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

研究新型非肌肉肌球蛋白 II 网络在肠上皮细胞顶端域组织中的作用

• 批准号: 9911150
• 负责人: Colbie Chinowsky
• 金额: $ 3.02万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2021-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9911150
• 关键词: Actins; Antibodies; Apical; Architecture; Brush Border; Cell Culture System; Cell Culture Techniques; Cell surface; Cells; Complex; Cytoskeleton; Data; Development; Disease; Distal; Enterocytes; Epithelial Cells; Eukaryotic Cell; Exhibits; Farming environment; Filament; Fingers; Functional disorder; Gastrointestinal Diseases; Genes; Growth; Head; Image; Individual; Internet; Intestines; Investigation; Kinetics; Knowledge; Lead; Length; Ligands; Light; Lighting; Link; Location; Longevity; Maintenance; Mechanics; Membrane; Microfilaments; Microscopy; Modeling; Morphology; Motor; Mus; Mutation; Myosin ATPase; Myosin Type II; N-terminal; Nonmuscle Myosin Type IIA; Organ; Organism; Perfusion; Pharmaceutical Preparations; Pharmacotherapy; Phosphotransferases; Play; Point Mutation; Positioning Attribute; Prevention; Process; Protein Isoforms; Proteins; Research; Resolution; Role; S-nitro-N-acetylpenicillamine; Scanning; Site; Small Intestines; Stains; Structure; Surface; System; Tail; Testing; Thick Filament; Tissues; base; blebbistatin; cellular microvillus; dimer; experimental study; fluorophore; gastrointestinal; gastrointestinal epithelium; gastrointestinal function; inhibitor/antagonist; insight; intestinal epithelium; intestinal homeostasis; knock-down; non-muscle myosin; novel; nutrient absorption; overexpression; pathogen; regenerative; response

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.

项目概要 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 肠细胞顶端表面的突起这些突起（称为微绒毛）也形成第一线。 刷状缘的正确形成取决于其正确的形成。 微绒毛是膜覆盖的突起，每个微绒毛含有一束 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 由于终末肠细胞不断更新，因此微绒毛的生长在整个过程中至关重要。 然而，对于驱动生长和发育的机制知之甚少。 微绒毛的组织，或终端网络的精确作用。 多年前的超微结构研究表明，微绒毛肌动蛋白束嵌入致密的结构中。 终端网络，尽管我们的初步研究对该结构的组成和功能仍然不明确。 表明该结构的主要成分是非肌肉肌球蛋白 II (NMII)，一种形成丝状的肌球蛋白马达 在所有真核细胞中均表达 NMII 的三种不同亚型：A、B 和 C； 我的初步超分辨率图像显示，NMII-A 和 NMII-C 在肠细胞中占主导地位。 NMII-C 在终端网络水平上形成一个跨肠细胞顶端域的新型网络。 沿微绒毛轴的观察表明，NMII-C 在微绒毛肌动蛋白束的尖端附近富集。 使用 Blebbistatin（一种肌球蛋白 II 抑制剂）的初步研究发现，小鼠肠道组织中的天然微绒毛 明显更短，并且似乎失去了细胞相对表面的直立方向。 初步数据显示，我发现跨越顶端域的非肌肉肌球蛋白 II 网络提供了 为了验证这个假设，我将： （目标 1）定义心尖下非肌肉肌球蛋白 II 网络的组织，以及（目标 2）确定作用 NMII-C 在微绒毛生长和组织中的作用对这种新型肌球蛋白 II 阵列的研究将具有重大意义。 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。