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: 10370436
• 负责人: MATTHEW J TYSKA
• 金额: $ 34.87万
• 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/10370436
• 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.

概括 肠细胞通过建造顶端优化其肠腔摄入的固体吸收的形态 专业：一系列密集的肌动蛋白束支撑的微绒毛，统称为“刷边框”。 通过脚手架顶膜，刷子微壁可显着增加住房的能力 膜结合的转运蛋白和驱动固体传输的通道。在立即下方的细胞质中 顶部表面，微绒毛核肌动蛋白束的根部固定在已知的细丝中 几十年前，“终端网”首先在经典的超微结构研究中可视化。富含中级和肌动蛋白 细丝，该网络足够密集，可以排除微管，蔬菜和其他大型细胞器。虽然 终端网络良好，以调节一系列关键的亚细胞活动，功能和 该领域的组成及其对肠道生理的贡献尚不清楚。在令人兴奋的初步中 研究，我们将非肌肉肌球蛋白-2C（MyO2C）确定为肠球末端网络的组成部分。 myo2 分子由结合肌动蛋白并产生力的N末端运动结构域组成，并且C末端杆样 尾巴，驱动细胞中收缩丝的形成。在三个非肌肉肌球蛋白-2同工型中 （2a，2b，2c），Myo2c是独一无二的，因为它的表达在很大程度上是肠上皮的特异性。此外， 单细胞RNASEQ分析表明，MyO2C相对于其他 肠道中的上皮细胞类型。我们发现Myo2c在绒毛的刷子边框的底部高度丰富 小鼠和人类小肠以及培养的肠上皮细胞系中的肠上皮细胞。极好的- 分辨率显微镜表明，Myo2c在终端平面上形成了广泛的点网络 Web，在空间上与微伏肌动蛋白束的根重叠。小分子和遗传 培养的上皮细胞中的扰动研究导致微绒毛的显着伸长，这表明这些 MyO2C可能会促进肌动蛋白拆卸的尖头末端。最后，我们发现myo2c ko小鼠有 不仅在微型组织中，而且在肠肠细胞和绒毛规模的组织结构中存在缺陷。基于 这些发现，我们假设MyO2C形成了控制刷边框的终端网络收缩网络 肌动蛋白结构并传播组织尺度的机械力，以实现有效的集体细胞迁移 地下村轴。为了检验这一假设，我们将采用最先进的超级分辨率 显微镜，电子显微镜的高级形式和晶格光片活成像至：（目标1）映射 终端Web myo2c网络的组织，（AIM 2）研究依赖MyO2C的机制 微绒毛长度调节，（AIM 3）在体内定义了MyO2C的功能。这些研究 将导致新的范式理解控制形态的基本机制 它们完成的肠上皮细胞及其上皮组织。