TI - RICH2 knockdown in Caco-2 cells leads to a similar phenotype to that arising after CD317 knockdown . AB - We reasoned that if RICH2 does provide a functional link between CD317 , Rac/Rho , and the actin cytoskeleton , knockdown of RICH2 expression should phenocopy knockdown of CD317 expression in Caco-2 cells . To test this hypothesis , the expression of RICH2 was knocked down in Caco-2 cells using the same plasmid-based method that was used to knock down expression of CD317 in these cells . Immunoblot analysis of lysate from control and RICH2 knockdown cells demonstrated that RICH2 levels in knockdown cells had been reduced to cells ( Fig 5 A and Fig S3 A ) . The stably transfected RICH2 knockdown Caco-2 cells were then grown to confluence to generate polarized monolayers . These polarized RICH2 knockdown cells the polarized CD317 knockdown cells ( i.e. , a loss of apical F-actin , the presence of distinct bundles of F-actin at the basal membrane , a decrease in the amount of apically localized ezrin [Fig 5 B] , a loss of apical microvilli [Fig 5 C] , and reduced cell height but no loss of transepithelial resistance or of tight junctions or any change in the distribution of apical and basolateral marker proteins [not depicted] ) . Lamin A/C knockdown cells were used as a control in these experiments and showed a wild-type phenotype with respect to actin organization , microvilli formation , ezrin localization and cell height . Knocking down expression of RICH2 also led to an increase in the level of active Rac in polarized Caco-2 cells ( Fig 5 D and Fig S2 B ) , an ~10-fold increase in phosphorylaTION of MLC ( Fig 5 E and Fig S2 A ) , and an approximately threefold increase in PHOSphorylated ezrin ( when compared with the situation in control cells ; Fig 5 F and Fig S3 B ) , closely paralleling what occurs when CD317 expression is knocked down . The fact that RICH2 knockdown cells are phenocopies of CD317 knockdown cells is consistent with the two proteins acting on the same pathway , whereas the increase in MLC PHOSphorylation observed in RICH2 and CD317 knockdown cells is indicative of Rac and Rho activation in these cells . Rac and Rho activities have previously been shown to be required for production of actin -dependent ruffles ( Hall , 1998 ) . Therefore , we reasoned that if the putative Rac GAP domain in RICH2 is indeed functioning as a Rac GAP in vivo ( as it has been shown to be in vitro ; Richnau and Aspenstrom , 2001 ) , elevated expression of RICH2 might lead to a reduction in active Rac within cells , which would , in turn , lead to a reduction in actin -dependent ruffles . To test this hypothesis , we expressed an RFP-tagged form of RICH2 ( RICH2-RFP ) in COS cells and assayed ruffle formation in these and in control , nontransfected cells . Over 90% of control cells exhibited ruffles , whereas cells expressing RICH2-RFP did so . An R288A mutation in the Rac GAP domain of RICH1 has previously been shown to destroy GAP activity in that protein ( Richnau and Aspenstrom , 2001 ) . Therefore , we generated the corresponding mutation ( R291A ) in RICH2-RFP and expressed the mutant protein in Caco-2 cells . Cells expressing R291A RICH2-RFP behaved as control cells , demonstrating that the Rac GAP activity of RICH2 is required to inhibit ruffle formation , which is a result consistent with the Rac GAP domain of RICH2 working as a Rac GAP in vivo .