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

描述(申请人提供)：许多生理过程要求细胞根据当地的环境因素和条件自适应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(CRP)来获得这种控制，这些蛋白共同作用于产生、维持和重塑不同形式的肌动蛋白和微管细胞骨架结构。在多种类型的肿瘤中，多种CRP的过度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。在聚焦于一类被认为是主要细胞骨架调节因子(IQGAP，WAVE)的CRP的同时，该项目将开发一种新的多尺度方法来剖析CRP网络的复合状态，并建立它们与形态动力学细胞行为之间的功能关系。我们的方法集成了合成生物学、DNA纳米技术、超分辨率显微镜和系统生物学领域的工具，以：(I)调节细胞中单个和多个CRP的状态；(Ii)表征它们的纳米尺度定位模式；以及(Iii)确定CRP网络状态和复合形态细胞表型如何对扰动做出机械响应。将使用新的基因表达技术引入基于表达的扰动，这些技术在对细胞群体中的哺乳动物蛋白质表达提供精确和统一的控制的同时，对细胞生理造成的干扰最小。这样的控制将打开新的机会，在高通量成像分析中筛选对特定CRP扰动的表型反应，同时我们调整单个和多个CRP的表达水平和空间分布(目标1)。一种新的单分子条形码超分辨率成像程序将使对CRP分布的空间描述、网络水平的分析成为可能，该程序提供了表征局部模式的机会 几十个CRP(可能更多)同时在同一细胞内，同时也允许肌动蛋白和微管网络的超结构特征被解析(目标2)。这些新技术将通过计算图像分析和细胞反应的状态机建模联系起来，以确定不同的细胞表型并预测其对C反应蛋白扰动的反应(目标3)。三位具有互补专业知识的研究人员的协同合作将提高对CRP网络功能的理解，从而促进新疾病治疗的发展。

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.