Role of actin bundlers during enterocyte differentiation

摘要 在分化过程中，肠细胞形成一个广泛的微绒毛顶端阵列，称为刷状边缘，它 用于放大可用于养分吸收的质膜表面积。单个微绒毛 结构简单，由从顶端突出的~25条肌动蛋白细丝组成的支持核心束组成。 用薄膜包裹的表面。除了作为唯一的营养吸收场所，刷状边缘的微绒毛 也为糖萼提供锚定点，并调节与管腔微生物的相互作用。尽管 刷子边缘是肠道的主要功能界面，是驱动及时 直到最近，肠细胞分化过程中微绒毛的形成仍不清楚。在我们的第一笔资金 期间，我们发现了在微绒毛形成过程中控制肌动蛋白细丝聚合的几个因素， 包括IRTKS/Eps8复合体。然而，构建稳定的微绒毛也需要肌动蛋白细丝 组织成芯束，这些芯束表现出足够高的弯曲刚性，足以使根尖表面变形。多么 新生的肠细胞协调肌动蛋白微丝聚合和空间捆绑的基本活动 建立稳定的微绒毛的时间尚不清楚。在最近的初步研究中，我们使用了邻近标记 在微绒毛组装过程中识别IRTKS/Eps8点~20 nm内蛋白质的方法；该筛查导致 为我们令人兴奋的发现有丝分裂纺锤体定位(MISP)作为一种新的肌动蛋白细丝束蛋白 画笔边框。MiSP沿着完整的隐窝绒毛轴表达，定位于根尖表面。拉近 用超分辨显微镜观察发现，MISP在岩心上表现出明显的特殊富集性 捆绑根。在培养的细胞中，我们发现MISP稳定和拉长了小根，并招募了其他 经典的肌动蛋白捆绑到这些部位。重要的是，我们发现纯化的MISP足以组织肌动蛋白 花丝在体外变成紧凑的线状束。最后，我们对MISP基因敲除小鼠的初步分析揭示了 根细丝显著丢失，微绒毛表面密度降低。根据我们的初步数据，我们建议 以下中心假设：在分化的肠细胞的顶端表面，MISP组织肌动蛋白 IRTKS/Eps8复合体产生的细丝形成支持刷子突起的芯束 边缘微绒毛。使用最先进的光学和电子显微镜技术和 新的生物模型系统，我们将：(目标1)确定MISP是否在 顶面，(目标2)定义MISP肌动蛋白结合和捆绑的机制，(目标3)阐明其功能 MISP在体内肠细胞分化中的作用。我们期望这些目标的实现将导致新的范例。 以了解肠上皮形态发生。

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.