Acquisition of a confocal microscope for imaging and controlling intracellular signals

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通路这样的通路，缺乏观察和剖析内源信号动力学的探针 在空间和时间上的精确度。这项提议的总体目标是开发能够 可视化和扰动内源信号动力学，重点关注Wnt/-连环蛋白途径。我们的建议是 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实现直接 WNT信号动力学的可视化。现有的WNT记者可能很昏暗，需要对 靶细胞，作用于缓慢的转录时间尺度，这可能会掩盖上游途径的动力学，并提供 没有关于输入WNT信号的空间信息。因为WNT刺激需要途径的聚集 共受体LRP6，观察LRP6寡聚可以提供时空分辨的读数 途径活性。因此，我们设想了一种新的生物传感器，它可以观察到 胞内蛋白质。我们的记者必须满足两个重要的设计标准。首先，它必须报告内生性 蛋白质聚集，以避免信号蛋白或基因组修饰的过度表达。第二，因为 生理蛋白质聚集体很小，往往低于可见光的衍射极限，我们的记者一定 “视觉放大”内生团，这样它们就可以用常规显微镜观察到。我们描述了计划 开发、表征和应用这样一种名为CLUMPS的记者。团块在视觉上放大小 通过蛋白质相分离原理实现内源蛋白质簇。同时使用实验和 模拟，我们将表征团块检测和放大细胞内蛋白质聚集体的能力， 以绿色荧光蛋白的光遗传聚类法作为模型分析物。然后，我们将应用丛集来检测 已知的形成生理聚集体的内源性蛋白质。最后，我们将应用簇来检测 内源性Wnt受体LRP6在细胞Wnt刺激下的聚集，我们将验证LRP6- 在细胞、组织和发育模型中聚集活动。簇的模块化将允许其自适应 以产生不同信号通路和细胞状态的传感器。在第二个项目中，我们将设计 第一个能够以时空精确度抑制内源信号通路的光遗传学工具。我们 将分别针对Wnt/-连环蛋白信号和RAS-ERK信号的抑制。我们将验证 在肿瘤细胞培养模型和体外肠道构型背景下成功的途径抑制 有机化合物。值得注意的是，我们所有的探测器都将以模块化方式设计，因此可以很容易地进行修改，以 观察或抑制感兴趣的不同目标。我们工作的成功将产生一套新的观察工具， 扰乱并理解构成正常生理学基础的基本生化过程及其 疾病分类，与NIGMS的中心使命密切一致。

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.