Genetic disruption of calpain correlates with loss of membrane blebbing and differential expression of RhoGDI-1, cofilin and tropomyosin
Résumé
Dynamic regulation of the actin-cytoskeleton is essential to cell motility, spreading, and the formation of membrane surface extensions like lamellipodia, ruffles and blebs. The ubiquitous calpains contribute to integrin-mediated cytoskeletal remodelling during cell migration and spreading, by cleavage of focal adhesion components and signalling molecules. In this study, the live-cell morphology of calpain-knockout and wild type cells was examined by time-lapse fluorescence microscopy and a role of calpain in mediating formation of sporadic membrane blebs was established. Membrane blebbing was significantly reduced in calpain-knockout cells, and genetic rescue fully restored the wild type phenotype in knockout cells. Proteomic comparison of wild type and knockout cells identified decreased levels of RhoGDI-1 and cofilin 1, and increased levels of tropomyosin in calpain knockout cells, suggesting a role of calpain in regulating membrane extensions involving these proteins. RhoGDI, cofilin and tropomyosin are known regulators of actin filament dymamics and membrane extensions. The reduced levels of RhoGDI-1 in calpain-knockout cells observed by proteome analysis were confirmed by immunoblotting. Genetic rescue of the calpain-knockout cells enhanced RhoGDI-1-expression 2-fold above that normally present in wild type cells. These data suggest a regulatory connection between calpain and RhoGDI-1 to promote formation of membrane blebs.
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