Shaping Cells Using Modular Actin Filament Bundles

肌动蛋白是真核细胞中关键的细胞骨架元件之一。肌动蛋白分子（单体）可逆聚合形成肌动蛋白微丝，在肌动蛋白相关捆绑蛋白的帮助下，肌动蛋白微丝进一步组装成束。微丝在活细胞中是动态的，可以以一种严格由各种其他肌动蛋白相关蛋白调节的方式缩短和伸长。微丝在细胞的生命中起着许多关键的作用，它们要么是细胞形状或弹性的结构元件，要么是肌动蛋白马达（肌球蛋白）转运的轨迹。本研究旨在了解肌动蛋白束是如何形成和维持使用果蝇刚毛细胞为模型。单极（平行）肌动蛋白丝的交联束在真核细胞中很常见，并提供维持细胞形状的支架。熟悉的例子包括肠上皮细胞的刷状边缘（微绒毛）和内耳的立体纤毛。在这些系统和其他系统中，有一系列肌动蛋白结合蛋白控制肌动蛋白聚合的动力学，将肌动蛋白细丝组装成束，并调节束的维持。果蝇的刚毛细胞在16个小时内发育出数百微米长的巨大细胞延伸，这是细胞如何利用其细胞骨架改变和保持其形状的一个极好的例子。这种细胞延伸是由长肌动蛋白束的支架支撑的，该支架由肌动蛋白丝与交联剂捆绑在一起的短模块端到端组装而成。该系统为肌动蛋白动力学研究提供了重要的实验优势，允许将遗传和分子方法应用于基本的细胞生物学特征，可以通过光学显微镜和高分辨率电子显微镜在活细胞中进行分析。Guild和Tilney计划确定肌动蛋白束的模块化成分是如何形成、维持并最终从细胞质中移除的。他们计划使用细长的刚毛细胞作为体内试管，并使用遗传和转基因工具来干扰和修改束的组装。这些变化的细胞骨架后果将通过共聚焦显微镜检查活细胞背景下的肌动蛋白细胞骨架和电子显微镜检查细胞骨架的详细结构来评估。该项目有两个目的。首先，研究人员将确定肌动蛋白封盖蛋白是否在模块长度调节中发挥作用，如果是的话，他们将测试是否可以用不同的封盖蛋白来设计模块长度。其次，他们将确定模块是如何分解的，以及肌动蛋白封顶蛋白是否在这一过程中发挥作用。此外，他们将测试肌动蛋白模块的逆行流动是否在细胞延伸和束破裂中起作用，以及肌凝蛋白马达是否驱动这种流动。肌动蛋白束的模块在许多专门的细胞中使用，以支持细胞扩展，在所有情况下模块长度都是一个问题。因此，在果蝇系统中发现的原理应该普遍适用于许多其他系统。

Actin is one of the key cytoskeletal elements in eukaryotic cells. Actin protein molecules (monomers) reversibly polymerize to form actin microfilaments, which can further assemble into bundles with the assistance of actin-associated bundling proteins. Microfilaments are dynamic in the living cell, and can shorten and elongate in a manner that is strictly regulated by a variety of other actin-associated proteins. Microfilaments serve many critical roles in the life of the cell, either as structural elements for cell shape or resiliency or as tracks along which the actin-based motor, myosin, translocates. This research is aimed at understanding how actin bundles are formed and maintained using the Drosophila bristle cell as a model. Cross-linked bundles of unipolar (parallel) actin filaments are common in eukaryotic cells and provide the scaffolding that maintains cell shape. Familiar examples include the brush border (microvilli) of intestinal epithelial cells and the stereocilia of the inner ear. In these and other systems there is an array of actin-binding proteins that serve to control the dynamics of actin polymerization, assemble actin filaments into bundles, and regulate bundle maintenance.The Drosophila bristle cell develops a gigantic cellular extension hundreds of microns long over a period of 16 hours and is an excellent example of how a cell uses its cytoskeleton to change and maintain its shape. This cellular extension is supported by a scaffold of long actin bundles constructed by the end-to-end assembly of short modules composed of actin filaments bundled together with cross-linkers. This system offers important experimental advantages for the study of actin dynamics, by allowing the application of genetic and molecular approaches to a fundamental cell biological feature that can be analyzed in the living cell by light microscopy and at high resolution by electron microscopy.Drs. Guild and Tilney plan to determine how the modular components of actin bundles are formed, maintained, and eventually removed from the cytoplasm. They plan to use elongating bristle cells as in vivo test tubes and to employ genetic and transgenic tools to perturb and modify bundle assembly. The cytoskeletal consequences of these changes will be assessed by examining the actin cytoskeleton in the context of living cells by confocal microscopy and by examining the detailed structure of the cytoskeleton by electron microscopy. The project has two aims. First, the investigators will determine whether actin-capping proteins play a role in module length regulation and, if so, they will test whether they can engineer module length with different capping proteins. Second, they will determine how modules break down and whether actin capping proteins play a role in this process. In addition, they will test whether retrograde flow of actin modules plays a role in cell extension and in bundle breakdown and whether myosin motors drive this flow. Modules of actin bundles are used in many specialized cells to support cellular extensions and in all cases module length is an issue. Thus, the principles that are uncovered in the Drosophila system should be generally applicable to many other systems.