FIMBRIN IN PODOSOMES OF MONOCYTE-DERIVED OSTEOCLASTS

Fimbrin, an actin-bundling protein, is a component of the osteoclast a dhesion complexes called podosomes. In this study, we (1) determined t he localization of fimbrin in the mature rabbit osteoclast as well as in differentiating osteoclasts using the avian monocyte-derived osteoc last differentiation model, (2) characterized the distribution and acc umulation of three fimbrin isotypes (T, L, and I) in avian monocytes a s they fused to form multinucleate osteoclast-like cells. and (3) repo rt for the first time, a close spatial relationship between podosomes and microtubules using fimbrin as a marker of the podosome. Immunofluo rescence using anti-T-fimbrin, anti-L-fimbrin, and pan-isotype-anti-fi mbrin antibodies, showed that fimbrin is an integral component of the podosome core in the mature rabbit osteoclast and in the monocyte-deri ved osteoclast throughout differentiation. Anti-I-fimbrin, however, di d not show immunoreactivity in these cultures. These studies also show that in the avian model of monocyte-derived osteoclast differentiatio n, day 2 cells (D2) are predominantly mononucleate and have few podoso mes. 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. Ana lysis by Western blot of protein accumulation showed that after an ini tial small rise from D2 to D4, L-fimbrin levels remained relatively co nstant from D3 to DG. However, T-fimbrin protein levels increase stead ily from D2 to D6, suggesting that it may be related to the increase i n podosome formation as monocytes fuse to form osteoclasts. Finally, w e examined the distribution of podosomes relative to other cytoskeleta l elements such as microtubules and intermediate filaments. Double imm unofluorescence labeling using anti-fimbrin and anti-tubulin showed po dosomes lying adjacent to microtubules at cell margins. When osteoclas ts were treated with nocodazole (1 x 10(-6) M) to disrupt microtubules , the distribution of podosomes became more random and was no longer c onfined to the cell periphery. These results suggest that microtubule- podosome interactions may play a role in osteoclast adhesion. (C) 1997 Wiley-Liss, Inc.