Cell adhesion: parallels between vertebrate and invertebrate focal adhesions

AbstractRas suppressor-1 (Rsu-1) is a leucine-rich repeat (LRR)-containing protein that is crucial for regulating fundamental cell adhesion processes and tumor development. Rsu-1 interacts with a zinc-finger type multi LIM domain-containing adaptor protein PINCH-1 involved in the integrin-mediated consensus adhesome but not with highly homologous isoform PINCH-2. However, the structural basis for such specific interaction and regulatory mechanism remains unclear. Here, we determined the crystal structures of Rsu-1 and its complex with the PINCH-1 LIM4-5 domains. Rsu-1 displays an arc-shaped solenoid architecture with eight LRRs shielded by the N- and C-terminal capping modules. We show that a large conserved concave surface of the Rsu-1 LRR domain recognizes the PINCH-1 LIM5 domain, and that the C-terminal non-LIM region of PINCH-2 but not PINCH-1 sterically disfavors the Rsu-1 binding. We further show that Rsu-1 can be assembled, via PINCH-1-binding, into a tight hetero-pentamer complex comprising Rsu-1, PINCH-1, ILK, Parvin, and Kindlin-2 that constitute a major consensus integrin adhesome crucial for focal adhesion assembly. Consistently, our mutagenesis and cell biological data consolidate the significance of the Rsu-1/PINCH-1 interaction in focal adhesion assembly and cell spreading. Our results provide a crucial molecular insight into Rsu-1-mediated cell adhesion with implication on how it may regulate tumorigenic growth.

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Multiparametric analysis of focal adhesion formation by RNAi-mediated gene knockdown

The Journal of Cell Biology ◽

10.1083/jcb.200901105 ◽

2009 ◽

Vol 186 (3) ◽

pp. 423-436 ◽

Cited By ~ 43

Author(s):

Sabina E. Winograd-Katz ◽

Shalev Itzkovitz ◽

Zvi Kam ◽

Benjamin Geiger

Keyword(s):

Cell Adhesion ◽

Adhesion Formation ◽

Focal Adhesions ◽

Gene Families ◽

Adaptor Proteins ◽

Small Interfering Rnas ◽

Comprehensive Information ◽

Focal Adhesion Formation ◽

Multiple Cell ◽

And Migration

Cell adhesion to the extracellular matrix is mediated by elaborate networks of multiprotein complexes consisting of adhesion receptors, cytoskeletal components, signaling molecules, and diverse adaptor proteins. To explore how specific molecular pathways function in the assembly of focal adhesions (FAs), we performed a high-throughput, high-resolution, microscopy-based screen. We used small interfering RNAs (siRNAs) to target human kinases, phosphatases, and migration- and adhesion-related genes. Multiparametric image analysis of control and of siRNA-treated cells revealed major correlations between distinct morphological FA features. Clustering analysis identified different gene families whose perturbation induced similar effects, some of which uncoupled the interfeature correlations. Based on these findings, we propose a model for the molecular hierarchy of FA formation, and tested its validity by dynamic analysis of FA formation and turnover. This study provides a comprehensive information resource on the molecular regulation of multiple cell adhesion features, and sheds light on signaling mechanisms regulating the formation of integrin adhesions.

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Contractility modulates cell adhesion strengthening through focal adhesion kinase and assembly of vinculin-containing focal adhesions

Journal of Cellular Physiology ◽

10.1002/jcp.22084 ◽

2010 ◽

pp. n/a-n/a ◽

Cited By ~ 14

Author(s):

David W. Dumbauld ◽

Heungsoo Shin ◽

Nathan D. Gallant ◽

Kristin E. Michael ◽

Harish Radhakrishna ◽

...

