Ashish; Kaushik, Jai Kumar; Saklani, Asha Chandola; Grover, Sunita; Batish, Virender Kumar

2013-12-14

Human feacal isolates were ascertain as genus Lactobacillus using specific primer LbLMA1/R16-1 and further identified as Lactobacillus plantarum with species specific primers Lpl-3/Lpl-2. 25 L. plantarum strains were further assessed for hydrophobicity following the microbial adhesion to hydrocarbons (MATH) method and colonization potentials based on their adherence to immobilized human collagen type-1. Surface proteins were isolated from selected L. plantarum 91(Lp91) strain. The purified collagen binding protein (Cbp) protein was assessed for its anti-adhesion activity against enteric Escherichia coli 0157:H7 pathogen on immobilized collagen. Four L. plantarum strains displayed high degree of hydrophobicity and significant adhesion to collagen. A 72 kDa protein was purified which reduced 59.71% adhesion of E. coli 0157:H7 on immobilized collagen as compared to control well during adhesion assay. Cbp protein is the major influencing factor in inhibition of E. coli 0157:H7 adhesion with extracellular matrix (ECM) components. Hydrophobicity and adhesion potential are closely linked attributes precipitating in better colonization potential of the lactobacillus strains. Cbp is substantiated as a crucial surface protein contributing in adhesion of lactobacillus strains. The study can very well be the platform for commercialization of indigenous probiotic strain once their functional attributes are clinically explored. Copyright © 2013 Elsevier GmbH. All rights reserved.

SU-F-SPS-08: Measuring the Interaction Of DDR Cell Receptors and Extracellular Matrix Collagen in Prostate Cells

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

Dong, J; Sarkar, A; Hoffmann, P

Purpose: Discoidin domain receptors (DDR) have recently been recognized as important players in cancer progression. DDRs are cell receptors that interact with collagen, an extracellular matrix (ECM) protein. However the detailed mechanism of their interaction is unclear. Here we attempted to examine their interaction in terms of structural (surface topography), mechanical (rupture force), and kinetic (binding probability) information on the single molecular scale with the use of atomic force microscopy (AFM). Methods: The Quantitative Nano-mechanical property Mapping (QNM) mode of AFM allowed to assess the cells in liquid growth media at their optimal physiological while being viable. Human benign prostatemore » hyperplasia (BPH-1) cell line was genetically regulated to suppress DDR expression (DDR- cells) and was compared with naturally DDR expressing cells (DDR+). Results: Binding force measurements (n = 1000) were obtained before and after the two groups were treated with fibronectin (FN), an integrin-inhibiting antibody to block the binding of integrin. The quantification indicates that cells containing DDR bind with collagen at a most probable force of 80.3–83.0 ±7.6 pN. The probability of them binding is 0.167 when other interactions (mainly due to integrin-collagen binding) are minimized. Conclusion: Together with further force measurements at different pulling speeds will determine dissociation rate, binding distance and activation barrier. These parameters in benign cells provides some groundwork in understanding DDR’s behavior in various cell microenvironments such as in malignant tumor cells. Funding supported by Richard Barber Interdisciplinary Research Program of Wayne State University.« less

Aberrant methylation patterns affect the molecular pathogenesis of rheumatoid arthritis.

PubMed

Lin, Yang; Luo, Zhengqiang

2017-05-01

This study aims to investigate DNA methylation signatures in fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis (RA), and to explore the relationship with transcription factors (TFs) that help to distinguish RA from osteoarthritis (OA). Microarray dataset of GSE46346, including six FLS samples from patients with RA and five FLS samples from patients with OA, was downloaded from the Gene Expression Omnibus database. RA and OA samples were screened for differentially methylated loci (DMLs). The corresponding differentially methylated genes (DMGs) were identified, followed by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) enrichment analysis. A transcriptional regulatory network was built with TFs and their corresponding DMGs. Overall, 280 hypomethylated loci and 561 hypermethylated loci were screened. Genes containing hypermethylated loci were enriched in pathways in cancer, ECM-receptor interaction, focal adhesion and neurotrophin signaling pathways. Genes containing hypomethylated loci were enriched in the neurotrophin signaling pathway. Moreover, we found that CCCTC-binding factor (CTCF), Yin Yang 1 (YY1), v-myc avian myelocytomatosis viral oncogene homolog (c-MYC), and early growth response 1 (EGR1) were important TFs in the transcriptional regulatory network. Therefore, DMGs might participate in the neurotrophin signaling pathway, pathways in cancer, ECM-receptor interaction and focal adhesion pathways in RA. Furthermore, CTCF, c-MYC, YY1, and EGR1 may play important roles in RA through regulating DMGs. Copyright © 2017 Elsevier B.V. All rights reserved.

Dentin extracellular matrix (ECM) proteins: comparison to bone ECM and contribution to dynamics of dentinogenesis.

PubMed

Butler, William T; Brunn, Jan C; Qin, Chunlin

2003-01-01

Dentinogenesis involves the initial odontoblastic synthesis of a collagen-rich extracellular matrix (ECM) and predentin that is converted to dentin when the collagen fibrils become mineralized. Since the width of predentin is rather uniform, we postulate that extracellular events regulate dentinogenesis. Similarly, osteogenesis involves an initial unmineralized osteoid that is mineralized and converted to bone. To gain insights into these two processes, we compared ECM proteins in bone with those in dentin, focusing upon the sialic acid (SA)-rich proteins. We observed qualitative similarities between the SA-rich proteins, but distinct differences in the amounts of osteopontin (OPN) and dentin sialoprotein (DSP). OPN, a predominant protein in bone, was found in much smaller amounts in dentin. Conversely, DSP was abundant in dentin ECM, but found sparingly in bone. Molecular cloning experiments indicate that coding sequences for DSP and dentin phosphoprotein (DPP) are found on the same mRNA. We believe that the initial form of the precursor protein DSPP is inactive in influencing the mineralization process and that it must be activated by cleavage of peptide bonds in conserved regions. Thus, unknown proteinases would act on DSPP, possibly at the mineralization front, and liberate active DPP, which plays an initiation and regulatory role in the formation of apatite crystals. This post-translational processing reaction would represent an important control point in dentinogenesis. Recently, we identified uncleaved DSPP in dentin extracts, which should allow us to test portions of our hypothesis.

Decellularized heart ECM hydrogel using supercritical carbon dioxide for improved angiogenesis.

PubMed

Seo, Yoojin; Jung, Youngmee; Kim, Soo Hyun

2018-02-01

Initial angiogenesis within the first 3 days is critical for healing ischemic diseases such as myocardial infarction. Recently, decellularized extracellular matrix (dECM) has been reported to provide tissue-derived ECM components and can be used as a scaffold for cell delivery for angiogenesis in tissue engineering. Decellularization by various detergents such as sodium dodecyl sulfate (SDS) and triton X-100 can remove the cell nuclei in tissue organs. However, this leads to ECM structure denaturation, decreased presence of various ECM proteins and cytokines, and loss of mechanical properties. To overcome these limitations, in this study, we developed a supercritical carbon dioxide and ethanol co-solvent (scCO 2 -EtOH) decellularization method, which is a detergent-free system that prevents ECM structure disruption and retains various angiogenic proteins in the heart dECM, and tested on rat heart tissues. The heart tissue was placed into the scCO 2 reactor and decellularized at 37 °C and 350 bar. After scCO 2 -EtOH treatment, the effects were evaluated by DNA, collagen, and glycosaminoglycan (GAG) quantification and hematoxylin and eosin and immunofluorescence staining to determine the absence of nucleic acids and preservation of heart ECM components. Similar to the native group, the scCO 2 -EtOH group contained more ECM components such as collagen, GAGs, collagen I, laminin, and fibronectin and angiogenic factors including vascular endothelial growth factor, fibroblast growth factor, and platelet-derived growth factor and others in comparison to the detergent group. In addition, to estimate angiogenesis of the dECM hydrogels, the neutralized dECM solution was injected in a rat subcutaneous layer (n = 6 in each group: collagen, scCO 2 -EOH, and detergent group), after which the solution naturally formed gelation in the subcutaneous layer. After 3 days, the gels were harvested and estimated by immunofluorescence staining and the ImageJ program for

Measuring two-dimensional receptor-ligand binding kinetics by micropipette.

PubMed Central

Chesla, S E; Selvaraj, P; Zhu, C

1998-01-01

We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2. PMID:9726957

Single-cell force spectroscopy as a technique to quantify human red blood cell adhesion to subendothelial laminin.

PubMed

Maciaszek, Jamie L; Partola, Kostyantyn; Zhang, Jing; Andemariam, Biree; Lykotrafitis, George

2014-12-18

Single-cell force spectroscopy (SCFS), an atomic force microscopy (AFM)-based assay, enables quantitative study of cell adhesion while maintaining the native state of surface receptors in physiological conditions. Human healthy and pathological red blood cells (RBCs) express a large number of surface proteins which mediate cell-cell interactions, or cell adhesion to the extracellular matrix. In particular, RBCs adhere with high affinity to subendothelial matrix laminin via the basal cell adhesion molecule and Lutheran protein (BCAM/Lu). Here, we established SCFS as an in vitro technique to study human RBC adhesion at baseline and following biochemical treatment. Using blood obtained from healthy human subjects, we recorded adhesion forces from single RBCs attached to AFM cantilevers as the cell was pulled-off of substrates coated with laminin protein. We found that an increase in the overall cell adhesion measured via SCFS is correlated with an increase in the resultant total force measured on 1 µm(2) areas of the RBC membrane. Further, we showed that SCFS can detect significant changes in the adhesive response of RBCs to modulation of the cyclic adenosine monophosphate (cAMP) and protein kinase A (PKA) pathway. Lastly, we identified variability in the RBC adhesion force to laminin amongst the human subjects, suggesting that RBCs maintain diverse levels of active BCAM/Lu adhesion receptors. By using single-cell measurements, we established a powerful new method for the quantitative measurement of single RBC adhesion with specific receptor-mediated binding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Regulation of T-lymphocyte motility, adhesion and de-adhesion by a cell surface mechanism directed by low density lipoprotein receptor-related protein 1 and endogenous thrombospondin-1.

PubMed

Talme, Toomas; Bergdahl, Eva; Sundqvist, Karl-Gösta

2014-06-01

T lymphocytes are highly motile and constantly reposition themselves between a free-floating vascular state, transient adhesion and migration in tissues. The regulation behind this unique dynamic behaviour remains unclear. Here we show that T cells have a cell surface mechanism for integrated regulation of motility and adhesion and that integrin ligands and CXCL12/SDF-1 influence motility and adhesion through this mechanism. Targeting cell surface-expressed low-density lipoprotein receptor-related protein 1 (LRP1) with an antibody, or blocking transport of LRP1 to the cell surface, perturbed the cell surface distribution of endogenous thrombospondin-1 (TSP-1) while inhibiting motility and potentiating cytoplasmic spreading on intercellular adhesion molecule 1 (ICAM-1) and fibronectin. Integrin ligands and CXCL12 stimulated motility and enhanced cell surface expression of LRP1, intact TSP-1 and a 130,000 MW TSP-1 fragment while preventing formation of a de-adhesion-coupled 110 000 MW TSP-1 fragment. The appearance of the 130 000 MW TSP-1 fragment was inhibited by the antibody that targeted LRP1 expression, inhibited motility and enhanced spreading. The TSP-1 binding site in the LRP1-associated protein, calreticulin, stimulated adhesion to ICAM-1 through intact TSP-1 and CD47. Shear flow enhanced cell surface expression of intact TSP-1. Hence, chemokines and integrin ligands up-regulate a dominant motogenic pathway through LRP1 and TSP-1 cleavage and activate an associated adhesion pathway through the LRP1-calreticulin complex, intact TSP-1 and CD47. This regulation of T-cell motility and adhesion makes pro-adhesive stimuli favour motile responses, which may explain why T cells prioritize movement before permanent adhesion.

The Cell Wall Protein Ecm33 of Candida albicans is Involved in Chronological Life Span, Morphogenesis, Cell Wall Regeneration, Stress Tolerance, and Host–Cell Interaction

PubMed Central

Gil-Bona, Ana; Reales-Calderon, Jose A.; Parra-Giraldo, Claudia M.; Martinez-Lopez, Raquel; Monteoliva, Lucia; Gil, Concha

2016-01-01

Ecm33 is a glycosylphosphatidylinositol-anchored protein in the human pathogen Candida albicans. This protein is known to be involved in fungal cell wall integrity (CWI) and is also critical for normal virulence in the mouse model of hematogenously disseminated candidiasis, but its function remains unknown. In this work, several phenotypic analyses of the C. albicans ecm33/ecm33 mutant (RML2U) were performed. We observed that RML2U displays the inability of protoplast to regenerate the cell wall, activation of the CWI pathway, hypersensitivity to temperature, osmotic and oxidative stresses and a shortened chronological lifespan. During the exponential and stationary culture phases, nuclear and actin staining revealed the possible arrest of the cell cycle in RML2U cells. Interestingly, a “veil growth,” never previously described in C. albicans, was serendipitously observed under static stationary cells. The cells that formed this structure were also observed in cornmeal liquid cultures. These cells are giant, round cells, without DNA, and contain large vacuoles, similar to autophagic cells observed in other fungi. Furthermore, RML2U was phagocytozed more than the wild-type strain by macrophages at earlier time points, but the damage caused to the mouse cells was less than with the wild-type strain. Additionally, the percentage of RML2U apoptotic cells after interaction with macrophages was fewer than in the wild-type strain. PMID:26870022

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps

PubMed Central

Aufschnaiter, Roland; Zamir, Evan A.; Little, Charles D.; Özbek, Suat; Münder, Sandra; David, Charles N.; Li, Li; Sarras, Michael P.; Zhang, Xiaoming

2011-01-01

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra ‘tissue movements’ are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues. PMID:22194305

In vivo imaging of basement membrane movement: ECM patterning shapes Hydra polyps.

PubMed

Aufschnaiter, Roland; Zamir, Evan A; Little, Charles D; Özbek, Suat; Münder, Sandra; David, Charles N; Li, Li; Sarras, Michael P; Zhang, Xiaoming

2011-12-01

Growth and morphogenesis during embryonic development, asexual reproduction and regeneration require extensive remodeling of the extracellular matrix (ECM). We used the simple metazoan Hydra to examine the fate of ECM during tissue morphogenesis and asexual budding. In growing Hydra, epithelial cells constantly move towards the extremities of the animal and into outgrowing buds. It is not known, whether these tissue movements involve epithelial migration relative to the underlying matrix or whether cells and ECM are displaced as a composite structure. Furthermore, it is unclear, how the ECM is remodeled to adapt to the shape of developing buds and tentacles. To address these questions, we used a new in vivo labeling technique for Hydra collagen-1 and laminin, and tracked the fate of ECM in all body regions of the animal. Our results reveal that Hydra 'tissue movements' are largely displacements of epithelial cells together with associated ECM. By contrast, during the evagination of buds and tentacles, extensive movement of epithelial cells relative to the matrix is observed, together with local ECM remodeling. These findings provide new insights into the nature of growth and morphogenesis in epithelial tissues.

Biological and MRI characterization of biomimetic ECM scaffolds for cartilage tissue regeneration.

PubMed

Ravindran, Sriram; Kotecha, Mrignayani; Huang, Chun-Chieh; Ye, Allen; Pothirajan, Padmabharathi; Yin, Ziying; Magin, Richard; George, Anne

2015-12-01

Osteoarthritis is the most common joint disorder affecting millions of people. Most scaffolds developed for cartilage regeneration fail due to vascularization and matrix mineralization. In this study we present a chondrogenic extracellular matrix (ECM) incorporated collagen/chitosan scaffold (chondrogenic ECM scaffold) for potential use in cartilage regenerative therapy. Biochemical characterization showed that these scaffolds possess key pro-chondrogenic ECM components and growth factors. MRI characterization showed that the scaffolds possess mechanical properties and diffusion characteristics important for cartilage tissue regeneration. In vivo implantation of the chondrogenic ECM scaffolds with bone marrow derived mesenchymal stem cells (MSCs) triggered chondrogenic differentiation of the MSCs without the need for external stimulus. Finally, results from in vivo MRI experiments indicate that the chondrogenic ECM scaffolds are stable and possess MR properties on par with native cartilage. Based on our results, we envision that such ECM incorporated scaffolds have great potential in cartilage regenerative therapy. Additionally, our validation of MR parameters with histology and biochemical analysis indicates the ability of MRI techniques to track the progress of our ECM scaffolds non-invasively in vivo; highlighting the translatory potential of this technology. Copyright © 2015 Elsevier Ltd. All rights reserved.

PROLACTIN-INDUCED TYROSINE PHOSPHORYLATION, ACTIVATION AND RECEPTOR ASSOCIATION OF FOCAL ADHESION KINASE (FAK) IN MAMMARY EPITHELIAL CELLS

EPA Science Inventory

Prolactin-Induced Tyrosine Phosphorylation, Activation and Receptor
Association of Focal Adhesion Kinase (FAK) in Mammary Epithelial Cells.
Suzanne E. Fenton1 and Lewis G. Sheffield2. 1U.S. Environmental Protection
Agency, MD-72, Research Triangle Park, NC 27711, and

Biological functionality and mechanistic contribution of extracellular matrix-ornamented three dimensional Ti-6Al-4V mesh scaffolds.

PubMed

Kumar, A; Nune, K C; Misra, R D K

2016-11-01

The 3D printed metallic implants are considered bioinert in nature because of the absence of bioactive molecules. Thus, surface modification of bioinert materials is expected to favorably promote osteoblast functions and differentiation. In this context, the objective of this study is to fundamentally elucidate the effect of cell-derived decellularized extracellular matrix (dECM) ornamented 3D printed Ti-6Al-4V scaffolds on biological functions, involving cell adhesion, proliferation, and synthesis of vinculin and actin proteins. To mimic the natural ECM environment, the mineralized ECM of osteoblasts was deposited on the Ti-6Al-4V porous scaffolds, fabricated by electron beam melting (EBM) method. The process comprised of osteoblast proliferation, differentiation, and freeze-thaw cycles to obtain decellularized extra cellular matrix (dECM), in vitro. The dECM provided a natural environment to restore the natural cell functionality of osteoblasts that were cultured on dECM ornamented Ti-6Al-4V scaffolds. In comparison to the bare Ti-6Al-4V scaffolds, a higher cell functionality such as cell adhesion, proliferation, and growth including cell-cell and cell-material interaction were observed on dECM ornamented Ti-6Al-4V scaffolds, which were characterized by using markers for focal adhesion and cytoskeleton such as vinculin and actin. Moreover, electron microscopy also indicated higher cell-material interaction and enhanced proliferation of cells on dECM ornamented Ti-6Al-4V scaffolds, supported by MTT assay. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2751-2763, 2016. © 2016 Wiley Periodicals, Inc.

New Tricks for “Old” Domains: How Novel Architectures and Promiscuous Hubs Contributed to the Organization and Evolution of the ECM

PubMed Central

Cromar, Graham; Wong, Ka-Chun; Loughran, Noeleen; On, Tuan; Song, Hongyan; Xiong, Xuejian; Zhang, Zhaolei; Parkinson, John

2014-01-01

The extracellular matrix (ECM) is a defining characteristic of metazoans and consists of a meshwork of self-assembling, fibrous proteins, and their functionally related neighbours. Previous studies, focusing on a limited number of gene families, suggest that vertebrate complexity predominantly arose through the duplication and subsequent modification of retained, preexisting ECM genes. These genes provided the structural underpinnings to support a variety of specialized tissues, as well as a platform for the organization of spatio-temporal signaling and cell migration. However, the relative contributions of ancient versus novel domains to ECM evolution have not been quantified across the full range of ECM proteins. Here, utilizing a high quality list comprising 324 ECM genes, we reveal general and clade-specific domain combinations, identifying domains of eukaryotic and metazoan origin recruited into new roles in approximately two-third of the ECM proteins in humans representing novel vertebrate proteins. We show that, rather than acquiring new domains, sampling of new domain combinations has been key to the innovation of paralogous ECM genes during vertebrate evolution. Applying a novel framework for identifying potentially important, noncontiguous, conserved arrangements of domains, we find that the distinct biological characteristics of the ECM have arisen through unique evolutionary processes. These include the preferential recruitment of novel domains to existing architectures and the utilization of high promiscuity domains in organizing the ECM network around a connected array of structural hubs. Our focus on ECM proteins reveals that distinct types of proteins and/or the biological systems in which they operate have influenced the types of evolutionary forces that drive protein innovation. This emphasizes the need for rigorously defined systems to address questions of evolution that focus on specific systems of interacting proteins. PMID:25323955

Mapping cell surface adhesion by rotation tracking and adhesion footprinting

NASA Astrophysics Data System (ADS)

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

2017-03-01

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

Opioid receptor trafficking and interaction in nociceptors

PubMed Central

Zhang, X; Bao, L; Li, S

2015-01-01

Opiate analgesics such as morphine are often used for pain therapy. However, antinociceptive tolerance and dependence may develop with long-term use of these drugs. It was found that μ-opioid receptors can interact with δ-opioid receptors, and morphine antinociceptive tolerance can be reduced by blocking δ-opioid receptors. Recent studies have shown that μ- and δ-opioid receptors are co-expressed in a considerable number of small neurons in the dorsal root ganglion. The interaction of μ-opioid receptors with δ-opioid receptors in the nociceptive afferents is facilitated by the stimulus-induced cell-surface expression of δ-opioid receptors, and contributes to morphine tolerance. Further analysis of the molecular, cellular and neural circuit mechanisms that regulate the trafficking and interaction of opioid receptors and related signalling molecules in the pain pathway would help to elucidate the mechanism of opiate analgesia and improve pain therapy. LINKED ARTICLES This article is part of a themed section on Opioids: New Pathways to Functional Selectivity. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2015.172.issue-2 PMID:24611685

ADAM12 induction by Twist1 promotes tumor invasion and metastasis via regulation of invadopodia and focal adhesions

PubMed Central

Eckert, Mark A.; Santiago-Medina, Miguel; Lwin, Thinzar M.; Kim, Jihoon; Courtneidge, Sara A.

2017-01-01

ABSTRACT The Twist1 transcription factor promotes tumor invasion and metastasis by inducing epithelial–mesenchymal transition (EMT) and invadopodia-mediated extracellular matrix (ECM) degradation. The critical transcription targets of Twist1 for mediating these events remain to be uncovered. Here, we report that Twist1 strongly induces expression of a disintegrin and metalloproteinase 12 (ADAM12). We observed that the expression levels of Twist1 mRNA and ADAM12 mRNA are tightly correlated in human breast tumors. Knocking down ADAM12 blocked cell invasion in a 3D mammary organoid culture. Suppression of ADAM12 also inhibited Twist1-induced tumor invasion and metastasis in human breast tumor xenografts, without affecting primary tumor formation. Mechanistically, knockdown of ADAM12 in breast cancer cells significantly reduced invadopodia formation and matrix degradation, and simultaneously increased overall cell adhesion to the ECM. Live-imaging analysis showed that knockdown of ADAM12 significantly inhibited focal adhesion turnover. Mechanistically, both the disintegrin and metalloproteinase domains of ADAM12 are required for its function at invadopodia, whereas the metalloproteinase domain is dispensable for its function at focal adhesions. Taken together, these data suggest that ADAM12 plays a crucial role in tumor invasion and metastasis by regulating both invadopodia and focal adhesions. PMID:28468988

Adhesion mechanisms in embryogenesis and in cancer invasion and metastasis.

