Myosin I in epithelial cell-cell contact and polarity

肌球蛋白 I 在上皮细胞-细胞接触和极性中的作用

• 批准号: 9333408
• 负责人: LYNNE M COLUCCIO
• 金额: $ 41.75万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-16 至 2019-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9333408
• 关键词: Abate; Accounting; Actin-Binding Protein; Actins; Address; Adherens Junction; Adhesions; Affect; Apical; Binding; Biological Assay; Biological Models; Biology; Blocking Antibodies; Cadherins; Canis familiaris; Carcinoma; Cell Fractionation; Cell Polarity; Cell-Cell Adhesion; Cells; Collagen; Complex; Cyst; Cytoplasm; Cytoskeleton; Data; Defect; Development; Diffuse; Dimensions; Disease; E-Cadherin; Employee Strikes; Epithelial; Epithelial Cells; Epithelium; Excision; Fluorescence Recovery After Photobleaching; Fractionation; Growth; Growth and Development function; Image; Kidney; Lasers; Lead; MDCK cell; Maintenance; Malignant Neoplasms; Mediating; Membrane; Methods; Microfilaments; Molecular Motors; Monitor; Morphogenesis; Morphology; Motor Activity; Myosin ATPase; Myosin Type I; Organ; Pathway interactions; Phenotype; Pilot Projects; Process; Proteins; Recruitment Activity; Recycling; Research; Resistance; Role; Staining method; Stains; Surface; Testing; Tissues; Vesicle; Work; apical membrane; base; basolateral membrane; chromophore; design; imaging approach; improved; in vivo; insight; kidney epithelial cell; knock-down; live cell imaging; monolayer; mutant; novel; novel strategies; photoactivation; polarized cell; prevent; public health relevance; therapy development; tissue regeneration; trafficking; tumor growth

 DESCRIPTION (provided by applicant): E-cadherin-based cell-cell contacts, or adherens junctions (AJs), mediate the formation and maintenance of polarized epithelial cells, which are essential for normal growth and development; loss of E-cadherin results in tumor growth. Cadherins work cooperatively with the actin cytoskeleton to mediate cell-cell contact, organize cells into sheets, and modulate morphological changes. This research group found that the actin- and membrane-associated molecular motor protein myosin 1c (Myo1c) localizes with E-cadherin at cell-cell contacts in polarized Madin-Darby canine kidney (MDCK) epithelial cells. Knock down (kd) of Myo1c expression disrupts E-cadherin localization and results in less well-polarized cells with reduced E-cadherin-mediated cell-cell contact. How Myo1c supports the formation of E-cadherin-based cell-cell contacts and mediates epithelial morphogenesis is addressed here. (1) Specific Aim 1 is to determine Myo1c localization and dynamics at developing and mature AJs; the effect of Myo1c kd and local Myo1c inactivation on recruitment of cadherin complexes; and how the actin cytoskeleton supports Myo1c function at developing and mature cell-cell contacts using advanced live-cell confocal imaging approaches including fluorescence recovery after photobleaching (FRAP), photoactivation, and chromophore-assisted laser inactivation (CALI). (2) E-cadherin at assembled cell-cell contacts is dynamic; moreover, the establishment of epithelial polarity relies on the translocation of E-cadherin to and from the basolateral membrane by endocytic and exocytic pathways. Intracellular vesicles positive for both E-cadherin and Myo1c are found in MDCK cells, and at 18ºC, which prevents vesicle recycling, more E-cadherin-positive vesicles accumulate in the cytoplasm of Myo1c-kd vs. control cells. Specific Aim 2 is to identify intracellular vesicles positive for E-cadherin and Myo1c, and to use Myo1c mutants, including those that affect motor activity and membrane binding, in conjunction with trafficking assays and fractionation studies to investigate the role o Myo1c in the intracellular trafficking of E- cadherin. (3) In collagen, MDCK cells form 3D cysts, spheres consisting of a monolayer of cells around a central hollow lumen with the apical membrane facing the lumen; the cysts can be induced to form tubules. In pilot studies, cysts formed with Myo1c-kd cells are large and dysmorphic with actin, normally prominent at the luminal surface, on the outside surface, suggesting that Myo1c mediates aspects of cell polarity, which is critical for epithelial growth and tissue formation. Specific Aim 3 is to investigate the role of Myo1c in cyst and tubule formation, the building blocks of epithelial organs, using MDCK cells grown in 3D organotypic culture. The studies are expected to provide new insight into how the cytoskeleton supports cadherin biology. Moreover, they could lead to the development of therapies to identify, prevent or treat epithelial cancers, which account for most cancer fatalitie, or to promote tissue regeneration.

 描述（由申请人提供）：基于E-钙粘蛋白的细胞间接触或粘附连接（AJ）介导极化上皮细胞的形成和维持，这对于正常生长和发育至关重要； E-钙粘蛋白的缺失导致肿瘤生长。钙粘蛋白与肌动蛋白细胞骨架协同作用，介导细胞与细胞的接触，将细胞组织成片，并调节形态变化。该研究小组发现，在极化的 Madin-Darby 犬肾 (MDCK) 上皮细胞中，肌动蛋白和膜相关分子运动蛋白肌球蛋白 1c (Myo1c) 与 E-钙粘蛋白一起定位在细胞与细胞接触处。 Myo1c 表达的敲低 (kd) 会破坏 E-钙粘蛋白的定位，导致细胞极化较差，同时 E-钙粘蛋白介导的细胞间接触减少。本文讨论了 Myo1c 如何支持基于 E-钙粘蛋白的细胞间接触的形成并介导上皮形态发生。 (1) 具体目标 1 是确定 Myo1c 在发育中和成熟 AJ 中的定位和动态； Myo1c kd 和局部 Myo1c 失活对钙粘蛋白复合物募集的影响；以及肌动蛋白细胞骨架如何使用先进的活细胞共聚焦成像方法（包括光漂白后荧光恢复 (FRAP)、光激活和发色团辅助激光灭活 (CALI)）支持发育和成熟细胞接触中的 Myo1c 功能。 (2)组装的细胞-细胞接触处的E-钙粘蛋白是动态的；此外，上皮极性的建立依赖于E-钙粘蛋白通过胞吞和胞吐途径进出基底外侧膜的易位。 MDCK 细胞中发现 E-钙粘蛋白和 Myo1c 均呈阳性的细胞内囊泡，在 18°C 时，阻止囊泡循环，与对照细胞相比，Myo1c-kd 的细胞质中积累了更多的 E-钙粘蛋白阳性囊泡。具体目标 2 是鉴定 E-cadherin 和 Myo1c 阳性的细胞内囊泡，并使用 Myo1c 突变体（包括影响运动活动和膜结合的突变体），结合运输测定和分级研究来研究 Myo1c 在 E-cadherin 细胞内运输中的作用。 (3) 在胶原蛋白中，MDCK 细胞形成 3D 囊肿，即由围绕中央中空管腔的单层细胞组成的球体，顶膜面向管腔；可以诱导囊肿形成小管。在初步研究中，Myo1c-kd 细胞形成的囊肿很大，并且肌动蛋白变形，通常在管腔表面和外表面上突出，表明 Myo1c 介导细胞极性的各个方面，这对于上皮生长和组织形成至关重要。具体目标 3 是使用在 3D 器官型培养中生长的 MDCK 细胞来研究 Myo1c 在囊肿和小管形成（上皮器官的组成部分）中的作用。这些研究有望为细胞骨架如何支持钙粘蛋白生物学提供新的见解。此外，它们可能会导致开发出识别、预防或治疗上皮癌（大多数癌症死亡原因）或促进组织再生的疗法。