Cooperative polarization of MCAM/CD146 and ERM family proteins in melanoma

Area of Science:

• Cell Biology
• Molecular Biology
• Biophysics

Background:

• WRAMP structures regulate cell migration dynamics, involving actomyosin contractility and membrane retraction.
• Melanoma cell adhesion molecule (MCAM) is a key marker for WRAMP structures, known to polarize to the cell rear during migration.
• The molecular mechanisms governing MCAM polarization remain largely uncharacterized.

Purpose of the Study:

• To identify factors mediating MCAM polarization to the cell rear.
• To characterize the role of ERM proteins in MCAM recruitment to WRAMP structures.
• To develop and validate a novel computational tool for quantitative analysis of protein polarization.

Main Methods:

• BioID proximity labeling using MCAM as bait.
• Development and application of the Protein Polarity by Percentile (3P) machine learning image analysis pipeline.
• CRISPR-mediated gene depletion of ERM proteins (moesin, ezrin).
• Live-cell imaging and analysis of MCAM and ERM protein dynamics.
• Site-directed mutagenesis of MCAM to disrupt ERM protein interactions.

Main Results:

• BioID identified ERM proteins (moesin, ezrin, radixin) as novel WRAMP structure components.
• Depletion of moesin and ezrin significantly reduced MCAM polarization.
• The 3P pipeline revealed subpopulations with distinct MCAM and ERM protein localization patterns.
• Live-cell imaging demonstrated coordinated but distinct temporal dynamics of MCAM and ERM polarization, with ERM proteins localizing peripherally first.
• Disruption of MCAM-ERM interaction domains impaired MCAM polarization.

Conclusions:

• ERM proteins are critical for the recruitment of MCAM to WRAMP structures.
• MCAM polarization is tightly regulated by its interaction with ERM proteins.
• The 3P computational tool provides a robust method for quantifying protein polarization in cell migration studies.