| Popis: |
The actin cytoskeleton plays a critical role in cell motility and the regulation of cell shape. One component of the actin cytoskeleton, the podosome, is involved in adhesion to the matrix, matrix degradation, cell migration, and mechanosensing of myeloid lineage cells. A podosome comprises of a cone-shaped actin core with a surrounding adhesion domain. The dynamics of the podosome depend on the directed polymerization of branched actin filaments via Arp2/3 in the actin core. Each podosome is connected with its neighbors via the F-actin cables that contribute to the collective behavior of podosomes. The assembly of podosomes in a specified area changes according to the developmental program, and in response to signals from the matrix and cytokines. However, the mechanisms underlying the organization of these podosome assemblies are largely unknown. When osteoclasts–bone-resorbing cells–are plated on glass, the assembly of podosomes evolves from the podosome cluster, the podosome ring, and the podosome belt, to a zipper-like structure (ZLS). To elucidate the pattern transition of the podosome assembly, we recorded the actin dynamics by live-cell imaging using osteoclasts differentiated from RAW 264.7 cells transfected with enhanced green fluorescent protein (EGFP)-actin. The time-lapse recordings of EGFP-actin signals indicated that bulk actin flow occurred in all four podosome assemblies. The intensity of fluorescence arising from EGFP-actin was lower in the actin flow than in the actin core. Actin flow appeared to be independent of the formation and deformation of the actin core. Interestingly, the ZLS exhibited consistently symmetrical actin flow during recording, whereas the actin flow observed in other structures was sporadic and the direction of flow was not constant. The Arp2/3 inhibitor CK-666 disrupted actin flow and disorganized the ZLS, but the myosin light chain kinase inhibitor ML-7 did not. The results suggest that actin flow, resulting from the Arp2/3-mediated branched elongation of actin filaments, is a previously unknown aspect of the ZLS. Self-organized actin flow in the podosome field probably determines the dynamics of podosome assemblies. |
