MOLECULAR DESIGN OF FILOPODIA, CELL'S SENSORY ORGANELLES

描述（由申请人提供）：本提案的总体目标是了解细胞如何以有意义的方式相互沟通以及与环境沟通。为了找到粘附的目标表面或迁移的最终目的地，细胞应该能够探索环境。丝状足是细长的细胞突起，被认为是细胞的感觉和引导细胞器。它们可以识别环境线索，探测合适的表面，并确定细胞运动的方向。丝状足对于正常组织细胞的导航和癌细胞的转移尤其重要，但对于特殊结构的初始化也很重要，如突触、连接和通信途径。本项目将重点研究丝状足在形成、突出和分化为特殊结构过程中的动态行为的结构基础。具体目标是深入研究丝状伪体机制，主要关注丝状伪体细胞骨架与质膜的相互作用；在广度上，通过分析不同细胞类型在不同条件和不同目的下形成的丝状伪体之间的结构和动态变化。我们将结合结构、动力学、功能和分子遗传学的方法来研究丝状结构机制的核心参与者IRSp53和mDia2在细胞骨架和质膜之间的界面上的作用。拟研究的丝状足相关结构包括控制细胞运动的前缘丝状足，在脑内建立突触并分化为树突棘的树突丝状足，以及参与内皮通透性屏障形成的连接丝状足。本项目的基本假设表明，在丝状足突出过程中，肌动蛋白细胞骨架组装和质膜动力学这两种分子机制相互紧密合作，这种平衡可以在不同方向上进行调节，从而产生各种基于丝状足的结构，服务于不同的功能。我们研究策略的一个关键要素是通过铂复制电子显微镜获得高分辨率的结构信息，并将其与先进光学显微镜记录的相同细胞的动态行为相关联。当与现代功能方法相结合时，该策略具有独特的能力，可以通过在亚细胞和超分子水平上提供桥接信息，填补单个分子特性与细胞行为之间的现有差距。

DESCRIPTION (provided by applicant): The broad objective of this proposal is to understand how cells communicate with each other and with the environment in a meaningful way. To find a target surface for adhesion or a final destination for migration, a cell should be able to explore the environment. Filopodia are long slender cellular protrusions which are believed to be the cell's sensory and guiding organelles. They can recognize environmental cues, detect appropriate surfaces and determine the direction of cell locomotion. Filopodia are especially important for navigation of normal tissue cells and for metastasis of cancer cells, but also for initialization of specialized structures, such as synapses, junctions, and communication pathways. This project will focus on studying the structural basis of the dynamic behavior of filopodia during their formation, protrusion, and differentiation into specialized structures. The specific aims are designed to investigate the filopodial machinery in depth with a major focus on interaction of the filopodial cytoskeleton with the plasma membrane, and in breadth, by analyzing the structural and dynamic variations among filopodia formed by different cell types in different conditions and for different purposes. Roles of central players of filopodial machinery, IRSp53 and mDia2, functioning at the interface between the cytoskeleton and plasma membrane will be investigated by combination of structural, kinetic, functional and molecular genetics approaches. The range of filopodia-related structures to be investigated includes leading edge filopodia that control cell motility, dendritic filopodia that establish synapses in brain and differentiate into dendritic spines, and junctional filopodia that are involved in formation of permeability barrier in endothelium. The underlying hypothesis for this project suggests that two molecular machineries, actin cytoskeleton assembly and plasma membrane dynamics, tightly cooperate with each other during filopodia protrusion and this balance can be tuned in different directions to produce a variety of filopodia-based structures serving different functions. A key element of our research strategy is to obtain high resolution structural information by platinum replica electron microscopy and correlate it with the dynamic behavior of the same cell recorded by advanced light microscopy. When additionally combined with modern functional approaches, this strategy has a unique ability to fill the existing gap between properties of individual molecules and behavior of a cell by providing bridging information at subcellular and supramolecular levels. PUBLIC HEALTH RELEVANCE: The results will contribute to understanding of the molecular mechanisms of filopodia formation and the roles of filopodia in such fundamental processes in development and disease as cell migration, cell-cell communication, and tissue morphogenesis. This information will help to design drugs, treatments and diagnostic tools.