Intermediate filaments interact with dormant ezrin in intestinal epithelial cells.
Ezrin connects the apical F-actin scaffold to membrane proteins in the apical brush border of intestinal epithelial cells. Yet, the mechanisms that recruit ezrin to the apical domain remain obscure. Using stable CACO-2 transfectants expressing keratin 8 (K8) antisense RNA under a tetracycline-responsive element, we showed that the actin-ezrin scaffold cannot assemble in the absence of intermediate filaments (IFs). Overexpression of ezrin partially rescued this phenotype. Overexpression of K8 in mice also disrupted the assembly of the brush border, but ezrin distributed away from the apical membrane in spots along supernumerary IFs. In cytochalasin D-treated cells ezrin localized to a subapical compartment and coimmunoprecipitated with IFs. Overexpression of ezrin in undifferentiated cells showed a Triton-insoluble ezrin compartment negative for phospho-T567 (dormant) ezrin visualized as spots along IFs. Pulse-chase analysis showed that Triton-insoluble, newly synthesized ezrin transiently coimmunoprecipitates with IFs during the first 30 min of the chase. Dormant, but not active (p-T567), ezrin bound in vitro to isolated denatured keratins in Far-Western analysis and to native IFs in pull-down assays. We conclude that a transient association to IFs is an early step in the polarized assembly of apical ezrin in intestinal epithelial cells.
Department of Cell Biology and Anatomy R-124, University of Miami School of Medicine, Miami, FL 33101, USA.
Flavia A
FA
Wald
Andrea S
AS
Oriolo
M Llanos
ML
Casanova
Pedro J I
PJ
Salas
D000818:Animals; D018938:Caco-2 Cells; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D003598:Cytoskeletal Proteins; D020895:Enterocytes / Q000166:cytology / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007382:Intermediate Filaments / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000166:cytology / Q000378:metabolism*; D053533:Keratin-8; D007633:Keratins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D017830:Octoxynol; D010750:Phosphoproteins / Q000378:metabolism*; D000099149:Ezrin
D003598:Cytoskeletal Proteins; D053533:Keratin-8; D007633:Keratins; D017830:Octoxynol; D010750:Phosphoproteins; D000099149:Ezrin; C486595:Krt8 protein, mouse