Disruption of dynamic cell surface architecture of NIH3T3 fibroblasts by the N-terminal domains of moesin and ezrin: in vivo imaging with GFP fusion proteins

Lamellipodia, filopodia, microspikes and retraction fibers are characterist ic features of a dynamic and continuously changing cell surface architectur e and moesin, ezrin and radixin are thought to function in these microexten sions as reversible links between plasma membrane proteins and actin microf ilaments, Full-length and truncated domains of the three proteins were fuse d to green fluorescent protein (GFP), expressed in NIH3T3 cells, and distri bution and behaviour of cells were analysed by using digitally enhanced dif ferential interference contrast (DIC) and fluorescence video microscopy, Th e amino-terminal (N-)domains of all three proteins localize to the plasma m embrane and fluorescence recordings parallel the dynamic changes in cell su rface morphology observed by DIC microscopy of cultured cells, Expression o f this domain, however, significantly affects cell surface architecture by the formation of abnormally long and fragile filopodia that poorly attach a nd retract abnormally, Even more striking are abundant irregular, branched and motionless membraneous structures that accumulate during retraction of lamellipodia, These are devoid of actin, endogenous moesin, ezrin and radix in, but contain the GFP-labeled domain, While a large proportion of endogen ous proteins can be extracted with non-ionic detergents as in untransfected control cells, >90% of N-moesin and >60% of N-ezrin and N-radixin remain i nsoluble, The minimal size of the domain of moesin required for membrane lo calization and change in behavior includes residues 1-320, Deletions of ami no acid residues from either end result in diffuse intracellular distributi on, but also in normal cell behavior, Expression of GFP-fusions of full-len gth moesin or its carboxy-terminal domain has no effect on cell behavior du ring the observation period of 6-8 hours, The data suggest that, in the abs ence of the carboxy-terminal domain, N-moesin, -ezrin and -radixin interact tightly with the plasma membrane and interfere with normal functions of en dogeneous proteins mainly during retraction.