Talin controls the exit of the integrin alpha 5 beta 1 from an early compartment of the secretory pathway

2000 ◽  
Vol 113 (11) ◽  
pp. 1951-1961 ◽  
Author(s):  
V. Martel ◽  
L. Vignoud ◽  
S. Dupe ◽  
P. Frachet ◽  
M.R. Block ◽  
...  
Keyword(s):  
Membrane Fraction ◽  
Secretory Pathway ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Integrin Receptors ◽  
Fibronectin Receptor ◽  
Integrin Alpha5beta1 ◽  
Beta1 Integrin ◽  
Intracellular Domains ◽  
Export Signal

Talin is a major cytosolic protein that links the intracellular domains of beta1 and beta3 integrins to the cytoskeleton. It is required for focal adhesion assembly. However, its downregulation not only slows down cell spreading and organization of focal adhesions but also impairs the maturation of some beta1 integrins, including the fibronectin receptor alpha5beta1. To investigate this, we characterized the beta1 integrin synthesized in cells expressing talin anti-sense RNA (AT22 cells). We identified a large intracellular pool of beta1 integrins that is abnormally accumulated in an earlier compartment of the secretory pathway. In this report, we show that in talin-deficient AT22 cells, the aberrant glycosylation of integrin receptors is accompanied by a delay in the export of the integrin alpha5beta1. In normal cells, talin was found associated with beta1 integrins in an enriched membrane fraction containing Golgi and endoplasmic reticulum. Finally, microinjection of anti-talin antibodies resulted in accumulation of the integrins within the cells. These data strongly suggest that talin plays a specific role in the export of newly synthesized integrins. We propose that talin binding to the integrin may disclose a diphenylalanine export signal, which is present in the membrane-proximal GFFKR motif conserved in all integrin alpha chains.

scholarly journals Initiation of Attachment and Generation of Mature Focal Adhesions by Integrin-containing Filopodia in Cell Spreading

2006 ◽  
Vol 17 (10) ◽  
pp. 4237-4248 ◽  
Author(s):  
Michael A. Partridge ◽  
Eugene E. Marcantonio
Keyword(s):  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Integrin Receptors ◽  
Cortical Actin ◽  
Cell Matrix ◽  
Cytoplasmic Proteins ◽  
Radial Projection ◽  
Anchor Points

Integrin receptors, and associated cytoplasmic proteins mediate adhesion, cell signaling and connections to the cytoskeleton. Using fluorescent protein chimeras, we analyzed initial integrin adhesion in spreading fibroblasts with Total Internal Reflection Fluorescence (TIRF) microscopy. Surprisingly, sequential radial projection of integrin and actin containing filopodia formed the initial cell-matrix contacts. These Cdc42-dependent, integrin-containing projections recruited cytoplasmic focal adhesion (FA) proteins in a hierarchical manner; initially talin with integrin and subsequently FAK and paxillin. Radial FA structures then anchored cortical actin bridges between them and subsequently cells reorganized their actin, a process promoted by Src, and characterized by lateral FA reorientation to provide anchor points for actin stress fibers. Finally, the nascent adhesions coalesced until they formed mature FAs.

scholarly journals EHD1 regulates beta1 integrin endosomal transport: effects on focal adhesions, cell spreading and migration

2007 ◽  
Vol 120 (5) ◽  
pp. 802-814 ◽  
Author(s):  
M. Jovic ◽  
N. Naslavsky ◽  
D. Rapaport ◽  
M. Horowitz ◽  
S. Caplan
Keyword(s):  
Focal Adhesions ◽  
Cell Spreading ◽  
Transport Effects ◽  
Beta1 Integrin ◽  
Endosomal Transport ◽  
And Migration

scholarly journals Integrin alpha 8 beta 1 promotes attachment, cell spreading, and neurite outgrowth on fibronectin.

