Caldesmon is an integral component of podosomes in smooth muscle cells

Abstract

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 actinbinding 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 PDBuinduced 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 Ca 2+ /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 Ca 2+ /calmodulin may be part of a regulatory mechanism in podosome formation.

Erk1/2 MAPK and caldesmon differentially regulate podosome dynamics in A7r5 vascular smooth muscle cells

“…Recent findings have implicated the involvement of podosomes in the invasion of vascular smooth muscle cells in proliferative vascular diseases such as atherosclerosis and restenosis [8]. A7r5 vascular smooth muscle cells have been studied extensively as a model system for investigating the mechanisms of podosome formation by several laboratories [3,4,9,10,11]. Conventional PKC has been found to mediate phorbol dibutyrate (PDBu)-stimulated podosome formation in A7r5 vascular smooth muscle cells [4].…”

“…Erk-dependent phosphorylation of caldesmon has been shown to reverse the ability of the actin-binding carboxyl-terminal fragment of caldesmon to stabilize actin filaments against actin-severing proteins [23]. Recently, Eves et al [10] showed that overexpression of caldesmon suppressed PDBu-stimulated podosome formation, whereas siRNA knock-down of caldesmon expression facilitated PDBu-stimulated podosome formation in A7r5 cells. However, their study did not address the role of Erk-dependent caldesmon phosphorylation in the regulation of podosome formation and dynamics.…”

Caldesmon and the Regulation of Cytoskeletal Functions

“…183,184 In A7r5 cells podosomes are formed upon stimulation with phorbol dibutyrate (PDBu), which activates PKC. Interestingly, l-CaD is present in such podosomes 185 and is phosphorylated at ERK sites. 186 By overexpressing a phosphorylation-deficient CaD-GFP mutant it was found that, although both the size and the lifetime of the podosomes are modulated by ERK-mediated CaD phosphorylation, the modes of ERK and CaD in their modulation of the formation and dynamics of podosomes are different.…”

“…155,156,171,185,[188][189][190][191][192] These interesting yet confusing results may have stemmed from (i) the difference of cell types used, (ii) different stages of cells and (iii) different levels of over-expression. Nevertheless, several observations are particularly noteworthy.…”

The Conformational State of Actin Filaments Regulates Branching by Actin-related Protein 2/3 (Arp2/3) Complex

“…Caldesmon (CaD) 3 is one such actin "side binder" that exists in nearly all vertebrate cells. Although the muscle-specific isoform of CaD plays a regulatory role in smooth muscle contractility (3), nonmuscle CaD is more broadly implicated in cell motility (4), including processes such as cell migration (5), focal adhesion assembly (6), podosome dynamics (7), and neointimal formation (8). Recently, CaD was also suggested to control a key structural transition occurring during actin polymerization, known as "maturation" (9,10).…”