Modulation of FAK and Src adhesion signaling occurs independently of adhesion complex composition

Self Cite

Low-intensity pulsed ultrasound (LIPUS) is a therapy used clinically to promote healing. Using live-cell imaging we show that LIPUS stimulation, acting through integrin-mediated cell-matrix adhesions, rapidly induces Rac1 activation associated with dramatic actin cytoskeleton rearrangements. Our study demonstrates that the mechanosensitive focal adhesion (FA) protein vinculin, and both focal adhesion kinase (FAK, also known as PTK2) and Rab5 (both the Rab5a and Rab5b isoforms) have key roles in regulating these effects. Inhibiting the link of vinculin to the actin-cytoskeleton abolished LIPUS sensing. We show that this vinculin-mediated link was not only critical for Rac1 induction and actin rearrangements, but was also important for the induction of a Rab5-dependent increase in the number of early endosomes. Expression of dominant-negative Rab5, or inhibition of endocytosis with dynasore, also blocked LIPUSinduced Rac1 signalling events. Taken together, our data show that LIPUS is sensed by cell matrix adhesions through vinculin, which in turn modulates a Rab5-Rac1 pathway to control ultrasound-mediated endocytosis and cell motility. Finally, we demonstrate that a similar FAK-Rab5-Rac1 pathway acts to control cell spreading upon fibronectin.

Section: I997asupporting

confidence: 80%

“…6B). We and others have recently shown that pretreatment with 3 µM FAKi for 1 h reduces tyrosine phosphorylation at FAs in NIH3T3 and HFF cells, without affecting the molecular composition of FAs or vinculin turnover (Horton et al, 2016;Stutchbury et al, 2017).…”

Section: I997amentioning

confidence: 99%

Low-intensity pulsed ultrasound promotes cell motility through vinculin-controlled Rac1 GTPase activity

Atherton

Lausecker

Harrison³

et al. 2017

Self Cite

“…Regulation of adhesion complexes by LAR is independent of FAK activity FAK and Src are non-receptor tyrosine kinases frequently linked to adhesion complex dynamics, cell adhesion and cell migration (Mitra and Schlaepfer, 2006), although recent data also report that adhesion complex composition is unaffected when Src and FAK are inhibited (Horton et al, 2016). Tyr 118 of paxillin is a known FAK substrate (Bellis et al, 1995), and we tested whether activation of FAK was affected in the absence of LAR activity.…”

Section: Resultsmentioning

confidence: 99%

LAR protein tyrosine phosphatase regulates focal adhesions through CDK1

Sarhan

Patel

Cowell

et al. 2016

Focal adhesions are complex multi-molecular structures that link the actin cytoskeleton to the extracellular matrix through integrin adhesion receptors and play a key role in regulation of many cellular functions. LAR (also known as PTPRF) is a receptor protein tyrosine phosphatase that regulates PDGF signalling and localises to focal adhesions. We have observed that loss of LAR phosphatase activity in mouse embryonic fibroblasts results in reduced numbers of focal adhesions and decreased adhesion to fibronectin. To understand how LAR regulates cell adhesion we used phosphoproteomic data, comparing global phosphorylation events in wild-type and LAR phosphatase-deficient cells, to analyse differential kinase activity. Kinase prediction analysis of LAR-regulated phosphosites identified a node of cytoskeleton-and adhesion-related proteins centred on cyclin-dependent kinase-1 (CDK1). We found that loss of LAR activity resulted in reduced activity of CDK1, and that CDK1 activity was required for LAR-mediated focal adhesion complex formation. We also established that LAR regulates CDK1 activity through c-Abl and Akt family proteins. In summary, we have identified a new role for a receptor protein tyrosine phosphatase in regulating CDK1 activity and hence cell adhesion to the extracellular matrix.

“…extensively characterized and lead to a strong inhibition of FAK and paxillin phosphorylation in the FA (Horton et al, 2016, Fig. S4A).…”

Section: Fak and Paxillin Phosphorylation In Mechanotransductionmentioning

confidence: 99%

“…NIH3T3 cells were trypsinized and suspended in 2 ml of supplemented DMEM containing 3 µM FAKi+3 µM Srci+50 µM Y-27632, or an equivalent volume of DMSO for 1 h. These concentrations and treatment times are sufficient for maximal FAK and Src inhibition (Horton et al, 2016). Cells were plated on FN-coated coverslips for 4 h prior to fixation and staining.…”

Section: Y-27632 Experimentsmentioning

confidence: 99%

Distinct focal adhesion protein modules control different aspects of mechanotransduction

Stutchbury

Atherton

Tsang

et al. 2017

Self Cite

156

142

Focal adhesions (FAs) are macromolecular complexes that regulate cell adhesion and mechanotransduction. By performing fluorescence recovery after photobleaching (FRAP) and fluorescence loss after photoactivation (FLAP) experiments, we found that the mobility of core FA proteins correlates with their function. Structural proteins such as tensin, talin and vinculin are significantly less mobile in FAs than signaling proteins such as FAK (also known as PTK2) and paxillin. The mobilities of the structural proteins are directly influenced by substrate stiffness, suggesting that they are involved in sensing the rigidity of the extracellular environment. The turnover rates of FAK and paxillin, as well as kindlin2 (also known as FERMT2), are not influenced by substrate stiffness. By using specific Src and FAK inhibitors, we reveal that force-sensing by vinculin occurs independently of FAK and paxillin phosphorylation. However, their phosphorylation is required for downstream Rac1-driven cellular processes, such as protrusion and cell migration. Overall, we show that the FA is composed of different functional modules that separately control mechanosensing and the cellular mechano-response.