1995 ◽  
Vol 6 (4) ◽  
pp. 433-448 ◽  
Author(s):  
U Müller ◽  
B Bossy ◽  
K Venstrom ◽  
L F Reichardt
Keyword(s):  
Neurite Outgrowth ◽  
Cell Attachment ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
K562 Cells ◽  
Integrin Subunit ◽  
Rgd Peptides ◽  
Fibronectin Receptor ◽  
Extensive Cell ◽  
Rgd Site

The integrin alpha 8 subunit, isolated by low stringency hybridization, is a novel integrin subunit that associates with beta 1. To identify ligands, we have prepared a function-blocking antiserum to the extracellular domain of alpha 8, and we have established by transfection K562 cell lines that stably express alpha 8 beta 1 heterodimers on the cell surface. We demonstrate here by cell adhesion and neurite outgrowth assays that alpha 8 beta 1 is a fibronectin receptor. Studies on fibronectin fragments using RGD peptides as inhibitors show that alpha 8 beta 1 binds to the RGD site of fibronectin. In contrast to the endogenous alpha 5 beta 1 fibronectin receptor in K562 cells, alpha 8 beta 1 not only promotes cell attachment but also extensive cell spreading, suggesting functional differences between the two receptors. In chick embryo fibroblasts, alpha 8 beta 1 is localized to focal adhesions. We conclude that alpha 8 beta 1 is a receptor for fibronectin and can promote attachment, cell spreading, and neurite outgrowth on fibronectin.

Myocilin binding to Hep II domain of fibronectin inhibits cell spreading and incorporation of paxillin into focal adhesions

2005 ◽  
Vol 303 (2) ◽  
pp. 218-228 ◽  
Author(s):  
Donna M. Peters ◽  
Kathleen Herbert ◽  
Brenda Biddick ◽  
Jennifer A. Peterson
Keyword(s):  
Focal Adhesions ◽  
Cell Spreading

Syndesmos, a protein that interacts with the cytoplasmic domain of syndecan-4, mediates cell spreading and actin cytoskeletal organization

2000 ◽  
Vol 113 (2) ◽  
pp. 315-324 ◽  
Author(s):  
P.C. Baciu ◽  
S. Saoncella ◽  
S.H. Lee ◽  
F. Denhez ◽  
D. Leuthardt ◽  
...  
Keyword(s):  
Plasma Membranes ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Cytoplasmic Domain ◽  
Cellular Protein ◽  
Actin Stress Fiber ◽  
Independent Manner ◽  
Intracellular Proteins ◽  
Central Regions ◽  
A Cell

Syndecan-4 is a cell surface heparan sulfate proteoglycan which, in cooperation with integrins, transduces signals for the assembly of focal adhesions and actin stress fibers in cells plated on fibronectin. The regulation of these cellular events is proposed to occur, in part, through the interaction of the cytoplasmic domains of these transmembrane receptors with intracellular proteins. To identify potential intracellular proteins that interact with the cytoplasmic domain of syndecan-4, we carried out a yeast two-hybrid screen in which the cytoplasmic domain of syndecan-4 was used as bait. As a result of this screen, we have identified a novel cellular protein that interacts with the cytoplasmic domain of syndecan-4 but not with those of the other three syndecan family members. The interaction involves both the membrane proximal and variable central regions of the cytoplasmic domain. We have named this cDNA and encoded protein syndesmos. Syndesmos is ubiquitously expressed and can be myristylated. Consistent with its myristylation and syndecan-4 association, syndesmos colocalizes with syndecan-4 in the ventral plasma membranes of cells plated on fibronectin. When overexpressed in NIH 3T3 cells, syndesmos enhances cell spreading, actin stress fiber and focal contact formation in a serum-independent manner.

scholarly journals Microinjection of antibodies against talin inhibits the spreading and migration of fibroblasts

