Excitable Networks in Directed Cell Migration

 描述(由申请人提供)：定向细胞迁移在发育和生理学中至关重要，也是许多疾病的潜在脆弱性，包括转移性癌症。整个过程由方向性感知、运动性和极性组成。虽然教科书上的观点暗示自主的细胞骨架活动是运动的基础，信号转导事件提供指导，但最近的研究表明，信号转导网络的可兴奋行为是驱动运动的必要的“起搏器”。外部梯度和内部极性信号对这种生化兴奋性的局部调制可以指导细胞，而大的全局扰动可以产生深刻的形态后果。我们正在解决由定向细胞迁移的这种“有偏见的可兴奋网络”观点提出的重要问题。是什么分子机制使信号转导网络变得兴奋？我们正在追求一个工作假说，即正反馈和延迟负反馈是兴奋性的基础，涉及对RasGTP酶、PI 3-激酶和膜局部区域PLC活性的控制。我们正在使用合成致动器、诱导隔离和生物传感器来扰动和监测酶的活性和膜状态，以找到反馈回路。初步实验表明，PIP2的逐渐减少或RAS信号的增加逐渐导致细胞从变形虫、扇形、振荡到持续展开的形式，如果细胞形态由可兴奋的网络控制的话。细胞如何感知、整合和适应时间和空间线索？为了解释细胞如何对差异做出反应并适应统一的趋化刺激，我们提出了局部激发-全局抑制方案使可兴奋网络产生偏差。以确定抑制物 为了平衡G蛋白的兴奋，我们专注于与生理相关的蛋白修饰、基因筛选和体外重建。趋化网络的整体复杂性是多少？在正向基因筛查中，我们已经确定了一大系列定向迁移的新型调控因子。结合已建立的生化和遗传分析，我们正在确定这些新基因与现有网络的联系，以及它们在定向感知、运动性和极性中的作用。这些基因中的许多都有人类同源物，我们将在中性粒细胞和乳腺细胞中进行靶向，以研究对趋化作用的影响。如何利用这些新概念来控制迁移和针对特定细胞？我们最近发现，多条平行迁移途径的持续激活会导致细胞过度扩散、碎裂和死亡。由于这些干扰中的许多也与肿瘤发生有关，我们正在追求一个公认的非传统概念，即最具侵袭性的癌细胞可以通过进一步的激活来靶向。作为原则的证明，我们正在测试遗传和化学扰动，这些扰动选择性地针对具有明确致癌突变的培养细胞，在包裹体和有机物中。

 DESCRIPTION (provided by applicant): Directed cell migration is vital in development and physiology and a potential point of vulnerability in numerous diseases, including metastatic cancer. The overall process is comprised of directional sensing, motility, and polarity. Whereas the textbook view implies that autonomous cytoskeletal activity underlies motility and that signal transduction events provide guidance, recent studies suggest that excitable behavior of the signal transduction network is an essential "pacemaker" that drives movement. Local modulation of this biochemical excitability by external gradients and internal polarity cues can guide cells and large global perturbations can have profound morphological consequences. We are addressing the important questions raised by this "biased excitable network" view of directed cell migration. What molecular mechanisms make the signal transduction network excitable? We are pursuing a working hypothesis that positive and delayed negative feedbacks, involving control of RasGTPase, PI 3- kinase, and PLC activities in local regions of the membrane, are the basis of excitability. We are using synthetic actuators, induced sequestration, and biosensors to perturb and monitor enzyme activities and membrane states to find the feedback loops. Initial experiments show that gradual decreases in PIP2 or increases in Ras signaling progressively cause cells to shift from amoeboid, to fan-like, to oscillatory, to persistently spread forms as would be expected if cell morphology is controlled by an excitable network. How can cells sense, integrate, and adapt to temporal and spatial cues? To explain how cells to respond to differences and adapt to uniform chemotactic stimuli, we proposed that a local excitation-global inhibition scheme biases the excitable network. To identify the inhibitor that balances G-protein excitation, we are focusing on physiological relevant protein modifications, genetic screens, and in vitro reconstitution. What is the overall complexity of the chemotactic networks? In forward genetic screens, we have identified a large series of novel regulators of directed migration. With a combination of established biochemical and genetic analyses we are defining the links of these new genes to the existing networks and their roles in directional sensing, motility, and polarity. Many of these genes have human homologues that we will target in neutrophils and mammary cells to investigate effects on chemotaxis. How can these new concepts be exploited to control migration and target specific cells? We recently discovered that persistent activation of multiple parallel migration pathways causes cells to spread excessively, fragment, and die. Since many of these perturbations are also involved in oncogenesis, we are pursuing the admittedly unconventional concept that the most aggressive cancer cells can be targeted by even further activation. As proof of principle, we are testing genetic and chemical perturbations, which selectively target cultured cells with defined oncogenic mutations, in xenographs and organoids.