PubMed

Thiery, J P; Boyer, B; Tucker, G; Gavrilovic, J; Valles, A M

1988-01-01

Cell-substratum and cell-cell adhesion mechanisms contribute to the development of animal form. The adhesive status of embryonic cells has been analysed during epithelial-mesenchymal cell interconversion and in cell migrations. Clear-cut examples of the modulation of cell adhesion molecules (CAMs) have been described at critical periods of morphogenesis. In chick embryos the three primary CAMs (N-CAM. L-CAM and N-cadherin) present early in embryogenesis are expressed later in a defined pattern during morphogenesis and histogenesis. The axial mesoderm derived from gastrulating cells expresses increasing amounts of N-cadherin and N-CAM. During metamerization these two adhesion molecules become abundant at somitic cell surfaces. Both CAMs are functional in an in vitro aggregation assay; however, the calcium-dependent adhesion molecule N-cadherin is more sensitive to perturbation by specific antibodies. Neural crest cells which separate from the neural epithelium lose their primary CAMs in a defined time-sequence. Adhesion to fibronectins via specific surface receptors becomes a predominant interaction during the migratory process, while some primary and secondary CAMs are expressed de novo during the ontogeny of the peripheral nervous system. In vitro, different fibronectin functional domains have been identified in the attachment, spreading and migration of neural crest cells. The fibronectin receptors which transduce the adhesive signals play a key role in the control of cell movement. All these results have prompted us to examine whether similar mechanisms operate in carcinoma cell invasion and metastasis. In vitro, rat bladder transitional carcinoma cells convert reversibly into invasive mesenchymal cells. A rapid modulation of adhesive properties is found during the epithelial-mesenchymal carcinoma cell interconversion. The different model systems analysed demonstrate that a limited repertoire of adhesion molecules, expressed in a well-defined spatiotemporal

Impaired P2X1 Receptor-Mediated Adhesion in Eosinophils from Asthmatic Patients.

PubMed

Wright, Adam; Mahaut-Smith, Martyn; Symon, Fiona; Sylvius, Nicolas; Ran, Shaun; Bafadhel, Mona; Muessel, Michelle; Bradding, Peter; Wardlaw, Andrew; Vial, Catherine

2016-06-15

Eosinophils play an important role in the pathogenesis of asthma and can be activated by extracellular nucleotides released following cell damage or inflammation. For example, increased ATP concentrations were reported in bronchoalveolar lavage fluids of asthmatic patients. Although eosinophils are known to express several subtypes of P2 receptors for extracellular nucleotides, their function and contribution to asthma remain unclear. In this article, we show that transcripts for P2X1, P2X4, and P2X5 receptors were expressed in healthy and asthmatic eosinophils. The P2X receptor agonist α,β-methylene ATP (α,β-meATP; 10 μM) evoked rapidly activating and desensitizing inward currents (peak 18 ± 3 pA/pF at -60 mV) in healthy eosinophils, typical of P2X1 homomeric receptors, which were abolished by the selective P2X1 antagonist NF449 (1 μM) (3 ± 2 pA/pF). α,β-meATP-evoked currents were smaller in eosinophils from asthmatic patients (8 ± 2 versus 27 ± 5 pA/pF for healthy) but were enhanced following treatment with a high concentration of the nucleotidase apyrase (17 ± 5 pA/pF for 10 IU/ml and 11 ± 3 pA/pF for 0.32 IU/ml), indicating that the channels are partially desensitized by extracellular nucleotides. α,β-meATP (10 μM) increased the expression of CD11b activated form in eosinophils from healthy, but not asthmatic, donors (143 ± 21% and 108 ± 11% of control response, respectively). Furthermore, α,β-meATP increased healthy (18 ± 2% compared with control 10 ± 1%) but not asthmatic (13 ± 1% versus 10 ± 0% for control) eosinophil adhesion. Healthy human eosinophils express functional P2X1 receptors whose activation leads to eosinophil αMβ2 integrin-dependent adhesion. P2X1 responses are constitutively reduced in asthmatic compared with healthy eosinophils, probably as the result of an increase in extracellular nucleotide concentration. Copyright © 2016 by The American Association of Immunologists, Inc.

Leptospira Immunoglobulin-Like Protein B Interacts with the 20th Exon of Human Tropoelastin Contributing to Leptospiral Adhesion to Human Lung Cells

PubMed Central

Hsieh, Ching-Lin; Tseng, Andrew; He, Hongxuan; Kuo, Chih-Jung; Wang, Xuannian; Chang, Yung-Fu

2017-01-01

Leptospira immunoglobulin-like protein B (LigB), a surface adhesin, is capable of mediating the attachment of pathogenic leptospira to the host through interaction with various components of the extracellular matrix (ECM). Human tropoelastin (HTE), the building block of elastin, confers resilience and elasticity to lung, and other tissues. Previously identified Ig-like domains of LigB, including LigB4 and LigB12, bind to HTE, which is likely to promote Leptospira adhesion to lung tissue. However, the molecular mechanism that mediates the LigB-HTE interaction is unclear. In this study, the LigB-binding site on HTE was further pinpointed to a N-terminal region of the 20th exon of HTE (HTE20N). Alanine mutants of basic and aromatic residues on HTE20N significantly reduced binding to the LigB. Additionally, HTE-binding site was narrowed down to the first β-sheet of LigB12. On this binding surface, residues F1054, D1061, A1065, and D1066 were critical for the association with HTE. Most importantly, the recombinant HTE truncates could diminish the binding of LigB to human lung fibroblasts (WI-38) by 68%, and could block the association of LigA-expressing L. biflexa to lung cells by 61%. These findings should expand our understanding of leptospiral pathogenesis, particularly in pulmonary manifestations of leptospirosis. PMID:28536676

Peptides Derived from a Phage Display Library Inhibit Adhesion and Protect the Host against Infection by Paracoccidioides brasiliensis and Paracoccidioides lutzii

PubMed Central

de Oliveira, Haroldo C.; Michaloski, Jussara S.; da Silva, Julhiany F.; Scorzoni, Liliana; de Paula e Silva, Ana C. A.; Marcos, Caroline M.; Assato, Patrícia A.; Yamazaki, Daniella S.; Fusco-Almeida, Ana M.; Giordano, Ricardo J.; Mendes-Giannini, Maria J. S.

2016-01-01

Paracoccidioides brasiliensis and Paracoccidioides lutzii are dimorphic fungi and are the etiological agents of paracoccidioidomycosis (PCM). Adhesion is one of the most important steps in infections with Paracoccidioides and is responsible for the differences in the virulence of isolates of these fungi. Because of the importance of adhesion to the establishment of an infection, this study focused on the preliminary development of a new therapeutic strategy to inhibit adhesion by Paracoccidioides, thus inhibiting infection and preventing the disease. We used two phage display libraries to select peptides that strongly bind to the Paracoccidioides cell wall to inhibit adhesion to host cells and extracellular matrix (ECM) components (laminin, fibronectin, and type I and type IV collagen). This approach allowed us to identify four peptides that inhibited up to 64% of the adhesion of Paracoccidioides to pneumocytes in vitro and inhibited the adhesion to the ECM components by up to 57%. Encouraged by these results, we evaluated the ability of these peptides to protect Galleria mellonella from Paracoccidioides infection by treating G. mellonella larvae with the different peptides prior to infection with Paracoccidioides and observing larval survival. The results show that all of the peptides tested increased the survival of the larvae infected with P. brasiliensis by up to 64% and by up to 60% in those infected with P. lutzii. These data may open new horizons for therapeutic strategies to prevent PCM, and anti-adhesion therapy could be an important strategy. PMID:28066254

Isolation of integrin-based adhesion complexes.

PubMed

Jones, Matthew C; Humphries, Jonathan D; Byron, Adam; Millon-Frémillon, Angélique; Robertson, Joseph; Paul, Nikki R; Ng, Daniel H J; Askari, Janet A; Humphries, Martin J

2015-03-02

The integration of cells with their extracellular environment is facilitated by cell surface adhesion receptors, such as integrins, which play important roles in both normal development and the onset of pathologies. Engagement of integrins with their ligands in the extracellular matrix, or counter-receptors on other cells, initiates the intracellular assembly of a wide variety of proteins into adhesion complexes such as focal contacts, focal adhesions, and fibrillar adhesions. The proteins recruited to these complexes mediate bidirectional signaling across the plasma membrane, and, as such, help to coordinate and/or modulate the multitude of physical and chemical signals to which the cell is subjected. The protocols in this unit describe two approaches for the isolation or enrichment of proteins contained within integrin-associated adhesion complexes, together with their local plasma membrane/cytosolic environments, from cells in culture. In the first protocol, integrin-associated adhesion structures are affinity isolated using microbeads coated with extracellular ligands or antibodies. The second protocol describes the isolation of ventral membrane preparations that are enriched for adhesion complex structures. The protocols permit the determination of adhesion complex components via subsequent downstream analysis by western blotting or mass spectrometry. Copyright © 2015 John Wiley & Sons, Inc.

Gold nanoparticles for cancer detection and treatment: The role of adhesion

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

Oni, Y.; Department of Mechanical and Aerospace Engineering, Princeton University, Princeton, New Jersey 08544; Hao, K.

2014-02-28

This paper presents the results of an experimental study of the effects of adhesion between gold nanoparticles and surfaces that are relevant to the potential applications in cancer detection and treatment. Adhesion is measured using a dip coating/atomic force microscopy (DC/AFM) technique. The adhesion forces are obtained for dip-coated gold nanoparticles that interact with peptide or antibody-based molecular recognition units (MRUs) that attach specifically to breast cancer cells. They include MRUs that attach specifically to receptors on breast cancer cells. Adhesion forces between anti-cancer drugs such as paclitaxel, and the constituents of MRU-conjugated Au nanoparticle clusters, are measured using forcemore » microscopy techniques. The implications of the results are then discussed for the design of robust gold nanoparticle clusters and for potential applications in localized drug delivery and hyperthermia.« less

Patterned layers of adsorbed extracellular matrix proteins: influence on mammalian cell adhesion.

PubMed

Dupont-Gillain, C C; Alaerts, J A; Dewez, J L; Rouxhet, P G

2004-01-01

Three patterned systems aiming at the control of mammalian cell behavior are presented. The determinant feature common to these systems is the spatial distribution of extracellular matrix (ECM) proteins (mainly collagen) on polymer substrates. This distribution differs from one system to another with respect to the scale at which it is affected, from the supracellular to the supramolecular scale, and with respect to the way it is produced. In the first system, the surface of polystyrene was oxidized selectively to form micrometer-scale patterns, using photolithography. Adsorption of ECM proteins in presence of a competitor was enhanced on the oxidized domains, allowing selective cell adhesion to be achieved. In the second system, electron beam lithography was used to engrave grooves (depth and width approximately 1 microm) on a poly(methyl methacrylate) (PMMA) substratum. No modification of the surface chemistry associated to the created topography could be detected. Cell orientation along the grooves was only observed when collagen was preadsorbed on the substratum. In the third system, collagen adsorbed on PMMA was dried in conditions ensuring the formation of a nanometer-scale pattern. Cell adhesion was enhanced on such patterned collagen layers compared to smooth collagen layers.

Associations of chemo- and radio-resistant phenotypes with the gap junction, adhesion and extracellular matrix in a three-dimensional culture model of soft sarcoma.

PubMed

Bai, Chujie; Yang, Min; Fan, Zhengfu; Li, Shu; Gao, Tian; Fang, Zhiwei

2015-06-10

Three-dimensional (3D) culture models are considered to recapitulate the cell microenvironment in solid tumors, including the extracellular matrix (ECM), cell-cell interactions, and signal transduction. These functions are highly correlated with cellular behaviors and contribute to resistances against chemo- and radio-therapies. However, the biochemical effects and mechanisms remain unknown in soft sarcoma. Therefore, we developed an in vitro 3D model of sarcoma to analyze the reasons of the chemo- and radio-resistance in therapies. Four soft sarcoma cell lines, HT1080, RD, SW872, and human osteosarcoma cell line 1 (HOSS1), a cell line established from a patient-derived xenograft, were applied to 3D culture and treated with growth factors in methylcellulose-containing medium. Spheroids were examined morphologically and by western blotting, RT-qPCR, and immunofluorescence staining to analyze cell adhesion, gap junctions, ECM genes, and related factors. Proliferation and colony formation assays were performed to assess chemo- and radio-resistances between 3D and two-dimensional (2D) cell cultures. Annexin V and Propidium Iodide staining was used to detect early apoptotic sarcoma cells treated with Doxorubicin, Gemcitabine, and Docetaxel in the 3D model. The four soft sarcoma cell lines formed spheres in vitro by culture in modified condition medium. Compared with 2D cell culture, expression of ECM genes and proteins, including COL1A1, LOX, SED1, FN1, and LAMA4, was significantly increased in 3D culture. Analysis of cadherin and gap junction molecules showed significant changes in the gene and protein expression profiles under 3D conditions. These changes affected cell-cell communication and were mainly associated with biological processes such as cell proliferation and apoptosis related to chemo- and radio-resistances. Our findings revealed significant differences between 3D and 2D cell culture systems, and indicated that cellular responsiveness to external stress

Critical Role of Transient Activity of MT1-MMP for ECM Degradation in Invadopodia

PubMed Central

Watanabe, Ayako; Hosino, Daisuke; Koshikawa, Naohiko; Seiki, Motoharu; Suzuki, Takashi; Ichikawa, Kazuhisa

2013-01-01

Focal degradation of extracellular matrix (ECM) is the first step in the invasion of cancer cells. MT1-MMP is a potent membrane proteinase employed by aggressive cancer cells. In our previous study, we reported that MT1-MMP was preferentially located at membrane protrusions called invadopodia, where MT1-MMP underwent quick turnover. Our computer simulation and experiments showed that this quick turnover was essential for the degradation of ECM at invadopodia (Hoshino, D., et al., (2012) PLoS Comp. Biol., 8: e1002479). Here we report on characterization and analysis of the ECM-degrading activity of MT1-MMP, aiming at elucidating a possible reason for its repetitive insertion in the ECM degradation. First, in our computational model, we found a very narrow transient peak in the activity of MT1-MMP followed by steady state activity. This transient activity was due to the inhibition by TIMP-2, and the steady state activity of MT1-MMP decreased dramatically at higher TIMP-2 concentrations. Second, we evaluated the role of the narrow transient activity in the ECM degradation. When the transient activity was forcibly suppressed in computer simulations, the ECM degradation was heavily suppressed, indicating the essential role of this transient peak in the ECM degradation. Third, we compared continuous and pulsatile turnover of MT1-MMP in the ECM degradation at invadopodia. The pulsatile insertion showed basically consistent results with the continuous insertion in the ECM degradation, and the ECM degrading efficacy depended heavily on the transient activity of MT1-MMP in both models. Unexpectedly, however, low-frequency/high-concentration insertion of MT1-MMP was more effective in ECM degradation than high-frequency/low-concentration pulsatile insertion even if the time-averaged amount of inserted MT1-MMP was the same. The present analysis and characterization of ECM degradation by MT1-MMP together with our previous report indicate a dynamic nature of MT1-MMP at

Simulated Microgravity Alters Actin Cytoskeleton and Integrin-Mediated Focal Adhesions of Cultured Human Mesenchymal Stromal Cells

NASA Astrophysics Data System (ADS)

Gershovich, P. M.; Gershovic, J. G.; Buravkova, L. B.

2008-06-01

Cytoskeletal alterations occur in several cell types including lymphocytes, glial cells, and osteoblasts, during spaceflight and under simulated microgravity (SMG) (3, 4). One potential mechanism for cytoskeletal gravisensitivity is disruption of extracellular matrix (ECM) and integrin interactions. Focal adhesions are specialized sites of cell-matrix interaction composed of integrins and the diversity of focal adhesion-associated cytoplasmic proteins including vinculin, talin, α-actinin, and actin filaments (4, 5). Integrins produce signals essential for proper cellular function, survival and differentiation. Therefore, we investigated the effects of SMG on F-actin cytoskeleton structure, vinculin focal adhesions, expression of some integrin subtypes and cellular adhesion molecules (CAMs) in mesenchymal stem cells derived from human bone marrow (hMSCs). Simulated microgravity was produced by 3D-clinostat (Dutch Space, Netherlands). Staining of actin fibers with TRITC-phalloidin showed reorganization even after 30 minutes of simulated microgravity. The increasing of cells number with abnormal F-actin was observed after subsequent terms of 3D-clinorotation (6, 24, 48, 120 hours). Randomization of gravity vector altered dimensional structure of stress fibers and resulted in remodeling of actin fibers inside the cells. In addition, we observed vinculin redistribution inside the cells after 6 hours and prolonged terms of clinorotation. Tubulin fibers in a contrast with F-actin and vinculin didn't show any reorganization even after long 3Dclinorotation (120 hours). The expression of integrin α2 increased 1,5-6-fold in clinorotated hMSCs. Also we observed decrease in number of VCAM-1-positive cells and changes in expression of ICAM-1. Taken together, our findings indicate that SMG leads to microfilament and adhesion alterations of hMSCs most probably associated with involvement of some integrin subtypes.

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells

PubMed Central

Hunt, Geoffrey C.; Singh, Purva; Schwarzbauer, Jean E.

2012-01-01

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. PMID:22710062

Endogenous production of fibronectin is required for self-renewal of cultured mouse embryonic stem cells.

PubMed

Hunt, Geoffrey C; Singh, Purva; Schwarzbauer, Jean E

2012-09-10

Pluripotent cells are attached to the extracellular matrix (ECM) as they make cell fate decisions within the stem cell niche. Here we show that the ubiquitous ECM protein fibronectin is required for self-renewal decisions by cultured mouse embryonic stem (mES) cells. Undifferentiated mES cells produce fibronectin and assemble a fibrillar matrix. Increasing the level of substrate fibronectin increased cell spreading and integrin receptor signaling through focal adhesion kinase, while concomitantly inducing the loss of Nanog and Oct4 self-renewal markers. Conversely, reducing fibronectin production by mES cells growing on a feeder-free gelatin substrate caused loss of cell adhesion, decreased integrin signaling, and decreased expression of self-renewal markers. These effects were reversed by providing the cells with exogenous fibronectin, thereby restoring adhesion to the gelatin substrate. Interestingly, mES cells do not adhere directly to the gelatin substrate, but rather adhere indirectly through gelatin-bound fibronectin, which facilitates self-renewal via its effects on cell adhesion. These results provide new insights into the mechanism of regulation of self-renewal by growth on a gelatin-coated surface. The effects of increasing or decreasing fibronectin levels show that self-renewal depends on an intermediate level of cell-fibronectin interactions. By providing cell adhesive signals that can act with other self-renewal factors to maintain mES cell pluripotency, fibronectin is therefore a necessary component of the self-renewal signaling pathway in culture. Copyright © 2012 Elsevier Inc. All rights reserved.

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

PubMed Central

Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

2008-01-01

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

Carbohydrate Coating Reduces Adhesion of Biofilm-Forming Bacillus subtilis to Gold Surfaces

PubMed Central

Kesel, S.; Mader, A.; Seeberger, P. H.; Lieleg, O.

2014-01-01

The growth of bacterial biofilms in pipes and food tanks causes severe problems in industry. Biofilms growing on medical implants or catheters are of great concern, as they can cause serious infections and decrease the functionality of the medical device. The prevention of bacterial adhesion—the first step in colonization and biofilm formation—is therefore very important. Current research comprises alterations in surface properties, the prevention of adhesin biosynthesis, inhibition with receptor analogs, or the development of anti-adhesive vaccines. We present a new approach that allows us to study bacterial adhesion with high sensitivity in real-time while testing several different surfaces in parallel. Using the cantilever-array technique we demonstrate that coating of gold surfaces with mono- or disaccharides results in a reduction of the bacterial adhesion of the biofilm-forming bacterium Bacillus subtilis NCIB 3610 to these gold surfaces. This reduction in bacterial adhesion is independent of the studied carbohydrate. Using several mutant strains, we investigate the underlying molecular interactions, and our results suggest that adhesion to gold surfaces is mediated by thiol groups present in proteins of the bacterial cell membrane or biofilm matrix proteins expressed at low levels by the wild-type strain. Furthermore, our data indicate that the adhesion of B. subtilis NCIB 3610 to carbohydrate-coated gold surfaces is facilitated by interactions between carbohydrates installed on the cantilever gold surface and an exopolysaccharide expressed by this strain. Understanding general and specific contributions of molecular interactions mediating bacterial adhesion will enable its prevention in the future. PMID:25038098

Proteinase-activated receptors (PARs) – focus on receptor-receptor-interactions and their physiological and pathophysiological impact

PubMed Central

2013-01-01

Proteinase-activated receptors (PARs) are a subfamily of G protein-coupled receptors (GPCRs) with four members, PAR1, PAR2, PAR3 and PAR4, playing critical functions in hemostasis, thrombosis, embryonic development, wound healing, inflammation and cancer progression. PARs are characterized by a unique activation mechanism involving receptor cleavage by different proteinases at specific sites within the extracellular amino-terminus and the exposure of amino-terminal “tethered ligand“ domains that bind to and activate the cleaved receptors. After activation, the PAR family members are able to stimulate complex intracellular signalling networks via classical G protein-mediated pathways and beta-arrestin signalling. In addition, different receptor crosstalk mechanisms critically contribute to a high diversity of PAR signal transduction and receptor-trafficking processes that result in multiple physiological effects. In this review, we summarize current information about PAR-initiated physical and functional receptor interactions and their physiological and pathological roles. We focus especially on PAR homo- and heterodimerization, transactivation of receptor tyrosine kinases (RTKs) and receptor serine/threonine kinases (RSTKs), communication with other GPCRs, toll-like receptors and NOD-like receptors, ion channel receptors, and on PAR association with cargo receptors. In addition, we discuss the suitability of these receptor interaction mechanisms as targets for modulating PAR signalling in disease. PMID:24215724

An easily accessible sulfated saccharide mimetic inhibits in vitro human tumor cell adhesion and angiogenesis of vascular endothelial cells

PubMed Central

Marano, Grazia; Gronewold, Claas; Frank, Martin; Merling, Anette; Kliem, Christian; Sauer, Sandra; Wiessler, Manfred; Frei, Eva

2012-01-01

Summary Oligosaccharides aberrantly expressed on tumor cells influence processes such as cell adhesion and modulation of the cell’s microenvironment resulting in an increased malignancy. Schmidt’s imidate strategy offers an effective method to synthesize libraries of various oligosaccharide mimetics. With the aim to perturb interactions of tumor cells with extracellular matrix proteins and host cells, molecules with 3,4-bis(hydroxymethyl)furan as core structure were synthesized and screened in biological assays for their abilities to interfere in cell adhesion and other steps of the metastatic cascade, such as tumor-induced angiogenesis. The most active compound, (4-{[(β-D-galactopyranosyl)oxy]methyl}furan-3-yl)methyl hydrogen sulfate (GSF), inhibited the activation of matrix-metalloproteinase-2 (MMP-2) as well as migration of the human melanoma cells of the lines WM-115 and WM-266-4 in a two-dimensional migration assay. GSF inhibited completely the adhesion of WM-115 cells to the extracellular matrix (ECM) proteins, fibrinogen and fibronectin. In an in vitro angiogenesis assay with human endothelial cells, GSF very effectively inhibited endothelial tubule formation and sprouting of blood vessels, as well as the adhesion of endothelial cells to ECM proteins. GSF was not cytotoxic at biologically active concentrations; neither were 3,4-bis{[(β-D-galactopyranosyl)oxy]methyl}furan (BGF) nor methyl β-D-galactopyranoside nor 3,4-bis(hydroxymethyl)furan, which were used as controls, eliciting comparable biological activity. In silico modeling experiments, in which binding of GSF to the extracellular domain of the integrin αvβ3 was determined, revealed specific docking of GSF to the same binding site as the natural peptidic ligands of this integrin. The sulfate in the molecule coordinated with one manganese ion in the binding site. These studies show that this chemically easily accessible molecule GSF, synthesized in three steps from 3,4-bis

A feedback mechanism converts individual cell features into a supracellular ECM structure in Drosophila trachea

PubMed Central

Öztürk-Çolak, Arzu; Moussian, Bernard; Araújo, Sofia J; Casanova, Jordi

2016-01-01

The extracellular matrix (ECM), a structure contributed to and commonly shared by many cells in an organism, plays an active role during morphogenesis. Here, we used the Drosophila tracheal system to study the complex relationship between the ECM and epithelial cells during development. We show that there is an active feedback mechanism between the apical ECM (aECM) and the apical F-actin in tracheal cells. Furthermore, we reveal that cell-cell junctions are key players in this aECM patterning and organisation and that individual cells contribute autonomously to their aECM. Strikingly, changes in the aECM influence the levels of phosphorylated Src42A (pSrc) at cell junctions. Therefore, we propose that Src42A phosphorylation levels provide a link for the ECM environment to ensure proper cytoskeletal organisation. DOI: http://dx.doi.org/10.7554/eLife.09373.001 PMID:26836303

Toll-like receptor (TLR)-1/2 triggering of multiple myeloma cells modulates their adhesion to bone marrow stromal cells and enhances bortezomib-induced apoptosis.

