# Acquisition of a confocal microscope for imaging and controlling intracellular signals

> **NIH NIH R35** · UNIVERSITY OF PENNSYLVANIA · 2022 · $250,000

## Abstract

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,
per...

## Key facts

- **NIH application ID:** 10582253
- **Project number:** 3R35GM138211-03S2
- **Recipient organization:** UNIVERSITY OF PENNSYLVANIA
- **Principal Investigator:** Lukasz Bugaj
- **Activity code:** R35 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $250,000
- **Award type:** 3
- **Project period:** 2020-08-01 → 2025-07-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10582253

## Citation

> US National Institutes of Health, RePORTER application 10582253, Acquisition of a confocal microscope for imaging and controlling intracellular signals (3R35GM138211-03S2). Retrieved via AI Analytics 2026-05-27 from https://api.ai-analytics.org/grant/nih/10582253. Licensed CC0.

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