Labeling proteins inside living cells using external fluorophores for microscopy

Area of Science:

• Cell Biology
• Biochemistry
• Microscopy

Background:

• Site-specific protein labeling is crucial for studying cellular processes.
• Existing methods often lack efficiency or compromise cell viability.
• Live-cell imaging requires robust and rapid labeling techniques.

Purpose of the Study:

• To develop a novel, efficient method for site-specific fluorescent labeling of proteins in live mammalian cells.
• To assess the impact of the labeling method on cell viability and function.
• To enhance imaging capabilities, including super-resolution microscopy.

Main Methods:

• Utilized Streptolysin O (SLO), a bacterial enzyme, to create temporary pores in cell membranes.
• Delivered various fluorescent probes (IgGs, ligands) with high efficiency (>85% of cells).
• Performed cell viability assays, including division, signaling response, and organelle morphology.
• Combined SLO labeling with Oxyrase for photostabilization and glutathione for super-resolution imaging.

Main Results:

• Achieved efficient (>85%) fluorescent labeling of proteins in live mammalian cells within 30 minutes.
• Demonstrated maintained cell viability, division capacity, and normal signaling responses post-labeling.
• Enhanced fluorescence photostability by approximately 20-fold using Oxyrase.
• Enabled super-resolution fluorescence imaging with 20-30 nm resolution over 30 minutes using glutathione.

Conclusions:

• Streptolysin O provides a rapid and efficient method for site-specific fluorescent protein labeling in live cells.
• The SLO method preserves cell viability and function, suitable for various downstream applications.
• The combined approach allows for advanced imaging techniques, including long-term super-resolution microscopy.