Dynamics of Membrane Rafts, Talin, and Actin at Nascent and Mechanically Perturbed Focal Adhesions

2011 ◽  
Author(s):  
Daniela E. Fuentes ◽  
Peter J. Butler
Keyword(s):  
Focal Adhesion ◽  
Focal Adhesions ◽  
Time Lapse ◽  
Membrane Rafts ◽  
Apical Surface ◽  
Electronic Signature ◽  
Mechanical Coupling ◽  
Focal Adhesion Proteins ◽  
Probe Site ◽  
Kinetics Of

A single endothelial cell was deformed at the apical surface by binding a functionalized nanoelectrode probe to a predetermined location on the surface of the cell. After identifying the point of contact, as recognized by the electronic signature of the nanoelectrode, and allowing binding to the cell of the fibronectin-functionalized tip, a focal adhesion site was induced at the probe site. The probe was displaced thereby applying a prescribed shear deformation to the surface of the cell. Locations of membrane rafts were identified by cholera toxin, and focal adhesion proteins were assessed using RFP-talin, and GFP-actin. Mechanical coupling and kinetics of assembly of these labeled proteins were measured using time-lapse fluorescent images taken under 60X with a multi-point confocal scanner. Raft marker GM1, Actin, and Talin were observed to sequentially accumulate at probe site with different kinetics not only upon probe contact but also upon deformation. Following deformation, later transient motion of rafts in the opposite direction of initial deformation was observed suggesting that rafts recoil. In conclusion, we report a novel nanoelectrode-based method for controlled manipulation of the cell surface and observed mechanical coupling of focal adhesions and cross-linked lipid rafts.

Role of tyrosine kinase pathways in ETB receptor activation of NHE3

1996 ◽  
Vol 271 (3) ◽  
pp. C763-C771 ◽  
Author(s):  
T. S. Chu ◽  
H. Tsuganezawa ◽  
Y. Peng ◽  
A. Cano ◽  
M. Yanagisawa ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Integral Membrane Protein ◽  
Receptor Activation ◽  
Kinase C ◽  
Focal Adhesion Proteins ◽  
Etb Receptor

Endothelin-1 (ET-1) binding to ETB receptors increases the activity of the apical membrane Na+/H+ antiporter (NHE3) of renal proximal tubule and cultured OKP cells. In OKPETB6 cells, a clonal cell line of OKP cells that overexpresses ETB receptors, ET-1-induced increases in Na+/H+ antiporter activity are mediated 50% by Ca2(+)-dependent pathways and 50% by tyrosine kinase pathways. ET-1 induces tyrosine phosphorylation of proteins of 68, 110, 125, 130, and 210 kDa. ET-1-induced tyrosine phosphorylation is mediated by the ETB receptor and is not dependent on increases in cell Ca2+ or protein kinase C. The 68-, 110-, 125-, and 130-kDa phosphoproteins are cytosolic, whereas the 210-kDa phosphoprotein is an integral membrane protein. Immunoprecipitation studies showed that the 68-kDa protein is paxillin and the 125-kDa protein is p125FAK (focal adhesion kinase). Cytochalasin D, which disrupts focal adhesions, prevented ET-1-induced tyrosine phosphorylation of paxillin, p110, p125FAK, and p130 but did not prevent tyrosine phosphorylation of p210 and did not prevent ET-1-induced increases in Na+/H+ antiporter activity. Thus 50% of ETB receptor-induced Na+/H+ antiporter activation is mediated by tyrosine kinase pathways, possibly involving p210. ETB receptor activation also induces tyrosine phosphorylation of focal adhesion proteins, but this is not required for antiporter activation.

scholarly journals Diverse cell junctions with unique molecular composition in tissues of a sponge (Porifera)

EvoDevo ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Jennyfer M. Mitchell ◽  
Scott A. Nichols
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Adherens Junction ◽  
Focal Adhesions ◽  
Molecular Composition ◽  
Cell Junctions ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins ◽  
Junction Protein ◽  
Cell Cell

