Immunofluorescence staining of actin-binding proteins in osteoblasts and osteocytes was performed. α-Actinin, myosin, and tropomyosin showed similar organization in both osteoblastic stress fibers and osteocyte processes. However, fimbrin, villin, filamin, and spectrin showed dramatic differences in distribution between osteoblasts and osteocytes. This study suggested that terminal differentiation of osteoblasts to osteocytes is accompanied by highly dramatic changes in the distribution of actin-binding proteins.
Introduction: We previously reported that osteocyte shape is dependent on actin filaments. To analyze the terminal differentiation from osteoblasts to osteocytes, we investigated the actin-binding proteins, which are the control elements in the dynamic organization of the actin cytoskeleton.
Materials and Methods: We used primary chick osteocytes and osteoblasts, the phenotypes of which were confirmed by use of OB7.3, a chick osteocyte-specific monoclonal antibody and by detection of alkaline phosphatase activity, respectively. Immunofluorescence staining was performed for visualizing actin-binding proteins. Furthermore, we applied shear stress at 12 dyns/cm2 to the cells and compared the changes in fimbrin distribution.
Results: Immunofluorescence staining of fimbrin and α-actinin showed their presence in the processes of osteocytes, with especially strong signals of fimbrin at the sites of divarication of the processes. Anti-villin was reactive with the osteocyte cytoplasm but not with the processes. Interestingly, anti-villin immunoreactivity was much stronger in osteocytes than in osteoblasts. Filamin was localized along the stress fibers of osteoblasts but was seen only in those in the proximal base of osteocyte processes. Myosin and tropomyosin were found to have a similar pattern in both stress fibers of osteoblasts and osteocyte processes. The difference in the distribution of anti-spectrin staining was highly dramatic. Osteoblasts immunostained with anti-spectrin showed punctate signals on their cytoplasmic membranes, whereas anti-spectrin in osteocytes detected a filamentous organization; and the spectrin was totally colocalized with actin from the distal portion of the cytoplasmic processes to the cell center. In osteoblasts, shear stress induced recruitment of fimbrin to the end of stress fibers. However, fimbrin in the osteocyte processes did not change its localization.
Conclusion: We found that terminal differentiation of osteoblasts to osteocytes was accompanied by highly dramatic changes in the distribution of actin-binding proteins, changes of which may affect cellular function.