Investigating the role of a novel non muscle myosin II network in apical domain organization of enterocytes

项目摘要 在肠道内，营养吸收发生在刷状缘，这是一个密集的肌动蛋白区域， 肠上皮细胞顶面的突起。这些突起，被称为微绒毛，也形成了第一条线 抵抗管腔病原体的能力。画笔边框的正确形成取决于正确的形成 和微绒毛聚集。微绒毛是被膜覆盖的突起，每个突起含有一束20-30个肌动蛋白 细丝，正端位于远端，负端锚定在细胞的已知区域中， 作为终端网络。肠细胞不断更新，因此微绒毛的生长在整个过程中至关重要。 一个有机体的整个生命周期。然而，人们对推动增长的机制知之甚少， 微绒毛的组织，或末端网的确切作用。 多年前的超微结构研究表明，微绒毛肌动蛋白束嵌在致密的 终端网，虽然这个结构的组成和功能仍然不明确。我们的初步研究 表明这种结构主要成分是非肌肉肌球蛋白II（NMII），一种形成顺应性的肌球蛋白马达 在所有真核细胞中表达。在肠中，表达三种不同的匪II同种型：A、B和C， 在肠上皮细胞中，匪II-A和匪II-C主导表达。我初步的超分辨率图像显示 NMII-C在末端网水平形成了一个跨越肠上皮细胞顶端结构域的新型网络。线扫描 沿着微绒毛轴的观察表明，在微绒毛肌动蛋白束的尖端附近，NMII-C富集。在 使用Blebbistatin（一种肌球蛋白II抑制剂）的初步研究， 明显更短，并且似乎失去了它们相对于细胞表面的直立方向。根据我 根据初步数据，我假设一个非肌肉肌球蛋白II网络跨越顶端域提供了 为微绒毛生长和维持提供必要的张力和机械支持。为了验证这个假设，我将： (Aim 1）定义亚顶端非肌肉肌球蛋白II网络的组织，并且（目的2）确定 NMII-C在微绒毛的生长和组织中的作用。这种新型肌球蛋白II阵列的研究将显着 加深我们对微绒毛形成和维持的物理机制的理解，以及 与非肌肉肌球蛋白II突变相关的疾病。

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.