Multiplex Imaging of Signaling Pathways in Cell Motility

摘要： 细胞生物学中的荧光生物传感器技术现在允许对分子过程进行实时询问 因为它们分别以微米和秒的空间和时间分辨率出现在活细胞内。我 有一个优先和持续的重点放在新的福斯特共振能量转移（FRET）的发展- 基于生物传感器技术，利用单体荧光蛋白获得卓越的灵敏度和探针 可逆性此外，单链结构用于方便定量分析。我最近开创了 近红外（NIR）-FRET生物传感器模式，其中包括第一个同时，正交可视化 蓝黄色荧光蛋白基FRET和NIR-FRET生物传感器在单个活细胞。所得 这些数据是对活细胞中两个重要分子开关Rac 1和RhoA的首次真正的多重分析。 GTP酶。该分析揭示了这些GTP酶在细胞迁移过程中的实时直接协调。 在此，我建议研究与重要信号通路相关的Rho GTP酶的协调作用， 通过为特定信号节点设计新的生物传感器并利用直接多重FRET成像， approach.具体来说，我将首先针对成纤维细胞中Rho GTdR信号传导的局部水平协调 在迁移、趋化性和定向引导过程中。RhoA与Rac 1 GTP酶在乳腺癌中的协同作用 将通过确定下游靶蛋白的作用来研究成纤维细胞， 假设在细胞运动过程中协调RhoA和Rac 1。直接多重成像方法将是 用于评估成对的生物传感器信号。此外，RhoA和Rac 1途径将受到干扰， 光遗传学工具来确定对控制细胞形态动力学重要的GTdR协调。 接下来，这些方法将应用于对人类健康有重要影响的两个系统， 疾病第一，巨噬细胞运动和定向引导，这需要Rho GTP酶的协调 在对炎性趋化因子的趋化反应期间，将进行研究。然后，多重成像和 微扰方法将应用于乳腺癌的侵袭和迁移，这对控制乳腺癌的侵袭和迁移至关重要。 肿瘤转移总的来说，Rho GTPases与相关分子信号传导之间的协调 控制细胞运动的基因将通过开发新的生物传感器来识别， 信令网络的探测。

Abstract: Fluorescent biosensor techniques in cell biology now allow for the real-time interrogation of molecular processes as they occur inside living cells at spatial and temporal resolutions of microns and seconds, respectively. I have a prior and continued focus on the development of novel Förster resonance energy transfer (FRET)- based biosensor technologies that utilize monomeric fluorescent proteins for exceptional sensitivity and probe reversibility. In addition, a single-chain construction is used to facilitate quantitative analysis. I recently pioneered the near-infrared (NIR)-FRET biosensor modality, which included the first simultaneous, orthogonal visualization of cyan-yellow fluorescent protein-based FRET and NIR-FRET biosensors in single living cells. The resulting data were the first true multiplex analysis of two important molecular switches in living cells, the Rac1 and RhoA GTPases. This analysis revealed the direct coordination of these GTPases during cell migration in real-time. Herein, I propose to study the coordination of Rho GTPases associated with important signaling pathways by designing new biosensors for specific signaling nodes and utilizing the direct multiplex FRET imaging approach. Specifically, I will first target the local-level coordination of Rho GTPase signaling in fibroblast cells during migration, chemotaxis, and directional guidance. The coordination of RhoA versus Rac1 GTPases in fibroblasts will be investigated by determining the role of a downstream target protein, the formin mDia1, which is hypothesized to coordinate RhoA and Rac1 during cell motility. The direct multiplex imaging approach will be used to evaluate pairwise biosensor signals. In addition, the RhoA and Rac1 pathways will be perturbed with optogenetic tools to determine the GTPase coordination that is important for controlling cellular morphodynamics. Next, these approaches will be applied to two systems that have important implications for human health and disease. First, macrophage motility and directional guidance, which requires the coordination of Rho GTPases during the chemotactic response to inflammatory chemokines, will be studied. Then, the multiplex imaging and perturbation approaches will be applied to breast cancer invasion and migration, which are critical to controlling tumor metastasis. Collectively, the coordination between Rho GTPases and the associated molecular signaling that governs cell motility will be identified through the development of new biosensors that enable direct multiplex probing of signaling networks.