1992 ◽  
Vol 102 (4) ◽  
pp. 753-762
Author(s):  
G.H. Nuckolls ◽  
L.H. Romer ◽  
K. Burridge
Keyword(s):  
Extracellular Matrix ◽  
Tissue Culture ◽  
Actin Filaments ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
And Migration ◽  
Extracellular Matrix Receptors

Talin is believed to be one of the key proteins involved in linking actin filaments to extracellular matrix receptors in focal adhesions. Our strategy for studying the function of talin has been to inactivate talin in living fibroblasts in tissue culture through the microinjection of affinity-purified, polyclonal anti-talin antibodies. The effect of the injected anti-talin antibodies on cell spreading was found to depend on how recently the cells had been plated. Cells that were in the process of spreading on a fibronectin substratum, and which had newly developed focal adhesions, were induced to round up and to disassemble many of the adhesions. However, if fibroblasts were allowed to spread completely before they were microinjected with the anti-talin antibody, focal adhesions remained intact and the flat morphology of the cells was unaffected. The percentage of cells that were able to maintain a spread morphology despite the injection of anti-talin antibodies increased during the first few hours after plating on fibronectin substrata. Fibroblasts that were allowed to spread completely before microinjection with the anti-talin antibody retained both intact focal adhesions and a flat, well-spread morphology, but failed to migrate effectively. Our experiments do not directly address the role of talin in mature focal adhesions, but they indicate that talin is essential for the spreading and migration of fibroblasts on fibronectin as well as for the development and initial maintenance of focal adhesions on this substratum.

scholarly journals Focal adhesions are sites of integrin extension

2010 ◽  
Vol 188 (6) ◽  
pp. 891-903 ◽  
Author(s):  
Janet A. Askari ◽  
Christopher J. Tynan ◽  
Stephen E.D. Webb ◽  
Marisa L. Martin-Fernandez ◽  
Christoph Ballestrem ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Resonance Energy Transfer ◽  
Focal Adhesions ◽  
Resonance Energy ◽  
Cell Spreading ◽  
Integrin Subunit ◽  
Adherent Cells ◽  
Extended Form ◽  
Integrin Α5β1 ◽  
Mediate Cell

Integrins undergo global conformational changes that specify their activation state. Current models portray the inactive receptor in a bent conformation that upon activation converts to a fully extended form in which the integrin subunit leg regions are separated to enable ligand binding and subsequent signaling. To test the applicability of this model in adherent cells, we used a fluorescent resonance energy transfer (FRET)–based approach, in combination with engineered integrin mutants and monoclonal antibody reporters, to image integrin α5β1 conformation. We find that restricting leg separation causes the integrin to adopt a bent conformation that is unable to respond to agonists and mediate cell spreading. By measuring FRET between labeled α5β1 and the cell membrane, we find extended receptors are enriched in focal adhesions compared with adjacent regions of the plasma membrane. These results demonstrate definitely that major quaternary rearrangements of β1-integrin subunits occur in adherent cells and that conversion from a bent to extended form takes place at focal adhesions.

scholarly journals Lipid droplets disrupt mechanosensing in human hepatocytes

2020 ◽  
Vol 319 (1) ◽  
pp. G11-G22
Author(s):  
LiKang Chin ◽  
Neil D. Theise ◽  
Abigail E. Loneker ◽  
Paul A. Janmey ◽  
Rebecca G. Wells
Keyword(s):  
Mechanical Stress ◽  
Nuclear Localization ◽  
Lipid Droplets ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Human Hepatocytes ◽  
Stress Fibers ◽  
Primary Hepatocytes ◽  
The Impact ◽  
Lipid Loading

This work examines the impact of lipid loading on mechanosensing by human hepatocytes. In cirrhotic livers, the presence of large (although not small) lipid droplets increased nuclear localization of the mechanotransducer YAP. In primary hepatocytes in culture, lipid droplets led to decreased stiffness-induced cell spreading and disrupted focal adhesions and stress fibers; the presence of large lipid droplets resulted in increased YAP nuclear localization. Collectively, the data suggest that lipid droplets induce intracellular mechanical stress.