PubMed

Abdi, Jahangir; Mutis, Tuna; Garssen, Johan; Redegeld, Frank A

2014-01-01

In multiple myeloma (MM), the malignant plasma cells usually localize to the bone marrow where they develop drug resistance due to adhesion to stromal cells and various environmental signals. Hence, modulation of this interaction is expected to influence drug sensitivity of MM cells. Toll-like receptor (TLR) ligands have displayed heterogeneous effects on B-cell malignancies and also on MM cells in a few recent studies, but effects on adhesion and drug sensitivity of myeloma cells in the context of bone marrow stromal cells (BMSCs) have never been investigated. In the present study, we explored the modulatory effects of TLR1/2 ligand (Pam3CSK4) on adhesion of human myeloma cells to BMSCs. It is shown that TLR1/2 triggering has opposite effects in different HMCLs on their adhesion to BMSCs. Fravel, L363, UM-6, UM-9 and U266 showed increased adhesion to BMSC in parallel with an increased surface expression of integrin molecules α4 and αVβ3. OPM-1, OPM-2 and NCI-H929 showed a dose-dependent decrease in adhesion upon TLR activation following a downregulation of β7 integrin expression. Importantly, TLR1/2 triggering increased cytotoxic and apoptotic effects of bortezomib in myeloma cells independent of the effect on stromal cell adhesion. Moreover, the apoptosis-enhancing effect of Pam3CSK4 paralleled induction of cleaved caspase-3 protein in FACS analysis suggesting a caspase-dependent mechanism. Our findings uncover a novel role of TLR activation in MM cells in the context of bone marrow microenvironment. Stimulation of TLR1/2 bypasses the protective shield of BMSCs and may be an interesting strategy to enhance drug sensitivity of multiple myeloma cells.

Metabotropic Glutamate Receptors and Interacting Proteins in Epileptogenesis

PubMed Central

Qian, Feng; Tang, Feng-Ru

2016-01-01

Neurotransmitter and receptor systems are involved in different neurological and neuropsychological disorders such as Parkinson's disease, depression, Alzheimer’s disease and epilepsy. Recent advances in studies of signal transduction pathways or interacting proteins of neurotransmitter receptor systems suggest that different receptor systems may share the common signal transduction pathways or interacting proteins which may be better therapeutic targets for development of drugs to effectively control brain diseases. In this paper, we reviewed metabotropic glutamate receptors (mGluRs) and their related signal transduction pathways or interacting proteins in status epilepticus and temporal lobe epilepsy, and proposed some novel therapeutical drug targets for controlling epilepsy and epileptogenesis. PMID:27030135

A neurokinin 1 receptor antagonist decreases postoperative peritoneal adhesion formation and increases peritoneal fibrinolytic activity.

PubMed

Reed, Karen L; Fruin, A Brent; Gower, Adam C; Stucchi, Arthur F; Leeman, Susan E; Becker, James M

2004-06-15

Fibrous adhesions remain a major sequela of abdominal surgery. The proinflammatory peptide substance P (SP), known to participate in inflammatory events, may play a key role in adhesion formation. This hypothesis was tested by using an antagonist, CJ-12,255 (Pfizer), that blocks the binding of SP to the neurokinin 1 receptor (NK-1R). Adhesion formation was surgically induced in the peritoneum of rats receiving daily doses of the NK-1R antagonist (NK-1RA; 5.0 or 10.0 mg/kg per day) or saline. On postoperative day 7, both the low and high doses of NK-1RA significantly (P < 0.05) reduced adhesion formation by 45% and 53%, respectively, compared with controls. Subsequently, the effect of NK-1RA administration on peritoneal fibrinolytic activity was investigated to determine a potential mechanism for SP action in the peritoneum. Samples were collected from nonoperated controls and from animals 24 h postsurgery that were administered either NK-1RA or saline. Fibrinolytic activity in peritoneal fluid was assayed by zymography, and expression of tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1, both regulators of fibrinolytic activity, was assessed in peritoneal tissue and fluid by RT-PCR and bioassay, respectively. NK-1RA administration led to a marked (P < 0.05) increase in tPA mRNA levels in peritoneal tissue compared with nonoperated and saline-administered animals. Likewise, NK-1RA administration significantly (P < 0.05) increased tPA in the peritoneal fluid. These data suggest that activation of the NK-1R promotes peritoneal adhesion formation by limiting fibrinolytic activity in the postoperative peritoneum, thus enabling fibrinous adhesions to persist.

ECM1 regulates tumor metastasis and CSC-like property through stabilization of β-catenin.

PubMed

Lee, K-m; Nam, K; Oh, S; Lim, J; Kim, R K; Shim, D; Choi, J-h; Lee, S-J; Yu, J-H; Lee, J W; Ahn, S H; Shin, I

2015-12-10

Extracellular Matrix Protein 1 (ECM1) is a marker for tumorigenesis and is correlated with invasiveness and poor prognosis in various types of cancer. However, the functional role of ECM1 in cancer metastasis is unclear. Here, we detected high ECM1 level in breast cancer patient sera that was associated with recurrence of tumor. The modulation of ECM1 expression affected not only cell migration and invasion, but also sphere-forming ability and drug resistance in breast cancer cell lines. In addition, ECM1 regulated the gene expression associated with the epithelial to mesenchymal transition (EMT) progression and cancer stem cell (CSC) maintenance. Interestingly, ECM1 increased β-catenin expression at the post-translational level through induction of MUC1, which was physically associated with β-catenin. Indeed, the association between β-catenin and the MUC1 cytoplasmic tail was increased by ECM1. Furthermore, forced expression of β-catenin altered the gene expression that potentiated EMT progression and CSC phenotype maintenance in the cells. These data provide evidence that ECM1 has an important role in cancer metastasis through β-catenin stabilization.

Pharmacological endothelin receptor interaction does not occur in veins from ET(B) receptor deficient rats.

PubMed

Thakali, Keshari; Galligan, James J; Fink, Gregory D; Gariepy, Cheryl E; Watts, Stephanie W

2008-07-01

Heterodimerization of G-protein coupled receptors can alter receptor pharmacology. ET A and ET B receptors heterodimerize when co-expressed in heterologous expression lines. We hypothesized that ET A and ET B receptors heterodimerize and pharmacologically interact in vena cava from wild-type (WT) but not ET B receptor deficient (sl/sl) rats. Pharmacological endothelin receptor interaction was assessed by comparing ET-1-induced contraction in rings of rat thoracic aorta and thoracic vena cava from male Sprague Dawley rats under control conditions, ET A receptor blockade (atrasentan, 10 nM), ET B receptor blockade (BQ-788, 100 nM) or ET B receptor desensitization (Sarafotoxin 6c, 100 nM) and ET A plus ET B receptor blockade or ET A receptor blockade plus ET B receptor desensitization. In addition, similar pharmacological ET receptor antagonism experiments were performed in rat thoracic aorta and vena cava from WT and sl/sl rats. ET A but not ET B receptor blockade or ET B receptor desensitization inhibited aortic and venous ET-1-induced contraction. In vena cava but not aorta, when ET B receptors were blocked (BQ-788, 100 nM) or desensitized (S6c, 100 nM), atrasentan caused a greater inhibition of ET-1-induced contraction. Vena cava from WT but not sl/sl rats exhibited similar pharmacological ET receptor interaction. Immunocytochemistry was performed on freshly dissociated aortic and venous vascular smooth muscle cells to determine localization of ET A and ET B receptors. ET A and ET B receptors qualitatively co-localized more strongly to the plasma membrane of aortic compared to venous vascular smooth muscle cells. Our data suggest that pharmacological ET A and ET B receptor interaction may be dependent on the presence of functional ET B receptors and independent of receptor location.

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells

PubMed Central

LECKEL, K; BEECKEN, W-D; JONAS, D; OPPERMANN, E; COMAN, M C; BECK, K-F; CINATL, J; HAILER, N P; AUTH, M K H; BECHSTEIN, W O; SHIPKOVA, M; BLAHETA, R A

2003-01-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell–endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 µm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 µm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy. PMID:14616783

The immunosuppressive drug mycophenolate mofetil impairs the adhesion capacity of gastrointestinal tumour cells.

PubMed

Leckel, K; Beecken, W-D; Jonas, D; Oppermann, E; Coman, M C; Beck, K-F; Cinatl, J; Hailer, N P; Auth, M K H; Bechstein, W O; Shipkova, M; Blaheta, R A

2003-11-01

Immunosuppression correlates with the development and recurrence of cancer. Mycophenolate mofetil (MMF) has been shown to reduce adhesion molecule expression and leucocyte recruitment into the donor organ. We have hypothesized that MMF might also prevent receptor-dependent tumour dissemination. Therefore, we have investigated the effects of MMF on tumour cell adhesion to human umbilical vein endothelial cells (HUVEC) and compared them with the effects on T cell-endothelial cell interactions. Influence of MMF on cellular adhesion to HUVEC was analysed using isolated CD4+ and CD8+ T cells, or WiDr colon adenocarcinoma cells as the model tumour. HUVEC receptors ICAM-1, VCAM-1, E-selectin and P-selectin were detected by flow cytometry, Western blot or Northern blot analysis. Binding activity of T cells or WiDr cells in the presence of MMF were measured using immobilized receptor globulin chimeras. MMF potently blocked both T cell and WiDr cell binding to endothelium by 80%. Surface expression of the endothelial cell receptors was reduced by MMF in a dose-dependent manner. E-selectin mRNA was concurrently reduced with a maximum effect at 1 microm. Interestingly, MMF acted differently on T cells and WiDr cells. Maximum efficacy of MMF was reached at 10 and 1 microm, respectively. Furthermore, MMF specifically suppressed T cell attachment to ICAM-1, VCAM-1 and P-selectin. In contrast, MMF prevented WiDr cell attachment to E-selectin. In conclusion, our data reveal distinct effects of MMF on both T cell adhesion and tumour cell adhesion to endothelial cells. This suggests that MMF not only interferes with the invasion of alloactivated T cells, but might also be of value in managing post-transplantation malignancy.

Advanced glycation end products and the progressive course of renal disease.

PubMed

Heidland, A; Sebekova, K; Schinzel, R

2001-10-01

In experimental and human diabetic nephropathy (DN), it has been shown that advanced glycation end products (AGEs), in particular, carboxymethyl-lysine and pentosidine, accumulate with malondialdehyde in glomerular lesions in relation to disease severity and in the presence of an upregulated receptor for AGE (RAGE) in podocytes. Toxic effects of AGEs result from structural and functional alterations in plasma and extracellular matrix (ECM) proteins, in particular, from cross-linking of proteins and interaction of AGEs with their receptors and/or binding proteins. In mesangial and endothelial cells, the AGE-RAGE interaction caused enhanced formation of oxygen radicals with subsequent activation of nuclear factor-kappaB and release of pro-inflammatory cytokines (interleukin-6, tumor necrosis factor-alpha), growth factors (transforming growth factor-beta1 [TGF-beta1], insulin-like growth factor-1), and adhesion molecules (vascular cell adhesion molecule-1, intercellular adhesion molecule-1). In tubular cells, incubation with AGE albumin was followed by stimulation of the mitogen-activating protein (MAP) kinase pathway and its downstream target, the activating protien-1 (AP-1) complex, TGF-beta1 overexpression, enhanced protein kinase C activity, decreased cell proliferation, and impaired protein degradation rate, in part caused by decreased cathepsin activities. The pathogenic relevance of AGEs was further verified by in vivo experiments in euglycemic rats and mice by the parenteral administration of AGE albumin, leading in the glomeruli to TGF-beta1 overproduction, enhanced gene expression of ECM proteins, and morphological lesions similar to those of DN. Evidence for the pathogenic relevance of AGEs in DN also comes from experimental studies in which the formation and/or action of AGEs was modulated by aminoguanidine, OPB-9195, pyridoxamine, soluble RAGEs, serine protease trypsin, and antioxidants, resulting in improved cell and/or renal function.

Fibronectin induces macrophage migration through a SFK-FAK/CSF-1R pathway.

PubMed

Digiacomo, Graziana; Tusa, Ignazia; Bacci, Marina; Cipolleschi, Maria Grazia; Dello Sbarba, Persio; Rovida, Elisabetta

2017-07-04

Integrins, following binding to proteins of the extracellular matrix (ECM) including collagen, laminin and fibronectin (FN), are able to transduce molecular signals inside the cells and to regulate several biological functions such as migration, proliferation and differentiation. Besides activation of adaptor molecules and kinases, integrins transactivate Receptor Tyrosine Kinases (RTK). In particular, adhesion to the ECM may promote RTK activation in the absence of growth factors. The Colony-Stimulating Factor-1 Receptor (CSF-1R) is a RTK that supports the survival, proliferation, and motility of monocytes/macrophages, which are essential components of innate immunity and cancer development. Macrophage interaction with FN is recognized as an important aspect of host defense and wound repair. The aim of the present study was to investigate on a possible cross-talk between FN-elicited signals and CSF-1R in macrophages. FN induced migration in BAC1.2F5 and J774 murine macrophage cell lines and in human primary macrophages. Adhesion to FN determined phosphorylation of the Focal Adhesion Kinase (FAK) and Src Family Kinases (SFK) and activation of the SFK/FAK complex, as witnessed by paxillin phosphorylation. SFK activity was necessary for FAK activation and macrophage migration. Moreover, FN-induced migration was dependent on FAK in either murine macrophage cell lines or human primary macrophages. FN also induced FAK-dependent/ligand-independent CSF-1R phosphorylation, as well as the interaction between CSF-1R and β1. CSF-1R activity was necessary for FN-induced macrophage migration. Indeed, genetic or pharmacological inhibition of CSF-1R prevented FN-induced macrophage migration. Our results identified a new SFK-FAK/CSF-1R signaling pathway that mediates FN-induced migration of macrophages.

Vinculin tension distributions of individual stress fibers within cell–matrix adhesions

PubMed Central

Chang, Ching-Wei; Kumar, Sanjay

2013-01-01

Summary Actomyosin stress fibers (SFs) enable cells to exert traction on planar extracellular matrices (ECMs) by tensing focal adhesions (FAs) at the cell–ECM interface. Although it is widely appreciated that the spatial and temporal distribution of these tensile forces play key roles in polarity, motility, fate choice, and other defining cell behaviors, virtually nothing is known about how an individual SF quantitatively contributes to tensile loads borne by specific molecules within associated FAs. We address this key open question by using femtosecond laser ablation to sever single SFs in cells while tracking tension across vinculin using a molecular optical sensor. We show that disruption of a single SF reduces tension across vinculin in FAs located throughout the cell, with enriched vinculin tension reduction in FAs oriented parallel to the targeted SF. Remarkably, however, some subpopulations of FAs exhibit enhanced vinculin tension upon SF irradiation and undergo dramatic, unexpected transitions between tension-enhanced and tension-reduced states. These changes depend strongly on the location of the severed SF, consistent with our earlier finding that different SF pools are regulated by distinct myosin activators. We critically discuss the extent to which these measurements can be interpreted in terms of whole-FA tension and traction and propose a model that relates SF tension to adhesive loads and cell shape stability. These studies represent the most direct and high-resolution intracellular measurements of SF contributions to tension on specific FA proteins to date and offer a new paradigm for investigating regulation of adhesive complexes by cytoskeletal force. PMID:23687380

α2,6 sialylation associated with increased beta 1,6-branched N-oligosaccharides influences cellular adhesion and invasion.

PubMed

Ranjan, Amit; Kalraiya, Rajiv D

2013-12-01

Expression of β1,6-branched N-linked oligosaccharides have a definite association with invasion and metastasis of cancer cells. However, the mechanism by which these oligosaccharides regulate these processes is not well understood. Invasive variants of B16 murine melanoma, B16F10 (parent) and B16BL6 (highly invasive variant) cell lines have been used for these studies. We demonstrate that substitution of α2,6-linked sialic acids on multiantennary structures formed as a result of β1,6-branching modulate cellular adhesion on both extracellular matrix (ECM) and basement membrane (BM) components. Removal of α2,6 sialic acids either by enzymatic desialylation or by stably down-regulating the ST6Gal-I (enzyme that catalyses the addition of α2,6-linked sialic acids on N-linked oligosaccharides) by lentiviral driven shRNA decreased the adhesion on both ECM and BM components and invasion through reconstituted BM matrigel.

Role of the ECM in notochord formation, function and disease.

PubMed

Trapani, Valeria; Bonaldo, Paolo; Corallo, Diana

2017-10-01

The notochord is a midline structure common to all chordate animals; it provides mechanical and signaling cues for the developing embryo. In vertebrates, the notochord plays key functions during embryogenesis, being a source of developmental signals that pattern the surrounding tissues. It is composed of a core of vacuolated cells surrounded by an epithelial-like sheath of cells that secrete a thick peri-notochordal basement membrane made of different extracellular matrix (ECM) proteins. The correct deposition and organization of the ECM is essential for proper notochord morphogenesis and function. Work carried out in the past two decades has allowed researchers to dissect the contribution of different ECM components to this embryonic tissue. Here, we will provide an overview of these genetic and mechanistic studies. In particular, we highlight the specific functions of distinct matrix molecules in regulating notochord development and notochord-derived signals. Moreover, we also discuss the involvement of ECM synthesis and its remodeling in the pathogenesis of chordoma, a malignant bone cancer that originates from remnants of notochord remaining after embryogenesis. © 2017. Published by The Company of Biologists Ltd.

IgG1-iS18 impedes the adhesive and invasive potential of early and late stage malignant melanoma cells.

PubMed

Munien, Carmelle; Rebelo, Thalia M; Ferreira, Eloise; Weiss, Stefan F T

2017-02-15

The 37kDa/67kDa laminin receptor (LRP/LR) is a non-integrin laminin receptor which is overexpressed in tumorigenic cells and supports progression of cancer via promoting metastasis, angiogenesis and telomerase activity and impediment of apoptosis. The present study investigates the role of LRP/LR on the metastatic potential of early (A375) and late (A375SM) stage malignant melanoma cells. Flow cytometry revealed that both early and late stage malignant melanoma cells display high levels of LRP/LR on their cell surface. Flow cytometry and western blot analysis showed that late stage malignant melanoma cells display significantly higher total and cell surface LRP/LR levels in comparison to early stage malignant melanoma cells and the poorly invasive breast cancer (MCF-7) control cell line. Targeting LRP/LR using the LRP/LR specific antibody IgG1-iS18 resulted in a significant reduction of the adhesive potential to laminin-1 and the invasive potential through the 'ECM-simulating' Matrigel™ of both early and late stage malignant melanoma cells. Furthermore, Pearson's correlation coefficient confirmed that increased LRP levels correlate with the increased invasive and adhesive potential in early and late stage melanoma cells. Thus, blocking LRP/LR using the IgG1-iS18 antibody may therefore be a promising therapeutic strategy for early and late stage malignant melanoma treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

Hacking macrophage-associated immunosuppression for regulating glioblastoma angiogenesis.

PubMed

Cui, Xin; Morales, Renee-Tyler Tan; Qian, Weiyi; Wang, Haoyu; Gagner, Jean-Pierre; Dolgalev, Igor; Placantonakis, Dimitris; Zagzag, David; Cimmino, Luisa; Snuderl, Matija; Lam, Raymond H W; Chen, Weiqiang

2018-04-01

Glioblastoma (GBM) is the most lethal primary adult brain tumor and its pathology is hallmarked by distorted neovascularization, diffuse tumor-associated macrophage infiltration, and potent immunosuppression. Reconstituting organotypic tumor angiogenesis models with biomimetic cell heterogeneity and interactions, pro-/anti-inflammatory milieu and extracellular matrix (ECM) mechanics is critical for preclinical anti-angiogenic therapeutic screening. However, current in vitro systems do not accurately mirror in vivo human brain tumor microenvironment. Here, we engineered a three-dimensional (3D), microfluidic angiogenesis model with controllable and biomimetic immunosuppressive conditions, immune-vascular and cell-matrix interactions. We demonstrate in vitro, GL261 and CT-2A GBM-like tumors steer macrophage polarization towards a M2-like phenotype for fostering an immunosuppressive and proangiogenic niche, which is consistent with human brain tumors. We distinguished that GBM and M2-like immunosuppressive macrophages promote angiogenesis, while M1-like pro-inflammatory macrophages suppress angiogenesis, which we coin "inflammation-driven angiogenesis." We observed soluble immunosuppressive cytokines, predominantly TGF-β1, and surface integrin (α v β 3 ) endothelial-macrophage interactions are required in inflammation-driven angiogenesis. We demonstrated tuning cell-adhesion receptors using an integrin (α v β 3 )-specific collagen hydrogel regulated inflammation-driven angiogenesis through Src-PI3K-YAP signaling, highlighting the importance of altered cell-ECM interactions in inflammation. To validate the preclinical applications of our 3D organoid model and mechanistic findings of inflammation-driven angiogenesis, we screened a novel dual integrin (α v β 3 ) and cytokine receptor (TGFβ-R1) blockade that suppresses GBM tumor neovascularization by simultaneously targeting macrophage-associated immunosuppression, endothelial-macrophage interactions, and

Effect of ECM2 expression on bovine skeletal muscle-derived satellite cell differentiation.

PubMed

Liu, Chang; Tong, Huili; Li, Shufeng; Yan, Yunqin

2018-05-01

Extracellular matrix components have important regulatory functions during cell proliferation and differentiation. In recent study, extracellular matrix were shown to have a strong effect on skeletal muscle differentiation. Here, we aimed to elucidate the effects of extracellular matrix protein 2 (ECM2), an extracellular matrix component, on the differentiation of bovine skeletal muscle-derived satellite cells (MDSCs). Western blot and immunofluorescence analyses were used to elucidate the ECM2 expression pattern in bovine MDSCs during differentiation in vitro. CRISPR/Cas9 technology was used to activate or inhibit ECM2 expression to study its effects on the in vitro differentiation of bovine MDSCs. ECM2 expression was shown to increase gradually during bovine MDSC differentiation, and the levels of this protein were higher in more highly differentiated myotubes. ECM2 activation promoted MDSC differentiation, whereas its suppression inhibited the differentiation of these cells. Here, for the first time, we demonstrated the importance of ECM2 expression during bovine MDSC differentiation; these results could lead to treatments that help to increase beef cattle muscularity. © 2018 International Federation for Cell Biology.

Interactions with nanoscale topography: adhesion quantification and signal transduction in cells of osteogenic and multipotent lineage.

PubMed

Biggs, Manus J P; Richards, R Geoff; Gadegaard, Nikolaj; McMurray, Rebecca J; Affrossman, Stanley; Wilkinson, Chris D W; Oreffo, Richard O C; Dalby, Mathew J

2009-10-01

Polymeric medical devices widely used in orthopedic surgery play key roles in fracture fixation and orthopedic implant design. Topographical modification and surface micro-roughness of these devices regulate cellular adhesion, a process fundamental in the initiation of osteoinduction and osteogenesis. Advances in fabrication techniques have evolved the field of surface modification; in particular, nanotechnology has allowed the development of nanoscale substrates for the investigation into cell-nanofeature interactions. In this study human osteoblasts (HOBs) were cultured on ordered nanoscale pits and random nano "craters" and "islands". Adhesion subtypes were quantified by immunofluorescent microscopy and cell-substrate interactions investigated via immuno-scanning electron microscopy. To investigate the effects of these substrates on cellular function 1.7 k microarray analysis was used to establish gene profiles of enriched STRO-1+ progenitor cell populations cultured on these nanotopographies. Nanotopographies affected the formation of adhesions on experimental substrates. Adhesion formation was prominent on planar control substrates and reduced on nanocrater and nanoisland topographies; nanopits, however, were shown to inhibit directly the formation of large adhesions. STRO-1+ progenitor cells cultured on experimental substrates revealed significant changes in genetic expression. This study implicates nanotopographical modification as a significant modulator of osteoblast adhesion and cellular function in mesenchymal populations.

