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 proteins currently comprises three genetic variants. Their functional roles implicated from in vitro studies include the regulation of actomyosin interactions in smooth muscle cells (h1 calponin), cytoskeletal organisation in non-muscle cells (h2 calponin) and the control of neurite outgrowth (acidic calponin). We have now investigated the effects of calponin (CaP) isoforms and their C-terminal deletion mutants on the actin cytoskeleton by time lapse video microscopy of GFP fusion proteins in living smooth muscle cells and fibroblasts. 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 protrusions of spreading cells. Cells expressing h2 CaP spread more efficiently than those expressing h1 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 cytoskeleton to the actin polymerization antagonists cytochalasin B and latrunculin B, as well as to the protein kinase inhibitors H7-dihydrochloride and rho-kinase inhibitor Y-27632. These data point towards a dual role of CaP in the stabilization and regulation of the actin cytoskeleton in vivo. Deletion studies further identify an autoregulatory role for the unique C-terminal tail sequences in the respective CaP isoforms.
Danninger
C
C
Institute of Molecular Biology, Department of Cell Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria.
Gimona
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M
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement; D003599:Cytoskeleton / Q000378:metabolism*; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism*; D051379:Mice; D008840:Microfilament Proteins; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008840:Microfilament Proteins; D020033:Protein Isoforms; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D000096985:Calponins