Calpain- and talin-dependent control of microvascular pericyte contractility and cellular stiffness

Abstract

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.

Fig. 1. Molecular control of pericyte contractility

Bovine retinal pericytes were electroporated either with plasmids encoding control EGFP and fusion of EGFP-talin, EGFP-talin L432G (calpain-resistant mutant) or EGFP-vinculin, or co-transfected (in 1:4 ratio) with plasmids encoding EGFP and RhoA Q63L (dominant active mutant). On next day, transfected pericytes were seeded onto deformable silicone substrata. After 24 h live GFP-positive cells were evaluated by microscopy. (A) A pericyte overexpressing EGFP-talin is shown wrinkling silicone substratum (left panel - phase contrast image, central panel – fluorescence image, right panel – overlay image of phase contrast and fluorescence pseudocolored red and green, respectively). An arrow points at cell contraction-driven deformations of the substrata. (B) A graph showing contractile force transduction, as measured by cell contractility index (CCI) and described in Methods, in pericytes overexpressing designated proteins, CCI for EGFP control was equaled to 1, to which other CCI values were normalized. Asterisk (*) indicates statistically significant difference between control EGFP and EGFP-talin (p<4E-9), and EGFP/RhoA Q63L (p<0.04), respectively. Double asterisk (**) indicates statistically significant difference between EGFP-talin and EGFP-talin L432G (p<2E-05). The CCI of EGFP-talin L432G and EGFP-vinculin were not statistically significantly different from control EGFP. Scale bar = 30 µm.

Fig. 2. Calpain regulates pericyte contractile force production

Untransfected pericyte force production, as measured by Cell Contractility Index (CCI, see Materials and Methods) was monitored and quantified as a function of calpain inhibition. CALPST and its inactive mutant CALPSTala were added at final concentrations of 0, 5, 25, 100 µM for 24, 48 and 96 hours of treatment. The inhibitors were added at the time of seeding pericytes onto deformable substrata and re-applied in a fresh medium at 24 hr in cultures scored at 48 h, and at 24 and 72 hr in cultures scored at 96 h. Asterisk (*) indicates statistically significant differences between pericytes treated with 100 µM CALPST for 48 h and either untreated (p<4E-04), treated with 100 µM CALPSTala for 48 h (p<0.01) or 25 µM CALPST for 48 h (p<0.01). Double asterisk (**) indicates statistically significant differences between pericytes treated with 100 µM CALPST for 96 h and either untreated (p<2E-07), treated with 100 µM CALPSTala for 96 h (p<6E-05), 100 µM CALPSTala for 96 h (p<5E-04) or 25 µM CALPST for 96 h (p<5E-04),

Fig. 3. Talin, but not RhoA-induced pericyte contractility is calpain-dependent

Bovine retinal pericytes were electroporated either with plasmids encoding control EGFP and fusion of EGFP-talin, EGFP-talin L432G (calpain-resistant mutant, or co-transfected (in 1:4 ratio) with plasmids encoding EGFP and RhoA Q63L (dominant active mutant). On next day, transfected pericytes were seeded onto deformable silicone substrata. After 24 h live GFP-positive cells were evaluated by microscopy. On next day, transfected pericytes were seeded onto deformable silicone substrata, and either left untreated or treated with calpain inhibitor CALPST at 25 µM final concentration for 24 h. Contractility of samples was analyzed for Cell Contractility Index (CCI) and normalized to untreated EGFP control equaled to 1. Asterisk (*) indicates statistically significant difference between CALPST treated EGFP-talin and untreated EGFP-talin (p<2E-6). CALPST treatment of other cultures does not cause statistically significant differences, as compared with their corresponding untreated controls.

Fig. 4. Assessment of local cell stiffness via atomic force microscope (AFM)-enabled nanoindentation

Left (A) and right (C) images show optical microscopy images of the cantilever positioned directly above and far from wrinkled regions of the silicone substrata, respectively (on-wrinkle and off-wrinkle, respectively). Black arrowheads indicate wrinkles observable in silicone substrate. Central diagram (B) demonstrates the experimental set-up, wherein pericytes were grown on deformable silicone substrata and exhibited actin stress fibers (also marked by the star in left inset); pericyte contractile forces deformed or wrinkled the substratum, and indentation of subcellular regions was conducted near and far from regions of visible substrata contraction. Insets in (A) – (C) show AFM deflection images of pericytes and underlying silocone substrata with cell-generated wrinkle deformations. (D) Subcellular stiffness expressed as indentation effective elastic moduli E_eff at on- and off-wrinkle positions, as a function of exposure to calpain inhibitor CALPST (100 µM final concentration, 24 h). At least five cells were analyzed per condition, and data are normalized by the value of E_eff for the untreated cells on-wrinkle stiffness. Asterisk (*) indicates statistically significant difference in E_eff between on-wrinkle and off-wrinkle locations (p<0.05); and plus sign (‡) indicates statistically significant differences in E_eff for CALPST-treated as compared to untreated or CALPST-ala cells (p<0.001); double asterisk (**) indicates statistically significant difference in E_eff between CALPST treated on-wrinkle and off-wrinkle (p<0.001). Scale bar = 20 µm.