# Harnessing genetic code expansion to measure in vivo actin dynamics

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2020 · $188,789

## Abstract

Summary
 Our goal is to establish tools to directly label actin in any model system that can utilize genetic code
expansion. Although we know a great deal about actin and the molecular components of cytoskeletal structures,
we still know very little about actin dynamics that are essential to the functions of these structures. Our ability to
establish mechanistic understandings of actin structures is fundamental to our knowledge of cell biology and
human disease. We are limited by the availability of research tools for quantitative measurement of in vivo actin
dynamics. Pinpointing a position on actin that will tolerate change is not easy due to the extensive intrafilament
interfaces and the surfaces that interact with the >100 actin binding proteins. Genetic tags as small as 12 amino
acids disrupt multiple cellular processes. In response to this need, we propose to take advantage of the exciting
new capabilities of genetic code expansion and recently published high resolution structures of actin filaments.
We will use orthogonal amber suppressor aminoacyl-tRNA synthetase/tRNA pairs to site-specifically incorporate
non-canonical amino acids (ncAAs) with reactive side chains at carefully chosen positions on actin. Using the
inverse demand Diels-Alder reaction (a significantly faster variant of metal-free click chemistry) we will add
fluorophores to the ncAA for in vivo imaging. We expect to be able to modify a single amino acid within actin,
without disrupting function, based on the fact that actin covalently labeled with a small fluorescent probe is
functional. Further, previous work shows that labeling only ~2% of actin is sufficient for visualization of most
structures. Thus slight perturbations and/or low incorporation efficiency will not be a hindrance to proof-of-
principle experiments.
 First, we will identify candidate positions for ncAA incorporation using a genetic screen. Initially, we will work
in the powerful genetic model organism budding yeast, Saccaromyces cerevisiae. Because of its 87% sequence
identity with skeletal actin, yeast actin has been studied for decades, providing extensive data about surface
residues and powerful, yet simple, assays of actin function. Genetic code expansion is established in yeast; and,
importantly, in the context of this grant, yeast work is fast. Once we have established proof-of-principle, we will
shift to the fruit fly, Drosophila melanogaster. The fly is another powerful model organism that offers a broad
range of genetic tools. Genetic code expansion has been demonstrated in both Drosophila-derived S2 cells and
the fly. Being able to work in a relatively high throughput manner with S2 cells before moving to whole animals
makes Drosophila an ideal system in which to expand. Success will result in a strategy to directly label actin in
essentially every model system and tools already working in yeast and S2 cells. Success in labeling actin, will
lead to major advances in our understanding of its dyna...

## Key facts

- **NIH application ID:** 10009413
- **Project number:** 5R21GM134473-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Margot E Quinlan
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $188,789
- **Award type:** 5
- **Project period:** 2019-09-15 → 2022-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10009413

## Citation

> US National Institutes of Health, RePORTER application 10009413, Harnessing genetic code expansion to measure in vivo actin dynamics (5R21GM134473-02). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10009413. Licensed CC0.

---

*[NIH grants dataset](/datasets/nih-grants) · CC0 1.0*