The calcium-sensing receptor and its interacting proteins

PubMed Central

Huang, Chunfa; Miller, R Tyler

2007-01-01

Abstract Seven membrane-spanning, or G protein-coupled receptors were originally thought to act through het-erotrimeric G proteins that in turn activate intracellular enzymes or ion channels, creating relatively simple, linear signalling pathways. Although this basic model remains true in that this family does act via a relatively small number of G proteins, these signalling systems are considerably more complex because the receptors interact with or are located near additional proteins that are often unique to a receptor or subset of receptors. These additional proteins give receptors their unique signalling ‘personalities’. The extracellular Ca-sensing receptor (CaR) signals via Gαi, Gαq and Gα12/13, but its effects in vivo demonstrate that the signalling pathways controlled by these subunits are not sufficient to explain all its biologic effects. Additional structural or signalling proteins that interact with the CaR may explain its behaviour more fully. Although the CaR is less well studied in this respect than other receptors, several CaR-interacting proteins such as filamin, a potential scaffolding protein, receptor activity modifying proteins (RAMPs) and potassium channels may contribute to the unique characteristics of the CaR. The CaR also appears to interact with additional proteins common to other G protein-coupled receptors such as arrestins, G protein receptor kinases, protein kinase C, caveolin and proteins in the ubiquitination pathway. These proteins probably represent a few initial members of CaR-based signalling complex. These and other proteins may not all be associated with the CaR in all tissues, but they form the basis for understanding the complete nature of CaR signalling. PMID:17979874

Modulation of adhesion-dependent cAMP signaling by echistatin and alendronate

NASA Technical Reports Server (NTRS)

Fong, J. H.; Ingber, D. E.

1996-01-01

We measured intracellular cAMP levels in cells during attachment and spreading on different extracellular matrix (ECM) proteins. Increases in cAMP were observed within minutes when cells attached to fibronectin, vitronectin, and a synthetic RGD-containing fibronectin peptide (Petite 2000), but not when they adhered to another integrin alpha nu beta 3 ligand, echistatin. Because echistatin also inhibits bone resorption, we measured the effects of adding another osteoporosis inhibitor, alendronate, in this system. Alendronate inhibited the cAMP increase induced by ligands that primarily utilize integrin alpha nu beta 3 (vitronectin, Peptite 2000), but not by fibronectin which can also use integrin alpha 5 beta 1. These results show that cell adhesion to ECM can increase intracellular cAPM levels and raise the possibility that inhibitors of osteoporosis may act, in part, by preventing activation of this pathway by integrins.

FES-related tyrosine kinase activates the insulin-like growth factor-1 receptor at sites of cell adhesion.

PubMed

Stanicka, Joanna; Rieger, Leonie; O'Shea, Sandra; Cox, Orla; Coleman, Michael; O'Flanagan, Ciara; Addario, Barbara; McCabe, Nuala; Kennedy, Richard; O'Connor, Rosemary

2018-06-01

IGF-1 receptor (IGF-1R) and integrin cooperative signaling promotes cancer cell survival, proliferation, and motility, but whether this influences cancer progression and therapy responses is largely unknown. Here we investigated the non-receptor tyrosine adhesion kinase FES-related (FER), following its identification as a potential mediator of sensitivity to IGF-1R kinase inhibition in a functional siRNA screen. We found that FER and the IGF-1R co-locate in cells and can be co-immunoprecipitated. Ectopic FER expression strongly enhanced IGF-1R expression and phosphorylation on tyrosines 950 and 1131. FER phosphorylated these sites in an IGF-1R kinase-independent manner and also enhanced IGF-1-mediated phosphorylation of SHC, and activation of either AKT or MAPK-signaling pathways in different cells. The IGF-1R, β1 Integrin, FER, and its substrate cortactin were all observed to co-locate in cell adhesion complexes, the disruption of which reduced IGF-1R expression and activity. High FER expression correlates with phosphorylation of SHC in breast cancer cell lines and with a poor prognosis in patient cohorts. FER and SHC phosphorylation and IGF-1R expression could be suppressed with a known anaplastic lymphoma kinase inhibitor (AP26113) that shows high specificity for FER kinase. Overall, we conclude that FER enhances IGF-1R expression, phosphorylation, and signaling to promote cooperative growth and adhesion signaling that may facilitate cancer progression.

A mucus adhesion promoting protein, MapA, mediates the adhesion of Lactobacillus reuteri to Caco-2 human intestinal epithelial cells.

PubMed

Miyoshi, Yukihiro; Okada, Sanae; Uchimura, Tai; Satoh, Eiichi

2006-07-01

Lactobacillus reuteri is one of the dominant lactobacilli found in the gastrointestinal tract of various animals. A surface protein of L. reuteri 104R, mucus adhesion promoting protein (MapA), is considered to be an adhesion factor of this strain. We investigated the relation between MapA and adhesion of L. reuteri to human intestinal (Caco-2) cells. Quantitative analysis of the adhesion of L. reuteri strains to Caco-2 cells showed that various L. reuteri strains bind not only to mucus but also to intestinal epithelial cells. In addition, purified MapA bound to Caco-2 cells, and this binding inhibited the adhesion of L. reuteri in a concentration-dependent manner. Based on these observations, the adhesion of L. reuteri appears due to the binding of MapA to receptor-like molecules on Caco-2 cells. Further, far-western analysis indicated the existence of multiple receptor-like molecules in Caco-2 cells.

Novel molecular insights into RhoA GTPase-induced resistance to aqueous humor outflow through the trabecular meshwork

PubMed Central

Zhang, Min; Maddala, Rupalatha; Rao, Ponugoti Vasantha

2008-01-01

Impaired drainage of aqueous humor through the trabecular meshwork (TM) culminating in increased intraocular pressure is a major risk factor for glaucoma, a leading cause of blindness worldwide. Regulation of aqueous humor drainage through the TM, however, is poorly understood. The role of RhoA GTPase-mediated actomyosin organization, cell adhesive interactions, and gene expression in regulation of aqueous humor outflow was investigated using adenoviral vector-driven expression of constitutively active mutant of RhoA (RhoAV14). Organ-cultured anterior segments from porcine eyes expressing RhoAV14 exhibited significant reduction of aqueous humor outflow. Cultured TM cells expressing RhoAV14 exhibited a pronounced contractile morphology, increased actin stress fibers, and focal adhesions and increased levels of phosphorylated myosin light chain (MLC), collagen IV, fibronectin, and laminin. cDNA microarray analysis of RNA extracted from RhoAV14-expressing human TM cells revealed a significant increase in the expression of genes encoding extracellular matrix (ECM) proteins, cytokines, integrins, cytoskeletal proteins, and signaling proteins. Conversely, various ECM proteins stimulated robust increases in phosphorylation of MLC, paxillin, and focal adhesion kinase and activated Rho GTPase and actin stress fiber formation in TM cells, indicating a potential regulatory feedback interaction between ECM-induced mechanical strain and Rho GTPase-induced isometric tension in TM cells. Collectively, these data demonstrate that sustained activation of Rho GTPase signaling in the aqueous humor outflow pathway increases resistance to aqueous humor outflow through the trabecular pathway by influencing the actomyosin assembly, cell adhesive interactions, and the expression of ECM proteins and cytokines in TM cells. PMID:18799648

Cell adhesion heterogeneity reinforces tumour cell dissemination: novel insights from a mathematical model.

PubMed

Reher, David; Klink, Barbara; Deutsch, Andreas; Voss-Böhme, Anja

2017-08-11

Cancer cell invasion, dissemination, and metastasis have been linked to an epithelial-mesenchymal transition (EMT) of individual tumour cells. During EMT, adhesion molecules like E-cadherin are downregulated and the decrease of cell-cell adhesion allows tumour cells to dissociate from the primary tumour mass. This complex process depends on intracellular cues that are subject to genetic and epigenetic variability, as well as extrinsic cues from the local environment resulting in a spatial heterogeneity in the adhesive phenotype of individual tumour cells. Here, we use a novel mathematical model to study how adhesion heterogeneity, influenced by intrinsic and extrinsic factors, affects the dissemination of tumour cells from an epithelial cell population. The model is a multiscale cellular automaton that couples intracellular adhesion receptor regulation with cell-cell adhesion. Simulations of our mathematical model indicate profound effects of adhesion heterogeneity on tumour cell dissemination. In particular, we show that a large variation of intracellular adhesion receptor concentrations in a cell population reinforces cell dissemination, regardless of extrinsic cues mediated through the local cell density. However, additional control of adhesion receptor concentration through the local cell density, which can be assumed in healthy cells, weakens the effect. Furthermore, we provide evidence that adhesion heterogeneity can explain the remarkable differences in adhesion receptor concentrations of epithelial and mesenchymal phenotypes observed during EMT and might drive early dissemination of tumour cells. Our results suggest that adhesion heterogeneity may be a universal trigger to reinforce cell dissemination in epithelial cell populations. This effect can be at least partially compensated by a control of adhesion receptor regulation through neighbouring cells. Accordingly, our findings explain how both an increase in intra-tumour adhesion heterogeneity and the

MENA Promotes Tumor-Intrinsic Metastasis through ECM Remodeling and Haptotaxis.

PubMed

Santiago-Medina, Miguel; Yang, Jing

2016-05-01

Oudin and colleagues report a novel and specific function of MENA in mediating directional migration of breast cancer cells toward a fibronectin gradient of increasing concentration. This MENA-mediated haptotactic response depends on the binding of MENA to the α5β1 integrin receptor, adhesion protein signaling, and fibronectin fibrillogenesis. Cancer Discov; 6(5); 474-6. ©2016 AACRSee related article by Oudin et al., p. 516. ©2016 American Association for Cancer Research.

Kon-tiki enhances PS2 integrin adhesion and localizes its ligand, Thrombospondin, in the myotendinous junction.

PubMed

Pérez-Moreno, Juan J; Espina-Zambrano, Agueda G; García-Calderón, Clara B; Estrada, Beatriz

2017-03-01

Cell-extracellular-matrix adhesion is mediated by cell receptors, mainly integrins and transmembrane proteoglycans, which can functionally interact. How these receptors are regulated and coordinated is largely unknown. We show that the conserved transmembrane Drosophila proteoglycan Kon-tiki (Kon, also known as Perdido) interacts with the αPS2βPS integrin (αPS2 is encoded by inflated and βPS by myospheroid ) to mediate muscle-tendon adhesion. kon and inflated double mutant embryos show a synergistic increase in muscle detachment. Furthermore, Kon modulates αPS2βPS signaling at the muscle attachment, since phosphorylated Fak is reduced in kon mutants. This reduction in integrin signaling can be rescued by the expression of a truncated Kon protein containing its transmembrane and extracellular domains, suggesting that these domains are sufficient to mediate this signaling. We show that these domains are sufficient to properly localize the αPS2βPS ligand, Thrombospondin, to the muscle attachment, and to partially rescue Kon-dependent muscle-tendon adhesion. We propose that Kon can engage in a protein complex with αPS2βPS and enhance integrin-mediated signaling and adhesion by recruiting its ligand, which would increase integrin-binding affinity to the extracellular matrix, resulting in the consolidation of the myotendinous junction. © 2017. Published by The Company of Biologists Ltd.

Revealing Early Steps of α2β1 Integrin-mediated Adhesion to Collagen Type I by Using Single-Cell Force Spectroscopy

PubMed Central

Taubenberger, Anna; Cisneros, David A.; Friedrichs, Jens; Puech, Pierre-Henri; Muller, Daniel J.

2007-01-01

We have characterized early steps of α2β1 integrin-mediated cell adhesion to a collagen type I matrix by using single-cell force spectroscopy. In agreement with the role of α2β1 as a collagen type I receptor, α2β1-expressing Chinese hamster ovary (CHO)-A2 cells spread rapidly on the matrix, whereas α2β1-negative CHO wild-type cells adhered poorly. Probing CHO-A2 cell detachment forces over a contact time range of 600 s revealed a nonlinear adhesion response. During the first 60 s, cell adhesion increased slowly, and forces associated with the smallest rupture events were consistent with the breakage of individual integrin–collagen bonds. Above 60 s, a fraction of cells rapidly switched into an activated adhesion state marked by up to 10-fold increased detachment forces. Elevated overall cell adhesion coincided with a rise of the smallest rupture forces above the value required to break a single-integrin–collagen bond, suggesting a change from single to cooperative receptor binding. Transition into the activated adhesion mode and the increase of the smallest rupture forces were both blocked by inhibitors of actomyosin contractility. We therefore propose a two-step mechanism for the establishment of α2β1-mediated adhesion as weak initial, single-integrin–mediated binding events are superseded by strong adhesive interactions involving receptor cooperativity and actomyosin contractility. PMID:17314408

Membrane receptor location defines receptor interaction with signaling proteins in a polarized epithelium.

PubMed

Amsler, K; Kuwada, S K

1999-01-01

Signal transduction from receptors is mediated by the interaction of activated receptors with proximate downstream signaling proteins. In polarized epithelial cells, the membrane is divided into subdomains: the apical and basolateral membranes. Membrane receptors may be present in one or both subdomains. Using a combination of immunoprecipitation and Western blot analyses, we tested the hypothesis that a tyrosine kinase growth factor receptor, epidermal growth factor receptor (EGFR), interacts with distinct signaling proteins when present at the apical vs. basolateral membrane of a polarized renal epithelial cell. We report here that tyrosine phosphorylation of phospholipase C-gamma (PLC-gamma) was induced only when basolateral EGFR was activated. In contrast, tyrosine phosphorylation of several other signaling proteins was increased by activation of receptor at either surface. All signaling proteins were distributed diffusely throughout the cytoplasm; however, PLC-gamma protein also displayed a concentration at lateral cell borders. These results demonstrate that in polarized epithelial cells the array of signaling pathways initiated by activation of a membrane receptor is defined, at least in part, by the membrane location of the receptor.

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

PubMed

Shinohara, M; Kodama, A; Matozaki, T; Fukuhara, A; Tachibana, K; Nakanishi, H; Takai, Y

2001-06-01

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Very late antigen integrins are involved in the adhesive interaction of lymphoid cells to human gingival fibroblasts.

PubMed Central

Murakami, S; Saho, T; Shimabukuro, Y; Isoda, R; Miki, Y; Okada, H

1993-01-01

To date, it is still unclear how the trafficking and retention of activated lymphocytes in periodontal lesions are regulated. In this study, we investigated the molecular basis for the adhesive interactions between lymphocytes and human gingival fibroblasts (HGF). Peripheral blood T lymphocytes (PBT) exhibited binding ability, but only when the calls were activated with phorbol 12-myristate 13-acetate (PMA). Among several human cell lines tested, PMA-stimulated Molt-4, a human T-cell leukaemia line, also displayed significant binding ability to HGF. In order to clarify the molecule(s) involved in this cell-cell interaction, a panel of monoclonal antibodies (mAb) was prepared to PMA-activated Molt-4 and one clone, 4-145, was selected on the basis of its ability to block the binding of PMA-activated Molt-4 to HGF. Moreover, 4-145 inhibited the binding of not only activated Molt-4 but also activated PBT and other cell types to HGF. Biochemical and flow cytometric analyses revealed that 4-145 probably recognizes the beta 1 chain of very late antigen (VLA) integrins. Blocking experiments using mAb specific for the alpha-chain of VLA integrins demonstrated the involvement of alpha 4 (VLA-4) and, to a lesser extent, alpha 5 (VLA-5) chains in the adhesive interactions between T cells and HGF. Despite the significant involvement of VLA integrins in the adhesive interaction between PBT and HGF, the binding of PBT to human dermal fibroblasts (HDF) was not abrogated by 4-145, suggesting that HGF and HDF differ in their requirement of VLA integrins for adhesion to activated PBT. Furthermore, the fact that vascular cell adhesion molecule-1 (VCAM-1), one of the ligands of VLA-4, was not detected on HGF by flow cytometry and anti-fibronectin (FN) Ab did not block the adhesive interaction to HGF suggests that not-yet-identified ligand(s) for VLA-4 might be present on HGF. Images Figure 4 PMID:8406571

Measurement of cell adhesion force by vertical forcible detachment using an arrowhead nanoneedle and atomic force microscopy

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

Ryu, Seunghwan; Hashizume, Yui; Mishima, Mari

Graphical abstract: - Highlights: • We developed a method to measure cell adhesion force by detaching cell using an arrowhead nanoneedle and AFM. • A nanofilm consisting of fibronectin and gelatin was formed on cell surface to reinforce the cell cortex. • By the nanofilm lamination, detachment efficiencies of strongly adherent cell lines were improved markedly. - Abstract: The properties of substrates and extracellular matrices (ECM) are important factors governing the functions and fates of mammalian adherent cells. For example, substrate stiffness often affects cell differentiation. At focal adhesions, clustered–integrin bindings link cells mechanically to the ECM. In order tomore » quantitate the affinity between cell and substrate, the cell adhesion force must be measured for single cells. In this study, forcible detachment of a single cell in the vertical direction using AFM was carried out, allowing breakage of the integrin–substrate bindings. An AFM tip was fabricated into an arrowhead shape to detach the cell from the substrate. Peak force observed in the recorded force curve during probe retraction was defined as the adhesion force, and was analyzed for various types of cells. Some of the cell types adhered so strongly that they could not be picked up because of plasma membrane breakage by the arrowhead probe. To address this problem, a technique to reinforce the cellular membrane with layer-by-layer nanofilms composed of fibronectin and gelatin helped to improve insertion efficiency and to prevent cell membrane rupture during the detachment process, allowing successful detachment of the cells. This method for detaching cells, involving cellular membrane reinforcement, may be beneficial for evaluating true cell adhesion forces in various cell types.« less

Load sharing in bioinspired fibrillar adhesives with backing layer interactions and interfacial misalignment

NASA Astrophysics Data System (ADS)

Bacca, Mattia; Booth, Jamie A.; Turner, Kimberly L.; McMeeking, Robert M.

2016-11-01

Bio-inspired fibrillar adhesives rely on the utilization of short-range intermolecular forces harnessed by intimate contact at fibril tips. The combined adhesive strength of multiple fibrils can only be utilized if equal load sharing (ELS) is obtained at detachment. Previous investigations have highlighted that mechanical coupling of fibrils through a compliant backing layer gives rise to load concentration and the nucleation and propagation of interfacial flaws. However, misalignment of the adhesive and contacting surface has not been considered in theoretical treatments of load sharing with backing layer interactions. Alignment imperfections are difficult to avoid for a flat-on-flat interfacial configuration. In this work we demonstrate that interfacial misalignment can significantly alter load sharing and the kinematics of detachment in a model adhesive system. Load sharing regimes dominated by backing layer interactions and misalignment are revealed, the transition between which is controlled by the misalignment angle, fibril separation, and fibril compliance. In the regime dominated by misalignment, backing layer deformation can counteract misalignment giving rise to improved load sharing when compared to an identical fibrillar array with a rigid backing layer. This result challenges the conventional belief that stiffer (and thinner) backing layers consistently reduce load concentration among fibrils. Finally, we obtain analytically the fibril compliance distribution required to harness backing layer interactions to obtain ELS. Through fibril compliance optimization, ELS can be obtained even with misalignment. However, since misalignment is typically not deterministic, it is of greater practical significance that the array optimized for perfect alignment exhibits load sharing superior to that of a homogeneous array subject to misalignment. These results inform the design of fibrillar arrays with graded compliance capable of exhibiting improved load sharing

Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

PubMed

El-Amin, S F; Lu, H H; Khan, Y; Burems, J; Mitchell, J; Tuan, R S; Laurencin, C T

2003-03-01

The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal

Macrophage Migration Inhibitory Factor-CXCR4 Receptor Interactions

PubMed Central

Rajasekaran, Deepa; Gröning, Sabine; Schmitz, Corinna; Zierow, Swen; Drucker, Natalie; Bakou, Maria; Kohl, Kristian; Mertens, André; Lue, Hongqi; Weber, Christian; Xiao, Annie; Luker, Gary; Kapurniotu, Aphrodite; Lolis, Elias; Bernhagen, Jürgen

2016-01-01

An emerging number of non-chemokine mediators are found to bind to classical chemokine receptors and to elicit critical biological responses. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that exhibits chemokine-like activities through non-cognate interactions with the chemokine receptors CXCR2 and CXCR4, in addition to activating the type II receptor CD74. Activation of the MIF-CXCR2 and -CXCR4 axes promotes leukocyte recruitment, mediating the exacerbating role of MIF in atherosclerosis and contributing to the wealth of other MIF biological activities. Although the structural basis of the MIF-CXCR2 interaction has been well studied and was found to engage a pseudo-ELR and an N-like loop motif, nothing is known about the regions of CXCR4 and MIF that are involved in binding to each other. Using a genetic strain of Saccharomyces cerevisiae that expresses a functional CXCR4 receptor, site-specific mutagenesis, hybrid CXCR3/CXCR4 receptors, pharmacological reagents, peptide array analysis, chemotaxis, fluorescence spectroscopy, and circular dichroism, we provide novel molecular information about the structural elements that govern the interaction between MIF and CXCR4. The data identify similarities with classical chemokine-receptor interactions but also provide evidence for a partial allosteric agonist compared with CXCL12 that is possible due to the two binding sites of CXCR4. PMID:27226569

The CD44-initiated pathway of T-cell extravasation uses VLA-4 but not LFA-1 for firm adhesion

PubMed Central

Siegelman, Mark H.; Stanescu, Diana; Estess, Pila

2000-01-01

Leukocytes extravasate from the blood in response to physiologic or pathologic demands by means of complementary ligand interactions between leukocytes and endothelial cells. The multistep model of leukocyte extravasation involves an initial transient interaction (“rolling” adhesion), followed by secondary (firm) adhesion. We recently showed that binding of CD44 on activated T lymphocytes to endothelial hyaluronan (HA) mediates a primary adhesive interaction under shear stress, permitting extravasation at sites of inflammation. The mechanism for subsequent firm adhesion has not been elucidated. Here we demonstrate that the integrin VLA-4 is used in secondary adhesion after CD44-mediated primary adhesion of human and mouse T cells in vitro, and by mouse T cells in an in vivo model. We show that clonal cell lines and polyclonally activated normal T cells roll under physiologic shear forces on hyaluronate and require VCAM-1, but not ICAM-1, as ligand for subsequent firm adhesion. This firm adhesion is also VLA-4 dependent, as shown by antibody inhibition. Moreover, in vivo short-term homing experiments in a model dependent on CD44 and HA demonstrate that superantigen-activated T cells require VLA-4, but not LFA-1, for entry into an inflamed peritoneal site. Thus, extravasation of activated T cells initiated by CD44 binding to HA depends upon VLA-4–mediated firm adhesion, which may explain the frequent association of these adhesion receptors with diverse chronic inflammatory processes. PMID:10712440

Nifedipine inhibits advanced glycation end products (AGEs) and their receptor (RAGE) interaction-mediated proximal tubular cell injury via peroxisome proliferator-activated receptor-gamma activation

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

Matsui, Takanori; Yamagishi, Sho-ichi, E-mail: shoichi@med.kurume-u.ac.jp; Takeuchi, Masayoshi

2010-07-23

Research highlights: {yields} Nifedipine inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma}. {yields} GW9662 treatment alone increased RAGE mRNA levels in tubular cells. {yields} Nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-{beta} gene expression in tubular cells, all of which were blocked by GW9662. -- Abstract: There is a growing body of evidence that advanced glycation end products (AGEs) and their receptor (RAGE) interaction evokes oxidative stress generation and subsequently elicits inflammatory and fibrogenicmore » reactions, thereby contributing to the development and progression of diabetic nephropathy. We have previously found that nifedipine, a calcium-channel blocker (CCB), inhibits the AGE-induced mesangial cell damage in vitro. However, effects of nifedipine on proximal tubular cell injury remain unknown. We examined here whether and how nifedipine blocked the AGE-induced tubular cell damage. Nifedipine, but not amlodipine, a control CCB, inhibited the AGE-induced up-regulation of RAGE mRNA levels in tubular cells, which was prevented by the simultaneous treatment of GW9662, an inhibitor of peroxisome proliferator-activated receptor-{gamma} (PPAR{gamma}). GW9662 treatment alone was found to increase RAGE mRNA levels in tubular cells. Further, nifedipine inhibited the AGE-induced reactive oxygen species generation, NF-{kappa}B activation and increases in intercellular adhesion molecule-1 and transforming growth factor-beta gene expression in tubular cells, all of which were blocked by GW9662. Our present study provides a unique beneficial aspect of nifedipine on diabetic nephropathy; it could work as an anti-oxidative and anti-inflammatory agent against AGEs in tubular cells by suppressing RAGE

Macrophage interleukin-6 and tumour necrosis factor-α are induced by coronavirus fixation to Toll-like receptor 2/heparan sulphate receptors but not carcinoembryonic cell adhesion antigen 1a

PubMed Central

Jacques, Alexandre; Bleau, Christian; Turbide, Claire; Beauchemin, Nicole; Lamontagne, Lucie

2009-01-01

A rapid antiviral immune response may be related to viral interaction with the host cell leading to activation of macrophages via pattern recognition receptors (PPRs) or specific viral receptors. Carcinoembryonic cell adhesion antigen 1a (CEACAM1a) is the specific receptor for the mouse hepatitis virus (MHV), a coronavirus known to induce acute viral hepatitis in mice. The objective of this study was to understand the mechanisms responsible for the secretion of high-pathogenic MHV3-induced inflammatory cytokines. We report that the induction of the pro-inflammatory cytokines interleukin (IL)-6 and tumour necrosis factor (TNF)-α in peritoneal macrophages does not depend on CEACAM1a, as demonstrated in cells isolated from Ceacam1a−/− mice. The induction of IL-6 and TNF-α production was related rather to the fixation of the spike (S) protein of MHV3 on Toll-like receptor 2 (TLR2) in regions enriched in heparan sulphate and did not rely on viral replication, as demonstrated with denatured S protein and UV-inactivated virus. High levels of IL-6 and TNF-α were produced in livers from infected C57BL/6 mice but not in livers from Tlr2−/− mice. The histopathological observations were correlated with the levels of those inflammatory cytokines. Depending on mouse strain, the viral fixation to heparan sulfate/TLR2 stimulated differently the p38 mitogen-activated protein kinase (MAPK) and nuclear factor (NF)-κB in the induction of IL-6 and TNF-α. These results suggest that TLR2 and heparan sulphate receptors can act as new viral PPRs involved in inflammatory responses. PMID:19740307

Macrophage polarization in response to ECM coated polypropylene mesh

PubMed Central

Wolf, MT; Dearth, CL; Ranallo, CA; LoPresti, S; Carey, LE; Daly, KA; Brown, BN; Badylak, SF

2015-01-01

The host response to implanted biomaterials is a highly regulated process that influences device functionality and clinical outcome. Non-degradable biomaterials, such as knitted polypropylene mesh, frequently elicit a chronic foreign body reaction with resultant fibrosis. Previous studies have shown that an extracellular matrix (ECM) hydrogel coating of polypropylene mesh reduces the intensity of the foreign body reaction, though the mode of action is unknown. Macrophage participation plays a key role in the development of the foreign body reaction to biomaterials, and therefore the present study investigated macrophage polarization following mesh implantation. Spatiotemporal analysis of macrophage polarization was conducted in response to uncoated polypropylene mesh and mesh coated with hydrated and dry forms of ECM hydrogels derived from either dermis or urinary bladder. Pro-inflammatory M1 macrophages (CD86+/CD68+), alternatively activated M2 macrophages (CD206+/CD68+), and foreign body giant cells were quantified between 3-35 days. Uncoated polypropylene mesh elicited a dominant M1 response at the mesh fiber surface, which was decreased by each ECM coating type beginning at 7 days. The diminished M1 response was accompanied by a reduction in the number of foreign body giant cells at 14 and 35 days, though there was a minimal effect upon the number of M2 macrophages at any time. These results show that ECM coatings attenuate the M1 macrophage response and increase the M2/M1 ratio to polypropylene mesh in vivo. PMID:24856104

Mechanisms of integrin-vascular endothelial growth factor receptor cross-activation in angiogenesis.

