# Probing Biomolecular Condensates in Live Cells using Microsecond-scale Single Molecule Rotational Diffusion Microscopy (µs-SiMRoD)

> **NIH NIH R21** · TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR · 2024 · $190,625

## Abstract

PROJECT SUMMARY
 The crowded conditions within cells present a complex physicochemical challenge for maintaining the
solubility and fluidity of thousands of distinct macromolecules. High macromolecular densities promote assembly
into a variety of distinct structures, which in turn can lead to the formation of insoluble solid aggregates. Though
solidified structures could indeed serve protective purposes or as quiescent storage sites, they are more
generally considered to be toxic to the cell. The physical constraints and properties generated by crowded cellular
environments, which contribute significantly to the functional behaviors of the resident molecules, are often
difficult to probe experimentally. In addition to membrane-bound compartments, which define the primary
structural organization of cells, numerous membraneless organelles (MLOs) populate the cytoplasm and the
nucleoplasm providing additional complexity. These biomolecular condensates (BMCs) form via liquid-liquid
phase separation (LLPS), a process that concentrates the resident macromolecules, but which in turn also can
promote additional phase states, including solid aggregates. Multiple BMC-resident proteins that undergo LLPS
in vitro are the dominant proteins in cytoplasmic inclusions found in patients with a range of devastating
neurological disorders. In one such example, the fused in sarcoma (FUS) protein accumulates in MLOs termed
stress granules (SGs), which form as a natural response to various cellular stress conditions. The high molecular
density of FUS in SGs is potentially an essential early step toward the formation of FUS-containing solid
inclusions linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Directly observing
the formation and growth of such inclusions within a cellular context is challenged by the lack of appropriate
tools. While genetic approaches to attach fluorescent tags are commonly used to determine spatiotemporal
behaviors, their attachment can often perturb function, there are generally only a few reasonable attachment
points, and such probes have inferior photophysical properties. Recent improvements in genetic code expansion
to incorporate non-canonical amino acids (ncAAs) for in cellulo labeling promises to significantly extend the types
of questions that may be addressed. This is particularly relevant for the application of multiple single molecule
biophysical strategies. The Specific Aim of the proposed work is to identify and characterize solid aggregates
within live cells. To accomplish this, designer MLOs will be used to specifically modify FUS via ncAA-mediated
fluorescent labeling in cells. Microsecond-scale single molecule rotational diffusion (µs-SiMRoD) microscopy will
then be used to probe for local environmental constraints such as increased viscosity or structural confinement.
The target application will be identifying and monitoring solidified FUS-containing aggregates. This strategy is
expected to be i...

## Key facts

- **NIH application ID:** 10986330
- **Project number:** 1R21NS141768-01A1
- **Recipient organization:** TEXAS A&M UNIVERSITY HEALTH SCIENCE CTR
- **Principal Investigator:** SIEGFRIED M MUSSER
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $190,625
- **Award type:** 1
- **Project period:** 2024-09-18 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10986330

## Citation

> US National Institutes of Health, RePORTER application 10986330, Probing Biomolecular Condensates in Live Cells using Microsecond-scale Single Molecule Rotational Diffusion Microscopy (µs-SiMRoD) (1R21NS141768-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10986330. Licensed CC0.

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