Collaboration between actin nucleators - Spire and Cappuccino

肌动蛋白成核剂 - Spire 和 Cappuccino 之间的合作

• 批准号: 10314737
• 负责人: Margot E Quinlan
• 金额: $ 31.57万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-01 至 2025-08-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10314737
• 关键词: Actin-Binding Protein; Actins; Address; Animal Model; Animals; Auxins; Binding; Biochemical; Biochemical Genetics; Biological; Biological Models; CRISPR/Cas technology; Caenorhabditis elegans; Cell Polarity; Cells; Chemotaxis; Collaborations; Complex; Contracts; Cytoskeleton; Data; Defect; Development; Diffuse; Drosophila genus; Embryo; Epithelial; Excision; Fertility; Filament; Genes; Genetic; Genetic Models; Genetic Screening; Goals; Health; Human; In Vitro; Kinesin; Knowledge; Learning; Maintenance; Mammals; Measures; Mediating; Membrane; Methods; Microtubules; Molecular Motors; Motion; Motor; Mus; Myosin Type V; Nutrient; Oocytes; Oogenesis; Ovary; Pharmacology; Physiological; Process; Protein Isoforms; Proteins; Proteomics; RNA; RNA Splicing; Role; Set protein; Somatic Cell; Starfish; Stream; Structure; System; Testing; Time; Translating; Vesicle; Work; base; cell motility; egg; experimental study; facsimile; fluid flow; fly; genetic approach; imaging platform; in vitro Assay; in vivo; insight; intravital imaging; melanocyte; paralogous gene; premature; reconstitution; tool; vacuolar H+-ATPase; vesicle transport

Project Summary/Abstract Cell polarity is essential to an array of processes, including transport of nutrients across epithelial layers, chemotaxis, and development. Thus, defects in polarization can have dire physiological consequences. The overarching goal of this proposal is to understand the role(s) of actin in polarity establishment in the developing egg. Cytoplasmic actin meshes are observed in oocytes of mouse, Drosophila, starfish, and C. elegans, which suggests meshes are a conserved feature that have diverged functionally, in some cases. The Drosophila oocyte is an ideal system in which to study the actin mesh because the ovaries are large, facilitating cell biological and biochemical examination in a genetically tractable model organism. The presence of the actin mesh and its correctly timed removal are critical to polarity establishment. Little is known about the mesh makeup beyond the two actin nucleators required to build it, Spire and Cappuccino, and a molecular motor that colocalizes with Spire on vesicles, myosin V. Our knowledge about the mesh is limited by two technical challenges: 1) several stages of oogenesis do not take place ex vivo and 2) many actin-binding proteins are essential to early oogenesis, diminishing the power of classical genetics to discover mesh components necessary during mid-oogenesis. We are developing an intra vital imaging platform to address both issues. Intra vital imaging will facilitate long-term, direct observation of the mesh and its disappearance within the animal. When combined with Crispr/Cas9- mediated gene editing and auxin-inducible-degradation, we will be able to observe changes in the mesh upon removal of specific proteins in a temporally controlled manner. We will use these tools to test the hypothesis that the mesh slows fluid flows to facilitate formation of a posterior anchoring structure that is necessary for polarity establishment and fails to form when fluid flows are prematurely accelerated, as is the case if the mesh is compromised. A comparable actin mesh, also built by Spire, Cappuccino, and myosin V, was recently discovered in a somatic cell, the dendritic melanocyte. Interestingly, in the mouse oocyte, the mesh moves vesicles towards each other and the periphery of the cell, whereas, in the melanocyte, the mesh moves vesicles apart, dispersing them throughout the cell. By combining in vivo experiments with in vitro reconstitution of the mesh, we will determine how the same set of proteins can drive vesicles in opposite directions in mouse oocytes and melanocytes and determine how the mesh is built in the Drosophila oocyte. Finally, we will examine the consequences of the recently-discovered interaction between Spir and myosin V, using complementary biochemical and genetic approaches. Coordination between an actin nucleator and an actin-based motor has exciting implications for how the mesh and other structures are built. Upon completion of this work, we will have established how the mesh, a structure essential to cell polarity, is built, identified previously unknown mesh components, and determined whether slow streaming is necessary for posterior anchoring. Now that we know that actin meshes are not exclusive to oocytes, the implications of our findings are even broader.

项目总结/摘要 细胞极性对一系列过程至关重要，包括营养物质跨上皮层的运输， 趋化性和发育。因此，极化的缺陷可能具有可怕的生理后果。的 本提案的总体目标是了解肌动蛋白在发展中极性建立中的作用 蛋在小鼠、果蝇、海星和C.优雅， 表明网格是一种保守的功能，在某些情况下，已经发散的功能。果蝇卵母细胞 是研究肌动蛋白网格的理想系统，因为卵巢很大，有利于细胞生物学， 在遗传学上易处理的模式生物中进行生化检查。肌动蛋白网格的存在及其 正确定时的移除对于极性建立是至关重要的。关于网眼化妆品， 两个肌动蛋白成核剂，Spire和Cappuccino，以及一个与Spire共定位的分子马达 我们对网状物的了解受到两个技术挑战的限制：1）几个阶段 卵子发生不发生离体和2）许多肌动蛋白结合蛋白是必需的早期卵子发生， 这削弱了经典遗传学发现卵子发生中期所需的网状成分的能力。我们 正在开发一个活体成像平台来解决这两个问题。活体成像将有助于长期， 直接观察补片及其在动物体内的消失。当与Crispr/Cas9结合时， 介导的基因编辑和生长素诱导的降解，我们将能够观察到网格的变化， 以时间控制的方式去除特定蛋白质。我们将使用这些工具来检验假设， 网状物减慢流体流动以便于形成极性所必需的后部锚定结构 当流体流动过早加速时，就像如果网被 暴露了一个类似的肌动蛋白网，也是由Spire，Cappuccino和肌球蛋白V构建的，最近被发现 树突状黑素细胞。有趣的是，在小鼠卵母细胞中，网格将囊泡移动到 而在黑素细胞中，网状物使囊泡分开，分散在细胞周围， 在整个细胞中。通过结合体内实验和体外重建补片，我们将 确定同一组蛋白质如何在小鼠卵母细胞中以相反的方向驱动囊泡， 黑色素细胞，并确定如何在果蝇卵母细胞中构建网格。最后，我们将研究 最近发现的Spir和肌球蛋白V之间的相互作用的后果，使用互补的 生物化学和遗传学方法。肌动蛋白成核剂和肌动蛋白基马达之间的协调， 这对如何构建网格和其他结构具有令人兴奋的意义。完成这项工作后，我们将 建立了如何建立网格，一个对细胞极性至关重要的结构，确定了以前未知的网格 组件，并确定缓慢的流动是否是必要的后方锚定。现在我们知道 肌动蛋白网格并不只限于卵母细胞，我们的发现的意义甚至更广泛。