PubMed

Mahabeleshwar, Ganapati H; Feng, Weiyi; Reddy, Kumar; Plow, Edward F; Byzova, Tatiana V

2007-09-14

The functional responses of endothelial cells are dependent on signaling from peptide growth factors and the cellular adhesion receptors, integrins. These include cell adhesion, migration, and proliferation, which, in turn, are essential for more complex processes such as formation of the endothelial tube network during angiogenesis. This study identifies the molecular requirements for the cross-activation between beta3 integrin and tyrosine kinase receptor 2 for vascular endothelial growth factor (VEGF) receptor (VEGFR-2) on endothelium. The relationship between VEGFR-2 and beta3 integrin appears to be synergistic, because VEGFR-2 activation induces beta3 integrin tyrosine phosphorylation, which, in turn, is crucial for VEGF-induced tyrosine phosphorylation of VEGFR-2. We demonstrate here that adhesion- and growth factor-induced beta3 integrin tyrosine phosphorylation are directly mediated by c-Src. VEGF-stimulated recruitment and activation of c-Src and subsequent beta3 integrin tyrosine phosphorylation are critical for interaction between VEGFR-2 and beta3 integrin. Moreover, c-Src mediates growth factor-induced beta3 integrin activation, ligand binding, beta3 integrin-dependent cell adhesion, directional migration of endothelial cells, and initiation of angiogenic programming in endothelial cells. Thus, the present study determines the molecular mechanisms and consequences of the synergism between 2 cell surface receptor systems, growth factor receptor and integrins, and opens new avenues for the development of pro- and antiangiogenic strategies.

A2A-D2 receptor-receptor interaction modulates gliotransmitter release from striatal astrocyte processes.

PubMed

Cervetto, Chiara; Venturini, Arianna; Passalacqua, Mario; Guidolin, Diego; Genedani, Susanna; Fuxe, Kjell; Borroto-Esquela, Dasiel O; Cortelli, Pietro; Woods, Amina; Maura, Guido; Marcoli, Manuela; Agnati, Luigi F

2017-01-01

Evidence for striatal A2A-D2 heterodimers has led to a new perspective on molecular mechanisms involved in schizophrenia and Parkinson's disease. Despite the increasing recognition of astrocytes' participation in neuropsychiatric disease vulnerability, involvement of striatal astrocytes in A2A and D2 receptor signal transmission has never been explored. Here, we investigated the presence of D2 and A2A receptors in isolated astrocyte processes prepared from adult rat striatum by confocal imaging; the effects of receptor activation were measured on the 4-aminopyridine-evoked release of glutamate from the processes. Confocal analysis showed that A2A and D2 receptors were co-expressed on the same astrocyte processes. Evidence for A2A-D2 receptor-receptor interactions was obtained by measuring the release of the gliotransmitter glutamate: D2 receptors inhibited the glutamate release, while activation of A2A receptors, per se ineffective, abolished the effect of D2 receptor activation. The synthetic D2 peptide VLRRRRKRVN corresponding to the receptor region involved in electrostatic interaction underlying A2A-D2 heteromerization abolished the ability of the A2A receptor to antagonize the D2 receptor-mediated effect. Together, the findings are consistent with heteromerization of native striatal astrocytic A2A-D2 receptors that via allosteric receptor-receptor interactions could play a role in the control of striatal glutamatergic transmission. These new findings suggest possible new pathogenic mechanisms and/or therapeutic approaches to neuropsychiatric disorders. © 2016 International Society for Neurochemistry.

Methodologies for screening of bacteria-carbohydrate interactions: anti-adhesive milk oligosaccharides as a case study.

PubMed

Lane, Jonathan A; Mariño, Karina; Rudd, Pauline M; Carrington, Stephen D; Slattery, Helen; Hickey, Rita M

2012-07-01

Many studies have demonstrated the capacity of glycan-based compounds to disrupt microbial binding to mucosal epithelia. Therefore, oligosaccharides have potential application in the prevention of certain bacterial diseases. However, current screening methods for the identification of anti-adhesive oligosaccharides have limitations: they are time-consuming and require large amounts of oligosaccharides. There is a need to develop analytical techniques which can quickly screen for, and structurally define, anti-adhesive oligosaccharides prior to using human cell line models of infection. Considering this, we have developed a rapid method for screening complex oligosaccharide mixtures for potential anti-adhesive activity against bacteria. Our approach involves the use of whole bacterial cells to "deplete" free oligosaccharides from solution. As a case study, the free oligosaccharides from the colostrum of Holstein Friesian cows were screened for interactions with whole Escherichia coli cells. Reductions in oligosaccharide concentrations were determined by High pH Anion Exchange Chromatography and Hydrophilic Interaction Liquid Chromatography (HILIC-HPLC). Oligosaccharide structures were confirmed by a combination of HILIC-HPLC, exoglycosidase digestion and off-line negative ion mode MS/MS. The depletion assay confirmed selective bacterial interaction with certain bovine oligosaccharides which in previous studies, by other methodologies, had been shown to interact with E. coli. In particular, the bacterial cells depleted the following oligosaccharides in a population dependent manner: 3'-sialyllactose, disialyllactose, and 6'-sialyllactosamine. The assay methodology was further validated by studies in which we demonstrated the inhibitory activity of 3'-sialyllactose, and a mixture of bovine colostrum oligosaccharides, on E. coli adhesion to differentiated HT-29 cells. Copyright © 2012 Elsevier B.V. All rights reserved.

Glutamine supplementation attenuates expressions of adhesion molecules and chemokine receptors on T cells in a murine model of acute colitis.

PubMed

Hou, Yu-Chen; Wu, Jin-Ming; Wang, Ming-Yang; Wu, Ming-Hsun; Chen, Kuen-Yuan; Yeh, Sung-Ling; Lin, Ming-Tsan

2014-01-01

Migration of T cells into the colon plays a major role in the pathogenesis in inflammatory bowel disease. This study investigated the effects of glutamine (Gln) supplementation on chemokine receptors and adhesion molecules expressed by T cells in mice with dextran sulfate sodium- (DSS-) induced colitis. C57BL/6 mice were fed either a standard diet or a Gln diet replacing 25% of the total nitrogen. After being fed the diets for 5 days, half of the mice from both groups were given 1.5% DSS in drinking water to induce colitis. Mice were killed after 5 days of DSS exposure. DSS colitis resulted in higher expression levels of P-selectin glycoprotein ligand- (PSGL-) 1, leukocyte function-associated antigen- (LFA-) 1, and C-C chemokine receptor type 9 (CCR9) by T helper (Th) and cytotoxic T (Tc) cells, and mRNA levels of endothelial adhesion molecules in colons were upregulated. Gln supplementation decreased expressions of PSGL-1, LFA-1, and CCR9 by Th cells. Colonic gene expressions of endothelial adhesion molecules were also lower in Gln-colitis mice. Histological finding showed that colon infiltrating Th cells were less in the DSS group with Gln administration. Gln supplementation may ameliorate the inflammation of colitis possibly via suppression of T cell migration.

Adhesion force interactions between cyclopentane hydrate and physically and chemically modified surfaces.

PubMed

Aman, Zachary M; Sloan, E Dendy; Sum, Amadeu K; Koh, Carolyn A

2014-12-07

Interfacial interactions between liquid-solid and solid-solid phases/surfaces are of fundamental importance to the formation of hydrate deposits in oil and gas pipelines. This work establishes the effect of five categories of physical and chemical modification to steel on clathrate hydrate adhesive force: oleamide, graphite, citric acid ester, nonanedithiol, and Rain-X anti-wetting agent. Hydrate adhesive forces were measured using a micromechanical force apparatus, under both dry and water-wet surface conditions. The results show that the graphite coating reduced hydrate-steel adhesion force by 79%, due to an increase in the water wetting angle from 42 ± 8° to 154 ± 7°. Two chemical surface coatings (nonanedithiol and the citric acid ester) induced rapid hydrate growth in the hydrate particles; nonanedithiol increased hydrate adhesive force by 49% from the baseline, while the citric acid ester coating reduced hydrate adhesion force by 98%. This result suggests that crystal growth may enable a strong adhesive pathway between hydrate and other crystalline structures, however this effect may be negated in cases where water-hydrocarbon interfacial tension is minimised. When a liquid water droplet was placed on the modified steel surfaces, the graphite and citric acid ester became less effective at reducing adhesive force. In pipelines containing a free water phase wetting the steel surface, chemical or physical surface modifications alone may be insufficient to eliminate hydrate deposition risk. In further tests, the citric acid ester reduced hydrate cohesive forces by 50%, suggesting mild activity as a hybrid anti-agglomerant suppressing both hydrate deposition and particle agglomeration. These results demonstrate a new capability to develop polyfunctional surfactants, which simultaneously limit the capability for hydrate particles to aggregate and deposit on the pipeline wall.

Perspectives on the Trypanosoma cruzi–host cell receptor interactions

PubMed Central

Villalta, Fernando; Scharfstein, Julio; Ashton, Anthony W.; Tyler, Kevin M.; Guan, Fangxia; Mukherjee, Shankar; Lima, Maria F.; Alvarez, Sandra; Weiss, Louis M.; Huang, Huan; Machado, Fabiana S.

2009-01-01

Chagas disease is caused by the parasite Trypanosoma cruzi. The critical initial event is the interaction of the trypomastigote form of the parasite with host receptors. This review highlights recent observations concerning these interactions. Some of the key receptors considered are those for thromboxane, bradykinin, and for the nerve growth factor TrKA. Other important receptors such as galectin-3, thrombospondin, and laminin are also discussed. Investigation into the molecular biology and cell biology of host receptors for T. cruzi may provide novel therapeutic targets. PMID:19283409

New advances in probing cell–extracellular matrix interactions

PubMed Central

2017-01-01

The extracellular matrix (ECM) provides structural and biochemical support to cells within tissues. An emerging body of evidence has established that the ECM plays a key role in cell mechanotransduction – the study of coupling between mechanical inputs and cellular phenotype – through either mediating transmission of forces to the cells, or presenting mechanical cues that guide cellular behaviors. Recent progress in cell mechanotransduction research has been facilitated by advances of experimental tools, particularly microtechnologies, engineered biomaterials, and imaging and analytical methods. Microtechnologies have enabled the design and fabrication of controlled physical microenvironments for the study and measurement of cell–ECM interactions. Advances in engineered biomaterials have allowed researchers to develop synthetic ECMs that mimic tissue microenvironments and investigate the impact of altered physicochemical properties on various cellular processes. Finally, advanced imaging and spectroscopy techniques have facilitated the visualization of the complex interaction between cells and ECM in vitro and in living tissues. This review will highlight the application of recent innovations in these areas to probing cell–ECM interactions. We believe cross-disciplinary approaches, combining aspects of the different technologies reviewed here, will inspire innovative ideas to further elucidate the secrets of ECM-mediated cell control. PMID:28352896

Microenvironment interactions and B-cell receptor signaling in Chronic Lymphocytic Leukemia: implications for disease pathogenesis and treatment

PubMed Central

ten Hacken, Elisa; Burger, Jan A.

2015-01-01

Chronic Lymphocytic Leukemia (CLL) is a malignancy of mature B lymphocytes which are highly dependent on interactions with the tissue microenvironment for their survival and proliferation. Critical components of the microenvironment are monocyte-derived nurselike cells (NLCs), mesenchymal stromal cells, T cells and NK cells, which communicate with CLL cells through a complex network of adhesion molecules, chemokine receptors, tumor necrosis factor (TNF) family members, and soluble factors. (Auto-) antigens and/or autonomous mechanisms activate the B-cell receptor (BCR) and its downstream signaling cascade in secondary lymphatic tissues, playing a central pathogenetic role in CLL. Novel small molecule inhibitors, including the Bruton’s tyrosine kinase (BTK) inhibitor ibrutinib and the phosphoinositide-3-kinase delta (PI3Kδ) inhibitor idelalisib, target BCR signaling and have become the most successful new therapeutics in this disease. We here review the cellular and molecular characteristics of CLL cells, and discuss the cellular components and key pathways involved in the cross-talk with their microenvironment. We also highlight the relevant novel treatment strategies, focusing on immunomodulatory agents and BCR signaling inhibitors and how these treatments disrupt CLL-microenvironment interactions. PMID:26193078

Dissecting the Impact of Matrix Anchorage and Elasticity in Cell Adhesion

PubMed Central

Pompe, Tilo; Glorius, Stefan; Bischoff, Thomas; Uhlmann, Ina; Kaufmann, Martin; Brenner, Sebastian; Werner, Carsten

2009-01-01

Abstract Extracellular matrices determine cellular fate decisions through the regulation of intracellular force and stress. Previous studies suggest that matrix stiffness and ligand anchorage cause distinct signaling effects. We show herein how defined noncovalent anchorage of adhesion ligands to elastic substrates allows for dissection of intracellular adhesion signaling pathways related to matrix stiffness and receptor forces. Quantitative analysis of the mechanical balance in cell adhesion using traction force microscopy revealed distinct scalings of the strain energy imparted by the cells on the substrates dependent either on matrix stiffness or on receptor force. Those scalings suggested the applicability of a linear elastic theoretical framework for the description of cell adhesion in a certain parameter range, which is cell-type-dependent. Besides the deconvolution of biophysical adhesion signaling, site-specific phosphorylation of focal adhesion kinase, dependent either on matrix stiffness or on receptor force, also demonstrated the dissection of biochemical signaling events in our approach. Moreover, the net contractile moment of the adherent cells and their strain energy exerted on the elastic substrate was found to be a robust measure of cell adhesion with a unifying power-law scaling exponent of 1.5 independent of matrix stiffness. PMID:19843448

Biomimetic modified clinical-grade POSS-PCU nanocomposite polymer for bypass graft applications: a preliminary assessment of endothelial cell adhesion and haemocompatibility.

PubMed

Solouk, Atefeh; Cousins, Brian G; Mirahmadi, Fereshteh; Mirzadeh, Hamid; Nadoushan, Mohammad Reza Jalali; Shokrgozar, Mohammad Ali; Seifalian, Alexander M

2015-01-01

To date, there are no small internal diameter (<5mm) vascular grafts that are FDA approved for clinical use due to high failure rates from thrombosis and unwanted cell proliferation. The ideal conditions to enhance bioengineered grafts would be the blood contacting lumen of the bypass graft fully covered by endothelial cells (ECs). As a strategy towards this aim, we hypothesized that by immobilising biomolecules on the surface of the polyhedral oligomeric silsesquioxane-poly(carbonate-urea)urethane (POSS-PCU) nanocomposite polymers, which contain binding sites and ligands for cell surface receptors similar to extracellular matrix (ECM) will positively influence the attachment and proliferation of ECs. Since, the surface of POSS-PCU is inert and not directly suitable for immobilisation of biomolecules, plasma graft polymerisation is a suitable method to modify the surface properties ready for immobilisation and biofunctionalisation. POSS-PCU was activated by plasma treatment in air/O2 to from hydroperoxides (-OH, -OOH), and then carboxylated via plasma polymerisation of a 30% acrylic acid solution (Poly-AA) using a two-step plasma treatment (TSPT) process. Collagen type I, a major component of ECM, was covalently immobilised to mimic the ECM structures to ECs (5mg/ml) using a two-step chemical reaction using EDC chemistry. Successful immobilisation of poly-AA and collagen on to the nanocomposites was confirmed using Toluidine Blue staining and the Bradford assay. Un-treated POSS-PCU served as a simple control. The impact of collagen grafting on the physical, mechanical and biological properties of POSS-PCU was evaluated via contact angle (θ) measurements, scanning electron microscopy (SEM), atomic force microscopy (AFM), dynamic mechanical thermal analysis (DMTA), ECs adhesion and proliferation followed by platelet adhesion and haemolysis ratio (HR) tests. Poly-AA content on each of the plasma treated nanocomposite films increased on Low, Med and High samples due

Energy of adhesion of human T cells to adsorption layers of monoclonal antibodies measured by a film trapping technique.

PubMed Central

Ivanov, I B; Hadjiiski, A; Denkov, N D; Gurkov, T D; Kralchevsky, P A; Koyasu, S

1998-01-01

A novel method for studying the interaction of biological cells with interfaces (e.g., adsorption monolayers of antibodies) is developed. The method is called the film trapping technique because the cell is trapped within an aqueous film of equilibrium thickness smaller than the cell diameter. A liquid film of uneven thickness is formed around the trapped cell. When observed in reflected monochromatic light, this film exhibits an interference pattern of concentric bright and dark fringes. From the radii of the fringes one can restore the shape of interfaces and the cell. Furthermore, one can calculate the adhesive energy between the cell membrane and the aqueous film surface (which is covered by a layer of adsorbed proteins and/or specific ligands), as well as the disjoining pressure, representing the force of interaction per unit area of the latter film. The method is applied to two human T cell lines: Jurkat and its T cell receptor negative (TCR-) derivative. The interaction of these cells with monolayers of three different monoclonal antibodies adsorbed at a water-air interface is studied. The results show that the adhesive energy is considerable (above 0.5 mJ/m2) when the adsorption monolayer contains antibodies acting as specific ligands for the receptors expressed on the cell surface. In contrast, the adhesive energy is close to zero in the absence of such a specific ligand-receptor interaction. In principle, the method can be applied to the study of the interaction of a variety of biological cells (B cells, natural killer cells, red blood cells, etc.) with adsorption monolayers of various biologically active molecules. In particular, film trapping provides a tool for the gentle micromanipulation of cells and for monitoring of processes (say the activation of a T lymphocyte) occurring at the single-cell level. PMID:9649417

Chronic Trypanosoma cruzi-elicited cardiomyopathy: from the discovery to the proposal of rational therapeutic interventions targeting cell adhesion molecules and chemokine receptors--how to make a dream come true.

PubMed

Lannes-Vieira, Joseli; Silverio, Jaline Coutinho; Pereira, Isabela Resende; Vinagre, Nathália Ferreira; Carvalho, Cristiano Marcelo Espinola; Paiva, Cláudia Neto; Silva da, Andréa Alice

2009-07-01

One hundred years ago, Carlos Chagas discovered a new disease, the American trypanosomiasis. Chagas and co-workers later characterised the disease's common manifestation, chronic cardiomyopathy, and suggested that parasitic persistence coupled with inflammation was the key underlying pathogenic mechanism. Better comprehension of the molecular mechanisms leading to clinical heart afflictions is a prerequisite to developing new therapies that ameliorate inflammation and improve heart function without hampering parasite control. Here, we review recent data showing that distinct cell adhesion molecules, chemokines and chemokine receptors participate in anti-parasite immunity and/or detrimental leukocyte trafficking to the heart. Moreover, we offer evidence that CC-chemokine receptors may be attractive therapeutic targets aiming to regain homeostatic balance in parasite/host interaction thereby improving prognosis, supporting that it is becoming a non-phantasious proposal.

Analysis of Ethylene Receptor Interactions by Co-immunoprecipitation Assays.

PubMed

Gao, Zhiyong; Schaller, G Eric

2017-01-01

Ethylene receptors are predominantly localized to the endoplasmic reticulum (ER) membrane, and coordinate ethylene signal output through protein-protein interactions with each other and additional signaling components. Here, we describe a co-immunoprecipitation (Co-IP) assay based on the use of the Tandem Affinity Purification (TAP) tag to examine the interactions of ethylene receptors in plant extracts. Human IgG-agarose beads are used to pull down TAP-tagged versions of the protein of interest from detergent extracts of Arabidopsis membranes, and the precipitate then is analyzed immunologically for co-purification of the ethylene receptors. This method has been successfully used to examine interactions of the receptors with each other as well as with the Raf-like kinase CTR1.

Dynamic expression patterns of ECM molecules in the developing mouse olfactory pathway

PubMed Central

Shay, Elaine L.; Greer, Charles A.; Treloar, Helen B.

