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
基本信息
- 批准号:10582253
- 负责人:
- 金额:$ 25万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:BehaviorBiochemical ProcessBiosensorCancer ModelCell Culture TechniquesCellsDevelopmentDiseaseEngineeringFutureGenetic TranscriptionGenomicsGoalsIn VitroInstructionIntestinesMalignant NeoplasmsMicroscopyMissionModelingModificationMolecular ProbesNational Institute of General Medical SciencesNerve DegenerationOrganoidsPathway interactionsPatternPhasePhysiologicalPhysiological ProcessesPhysiologyProteinsReporterReportingSignal PathwaySignal TransductionSignaling ProteinTimeTissuesVisible RadiationVisualizationWNT Signaling PathwayWorkaggregation pathwaydesignexperimental studyhuman diseaseinterestmicroscopic imagingnoveloptogeneticsoverexpressionprotein aggregationreceptorresponsesensorsimulationspatiotemporalsuccesstool
项目摘要
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
项目成果
期刊论文数量(0)
专著数量(0)
科研奖励数量(0)
会议论文数量(0)
专利数量(0)
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Lukasz Bugaj其他文献
Lukasz Bugaj的其他文献
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{{ truncateString('Lukasz Bugaj', 18)}}的其他基金
Dynamic probes of endogenous protein aggregation and cell signaling
内源蛋白聚集和细胞信号传导的动态探针
- 批准号:
10029409 - 财政年份:2020
- 资助金额:
$ 25万 - 项目类别:
Dynamic probes of endogenous protein aggregation and cell signaling
内源蛋白聚集和细胞信号传导的动态探针
- 批准号:
10457270 - 财政年份:2020
- 资助金额:
$ 25万 - 项目类别:
Dynamic probes of endogenous protein aggregation and cell signaling
内源蛋白聚集和细胞信号传导的动态探针
- 批准号:
10414499 - 财政年份:2020
- 资助金额:
$ 25万 - 项目类别:
Dynamic probes of endogenous protein aggregation and cell signaling
内源蛋白聚集和细胞信号传导的动态探针
- 批准号:
10655952 - 财政年份:2020
- 资助金额:
$ 25万 - 项目类别:
Developing CluMPS reporters to visualize aggregation dynamics of receptor tyrosine kinase fusions
开发 CluMPS 报告基因以可视化受体酪氨酸激酶融合的聚集动态
- 批准号:
10321062 - 财政年份:2020
- 资助金额:
$ 25万 - 项目类别:
Dynamic probes of endogenous protein aggregation and cell signaling
内源蛋白聚集和细胞信号传导的动态探针
- 批准号:
10225592 - 财政年份:2020
- 资助金额:
$ 25万 - 项目类别:
Dynamic probes of endogenous protein aggregation and cell signaling
内源蛋白聚集和细胞信号传导的动态探针
- 批准号:
10669601 - 财政年份:2020
- 资助金额:
$ 25万 - 项目类别:
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