Spatially-delineated System-level Analyses and Control of Cytoskeletal Regulation

描述（由申请人提供）：许多生理过程需要细胞根据局部环境因素和条件适应性地调节其形态和运动特性。细胞通过使用大量的细胞骨架调节蛋白（CRPs）来获得这种控制，这些蛋白共同产生、维持和重塑不同形式的肌动蛋白和微管细胞骨架结构。几种crp在多种癌症中的过表达与预后不良有关。然而，这些扰动对细胞骨架调节和致癌细胞行为的影响在很大程度上是未知的。该项目将重点关注一类被认为是主要细胞骨架调节因子的CRP (IQGAPs, WAVEs)，并将开发一种新的多尺度方法来分析CRP网络的复合状态，并建立与细胞形态动力学行为相关的功能关系。我们的方法整合了合成生物学、DNA纳米技术、超分辨率显微镜和系统生物学领域的工具，以便：(i)调节细胞中单个和多个crp的状态；（ii）表征其纳米尺度的局部化模式；（iii）确定CRP网络状态和复合形态细胞表型如何对扰动作出机制反应。基于表达的扰动将使用新的基因表达技术引入，该技术在细胞群体中提供对哺乳动物蛋白质表达的精确和统一的控制，同时对细胞生理学引入最小的干扰。当我们调整单个和多个CRP的表达水平和空间分布时，这种控制将为在高通量成像分析中筛选特定CRP扰动的表型反应提供新的机会（目的1）。一种新的单分子“条形码”超分辨率成像程序将使CRP分布的空间描绘，网络级分析成为可能，该程序提供了表征定位模式的机会

DESCRIPTION (provided by applicant): Many physiological processes require cells to adaptively regulate their morphological and motile properties in response to local environmental factors and conditions. Cells gain such control by employing numerous cytoskeletal regulatory proteins (CRPs) that function collectively to generate, maintain and remodel different forms of actin and microtubule cytoskeletal structures. The overexpression of several CRPs in many forms of cancer been associated with poor prognosis. Yet, the impact of these perturbations on cytoskeletal regulation and oncogenic cell behaviors is largely unknown. While focusing on a class of CRPs believed to function as master cytoskeletal regulators (IQGAPs, WAVEs), this project will develop a new multi-scale approach to dissect composite states of CRP networks and establish functional relationships relating them to morphodynamic cell behaviors. Our approach integrates tools from the fields of synthetic biology, DNA nanotechnology, super-resolution microscopy, and systems biology in order to: (i) modulate the states of individual and multiple CRPs in cells; (ii) characterize their nanometer-scale localization patterns; and (iii) determine how CRP network states and composite morphological cell phenotypes respond mechanistically to perturbations. Expression-based perturbations will be introduced using novel gene expression technologies that provide precise and uniform control over mammalian protein expression in a cell population while introducing minimal disruptions to cell physiology. Such control will open new opportunities to screen phenotypic responses to specific CRP perturbations in high-throughput imaging assays while we adjust the expression levels and spatial distributions of single and multiple CRPs (Aim 1). Spatially-delineated, network- level analyses of CRP distributions will be enabled by a new, single-molecule 'barcoding' super resolution imaging procedure that offers opportunities to characterize the localization patterns of several dozens of CRPs (and potentially many more) simultaneously within the same cell, while also allowing ultra-structural features of actin and microtubule networks to be resolved (Aim 2). These new technologies will be linked through computational image analyses and state machine modeling of cell responses in order to identify distinct cell phenotypes and predict their responses to CRP perturbations (Aim 3). The synergistic collaborative effort of three investigators with complementary expertise will improve the understanding of CRP network function, thereby promoting the development of new disease treatments.