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实时询问分子过程 当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时。我 优先并继续关注新型Förster共振能量转移(FRET)的开发- 基于生物传感器技术，利用单体荧光蛋白实现非凡的灵敏度和探针 可逆性。此外，为了便于定量分析，还使用了单链结构。我最近开创了 近红外(NIR)-FRET生物传感器模式，包括第一个同时、正交可视化 基于蓝黄色荧光蛋白的FRET和近红外FRET生物传感器在单个活细胞中的应用。由此产生的 这些数据是对活细胞中两个重要的分子开关--rac1和RhoA--的首次真正的多重分析 GTP酶。这一分析揭示了这些GTP酶在细胞迁移过程中的直接协调。 在此，我建议研究与重要信号通路相关的Rho GTP酶的协调 通过为特定的信令节点设计新的生物传感器并利用直接多路FRET成像 接近。具体地说，我将首先针对成纤维细胞中Rho GTPase信号的局部水平协调 在迁徙、趋化和定向引导过程中。RhoA和rac1 GTP酶的协同作用 将通过确定下游靶蛋白Forin mDia1的作用来研究成纤维细胞。 假设是为了在细胞运动过程中协调RhoA和rac1。直接多路成像方法将是 用于评估成对生物传感器信号。此外，RhoA和rac1通路将受到干扰 光遗传学工具来确定对控制细胞形态动力学很重要的GTP酶的配位。 接下来，这些方法将应用于对人类健康具有重要影响的两个系统和 疾病。第一，巨噬细胞的运动和定向引导，这需要Rho GTP酶的协调 在趋化期间对炎性趋化因子的反应，将被研究。然后，多路成像和 微扰方法将应用于乳腺癌的侵袭和转移，这是控制乳腺癌的关键 肿瘤转移。总体而言，Rho GTP酶与相关分子信号之间的协调 将通过开发能够实现直接多路传输的新型生物传感器来确定控制细胞运动的因素 信令网络探测。

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.