| dc.description.abstract | Microvilli are actin-based surface protrusions that have been repurposed throughout evolution to fulfill diverse cell functions including mechanosensation in inner ear hair cells and nutrient absorption in transporting epithelia. In the latter case, microvilli are supported by a core of actin filaments bundled in parallel by villin, fimbrin, and espin. Remarkably, microvilli biogenesis persists in mice lacking all three of these factors, suggesting the existence of unknown bundlers. We identified Mitotic Spindle Positioning (MISP) as an actin binding factor that localizes specifically to the basal end of the microvillar core bundle – the rootlet – in tissue and cell culture models. Gain-and-loss of function assays reveal that MISP controls rootlet length and stabilizes microvilli. We also found that MISP drives the recruitment of fimbrin to actin bundles, which further elongates and stabilizes microvilli. Consistent with its ability to elongate rootlets in cells, purified MISP exhibits potent filament bundling activity in vitro. We next sought to investigate the determinants for MISP specificity to the rootlet of epithelial microvilli. We found that MISP confinement to the rootlet is partially enforced by ezrin, which prevents decoration of the membrane-wrapped distal end of the core bundle. Given that the rootlets of microvilli are also the sites where the pointed ends of filaments converged, we sought to determine whether MISP sorts to these ends of actin filaments independent of extrinsic factors using in vitro TIRF microscopy. We found that MISP displays preferential binding to the pointed ends of aged actin filaments (i.e., ADP F-actin). Furthermore, although MISP assembles two-filament bundles in parallel and antiparallel configurations, the ability of MISP to generate polarized parallel bundles persists in multi-filament arrays. Taken together, our findings reveal that MISP is restricted to rootlets by extrinsic and intrinsic mechanisms, which contribute to the assembly and maintenance of the brush border microvilli. Moreover, MISP selective binding to the pointed ends, along with its bundling capacity, sheds light on a potential mechanism to establish the polarity of core actin bundles at the early stages of their assembly. | |
