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.

项目摘要 在肠内，营养滥用发生在刷子边界，这是一个基于肌动蛋白的挤压区域 肠形细胞顶部表面的突起。这些突出被称为微绒毛，也形成了第一行 针对腔病原体的防御。刷子边框的正确形成取决于正确的形成 和微绒毛的聚类。微绒毛是膜覆盖的蛋白 细丝，正末端位于远端尖端，负端锚定在已知的细胞区域 作为终端网。肠细胞不断更新，因此微绒毛的生长至关重要 有机体的整个生命。但是，关于推动增长和的机制知之甚少 微维利的组织或终端网的精确作用。 超微结构研究表明，许多年前，微绒毛肌动蛋白束嵌入了密集 终端网，尽管该结构的组成和功能仍然模棱两可。我们的初步研究 建议该结构的主要结构是非肌肉肌球蛋白II（NMII），这是一种形成细丝的肌球蛋白运动 在所有真核细胞中表达。在肠道中，表达了三种不同的NMII同工型。 A，B和C，带有 NMII-A和NMII-C在肠上皮细胞中占主导地位。我的初步超分辨率图像表明 NMII-C在终端Web的级别上跨肠球细胞顶端域形成一个新型网络。线扫描 沿微绒毛轴显示NMII-C在微伏肌动蛋白束的尖端附近富集。 使用肌球蛋白II抑制剂BLEBBISTATIN的初步研究，小鼠肠道组织中的天然微绒毛 明显短，并且似乎失去了与细胞表面相对的直立定向。基于我 初步数据，我假设跨越顶端域的非肌肉肌球蛋白II网络提供 对微型维尔的生长和维护的必要张力和机械支持。为了检验这一假设，我将： （AIM 1）定义次级非肌肉肌球蛋白II网络的组织，并确定角色 NMII-C在微绒毛的增长和组织中的。对这部小说的肌球蛋白II阵列的调查将大大显着 加深我们对微伏特形成和维护的物理机制的理解，以及 与非肌肉肌球蛋白IIS突变有关的疾病。