scholarly journals Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates

2018 ◽  
Vol 115 (12) ◽  
pp. E2686-E2695 ◽  
Author(s):  
Ze Gong ◽  
Spencer E. Szczesny ◽  
Steven R. Caliari ◽  
Elisabeth E. Charrier ◽  
Ovijit Chaudhuri ◽  
...  
Keyword(s):  
Focal Adhesions ◽  
Cell Spreading ◽  
Optimal Level ◽  
Cellular Adhesion ◽  
Direct Monte Carlo Simulation ◽  
Material Parameters ◽  
Wide Range ◽  
Maximum Cell ◽  
And Function ◽  
Characteristic Binding

Recent evidence has shown that, in addition to rigidity, the viscous response of the extracellular matrix (ECM) significantly affects the behavior and function of cells. However, the mechanism behind such mechanosensitivity toward viscoelasticity remains unclear. In this study, we systematically examined the dynamics of motor clutches (i.e., focal adhesions) formed between the cell and a viscoelastic substrate using analytical methods and direct Monte Carlo simulation. Interestingly, we observe that, for low ECM rigidity, maximum cell spreading is achieved at an optimal level of viscosity in which the substrate relaxation time falls between the timescale for clutch binding and its characteristic binding lifetime. That is, viscosity serves to stiffen soft substrates on a timescale faster than the clutch off-rate, which enhances cell−ECM adhesion and cell spreading. On the other hand, for substrates that are stiff, our model predicts that viscosity will not influence cell spreading, since the bound clutches are saturated by the elevated stiffness. The model was tested and validated using experimental measurements on three different material systems and explained the different observed effects of viscosity on each substrate. By capturing the mechanism by which substrate viscoelasticity affects cell spreading across a wide range of material parameters, our analytical model provides a useful tool for designing biomaterials that optimize cellular adhesion and mechanosensing.

scholarly journals Lateral Spacing of TiO2 Nanotubes Modulates Osteoblast Behavior

Materials ◽  
2019 ◽  
Vol 12 (18) ◽  
pp. 2956 ◽  
Author(s):  
Madalina Georgiana Necula ◽  
Anca Mazare ◽  
Raluca Nicoleta Ion ◽  
Selda Ozkan ◽  
Jung Park ◽  
...  
Keyword(s):  
Tio2 Nanotubes ◽  
Focal Adhesions ◽  
Cell Spreading ◽  
Tio2 Nanotube ◽  
Local Drug Delivery ◽  
Cell Behavior ◽  
Osteoblast Cells ◽  
Differentiation Potential ◽  
Nanostructured Surfaces ◽  
Biological Performance

Titanium dioxide (TiO2) nanotube coated substrates have revolutionized the concept of implant in a number of ways, being endowed with superior osseointegration properties and local drug delivery capacity. While accumulating reports describe the influence of nanotube diameter on cell behavior, little is known about the effects of nanotube lateral spacing on cells involved in bone regeneration. In this context, in the present study the MC3T3-E1 murine pre-osteoblast cells behavior has been investigated by using TiO2 nanotubes of ~78 nm diameter and lateral spacing of 18 nm and 80 nm, respectively. Both nanostructured surfaces supported cell viability and proliferation in approximately equal extent. However, obvious differences in the cell spreading areas, morphologies, the organization of the actin cytoskeleton and the pattern of the focal adhesions were noticed. Furthermore, investigation of the pre-osteoblast differentiation potential indicated a higher capacity of larger spacing nanostructure to enhance the expression of the alkaline phosphatase, osteopontin and osteocalcin osteoblast specific markers inducing osteogenic differentiation. These findings provide the proof that lateral spacing of the TiO2 nanotube coated titanium (Ti) surfaces has to be considered in designing bone implants with improved biological performance.

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