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

Abstract

Molting is an essential developmental process in nematodes during which the epidermal apical extracellular matrix, the cuticle, is remodeled to accommodate further growth. Using genetic approaches, we identified a requirement for three conserved ankyrin repeat-rich proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, in Caenorhabditis elegans molting. Loss of mlt function resulted in severe defects in the ability of larvae to shed old cuticle and led to developmental arrest. Genetic analyses demonstrated that MLT proteins functionally cooperate with the conserved NIMA kinase family members NEKL-2/NEK8 and NEKL-3/NEK6/NEK7 to promote cuticle shedding. MLT and NEKL proteins were specifically required within the hyp7 epidermal syncytium, and fluorescently tagged mlt and nekl alleles were expressed in puncta within this tissue. Expression studies further showed that NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3 colocalize within largely distinct assemblies of apical foci. MLT-2 and MLT-4 were required for the normal accumulation of NEKL-2 at the hyp7-seam cell boundary, and loss of mlt-2 caused abnormal nuclear accumulation of NEKL-2. Correspondingly, MLT-3, which bound directly to NEKL-3, prevented NEKL-3 nuclear localization, supporting the model that MLT proteins may serve as molecular scaffolds for NEKL kinases. Our studies additionally showed that the NEKL-MLT network regulates early steps in clathrin-mediated endocytosis at the apical surface of hyp7, which may in part account for molting defects observed in nekl and mlt mutants. This study has thus identified a conserved NEKL-MLT protein network that regulates remodeling of the apical extracellular matrix and intracellular trafficking, functions that may be conserved across species.KEYWORDS C. elegans; molting; NIMA kinase; endocytosis; ankyrin repeat proteins M OLTING is a ubiquitous process in nematode species, including Caenorhabditis elegans, and is required for organismal growth and development. Although the observable biomechanics and major morphological features of C. elegans molting were first described nearly 40 years ago (Hirsh et al. 1976;Singh and Sulston 1978), the molecular and cellular mechanisms underlying this complex process are largely unknown. Notably, molting occurs in both pathogenic and nonpathogenic nematode species, and molting factors have been proposed as potential targets for antiparasitic drugs (Page et al. 2014;Gooyit et al. 2015).Mechanistically, molting is the process by which nematodes remodel their epidermal apical extracellular matrix (ECM), termed the cuticle. In C. elegans larvae and adults, the cuticle serves as a mechanical barrier and provides physical and chemical protection from the environment (Page and Johnstone 2007). In addition, similar to the chitin-based exoskeleton of insects and other arthropods, the cuticle is required to facilitate organismal movement in conjunction with attached underlying muscles. Unlike arthropods, however, in nematodes the cuticle is comprised primarily of collagens (Page and Johnstone 20...

Allosteric feed-forward activation of AP-2viaFCHO-BMP2K axis promotes clathrin-mediated endocytosis

“…Since our current investigation is limited to qualitative analyses of 411 endocytosis phenotypes, these possibilities are speculative and require future investigations to delineate 412 precise mechanisms. However, such effects of kinase and AP-2 phosphorylation are very well consistent 413 with our own results as well as with similar phenotypes observed in C. elegans mutants of related NIMA 414 family of kinases (Yochem et al, 2015;Lazetic and Fay, 2017;Lazetic et al, 2018) or in mammalian 415 cells upon depletion of late endocytic effectors (NECAP/ SNX9/ Amphiphysin) (Wrobel et al, 2019). 416…”

A multi-layered and dynamic apical extracellular matrix shapes the vulva lumen inCaenorhabditis elegans

“…Here we show that a spatially and temporally dynamic aECM assembles and disassembles within the vulva lumen during morphogenesis. This aECM shares components with the glycocalyx-like sheath or pre-cuticle matrix that coats other apical surfaces in C. elegans prior to each round of cuticle secretion (Forman-Rubinsky, Cohen, & Sundaram, 2017;Gill et al, 2016;Katz, Maybrun, Maul-Newby, & Frand, 2018;Kelley et al, 2015;Labouesse, 2012;Lazetic & Fay, 2017;Mancuso et al, 2012;Priess & Hirsh, 1986;Vuong-Brender, Suman, & Labouesse, 2017). It contains both fibrillar and gel-like or granular components, and also extracellular vesicles, as observed at the ultrastructural level.…”

Control of clathrin-mediated endocytosis by NIMA family kinases

“…We previously described a genetic and bioinformatic approach to identify suppressors of larval lethality in strains deficient for NEKL kinase activity [72]. Our screen makes use of weak aphenotypic alleles of nekl-2(fd81) and nekl-3(gk894345) , which, when combined in double mutants, lead to penetrant larval arrest due to molting defects [72, 73]. Homozygous nekl-2(fd81); nekl-3(gk894345) mutants (hereafter referred to as nekl-2; nekl-3 mutants) can be propagated only in the presence of a nekl-2 + or nekl-3 + rescuing extrachromosomal array, whereas strains that acquire a suppressor mutation no longer require the array for viability (Fig 1A,B).…”

“…In addition, loss of function in the conserved ankyrin repeat proteins MLT-2 (human ANKS6), MLT-3 (human ANKS3), and MLT-4 (human INVS), leads to molting defects that are identical to those of nekl mutants. The NEKL–MLTs form two distinct complexes (NEKL-2 with MLT-2–MLT-4 and NEKL-3 with MLT-3), and the MLTs are required for the correct subcellular localization of the NEKLs [21]. Importantly, these physical and functional interactions between NEKLs and MLTs appear to be highly conserved [22-25].…”