Keyword(s):

Cell Adhesion ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Focal Adhesions

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Aurora B spatially regulates EB3 phosphorylation to coordinate daughter cell adhesion with cytokinesis

The Journal of Cell Biology ◽

10.1083/jcb.201301131 ◽

2013 ◽

Vol 201 (5) ◽

pp. 709-724 ◽

Cited By ~ 40

Author(s):

Jorge G. Ferreira ◽

António J. Pereira ◽

Anna Akhmanova ◽

Helder Maiato

Keyword(s):

Cell Adhesion ◽

Daughter Cell ◽

Cortical Microtubule ◽

Focal Adhesions ◽

Mitotic Exit ◽

Aurora B ◽

Microtubule Stability ◽

Spatiotemporal Regulation ◽

Cytoskeleton Remodeling ◽

Microtubule Growth

During mitosis, human cells round up, decreasing their adhesion to extracellular substrates. This must be quickly reestablished by poorly understood cytoskeleton remodeling mechanisms that prevent detachment from epithelia, while ensuring the successful completion of cytokinesis. Here we show that the microtubule end-binding (EB) proteins EB1 and EB3 play temporally distinct roles throughout cell division. Whereas EB1 was involved in spindle orientation before anaphase, EB3 was required for stabilization of focal adhesions and coordinated daughter cell spreading during mitotic exit. Additionally, EB3 promoted midbody microtubule stability and, consequently, midbody stabilization necessary for efficient cytokinesis. Importantly, daughter cell adhesion and cytokinesis completion were spatially regulated by distinct states of EB3 phosphorylation on serine 176 by Aurora B. This EB3 phosphorylation was enriched at the midbody and shown to control cortical microtubule growth. These findings uncover differential roles of EB proteins and explain the importance of an Aurora B phosphorylation gradient for the spatiotemporal regulation of microtubule function during mitotic exit and cytokinesis.

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Mice Devoid of Fer Protein-Tyrosine Kinase Activity Are Viable and Fertile but Display Reduced Cortactin Phosphorylation

Molecular and Cellular Biology ◽

10.1128/mcb.21.2.603-613.2001 ◽

2001 ◽

Vol 21 (2) ◽

pp. 603-613 ◽

Cited By ~ 67

Author(s):

Andrew W. B. Craig ◽

Ralph Zirngibl ◽

Karen Williams ◽

Lesley-Ann Cole ◽

Peter A. Greer

Keyword(s):

Cell Adhesion ◽

Growth Factor ◽

Cell Growth ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Kinase Activity ◽

Focal Adhesions ◽

Embryonic Fibroblasts ◽

Protein Tyrosine ◽

Phenotypic Differences

ABSTRACT The ubiquitous Fer protein-tyrosine kinase has been proposed to regulate diverse processes such as cell growth, cell adhesion, and neurite outgrowth. To gain insight into the biological function of Fer, we have targeted the fer locus with a kinase-inactivating missense mutation (ferD743R ). Mice homozygous for this mutation develop normally, have no overt phenotypic differences from wild-type mice, and are fertile. Since these mice lack both Fer and the testis-specific FerT kinase activities, these proteins are clearly not essential for development and survival. No differences were observed in overall cellularity of bone marrow, spleen, or thymus in the absence of Fer activity. While most platelet-derived growth factor (PDGF)-induced tyrosine phosphorylation was unchanged inferD743R homozygous embryonic fibroblasts, cortactin phosphorylation was reduced. However, Fer kinase activity was not required for PDGF-induced Stat3, p120ctn, or epidermal growth factor (EGF)-induced β-catenin phosphorylation. Also, no defects were observed in changes to the actin cytoskeleton, adherens junctions, or focal adhesions in PDGF- or EGF-stimulatedferD743R homozygous embryonic fibroblasts. Therefore, Fer likely serves a redundant role in regulating cell growth, cell adhesion, retinal development, and spermatogenesis but is required for efficient phosphorylation of cortactin.

