Conserved NIMA kinases regulate multiple steps of endocytic trafficking

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• NIMA-related kinases (NEKs) are crucial for cell cycle, DNA damage control, and ciliogenesis.
• NEKL-2 and NEKL-3 in C. elegans regulate clathrin-mediated endocytosis and molting.
• NEKs have diverse cellular functions, but their roles in endosomal trafficking are less understood.

Purpose of the Study:

• To investigate the distinct roles of NEKL-2 and NEKL-3 in endosome function and morphology.
• To determine the impact of NEKL depletion on cargo trafficking and endocytosis in C. elegans and human cells.
• To explore the conserved functions of NEKs in endosomal pathways across species.

Main Methods:

• RNA interference (RNAi) to deplete NEKL-2 and NEKL-3 in C. elegans.
• Confocal microscopy to analyze endosome morphology and localization.
• Analysis of cargo trafficking for TGN-resident proteins and basolateral receptors.
• siRNA knockdown of NEK6 and NEK7 in human cell lines.
• Mannose 6-phosphate receptor localization studies.

Main Results:

• NEKL-2 depletion enlarged early endosomes with tubular extensions; NEKL-3 depletion affected early, late, and recycling endosomes.
• NEKL-2 localized to early endosomes, while NEKL-3 localized to multiple endosomal compartments.
• NEKL depletion caused missorting of TGN cargoes (MIG-14/Wntless, TGN-38/TGN38) to lysosomes.
• Defects in clathrin-dependent and independent cargo uptake from the basolateral surface were observed.
• Human NEK6/NEK7 knockdown led to missorting of mannose 6-phosphate receptor and disrupted early/recycling endosomes, including tubulation.

Conclusions:

• NIMA family kinases (NEKs) play conserved, multifaceted roles in endocytosis and endosomal trafficking in both worms and humans.
• NEKL-2 and NEKL-3 exhibit distinct but overlapping functions in regulating endosome morphology and cargo sorting.
• Trafficking defects mediated by NEKs may underlie their involvement in various human diseases.