Abstract
Membrane trafficking, including endocytosis and exocytosis, is a complex process that is coordinated by trafficking-associated proteins, cargo molecules, the cytoskeleton, and membrane lipid composition. The NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking in Caenorhabditis elegans and are required for successful molting. Through a genetic approach, we isolated reduction-of-function mutations in piki-1 that suppress nekl-associated molting defects. piki-1 encodes the sole predicted C. elegans Class II phosphatidylinositol 3-kinase (PI3Ks), an understudied class of lipid modifiers that contribute to the production of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P(2)). Using a set of genetically encoded lipid sensors, we found that PIKI-1 was responsible for the production of PI(3,4)P(2) in the C. elegans epidermis but played only a minor role in the control of PI(3)P levels. Consistent with this, both PI(3,4)P(2) and PIKI-1 colocalized to early endosomes, and reduction of PIKI-1 function strongly affected early endosomal morphology and protein composition. Additionally, reduced PIKI-1 function led to excess tubulation of endosomal compartments associated with recycling or the degradation of cellular debris. In contrast to previous studies using mammalian cell culture, PIKI-1 was largely dispensable for clathrin-mediated endocytosis in the context of the worm epidermis, which is a polarized epithelium. Notably, reduction of PIKI-1 function strongly mitigated defects in early endosomes associated with the depletion of NEKL-2. We propose that reduction of PIKI-1 function may suppress nekl molting defects by partially restoring endocytic trafficking within specific compartments, including the early endosome. We also show that inhibition of the PI(3,4)P(2)-binding protein HIPR-1 (HIP1/HIPR1) suppresses nekl molting defects, suggesting that reduced PI(3,4)P(2) levels alter endosomal protein recruitment in a manner that antagonizes NEKL-2 function.