Mechanisms of filopodia initiation and actin transport

描述（由申请人提供）： 肌动蛋白丝和微管是动态的细胞骨架聚合物，在细胞生理学中具有重要作用，并决定细胞形态。神经元复杂的细胞形态与神经系统中神经元的功能直接相关。因此，神经元提供了一个很好的模型系统，研究动态的细胞骨架，因为它涉及到细胞功能。肌动蛋白丝对于神经元之间的连接模式的发展和维持以及突触的形成和功能具有根本的重要性。神经元不断地通过轴突运输将在细胞体中合成的新的细胞骨架蛋白递送到远端轴突。轴突运输的过程是维持功能性神经系统所必需的，并且在各种疾病状态中受损。丝状伪足是细长的指状细胞突起，严格依赖于肌动蛋白丝，是轴突引导和突触形成所必需的。因此，了解丝足起始的机制与了解神经系统的再生和维持功能直接相关。该提案提出了旨在（1）确定形式（例如，纤维与单体）的肌动蛋白运输的轴突，和（2）确定肌动蛋白为基础的机制丝状伪足启动。细胞骨架蛋白的转运形式是有争议的。从体外研究中使用的神经元转染EGFP-肌动蛋白，我们提供了初步的描述性证据表明运输的肌动蛋白丝和实验证据有利于运输的单体肌动蛋白。一系列的研究是详细的实验测试的假设，肌动蛋白丝是肌动蛋白运输的形式。此外，基于在轴突和生长锥中的EGFP-肌动蛋白的实时成像，我们已经确定了作为丝状伪足形成的前体的自发形成的肌动蛋白斑点的存在。这些观察结果提供了一个模型系统，研究动态的细胞骨架基础的丝状伪足形成在活的神经元。我们目前的研究，旨在确定的作用，细胞外信号，信号通路，微管在调节最早的事件在丝状伪足启动。

DESCRIPTION (provided by applicant): Actin filaments and microtubules are dynamic cytoskeletal polymers that have important roles in cell physiology and determine cell morphology. The complex cellular morphology of neurons is directly related to the function of neurons in the nervous system. Thus, neurons provide an excellent model system for investigating the dynamics of the cytoskeleton as it relates to cellular functions. Actin filaments are of fundamental importance to the development and maintenance of connectivity patterns between neurons, and the formation and function of synapses. Neurons constantly deliver new cytoskeletal proteins synthesized in the cell body to the distal axon through axonal transport. The process of axonal transport is required for maintenance of a functional nervous system and is impaired in a variety of disease states. Filopodia are slender finger-like cellular projections that are strictly dependent on actin filaments and are required for axon guidance and synapse formation. Understanding the mechanisms responsible for filopodial initiation is thus of direct relevance to the understanding of regeneration and the maintenance of nervous system function. This proposal presents studies aimed at (1) determining the form (e.g., filaments versus monomers) of actin transport in axons, and (2) determining the actin-based mechanism of filopodia initiation. The form in which cytoskeletal proteins are transported is controversial. From in vitro studies using neurons transfected with EGFP-actin, we provide preliminary descriptive evidence suggestive of the transport of actin filaments and experimental evidence in favor of the transport of monomeric actin. A series of studies is detailed to experimentally test the hypothesis that actin filaments are the form of actin transport. Additionally, based on live imaging of EGFP-actin in axons and growth cones we have identified the presence of spontaneously formed actin puncta that serve as precursors to filopodial formation. These observations provide a model system for investigating the dynamic cytoskeletal basis of filopodial formation in living neurons. We present studies aimed at determining the role of extra-cellular signals, signaling pathways, and microtubules in regulating the earliest events in filopodial initiation.