A quantative understanding of the regulation of actin dynamics in live T cells

描述(由申请人提供)：肌动蛋白动力学关键地调节细胞功能的许多方面。调节肌动蛋白周转的蛋白质已经在很大的生化细节中得到了表征。然而，这些生化特性如何在活的原代细胞中发挥作用，以控制肌动蛋白的动态，人们却知之甚少。为了这样的理解，我们需要全球细胞内肌动蛋白调节剂的浓度，因为蛋白质的相互作用是由质量作用控制的。我们需要局部浓度的肌动蛋白调节剂，因为活细胞内蛋白质的浓度在时间和空间上变化很大，而且肌动蛋白的动态是局部调控的，而不是全球调控的。我们需要肌动蛋白调节剂的扩散常数，因为它们将影响局部浓度随时间的变化。这三个数据集，我们允许生成预测性数学模型，描述如何在活细胞中调节肌动蛋白动态。鉴于肌动蛋白调控在不同细胞类型中的普遍性质，这样的模型将引起普遍的兴趣。在这里，我们建议发展方法，并获得在原代T细胞中产生这样一个模型所需的数据。明确的时空限制，关于上游信号的广泛数据，以及高度的生理学相关性，使T细胞成为阐明活的原代细胞中肌动蛋白动态调节的实验系统。肌动蛋白的周转是由新的肌动蛋白细丝的成核、它们通过封顶而稳定、它们通过切断而被破坏来驱动的。由于T细胞主要依赖于一个肌动蛋白核因子，即Arp2/3复合体，我们将研究Arp2/3复合体、其三个激活物Wasp、WAVE2和造血皮质蛋白同源物HS1、Capping蛋白、肌动蛋白细丝切断蛋白cofilin和肌动蛋白本身。我们将在两个目标上建立肌动蛋白动力学模型的定量基础：目标1：确定肌动蛋白调节蛋白在初始T细胞激活中的全球浓度。我们将使用T细胞提取物的定量免疫印迹来确定初级T细胞中肌动蛋白调节蛋白的全球浓度。我们还将确定当GFP标记的调节剂版本与内源蛋白共表达时，这些浓度将如何变化。目的2：确定肌动蛋白调节剂在T细胞初始激活过程中的局部浓度。我们将使用GFP标记的肌动蛋白调节剂版本、旋转圆盘共聚焦显微镜和定量三维图像分析来确定肌动蛋白调节器浓度的时空变化。这些数据将通过内源性蛋白的染色得到证实。我们还将在FRAP实验中使用肌动蛋白调节器的GFP标记版本来确定肌动蛋白调节器在活的原代T细胞中的扩散常数。与公共健康相关：T细胞是免疫系统的中央调节细胞，是我们身体中对抗感染和癌症的部分，在自身免疫性疾病中受到损害。因此，T细胞激活的调节是至关重要的。然而，这很难理解，因为这是一个高度复杂的过程，涉及数十种不同的分子，它们的活性在时间和空间上不同。这种复杂的T细胞组织是由肌动蛋白动力学驱动的。在这里，我们结合生化信息和包含在肌动蛋白调节蛋白时空模式中的信息来阐明活的原代T细胞在激活时肌动蛋白动力学是如何调节的。

DESCRIPTION (provided by applicant): Actin dynamics critically regulate numerous aspects of cellular function. The proteins mediating actin turnover have been characterized in great biochemical detail. However, how these biochemical properties play out inside a live primary cell to control actin dynamics is poorly understood. For such an understanding we need global cellular concentrations of actin regulators, as protein interactions are controlled by mass action. We need local concentrations of actin regulators, as concentrations of proteins inside a live cell vary substantially in time and space and as actin dynamics are regulated locally not globally. We need diffusion constants of actin regulators, as they will influence how local concentrations change over time. These three datasets we allow the generation of predictive mathematical models that describe how actin dynamics are regulated in live cells. Given the pervasive nature of actin regulation throughout different cell types, such models will be of universal interest. Here we propose to develop the methods and acquire the data necessary for generating such a model in primary T cells. Well-defined spatiotemporal constraints, extensive data on upstream signaling, and high physiological relevance make T cells interesting as an experimental system to elucidate the regulation of actin dynamics in live primary cells. Actin turnover is driven by the nucleation of new actin filaments, their stabilization through capping, and their destruction through severing. As T cells predominantly rely on one actin nucleator, the Arp2/3 complex, we will investigate the Arp2/3 complex, its three activators WASP, WAVE2, and the hematopoietic cortactin homolog HS1, capping protein, the actin filament severing protein cofilin, and actin itself. We will build the quantitative foundation for a model of actin dynamics primary T cell activation in two aims: Aim 1: To determine global concentrations of actin regulators in primary T cell activation. We will use quantitative immunoblotting of T cell extracts to determine the global concentrations of actin regulators in primary T cells. We will also determine how such concentrations change when GFP- tagged versions of the regulators are co-expressed with the endogenous proteins. Aim 2: To determine local concentrations of actin regulators in primary T cell activation. We will use GFP-tagged versions of the actin regulators, spinning disk confocal microscopy, and quantitative three-dimensional image analysis to determine the spatiotemporal variations in the concentrations of the actin regulators. These data will be corroborated by staining of endogenous proteins. We will also use GFP-tagged versions of the actin regulators in FRAP experiments to determine the diffusion constants of the actin regulators inside live primary T cells. PUBLIC HEALTH RELEVANCE: T cells are the central regulatory cells of the immune system, the part of our body that fights infections and cancer and that is compromised in autoimmune disease. The regulation of T cell activation is thus critical. Yet it is difficult to understand, as it is a highly complex process involving dozens of different molecules whose activity varies in time and space. This complex T cell organization is driven by actin dynamics. Here we combine biochemical information and information contained in spatiotemporal patterns of actin regulators to elucidate how actin dynamics are regulated inside a live primary T cells upon activation.