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

LIM 结构域蛋白 Smallish 调节 ZA 肌动球蛋白收缩性和连接动力学的功能分析

• 批准号: 413909300
• 负责人: Professor Dr. Andreas Wodarz
• 金额: --
• 依托单位: Institut I für Anatomie
• 依托单位国家: 德国
• 项目类别: Priority Programmes
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2023-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/413909300?language=en
• 关键词: Functional; analysis; LIM; domain; protein

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.

在上皮细胞中，细胞以动态的方式相互粘连，使细胞在形态发生过程中改变自己的形状并沿着彼此移动。黏附的调节发生在带状黏附连接(AJ)和贴壁带(ZA)处。ZA的形成取决于极性调节剂的PAR-aPKC复合体的组成。我们已经确定LIM蛋白Smash(Smash)是脊椎动物LMO7的同源基因，是PAR-aPKC复合体的核心成分Bazooka/PAR-3(Baz)的结合伙伴。Smash还与Canoe/Afadin和酪氨酸激酶Src42a结合，并以平面极化的方式定位于Za。缺乏SMASH的动物表现为胚胎表皮平面细胞极性(PCP)的丧失和细胞结合张力的降低，导致胚胎上皮组织和器官的形态发生严重缺陷。Smash的过度表达会导致上皮细胞的顶端收缩。我们认为，Smash是ZA形态发生的关键调节因子，协调PCP和肌动球蛋白的收缩。在拟议的项目中，我们将通过实时成像和定量分析果蝇生殖带延伸和气管系统管状形态发生过程中的连接动力学和形态发生运动，分析SMASH功能丧失表型的细胞学基础。我们将在成像盘和卵泡上皮细胞中进行SMASH功能丧失的克隆分析，并研究SMASH突变对几个ZA相关蛋白亚细胞定位的影响。激光消融实验将提供粉碎、功能丧失和过度表达时细胞结合张力的信息。分析Smash在参与平面细胞极性和连接动力学调控的基因突变体中的亚细胞定位，将为Smash在控制形态发生的蛋白质相互作用网络中的位置提供信息。我们还将通过体内成像来研究不同Smash异构体的亚细胞定位的动力学，以将这些数据与已经分析的关键蛋白质的动力学相关联，例如肌球蛋白II和肌动蛋白。为了对Smash的结合伙伴有一个概述，我们将进行共同IP实验，然后进行质谱分析。确定的结合伙伴将在组织培养中通过共IP验证，并在突变或敲除时进行表型分析。最后，我们将讨论Smash是否作为影响其激酶活性或底物特异性的激酶Rok和Src42a的辅助因子。同时，我们希望通过这个项目进一步了解Smash的功能，Smash是一种新的ZA相关蛋白，是正常的上皮形态发生所必需的。