Myosin I in epithelial cell-cell contact and polarity

 描述(申请人提供)：基于E-钙粘素的细胞-细胞接触，或黏附连接(AJ)，介导极化上皮细胞的形成和维持，这是正常生长和发育所必需的；E-钙粘素的丢失导致肿瘤生长。钙粘附素与肌动蛋白细胞骨架协同工作，介导细胞与细胞之间的接触，将细胞组织成片状，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，肌动蛋白和膜相关的分子马达蛋白Myo1c(Myo1c)与E-cadherin一起定位于细胞-细胞接触处。Myo1c表达下调(Kd)扰乱了E-钙粘蛋白的定位，导致细胞极化较差，E-钙粘蛋白介导的细胞与细胞之间的接触减少。Myo1c如何支持基于E-钙粘附素的细胞-细胞接触的形成和介导上皮形态发生在这里被讨论。(1)具体目标1是确定Myo1c在发育和成熟AJ中的定位和动力学；Myo1c kd和局部Myo1c失活对钙粘素复合体招募的影响；以及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)、光激活和生色团辅助激光失活(CALI)，肌动蛋白细胞骨架如何支持Myo1c在发育和成熟的细胞-细胞接触中的功能。(2)E-钙粘蛋白在组装的细胞-细胞接触处是动态的；此外，上皮极性的建立依赖于E-钙粘蛋白通过胞内和胞外途径向基底膜和从基底膜移位。在MDCK细胞中发现E-cadherin和Myo1c阳性的细胞内小泡，在阻止囊泡循环的18℃下，与对照细胞相比，在Myo1c-kd细胞的细胞质中积累了更多E-cadherin阳性小泡。具体目的2是鉴定E-钙粘蛋白和Myo1c阳性的细胞内小泡，并使用Myo1c突变体，包括那些影响运动活性和膜结合的突变体，结合运输分析和分级研究来研究Myo1c在E-钙粘素细胞内运输中的作用。(3)在胶原蛋白中，MDCK细胞形成3D囊，即由单层细胞围绕中央中空腔组成的球体，顶膜朝向管腔；囊可被诱导形成小管。在初步研究中，由Myo1c-kd细胞形成的囊泡很大，变形，肌动蛋白通常突出在管腔表面的外表面，这表明Myo1c介导了细胞极性的各个方面，这对上皮生长和组织形成至关重要。具体目标3是利用在3D器官培养中生长的MDCK细胞，研究Myo1c在上皮器官的构建块--囊和小管形成中的作用。这些研究有望为细胞骨架如何支持钙粘素生物学提供新的见解。此外，它们可能导致开发出识别、预防或治疗上皮癌的疗法，或促进组织再生。上皮癌是导致癌症死亡的主要原因。

 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.