Control of clathrin-mediated endocytosis by NIMA family kinases

Abstract

Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated in vivo. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases.

Epidermal PAR-6 and PKC-3 are essential for larval development of C. elegans and organize non-centrosomal microtubules

“…To confirm that abnormalities in the hypodermis can affect seam-cell divisions, we used a CRISPR-tagged NEKL-2::AID strain that arrests growth and molting upon auxin addition ( Joseph et al, 2020 ). Indeed, NEKL-2 depletion caused defects in the morphology of seam cells, as well as a partially penetrant reduction in seam-cell divisions, confirming that abnormalities in the hypodermis can affect the seam cells ( Figure 3—figure supplement 1E,F ).…”

C. elegans Apical Extracellular Matrices Shape Epithelia

“…Molting involves the coordinated assembly and disassembly of pre-cuticles and cuticles. Many gene products are required for molting [ 156 ], including proteases [ 157 , 158 , 159 ], pre-cuticle [ 53 ] and cuticle components [ 131 ], endocytic proteins [ 160 ], metabolic enzymes [ 103 , 118 , 161 ], secretory pathway genes [ 69 , 162 ], and Hedgehog-and Patched-related genes [ 69 , 163 ]. The expression of many genes rises and falls in accordance with distinct phases of the molt cycle [ 87 , 88 , 103 , 104 ].…”

Mutation of NEKL-4/NEK10 and TTLL genes opposes loss of the CCPP-1 deglutamylase and prevents neuronal ciliary degeneration

“…Recent studies have ascribed a role for NEKs in regulating endocytosis. C. elegans NEKL-2 and NEKL-3 control clathrin-mediated endocytosis and trafficking in non-ciliated cells [55][56][57]. NEKL-4 localization to the periciliary membrane compartment -a site of endocytosis that regulates IFT, ciliary membrane volumes, and ciliary protein levels -hints at a role in endocytic trafficking at the ciliary base [58].…”