Abstract The integrity and organization of animal tissues depend upon specialized protein complexes that mediate adhesion between cells with each other (cadherin-based adherens junctions), and with the extracellular matrix (integrin-based focal adhesions). Reconstructing how and when these cell junctions evolved is central to understanding early tissue evolution in animals. We examined focal adhesion protein homologs in tissues of the freshwater sponge, Ephydatia muelleri (phylum Porifera; class Demospongiae). Our principal findings are that (1) sponge focal adhesion homologs (integrin, talin, focal adhesion kinase, etc.) co-precipitate as a complex, separate from adherens junction proteins; (2) that actin-based structures resembling focal adhesions form at the cell–substrate interface, and their abundance is dynamically regulated in response to fluid shear; (3) focal adhesion proteins localize to both cell–cell and cell–extracellular matrix adhesions, and; (4) the adherens junction protein β-catenin is co-distributed with focal adhesion proteins at cell–cell junctions everywhere except the choanoderm, and at novel junctions between cells with spicules, and between cells with environmental bacteria. These results clarify the diversity, distribution and molecular composition of cell junctions in tissues of E. muelleri, but raise new questions about their functional properties and ancestry.

scholarly journals Investigating lasp-2 in cell adhesion: new binding partners and roles in motility

2013 ◽  
Vol 24 (7) ◽  
pp. 995-1006 ◽  
Author(s):  
Katherine T. Bliss ◽  
Miensheng Chu ◽  
Colin M. Jones-Weinert ◽  
Carol C. Gregorio
Keyword(s):  
Focal Adhesion ◽  
Cell Attachment ◽  
Protein Complexes ◽  
Metastatic Cancer ◽  
Focal Adhesions ◽  
Actin Binding ◽  
Binding Partners ◽  
Focal Adhesion Proteins ◽  
Metastatic Cancer Cell

Focal adhesions are intricate protein complexes that facilitate cell attachment, migration, and cellular communication. Lasp-2 (LIM-nebulette), a member of the nebulin family of actin-binding proteins, is a newly identified component of these complexes. To gain further insights into the functional role of lasp-2, we identified two additional binding partners of lasp-2: the integral focal adhesion proteins vinculin and paxillin. Of interest, the interaction of lasp-2 with its binding partners vinculin and paxillin is significantly reduced in the presence of lasp-1, another nebulin family member. The presence of lasp-2 appears to enhance the interaction of vinculin and paxillin with each other; however, as with the interaction of lasp-2 with vinculin or paxillin, this effect is greatly diminished in the presence of excess lasp-1. This suggests that the interplay between lasp-2 and lasp-1 could be an adhesion regulatory mechanism. Lasp-2’s potential role in metastasis is revealed, as overexpression of lasp-2 in either SW620 or PC-3B1 cells—metastatic cancer cell lines—increases cell migration but impedes cell invasion, suggesting that the enhanced interaction of vinculin and paxillin may functionally destabilize focal adhesion composition. Taken together, these data suggest that lasp-2 has an important role in coordinating and regulating the composition and dynamics of focal adhesions.

scholarly journals Requirements for localization of p130cas to focal adhesions.

1997 ◽  
Vol 17 (7) ◽  
pp. 3884-3897 ◽  
Author(s):  
T Nakamoto ◽  
R Sakai ◽  
H Honda ◽  
S Ogawa ◽  
H Ueno ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Src Kinase ◽  
Focal Adhesions ◽  
Sh3 Domain ◽  
Transformed Cells ◽  
Binding Motifs ◽  
Adhesion Proteins ◽  
C Terminus ◽  
Focal Adhesion Proteins ◽  
Tyrosine Phosphorylated Protein

p130cas (Cas) is an adapter protein that has an SH3 domain followed by multiple SH2 binding motifs in the substrate domain. It also contains a tyrosine residue and a proline-rich sequence near the C terminus, which are the binding sites for the SH2 and SH3 domains of Src kinase, respectively. Cas was originally identified as a major tyrosine-phosphorylated protein in v-Crk- and v-Src-transformed cells. Subsequently, Cas was shown to be inducibly tyrosine phosphorylated upon integrin stimulation; it is therefore regarded as one of the focal adhesion proteins. Using an immunofluorescence study, we examined the subcellular localization of Cas and determined the regions required for its localization to focal adhesions. In nontransformed cells, Cas was localized predominantly to the cytoplasm and partially to focal adhesions. However, in 527F-c-Src-transformed cells, Cas was localized mainly to podosomes, where the focal adhesion proteins are assembled. The localization of Cas to focal adhesions was also observed in cells expressing the kinase-negative 527F/295M-c-Src. A series of analyses with deletion mutants expressed in various cells revealed that the SH3 domain of Cas is necessary for its localization to focal adhesions in nontransformed cells while both the SH3 domain and the C-terminal Src binding domain of Cas are required in 527F-c-Src-transformed cells and fibronectin-stimulated cells. In addition, the localization of Cas to focal adhesions was abolished in Src-negative cells. These results demonstrate that the SH3 domain of Cas and the association of Cas with Src kinase play a pivotal role in the localization of Cas to focal adhesions.

