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（AJs）介导极化上皮细胞的形成和维持，这对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，介导细胞间的接触，将细胞组织成片状，并调节形态学变化。该研究小组发现，肌动蛋白和膜相关分子马达蛋白肌球蛋白1c（Myo 1c）与E-钙粘蛋白在极化的Madin-Darby犬肾（MDCK）上皮细胞中的细胞-细胞接触处定位。Myo 1c表达的敲低（kd）破坏了E-钙粘蛋白的定位，并导致极化程度较低的细胞，E-钙粘蛋白介导的细胞-细胞接触减少。Myo 1c如何支持形成基于E-钙粘蛋白的细胞-细胞接触和介导上皮形态发生在这里解决。(1)具体目标1是确定Myo 1c在发育和成熟AJs中的定位和动力学; Myo 1c kd和局部Myo 1c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），光活化，和发色团辅助激光灭活（卡利）。(2)在组装的细胞-细胞接触的E-钙粘蛋白是动态的，此外，上皮极性的建立依赖于E-钙粘蛋白的易位和从基底外侧膜的内吞和外吞途径。在MDCK细胞中发现了对E-cadherin和Myo 1c均呈阳性的细胞内囊泡，并且在18ºC下，这阻止了囊泡再循环，与对照细胞相比，更多的E-cadherin阳性囊泡在Myo 1c-kd的细胞质中积累。具体目标2是鉴定E-钙粘蛋白和Myo 1c阳性的细胞内囊泡，并使用Myo 1c突变体，包括影响运动活性和膜结合的突变体，结合运输试验和分馏研究，以研究Myo 1c在E-钙粘蛋白细胞内运输中的作用。(3)在胶原蛋白中，MDCK细胞形成3D囊肿，由围绕中心中空腔的单层细胞组成的球体，顶膜面向腔;囊肿可以被诱导形成小管。在初步研究中，Myo 1c-kd细胞形成的囊肿很大，并且在外表面上具有肌动蛋白，通常在腔表面突出，这表明Myo 1c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器官型培养物中生长的MDCK细胞，研究Myo 1c在囊肿和小管形成（上皮器官的构建块）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，它们可能导致开发用于识别、预防或治疗上皮癌（占大多数癌症死亡率）或促进组织再生的疗法。