ACTIN CABLE FORMATION COORDINATED BY APC, FORMINS AND MICROTUBULES IN ANIMAL DEVELOPMENT

项目摘要 该建议的目标是定义控制细胞的动态组装和功能的机制， 肌动蛋白结构在一个活的，发展中的动物。单分子离体TIRF显微术的研究进展 为我们理解控制肌动蛋白的分子机制做出了重大贡献， 微管重排然而，该领域在整合这些体外 通过在动物模型中进行测试，将其机制纳入生理环境。为了填补这一空白，我们将使用 结合“自上而下”和“自下而上”的战略，将遗传和活体成像的力量结合在一起， 果蝇体外定量重组和单分子分析。具体来说，我们专注于一个 一组相互作用的肌动蛋白装配因子，包括腺瘤性结肠息肉病蛋白1和2 (APC1和APC 2）和透明（Dia），以及他们如何共同努力，建立一个惊人的阵列， 在卵子发生的特定阶段，果蝇滋养细胞中的长肌动蛋白电缆。我们的核心假设是， APC 1、APC 2和Dia各自对肌动蛋白索的形成做出了机制上独特和重要的贡献， 并且，电缆组装的适当的空间和时间模式来自于这些蛋白质的工作， 通过与微管和微管相关蛋白的相互作用在严格的调节下协调一致（例如，EB1 和CLIP-170），并受GSK 3 β激酶调节。通过直接在单一的可视化这些机制， 分子水平，并利用这些信息来指导在活体果蝇中进行严格的多方面测试 卵巢，我们将达到一个前所未有的水平的机械理解肌动蛋白组装， 微管-肌动蛋白的交互作用。具体目的是：（1）确定肌动蛋白索阵列 在卵子发生过程中，通过严格控制的多组分机制组装， 肌动蛋白丝成核和伸长因子;（2）定义微管触发的机制 肌动蛋白电缆的出现和相互作用，与不断增长的电缆，以指导他们的形态和他们的接触， 原子核

Project Summary The goal of this proposal is to define the mechanisms controlling dynamic assembly and function of cellular actin structures in a living, developing animal. Recent advances in single molecule in vitro TIRF microscopy have made major contributions to our understanding of the molecular mechanisms controlling actin and microtubule rearrangements. However, the field is lagging behind in the integration of these in vitro mechanisms into a physiological context by testing them in animal models. To fill this gap, we will use a combined `top-down' and `bottom-up' strategy, bringing together the genetic and live imaging power of Drosophila with quantitative in vitro reconstitution and single molecule analysis. Specifically, we focus on a group of interacting actin assembly factors, which includes Adenomatous polyposis coli proteins 1 and 2 (APC1 and APC2) and the formin Diaphanous (Dia), and how they work together to build a striking array of long actin cables in Drosophila nurse cells at a specific stage of oogenesis. Our central hypothesis is that APC1, APC2, and Dia each make a mechanistically distinct and essential contribution to actin cable formation, and that the proper spatial and temporal pattern of cable assembly arises from these proteins working in concert under tight regulation by interactions with microtubules and microtubule-associated proteins (e.g., EB1 and CLIP-170), and regulated by GSK3ß kinase. By directly visualizing these mechanisms at the single molecule level in vitro, and using this information to guide rigorous multi-faceted tests in living Drosophila ovaries, we will arrive at an unprecedented level of mechanistic understanding of actin assembly and microtubule-actin cross-talk in the living animal. The specific aims are: (1) Determine how actin cable arrays are assembled during oogenesis by tightly controlled multi-component mechanisms involving collaborative actin filament nucleation and elongation factors; and (2) Define the mechanisms by which microtubules trigger actin cable emergence and interact with growing cables to guide their morphogenesis and their contact with the nucleus.