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

在上皮中，细胞以动态方式彼此粘附，允许细胞在形态发生期间改变其形状并彼此沿着移动。粘附的调节发生在带状粘附连接（AJ），即粘附小带（ZA）。ZA的形成取决于极性调节剂的Par-aPKC复合物的组分。我们已经确定了LIM蛋白Smallish（Smash），脊椎动物LMO 7的直系同源物，作为Bazooka/Par-3（Baz）的结合伴侣，Par-aPKC复合物的核心成分。Smash还与Canoe/Afadin和酪氨酸激酶Src 42 A结合，并以平面极化方式定位于ZA。缺乏Smash的动物表现出胚胎表皮中平面细胞极性（PCP）的丧失和细胞键张力的降低，导致上皮组织和器官的胚胎形态发生期间的严重缺陷。Smash的过表达引起上皮细胞的顶端收缩。我们建议，粉碎是一个关键的调节器的形态协调PCP和肌动球蛋白收缩在ZA。 在拟议的项目中，我们将分析粉碎功能丧失表型的细胞基础，通过实时成像和定量分析的连接动力学和形态发生运动在果蝇生殖带延伸和管状形态发生的气管系统。我们将进行克隆分析粉碎损失的功能，在成虫盘和滤泡上皮，我们将研究粉碎突变的影响，几个ZA相关蛋白的亚细胞定位。激光烧蚀实验将提供粉碎功能丧失和过表达后细胞键张力的信息。Smash亚细胞定位的突变体中的基因参与调节平面细胞极性和连接动力学的分析将提供信息的Smash在蛋白质相互作用网络控制形态发生的位置。我们还将通过体内成像研究不同Smash亚型的亚细胞定位动态，以将这些数据与已经分析的关键蛋白质的动态相关联，例如。G.肌球蛋白II和肌动蛋白。为了获得关于Smash的结合伴侣的概述，我们将进行co-IP实验，然后进行质谱分析。将通过组织培养中的co-IP和突变或敲低后的表型分析验证鉴定的结合配偶体。最后，我们将讨论Smash是否作为激酶Rok和Src 42 A的辅因子发挥作用，影响其激酶活性或底物特异性的问题。我们希望通过该项目进一步了解Smash的功能，Smash是一种新的上皮形态发生所需的ZA相关蛋白。

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.