Live dynamics of GFP-calponin: isoform-specific modulation of the actin cytoskeleton and autoregulation by C-terminal sequences

The calponin family of F-actin-, tropomyosin- and calmodulin-binding protei ns currently comprises three genetic variants. Their functional roles impli cated from in vitro studies include the regulation of actomyosin interactio ns in smooth muscle cells (h1 calponin), cytoskeletal organisation in non-m uscle cells (h2 calponin) and the control of neurite outgrowth (acidic calp onin), We have now investigated the effects of calponin (CaP) isoforms and their C-terminal deletion mutants on the actin cytoskeleton by time lapse v ideo microscopy of GFP fusion proteins in living smooth muscle cells and fi broblasts. It is shown that h1 Cap associates with the actin stress fibers in the more central part of the cell, whereas h2 Cap localizes to the ends of stress fibres and in the motile lamellipodial spread more efficiently th an those expressing hi CaP and expression of GFP h1 Cap resulted in reduced cell motility in wound healing experiments. Notably, expression of GFP h1 CaP, but not GFP h2 CaP, conferred increased resistance of the actin cytosk eleton to the actin polymerization antagonists cytochalasin B and latruncul in B, as well as to the protein kinase inhibitors H7-dihydrochloride and rh o-kinase inhibitor Y-27632, These data point towards a dual role of CaP in the stabilization and regulation of the actin cytoskeleton in vivo. Deletio n studies further identify an autoregulatory role for the unique C-terminal tail sequences in the respective Cap isoforms.