Calpain- and talin-dependent control of microvascular pericyte contractility and cellular stiffness.
Pericytes surround capillary endothelial cells and exert contractile forces modulating microvascular tone and endothelial growth. We previously described pericyte contractile phenotype to be Rho GTPase- and α-smooth muscle actin (αSMA)-dependent. However, mechanisms mediating adhesion-dependent shape changes and contractile force transduction remain largely equivocal. We now report that the neutral cysteine protease, calpain, modulates pericyte contractility and cellular stiffness via talin, an integrin-binding and F-actin associating protein. Digital imaging and quantitative analyses of living cells reveal significant perturbations in contractile force transduction detected via deformation of silicone substrata, as well as perturbations of mechanical stiffness in cellular contractile subdomains quantified via atomic force microscope (AFM)-enabled nanoindentation. Pericytes overexpressing GFP-tagged talin show significantly enhanced contractility (~two-fold), which is mitigated when either the calpain-cleavage resistant mutant talin L432G or vinculin are expressed. Moreover, the cell-penetrating, calpain-specific inhibitor termed CALPASTAT reverses talin-enhanced, but not Rho GTP-dependent, contractility. Interestingly, our analysis revealed that CALPASTAT, but not its inactive mutant, alters contractile cell-driven substrata deformations while increasing mechanical stiffness of subcellular contractile regions of these pericytes. Altogether, our results reveal that calpain-dependent cleavage of talin modulates cell contractile dynamics, which in pericytes may prove instrumental in controlling normal capillary function or microvascular pathophysiology.
Kotecki
Maciej
M
Department of Physiology, and The Center for Innovations in Wound Healing Research, Tufts University School of Medicine, Boston, MA 02111, USA.
Zeiger
Adam S
AS
Van Vliet
Krystyn J
KJ
Herman
Ira M
IM
D000818:Animals; D002154:Calpain / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D048430:Cell Shape; D002478:Cells, Cultured; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004548:Elasticity; D040542:Mechanotransduction, Cellular; D051379:Mice; D018625:Microscopy, Atomic Force; D055806:Microvessels / Q000187:drug effects / Q000378:metabolism*; D009154:Mutation; D020286:Pericytes / Q000187:drug effects / Q000378:metabolism*; D010641:Phenotype; D011993:Recombinant Fusion Proteins / Q000378:metabolism / Q000494:pharmacology; D012171:Retinal Vessels / Q000187:drug effects / Q000378:metabolism*; D016608:Talin / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection; D016596:Vinculin / Q000378:metabolism; D020742:rhoA GTP-Binding Protein / Q000378:metabolism
D015853:Cysteine Proteinase Inhibitors; D011993:Recombinant Fusion Proteins; D016608:Talin; C503097:calpastat; C496077:Tln1 protein, mouse; D016596:Vinculin; D002154:Calpain; D020742:rhoA GTP-Binding Protein