Dynamic probes of endogenous protein aggregation and cell signaling

项目 摘要/摘要： 细胞信号的动力学是指导细胞和组织行为的有益特征。然而，许多重要的 像Wnt/-Catenin通路一样，缺乏观察和剖析内源信号动力学的探针 在空间和时间上都是精确的。这项提议的总体目标是开发新的分子探针， 可以可视化和扰乱内源信号动力学，重点是Wnt/-Catenin途径。我们的 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来使 WNT信号动力学的直接可视化。现有的WNT记者可能很昏暗，需要基因组 目标细胞的工程化，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LRP6的聚集，观察LRP6的寡聚可以提供一个 时空分辨的途径活性读数。因此，我们设想了一种新的生物传感器，它可以 细胞内蛋白质聚集的观察。我们的记者必须满足两个重要的设计标准。 首先，它必须报告内源蛋白质聚集，以避免信号蛋白或基因组的过度表达 修改。第二，因为生理蛋白质聚集体很小，通常低于衍射极限 对于可见光，我们的记者必须“视觉放大”内源星团，这样它们才能被 常规显微镜。我们描述了开发、描述和应用这样一个记者的计划，称为 成团的。团簇通过蛋白质相原理在视觉上放大内源小蛋白质簇 分离。使用实验和模拟，我们将表征团块检测和 利用GFP的光遗传聚集性作为模型分析物，放大细胞内的蛋白质聚集体。到时候我们会的 应用丛集来检测已知形成生理聚集体的内源蛋白质簇。最后，我们 将应用丛集来检测内源性Wnt受体LRP6对细胞Wnt的反应 刺激，我们将验证LRP6-Clumps在细胞、组织和发育模型中的活性。这个 团簇的模块化将使其适应产生不同信号通路和细胞的传感器 各州。在第二个项目中，我们将设计第一个光遗传工具，以允许抑制内源 具有时空精确度的信号通路。我们的目标是抑制Wnt/-连环蛋白信号和， 另外，RAS-ERK信号。我们将在细胞培养的背景下验证成功的通路抑制 癌症模型和体外肠道器官构型。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，因此可以很容易地修改，以观察或抑制不同的感兴趣的目标。在以下方面取得成功 我们的工作将产生一套新的工具来观察、扰乱和理解基本的生化 正常生理的基础过程及其在疾病中的分解，与中枢密切相关。 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.