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.

PUBLIC HEALTH RELEVANCE: Polarized epithelial cells, which line the major organs in our bodies and are critical for normal growth and development, are characterized by E-cadherin-based cell-cell contacts. This study addresses the role of myosin 1c in the formation and maintenance of E-cadherin-based cell-cell contacts and the establishment of cell polarity. Understanding the basis of these fundamental processes can result in the design of rational therapies to identify, treat or prevent epithelial cancers, which account for the majority of cance fatalities, or to stimulate tissue regeneration following disease or wounding.