2009-01-01

Olfactory sensory neuron (OSN) axons follow stereotypic spatio-temporal paths in the establishment of the olfactory pathway. Extracellular matrix (ECM) molecules are expressed early in the developing pathway and are proposed to have a role in its initial establishment. During later embryonic development, OSNs sort out and target specific glomeruli to form precise, complex topographic projections. We hypothesized that ECM cues may help to establish this complex topography. The aim of this study was to characterize expression of ECM molecules during the period of glomerulogenesis, when synaptic contacts are forming. We examined expression of laminin-1, perlecan, tenascin-C and CSPGs and found a coordinated pattern of expression of these cues in the pathway. These appear to restrict axons to the pathway while promoting axon outgrowth within. Thus, ECM molecules are present in dynamic spatio-temporal positions to affect OSN axons as they navigate to the olfactory bulb and establish synapses. PMID:18570250

Three-dimensional multi-scale model of deformable platelets adhesion to vessel wall in blood flow

PubMed Central

Wu, Ziheng; Xu, Zhiliang; Kim, Oleg; Alber, Mark

2014-01-01

When a blood vessel ruptures or gets inflamed, the human body responds by rapidly forming a clot to restrict the loss of blood. Platelets aggregation at the injury site of the blood vessel occurring via platelet–platelet adhesion, tethering and rolling on the injured endothelium is a critical initial step in blood clot formation. A novel three-dimensional multi-scale model is introduced and used in this paper to simulate receptor-mediated adhesion of deformable platelets at the site of vascular injury under different shear rates of blood flow. The novelty of the model is based on a new approach of coupling submodels at three biological scales crucial for the early clot formation: novel hybrid cell membrane submodel to represent physiological elastic properties of a platelet, stochastic receptor–ligand binding submodel to describe cell adhesion kinetics and lattice Boltzmann submodel for simulating blood flow. The model implementation on the GPU cluster significantly improved simulation performance. Predictive model simulations revealed that platelet deformation, interactions between platelets in the vicinity of the vessel wall as well as the number of functional GPIbα platelet receptors played significant roles in platelet adhesion to the injury site. Variation of the number of functional GPIbα platelet receptors as well as changes of platelet stiffness can represent effects of specific drugs reducing or enhancing platelet activity. Therefore, predictive simulations can improve the search for new drug targets and help to make treatment of thrombosis patient-specific. PMID:24982253

Bio-inspired reversible underwater adhesive.

PubMed

Zhao, Yanhua; Wu, Yang; Wang, Liang; Zhang, Manman; Chen, Xuan; Liu, Minjie; Fan, Jun; Liu, Junqiu; Zhou, Feng; Wang, Zuankai

2017-12-20

The design of smart surfaces with switchable adhesive properties in a wet environment has remained a challenge in adhesion science and materials engineering. Despite intense demands in various industrial applications and exciting progress in mimicking the remarkable wet adhesion through the delicate control of catechol chemistry, polyelectrolyte complex, and supramolecular architectures, the full recapitulation of nature's dynamic function is limited. Here, we show a facile approach to synthesize bioinspired adhesive, which entails the reversible, tunable, and fast regulation of the wet adhesion on diverse surfaces. The smart wet adhesive takes advantage of the host-guest molecular interaction and the adhesive nature of catechol chemistry, as well as the responsive polymer, allowing for screening and activation of the interfacial interaction simply by a local temperature trigger in an on-demand manner. Our work opens up an avenue for the rational design of bioinspired adhesives with performances even beyond nature.

Growth Factor-Reinforced ECM Fabricated from Chemically Hypoxic MSC Sheet with Improved In Vivo Wound Repair Activity.

PubMed

Du, Hui-Cong; Jiang, Lin; Geng, Wen-Xin; Li, Jing; Zhang, Rui; Dang, Jin-Ge; Shu, Mao-Guo; Li, Li-Wen

2017-01-01

MSC treatment can promote cutaneous wound repair through multiple mechanisms, and paracrine mediators secreted by MSC are responsible for most of its therapeutic benefits. Recently, MSC sheet composed of live MSCs and their secreted ECMs was reported to promote wound healing; however, whether its ECM alone could accelerate wound closure remained unknown. In this study, Nc-ECM and Cc-ECM were prepared from nonconditioned and CoCl 2 -conditioned MSC sheets, respectively, and their wound healing properties were evaluated in a mouse model of full-thickness skin defect. Our results showed that Nc-ECM can significantly promote wound repair through early adipocyte recruitment, rapid reepithelialization, enhanced granulation tissue growth, and augmented angiogenesis. Moreover, conditioning of MSC sheet with CoCl 2 dramatically enriched its ECM with collagen I, collagen III, TGF- β 1, VEGF, and bFGF via activation of HIF-1 α and hence remarkably improved its ECM's in vivo wound healing potency. All the Cc-ECM-treated wounds completely healed on day 7, while Nc-ECM-treated wounds healed about 85.0% ± 8.6%, and no-treatment wounds only healed 69.8% ± 9.6% ( p < 0.05). Therefore, we believe that such growth factor-reinforced ECM fabricated from chemically hypoxic MSC sheet has the potential for clinical translation and will lead to a MSC-derived, cost-effective, bankable biomaterial for wound management.

Human mesenchymal stem cell osteoblast differentiation, ECM deposition, and biomineralization on PAH/PAA polyelectrolyte multilayers.

PubMed

Pattabhi, Sudhakara Rao; Lehaf, Ali M; Schlenoff, Joseph B; Keller, Thomas C S

2015-05-01

Polyelectrolyte multilayer (PEMU) coatings built layer by layer with alternating pairs of polyelectrolytes can be tuned to improve cell interactions with surfaces and may be useful as biocompatible coatings to improve fixation between implants and tissues. Here, we show that human mesenchymal stromal cells (hMSCs) induced with bone differentiation medium (BDM) to become osteoblasts biomineralize crosslinked PEMUs built with the polycation poly(allylamine hydrochloride) (PAH) and the polyanion poly(acrylic acid) (PAA). Degrees of hMSC osteoblast differentiation and surface biomineralization on the smooth PAH-terminated PEMUs (PAH-PEMUs) and microstructured PAA-terminated PEMUs (PAA-PEMUs) reflect differences in cell-deposited extracellular matrix (ECM). BDM-induced hMSCs expressed higher levels of the early osteoblast differentiation marker alkaline phosphatase and collagen 1 (COL1) sooner on PAA-PEMUs than on PAH-PEMUs. Cells on both types of PEMUs proceeded to express the later stage osteoblast differentiation marker bone sialoprotein (BSP), but the BDM-induced cells organized a more amorphous Collagen I and denser BSP localization on PAA-PEMUs than on PAH-PEMUs. These ECM properties correlated with greater biomineralization on the PAA-PEMUs than on PAH-PEMUs. Together, these results confirm the suitability of PAH/PAA PEMUs as a substrate for hMSC osteogenesis and highlight the importance of substrate effects on ECM organization and BSP presentation on biomineralization. © 2014 Wiley Periodicals, Inc.

The N terminus of SKAP55 enables T cell adhesion to TCR and integrin ligands via distinct mechanisms

PubMed Central

Ophir, Michael J.; Liu, Beiyun C.

2013-01-01

The T cell receptor (TCR) triggers the assembly of “SLP-76 microclusters,” which mediate signals required for T cell activation. In addition to regulating integrin activation, we show that Src kinase–associated phosphoprotein of 55 kD (SKAP55) is required for microcluster persistence and movement, junctional stabilization, and integrin-independent adhesion via the TCR. These functions require the dimerization of SKAP55 and its interaction with the adaptor adhesion and degranulation-promoting adaptor protein (ADAP). A “tandem dimer” containing two ADAP-binding SKAP55 Src homology 3 (SH3) domains stabilized SLP-76 microclusters and promoted T cell adhesion via the TCR, but could not support adhesion to integrin ligands. Finally, the SKAP55 dimerization motif (DM) enabled the coimmunoprecipitation of the Rap1-dependent integrin regulator Rap1-GTP–interacting adaptor molecule (RIAM), the recruitment of talin into TCR-induced adhesive junctions, and “inside-out” signaling to β1 integrins. Our data indicate that SKAP55 dimers stabilize SLP-76 microclusters, couple SLP-76 to the force-generating systems responsible for microcluster movement, and enable adhesion via the TCR by mechanisms independent of RIAM, talin, and β1 integrins. PMID:24368808

Mathematical modeling of cell adhesion in shear flow: application to targeted drug delivery in inflammation and cancer metastasis.

PubMed

Jadhav, Sameer; Eggleton, Charles D; Konstantopoulos, Konstantinos

2007-01-01

Cell adhesion plays a pivotal role in diverse biological processes that occur in the dynamic setting of the vasculature, including inflammation and cancer metastasis. Although complex, the naturally occurring processes that have evolved to allow for cell adhesion in the vasculature can be exploited to direct drug carriers to targeted cells and tissues. Fluid (blood) flow influences cell adhesion at the mesoscale by affecting the mechanical response of cell membrane, the intercellular contact area and collisional frequency, and at the nanoscale level by modulating the kinetics and mechanics of receptor-ligand interactions. Consequently, elucidating the molecular and biophysical nature of cell adhesion requires a multidisciplinary approach involving the synthesis of fundamentals from hydrodynamic flow, molecular kinetics and cell mechanics with biochemistry/molecular cell biology. To date, significant advances have been made in the identification and characterization of the critical cell adhesion molecules involved in inflammatory disorders, and, to a lesser degree, in cancer metastasis. Experimental work at the nanoscale level to determine the lifetime, interaction distance and strain responses of adhesion receptor-ligand bonds has been spurred by the advent of atomic force microscopy and biomolecular force probes, although our current knowledge in this area is far from complete. Micropipette aspiration assays along with theoretical frameworks have provided vital information on cell mechanics. Progress in each of the aforementioned research areas is key to the development of mathematical models of cell adhesion that incorporate the appropriate biological, kinetic and mechanical parameters that would lead to reliable qualitative and quantitative predictions. These multiscale mathematical models can be employed to predict optimal drug carrier-cell binding through isolated parameter studies and engineering optimization schemes, which will be essential for developing

Analysis of a vinculin homolog in a sponge (phylum Porifera) reveals that vertebrate-like cell adhesions emerged early in animal evolution.

PubMed

Miller, Phillip W; Pokutta, Sabine; Mitchell, Jennyfer M; Chodaparambil, Jayanth V; Clarke, D Nathaniel; Nelson, William; Weis, William I; Nichols, Scott A

2018-06-07

The evolution of cell adhesion mechanisms in animals facilitated the assembly of organized multicellular tissues. Studies in traditional animal models have revealed two predominant adhesion structures, the adherens junction (AJ) and focal adhesions (FAs), which are involved in the attachment of neighboring cells to each other and to the secreted extracellular matrix (ECM), respectively. The AJ (containing cadherins and catenins) and FAs (comprising integrins, talin, and paxillin) differ in protein composition, but both junctions contain the actin-binding protein vinculin. The near ubiquity of these structures in animals suggests that AJ and FAs evolved early, possibly coincident with multicellularity. However, a challenge to this perspective is that previous studies of sponges-a divergent animal lineage-indicate that their tissues are organized primarily by an alternative, sponge-specific cell adhesion mechanism called "aggregation factor." In this study, we examined the structure, biochemical properties, and tissue localization of a vinculin ortholog in the sponge Oscarella pearsei ( Op ). Our results indicate that Op vinculin localizes to both cell-cell and cell-ECM contacts and has biochemical and structural properties similar to those of vertebrate vinculin. We propose that Op vinculin played a role in cell adhesion and tissue organization in the last common ancestor of sponges and other animals. These findings provide compelling evidence that sponge tissues are indeed organized like epithelia in other animals and support the notion that AJ- and FA-like structures extend to the earliest periods of animal evolution. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.

Towards a defined ECM and small molecule based monolayer culture system for the expansion of mouse and human intestinal stem cells.

PubMed

Tong, Zhixiang; Martyn, Keir; Yang, Andy; Yin, Xiaolei; Mead, Benjamin E; Joshi, Nitin; Sherman, Nicholas E; Langer, Robert S; Karp, Jeffrey M

2018-02-01

Current ISC culture systems face significant challenges such as animal-derived or undefined matrix compositions, batch-to-batch variability (e.g. Matrigel-based organoid culture), and complexity of assaying cell aggregates such as organoids which renders the research and clinical translation of ISCs challenging. Here, through screening for suitable ECM components, we report a defined, collagen based monolayer culture system that supports the growth of mouse and human intestinal epithelial cells (IECs) enriched for an Lgr5 + population comparable or higher to the levels found in a standard Matrigel-based organoid culture. The system, referred to as the Bolstering Lgr5 Transformational (BLT) Sandwich culture, comprises a collagen IV-coated porous substrate and a collagen I gel overlay which sandwich an IEC monolayer in between. The distinct collagen cues synergistically regulate IEC attachment, proliferation, and Lgr5 expression through maximizing the engagement of distinct cell surface adhesion receptors (i.e. integrin α2β1, integrin β4) and cell polarity. Further, we apply our BLT Sandwich system to identify that the addition of a bone morphogenetic protein (BMP) receptor inhibitor (LDN-193189) improves the expansion of Lgr5-GFP + cells from mouse small intestinal crypts by nearly 2.5-fold. Notably, the BLT Sandwich culture is capable of expanding human-derived IECs with higher LGR5 mRNA levels than conventional Matrigel culture, providing superior expansion of human LGR5 + ISCs. Considering the key roles Lgr5 + ISCs play in intestinal epithelial homeostasis and regeneration, we envision that our BLT Sandwich culture system holds great potential for understanding and manipulating ISC biology in vitro (e.g. for modeling ISC-mediated gut diseases) or for expanding a large number of ISCs for clinical utility (e.g. for stem cell therapy). Copyright © 2017 Elsevier Ltd. All rights reserved.

N-glycosylation at the SynCAM (synaptic cell adhesion molecule) immunoglobulin interface modulates synaptic adhesion.

PubMed

Fogel, Adam I; Li, Yue; Giza, Joanna; Wang, Qing; Lam, Tukiet T; Modis, Yorgo; Biederer, Thomas

2010-11-05

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn(60). Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn(70)/Asn(104) flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn(60) reduces adhesion, N-glycans at Asn(70)/Asn(104) of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion.

N-Glycosylation at the SynCAM (Synaptic Cell Adhesion Molecule) Immunoglobulin Interface Modulates Synaptic Adhesion*

PubMed Central

Fogel, Adam I.; Li, Yue; Giza, Joanna; Wang, Qing; Lam, TuKiet T.; Modis, Yorgo; Biederer, Thomas

2010-01-01

Select adhesion molecules connect pre- and postsynaptic membranes and organize developing synapses. The regulation of these trans-synaptic interactions is an important neurobiological question. We have previously shown that the synaptic cell adhesion molecules (SynCAMs) 1 and 2 engage in homo- and heterophilic interactions and bridge the synaptic cleft to induce presynaptic terminals. Here, we demonstrate that site-specific N-glycosylation impacts the structure and function of adhesive SynCAM interactions. Through crystallographic analysis of SynCAM 2, we identified within the adhesive interface of its Ig1 domain an N-glycan on residue Asn60. Structural modeling of the corresponding SynCAM 1 Ig1 domain indicates that its glycosylation sites Asn70/Asn104 flank the binding interface of this domain. Mass spectrometric and mutational studies confirm and characterize the modification of these three sites. These site-specific N-glycans affect SynCAM adhesion yet act in a differential manner. Although glycosylation of SynCAM 2 at Asn60 reduces adhesion, N-glycans at Asn70/Asn104 of SynCAM 1 increase its interactions. The modification of SynCAM 1 with sialic acids contributes to the glycan-dependent strengthening of its binding. Functionally, N-glycosylation promotes the trans-synaptic interactions of SynCAM 1 and is required for synapse induction. These results demonstrate that N-glycosylation of SynCAM proteins differentially affects their binding interface and implicate post-translational modification as a mechanism to regulate trans-synaptic adhesion. PMID:20739279

Receptors, mediators, and mechanisms involved in bacterial sepsis and septic shock.

PubMed

Van Amersfoort, Edwin S; Van Berkel, Theo J C; Kuiper, Johan

2003-07-01

Bacterial sepsis and septic shock result from the overproduction of inflammatory mediators as a consequence of the interaction of the immune system with bacteria and bacterial wall constituents in the body. Bacterial cell wall constituents such as lipopolysaccharide, peptidoglycans, and lipoteichoic acid are particularly responsible for the deleterious effects of bacteria. These constituents interact in the body with a large number of proteins and receptors, and this interaction determines the eventual inflammatory effect of the compounds. Within the circulation bacterial constituents interact with proteins such as plasma lipoproteins and lipopolysaccharide binding protein. The interaction of the bacterial constituents with receptors on the surface of mononuclear cells is mainly responsible for the induction of proinflammatory mediators by the bacterial constituents. The role of individual receptors such as the toll-like receptors and CD14 in the induction of proinflammatory cytokines and adhesion molecules is discussed in detail. In addition, the roles of a number of other receptors that bind bacterial compounds such as scavenger receptors and their modulating role in inflammation are described. Finally, the therapies for the treatment of bacterial sepsis and septic shock are discussed in relation to the action of the aforementioned receptors and proteins.

Osteopontin and fibronectin levels are decreased in vitreous of autoimmune uveitis and retinal expression of both proteins indicates ECM re-modeling.

PubMed

Deeg, Cornelia A; Eberhardt, Christina; Hofmaier, Florian; Amann, Barbara; Hauck, Stefanie M

2011-01-01

Autoimmune uveitis is an intraocular inflammation that arises through autoreactive T-cells attacking the inner eye, eventually leading to blindness. However, the contributing molecular pathomechanisms within the affected tissues remain as yet elusive. The extracellular matrix (ECM) is a highly dynamic structure that varies tremendously and influences the encompassing tissue. In order to assess ECM re-modeling in autoimmune uveitis, we investigated the expression of ECM molecules fibronectin and osteopontin in vitreous and retina samples. This was carried out in the only spontaneous animal model for human autoimmue uveitis, namely equine recurrent uveitis (ERU) that resembles the human disease in clinical as well as in immunopathological aspects. ERU is a naturally occurring autoimmune disease in horses that develops frequently and has already proved its value to study disease-related pathomechanisms. Western blot analysis of fibronectin and osteopontin in healthy and uveitic vitreous revealed significant reduction of both proteins in uveitis. Immunohistochemical expression of fibronectin in healthy retinas was restricted to the inner limiting membrane abutting vimentin positive Müller cell endfeet, while in uveitic sections, a disintegration of the ILM was observed changing the fibronectin expression to a dispersed pattern extending toward the vitreous. Retinal expression of osteopontin in control tissue was found in a characteristic Müller cell pattern illustrated by co-localization with vimentin. In uveitic retinas, the immunoreactivity of osteopontin in gliotic Müller cells was almost absent. The ability of Müller cells to express fibronectin and osteopontin was additionally shown by immunocytochemistry of primary cultured equine Müller cells and the equine Müller cell line eqMC-7. In conclusion, severe ECM re-modeling in autoimmune uveitis reported here, might affect the adhesive function of fibronectin and thus the anchoring of Müller cell endfeet to

MAPs/bFGF-PLGA microsphere composite-coated titanium surfaces promote increased adhesion and proliferation of fibroblasts.

PubMed

Wang, Zhongshan; Wu, Guofeng; Bai, Shizhu; Feng, Zhihong; Dong, Yan; Zhou, Jian; Qin, Haiyan; Zhao, Yimin

2014-06-01

Infection and epithelial downgrowth are two major problems with maxillofacial transcutaneous implants, and both are mainly due to lack of stable closure of soft tissues at transcutaneous sites. Fibroblasts have been shown to play a key role in the formation of biological seals. In this work, titanium (Ti) model surfaces were coated with mussel adhesive proteins (MAPs) utilizing its unique adhesion ability on diverse inorganic and organic surfaces in wet environments. Prepared basic fibroblast growth factor (bFGF)-poly(lactic-co-glycolic acid) (PLGA) microspheres can be easily synthesized and combined onto MAPs-coated Ti surfaces, due to the negative surface charges of microspheres in aqueous solution, which is in contrast to the positive charges of MAPs. Titanium model surfaces were divided into three groups. Group A: MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces. Group B: MAPs-coated Ti surfaces. Group C: uncoated Ti surfaces. The effects of coated Ti surfaces on adhesion of fibroblasts, cytoskeletal organization, proliferation, and extracellular matrix (ECM)-related gene expressions were examined. The results revealed increased adhesion (P < 0.05), enhanced actin cytoskeletal organization, and up-regulated ECM-related gene expressions in groups A and B compared with group C. Increased proliferation of fibroblasts during five days of incubation was observed in group A compared with groups B and C (P < 0.05). Collectively, the results from this in vitro study demonstrated that MAPs/bFGF-PLGA microspheres composite-coated Ti surfaces had the ability to increase fibroblast functionality. In addition, MAPs/bFGF-PLGA microsphere composite-coated Ti surfaces should be studied further as a method of promoting formation of stable biological seals around transcutaneous sites.

New functions and signaling mechanisms for the class of adhesion G protein–coupled receptors

PubMed Central

Liebscher, Ines; Ackley, Brian; Araç, Demet; Ariestanti, Donna M.; Aust, Gabriela; Bae, Byoung-il; Bista, Bigyan R.; Bridges, James P.; Duman, Joseph G.; Engel, Felix B.; Giera, Stefanie; Goffinet, André M.; Hall, Randy A.; Hamann, Jörg; Hartmann, Nicole; Lin, Hsi-Hsien; Liu, Mingyao; Luo, Rong; Mogha, Amit; Monk, Kelly R.; Peeters, Miriam C.; Prömel, Simone; Ressl, Susanne; Schiöth, Helgi B.; Sigoillot, Séverine M.; Song, Helen; Talbot, William S.; Tall, Gregory G.; White, James P.; Wolfrum, Uwe; Xu, Lei; Piao, Xianhua

2014-01-01

The class of adhesion G protein–coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix—a structural feature of all GPCRs—the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region. In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis–inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site (GPS) motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF are associated non-covalently as a heterodimer at the plasma membrane. While the biological function of the GAIN domain–mediated autocleavage is not fully understood, mounting evidence suggests that the NTF and CTF possess distinct biological activities in addition to their function as a receptor unit. We discuss recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs. PMID:25424900

Harnessing nanotopography and integrin-matrix interactions to influence stem cell fate

NASA Astrophysics Data System (ADS)

Dalby, Matthew J.; Gadegaard, Nikolaj; Oreffo, Richard O. C.

2014-06-01

Stem cells respond to nanoscale surface features, with changes in cell growth and differentiation mediated by alterations in cell adhesion. The interaction of nanotopographical features with integrin receptors in the cells' focal adhesions alters how the cells adhere to materials surfaces, and defines cell fate through changes in both cell biochemistry and cell morphology. In this Review, we discuss how cell adhesions interact with nanotopography, and we provide insight as to how materials scientists can exploit these interactions to direct stem cell fate and to understand how the behaviour of stem cells in their niche can be controlled. We expect knowledge gained from the study of cell-nanotopography interactions to accelerate the development of next-generation stem cell culture materials and implant interfaces, and to fuel discovery of stem cell therapeutics to support regenerative therapies.

ECM-Based Materials in Cardiovascular Applications: Inherent Healing Potential and Augmentation of Native Regenerative Processes

PubMed Central

Piterina, Anna V.; Cloonan, Aidan J.; Meaney, Claire L.; Davis, Laura M.; Callanan, Anthony; Walsh, Michael T.; McGloughlin, Tim M.

2009-01-01

The in vivo healing process of vascular grafts involves the interaction of many contributing factors. The ability of vascular grafts to provide an environment which allows successful accomplishment of this process is extremely difficult. Poor endothelisation, inflammation, infection, occlusion, thrombosis, hyperplasia and pseudoaneurysms are common issues with synthetic grafts in vivo. Advanced materials composed of decellularised extracellular matrices (ECM) have been shown to promote the healing process via modulation of the host immune response, resistance to bacterial infections, allowing re-innervation and reestablishing homeostasis in the healing region. The physiological balance within the newly developed vascular tissue is maintained via the recreation of correct biorheology and mechanotransduction factors including host immune response, infection control, homing and the attraction of progenitor cells and infiltration by host tissue. Here, we review the progress in this tissue engineering approach, the enhancement potential of ECM materials and future prospects to reach the clinical environment. PMID:20057951

Structural Insights into Selective Ligand-Receptor Interactions Leading to Receptor Inactivation Utilizing Selective Melanocortin 3 Receptor Antagonists.