scholarly journals Retrograde Fluxes of Focal Adhesion Proteins in Response to Cell Migration and Mechanical Signals

2007 ◽  
Vol 18 (11) ◽  
pp. 4519-4527 ◽  
Author(s):  
Wei-hui Guo ◽  
Yu-li Wang
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Mechanical Load ◽  
Focal Adhesions ◽  
Force Production ◽  
Retrograde Transport ◽  
Mechanical Stimuli ◽  
Adhesion Proteins ◽  
Mechanical Signals ◽  
Focal Adhesion Proteins

Recent studies suggest that mechanical signals mediated by the extracellular matrix play an essential role in various physiological and pathological processes; yet, how cells respond to mechanical stimuli remains elusive. Using live cell fluorescence imaging, we found that actin filaments, in association with a number of focal adhesion proteins, including zyxin and vasodilator-stimulated phosphoprotein, undergo retrograde fluxes at focal adhesions in the lamella region. This flux is inversely related to cell migration, such that it is amplified in fibroblasts immobilized on micropatterned islands. In addition, the flux is regulated by mechanical signals, including stretching forces applied to flexible substrates and substrate stiffness. Conditions favoring the flux share the common feature of causing large retrograde displacements of the interior actin cytoskeleton relative to the substrate anchorage site, which may function as a switch translating mechanical input into chemical signals, such as tyrosine phosphorylation. In turn, the stimulation of actin flux at focal adhesions may function as part of a feedback mechanism, regulating structural assembly and force production in relation to cell migration and mechanical load. The retrograde transport of associated focal adhesion proteins may play additional roles in delivering signals from focal adhesions to the interior of the cell.

scholarly journals Migration regulates cellular mechanical states

2019 ◽  
Vol 30 (26) ◽  
pp. 3104-3111 ◽  
Author(s):  
Stephanie S. Chang ◽  
Andrew D. Rape ◽  
Stephanie A. Wong ◽  
Wei-hui Guo ◽  
Yu-li Wang
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Traction Forces ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins

Cell migration has a profound effect on the generation of traction forces and the phosphorylation of focal adhesion proteins. The mechanism may involve the dynamic turnover of focal adhesions during cell migration and mechanical interactions between nascent and preexisting focal adhesions.

Focal adhesions disassemble during early pregnancy in rat uterine epithelial cells

2008 ◽  
Vol 20 (8) ◽  
pp. 892 ◽  
Author(s):  
Yui Kaneko ◽  
Laura A. Lindsay ◽  
Christopher R. Murphy
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Focal Adhesion ◽  
Basal Cell ◽  
Early Pregnancy ◽  
Focal Adhesions ◽  
Uterine Epithelial Cells ◽  
Focal Adhesion Proteins ◽  
Decidual Cells ◽  
Implantation Sites

During early pregnancy in rodents, invasion of the blastocyst into the endometrial decidual cells is accompanied by the removal of uterine epithelial cells around the implantation sites. The present study investigated the distribution and expression of two focal adhesion proteins, namely talin and paxillin, in rat uterine epithelial cells during early pregnancy and their role in the loss of these cells at the time of implantation. A major distributional change of talin and paxillin was demonstrated in uterine epithelial cells during early pregnancy. From a highly concentrated expression along the basal cell surface on Day 1 of pregnancy, talin and paxillin were lost from the basal cell surface at the time of implantation. There was also a corresponding statistically significant decrease in paxillin seen through western blotting analysis. Together, these observations suggest that uterine epithelial cells are less adherent to the underlying basal lamina due to the disassembly of talin and paxillin from focal adhesions, facilitating removal of these cells at the time of implantation. This phenomenon was restricted to the period of receptivity because talin and paxillin reappeared along the basal cell surface soon after implantation.

scholarly journals Diverse cell junctions with unique molecular composition in tissues of a sponge (Porifera)

2019 ◽  
Author(s):  
Jennyfer M. Mitchell ◽  
Scott A. Nichols
Keyword(s):  
Extracellular Matrix ◽  
Focal Adhesion ◽  
Protein Complexes ◽  
Adherens Junction ◽  
Focal Adhesions ◽  
Molecular Composition ◽  
Cell Junctions ◽  
Adhesion Proteins ◽  
Focal Adhesion Proteins ◽  
Junction Protein

