Simple visualization of submicroscopic protein clusters with a phase-separation-based fluorescent reporter

Area of Science:

• Cell Biology
• Biophysics
• Molecular Imaging

Background:

• Protein clustering is crucial for cellular functions and disease pathogenesis.
• Detecting small protein oligomers is challenging with conventional fluorescence microscopy.

Purpose of the Study:

• To introduce a sensitive fluorescent reporter strategy, CluMPS, for detecting and amplifying protein clusters.
• To validate CluMPS's ability to visualize small protein aggregates and higher-order protein assembly.

Main Methods:

• Developed a fluorescent reporter strategy named CluMPS (clusters magnified by phase separation).
• Employed computational modeling and optogenetic clustering for system validation.
• Applied CluMPS to detect pathological protein aggregates and endogenous protein clusters.

Main Results:

• CluMPS effectively detects and visually amplifies small protein clusters into quantifiable condensates.
• The system demonstrates rational behavior based on key parameters.
• CluMPS identified small aggregates of pathological proteins missed by GFP fusions and tracked endogenous protein clusters.
• Multiplexed orthogonal CluMPS probes were successfully used in cells.

Conclusions:

• CluMPS offers a powerful and accessible method for observing higher-order protein assembly in native cellular environments.
• This reporter strategy enhances the sensitivity for detecting protein clusters, aiding in understanding cellular physiology and disease.