THERE IS A GROWING consensus that the network of interconnected osteocytes provides a cellular system that senses changes in bone loading and subsequently activates osteoclasts and osteoblasts to resorb or produce bone matrix.(1, 2) The formation of the network results from the morphological changes from the cuboidal shape of osteoblasts to the stellate shape of the osteocyte, with its multiple, slender cytoplasmic processes.(3–5) Changes in cell shape are the results of regulated changes in the assembly and disassembly kinetics of the cytoskeleton. Furthermore, it is generally agreed that the cytoskeleton is somehow involved in the mechanism by which individual cells sense mechanical signals.(6-8) We previously reported that actin filaments were crucial for the maintenance of osteocyte processes and pointed out that two actin-binding proteins, fimbrin and α-actinin, were present in the osteocyte processes.(9) In general, actin-binding proteins are the control element for the dynamic reorganization of the actin cytoskeleton. Therefore, understanding how the actin-binding proteins are organized in osteoblasts and osteocytes will provide critical information as to how the osteoblast differentiates into the osteocyte as well as about the mechanosensory capabilities of osteocytes.
The success of this study stemmed from the successful isolation and maintenance of osteocytes in culture, which allowed us to conduct extensive analysis of the osteocyte cytoskeleton. Without the problematic autofluorescence from bone matrix, we used immunofluorescence microscopy to examine the relative distribution of actin filaments and actin-binding proteins. The actin-binding proteins examined were actin-bundling proteins including fimbrin, α-actinin, and villin(10); the gel-forming protein filamin(11); and the plasma membrane-anchored cytoskeletal protein spectrin.(12, 13) Furthermore, myosin, which has motor activity on actin filaments,(14, 15) and tropomyosin, which is localized along the groove of the F-actin filament and regulates the actin-myosin interaction,(16) were also analyzed. α-Actinin, myosin, and tropomyosin showed similar organization in both osteoblastic stress fiber and osteocyte processes. However, other actin-binding proteins analyzed in this study showed dramatic differences in distribution between osteoblasts and osteocytes.