Discerning protein pools by selective staining with self-labeling tags

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Transmembrane proteins mature from synthesis sites (endoplasmic reticulum) to the plasma membrane and can be endocytosed.
• Microscopy is crucial for visualizing protein populations at the cell surface and intracellularly.
• Distinguishing between surface and internalized proteins is vital for understanding protein trafficking.

Purpose of the Study:

• To present advanced microscopy methods for differentiating and quantifying intra- and extracellular protein pools.
• To highlight the utility of protein tags (SNAP-tag, HTP, FAST) and fluorescent ligands for protein localization studies.
• To explore novel approaches for analyzing endocytosed protein populations.

Main Methods:

• Utilizing protein tags (SNAP-tag, HTP, FAST) conjugated to fluorescent dyes.
• Employing (im)permeable ligands to differentiate extracellular and intracellular protein pools.
• Introducing cleavable groups in dye-ligands to selectively remove surface protein signals.
• Applying advanced microscopy techniques like fluorescence lifetime imaging microscopy and brightness demixing.

Main Results:

• Demonstrated ability to visually separate and quantify cell surface and intracellular protein populations.
• Successfully distinguished between newly synthesized and endocytosed protein pools.
• Showcased the effectiveness of cleavable dye-ligands in interrogating endocytosed protein trafficking.
• Highlighted enhanced resolution and quantification through advanced imaging modalities.

Conclusions:

• Novel microscopy strategies with fluorescent protein tags and cleavable ligands offer powerful tools for studying cell surface protein dynamics.
• These methods provide unprecedented insights into protein trafficking, localization, and abundance within cellular compartments.
• Advanced imaging techniques further refine the ability to dissect complex protein populations and their movements.