Experimental results derived from studies described in this five year research plan should provide essential new insights into isoactin functional diversity and the molecular mechanisms regulating isoactin-based control of cell motility. Preliminary data presented in the proposal and recent published results identify a complex of proteins, b actin, ezrin and a b actin-specific binding protein named bcap73, which play a role in membrane protrusion.
In specific aim 1, the investigator will use cell microinjection and fluorescence photoactivation to quantify in vivo isoactin filament dynamics/function in wild-type and mutant cell lines which are deficient or defective in b actin. To assess critically whether non-muscle isoactin filaments perform unique cell-specific functions, the investigator will introduce into cellsfunction-blocking isoactin-specific antibodies that can discriminate b vs g actin. He anticipates that the combined results of fluorescent actin isoprotein photoactivation and anti-isoactin 'loss-of-function' studies in wild type and mutant cells will reveal whether b actin filament dynamics is sufficient to drive cytoplasmic expansion at the leading edge.
In specific aim 2, the investigator will test the specific hypothesis that bcap73, either alone or with ezrin, regulates b actin nucleation and filament assembly. These activities are essential for cytoplasmic remodelling during cell motility. Quantitative iso-f-actin binding assays using combinations of full length or truncated bcap73 and ezrin, will identify the domains that nucleate or cap b actin but not other actin isoforms. Data from these in vitro assays will be compared with results of experiments from two novel fluorescent-phalloidin assays designed to reveal whether isoactin polymerization kinetics and flexibility are unique; and whether bcap73 or other actin capping/severing proteins influence these behaviors. Concomitantly, they will characterize bcap73 in wild type and mutant cell lines. These results should offer novel insights into interactions between isoactin, their binding proteins, and the membrane which give rise to functional cell motility during development and disease.
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