The cytoskeleton is a filamentous protein network that extends throughout the cytoplasm of eukaryotic cells. Intermediate filaments are tissue-specific cytoskeletal proteins that are thought to play a structural or tension bearing role in the cell. To test this possibility, we examined vimentin an intermediate filament protein that is expressed in a variety of a cells including osteoblasts, by immunopreciptation and immunofluorescent confocial microscopy. Calvarial explants were dissected from 12-day old chick embryos and grown for 14 days in vitro. Osteoblasts were plated onto control or flexible bottomed cell culture plates. The Flexercell(TM) Strain Unit was utilized to deliver the strain regimen which consisted of 3 control cells were either fixed and immunostained for scanning confocal laser microscopy, or pulse-labeled with methionine and immunoprecipitated with an antivimentin monclonal antibody. TCA precipitation was also performed to analyze total protein synthesis. Immunostaining reveals an increase in the vimentin protein throughout the cells following 24, 48, and 72 hours of strain. Furthermore, the vimentin compared with controls. Quantitatively, total protein synthesis increased after 24, 48, and 72 hours of strain. Vimentin protein synthesis also increased after 24 and 48 hours of strain, while after 72 hours of strain, a decrease in vimetin was noted. These studies indicate that changes in vimentin synthesis and organization may play a role in osteoblast response to strain. This model system will allow us to explore the potential of a direct structure-function relationship of various cytoskeletal proteins in bone response to stress.
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