Investigating FGF Signaling Dynamics in migrating cells

项目摘要 信号动力学在空间和时间上受到严格调控，以控制细胞迁移、增殖和分化。 分化全面了解细胞外输入如何影响下游信号传导， 细胞行为首先需要能够可视化关键的内源性信号蛋白，细胞结构， 体内的细胞骨架调节剂。其次，利用精确的工具来操纵蛋白质是必不可少的 定位和信号活性，以揭示机制。为了探索分泌的信号蛋白如何分散 并调节细胞行为，我们使用的是一种C线虫肌肉祖细胞（SMs）作为一个易处理的模型 用于体内细胞生物学。在幼虫发育过程中，SM从蠕虫的尾部附近迁移到中心 并且是研究细胞迁移机制的经典系统。SM迁移需要成纤维细胞生长 因子（FGF）信号传导，其被假设为充当长程化学引诱物。但 将FGF信号传导转化为定向迁移的关键分子和细胞生物学机制并不 知道的FGF通常被认为是可扩散的信号蛋白， 细胞外分散，但这种现象从未被证明为内源性FGF。到 为了研究FGF蛋白如何在细胞间移动以调节SM迁移，我标记了内源性的 FGF配体和受体参与SM迁移与荧光蛋白，并成像其定位在 vivo.出乎意料的是，在SM迁移过程中，我没有观察到FGF蛋白的梯度，而是观察到低浓度的FGF蛋白。 位于迁移性SM附近的腹侧中线细胞系中FGF表达水平。拟议 我的工作建立在这些发现和其他新的工具，我已经可视化和操纵关键FGF 研究FGF信号通路蛋白，以了解FGF信号通路如何调节细胞迁移。目标1将描述 内源性FGF在活体动物中的动力学，并使用基因组技术测试FGF如何在体内细胞间移动。 工程和实时成像方法。目的2将研究迁移细胞如何响应细胞外 通过测试FGF是许可或指导性信号的程度，表征空间组织， FGF下游的细胞内信号传导，并使用 光遗传学这些实验将为分泌信号蛋白如何移动提供新的见解 以及迁移细胞如何解释和响应动态细胞外信号。

Project Summary Signaling dynamics are tightly regulated in space and time to control cell migration, proliferation, and differentiation. A comprehensive understanding of how extracellular inputs influence downstream signaling and cell behaviors will first require the ability to visualize key endogenous signaling proteins, cell architectures, and cytoskeletal regulators in vivo. Second, it will be essential to utilize precise tools to manipulate protein localization and signaling activity to reveal mechanisms. To explore how secreted signaling proteins disperse and modulate cell behavior, we are using a type of C. elegans muscle progenitors (SMs) as a tractable model for in vivo cell biology. The SMs migrate from near the tail of the worm to the center during larval development and are a classical system to investigate cell migration mechanisms. SM migration requires Fibroblast Growth Factor (FGF) signaling, which has been hypothesized to act as a long-range chemoattractant. However, the key molecular and cell biological mechanisms that translate FGF signaling into directed migration are not known. FGFs are often thought of as diffusible signaling proteins that can form gradients through free, extracellular dispersal, but this phenomenon has never been demonstrated for an endogenous FGF. To investigate how FGF proteins move between cells to regulate SM migration, I have tagged the endogenous FGF ligand and receptor involved in SM migration with fluorescent proteins and imaged their localization in vivo. Unexpectedly, I did not observe a gradient of FGF protein during SM migration, but instead observed low levels of FGF expression in a line of ventral midline cells that are located near migrating SMs. The proposed work builds on these findings and other new tools that I have made to visualize and manipulate key FGF pathway proteins in order to understand how FGF signaling regulates cell migration. Aim 1 will characterize endogenous FGF dynamics in a living animal and test how FGF moves between cells in vivo using genome engineering and live imaging approaches. Aim 2 will investigate how migrating cells respond to extracellular FGF by testing the extent to which FGF is a permissive or instructive signal, characterizing spatial organization of intracellular signaling downstream of FGF, and testing functions for localized pathway activation using optogenetics. These experiments will provide novel insights into how secreted signaling proteins move between cells and how migrating cells interpret and respond to dynamic extracellular cues.