C. elegans NIMA-related kinases NEKL-2 and NEKL-3 are required for the completion of molting

Abstract

C. elegans molting is a process during which the apical extracellular matrix of the epidermis, the cuticle, is remodeled through a process of degradation and re-synthesis. Using a genetic approach, we identified nekl-3 as essential for the completion of molting. NEKL-3 is highly similar to the mammalian NEK kinase family members NEK6 and NEK7. Animals homozygous for a hypomorphic mutation in nekl-3, sv3, had a novel molting defect in which the central body region, but not the head or tail, was unable to shed the old cuticle. In contrast, a null mutation in nekl-3, gk506, led to complete enclosure within the old cuticle. nekl-2, which is most similar to mammalian NEK8, was also essential for molting. Mosaic analyses demonstrated that NEKL-2 and NEKL-3 were specifically required within the large epidermal syncytium, hyp7, to facilitate molting. Consistent with this, NEKL-2 and NEKL-3 were expressed at the apical surface of hyp7 where they localized to small spheres or tubular structures. Inhibition of nekl-2, but not nekl-3, led to the mislocalization of LRP-1/megalin, a cell surface receptor for low-density lipoprotein (LDL)-binding proteins. In addition, nekl-2 inhibition led to the mislocalization of several other endosome-associated proteins. Notably, LRP-1 acts within hyp7 to facilitate completion of molting, suggesting at least one mechanism by which NEKL-2 may influence molting. Notably, our studies failed to reveal a requirement for NEKL-2 or NEKL-3 in cell division, a function reported for several mammalian NEKs including NEK6 and NEK7. Our findings provide the first genetic and in vivo evidence for a role of NEK family members in endocytosis, which may be evolutionarily conserved.

Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking inCaenorhabditis elegans

“…As for the mlt genes, we used CRISPR-Cas9 to generate fluorophore-tagged versions of nekl-2 and nekl-3. Consistent with our previous study (Yochem et al 2015), these reporters were expressed strongly within the epidermis beginning in late embryonic development (Figure 7 and data not shown). NEKL-2::NeonGreen was present in cytosolic puncta that were enriched at the hyp7-seam cell boundary, similar to our observations for MLT-2 and MLT-4 ( Figure 4A, Figure 7A, and data not shown).…”

“…However, NEKL-3 puncta were observed in some animals near the hyp7-seam cell boundary and showed partial colocalization with MLT-4 in this region (Figure 7B). Likewise, NEKL-2 and NEKL-3 did not generally colocalize, consistent with our previous results using multicopy reporters (Yochem et al 2015). Nevertheless, we did observe occasional overlap of NEKL-2 and NEKL-3 at the hyp7-seam cell boundary and, in some animals, we observed moderate-to-extensive colocalization of puncta in other regions of hyp7 ( Figure S6 and data not shown).…”

“…In most sv9 homozygotes, the central body and tail regions are trapped within two layers of cuticle, whereas the head region contains only a single layer ( Figure 1A). Other sv9 homozygotes display the "corset" phenotype in which both the head and tail regions are free of old cuticle ( Figure 1B), similar to what is observed for nekl-3(sv3) mutants (Yochem et al 2015). sv9 was mapped to a region on LG V, and microinjection of cosmid ZC15, which contains a genomic insert corresponding to a portion of the implicated region, fully rescued the sv9 molting defect (see Materials and Methods).…”

“…Accordingly, dietary cholesterol promotes normal molting (Yochem et al 1999;Merris et al 2003;Entchev and Kurzchalia 2005;Roudier et al 2005), along with the LRP-1/megalin lipoprotein receptor, which is thought to support lipid uptake by the epidermis (Yochem et al 1999;May et al 2007). Although these and other identified molting factors have provided insights into the general processes involved in C. elegans molting, the specific molecular and cellular mechanisms that underlie molting remain poorly understood.We recently described functions for two highly conserved members of the NIMA kinase family, NEKL-2 and NEKL-3, in C. elegans molting (Yochem et al 2015). NEKL-2 is orthologous to mammalian NEK8, which has been implicated in ciliogenesis Quarmby and Mahjoub 2005;Otto et al 2008;Shiba et al 2010;Zalli et al 2012).…”

“…We recently described functions for two highly conserved members of the NIMA kinase family, NEKL-2 and NEKL-3, in C. elegans molting (Yochem et al 2015). NEKL-2 is orthologous to mammalian NEK8, which has been implicated in ciliogenesis Quarmby and Mahjoub 2005;Otto et al 2008;Shiba et al 2010;Zalli et al 2012).…”