Myosin-2 function in the enterocyte terminal web

肌球蛋白 2 在肠细胞终末网中的功能

• 批准号: 10211464
• 负责人: MATTHEW J TYSKA
• 金额: $ 34.83万
• 依托单位: VANDERBILT UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-03-15 至 2025-02-28
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10211464
• 关键词: ATP phosphohydrolase; Actins; Apical; Architecture; Binding; Biochemical; Brush Border; C-terminal; Cell Culture Techniques; Cell Line; Cells; Cytoplasm; Cytoskeleton; Data; Defect; Dimensions; Electron Microscopy; Enterocytes; Epithelial; Epithelial Cells; Eukaryotic Cell; Exhibits; Filament; Fingers; Generations; Genetic; Housing; Human; Image; Impairment; Individual; Intercellular Junctions; Intermediate Filaments; Internet; Intestines; Knockout Mice; Length; Light; Maps; Measures; Mechanics; Membrane; Microfilaments; Microscopy; Microtubules; Modeling; Morphology; Motor; Mus; Muscle; Myosin ATPase; Myosin S-2; N-terminal; Nutrient; Organelles; Organoids; Physiological; Physiology; Positioning Attribute; Process; Property; Protein Isoforms; Regulation; Resolution; Rod; Sarcomeres; Shapes; Small Intestines; Structure; Surface; Tail; Testing; Tissues; Vesicle; Villus; Work; actin depolymerizing factor; apical membrane; base; cell motility; cell type; cellular microvillus; human disease; in vivo; insight; intestinal epithelium; light microscopy; mechanical force; migration; mouse model; non-muscle myosin; regenerative; scaffold; single-cell RNA sequencing; small molecule; solute; uptake

SUMMARY Enterocytes optimize their morphology for solute uptake from the intestinal lumen by building an apical specialization: a dense array of actin bundle-supported microvilli, collectively referred to as the “brush border”. By scaffolding apical membrane, brush border microvilli significantly increase the capacity for housing membrane-bound transporters and channels that drive solute transport. In the cytoplasm immediately beneath the apical surface, the rootlets of microvillar core actin bundles are anchored in a meshwork of filaments known as the “terminal web” first visualized in classic ultrastructural studies decades ago. Rich in intermediate and actin filaments, this network is dense enough to exclude microtubules, vesicles and other large organelles. Although the terminal web is well-positioned to regulate a range of critical subcellular activities, the function and composition of this domain and its contribution to intestinal physiology remain unclear. In exciting preliminary studies, we identified non-muscle myosin-2C (MYO2C) as a component of the enterocyte terminal web. MYO2 molecules consist of an N-terminal motor domain that binds actin and generates force, and a C-terminal rod-like tail, which drives the formation of contractile filaments in cells. Among the three non-muscle myosin-2 isoforms (2A,2B,2C), MYO2C is unique in that its expression is largely specific to the intestinal epithelium. Additionally, single cell RNAseq analysis indicates that MYO2C demonstrates clear enrichment in enterocytes relative to other epithelial cell types in the gut. We found that MYO2C is highly enriched at the base of the brush border in villus enterocytes from mouse and human small intestine, and in cultured intestinal epithelial cell lines. Super- resolution microscopy revealed that MYO2C forms an extensive network of puncta in the plane of the terminal web, which spatially overlaps with the rootlets of microvillar actin bundles. Small molecule and genetic perturbation studies in cultured epithelial cells leads to dramatic elongation of microvilli, suggesting these MYO2C may promote actin disassembly from rootlet pointed ends. Finally, we found that MYO2C KO mice have defects not only in microvillar organization, but also in enterocyte- and villus-scale tissue structure. Based on these findings, we hypothesize that MYO2C forms a terminal web contractile network that controls brush border actin architecture and propagates tissue-scale mechanical forces to enable efficient collective cell migration up the crypt-villus axis. To test this hypothesis, we will employ a combination of state-of-the-art super-resolution microscopy, advanced forms of electron microscopy, and lattice light sheet live imaging to: (Aim 1) map the organization of the terminal web MYO2C network, (Aim 2) investigate the mechanism of MYO2C-dependent microvillar length regulation, and (Aim 3) define the function of MYO2C in the terminal web in vivo. These studies will lead to new paradigms for understanding the fundamental mechanisms that control the morphology of enterocytes and the epithelial tissue they comprise.

摘要 肠细胞通过建立一个根尖细胞来优化其形态，以便从肠腔吸收溶质。 特化：肌动蛋白束支持的微绒毛密集排列，统称为“刷缘”。 通过支架顶膜，刷状边缘微绒毛显著增加了住房的容量 驱动溶质运输的膜结合转运体和通道。在紧邻其下的细胞质中 在顶端表面，微绒毛核心肌动蛋白束根被固定在一个已知的细丝网络中。 就像几十年前在经典的超微结构研究中首次出现的“终端网”一样。富含中间体和肌动蛋白 细丝，这个网络的密度足以排除微管、小泡和其他大细胞器。虽然 终端网处于很好的位置，可以调节一系列关键的亚细胞活动，功能和 该结构域的组成及其对肠道生理学的贡献尚不清楚。在激动人心的预赛中 研究发现，非肌肉肌球蛋白-2C(MYO2C)是肠细胞终末网络的一个组成部分。MYO2 分子由结合肌动蛋白并产生力的N-末端马达结构域和C-末端棒状组成 尾巴，它驱动细胞中收缩细丝的形成。在三种非肌肉肌球蛋白-2亚型中 (2A，2B，2C)，MYO2C的独特之处在于其主要表达于肠道上皮细胞。另外， 单细胞RNAseq分析表明，MYO2C在肠上皮细胞中明显富含 肠道上皮细胞类型。我们发现MYO2C在绒毛的灌木丛边缘的底部高度富含 小鼠和人小肠以及培养的肠上皮细胞系中的肠上皮细胞。超级- 分辨率显微镜显示，MYO2C在终端平面上形成了一个广泛的点状网络 WEB，在空间上与微绒毛肌动蛋白束的根重叠。小分子与遗传学 在培养的上皮细胞中的扰动研究导致微绒毛显著延长，表明这些 MYO2C可促进根尖端肌动蛋白的分解。最后，我们发现MYO2C KO小鼠有 不仅在微绒毛组织上存在缺陷，而且在肠细胞和绒毛规模的组织结构上也存在缺陷。基于 这些发现，我们假设MYO2C形成了一个控制刷子边缘的末端网络收缩网络 肌动蛋白结构，并传播组织规模的机械力，以实现高效的集体细胞向上迁移 地穴-绒毛轴。为了验证这一假设，我们将使用最先进的超分辨率组合 显微镜、高级电子显微镜和点阵光片实时成像：(目标1)绘制 终端网络MYO2C网络的组织，(目标2)研究MYO2C依赖的机制 微绒毛长度的调节，以及(目的3)确定MYO2C在体内终末网络中的功能。这些研究 将导致新的范式来理解控制形态的基本机制 肠细胞及其构成的上皮组织。