This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We have defined a number of different types of motility that occur at the edges of cells based upon the morphology and kinematics of the edge as well as the proteins present (Dubin-Thaler, submitted;(Dobereiner et al., 2005)). Because the leading edge is typically very thin (<200 nm), it should be possible to visualize the molecular-level morphology of the complexes at the leading edge in the cryo-em. Many of the complexes have been visualized in vitro;therefore, it may be possible to identify the in vivo complexes, which will enable a much better understanding of the different types of motility at the molecular level. In particular, we propose to prepare cytoplasts (enucleated portions of cytoplasm) and characterize their spreading on fibronectin surfaces. Cytoplasts would then be spread as we recorded them in the light microscope. This would enable us to define the type of motility that was occurring. At the appropriate time, the sample would be fixed with formaldehyde and would subsequently be frozen in a very thin layer of vitreous ice. Alternatively, the samples would be allowed to spread for a defined period and then frozen without fixation.
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