Tracking protein dynamics with photoconvertible Dendra2 on spinning disk confocal systems

Area of Science:

• Cell Biology
• Microscopy Techniques
• Protein Dynamics

Background:

• Understanding protein function requires real-time analysis of their dynamic properties in live cells.
• Photoconvertible fluorescent proteins offer unique capabilities for tracking protein movement.
• Advanced microscopy is crucial for high-resolution imaging of cellular processes.

Purpose of the Study:

• To introduce and validate a robust platform for analyzing protein subcellular trafficking in living cells.
• To exploit the properties of the Dendra2 photoconvertible fluorescent protein for dynamic imaging.
• To characterize the differential motion of fast-trafficking proteins between subnuclear structures.

Main Methods:

• Utilized a Fluorescence Recovery After Photobleaching-Photoactivation unit combined with a Nikon Eclipse Ti E Spinning Disk confocal microscope.
• Employed the Dendra2 photoconvertible fluorescent protein for tracking protein dynamics.
• Adapted experimental settings to track fast-trafficking proteins like UBC9 and Fibrillarin.

Main Results:

• Demonstrated the platform's capability for high temporal and spatial resolution imaging.
• Successfully characterized the differential migration of UBC9 and Fibrillarin between subnuclear structures.
• Showcased reduced photobleaching and increased cell viability due to minimized light exposure.

Conclusions:

• The presented platform is advantageous and robust for studying protein subcellular trafficking in live cells.
• The system enables detailed characterization of dynamic protein motion at high resolution.
• This methodology facilitates the investigation of protein trafficking pathways.