Regulation of focal complex composition and disassembly by the calcium-dependent protease calpain.
Cell migration requires the regulated and dynamic turnover of adhesive complexes. We have previously demonstrated that the calcium-dependent protease, calpain, regulates the organization of adhesive complexes and cell detachment during cell migration. Evidence is now provided that inhibiting calpain through over-expression of the endogenous inhibitor of calpain, calpastatin, and pharmacological inhibitors results in an inhibition of adhesive complex disassembly with stabilization of GFP-vinculin and GFP/RFP-zyxin at the cell periphery. Calpain was also required for the microtubule-mediated turnover of adhesive complex sites after nocodazole wash-out, suggesting that calpain may mediate focal complex disassembly downstream of microtubules. Using dual imaging of RFP-zyxin and GFP-alpha-actinin, we observed a temporal and spatial relationship between alpha-actinin localization to focal contacts and the subsequent disassembly or translocation of RFP-zyxin containing focal complexes in areas of cell retraction. Calpain inhibition disrupted alpha-actinin localization to zyxin-containing focal contacts and focal complex disassembly or translocation to the cell center. In addition, disrupting alpha-actinin localization to focal complexes through expression of the alpha-actinin rod domain, but not the head domain, resulted in inhibition of focal adhesion disassembly similar to calpain inhibition. Our studies suggest a novel mechanism of action whereby calpain may modulate alpha-actinin localization into focal complexes and their subsequent disassembly or translocation.
Bhatt
Amit
A
Department of Pediatrics and Pharmacology, University of Wisconsin, 1300 University Avenue, University of Wisconsin Medical School, Madison, WI 53706, USA.
Kaverina
Irina
I
Otey
Carol
C
Huttenlocher
Anna
A
D000185:Actinin / Q000235:genetics / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002154:Calpain / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D006224:Cricetinae; D015853:Cysteine Proteinase Inhibitors / Q000378:metabolism; D003598:Cytoskeletal Proteins; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D022001:Focal Adhesions / Q000378:metabolism*; D006023:Glycoproteins; D006054:Goldfish; D049452:Green Fluorescent Proteins; D006801:Humans; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008667:Metalloproteins / Q000235:genetics / Q000378:metabolism; D018715:Microscopy, Video; D008870:Microtubules / Q000378:metabolism; D015739:Nocodazole / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D016596:Vinculin / Q000235:genetics / Q000378:metabolism; D060589:Zyxin; D000097573:Red Fluorescent Protein
D000185:Actinin; D002118:Calcium; D002135:Calcium-Binding Proteins; D002154:Calpain; D015853:Cysteine Proteinase Inhibitors; D003598:Cytoskeletal Proteins; D006023:Glycoproteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D008667:Metalloproteins; D015739:Nocodazole; D011993:Recombinant Fusion Proteins; D016596:Vinculin; D060589:Zyxin; D000097573:Red Fluorescent Protein; C092842:ZYX protein, human; C033668:calpastatin