PROJECT SUMMARY Within the intestine, nutrient absorption occurs at the brush border, a region of densely packed actin based protrusions on the apical surface of enterocytes. These protrusions, known as microvilli, also form the first line of defense against luminal pathogens. Proper formation of the brush border is dependent on the correct formation and clustering of microvilli. Microvilli are membrane covered protrusions, each containing a bundle of 20-30 actin filaments, with the plus ends located at the distal tips, and the minus ends anchored in a region of the cell known as the terminal web. Enterocytes are continually being renewed, thus growth of microvilli is critical throughout an organism’s entire lifespan. However, little is known about the mechanisms that drive the growth and organization of microvilli, or the precise role of the terminal web. Ultrastructural studies revealed many years ago that microvillar actin bundles are embedded in the dense terminal web, although the composition and function of this structure remain ambiguous. Our preliminary studies suggest that a major constituent of this structure is non-muscle myosin II (NMII), a filament-forming myosin motor expressed in all eukaryotic cells. In the intestine, three different isoforms of NMII are expressed; A, B and C, with NMII-A and NMII-C dominating expression in enterocytes. My preliminary super-resolution images reveal that NMII-C forms a novel network across the enterocyte apical domain at the level of the terminal web. Line scans along the microvillar axis show that NMII-C is enriched near the pointed-ends of microvillar actin bundles. In preliminary studies using Blebbistatin, a myosin II inhibitor, native microvilli in mouse intestinal tissue get noticeably shorter, and appear to lose their upright orientation relative the surface of the cell. Based on my preliminary data, I hypothesize that a non-muscle myosin II network spanning the apical domain provides necessary tension and mechanical support for microvillar growth and maintenance. To test this hypothesis, I will: (Aim 1) define the organization of the sub-apical non-muscle myosin II network, and (Aim 2) determine the role of NMII-C in the growth and organization of microvilli. Investigation of this novel myosin II array will significantly deepen our understanding of the physical mechanisms of microvillar formation and maintenance, as well as diseases linked to mutations in non-muscle myosin IIs.