Investigating a Role for the Actin Nucleator Cordon-Bleu in Brush Border Assembly

Abstract

Enterocytes, epithelial cells of the small intestine, exhibit remarkable apical-basal polarity. The apical surfaces of enterocytes display an array of tightly packed microvilli termed the brush border. Microvilli are composed of membrane supported by a linear actin bundle, with the plus ends of the actin filaments at the microvillar tips. Despite the importance of the brush border in nutrient absorption and host defense, the mechanism of brush border assembly is unclear. Because of the central role of actin in microvilli, the goal of this thesis is to provide molecular detail as to how microvillar actin bundles form. A proteomic analysis of the brush border by our laboratory identified two actin nucleators in the brush border: the Arp2/3 complex and Cordon-Bleu (COBL). Small molecule inhibition of the Arp2/3 complex did not have an effect on brush border assembly in Ls174T-W4 (W4) cells, which act as a single cell model of enterocyte polarization and brush border formation. Therefore, this work focused on the linear actin nucleator COBL. We show that COBL localizes to the base of the brush border in mouse small intestine and in W4 cells. COBL is necessary and sufficient to induce microvillar growth using a mechanism that requires functional WH2 domains. COBL functions downstream of the F-BAR domain containing protein syndapin-2, which drives targeting to the apical domain of enterocytes. In the syndapin-2 knockout mouse, COBL enrichment at the apical domain of enterocytes is impaired, and microvilli are significantly shorter as compared to wild-type control mice. In cells that do not normally build microvilli, exogenous COBL drives the aberrant formation of dynamic cytoplasmic actin bundles that grow and shrink over the course of minutes; stabilization of COBL-induced bundles by the actin bundling protein espin leads to robust microvillus-like protrusions. This study provides novel insight on mechanisms that control microvillar growth and thus, the maintenance of intestinal homeostasis. This work also reveals a novel assembly paradigm for actin-based protrusions that do not emerge from a dendritic array.