Control of clathrin-mediated endocytosis by NIMA family kinases Braveen B. Joseph*, Yu Wang+, Phil Edeen*, Vladimir Lažetić*, Barth D. Grant+ and David S. Fay* *Department of Molecular Biology, College of Agriculture and Natural Resources, University of Wyoming, Laramie, WY 82071 +Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854 Abstract Suppressor screen NEKL kinases are highly conserved members of the NIMA-kinase family, which have been predominantly implicated in cell division and ciliogenesis. We previously reported, that NEKL-2/NEK8/9 and NEKL-3/NEK6/7 are required within the C. elegans epidermis, and that their knockdown causes molting defects that results in larval lethality. In addition, we discovered a conserved set of ankyrin repeat proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, that partner with the NEKLs to regulate their intracellular localization. To better understand the mechanistic functions of NEKL-MLTs we obtained genetic suppressors of molting defects in nekl-2; nekl-3 double mutants. Strikingly, our screen identified several mutations that directly affect the activity of AP2, a conserved heterotetrametric adaptor complex required for clathrin mediated endocytosis. Knockdown of individual AP2 subunits, or an allosteric activator of AP2, FCHO-1, strongly suppress molting defects in nekl mutants. Conversely, mutations that increase the levels of active AP2 strongly enhance nekl molting defects. In addition, inhibition of nekls suppress morphological defects associated with reduced AP2 function, attesting to the tight functional connection between the NEKLs and AP2. To determine the functions of NEKLs in endocytosis, while avoiding potential confounding effects caused by nekl- mutant larval arrest, we used auxin inducible degradation methods to deplete NEKLs specifically at the adult stage. Loss of NEKLs led to greatly elevated levels of clathrin at the epidermal apical membrane and to a profound reduction in apical clathrin mobility. Adult NEKL depletion also adversely affected uptake of LRP-1, an epidermal cargo critical for molting. These results are consistent with depletion of NEKLs leading to a defect in the ability of vesicles to release clathrin after scission and suggest that NEKLs promote clathrin uncoating. Notably, both defects were largely alleviated by a reduction in AP2 or FCHO-1 activities. These findings suggest that reduced AP2 activity may facilitate clathrin uncoating and indicate that reduced AP2 activity suppresses nekl molting defects through the restoration of normal trafficking. Lastly, we show that human NEK6 and NEK7, orthologs of NEKL-3, can rescue both molting and trafficking defects in NEKL-3 depleted strains, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases. Individual knock down of µ, a, and d subunits Model that NEKLs may promote closed AP2 Loss of NEKL changes epidermal clathrin localization Cargo trafficking is disrupted in NEKL-depleted adults ⍺-Flag ⍺-actin + + – – NEKL-2 ::AID NEKL-3 ::AID Auxin (20h) Auxin- Inducible Degron NEKL Auxin NEKL NEKL Ub Poly- ubiquitin TIR1 SCF Ub Ub Several suppressors connected to clathrin mediated endocytosis Dynamin Closed AP2 Open AP2 Cargo Clathrin Actin Auxilin Hsc70 Uncoating Plasma Membrane FCHO-1 FCHO-1 σ nekl-2; nekl-3; fcho-1(fd131) nekl-2; nekl-3; dpy-23(fd155) nekl-2(fd81); nekl-3(gk894345); Ex nekl-3+, GFP+ Allele nekl-2(fd81) nekl-2(fd90) nekl-2(fd91) nekl-2(gk839) nekl-3(gk894345) nekl-3(sv3) nekl-3(gk506) mlt-3(fd72) mlt-4(sv9) Change Y84L G88A Y84L G87A G88A Y84L G87A Deletion D228N P194L Deletion Deletion Splice Loss of function Weak Strong Moderate Null Weak Moderate Null Null Moderate MLT-4 Larval Molting MLT-3 NEKL-3 NEKL-2 MLT-2 Old Cuticle New Cuticle Shed Cuticle Synthesis α β σ μ NEKL-3 NEKL-2 NEKL-3 NEKL-2 μ α σ nekl molting defects are suppressed by decreased function of the AP2 clathrin-adapter comple