Functional analysis of the LIM domain protein Smallish in regulation of actomyosincontractility and junctional dynamics at the ZA

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In epithelia, cells adhere to each other in a dynamic fashion, allowing the cells to change their shape and to move along each other during morphogenesis. The regulation of adhesion occurs at the belt-shaped adherens junction (AJ), the zonula adherens (ZA). Formation of the ZA depends on components of the Par-aPKC complex of polarity regulators. We have identified the LIM protein Smallish (Smash), the ortholog of vertebrate LMO7, as a binding partner of Bazooka/Par-3 (Baz), a core component of the Par-aPKC complex. Smash also binds to Canoe/Afadin and the tyrosine kinase Src42A and localizes to the ZA in a planar polarized fashion. Animals lacking Smash show loss of planar cell polarity (PCP) in the embryonic epidermis and reduced cell bond tension, leading to severe defects during embryonic morphogenesis of epithelial tissues and organs. Overexpression of Smash causes apical constriction of epithelial cells. We propose that Smash is a key regulator of morphogenesis coordinating PCP and actomyosin contractility at the ZA. In the proposed project we will analyze the cellular basis of the smash loss-of-function phenotype by live imaging and quantitative analysis of junction dynamics and morphogenetic movements during Drosophila germ band extension and tubular morphogenesis of the tracheal system. We will pursue clonal analysis of smash loss-of-function in imaginal discs and in the follicular epithelium and we will study the effect of smash mutation on the subcellular localization of several ZA-associated proteins. Laser ablation experiments will provide information on cell bond tension upon smash loss-of-function and overexpression. Analyses of Smash subcellular localization in mutants for genes involved in regulation of planar cell polarity and junction dynamics will provide information on the position of Smash in the protein interaction network controlling morphogenesis. We will also study the dynamics of the subcellular localization of different Smash isoforms by in vivo imaging to correlate these data with the dynamics of key proteins that have already been analyzed, e. g. myosin II and actin. To get an overview on binding partners of Smash we will pursue co-IP experiments followed by mass-spectrometry. Identified binding partners will be validated by co-IP in tissue culture and phenotypic analysis upon mutation or knockdown. Finally, we will address the question whether Smash functions as a cofactor for the kinases Rok and Src42A that affects their kinase activity or substrate specificity.Together, we expect from this project to further our understanding of the function of Smash, a new ZA-associated protein required for proper epithelial morphogenesis.