A combined in vitro and in vivo study of the assembly mechanism of actin bundles in Drosophila bristles.

平行肌动蛋白束由紧密排列的细丝组成，这些细丝具有相同的极性，并由肌动蛋白束蛋白交联，构成真核细胞骨架结构的关键组成部分。它们的作用是加强细胞抵抗机械变形，并允许局部力产生。在果蝇的刚毛细胞中发现了一个突出的肌动蛋白束组织。刚毛肌动蛋白束是膜状的，呈三角形，并沿其长度排列成模块。它们均匀分布在猪鬃的外周长，从猪鬃的底部延伸到其尖端，模块之间有明确的空间。鬃毛肌动蛋白束的形成已经揭示了生长过程中一个迷人的机制。然而，决定和控制猪鬃肌动蛋白束长度和形状的分子机制尚不清楚。本课题拟开展跨学科研究，结合体内和体外实验，探讨果蝇刚毛形成的机制。我们将确定猪鬃肌动蛋白束机制的必要基因，并使用高分辨率光学显微镜使用转基因果蝇，揭示生长速率限制步骤。我们的假设是肌动蛋白成核、交联和解聚蛋白之间复杂的相互作用，决定了观察到的肌动蛋白束长度。为了测试这一机制，将开发肌动蛋白束形成的体外系统。我们将测试这些束的三角形形状是如何依赖于细胞质中自发成核细丝的纵向结合的。体内和体外实验都将用于确定猪鬃横向生长的机制和几何形状。据信，在各种系统中，如微绒毛或立体纤毛，共同的机制负责控制束的形状，这项研究的结果将影响我们的理解，远远超出了刚毛束的形成。

Parallel actin bundles are composed of tightly packed filaments that all have the same polarity and are crosslinked by an actin-bundling protein and constitute a key component of eukaryotic cytoskeleton structures. Their function is to reinforce the cell against mechanical deformation and to allow localized force generation. One prominent actin bundle organization is found in Drosophila bristle cells. The bristle actin bundles are membrane-bound, triangular-shaped, and are organized into modules along their length. They are evenly spaced around the outer perimeter of the bristle, extending from the base of the bristle to its tip with well-defined spaces between modules. The formation of bristle actin bundles has already revealed a fascinating machinery of the growth process. However, the molecular mechanism determining and controlling the length and the shape of bristle actin bundles is still unknown. In this proposal we plan an inter-disciplinary study to identify the mechanism of Drosophila bristle formation by combining in vivo and in vitro experiments. We will identify the essential genes that are part of the bristle actin bundle machinery and use high-resolution light microscopy using transgenic flies, to reveal the growth rate limiting steps. Our hypothesis is that an intricate interplay between actin nucleation, crosslinking and depolymerization proteins, set the observed actin bundles length. To test this mechanism, an in vitro system of actin bundle formation will be developed. We will test how the triangular shape of the bundles depends on the longitudinal association of spontaneously nucleated filaments in the cytosol. Both in vivo as in in vitro experiments will be used to pin down the mechanism of the lateral growth and geometry of the bristles. As it is believed that common mechanisms are responsible for control of bundle shapes in a variety of systems, such as microvilli or stereocilia, the results of this study will affect our understanding well beyond the formation of bristles bundles.