# A comprehensive toolkit to visualize endogenously tagged fluorescent basement membrane components in a living animal

> **NIH NIH R21** · DUKE UNIVERSITY · 2021 · $193,425

## Abstract

PROJECT SUMMARY
Basement membranes (BMs) are dense, sheet-like extracellular matrices that surround most animal tissues and
provide mechanical strength and signaling cues that control growth, differentiation, and survival. Genetic and
regulatory disruptions in BM components underlie numerous diseases, including cancer, fibrosis, and diabetes
as well as eye, kidney, vasculature, and skin disorders. Despite the importance of BM to human health, there
are currently no animal models that allow comprehensive real time visualization of BM components in vivo, which
limits the understanding of fundamental properties of BMs and hinders development of therapies to treat BM
disorders. The overall objective of this proposal is to create a complete toolkit of endogenously fluorescently
tagged BM components in C. elegans with methodologies to advance our understanding of BM regulation and
renewal in normal and disease states. These tagged proteins can be visualized in vivo because C. elegans is
optically clear. C. elegans also has single genes encoding most major BM protein families and receptors and
conditional knockdown approaches to disrupt their activity. Preliminary work has pioneered Cas9-mediated
homologous recombination to insert the mNeonGreen fluorophore in-frame with 27 BM-associated genes,
including all core matrix components, most matricellular proteins and all BM-associated receptors. These strains
have been verified for full length protein expression, BM localization and viability. To complete the objective of
developing a new BM toolkit animal model the following two specific aims will establish methods: (1) to quantify
dynamic alterations of BM components and receptors in distinct tissues, and (2) to follow BM component turnover
for the first time in vivo. Under Aim 1, experimental approaches will be developed to reveal BM component
variation in different organs during development and aging, how BM composition variation is mediated by BM
receptors, and how BMs adapt after loss of a key component (modeling human disease). In Aim 2, matrix
components tagged with the photoconvertible fluorophore mEos2 will be made and fluorescent recovery after
photobleaching (FRAP) and optical highlighting techniques will be developed to determine how BMs are renewed
during normal maintenance and rapid growth. The proposed study will powerfully advance our understanding of
BMs by providing the first model to dynamically track BM levels to understand how BMs are assembled, adapt
in disease states, and change over time. Further, preliminary FRAP and photoconversion studies of BM
components are already revealing that most matrix components are remarkably dynamic and some even flow
along the BM. This unexpected dynamic nature implies that BM structural and signaling properties can be altered
on the order of minutes—a profound change in our view of BM regulation. The proposed research is thus
significant, as the tools and methods created by completion of this work wi...

## Key facts

- **NIH application ID:** 10145827
- **Project number:** 5R21OD028766-02
- **Recipient organization:** DUKE UNIVERSITY
- **Principal Investigator:** David R Sherwood
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2021
- **Award amount:** $193,425
- **Award type:** 5
- **Project period:** 2020-04-15 → 2023-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10145827

## Citation

> US National Institutes of Health, RePORTER application 10145827, A comprehensive toolkit to visualize endogenously tagged fluorescent basement membrane components in a living animal (5R21OD028766-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/10145827. Licensed CC0.

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