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Interaction With Nanoscale Topography: The Use of Nanowell-Trapped Charged Ligand-Bearing Nanoparticle Surfaces To Modulate Physiological Focal Adhesions in Endothelial Cells

ASME 2013 2nd Global Congress on NanoEngineering for Medicine and Biology ◽

10.1115/nemb2013-93345 ◽

2013 ◽

Author(s):

Phat L. Tran ◽

Jessica R. Gamboa ◽

Katherine E. McCracken ◽

Jeong-Yeol Yoon ◽

Marvin J. Slepian

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Self Assembly ◽

Cytochalasin B ◽

Focal Adhesions ◽

Cellular Adhesion ◽

Spatial Cues ◽

Rgd Peptides ◽

Physical Constraints ◽

Nanoscale Topography

Achieving cell adhesion, growth and homeostasis on an underlying biomaterial surface may be a desirable feature in implant device design and tissue engineering. Insight has been gained from numerous cell patterning strategies where spatial cues and physical constraints have been shown to regulate the structure and function of cells. Despite significant advances in modifying substrates for cellular attachment, migration and proliferation, the achievement of confluent and aligned growth of functional endothelial cells on cardiovascular blood-contacting implants under physiologically significant wall shear stress has proven difficult. Recently we have reported on a method that enhances cellular adhesion under flow conditions on synthetic polymer surfaces, without reliance on pro-adhesive protein biomaterials, which are often thrombogenic. In this method we utilize electron beam lithography and size-dependent self-assembly to fabricate line arrays of nanowells allowing entrapment and retention of charged nanoparticles, covalently conjugated with a RGD adhesive ligand, GRGDSPK. This approach is an additive strategy of combining substrata topographic alteration, electrostatic charge and biochemical ligands, all uniquely incorporated as an ensemble of charged, ligand-bearing nanoparticles entrapped in arrays of nanowells. However, the modulation of endothelial cell physiologic mechanisms as a result of ensemble surface exposure remains to be characterized. In this report, we extend our studies and probe cell physiologic mechanisms altered as a result of nanofeatured surface exposure. We first examined the functional intactness or normalcy of endothelial cells adherent to the nanofeatured ensemble surface utilizing standard immunostaining and flow cytometry methods. We found β1-integrin expression dominated quiescent adherent endothelial cells while αVβ3-integrins expression was more common in migratory cells. Endothelial cells were noted to express high levels of PECAM-1 over time when exposed to nanofeatured surface and RGD peptides. For understanding the contribution of the nanofeatured surface (entrapped RGD conjugated nanoparticles) to cell adhesion, cytochalasin B was used to alter cell spreading. Confocal microscopy illustrated the uptake of nanoparticles in endothelial cells on composite surfaces, as well as the inhibition of this endocytosis by cytochalasin B. After prohibiting the cells from engulfing nanoparticles, we found an 80% reduction in cell adhesion; suggesting that an endocytic mechanism might play a role in maintaining cell adhesion. Nanofeatured ensemble surfaces appear to be good substrates for achieving a high level of EC adhesion, with maintained growth and stability.

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A synthetic peptide from the heparin-binding domain III (repeats III4-5) of fibronectin promotes stress-fibre and focal-adhesion formation in melanoma cells

Biochemical Journal ◽

10.1042/bj20021344 ◽

2003 ◽

Vol 371 (2) ◽

pp. 565-571 ◽

Cited By ~ 14

Author(s):

José V. MOYANO ◽

Alfredo MAQUEDA ◽

Juan P. ALBAR ◽

Angeles GARCIA-PARDO

Keyword(s):

