Fimbrin in podosomes of monocyte‐derived osteoclasts

Abstract

Fimbrin, an actin-bundling protein, is a component of the osteoclast adhesion complexes called podosomes. In this study, we (1) determined the localization of fimbrin in the mature rabbit osteoclast as well as in differentiating osteoclasts using the avian monocyte-derived osteoclast differentiation model, (2) characterized the distribution and accumulation of three fimbrin isotypes (T, L, and I) in avian monocytes as they fused to form multinucleate osteoclast-like cells, and (3) report for the first time, a close spatial relationship between podosomes and microtubules using fimbrin as a marker of the podosome. Immunofluorescence using anti-T-fimbrin, anti-L-fimbrin, and pan-isotype-anti-fimbrin antibodies, showed that fimbrin is an integral component of the podosome core in the mature rabbit osteoclast and in the monocyte-derived osteoclast throughout differentiation. Anti-I-fimbrin, however, did not show immunoreactivity in these cultures. These studies also show that in the avian model of monocyte-derived osteoclast differentiation, day 2 cells (D2) are predominantly mononucleate and have few podosomes. By days 4 and 6 in culture (D4 and D6), many cells have fused and punctate rows of podosomes are commonly observed at cell margins. Analysis by Western blot of protein accumulation showed that after an initial small rise from D2 to D4, L-fimbrin levels remained relatively constant from D4 to D6. However, T-fimbrin protein levels increase steadily from D2 to D6, suggesting that it may be related to the increase in podosome formation as monocytes fuse to form osteoclasts. Finally, we examined the distribution of podosomes relative to other cytoskeletal elements such as microtubules and intermediate filaments. Double immunofluorescence labeling using anti-fimbrin and anti-tubulin showed podosomes lying adjacent to microtubules at cell margins. When osteoclasts were treated with nocodazole (1 X 10(-6) M) to disrupt microtubules, the distribution of podosomes became more random and was no longer confined to the cell periphery. These results suggest that microtubule-podosome interactions may play a role in osteoclast adhesion.

Regulation of Sealing Ring Formation by L-plastin and Cortactin in Osteoclasts

“…Actin aggregates may function as an assembly center in the localization of integrin and other signaling molecules that are critical in the maturation of actin aggregates to functional sealing rings. L-plastin was described to be homologous with the F-actin-bundling protein fimbrin (59), and it is present in the podosomes of osteoclasts (2,60). However, its function in osteoclasts is highly unknown.…”

“…Cells were then washed with PBS containing 5 mM EGTA (PBS/EGTA) and stained for actin with rhodamine-phalloidin (1:1000 dilution; Sigma) as described previously (2). Actin-stained cells were viewed and photographed on a Bio-Rad confocal laser-scanning microscope.…”

“…ABPs assist in stabilizing and rearranging the organization of the actin cytoskeleton in response to external stimuli or during cell migration and adhesion. A few of the ABPs have been identified as having a role in osteoclast function (1)(2)(3)(4)(5)(6).…”

Enoxacin Directly Inhibits Osteoclastogenesis without Inducing Apoptosis

“…L-plastin is an actin-cross-linking protein that has been shown to be present in the core of podosomes and in actin rings (50). Regulation of L-plastin levels has been tied to the capacity of osteoclasts to form actin rings and resorb bone (41).…”

Podosomes at a glance

“…Moreover, they have been shown to stabilize podosome patterns such as the marginal belts in osteoclasts (Babb et al, 1997), to influence the fusion and fission rates of podosome precursors in murine macrophages (Evans et al, 2003) and to regulate podosome formation in human macrophages (Linder et al, 2000b).…”