Suppression of nekl-associated molting defects by induction of L2d
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Molting is required for C. elegans to develop through larval stages into the adult. In addition, the timing of molting must be tightly coordinated with developmental progression. We have shown that the NIMA-related kinases, NEKL-2 and NEKL-3 (the NEKLs), promote molting through their involvement in the trafficking of epidermal cargos. This includes LRP-1/megalin, which is involved in the internalization of cholesterols, which can serve as precursors in steroid hormone synthesis. To better understanding the molting process and the functions of NEKLs, we screened for mutations that suppress molting defects in nekl mutants. Through this screen we identified catp-1, which encodes a predicted ATP-dependent Na+/K+ pump. Previous studies indicate that CATP-1 functions in the epidermis and may be involved in L2 developmental timing, as its loss of function results in a lengthened L2 stage. Notably, L1 larvae that are starved or exposed to dauer pheromone enter a lengthened L2 stage termed L2d. L2d comprises a predauer stage from which worms can later molt into dauers (L3d) or proceed with normal development (L3), depending on environmental conditions. Our findings indicate that loss of catp-1 induces a L2d-like state and that its pump activity is critical for CATP-1 function. Consistent with this, mutations that inhibit entry into L2d, such as daf-5/SKI loss of function, strongly reduce suppression by catp-1. In addition, we find that starvation, exposure to dauer pheromone, growth on heat-killed bacteria, and daf-C RNAi, conditions which induce L2d, can also suppress nekl molting defects. Notably, suppression by L2d occurs only with weak or partial loss-of-function (LOF) nekl alleles, which typically arrest at the L2/L3 molt, but not stronger LOF alleles, which typically arrest at ~L1/L2.Notably, previous studies by Bessereau and colleagues demonstrated that loss of catp-1 suppressed a lethal L2/L3 heterochronic phenotype induced by dimethylphenylpiperazinium, which causes cell divisions to become slowed and uncoupled from the molting timer. More recently, Ilbay and Ambros showed that induction of L2d or diapause suppressed abnormal cell divisions in heterochronic backgrounds that reiterate the L2 stage. We are currently testing the model that that nekl mutants may suffer from heterochronic defects that stem from an inability to endocytose precursors required for the synthesis of steroid hormones that control developmental timing.