Acquisition of a confocal microscope for imaging and controlling intracellular signals

获取用于成像和控制细胞内信号的共焦显微镜

• 批准号: 10582253
• 负责人: Lukasz Bugaj
• 金额: $ 25万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10582253
• 关键词: Behavior; Biochemical Process; Biosensor; Cancer Model; Cell Culture Techniques; Cells; Development; Disease; Engineering; Future; Genetic Transcription; Genomics; Goals; In Vitro; Instruction; Intestines; Malignant Neoplasms; Microscopy; Mission; Modeling; Modification; Molecular Probes; National Institute of General Medical Sciences; Nerve Degeneration; Organoids; Pathway interactions; Pattern; Phase; Physiological; Physiological Processes; Physiology; Proteins; Reporter; Reporting; Signal Pathway; Signal Transduction; Signaling Protein; Time; Tissues; Visible Radiation; Visualization; WNT Signaling Pathway; Work; aggregation pathway; design; experimental study; human disease; interest; microscopic imaging; novel; optogenetics; overexpression; protein aggregation; receptor; response; sensor; simulation; spatiotemporal; success; tool

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.

项目 摘要/摘要： 细胞信号的动态是指导细胞和组织行为的指导性特征，但还有许多重要的特征。 途径，如 Wnt/-catenin 途径，缺乏探针来观察和剖析内源信号动态 该提案的总体目标是开发能够实现空间和时间精度的新型分子探针。 可视化和扰乱内源信号动态，重点关注 Wnt/-catenin 通路。 在第一个项目中，我们将开发一种新型荧光生物传感器，以实现直接 现有 Wnt 信号动态的可视化可能很暗淡，需要对 Wnt 进行基因组工程。 靶细胞，作用于缓慢的转录时间尺度，可以掩盖上游途径动态，并提供 没有关于输入 Wnt 信号的空间信息，因为 Wnt 刺激需要通路的聚合。 LRP6 共受体，观察 LRP6 寡聚化可以提供时空分辨的读数 因此，我们设想了一种新型生物传感器，可以观察 我们的报告器必须满足两个重要的设计标准，首先，它必须报告内源性。 蛋白质聚类以避免信号蛋白过度表达或基因组修饰。 生理蛋白质聚集体很小并且通常低于可见光的衍射极限，我们的记者必须 “视觉放大”内源性簇，以便通过传统显微镜观察它们。我们描述了计划。 开发、表征和应用这样一个称为 CluMPS 的报告器，可以在视觉上放大小尺寸。 利用实验和蛋白质相分离原理分离内源蛋白质簇。 模拟，我们将表征 CluMPS 检测和放大细胞内蛋白质聚集体的能力， 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 来检测 GFP 的簇。 最后，我们将应用 CluMPS 来检测已知的形成生理聚集体的内源性蛋白质。 内源性 Wnt 受体 LRP6 响应细胞 Wnt 刺激而聚集，我们将验证 LRP6- CluMPS 在细胞、组织和发育模型中的活性 CluMPS 的模块化将使其适应。 生成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度抑制内源信号通路的光遗传学工具。 将分别针对 Wnt/-catenin 信号传导和 Ras-Erk 信号传导的抑制。 在癌症细胞培养模型和体外肠道模式中成功抑制通路 值得注意的是，我们所有的探针都将以模块化方式设计，因此可以很容易地进行修改。 观察或抑制不同的感兴趣目标。我们工作的成功将产生一套新的观察工具， 扰乱并了解正常生理学及其基础的基本生化过程 疾病细分，与 NIGMS 的中心使命密切相关。