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Abstract
Tau and radixin are two proteins that have been implicated in organizing the microtubule- and microfilament-based cytoskeletons respectively. Here we have examined the structure and function of these proteins in cultured mammalian cells. To test tau function, we inhibited its expression in embryonal carcinoma P19 cells with antisense RNA. The results show that inhibition of tau expression does not inhibit the ability of P19 cells to undergo morphological differentiation into neurons--a process that is known to depend on microtubule function.
Radixin is a member of the ezrin-radixin-moesin (ERM) protein family and is known to localize in cells to cortical structures in which there is a close apposition between the plasma membrane and underlying microfilament-rich cytoskeleton. To define the regions of the radixin molecule that specify its subcellular localization, we expressed full-length and truncated versions of the molecule in cultured mammalian cells and determined their localization. Exogenous full-length radixin localized in a manner similar to endogenous ERM proteins. Moreover, expression of full-length radixin was correlated with the disappearance of endogenous moesin from cortical structures suggesting that these two ERM proteins compete for localization in cortical structures. Localization of the full-length molecule depended on distinct determinants in both the carboxy- and amino-terminal domains of the protein. High level expression of the carboxy-terminal domain of radixin had deleterious effects on cells including the induction of abnormal cortical processes. Neither the full-length molecule nor its amino-terminal domain had these effects on cells. These results suggested that the amino-terminal domain of radixin modulated the function of the carboxy-terminal domain in the context of the full-length molecule. This hypothesis was tested in vitro. We found that the amino- and carboxy-terminal domains interact with one another with high affinity in solution. This inter-domain interaction could inhibit intermolecular binding of other proteins from cell extracts. Taken together, these studies have suggested an overall model for the molecular organization of radixin which might explain its localization and function in dynamic cortical cytoskeletal structures involved in cell motility. (Copies available exclusively from MIT Libraries, Rm. 14-0551, Cambridge, MA 02139-4307. Ph. 617-253-5668; Fax 617-253-1690.)
