Intermediate filaments interact with dormant ezrin in intestinal epithelial cells

Area of Science:

• Cell biology
• Epithelial cell biology
• Cytoskeletal dynamics

Background:

• Ezrin links the apical F-actin scaffold to membrane proteins in intestinal epithelial cells.
• Mechanisms of ezrin recruitment to the apical domain are not fully understood.

Purpose of the Study:

• To investigate the role of intermediate filaments (IFs) in the recruitment and assembly of ezrin at the apical brush border.
• To elucidate the early steps in the polarized assembly of ezrin in intestinal epithelial cells.

Main Methods:

• Utilized CACO-2 cell transfectants with keratin 8 (K8) antisense RNA under a tetracycline-responsive element.
• Investigated ezrin localization and interactions in response to K8 manipulation, cytochalasin D treatment, and overexpression.
• Performed pulse-chase analysis and in vitro binding assays (Far-Western, pull-down) with isolated keratins and IFs.

Main Results:

• Actin-ezrin scaffold assembly failed in the absence of IFs, with partial rescue by ezrin overexpression.
• K8 overexpression in mice disrupted brush border assembly, causing ezrin mislocalization along supernumerary IFs.
• Ezrin localized to a subapical compartment and coimmunoprecipitated with IFs in cytochalasin D-treated cells.
• Newly synthesized, dormant ezrin transiently coimmunoprecipitated with IFs and bound to keratins/IFs in vitro.

Conclusions:

• A transient association with intermediate filaments (IFs) is an essential early step for the polarized assembly of apical ezrin.
• IFs play a critical role in recruiting and organizing ezrin at the apical brush border of intestinal epithelial cells.