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: 10469526
• 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/10469526
• 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.

项目摘要/摘要 细胞极性对一系列过程是必不可少的，包括营养物质跨上皮层的运输， 趋化性和发育。因此，极化缺陷可能会产生可怕的生理后果。这个 这项建议的首要目标是理解肌动蛋白(S)在发育过程中的极性建立中的作用 蛋。在小鼠、果蝇、海星和线虫的卵母细胞中观察到细胞质肌动蛋白网。 这表明网格是一种保守的特征，在某些情况下在功能上存在分歧。果蝇卵母细胞 是研究肌动蛋白网的理想系统，因为卵巢很大，有利于细胞生物学和 在遗传易驯化的模式生物中进行生化检测。肌动蛋白网状结构的存在及其 正确的定时移除对于极性的建立至关重要。人们对网状结构知之甚少 构建它所需的两个肌动蛋白核素，Spire和Cappuccino，以及一个与Spire共存的分子马达 关于囊泡，肌球蛋白V。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 2)许多肌动蛋白结合蛋白对早期卵子发生是必不可少的， 削弱了经典遗传学在卵子发生中期发现必要网状成分的能力。我们 正在开发一种生命内成像平台来解决这两个问题。生命内成像将促进长期的， 直接观察网眼及其在动物体内的消失情况。当与Crispr/Cas9结合使用时- 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上的变化 以时间可控的方式去除特定的蛋白质。我们将使用这些工具来检验假设 网状物减缓流体流动，以促进极性所必需的后部锚定结构的形成 当流体流动过早加速时，无法形成，就像网格的情况一样 妥协了。最近发现了一种类似的肌动蛋白网状物，也是由Spire、Cappuccino和Myosin V构建的 在体细胞中，树突状黑素细胞。有趣的是，在小鼠卵母细胞中，网状物将小泡移向 而在黑素细胞中，网状物使囊泡分开，从而分散 它们遍布整个细胞。通过将体内实验与网状物的体外重建相结合，我们将 确定同一组蛋白质如何驱动小鼠卵母细胞中的小泡朝着相反的方向 并确定如何在果蝇卵母细胞中构建网状结构。最后，我们将研究 最近发现的Spir和肌球蛋白V之间相互作用的后果，使用互补 生物化学和遗传方法。肌动蛋白核子和基于肌动蛋白的马达之间的协调 对于网格和其他结构是如何构建的，这是令人兴奋的暗示。这项工作完成后，我们将拥有 确定了对细胞极性至关重要的网状结构是如何构建的，确定了以前未知的网状结构 组件，并确定后部锚定是否需要慢流。现在我们知道了 肌动蛋白网状结构并不是卵母细胞所独有的，我们的发现的意义甚至更广泛。