Collaboration between actin nucleators - Spire and Cappuccino

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，细胞分裂和运动就是最好的例子。这项建议的首要目标是了解两种极性因子，Spire(Spir)和Cappuccino(Capu)在调节细胞骨架和建立果蝇早期发育体轴中的作用。Spir和Capu是不同类型的肌动蛋白核因子，它们是从头开始形成新的肌动蛋白细丝的因素。任何一种基因的突变都会导致女性不育，原因是极性和细胞骨架缺陷，这表明它们参与了相同的生物途径。最显著的细胞骨架缺陷是失去了穿过果蝇卵母细胞的肌动蛋白网状结构，直到一个称为细胞质流动的动态过程开始。有趣的是，最近发现CAPU同源物FMN-2在哺乳动物卵母细胞中建立了一种基本的肌动蛋白结构，这表明CAPU和可能的Spir具有功能保守。在这项拟议的工作中，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和建立极性的三种模型：1)共核模型，在该模型中，Spir和Capu协同成核以构建肌动蛋白网状结构；2)协同模型，在该模型中，Spir不是核因子，但通过Capu间接促进成核；3)交联模型，在该模型中，Spir和Capu通过使肌动蛋白和微管细胞骨架交联来调节流动，而不是成核网状结构。第一种模型和第二种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对肌动蛋白细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细丝，以确定Spir主要是使新的细丝成核，还是切断现有的细丝或改变细丝的动力学。第一个和第三个模型将使用从体外实验中获得的知识来区分，以设计Spir和Capu的合理突变。这些突变将被引入果蝇体内，以确定这两种蛋白质的哪些活性对它们在体内的活性是必不可少的。还将进行其他实验，以确定这些蛋白质的活性是如何控制的。区分这些模型将导致对两种蛋白质的机械性理解，这两种蛋白质在后生动物物种中都是保守的。它将促进我们对细胞骨架及其控制方式的了解。鉴于这对蛋白质在哺乳动物的极性细胞(包括神经元和上皮细胞)中的共存，有人认为它们作为极性因子在果蝇中的作用是保守的。因此，我们预计，在果蝇卵子发生中所了解的Spir和Capu将适用于我们对许多动物的细胞骨架、细胞极性、生育、发育和健康的理解，从果蝇到人类。)) 与公共健康相关：细胞骨架对生活的许多方面都是必不可少的，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体轴方面的作用，主体轴的失败会导致出生缺陷和不育。通过研究细胞骨架在发育早期的正常功能和调节，我们将获得能够诊断和治疗广泛疾病的理解和工具。

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.