Mechanisms of filopodia initiation and actin transport

描述(由申请人提供)： 肌动蛋白细丝和微管是一种动态的细胞骨架聚合物，在细胞生理学和决定细胞形态方面具有重要作用。神经元复杂的细胞形态直接关系到神经系统中神经元的功能。因此，神经元为研究细胞骨架的动力学提供了一个很好的模型系统，因为它与细胞功能有关。肌动蛋白细丝对于神经元之间连接模式的发展和维持，以及突触的形成和功能都是至关重要的。神经元不断地通过轴突运输将细胞体中合成的新的细胞骨架蛋白输送到远端轴突。轴突运输过程是维持神经系统功能所必需的，在各种疾病状态下都会受到损害。丝状足突是细长的指状细胞突起，严格依赖于肌动蛋白细丝，是轴突引导和突触形成所必需的。因此，了解丝状突起的机制与了解再生和维持神经系统功能有直接关系。这项建议提供的研究旨在(1)确定肌动蛋白在轴突中的运输形式(例如，细丝与单体)，以及(2)确定基于肌动蛋白的丝足起始机制。细胞骨架蛋白的运输方式存在争议。通过使用转EGFP-肌动蛋白的神经元的体外研究，我们提供了提示肌动蛋白细丝运输的初步描述性证据和有利于单体肌动蛋白运输的实验证据。为了从实验上验证肌动蛋白细丝是肌动蛋白运输的形式这一假设，我们进行了一系列的研究。此外，基于轴突和生长锥体中EGFP-肌动蛋白的实时成像，我们已经确定了自发形成的点状肌动蛋白的存在，这些点状肌动蛋白是丝状突形成的前体。这些观察结果为研究活神经元中丝状突起形成的动态细胞骨架基础提供了一个模型系统。我们提出的研究旨在确定细胞外信号、信号通路和微管在调节丝状茎起始的最早事件中的作用。

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.