Abstract
Podosomes are dynamic cell adhesion structures that degrade the extracellular matrix, permitting extracellular matrix remodeling. Accumulating evidence suggests that actin and its associated proteins play a crucial role in podosome dynamics. Caldesmon is localized to the podosomes, and its expression is down-regulated in transformed and cancer cells. Here we studied the regulatory mode of caldesmon in podosome formation in Rous sarcoma virus-transformed fibroblasts. Exogenous expression analyses revealed that caldesmon represses podosome formation triggered by the N-WASP-Arp2/3 pathway. Conversely, depletion of caldesmon by RNA interference induces numerous small-sized podosomes with high dynamics. Caldesmon competes with the Arp2/3 complex for actin binding and thereby inhibits podosome formation. p21-activated kinases (PAK)1 and 2 are also repressors of podosome formation via phosphorylation of caldesmon. Consequently, phosphorylation of caldesmon by PAK1/2 enhances this regulatory mode of caldesmon. Taken together, we conclude that in Rous sarcoma virus-transformed cells, changes in the balance between PAK1/2-regulated caldesmon and the Arp2/3 complex govern the formation of podosomes.
Highlights
Degrade the extracellular matrix (ECM) with matrix metalloproteinases, which implicates remodeling of ECM [6]
Two different molecular weight (Mr) isoforms of CaD are identified: high molecular weight CaD (h-CaD; 120 –150 kDa) and low molecular weight CaD (l-CaD; 70 – 80 kDa). They are generated from a single gene by alternative splicing. h-CaD is exclusively expressed in smooth muscle cells, but l-CaD is widely distributed in non-muscle cells [15]
Expression and Localization of Caldesmon, Tropomyosin, Neural WASP (N-WASP), and the Arp2/3 Complex in Fibroblasts and Their RSV Transformants—CaD and TM bind to parallel actin filaments, stabilizing their alignment [16], whereas the N-WASP-Arp2/3 pathway induces the branching of actin filaments [29]
Summary
Degrade the extracellular matrix (ECM) with matrix metalloproteinases, which implicates remodeling of ECM [6]. To further compare the localizations previous report [12], the depletion of p34 Arc from BY1 cells of the Arp2/3 complex and CaD, we co-expressed trace resulted in the disassembly of podosomes and the formation of amounts of GFP-Arp3 and DsRed-CaD in BY1 cells, and we cortical actin bundles that co-localized with CaD (Fig. 3, B–D). CaD expression was markedly down-regulated in BY1 cells, we plex for actin binding and nucleation, we performed an in vitro examined the effect of HA-tagged CaD overexpression on co-sedimentation assay and actin polymerization assay using podosome formation.
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