Dynamic probes of endogenous protein aggregation and cell signaling

项目 总结/摘要： 细胞信号的动力学是指导细胞和组织行为的指导性特征。然而，许多重要的 像Wnt/β-连环蛋白通路这样的信号通路，缺乏观察和分析内源性信号动力学的探针 在空间和时间上都是精确的。该提案的总体目标是开发新的分子探针， 可以可视化和干扰内源性信号动力学，重点是Wnt/β-连环蛋白途径。我们 提案分为两个项目。在第一个项目中，我们将开发一种新型的荧光生物传感器， Wnt信号动力学的直接可视化。现有的Wnt报告基因可能是暗淡的，并且需要基因组测序。 靶细胞的工程化，作用于缓慢的转录时间尺度，这可能会掩盖上游途径 动态，并且不提供关于输入Wnt信号的空间信息。因为Wnt刺激需要 途径共受体LRP 6的聚集，LRP 6寡聚化的观察可以提供 时空分辨读出通路活性。因此，我们设想了一类新的生物传感器， 观察细胞内蛋白质的聚集。我们的记者必须满足两个重要的设计标准。 首先，它必须报告内源性蛋白质聚集，以避免信号蛋白或基因组蛋白的过度表达。 修改.第二，因为生理蛋白质聚集体很小，常常低于衍射极限 在可见光下，我们的记者必须“视觉放大”内源性集群，使它们可见， 常规显微镜。我们描述了开发、表征和应用这样一种报告器的计划，称为 Clumps。CluMPS通过蛋白质相位原理，在视觉上放大了小的内源性蛋白质簇 分居使用实验和模拟，我们将描述CluMPS的检测能力， 扩增细胞内蛋白质聚集体，使用GFP的光遗传学聚类作为模型分析物。然后我们将 应用CluMPS检测已知形成生理聚集体的内源性蛋白质簇。最后我们 将应用CluMPS检测响应于细胞Wnt的内源性Wnt受体LRP 6的聚集 刺激，我们将在细胞、组织和发育模型中验证LRP 6-CluMPS的活性。的 CluMPS的模块化将允许其适应产生不同信号传导途径和细胞的传感器。 states.在第二个项目中，我们将设计第一个光遗传学工具，以允许抑制内源性 时空精确的信号通路。我们将靶向抑制Wnt/β-连环蛋白信号传导， 分别是Ras-Erk信号我们将在细胞培养的背景下验证成功的途径抑制 癌症模型和体外肠类器官的模式化。值得注意的是，我们所有的探测器都将设计成 模块化的方式，因此可以容易地进行修改，以观察或抑制不同的目标。成功 我们的工作将产生一套新的工具来观察，扰动和理解基本的生物化学 这些过程是正常生理学的基础，在疾病中也是崩溃的基础，与中枢神经系统密切相关。 NIGMS的使命。

Project Summary/Abstract: The dynamics of cell signals are instructive features that guide cell and tissue behavior. Yet, many important pathways, like the Wnt/-catenin pathway, lack probes to observe and dissect endogenous signal dynamics with precision in space and in time. The overall goal of this proposal is to develop novel molecular probes that can visualize and perturb endogenous signal dynamics, with a focus on the Wnt/-catenin pathway. Our proposal is divided into two projects. In the first project, we will develop a novel fluorescent biosensor to enable direct visualization of Wnt signal dynamics. Existing Wnt reporters can be dim and require genomic engineering of the target cell, act on slow transcriptional timescales that can obscure the upstream pathway dynamics, and provide no spatial information about the input Wnt signal. Because Wnt stimulation requires the aggregation of pathway co-receptor LRP6, observation of LRP6 oligomerization could provide a spatiotemporally resolved readout of pathway activity. We thus envision a new class of biosensor that allows observation of the aggregation of intracellular proteins. Our reporter must meet two important design criteria. First, it must report on endogenous protein clustering to avoid overexpression of signaling proteins or genomic modifications. Second, because physiological protein aggregates are small and often below the diffraction limit of visible light, our reporter must “visually amplify” endogenous clusters such that they are visible by conventional microscopy. We describe plans to develop, characterize, and apply such a reporter, called CluMPS. CluMPS visually magnifies small endogenous protein clusters through principles of protein phase separation. Using both experiments and simulations, we will characterize the ability of CluMPS to detect and amplify intracellular protein aggregates, using optogenetic clustering of GFP as a model analyte. We will then apply CluMPS to detect clusters of endogenous proteins known to form physiological aggregates. Finally, we will apply CluMPS to detect the clustering of endogenous Wnt receptor LRP6 in response to cellular Wnt stimulation, and we will validate LRP6-CluMPS activity in cell, tissue, and developmental models. The modularity of CluMPS will allow its adaptation to generate sensors of diverse signaling pathways and cell states. In the second project, we will engineer the first optogenetic tools to allow inhibition of endogenous signaling pathways with spatiotemporal precision. We will target inhibition of both Wnt/-catenin signaling and, separately, Ras-Erk signaling. We will validate successful pathway inhibition in the context of cell culture models of cancer and patterning of in vitro intestinal organoids. Notably, all of our probes will be designed in a modular fashion and thus could be readily modified to observe or inhibit diverse targets of interest. Success in our work will result in a suite of new tools to observe, perturb, and understand the fundamental biochemical processes that underlie normal physiology and its breakdown in disease, in close alignment with the central mission of NIGMS.