AbstractThe integrity and organization of animal tissues depends upon specialized protein complexes that mediate adhesion between cells with each other (cadherin-based adherens junctions), and with the extracellular matrix (integrin-based focal adhesions). Reconstructing how and when these cell junctions evolved is central to understanding early tissue evolution in animals. We examined focal adhesion protein homologs in tissues of the freshwater sponge, Ephydatia muelleri (phylum Porifera). We found that sponge homologs of focal adhesion proteins co-precipitate as a complex and localize to cell junctions in sponge tissues. These data support that the adhesion roles of these proteins evolved early, prior to the divergence of sponges and other animals. However, in contrast to the spatially partitioned distribution of cell junctions in epithelia of other animals, focal adhesion proteins were found to be co-distributed with the adherens junction protein Emβ-catenin in sponge tissues; both at certain cell-cell and cell-extracellular matrix (ECM) adhesions. Sponge adhesion structures were found to be unique in other ways, too. The basopinacoderm (substrate-attachment epithelium) lacks typical polarity in that cell-ECM adhesions form on both basal and apical surfaces, and compositionally unique cell junctions form at the interface between cells with spicules (siliceous skeletal elements) and between cells and environmental bacteria. These results clarify the diversity, distribution and molecular composition of cell junctions in tissues of E. muelleri, but raise new questions about their function and homology with cell junctions in other animals.

scholarly journals Calpain-mediated Proteolysis of Paxillin Negatively Regulates Focal Adhesion Dynamics and Cell Migration

2011 ◽  
Vol 286 (12) ◽  
pp. 9998-10006 ◽  
Author(s):  
Christa L. Cortesio ◽  
Lindsy R. Boateng ◽  
Timothy M. Piazza ◽  
David A. Bennin ◽  
Anna Huttenlocher
Keyword(s):  
Cell Migration ◽  
Cell Motility ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Adaptor Protein ◽  
Time Lapse ◽  
Negative Regulator ◽  
Proteolytic Fragment ◽  
Proteolytic Site ◽  
And Migration

The dynamic turnover of integrin-mediated adhesions is important for cell migration. Paxillin is an adaptor protein that localizes to focal adhesions and has been implicated in cell motility. We previously reported that calpain-mediated proteolysis of talin1 and focal adhesion kinase mediates adhesion disassembly in motile cells. To determine whether calpain-mediated paxillin proteolysis regulates focal adhesion dynamics and cell motility, we mapped the preferred calpain proteolytic site in paxillin. The cleavage site is between the paxillin LD1 and LD2 motifs and generates a C-terminal fragment that is similar in size to the alternative product paxillin delta. The calpain-generated proteolytic fragment, like paxillin delta, functions as a paxillin antagonist and impairs focal adhesion disassembly and migration. We generated mutant paxillin with a point mutation (S95G) that renders it partially resistant to calpain proteolysis. Paxillin-deficient cells that express paxillin S95G display increased turnover of zyxin-containing adhesions using time-lapse microscopy and also show increased migration. Moreover, cancer-associated somatic mutations in paxillin are common in the N-terminal region between the LD1 and LD2 motifs and confer partial calpain resistance. Taken together, these findings suggest a novel role for calpain-mediated proteolysis of paxillin as a negative regulator of focal adhesion dynamics and migration that may function to limit cancer cell invasion.

scholarly journals The ubiquitin-proteasome system regulates focal adhesions at the leading edge of migrating cells

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Anjali Teckchandani ◽  
Jonathan A Cooper
Keyword(s):  
Cell Migration ◽  
Focal Adhesion ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Ubiquitin Proteasome System ◽  
Cytokine Signaling ◽  
Focal Adhesion Proteins ◽  
Cullin 5 ◽  
Ubiquitin Proteasome ◽  
Spatio Temporal

Cell migration requires the cyclical assembly and disassembly of focal adhesions. Adhesion induces phosphorylation of focal adhesion proteins, including Cas (Crk-associated substrate/p130Cas/BCAR1). However, Cas phosphorylation stimulates adhesion turnover. This raises the question of how adhesion assembly occurs against opposition from phospho-Cas. Here we show that suppressor of cytokine signaling 6 (SOCS6) and Cullin 5, two components of the CRL5SOCS6 ubiquitin ligase, inhibit Cas-dependent focal adhesion turnover at the front but not rear of migrating epithelial cells. The front focal adhesions contain phospho-Cas which recruits SOCS6. If SOCS6 cannot access focal adhesions, or if cullins or the proteasome are inhibited, adhesion disassembly is stimulated. This suggests that the localized targeting of phospho-Cas within adhesions by CRL5SOCS6 and concurrent cullin and proteasome activity provide a negative feedback loop, ensuring that adhesion assembly predominates over disassembly at the leading edge. By this mechanism, ubiquitination provides a new level of spatio-temporal control over cell migration.

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