TI - Ezrin is mislocalized in CD317 knockdown cells . AB - Given that knocking down expression of CD317 in polarized Caco-2 cells leads to a loss of apical microvilli and a change in the regulation of Rho activity , we speculated that ERM proteins might be mislocalized in CD317 knockdown cells . Members of the ERM family of closely related proteins function as cross-linkers between the plasma membrane and actin filaments , and such cross-links are fundamental to the making and maintenance of microvilli . Soluble ERM proteins in the cytosol are inactive in terms of their cross-linking activity , but when ERM proteins are activated ( involving PHOSphorylation ) , they undergo a conformational change that allows them to interact with both actin and , directly or indirectly , with the cytosolic domains of specific integral membrane proteins ( Bretscher et al. , 2000 , 2002 . Rho activity has also been implicated in the activation of ERM proteins . Because ezrin is normally enriched in apical microvilli in polarized epithelial cells , where it plays a critical role in coupling the actin cytoskeleton to the plasma membrane ( Bretscher , 1983 ; Berryman , 1993 ) , we reasoned that it would be mislocalized in CD317 knockdown cells lacking apical microvilli . As expected , we observed that ezrin localizes to the apical membrane in control polarized Caco-2 cells where there is some colocalization with F-actin ( Fig 3 D , top ) . However , in CD317 knockdown cells , this is not the case ( Fig 3 D , bottom ) . It is noteworthy that PHOSphorylation of ezrin is raised threefold in knockdown cells compared with control cells ( both lamin A/C knockdown and rescued CD317 knockdown ; Fig. 3 E and Fig . S3 B , available at http : . . . . . . /www.jcb.org/cgi/content/full/jcb. lt @@@@@ gt 200804154/DC ) , implying that the block on the formation of microvilli is not at the level of ezrin activation . These data also demonstrate that reexpression of CD317 in the knockdown cells restores the level of ezrin PHOSphorylation to that seen in control cells .