SUMMARY During differentiation, enterocytes build an extensive apical array of microvilli known as the brush border, which serves to amplify the plasma membrane surface area available for nutrient absorption. An individual microvillus is simple in structure, consisting of a supporting core bundle of ~25 actin filaments that protrudes from the apical surface wrapped in membrane. In addition to serving as the sole site of nutrient uptake, brush border microvilli also provide an anchoring point for the glycocalyx and regulate interactions with luminal microbes. Although the brush border serves as the primary functional interface of the intestinal tract, mechanisms that drive the timely formation of microvilli during enterocyte differentiation remained unclear until recently. During our first funding period, we discovered several factors that control actin filament polymerization during microvilli formation, including the IRTKS/EPS8 complex. However, building stable microvilli also requires that actin filaments are organized into core bundles, which exhibit flexural rigidities high enough to deform the apical surface. How nascent enterocytes coordinate the fundamental activities of actin filament polymerization and bundling in space and time to build stable microvilli remains unknown. In recent preliminary studies, we used a proximity labeling approach to identify proteins within ~20 nm of IRTKS/EPS8 puncta during microvillus assembly; this screen led to our exciting discovery of Mitotic Spindle Positioning (MISP) as a new actin filament bundling protein in the brush border. MISP is expressed along the full crypt-villus axis, where it localizes to the apical surface. Closer inspection with super-resolution microscopy revealed that MISP exhibits strikingly specific enrichment on core bundle rootlets. In cultured cells, we found that MISP stabilizes and elongates rootlets, and recruits other canonical actin bundlers to these sites. Importantly, we found that purified MISP is sufficient to organize actin filaments into tight linear bundles in vitro. Finally, our preliminary analysis of MISP knockout mice revealed a striking loss of rootlets and decrease in microvillar surface density. Based on our preliminary data, we propose the following CENTRAL HYPOTHESIS: At the apical surface of differentiating enterocytes, MISP organizes actin filaments generated by the IRTKS/EPS8 complex to form core bundles that support the protrusion of brush border microvilli. Using a unique combination of state-of-the-art light and electron microscopy technology and novel biological model systems, we will: (Aim 1) determine if MISP specifies sites of microvillar growth at the apical surface, (Aim 2) define the mechanism of MISP actin binding and bundling, (Aim 3) elucidate the function of MISP in enterocyte differentiation in vivo. We expect that completion of these Aims will lead to new paradigms for understanding intestinal epithelial morphogenesis.