PubMed

Cai, Minying; Marelli, Udaya Kiran; Mertz, Blake; Beck, Johannes G; Opperer, Florian; Rechenmacher, Florian; Kessler, Horst; Hruby, Victor J

2017-08-15

Systematic N-methylated derivatives of the melanocortin receptor ligand, SHU9119, lead to multiple binding and functional selectivity toward melanocortin receptors. However, the relationship between N-methylation-induced conformational changes in the peptide backbone and side chains and melanocortin receptor selectivity is still unknown. We conducted comprehensive conformational studies in solution of two selective antagonists of the third isoform of the melanocortin receptor (hMC3R), namely, Ac-Nle-c[Asp-NMe-His 6 -d-Nal(2') 7 -NMe-Arg 8 -Trp 9 -Lys]-NH 2 (15) and Ac-Nle-c[Asp-His 6 -d-Nal(2') 7 -NMe-Arg 8 -NMe-Trp 9 -NMe-Lys]-NH 2 (17). It is known that the pharmacophore (His 6 -DNal 7 -Arg 8 -Trp 9 ) of the SHU-9119 peptides occupies a β II-turn-like region with the turn centered about DNal 7 -Arg 8 . The analogues with hMC3R selectivity showed distinct differences in the spatial arrangement of the Trp 9 side chains. In addition to our NMR studies, we also carried out molecular-level interaction studies of these two peptides at the homology model of hMC3R. Earlier chimeric human melanocortin 3 receptor studies revealed insights regarding the binding and functional sites of hMC3R selectivity. Upon docking of peptides 15 and 17 to the binding pocket of hMC3R, it was revealed that Arg 8 and Trp 9 side chains are involved in a majority of the interactions with the receptor. While Arg 8 forms polar contacts with D154 and D158 of hMC3R, Trp 9 utilizes π-π stacking interactions with F295 and F298, located on the transmembrane domain of hMC3R. It is hypothesized that as the frequency of Trp 9 -hMC3R interactions decrease, antagonistic activity increases. The absence of any interactions of the N-methyl groups with hMC3R suggests that their primary function is to modulate backbone conformations of the ligands.

Mycophenolate mofetil increases adhesion capacity of tumor cells in vitro.

PubMed

Blaheta, Roman A; Bogossian, Harilaos; Beecken, Wolf-Dietrich; Jonas, Dietger; Hasenberg, Christoph; Makarevic, Jasmina; Ogbomo, Henry; Bechstein, Wolf O; Oppermann, Elsie; Leckel, Kerstin; Cinatl, Jindrich

2003-12-27

The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the immunoglobulin superfamily which, however, act as homophilic binding elements. Because decrease of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumors. The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell monolayer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, Western blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides. MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 microM, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumor cell adhesion. MMF decreases NCAM receptors, which is associated with enhanced tumor cell invasiveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.

The aryl hydrocarbon receptor and glucocorticoid receptor interact to activate human metallothionein 2A

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

Sato, Shoko, E-mail: satosho@rs.tus.ac.jp; Shirakawa, Hitoshi, E-mail: shirakah@m.tohoku.ac.jp; Tomita, Shuhei, E-mail: tomita@med.tottori-u.ac.jp

2013-11-15

Although the aryl hydrocarbon receptor (AHR) and glucocorticoid receptor (GR) play essential roles in mammalian development, stress responses, and other physiological events, crosstalk between these receptors has been the subject of much debate. Metallothioneins are classic glucocorticoid-inducible genes that were reported to increase upon treatment with AHR agonists in rodent tissues and cultured human cells. In this study, the mechanism of human metallothionein 2A (MT2A) gene transcription activation by AHR was investigated. Cotreatment with 3-methylcholanthrene and dexamethasone, agonists of AHR and GR respectively, synergistically increased MT2A mRNA levels in HepG2 cells. MT2A induction was suppressed by RNA interference against AHRmore » or GR. Coimmunoprecipitation experiments revealed a physical interaction between AHR and GR proteins. Moreover, chromatin immunoprecipitation assays indicated that AHR was recruited to the glucocorticoid response element in the MT2A promoter. Thus, we provide a novel mechanism whereby AHR modulates expression of human MT2A via the glucocorticoid response element and protein–protein interactions with GR. - Highlights: • Aryl hydrocarbon receptor forms a complex with glucocorticoid receptor in cells. • Human metallothionein gene is regulated by the AHR and GR interaction. • AHR–GR complex binds to glucocorticoid response element in metallothionein gene. • We demonstrated a novel transcriptional mechanism via AHR and GR interaction.« less

Disease-associated extracellular loop mutations in the adhesion G protein-coupled receptor G1 (ADGRG1; GPR56) differentially regulate downstream signaling.

PubMed

Kishore, Ayush; Hall, Randy A

2017-06-09

Mutations to the adhesion G protein-coupled receptor ADGRG1 (G1; also known as GPR56) underlie the neurological disorder bilateral frontoparietal polymicrogyria. Disease-associated mutations in G1 studied to date are believed to induce complete loss of receptor function through disruption of either receptor trafficking or signaling activity. Given that N-terminal truncation of G1 and other adhesion G protein-coupled receptors has been shown to significantly increase the receptors' constitutive signaling, we examined two different bilateral frontoparietal polymicrogyria-inducing extracellular loop mutations (R565W and L640R) in the context of both full-length and N-terminally truncated (ΔNT) G1. Interestingly, we found that these mutations reduced surface expression of full-length G1 but not G1-ΔNT in HEK-293 cells. Moreover, the mutations ablated receptor-mediated activation of serum response factor luciferase, a classic measure of Gα 12/13 -mediated signaling, but had no effect on G1-mediated signaling to nuclear factor of activated T cells (NFAT) luciferase. Given these differential signaling results, we sought to further elucidate the pathway by which G1 can activate NFAT luciferase. We found no evidence that ΔNT activation of NFAT is dependent on Gα q/11 -mediated or β-arrestin-mediated signaling but rather involves liberation of Gβγ subunits and activation of calcium channels. These findings reveal that disease-associated mutations to the extracellular loops of G1 differentially alter receptor trafficking, depending on the presence of the N terminus, and differentially alter signaling to distinct downstream pathways. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Selective Adhesion of Thiobacillus ferrooxidans to Pyrite

PubMed Central

Ohmura, Naoya; Kitamura, Keiko; Saiki, Hiroshi

1993-01-01

Bacterial adhesion to mineral surfaces plays an important role not only in bacterial survival in natural ecosystems, but also in mining industry applications. Selective adhesion was investigated with Thiobacillus ferrooxidans by using four minerals, pyrite, quartz, chalcopyrite, and galena. Escherichia coli was used as a control bacterium. Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria. The contact angle of E. coli in a 0.5% sodium chloride solution was 31°, and the contact angle of T. ferrooxidans in a pH 2.0 sulfuric acid solution was 23°. E. coli tended to adhere to more hydrophobic minerals by hydrophobic interaction, while T. ferrooxidans selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite. Ferrous ion inhibited the selective adhesion of T. ferrooxidans to pyrite competitively, while ferric ion scarcely inhibited such adhesion. When selective adhesion was quenched by ferrous ion completely, adhesion of T. ferrooxidans was controlled by hydrophilic interactions. Adhesion of E. coli to pyrite exhibited a liner relationship on langmuir isotherm plots, but adhesion of T. ferrooxidans did not. T. ferrooxidans recognized the reduced iron in minerals and selectively adhered to pyrite and chalcopyrite by a strong interaction other than the physical interaction. PMID:16349106

Multidimensional immunolabeling and 4D time-lapse imaging of vital ex vivo lung tissue

PubMed Central

Vierkotten, Sarah; Lindner, Michael; Königshoff, Melanie; Eickelberg, Oliver

2015-01-01

During the last decades, the study of cell behavior was largely accomplished in uncoated or extracellular matrix (ECM)-coated plastic dishes. To date, considerable cell biological efforts have tried to model in vitro the natural microenvironment found in vivo. For the lung, explants cultured ex vivo as lung tissue cultures (LTCs) provide a three-dimensional (3D) tissue model containing all cells in their natural microenvironment. Techniques for assessing the dynamic live interaction between ECM and cellular tissue components, however, are still missing. Here, we describe specific multidimensional immunolabeling of living 3D-LTCs, derived from healthy and fibrotic mouse lungs, as well as patient-derived 3D-LTCs, and concomitant real-time four-dimensional multichannel imaging thereof. This approach allowed the evaluation of dynamic interactions between mesenchymal cells and macrophages with their ECM. Furthermore, fibroblasts transiently expressing focal adhesions markers incorporated into the 3D-LTCs, paving new ways for studying the dynamic interaction between cellular adhesions and their natural-derived ECM. A novel protein transfer technology (FuseIt/Ibidi) shuttled fluorescently labeled α-smooth muscle actin antibodies into the native cells of living 3D-LTCs, enabling live monitoring of α-smooth muscle actin-positive stress fibers in native tissue myofibroblasts residing in fibrotic lesions of 3D-LTCs. Finally, this technique can be applied to healthy and diseased human lung tissue, as well as to adherent cells in conventional two-dimensional cell culture. This novel method will provide valuable new insights into the dynamics of ECM (patho)biology, studying in detail the interaction between ECM and cellular tissue components in their natural microenvironment. PMID:26092995

Hydrogels with tunable stress relaxation regulate stem cell fate and activity

NASA Astrophysics Data System (ADS)

Chaudhuri, Ovijit; Gu, Luo; Klumpers, Darinka; Darnell, Max; Bencherif, Sidi A.; Weaver, James C.; Huebsch, Nathaniel; Lee, Hong-Pyo; Lippens, Evi; Duda, Georg N.; Mooney, David J.

2016-03-01

Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel's initial elastic modulus, degradation, and cell-adhesion-ligand density. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture.

Hydrogels with tunable stress relaxation regulate stem cell fate and activity

PubMed Central

Chaudhuri, Ovijit; Gu, Luo; Klumpers, Darinka; Darnell, Max; Bencherif, Sidi A.; Weaver, James C.; Huebsch, Nathaniel; Lee, Hong-pyo; Lippens, Evi; Duda, Georg N.; Mooney, David J.

2015-01-01

Natural extracellular matrices (ECMs) are viscoelastic and exhibit stress relaxation. However, hydrogels used as synthetic ECMs for three-dimensional (3D) culture are typically elastic. Here, we report a materials approach to tune the rate of stress relaxation of hydrogels for 3D culture, independently of the hydrogel’s initial elastic modulus, cell-adhesion-ligand density and degradation. We find that cell spreading, proliferation, and osteogenic differentiation of mesenchymal stem cells (MSCs) are all enhanced in cells cultured in gels with faster relaxation. Strikingly, MSCs form a mineralized, collagen-1-rich matrix similar to bone in rapidly relaxing hydrogels with an initial elastic modulus of 17 kPa. We also show that the effects of stress relaxation are mediated by adhesion-ligand binding, actomyosin contractility and mechanical clustering of adhesion ligands. Our findings highlight stress relaxation as a key characteristic of cell-ECM interactions and as an important design parameter of biomaterials for cell culture. PMID:26618884

Hyperoxaluria Requires TNF Receptors to Initiate Crystal Adhesion and Kidney Stone Disease.

PubMed

Mulay, Shrikant R; Eberhard, Jonathan N; Desai, Jyaysi; Marschner, Julian A; Kumar, Santhosh V R; Weidenbusch, Marc; Grigorescu, Melissa; Lech, Maciej; Eltrich, Nuru; Müller, Lisa; Hans, Wolfgang; Hrabě de Angelis, Martin; Vielhauer, Volker; Hoppe, Bernd; Asplin, John; Burzlaff, Nicolai; Herrmann, Martin; Evan, Andrew; Anders, Hans-Joachim

2017-03-01

Intrarenal crystals trigger inflammation and renal cell necroptosis, processes that involve TNF receptor (TNFR) signaling. Here, we tested the hypothesis that TNFRs also have a direct role in tubular crystal deposition and progression of hyperoxaluria-related CKD. Immunohistochemical analysis revealed upregulated tubular expression of TNFR1 and TNFR2 in human and murine kidneys with calcium oxalate (CaOx) nephrocalcinosis-related CKD compared with controls. Western blot and mRNA expression analyses in mice yielded consistent data. When fed an oxalate-rich diet, wild-type mice developed progressive CKD, whereas Tnfr1-, Tnfr2- , and Tnfr1/2- deficient mice did not. Despite identical levels of hyperoxaluria, Tnfr1-, Tnfr2- , and Tnfr1/2 -deficient mice also lacked the intrarenal CaOx deposition and tubular damage observed in wild-type mice. Inhibition of TNFR signaling prevented the induced expression of the crystal adhesion molecules, CD44 and annexin II, in tubular epithelial cells in vitro and in vivo , and treatment with the small molecule TNFR inhibitor R-7050 partially protected hyperoxaluric mice from nephrocalcinosis and CKD. We conclude that TNFR signaling is essential for CaOx crystal adhesion to the luminal membrane of renal tubules as a fundamental initiating mechanism of oxalate nephropathy. Furthermore, therapeutic blockade of TNFR might delay progressive forms of nephrocalcinosis in oxalate nephropathy, such as primary hyperoxaluria. Copyright © 2017 by the American Society of Nephrology.

Hyperoxaluria Requires TNF Receptors to Initiate Crystal Adhesion and Kidney Stone Disease

PubMed Central

Mulay, Shrikant R.; Eberhard, Jonathan N.; Desai, Jyaysi; Marschner, Julian A.; Kumar, Santhosh V.R.; Weidenbusch, Marc; Grigorescu, Melissa; Lech, Maciej; Eltrich, Nuru; Müller, Lisa; Hans, Wolfgang; Hrabě de Angelis, Martin; Vielhauer, Volker; Hoppe, Bernd; Asplin, John; Burzlaff, Nicolai; Herrmann, Martin; Evan, Andrew

2017-01-01

Intrarenal crystals trigger inflammation and renal cell necroptosis, processes that involve TNF receptor (TNFR) signaling. Here, we tested the hypothesis that TNFRs also have a direct role in tubular crystal deposition and progression of hyperoxaluria-related CKD. Immunohistochemical analysis revealed upregulated tubular expression of TNFR1 and TNFR2 in human and murine kidneys with calcium oxalate (CaOx) nephrocalcinosis-related CKD compared with controls. Western blot and mRNA expression analyses in mice yielded consistent data. When fed an oxalate-rich diet, wild-type mice developed progressive CKD, whereas Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice did not. Despite identical levels of hyperoxaluria, Tnfr1-, Tnfr2-, and Tnfr1/2-deficient mice also lacked the intrarenal CaOx deposition and tubular damage observed in wild-type mice. Inhibition of TNFR signaling prevented the induced expression of the crystal adhesion molecules, CD44 and annexin II, in tubular epithelial cells in vitro and in vivo, and treatment with the small molecule TNFR inhibitor R-7050 partially protected hyperoxaluric mice from nephrocalcinosis and CKD. We conclude that TNFR signaling is essential for CaOx crystal adhesion to the luminal membrane of renal tubules as a fundamental initiating mechanism of oxalate nephropathy. Furthermore, therapeutic blockade of TNFR might delay progressive forms of nephrocalcinosis in oxalate nephropathy, such as primary hyperoxaluria. PMID:27612997

Surface charges promote nonspecific nanoparticle adhesion to stiffer membranes

NASA Astrophysics Data System (ADS)

Sinha, Shayandev; Jing, Haoyuan; Sachar, Harnoor Singh; Das, Siddhartha

2018-04-01

This letter establishes the manner in which the electric double layer induced by the surface charges of the plasma membrane (PM) enhances the nonspecific adhesion (NSA) of a metal nanoparticle (NP) to stiffer PMs (i.e., PMs with larger bending moduli). The NSA is characterized by the physical attachment of the NP to the membrane and occurs when the decrease in the surface energy (or any other mechanism) associated with the attachment process provides the energy for bending the membrane. Such an attachment does not involve receptor-ligand interactions that characterize the specific membrane-NP adhesion. Here, we demonstrate that a significant decrease in the electrostatic energy caused by the NP-attachment-induced destruction of the charged-membrane-electrolyte interface is responsible for providing the additional energy needed for bending the membrane during the NP adhesion to stiffer membranes. A smaller salt concentration and a larger membrane charge density augment this effect, which can help to design drug delivery to cells with stiffer membranes due to pathological conditions, fabricate NPs with biomimetic cholesterol-rich lipid bilayer encapsulation, etc.

Modeling Multivalent Ligand-Receptor Interactions with Steric Constraints on Configurations of Cell-Surface Receptor Aggregates

PubMed Central

Monine, Michael I.; Posner, Richard G.; Savage, Paul B.; Faeder, James R.; Hlavacek, William S.

2010-01-01

Abstract We use flow cytometry to characterize equilibrium binding of a fluorophore-labeled trivalent model antigen to bivalent IgE-FcεRI complexes on RBL cells. We find that flow cytometric measurements are consistent with an equilibrium model for ligand-receptor binding in which binding sites are assumed to be equivalent and ligand-induced receptor aggregates are assumed to be acyclic. However, this model predicts extensive receptor aggregation at antigen concentrations that yield strong cellular secretory responses, which is inconsistent with the expectation that large receptor aggregates should inhibit such responses. To investigate possible explanations for this discrepancy, we evaluate four rule-based models for interaction of a trivalent ligand with a bivalent cell-surface receptor that relax simplifying assumptions of the equilibrium model. These models are simulated using a rule-based kinetic Monte Carlo approach to investigate the kinetics of ligand-induced receptor aggregation and to study how the kinetics and equilibria of ligand-receptor interaction are affected by steric constraints on receptor aggregate configurations and by the formation of cyclic receptor aggregates. The results suggest that formation of linear chains of cyclic receptor dimers may be important for generating secretory signals. Steric effects that limit receptor aggregation and transient formation of small receptor aggregates may also be important. PMID:20085718

Junctional Adhesion Molecule A Serves as a Receptor for Prototype and Field-Isolate Strains of Mammalian Reovirus

PubMed Central

Campbell, Jacquelyn A.; Schelling, Pierre; Wetzel, J. Denise; Johnson, Elizabeth M.; Forrest, J. Craig; Wilson, Greame A. R.; Aurrand-Lions, Michel; Imhof, Beat A.; Stehle, Thilo; Dermody, Terence S.

2005-01-01

Reovirus infections are initiated by the binding of viral attachment protein σ1 to receptors on the surface of host cells. The σ1 protein is an elongated fiber comprised of an N-terminal tail that inserts into the virion and a C-terminal head that extends from the virion surface. The prototype reovirus strains type 1 Lang/53 (T1L/53) and type 3 Dearing/55 (T3D/55) use junctional adhesion molecule A (JAM-A) as a receptor. The C-terminal half of the T3D/55 σ1 protein interacts directly with JAM-A, but the determinants of receptor-binding specificity have not been identified. In this study, we investigated whether JAM-A also mediates the attachment of the prototype reovirus strain type 2 Jones/55 (T2J/55) and a panel of field-isolate strains representing each of the three serotypes. Antibodies specific for JAM-A were capable of inhibiting infections of HeLa cells by T1L/53, T2J/55, and T3D/55, demonstrating that strains of all three serotypes use JAM-A as a receptor. To corroborate these findings, we introduced JAM-A or the structurally related JAM family members JAM-B and JAM-C into Chinese hamster ovary cells, which are poorly permissive for reovirus infection. Both prototype and field-isolate reovirus strains were capable of infecting cells transfected with JAM-A but not those transfected with JAM-B or JAM-C. A sequence analysis of the σ1-encoding S1 gene segment of the strains chosen for study revealed little conservation in the deduced σ1 amino acid sequences among the three serotypes. This contrasts markedly with the observed sequence variability within each serotype, which is confined to a small number of amino acids. Mapping of these residues onto the crystal structure of σ1 identified regions of conservation and variability, suggesting a likely mode of JAM-A binding via a conserved surface at the base of the σ1 head domain. PMID:15956543

Streptococcal Adhesin P (SadP) contributes to Streptococcus suis adhesion to the human intestinal epithelium.

PubMed

Ferrando, Maria Laura; Willemse, Niels; Zaccaria, Edoardo; Pannekoek, Yvonne; van der Ende, Arie; Schultsz, Constance

2017-01-01

Streptococcus suis is a zoonotic pathogen, causing meningitis and septicemia. We previously demonstrated that the gastrointestinal tract (GIT) is an entry site for zoonotic S. suis infection. Here we studied the contribution of Streptococcal adhesin Protein (SadP) to host-pathogen interaction at GIT level. SadP expression in presence of Intestinal Epithelial Cells (IEC) was compared with expression of other virulence factors by measuring transcript levels using quantitative Real Time PCR (qRT-PCR). SadP variants were identified by phylogenetic analysis of complete DNA sequences. The interaction of SadP knockout and complementation mutants with IEC was tested in vitro. Expression of sadP was significantly increased in presence of IEC. Sequence analysis of 116 invasive strains revealed five SadP sequence variants, correlating with genotype. SadP1, present in zoonotic isolates of clonal complex 1, contributed to binding to both human and porcine IEC and translocation across human IEC. Antibodies against the globotriaosylceramide Gb3/CD77 receptor significantly inhibited adhesion to human IEC. SadP is involved in the host-pathogen interaction in the GIT. Differences between SadP variants may determine different affinities to the Gb3/CD77 host-receptor, contributing to variation in adhesion capacity to host IEC and thus to S. suis zoonotic potential.

ATP release due to Thy-1–integrin binding induces P2X7-mediated calcium entry required for focal adhesion formation

PubMed Central

Henríquez, Mauricio; Herrera-Molina, Rodrigo; Valdivia, Alejandra; Alvarez, Alvaro; Kong, Milene; Muñoz, Nicolás; Eisner, Verónica; Jaimovich, Enrique; Schneider, Pascal; Quest, Andrew F. G.; Leyton, Lisette

2011-01-01

Thy-1, an abundant mammalian glycoprotein, interacts with αvβ3 integrin and syndecan-4 in astrocytes and thus triggers signaling events that involve RhoA and its effector p160ROCK, thereby increasing astrocyte adhesion to the extracellular matrix. The signaling cascade includes calcium-dependent activation of protein kinase Cα upstream of Rho; however, what causes the intracellular calcium transients required to promote adhesion remains unclear. Purinergic P2X7 receptors are important for astrocyte function and form large non-selective cation pores upon binding to their ligand, ATP. Thus, we evaluated whether the intracellular calcium required for Thy-1-induced cell adhesion stems from influx mediated by ATP-activated P2X7 receptors. Results show that adhesion induced by the fusion protein Thy-1-Fc was preceded by both ATP release and sustained intracellular calcium elevation. Elimination of extracellular ATP with Apyrase, chelation of extracellular calcium with EGTA, or inhibition of P2X7 with oxidized ATP, all individually blocked intracellular calcium increase and Thy-1-stimulated adhesion. Moreover, Thy-1 mutated in the integrin-binding site did not trigger ATP release, and silencing of P2X7 with specific siRNA blocked Thy-1-induced adhesion. This study is the first to demonstrate a functional link between αvβ3 integrin and P2X7 receptors, and to reveal an important, hitherto unanticipated, role for P2X7 in calcium-dependent signaling required for Thy-1-stimulated astrocyte adhesion. PMID:21502139

Effects on in vitro and in vivo angiogenesis induced by small peptides carrying adhesion sequences.

PubMed

Conconi, Maria Teresa; Ghezzo, Francesca; Dettin, Monica; Urbani, Luca; Grandi, Claudio; Guidolin, Diego; Nico, Beatrice; Di Bello, Carlo; Ribatti, Domenico; Parnigotto, Pier Paolo

2010-07-01

It is well known that tumor growth is strictly dependent on neo-vessel formation inside the tumor mass and that cell adhesion is required to allow EC proliferation and migration inside the tumor. In this work, we have evaluated the in vitro and in vivo effects on angiogenesis of some peptides, originally designed to promote cell adhesion on biomaterials, containing RGD motif mediating cell adhesion via integrin receptors [RGD, GRGDSPK, and (GRGDSP)(4)K] or the heparin-binding sequence of human vitronectin that interacts with HSPGs [HVP(351-359)]. Cell adhesion, proliferation, migration, and capillary-like tube formation in Matrigel were determined on HUVECs, whereas the effects on in vivo angiogenesis were evaluated using the CAM assay. (GRGDSP)(4)K linear sequence inhibited cell adhesion, decreased cell proliferation, migration and morphogenesis in Matrigel, and induced anti-angiogenic responses on CAM at higher degree than that determined after incubation with RGD or GRGDSPK. Moreover, it counteracted both in vitro and in vivo the pro-angiogenic effects induced by the Fibroblast growth factor (FGF-2). On the other hand, HVP was not able to affect cell adhesion and appeared less effective than (GRGDSP)(4)K. Our data indicate that the activity of RGD-containing peptides is related to their adhesive properties, and their effects are modulated by the number of cell adhesion motifs and the aminoacidic residues next to these sequences. The anti-angiogenic properties of (GRGDSP)(4)K seem to depend on its interaction with integrins, whereas the effects of HVP may be partially due to an impairment of HSPGs/FGF-2.

