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Updated: Jan 16, 2026
FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
Published on: August 20, 2012
FRET-based biosensor moxCRONOS enables quantitative monitoring of macromolecular crowding in organelles and protein
Yurina Nakajima1, Hiroaki Suzuki1, Tamami Miyagi1
1Department of Pharmacology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan.
Abstract:
Macromolecular crowding is a fundamental property of the intracellular environment that influences protein folding, enzymatic activity, and phase behavior. Disruptions to the homeostasis of macromolecular crowding can drive pathological processes, such as aberrant liquid-liquid phase separation and protein aggregation, which are central features of several neurodegenerative diseases. However, tools for quantifying crowding and aggregation remain limited. Here, we describe moxCRONOS, a Förster resonance energy transfer (FRET)-based biosensor that enables the quantitative measurement of macromolecular crowding and protein condensation. moxCRONOS retains the optical properties of the original CRONOS sensor but offers enhanced stability in oxidative environments, such as within the endoplasmic reticulum or under sodium arsenite treatment, allowing for direct comparison of crowding levels across organelles regardless of redox conditions. Moreover, when fused to dipeptide repeat proteins associated with C9ORF72-linked neurodegeneration, moxCRONOS detects aggregation-prone states-especially in cells expressing glycine-alanine (GA) repeats. Using fluorescence-activated cell sorting, we achieved sensitive and quantitative detection of heterogeneous high-FRET cell populations containing GA aggregates. FRET signal intensity increased upon treatment with a molecular crowding agent or a proteasome inhibitor. These findings establish moxCRONOS as a versatile biosensor for investigating both physiological macromolecular crowding and pathological protein aggregation, with significant potential for disease modeling and therapeutic screening.
Insights
Researchers developed moxCRONOS, a new biosensor to measure macromolecular crowding and protein aggregation. This tool aids in understanding neurodegenerative diseases and screening potential therapies.
Area of Science:
- Cellular Biology
- Biophysics
- Neuroscience
Background:
- Macromolecular crowding is vital for intracellular processes, and its disruption is linked to neurodegenerative diseases.
- Current tools for quantifying cellular crowding and protein aggregation are limited.
- Aberrant liquid-liquid phase separation and protein aggregation are hallmarks of neurodegenerative conditions.
Purpose of the Study:
- To introduce moxCRONOS, a Förster resonance energy transfer (FRET)-based biosensor for quantitative measurement of macromolecular crowding and protein condensation.
- To enhance the stability of the CRONOS sensor in oxidative environments for cross-organelle comparisons.
- To detect aggregation-prone states in dipeptide repeat proteins associated with C9ORF72-linked neurodegeneration.
Main Methods:
- Development of a Förster resonance energy transfer (FRET)-based biosensor, moxCRONOS.
- Fusion of moxCRONOS to dipeptide repeat proteins, including glycine-alanine (GA) repeats.
- Utilizing fluorescence-activated cell sorting (FACS) for sensitive detection of high-FRET populations.
- Treatment with molecular crowding agents and proteasome inhibitors to validate sensor response.
Main Results:
- moxCRONOS demonstrates enhanced stability in oxidative environments, enabling comparisons across organelles.
- The biosensor successfully detects aggregation-prone states in cells expressing GA repeats associated with C9ORF72 neurodegeneration.
- Fluorescence-activated cell sorting enabled quantitative detection of heterogeneous cell populations with GA aggregates.
- FRET signal intensity correlated with treatments involving molecular crowding agents or proteasome inhibitors.
Conclusions:
- moxCRONOS is a stable and versatile biosensor for quantifying macromolecular crowding and pathological protein aggregation.
- The sensor facilitates direct comparison of crowding levels across different cellular compartments and redox conditions.
- moxCRONOS shows promise for disease modeling of neurodegenerative conditions and therapeutic screening.
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