Proximity Labeling of NIMA Kinase Complex Components in C. elegans.

Area of Science:

• Molecular Biology
• Cell Biology
• Proteomics

Background:

• Proximity labeling identifies protein-protein interactions, especially weak or transient ones.
• NEKL-MLT subcomplexes regulate membrane trafficking and actin dynamics in C. elegans.

Purpose of the Study:

• To comprehensively map the interactomes of NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4 using proximity labeling.
• To develop and validate quantitative metrics for assessing proximity labeling experiments.
• To establish a scalable strategy for probing endogenous protein networks in multicellular systems.

Main Methods:

• Utilized endogenously expressed TurboID fusions in C. elegans.
• Employed a data-independent acquisition (DIA) mass spectrometry (MS) pipeline across 23 experiments.
• Applied stringent controls, filtering strategies, and quantitative metrics for data analysis.

Main Results:

• Identified known NEKL-MLT binding partners and genetic suppressors.
• Discovered novel interactors involved in membrane trafficking, cytoskeletal regulation, and cell adhesion.
• Demonstrated the sensitivity and physiological relevance of DIA-MS workflows in proximity labeling.

Conclusions:

• DIA-MS is a powerful tool for proximity labeling, even with experimental variability.
• Internal controls, quantitative metrics, and biological validation enhance confidence in identified interactors.
• This study provides a robust framework for investigating endogenous protein networks in multicellular organisms.