Caldesmon is an integral component of podosomes in smooth muscle cells.
Podosomes are highly dynamic actin-based structures commonly found in motile and invasive cells such as macrophages, osteoclasts and vascular smooth muscle cells. Here, we have investigated the role of caldesmon, an actin-binding protein, in the formation of podosomes in aortic smooth muscle A7r5 cells induced by the phorbol ester PDBu. We found that endogenous low molecular weight caldesmon (l-caldesmon), which was normally localised to actin-stress fibres and membrane ruffles, was recruited to the actin cores of PDBu-induced podosomes. Overexpression of l-caldesmon in A7r5 cells caused dissociation of actin-stress fibres and disruption of focal adhesion complexes, and significantly reduced the ability of PDBu to induce podosome formation. By contrast, siRNA interference of caldesmon expression enhanced PDBu-induced formation of podosomes. The N-terminal fragment of l-caldesmon, CaD40, which contains the myosin-binding site, did not label stress fibres and was not translocated to PDBu-induced podosomes. Cad39, the C-terminal fragment housing the binding sites for actin, tropomyosin and calmodulin, was localised to stress fibres and was translocated to podosomes induced by PDBu. The caldesmon mutant, CadCamAB, which does not interact with Ca2+/calmodulin, was not recruited to PDBu-induced podosomes. These results show that (1) l-caldesmon is an integral part of the actin-rich core of the podosome; (2) overexpression of l-caldesmon suppresses podosome formation, whereas siRNA knock-down of l-caldesmon facilitates its formation; and (3) the actin-binding and calmodulin-binding sites on l-caldesmon are essential for the translocation of l-caldesmon to the podosomes. In summary, this data suggests that caldesmon may play a role in the regulation of the dynamics of podosome assembly and that Ca2+/calmodulin may be part of a regulatory mechanism in podosome formation.
Eves
Robert
R
Department of Biochemistry, and Protein Function Discovery Program, Queen's University, Kingston, ON K7L 3N6, Canada.
Webb
Bradley A
BA
Zhou
Shutang
S
Mak
Alan S
AS
D000199:Actins / Q000378:metabolism; D000818:Animals; D001011:Aorta / Q000166:cytology; D002147:Calmodulin / Q000378:metabolism; D002148:Calmodulin-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D003599:Cytoskeleton / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D015240:Phorbol 12,13-Dibutyrate / Q000494:pharmacology; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D022502:Stress Fibers / Q000378:metabolism / Q000648:ultrastructure
D000199:Actins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D020033:Protein Isoforms; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D015240:Phorbol 12,13-Dibutyrate