Actin Nucleators In Microvillar Assembly: Cordon Bleu As A Novel Microvillar Protein

Abstract

Epithelial cells polarize through reorganization of the actin cytoskeleton which results in distinct apical and basolateral domains. At the apical domain are structures called microvilli, which are plasma membrane protrusions tethered to a bundled actin filament core. It has been shown that ERM proteins, specifically Ezrin, are important for microvilli formation in these polarized cells. Ezrin is responsible for tethering the actin core to plasma membrane proteins both directly and indirectly through the scaffolding protein EBP50. What is unclear, however, is how the actin filament core of microvilli is nucleated and assembled. There are several classes of unbranched actin nucleators and regulators, including formins, Spire, VASp, and Cordon Bleu. Formins are involved in the nucleation and polymerization of other actin based membrane protrusions like filopodia, structures similar to microvilli. To determine a potential role for formins in microvilli formation, I used quantitative RT-PCR to quantify relative formin transcript levels in an epithelial cell line that displays abundant microvilli. The top formin candidates were then knocked down using siRNA to explore their role in microvilli formation. I saw minimal effects on microvilli and upon expression of GFP fusion proteins I found that formins were not localized to microvilli. I therefore looked at other actin regulators by examining their localization in epithelial cells. Cordon Bleu (Cobl) is a WH2-containing protein believed to act as an actin nucleator. I show that it has a very specific localization in epithelial cells at the basal region of microvilli, a localization unlikely to be involved in actin nucleation. The protein is localized by a central region between the N-terminal COBL domain and the three C-terminal WH2 domains. Ectopic expression of Cobl shortens apical microvilli, and this requires functional WH2 domains. Proteomic studies reveal that the COBL domain binds several BAR-containing proteins, including SNX9, PACSIN2/Syndapin-2 and ASAP1. ASAP1 is recruited to the base of microvilli by binding the COBL domain through its SH3 domain. I propose that Cobl is localized to the basal region of microvilli to participate in both length regulation and to recruit BAR proteins that associate with the curved membrane found at the microvillar base.