Collaboration between actin nucleators - Spire and Cappuccino

描述（由申请人提供）：细胞骨架对于生命的许多方面至关重要，细胞分裂和运动就是最好的例子。该提案的总体目标是了解两种极性因子 Spire (Spir) 和 Cappuccino (Capu) 在果蝇早期发育过程中调节细胞骨架和建立身体轴的作用。 Spir 和 Capu 是不同类型的肌动蛋白成核剂，是从头构建新肌动蛋白丝的因子。任一基因的突变都会因极性和细胞骨架缺陷而导致雌性不育，这表明它们参与相同的生物途径。最显着的细胞骨架缺陷是肌动蛋白网的丢失，该肌动蛋白网穿过果蝇卵母细胞，直到称为细胞质流的动态过程开始。有趣的是，最近发现 Capu 同源物 Fmn-2 在哺乳动物卵母细胞中构建了必需的肌动蛋白结构，这表明 Capu 甚至 Spir 的功能保守。在拟议的工作中，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）共核模型，其中 Spir 和 Capu 协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，而是通过Capu间接增强成核； 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，而不是使网格成核。第一个和第二个模型将通过使用荧光显微镜、电子显微镜和其他生化方法来研究 Spir 对肌动蛋白丝动力学的影响来区分。这些实验将允许直接观察肌动蛋白丝，以确定 Spir 是否主要使新丝成核、切断现有丝或改变丝动态。第一个和第三个模型将利用从体外实验中获得的知识来设计 Spir 和 Capu 的合理突变来区分。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。将进行额外的实验来确定如何控制这些蛋白质的活性。区分这些模型将导致对在后生动物物种中保守的两种蛋白质的机械理解。它将增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）中共存，有人提出它们作为果蝇极性因子的作用是保守的。因此，我们预计，在果蝇卵子发生过程中对 Spir 和 Capu 的了解将适用于我们对从果蝇到人类的许多动物的细胞骨架、细胞极性、生育力、发育和健康的理解。 ) ) 公共卫生相关性：细胞骨架对于生命的许多方面至关重要，细胞分裂和运动就是最好的例子。我们正在研究细胞骨架在建立主要身体轴中的作用，其失败会导致出生缺陷和不孕不育。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多种疾病的理解和工具。

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.