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: 8027386
• 负责人: Margot E Quinlan
• 金额: $ 25.62万
• 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 2016-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8027386
• 关键词: Actins; Address; Animals; Binding; Biochemical; Biological; Biological Assay; Bundling; Cell Polarity; Cell division; Cells; Collaborations; Complex; Confocal Microscopy; Congenital Abnormality; Cytoplasmic streaming; Cytoskeleton; Data; Defect; Development; Diagnosis; Disease; Drosophila genus; Electron Microscopy; Elements; Embryo; Epithelial Cells; Failure; Female sterility; Fertility; Filament; Fluorescence Microscopy; Fluorescence Resonance Energy Transfer; Genes; Genetic; Goals; Health; Homologous Gene; Human; In Vitro; Infertility; Kinetics; Knowledge; Lead; Learning; Life; Mammals; Microfilaments; Microinjections; Microtubules; Modeling; Mutation; Neurons; Oocytes; Oogenesis; Pathway interactions; Physiological; Play; Process; Proteins; Publishing; RNA Splicing; Regulation; Reporting; Role; Series; Signal Transduction; Stream; Structure; Testing; Time; Variant; Western Blotting; Work; base; cell motility; crosslink; design; fluid flow; in vitro activity; in vivo; model design; polymerization; profilin; research study; rho; tool

DESCRIPTION (provided by applicant): The cytoskeleton is essential to many aspects of life, cell division and motility being prime examples. The overarching goal of this proposal is to understand the roles of two polarity factors, Spire (Spir) and Cappuccino (Capu), in regulating the cytoskeleton and establishing the body axes in early Drosophila development. Spir and Capu are distinct types of actin nucleators, factors that build new actin filaments de novo. Mutations in either gene cause female sterility due to polarity and cytoskeletal defects, which indicates that they are involved in the same biological pathway. The most notable cytoskeletal defect is the loss of an actin mesh that traverses the Drosophila oocyte up until a dynamic process called cytoplasmic streaming begins. Interestingly, the Capu homolog, Fmn-2, was recently found to build an essential actin structure in mammalian oocytes suggesting functional conservation of Capu and perhaps Spir. In the proposed work, we will test three models of how Spir and Capu build actin structures and establish polarity in Drosophila oocytes: 1) the co-nucleation model, in which Spir and Capu nucleate collaboratively to build the actin mesh; 2) the synergy model, in which Spir is not a nucleator but enhances nucleation by Capu indirectly; 3) the crosslinking model, in which Spir and Capu regulate streaming by crosslinking the actin and microtubule cytoskeletons, as opposed to nucleating a mesh. The first and second models will be distinguished by using fluorescence microscopy, electron microscopy and other biochemical approaches to study the effects of Spir on actin filament dynamics. These experiments will allow direct observation of actin filaments to determine whether Spir primarily nucleates new filaments, severs existing filaments or alters filament dynamics. The first and third models will be distinguished using knowledge gained from in vitro experiments to design rational mutations in Spir and Capu. The mutations will be introduced into Drosophila to determine which activities of these two proteins are essential for their activities in vivo. Additional experiments will be performed to determine how the activities of these proteins are controlled. Distinguishing between these models will lead to a mechanistic understanding of two proteins, conserved throughout metazoan species. It will advance our knowledge of the cytoskeleton and how it is controlled. Given the co-existence of this pair of proteins in polar cells, including neurons and epithelial cells in mammals, it has been proposed that their role as polarity factors in Drosophila is conserved. Thus we anticipate that what is learned about Spir and Capu in Drosophila oogenesis will be applicable to our understanding of the cytoskeleton, cell polarity, fertility, development and health in many animals, ranging from Drosophila to humans. ) ) PUBLIC HEALTH RELEVANCE: The cytoskeleton is essential to many aspects of life, cell division and motility being prime examples. We are studying the role of the cytoskeleton in establishing the major body axes, the failure of which leads to birth defects and infertility. By studying normal function and regulation of the cytoskeleton during early development we will gain understanding and tools that enable diagnosis and treatment of a broad spectrum of diseases.

描述（由申请人提供）：细胞骨架对生命的许多方面都是必不可少的，细胞分裂和运动是主要的例子。该建议的总体目标是了解两个极性因子Spire（Spir）和Cappuccino（Capu）在调节细胞骨架和建立早期果蝇发育中的体轴中的作用。Spir和Capu是不同类型的肌动蛋白成核剂，它们是从头构建新肌动蛋白丝的因子。由于极性和细胞骨架缺陷，任何一个基因的突变都会导致雌性不育，这表明它们参与了相同的生物学途径。最显著的细胞骨架缺陷是肌动蛋白网的丢失，肌动蛋白网穿过果蝇卵母细胞，直到称为细胞质流动的动态过程开始。有趣的是，最近发现的Capu同源物，Fn-2，建立一个重要的肌动蛋白结构在哺乳动物卵母细胞表明Capu的功能保护，也许Spir。在本研究中，我们将测试Spir和Capu如何在果蝇卵母细胞中构建肌动蛋白结构并建立极性的三种模型：1）共成核模型，其中Spir和Capu合作成核以构建肌动蛋白网格; 2）协同模型，其中Spir不是成核剂，但间接地增强Capu的成核作用; 3）交联模型，其中Spir和Capu通过交联肌动蛋白和微管细胞骨架来调节流动，而不是使网状物成核。第一个和第二个模型将区分使用荧光显微镜，电子显微镜和其他生化方法来研究的影响，螺旋肌动蛋白丝动力学。这些实验将允许直接观察肌动蛋白丝，以确定是否Spir主要成核新的丝，切断现有的丝或改变丝动力学。将使用从体外实验获得的知识来区分第一和第三模型，以设计Spir和Capu中的合理突变。这些突变将被引入果蝇中，以确定这两种蛋白质的哪些活性对其体内活性至关重要。将进行额外的实验以确定如何控制这些蛋白质的活性。区分这些模型将导致两个蛋白质的机械理解，保守的后生动物物种。它将促进我们对细胞骨架及其控制方式的了解。鉴于这对蛋白质在极性细胞（包括哺乳动物的神经元和上皮细胞）中的共存，已经提出它们在果蝇中作为极性因子的作用是保守的。因此，我们预计，什么是了解Spir和Capu在果蝇卵子发生将适用于我们的理解细胞骨架，细胞极性，生育力，发育和健康的许多动物，从果蝇到人类。) ) 公共卫生相关性：细胞骨架对生命的许多方面都是必不可少的，细胞分裂和运动是主要的例子。我们正在研究细胞骨架在建立主要身体轴方面的作用，这一作用的失败会导致出生缺陷和不育。通过研究早期发育过程中细胞骨架的正常功能和调节，我们将获得能够诊断和治疗广泛疾病的理解和工具。