Reversible Live-Cell Labeling with Retro-engineered HaloTags Enables Long-Term High- and Super-Resolution Imaging

Area of Science:

• Biophysics
• Cell Biology
• Microscopy

Background:

• Self-labeling enzymes (SLE), like HaloTag, are vital for fluorescence microscopy.
• Fluorogenic SLE substrates allow imaging even with excess dye.
• Reversible labeling is crucial for advanced imaging techniques.

Purpose of the Study:

• To engineer HaloTag variants with restored dehalogenase activity for reversible labeling.
• To evaluate the performance of these engineered variants in advanced fluorescence microscopy.

Main Methods:

• Engineering of HaloTag7 variants (reHaloTagS and reHaloTagF) with restored dehalogenase activity.
• In vitro kinetic studies to determine turnover rates.
• Confocal and stimulated emission depletion (STED) microscopy for imaging and photostability assessment.
• Single molecule imaging and structured illumination microscopy for diffusion and dynamics studies.

Main Results:

• reHaloTagS and reHaloTagF exhibited distinct in vitro turnover kinetics (≈0.006 s⁻¹ and ≈0.055 s⁻¹, respectively).
• reHaloTag labeling demonstrated 3-5 times enhanced photostability compared to standard HaloTag.
• Single molecule imaging with reHaloTags achieved controlled and stable labeling density over extended periods.
• Simultaneous visualization of single molecule diffusion and organellar dynamics was successfully demonstrated.

Conclusions:

• Engineered reHaloTags facilitate reversible labeling, enhancing photostability and control in fluorescence microscopy.
• These variants enable advanced applications like simultaneous observation of molecular diffusion and cellular dynamics.
• reHaloTag labeling represents a significant advancement for high and super-resolution fluorescence microscopy techniques.