Cell Adhesion ◽

Focal Adhesion ◽

Adhesion Formation ◽

Focal Adhesions ◽

Melanoma Cells ◽

Small Gtpase ◽

Binding Domain ◽

Heparin Binding ◽

Chondroitin Sulphate Proteoglycans ◽

Heparin Binding Domain

Cell adhesion to fibronectin results in formation of actin stress fibres and focal adhesions. In fibroblasts, this response requires two co-operative signals provided by interactions of the RGD sequence with α5β1 integrin and the heparin-binding domain II (Hep II) domain with syndecan-4. Within Hep II, this activity was mapped to repeat III13 and to the peptide FN-C/H-V(WQPPRARITGY, repeat III14). We previously described that the synthetic heparin-binding peptide/III5 (HBP/III5) (WTPPRAQITGYRLTVGLTRR, repeat III5) binds heparin and mediates cell adhesion via chondroitin sulphate proteoglycans. We have now studied whether HBP/III5 co-operates with α5β1 and drives a full cytoskeletal response in melanoma cells. SKMEL-178 cells attached and spread on the RGD-containing FNIII7–FNIII10 (FNIII7–10) fragment, but did not form stress fibres or focal adhesions. Co-immobilization of HBP/III5 with FNIII7–10 or adding soluble HBP/III5 to cells prespread on FNIII7–10, effectively induced these structures. Cell transfection with dominant-negative N19RhoA, a member of the small GTPase family, abolished the HBP/III5 effect. Both chondroitinase and heparitinase diminished focal adhesions, indicating that both types of proteoglycans bound HBP/III5 in melanoma cells. We have mapped the active sequence of HBP/III5 to YRLTVGLTRR, which is a novel sequence in fibronectin with focal-adhesion-promoting activity. The last two arginine (R) residues of this sequence are required for activity, since their replacement by alanine completely abrogated the HBP/III5 cytoskeletal effect. Moreover, this sequence is also active in the context of large fibronectin fragments. Our results establish that the Hep III region provides co-operative signals to α5β1 for the progression of the cytoskeletal response and that these include activation of RhoA.

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Mena binds α5 integrin directly and modulates α5β1 function

The Journal of Cell Biology ◽

10.1083/jcb.201202079 ◽

2012 ◽

Vol 198 (4) ◽

pp. 657-676 ◽

Cited By ~ 41

Author(s):

Stephanie L. Gupton ◽

Daisy Riquelme ◽

Shannon K. Hughes-Alford ◽

Jenny Tadros ◽

Shireen S. Rudina ◽

...

Keyword(s):

Cell Migration ◽

Cell Adhesion ◽

Growth Factor Receptor ◽

Focal Adhesions ◽

Fibronectin Receptor ◽

Focal Adhesion Proteins ◽

Carboxy Terminal ◽

Bidirectional Interactions ◽

Dependent Processes ◽

Α5β1 Integrin

Mena is an Ena/VASP family actin regulator with roles in cell migration, chemotaxis, cell–cell adhesion, tumor cell invasion, and metastasis. Although enriched in focal adhesions, Mena has no established function within these structures. We find that Mena forms an adhesion-regulated complex with α5β1 integrin, a fibronectin receptor involved in cell adhesion, motility, fibronectin fibrillogenesis, signaling, and growth factor receptor trafficking. Mena bound directly to the carboxy-terminal portion of the α5 cytoplasmic tail via a 91-residue region containing 13 five-residue “LERER” repeats. In fibroblasts, the Mena–α5 complex was required for “outside-in” α5β1 functions, including normal phosphorylation of FAK and paxillin and formation of fibrillar adhesions. It also supported fibrillogenesis and cell spreading and controlled cell migration speed. Thus, fibroblasts require Mena for multiple α5β1-dependent processes involving bidirectional interactions between the extracellular matrix and cytoplasmic focal adhesion proteins.

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Integrin alpha 2 cytoplasmic domain deletion effects: loss of adhesive activity parallels ligand-independent recruitment into focal adhesions.