Trimethylamine N-oxide in atherogenesis: impairing endothelial self-repair capacity and enhancing monocyte adhesion.

PubMed

Ma, GuoHua; Pan, Bing; Chen, Yue; Guo, CaiXia; Zhao, MingMing; Zheng, LeMin; Chen, BuXing

2017-04-30

Several studies have reported a strong association between high plasma level of trimethylamine N-oxide (TMAO) and atherosclerosis development. However, the exact mechanism underlying this correlation is unknown. In the present study, we try to explore the impact of TMAO on endothelial dysfunction. After TMAO treatment, human umbilical vein endothelial cells (HUVECs) showed significant impairment in cellular proliferation and HUVECs-extracellular matrix (ECM) adhesion compared with control. Likewise, TMAO markedly suppressed HUVECs migration in transwell migration assay and wound healing assay. In addition, we found TMAO up-regulated vascular cell adhesion molecule-1 (VCAM-1) expression, promoted monocyte adherence, activated protein kinase C (PKC) and p-NF-κB. Interestingly, TMAO-stimulated VCAM-1 expression and monocyte adherence were diminished by PKC inhibitor. These results demonstrate that TMAO promotes early pathological process of atherosclerosis by accelerating endothelial dysfunction, including decreasing endothelial self-repair and increasing monocyte adhesion. Furthermore, TMAO-induced monocyte adhesion is partly attributable to activation of PKC/NF-κB/VCAM-1. © 2017 The Author(s).

Receptor recruitment: A mechanism for interactions between G protein-coupled receptors

PubMed Central

Holtbäck, Ulla; Brismar, Hjalmar; DiBona, Gerald F.; Fu, Michael; Greengard, Paul; Aperia, Anita

1999-01-01

There is a great deal of evidence for synergistic interactions between G protein-coupled signal transduction pathways in various tissues. As two specific examples, the potent effects of the biogenic amines norepinephrine and dopamine on sodium transporters and natriuresis can be modulated by neuropeptide Y and atrial natriuretic peptide, respectively. Here, we report, using a renal epithelial cell line, that both types of modulation involve recruitment of receptors from the interior of the cell to the plasma membrane. The results indicate that recruitment of G protein-coupled receptors may be a ubiquitous mechanism for receptor sensitization and may play a role in the modulation of signal transduction comparable to that of the well established phenomenon of receptor endocytosis and desensitization. PMID:10377404

Probing receptor-ligand interactions by sedimentation equilibrium

NASA Astrophysics Data System (ADS)

Philo, John S.

1997-05-01

While sedimentation equilibrium is most commonly used to characterize the molecular weight and state of association of single proteins, this technique is also a very powerful tool for probing the interactions between two or more different proteins, and can characterize both the binding stoichiometry and the equilibrium constants. To resolve the complex binding interactions that can occur in such systems, it is crucial to globally fit data from many experiments to a common binding model, including samples made with different mixing ratios and a wide range of total concentration. It is often also essential to constrain the parameters during fitting so that the fits correctly reproduce the molar ratio of proteins used in making each sample. We have applied this methodology to probe mechanisms of receptor activation for a number of hematopoietic receptors and their cognate ligands, using receptor extracellular domains expressed as soluble proteins. Such data can potentially help in the design of improved or new protein therapeutics, as well as in efforts to create small- molecular mimetics of protein hormones through structure- based drug design. Sedimentation equilibrium has shown that stem cell factor, erythropoietin, and granulocyte-colony stimulating factor can each dimerize their respective receptors in solution, but the mechanism of ligand-induced receptor dimerization for these three systems are strikingly different.

ADMA induces monocyte adhesion via activation of chemokine receptors in cultured THP-1 cells.

PubMed

Chen, Meifang; Li, Yuanjian; Yang, Tianlun; Wang, Yongjin; Bai, Yongping; Xie, Xiumei

2008-08-01

Asymmetric dimethylarginine (ADMA), an endogenous NOS inhibitor, is also an important inflammatory factor contributing to the development of atherosclerosis (AS). The present study was to test the effect of ADMA on angiotensin (Ang) II-induced monocytic adhesion. Human monocytoid cells (THP-1) or isolated peripheral blood monocyte cells (PBMCs) were incubated with Ang II (10(-6)M) or exogenous ADMA (30 microM) for 4 or 24h in the absence or presence of losartan or antioxidant PDTC. In cultured THP-1 cells, Ang II (10(-6)M) for 24h elevated the level of ADMA in the medium, upregulated the protein expression of protein arginine methyltransferase (PRMT) and decreased the activity of dimethylarginine dimethylaminohydrolase (DDAH). Both of Ang II and ADMA increased monocytic adhesion to human umbilical vein endothelial cells (HUVECs), elevated the levels of monocyte chemoattractant protein (MCP)-1, interleukin (IL)-8 and tumor necrosis factor (TNF)-alpha and upregulated CCR(2) and CXCR(2) mRNA expression, concomitantly with increase in reactive oxygen species (ROS) generation and activation of nuclear factor (NF)-kappaB. Pretreatment with losartan (10 microM) or PDTC (10 microM) abolished the effects mediated by Ang II or ADMA. In isolated PBMCs from healthy individuals, ADMA upregulated the expression of CXCR(2) mRNA, which was attenuated by losartan (10 microM), however, ADMA had no effect on surface protein expression of CCR(2). The present results suggest that ADMA may be involved in monocytic adhesion induced by Ang II via activation of chemokine receptors by ROS/NF-kappaB pathway.

Epidermal Notch signalling: differentiation, cancer and adhesion.

PubMed

Watt, Fiona M; Estrach, Soline; Ambler, Carrie A

2008-04-01

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

A non-canonical role for Rgnef in promoting integrin-stimulated focal adhesion kinase activation

PubMed Central

Miller, Nichol L. G.; Lawson, Christine; Kleinschmidt, Elizabeth G.; Tancioni, Isabelle; Uryu, Sean; Schlaepfer, David D.

2013-01-01

Summary Rgnef (also known as p190RhoGEF or ARHGEF28) is a Rho guanine-nucleotide-exchange factor (GEF) that binds focal adhesion kinase (FAK). FAK is recruited to adhesions and activated by integrin receptors binding to matrix proteins, such as fibronectin (FN). Canonical models place Rgnef downstream of integrin–FAK signaling in regulating Rho GTPase activity and cell movement. Herein, we establish a new, upstream role for Rgnef in enhancing FAK localization to early peripheral adhesions and promoting FAK activation upon FN binding. Rgnef-null mouse embryo fibroblasts (MEFs) exhibit defects in adhesion formation, levels of FAK phosphotyrosine (pY)-397 and FAK localization to peripheral adhesions upon re-plating on FN. Rgnef re-expression rescues these defects, but requires Rgnef–FAK binding. A mutation in the Rgnef pleckstrin homology (PH) domain inhibits adhesion formation, FAK localization, and FAK-Y397 and paxillin-Y118 phosphorylation without disrupting the Rgnef–FAK interaction. A GEF-inactive Rgnef mutant rescues FAK-Y397 phosphorylation and early adhesion localization, but not paxillin-Y118 phosphorylation. This suggests that, downstream of FN binding, paxillin-pY118 requires Rgnef GEF activity through a mechanism distinct from adhesion formation and FAK activation. These results support a scaffolding role for Rgnef in FAK localization and activation at early adhesions in a PH-domain-dependent but GEF-activity-independent manner. PMID:24006257

Oligosaccharide receptor mimics inhibit Legionella pneumophila attachment to human respiratory epithelial cells.

PubMed

Thomas, Richard J; Brooks, Tim J

2004-02-01

Legionnaire's disease is caused by the intracellular pathogen Legionella pneumophila, presenting as an acute pneumonia. Attachment is the key step during infection, often relying on an interaction between host cell oligosaccharides and bacterial adhesins. Inhibition of this interaction by receptor mimics offers possible novel therapeutic treatments. L. pneumophila attachment to the A549 cell line was significantly reduced by treatment with tunicamycin (73.6%) and sodium metaperiodate (63.7%). This indicates the importance of cell surface oligosaccharide chains in adhesion. A number of putative anti-adhesion compounds inhibited attachment to the A549 and U937 cell lines. The most inhibitory compounds were polymeric saccharides, GalNAcbeta1-4Gal, Galbeta1-4GlcNAc and para-nitrophenol. These compounds inhibited adhesion to a range of human respiratory cell lines, including nasal epithelial, bronchial epithelial and alveolar epithelial cell lines and the human monocytic cell line, U937. Some eukaryotic receptors for L. pneumophila were determined to be the glycolipids, asialo-GM1 and asialo-GM2 that contain the inhibitory saccharide moiety, GalNAcbeta1-4Gal. The identified compounds have the potential to be used as novel treatments for Legionnaire's disease.

Using engineered single-chain antibodies to correlate molecular binding properties and nanoparticle adhesion dynamics.

PubMed

Haun, Jered B; Pepper, Lauren R; Boder, Eric T; Hammer, Daniel A

2011-11-15

Elucidation of the relationship between targeting molecule binding properties and the adhesive behavior of therapeutic or diagnostic nanocarriers would aid in the design of optimized vectors and lead to improved efficacy. We measured the adhesion of 200-nm-diameter particles under fluid flow that was mediated by a diverse array of molecular interactions, including recombinant single-chain antibodies (scFvs), full antibodies, and the avidin/biotin interaction. Within the panel of scFvs, we used a family of mutants that display a spectrum of binding kinetics, allowing us to compare nanoparticle adhesion to bond chemistry. In addition, we explored the effect of molecular size by inserting a protein linker into the scFv fusion construct and by employing scFvs that are specific for targets with vastly different sizes. Using computational models, we extracted multivalent kinetic rate constants for particle attachment and detachment from the adhesion data and correlated the results to molecular binding properties. Our results indicate that the factors that increase encounter probability, such as adhesion molecule valency and size, directly enhance the rate of nanoparticle attachment. Bond kinetics had no influence on scFv-mediated nanoparticle attachment within the kinetic range tested, however, but did appear to affect antibody/antigen and avidin/biotin mediated adhesion. We attribute this finding to a combination of multivalent binding and differences in bond mechanical strength between recombinant scFvs and the other adhesion molecules. Nanoparticle detachment probability correlated directly with adhesion molecule valency and size, as well as the logarithm of the affinity for all molecules tested. On the basis of this work, scFvs can serve as viable targeting receptors for nanoparticles, but improvements to their bond mechanical strength would likely be required to fully exploit their tunable kinetic properties and maximize the adhesion efficiency of nanoparticles that

Growth cones are actively influenced by substrate-bound adhesion molecules.

PubMed

Burden-Gulley, S M; Payne, H R; Lemmon, V

1995-06-01

As axons advance to appropriate target tissues during development, their growth cones encounter a variety of cell adhesion molecules (CAMs) and extracellular matrix molecules (ECM molecules). Purified CAMs and ECM molecules influence neurite outgrowth in vitro and are thought to have a similar function in vivo. For example, when retinal ganglion cell (RGC) neurons are grown on different CAM and ECM molecule substrates in vitro, their growth cones display distinctive morphologies (Payne et al., 1992). Similarly, RGC growth cones in vivo have distinctive shapes at different points in the pathway from the eye to the tectum, suggesting the presence of localized cues that determine growth cone behaviors such as pathway selection at choice points. In this report, time-lapse video microscopy was utilized to examine dynamic transformations of RGC growth cones as they progressed from L1/8D9, N-cadherin, or laminin onto a different substrate. Contact made by the leading edge of a growth cone with a new substrate resulted in a rapid and dramatic alteration in growth cone morphology. In some cases, the changes encompassed the entire growth cone including those regions not in direct contact with the new substrate. In addition, the growth cones displayed a variety of behavioral responses that were dependent upon the order of substrate contact. These studies demonstrate that growth cones are actively affected by the substrate, and suggest that abrupt changes in the molecular composition of the growth cone environment are influential during axonal pathfinding.

Modeling and simulation of the fluid flow in wire electrochemical machining with rotating tool (wire ECM)

NASA Astrophysics Data System (ADS)

Klocke, F.; Herrig, T.; Zeis, M.; Klink, A.

2017-10-01

Combining the working principle of electrochemical machining (ECM) with a universal rotating tool, like a wire, could manage lots of challenges of the classical ECM sinking process. Such a wire-ECM process could be able to machine flexible and efficient 2.5-dimensional geometries like fir tree slots in turbine discs. Nowadays, established manufacturing technologies for slotting turbine discs are broaching and wire electrical discharge machining (wire EDM). Nevertheless, high requirements on surface integrity of turbine parts need cost intensive process development and - in case of wire-EDM - trim cuts to reduce the heat affected rim zone. Due to the process specific advantages, ECM is an attractive alternative manufacturing technology and is getting more and more relevant for sinking applications within the last few years. But ECM is also opposed with high costs for process development and complex electrolyte flow devices. In the past, few studies dealt with the development of a wire ECM process to meet these challenges. However, previous concepts of wire ECM were only suitable for micro machining applications. Due to insufficient flushing concepts the application of the process for machining macro geometries failed. Therefore, this paper presents the modeling and simulation of a new flushing approach for process assessment. The suitability of a rotating structured wire electrode in combination with an axial flushing for electrodes with high aspect ratios is investigated and discussed.

[Effect of Spatholobus suberctus on adhesion, invasion, migration and metastasis of melanoma cells].

PubMed

Xu, Jian-Ya; Gu, Qin; Xia, Wei-Jun

2010-10-01

To study the effect of Spatholobus suberctus, a kind of Chinese Traditional Medicine which can dissolve the stasis by activating the blood circulation, on invasion, adhesion, migration and metastasis of B16-BL6 metastatic mouse melanoma cells and its mechanism. The proliferation, adhesion, invasion and migration capacity of B16-BL6 metastatic cells was evaluated by MTP assay, adhesion assay and reconstituted basement membrane invasion and migration assay in vitro respectively. Mouse spontaneous motility melanoma model was used to study the effect of Spatholobus suberctus on metastasis in vivo. At the highest innoxious concentration, the extracts of Spatholobus suberctus inhibited the adhesion and invasion capacity of B16-BL6 metastatic cells significantly. In the mouse spontaneous melanoma model, the lung metastatic nodes number and its volume were significantly decreased after continuously treated with the extracts of Spatholobus suberctu. The extracts of Spatholobus suberctu can inhibit the metastasis of of B16-BI6 metastatic mouse melanoma cells and its mechanism may be inhibiting the capability of B16-BL6 cells in adhering to the ECM and invading the basement membrane.

Correlation between ECM guidance and actin polymerization on osteogenic differentiation of human adipose-derived stem cells

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

Keller, Vivian; Deiwick, Andrea; Pflaum, Michael

The correlation between extracellular matrix (ECM) components, cell shape, and stem cell guidance can shed light in understanding and mimicking the functionality of stem cell niches for various applications. This interplay on osteogenic guidance of human adipose-derived stem cells (hASCs) was focus of this study. Proliferation and osteogenic markers like alkaline phosphatase activity and calcium mineralization were slightly increased by the ECM components laminin (LA), collagen I (COL), and fibronectin (FIB); with control medium no differentiation occurred. ECM guided differentiation was rather dependent on osterix than on Runx2 pathway. FIB significantly enhanced cell elongation even in presence of actin polymerizationmore » blockers cytochalasin D (CytoD) and ROCK inhibitor Y-27632, which generally caused more rounded cells. Except for the COL surface, both inhibitors increased the extent of osterix, while the Runx2 pathway was more sensitive to the culture condition. Both inhibitors did not affect hASC proliferation. CytoD enabled osteogenic differentiation independently from the ECM, while it was rather blocked via Y-27632 treatment; on FIB the general highest extent of differentiation occurred. Taken together, the ECM effect on hASCs occurs indirectly and selectively via a dominant role of FIB: it sustains osteogenic differentiation in case of a tension-dependent control of actin polymerization. - Highlights: • Interplay of ECM and cell shape guides osteogenic differentiation of hASCs. • ECM components only present a promotive but not stimulative effect. • No direct correlation between ECM-enhanced cell elongation and differentiation. • Suppression of differentiation depends on a specific actin polymerization blocking. • Fibronectin sustains cell elongation and differentiation in case of blocking actin.« less

"Features of two proteins of Leptospira interrogans with potential role in host-pathogen interactions"

PubMed Central

2012-01-01

Background Leptospirosis is considered a re-emerging infectious disease caused by pathogenic spirochaetes of the genus Leptospira. Pathogenic leptospires have the ability to survive and disseminate to multiple organs after penetrating the host. Leptospires were shown to express surface proteins that interact with the extracellular matrix (ECM) and to plasminogen (PLG). This study examined the interaction of two putative leptospiral proteins with laminin, collagen Type I, collagen Type IV, cellular fibronectin, plasma fibronectin, PLG, factor H and C4bp. Results We show that two leptospiral proteins encoded by LIC11834 and LIC12253 genes interact with laminin in a dose - dependent and saturable mode, with dissociation equilibrium constants (KD) of 367.5 and 415.4 nM, respectively. These proteins were named Lsa33 and Lsa25 (Leptospiral surface adhesin) for LIC11834 and LIC12253, respectively. Metaperiodate - treated laminin reduced Lsa25 - laminin interaction, suggesting that sugar moieties of this ligand participate in this interaction. The Lsa33 is also PLG - binding receptor, with a KD of 23.53 nM, capable of generating plasmin in the presence of an activator. Although in a weak manner, both proteins interact with C4bp, a regulator of complement classical route. In silico analysis together with proteinase K and immunoflorescence data suggest that these proteins might be surface exposed. Moreover, the recombinant proteins partially inhibited leptospiral adherence to immobilized laminin and PLG. Conclusions We believe that these multifunctional proteins have the potential to participate in the interaction of leptospires to hosts by mediating adhesion and by helping the bacteria to escape the immune system and to overcome tissue barriers. To our knowledge, Lsa33 is the first leptospiral protein described to date with the capability of binding laminin, PLG and C4bp in vitro. PMID:22463075

RIC8A is essential for the organisation of actin cytoskeleton and cell-matrix interaction.

PubMed

Ruisu, Katrin; Meier, Riho; Kask, Keiu; Tõnissoo, Tambet; Velling, Teet; Pooga, Margus

2017-08-15

RIC8A functions as a chaperone and guanine nucleotide exchange factor for a subset of G protein α subunits. Multiple G protein subunits mediate various signalling events that regulate cell adhesion and migration and the involvement of RIC8A in some of these processes has been demonstrated. We have previously shown that the deficiency of RIC8A causes a failure in mouse gastrulation and neurogenesis - major events in embryogenesis that rely on proper association of cells with the extracellular matrix (ECM) and involve active cell migration. To elaborate on these findings, we used Ric8a -/- mouse embryonic stem cells and Ric8a-deficient mouse embryonic fibroblasts, and found that RIC8A plays an important role in the organisation and remodelling of actin cytoskeleton and cell-ECM association. Ric8a-deficient cells were able to attach to different ECM components, but were unable to spread correctly, and did not form stress fibres or focal adhesion complexes. We also found that the presence of RIC8A is necessary for the activation of β1 integrins and integrin-mediated cell migration. Copyright © 2017 Elsevier Inc. All rights reserved.

Neural ECM proteases in learning and synaptic plasticity.

PubMed

Tsilibary, Effie; Tzinia, Athina; Radenovic, Lidija; Stamenkovic, Vera; Lebitko, Tomasz; Mucha, Mariusz; Pawlak, Robert; Frischknecht, Renato; Kaczmarek, Leszek

2014-01-01

Recent studies implicate extracellular proteases in synaptic plasticity, learning, and memory. The data are especially strong for such serine proteases as thrombin, tissue plasminogen activator, neurotrypsin, and neuropsin as well as matrix metalloproteinases, MMP-9 in particular. The role of those enzymes in the aforementioned phenomena is supported by the experimental results on the expression patterns (at the gene expression and protein and enzymatic activity levels) and functional studies, including knockout mice, specific inhibitors, etc. Counterintuitively, the studies have shown that the extracellular proteolysis is not responsible mainly for an overall degradation of the extracellular matrix (ECM) and loosening perisynaptic structures, but rather allows for releasing signaling molecules from the ECM, transsynaptic proteins, and latent form of growth factors. Notably, there are also indications implying those enzymes in the major neuropsychiatric disorders, probably by contributing to synaptic aberrations underlying such diseases as schizophrenia, bipolar, autism spectrum disorders, and drug addiction.

Thermomechanical analysis of freezing-induced cell-fluid-matrix interactions in engineered tissues

PubMed Central

Han, Bumsoo; Teo, Ka Yaw; Ghosh, Soham; Dutton, J. Craig; Grinnell, Frederick

2012-01-01

Successful cryopreservation of functional engineered tissues (ETs) is significant to tissue engineering and regenerative medicine, but it is extremely challenging to develop a successful protocol because the effects of cryopreservation parameters on the post-thaw functionality of ETs are not well understood. Particularly, the effects on the microstructure of their extracellular matrix (ECM) have not been well studied, which determines many functional properties of the ETs. In this study, we investigated the effects of two key cryopreservation parameters – i) freezing temperature and corresponding cooling rate; and ii) the concentration of cryoprotective agent (CPA) on the ECM microstructure as well as the cellular viability. Using dermal equivalent as a model ET and DMSO as a model CPA, freezing-induced spatiotemporal deformation and post-thaw ECM microstructure of ETs was characterized while varying the freezing temperature and DMSO concentrations. The spatial distribution of cellular viability and the cellular actin cytoskeleton was also examined. The results showed that the tissue dilatation increased significantly with reduced freezing temperature (i.e., rapid freezing). A maximum limit of tissue deformation was observed for preservation of ECM microstructure, cell viability and cell-matrix adhesion. The dilatation decreased with the use of DMSO, and a freezing temperature dependent threshold concentration of DMSO was observed. The threshold DMSO concentration increased with lowering freezing temperature. In addition, an analysis was performed to delineate thermodynamic and mechanical components of freezing-induced tissue deformation. The results are discussed to establish a mechanistic understanding of freezing-induced cell-fluid-matrix interaction and phase change behavior within ETs in order to improve cryopreservation of ETs. PMID:23246556

Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction.

PubMed

Yan, Meiping; Zhang, Xinhua; Chen, Ao; Gu, Wei; Liu, Jie; Ren, Xiaojiao; Zhang, Jianping; Wu, Xiaoxiong; Place, Aaron T; Minshall, Richard D; Liu, Guoquan

2017-11-01

Intercellular adhesion molecule-1 (ICAM-1) mediates the firm adhesion of leukocytes to endothelial cells and initiates subsequent signaling that promotes their transendothelial migration (TEM). Vascular endothelial (VE)-cadherin plays a critical role in endothelial cell-cell adhesion, thereby controlling endothelial permeability and leukocyte transmigration. This study aimed to determine the molecular signaling events that originate from the ICAM-1-mediated firm adhesion of neutrophils that regulate VE-cadherin's role as a negative regulator of leukocyte transmigration. We observed that ICAM-1 interacts with Src homology domain 2-containing phosphatase-2 (SHP-2), and SHP-2 down-regulation via silencing of small interfering RNA in endothelial cells enhanced neutrophil adhesion to endothelial cells but inhibited neutrophil transmigration. We also found that VE-cadherin associated with the ICAM-1-SHP-2 complex. Moreover, whereas the activation of ICAM-1 leads to VE-cadherin dissociation from ICAM-1 and VE-cadherin association with actin, SHP-2 down-regulation prevented ICAM-1-VE-cadherin association and promoted VE-cadherin-actin association. Furthermore, SHP-2 down-regulation in vivo promoted LPS-induced neutrophil recruitment in mouse lung but delayed neutrophil extravasation. These results suggest that SHP-2- via association with ICAM-1-mediates ICAM-1-induced Src activation and modulates VE-cadherin switching association with ICAM-1 or actin, thereby negatively regulating neutrophil adhesion to endothelial cells and enhancing their TEM.-Yan, M., Zhang, X., Chen, A., Gu, W., Liu, J., Ren, X., Zhang, J., Wu, X., Place, A. T., Minshall, R. D., Liu, G. Endothelial cell SHP-2 negatively regulates neutrophil adhesion and promotes transmigration by enhancing ICAM-1-VE-cadherin interaction. © FASEB.

Involvement of lipid rafts in adhesion-induced activation of Met and EGFR.

PubMed