moxDendra2: an inert photoswitchable protein for oxidizing environments

Area of Science:

• Biochemistry
• Cell Biology
• Microscopy

Background:

• Fluorescent proteins (FPs) are crucial tools for visualizing cellular processes using techniques like PALM super-resolution microscopy.
• Most FPs are optimized for cytoplasmic environments, leading to folding issues in distinct subcellular organelle conditions.
• Subcellular organelles possess unique chemical environments that challenge the stability and functionality of conventional FPs.

Purpose of the Study:

• To develop a novel fluorescent protein suitable for use in oxidizing subcellular environments.
• To create a photoswitchable FP that can be reliably used for advanced imaging techniques in organelles.
• To overcome the limitations of existing FPs in non-cytoplasmic cellular compartments.

Main Methods:

• Engineering and characterization of a novel monomeric fluorescent protein.
• Testing the photophysical properties and stability of the FP in oxidizing conditions.
• Demonstration of the FP's utility in super-resolution microscopy and pulse-chase experiments within organelles.

Main Results:

• Successfully engineered a monomeric photoswitchable FP with green-to-red fluorescence.
• The novel FP demonstrates robust folding and functionality in oxidizing environments.
• The FP is suitable for PALM super-resolution microscopy and pulse-chase experiments in subcellular organelles.

Conclusions:

• This new FP expands the toolkit for live-cell imaging in challenging subcellular locations.
• The development enables more accurate and detailed studies of molecular dynamics within organelles.
• Addresses a critical need for FPs functional in diverse cellular compartments.