Molecular Biology of the Cell ◽

10.1091/mbc.5.9.977 ◽

1994 ◽

Vol 5 (9) ◽

pp. 977-988 ◽

Cited By ~ 28

Author(s):

S Kawaguchi ◽

J M Bergelson ◽

R W Finberg ◽

M E Hemler

Keyword(s):

Amino Acids ◽

Cell Adhesion ◽

Focal Adhesion ◽

Cho Cells ◽

Chinese Hamster ◽

Focal Adhesions ◽

Inverse Correlation ◽

Cytoplasmic Domain ◽

Negative Regulator ◽

Wild Type

Chinese hamster ovary (CHO) cells transfected with the integrin alpha 2 subunit formed a stable VLA-2 heterodimer that mediated cell adhesion to collagen. Within CHO cells spread on collagen, but not fibronectin, wild-type alpha 2 subunit localized into focal adhesion complexes (FACs). In contrast, alpha 2 with a deleted cytoplasmic domain was recruited into FACs whether CHO cells were spread on collagen or fibronectin. Thus, as previously seen for other integrins, the alpha 2 cytoplasmic domain acts as a negative regulator, preventing indiscriminate integrin recruitment into FACs. Notably, ligand-independent localization of the VLA-2 alpha 2 subunit into FACs was partially prevented if only one or two amino acids were present in the alpha 2 cytoplasmic domain (beyond the conserved GFFKR motif) and was completely prevented by four to seven amino acids. The addition of two alanine residues (added to GFFKR) also partially prevented ligand-independent localization. In a striking inverse correlation, the same mutants showing increased ligand-independent recruitment into FACs exhibited diminished alpha 2-dependent adhesion to collagen. Thus, control of VLA-2 localization may be closely related to the suppression of cell adhesion to collagen. In contrast to FAC localization and collagen adhesion results, VLA-2-dependent binding and infection by echovirus were unaffected by either alpha 2 cytoplasmic domain deletion or exchange with other cytoplasmic domains.

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Transcriptional profiling of human cavernosal endothelial cells reveals distinctive cell adhesion phenotype and role for claudin 11 in vascular barrier function

Physiological Genomics ◽

10.1152/physiolgenomics.90354.2008 ◽

2009 ◽

Vol 39 (2) ◽

pp. 100-108 ◽

Cited By ~ 21

Author(s):

Hunter Wessells ◽

Chris J. Sullivan ◽

Yoshiaki Tsubota ◽

Karen L. Engel ◽

Bryan Kim ◽

...

Keyword(s):

Endothelial Cells ◽

Cell Adhesion ◽

Barrier Function ◽

Umbilical Vein ◽

Transcriptional Profiling ◽

Corpus Cavernosum ◽

Focal Adhesions ◽

Receptor Interaction ◽

Molecular Features ◽

Junction Protein

To determine specific molecular features of endothelial cells (ECs) relevant to the physiological process of penile erection we compared gene expression of human EC derived from corpus cavernosum of men with and without erectile dysfunction (HCCEC) to coronary artery (HCAEC) and umbilical vein (HUVEC) using Affymetrix GeneChip microarrays and GeneSifter software. Genes differentially expressed across samples were partitioned around medoids to identify genes with highest expression in HCCEC. A total of 190 genes/transcripts were highly expressed only in HCCEC. Gene Ontology classification indicated cavernosal enrichment in genes related to cell adhesion, extracellular matrix, pattern specification and organogenesis. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed high expression of genes relating to ECM-receptor interaction, focal adhesions, and cytokine-cytokine receptor interaction. Real-time PCR confirmed expression differences in cadherins 2 and 11, claudin 11 (CLDN11), desmoplakin, and versican. CLDN11, a component of tight junctions not previously described in ECs, was highly expressed only in HCCEC and its knockdown by siRNA significantly reduced transendothelial electrical resistance in HCCEC. Overall, cavernosal ECs exhibited a transcriptional profile encoding matrix and adhesion proteins that regulate structural and functional characteristics of blood vessels. Contribution of the tight junction protein CLDN11 to barrier function in endothelial cells is novel and may reflect hemodynamic requirements of the corpus cavernosum.

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