Role of actin bundlers during enterocyte differentiation

总结 在分化过程中，肠上皮细胞形成广泛的顶端微绒毛阵列，称为刷状缘， 用于扩大可用于营养吸收的质膜表面积。单个微绒毛 结构简单，由一个由约25根肌动蛋白丝组成的支撑核心束组成， 表面被膜包裹。刷状缘微绒毛除了作为营养吸收的唯一场所外， 还为糖萼提供锚定点，并调节与管腔微生物的相互作用。虽然 刷状缘作为肠道的主要功能界面， 肠上皮细胞分化过程中微绒毛的形成直到最近仍不清楚。在我们第一次融资时， 在此期间，我们发现了在微绒毛形成过程中控制肌动蛋白丝聚合的几个因素， 包括IRTKS/EPS8复合物。然而，建立稳定的微绒毛也需要肌动蛋白丝， 组织成核心束，其表现出足够高的弯曲刚度以使顶端表面变形。如何 新生肠细胞协调肌动蛋白丝聚合和空间成束的基本活动 而建立稳定微绒毛的时间仍然未知。在最近的初步研究中，我们使用了邻近标记， 在微绒毛组装期间鉴定IRTKS/EPS8斑点~20 nm内的蛋白质的方法;该筛选导致 有丝分裂纺锤体定位（MISP）是一种新的肌动蛋白丝捆绑蛋白， 灌木丛边缘MISP沿着完整的隐窝-绒毛轴表达，在此其定位于顶端表面。近 用超分辨率显微镜观察发现，MISP在核上表现出惊人的特异性富集 维管束细根。在培养的细胞中，我们发现MISP稳定并延长了小根，并招募了其他细胞。 这些位点的典型肌动蛋白复制物。重要的是，我们发现纯化的MISP足以组织肌动蛋白 花丝在离体培养中形成紧密的线状束。最后，我们对MISP基因敲除小鼠的初步分析显示， 小根的显著损失和微绒毛表面密度的降低。根据我们的初步数据，我们建议 以下中心假设：在分化中的肠上皮细胞的顶面，MISP组织肌动蛋白 由IRTKS/EPS8复合物产生的细丝，以形成支撑刷突起的核心束 边缘微绒毛使用最先进的光学和电子显微镜技术的独特组合， 新的生物模型系统，我们将：（目的1）确定MISP是否指定微绒毛生长的网站在 目的2：阐明MISP肌动蛋白结合和成束的机制，目的3：阐明MISP肌动蛋白与肌动蛋白结合和成束的功能 MISP在体内肠上皮细胞分化中的作用。我们期望这些目标的完成将导致新的范例 来了解肠上皮的形态发生。

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.