Harnessing molecular motors for nanoscale pulldown in live cells

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• Protein-protein interactions (PPIs) are crucial for cellular functions but challenging to study in their native cytoplasmic environment.
• Existing methods often require cell lysis, disrupting native complex formation and localization.

Purpose of the Study:

• To develop a novel method for interrogating PPIs within living cells.
• To miniaturize the affinity pulldown technique for in vivo applications.
• To enable the study of PPIs in their native cellular context.

Main Methods:

• Developed nanoscale pulldowns (NanoSPDs) by hijacking intracellular myosin motor trafficking along filopodial actin filaments.
• Utilized dual-color total internal reflection fluorescence microscopy to visualize and quantify complex formation.
• Employing a rigorous quantification framework and software tool for data analysis.

Main Results:

• Demonstrated successful PPI detection through simultaneous trafficking and concentration of tagged proteins at filopodial tips.
• Observed amplified fluorescence signals due to molecular 'traffic jams' at filopodial tips.
• Validated NanoSPDs for studying nuclear and cytoplasmic PPIs, including those linked to human deafness, via domain mapping and mutagenesis.

Conclusions:

• NanoSPDs offer a powerful, miniaturized approach to study PPIs in real-time within living cells.
• The technique allows for dissection of molecular interactions and is adaptable for various fluorescent molecules and high-throughput screening.