Surface delivery quantification reveals distinct trafficking efficiencies among clustered protocadherin isoforms

Area of Science:

• Cell Biology
• Molecular Biology
• Neuroscience

Background:

• Cell-surface proteins are vital for cellular communication and environmental sensing.
• Proper folding and trafficking of these proteins to the plasma membrane in heterologous systems is a significant challenge.
• Clustered protocadherins (CPs) are crucial for cell-cell recognition in neuronal development.

Purpose of the Study:

• To develop a quantitative strategy for assessing cell surface protein localization using fluorescence microscopy.
• To investigate the surface delivery levels of different clustered protocadherin isoforms and engineered variants.
• To elucidate the regulatory mechanisms governing CP surface trafficking.

Main Methods:

• Developed a quantitative strategy for measuring surface protein localization from fluorescence microscopy images of surface-stained cells.
• Utilized epitope tag-based surface staining methods for quantitative comparisons.
• Employed natural and engineered variants of clustered protocadherins in experiments.

Main Results:

• Established a generalizable framework for screening proteins for proper cell surface localization.
• Demonstrated varying surface delivery levels among different CP isoforms and variants.
• Provided evidence that cis dimerization is not tightly coupled to surface delivery for CPs.
• Showed that an inhibitory domain affects surface delivery without relying on its protein-protein interface.

Conclusions:

• The developed framework enables quantitative screening of protein surface delivery.
• Findings challenge previous understandings of CP regulation, particularly regarding cis dimerization and inhibitory domains.
• This work facilitates the interpretation of mutational effects on protein trafficking and regulatory mechanisms.