The retromer complex regulates C. elegans development and mammalian ciliogenesis

Abstract

The mammalian retromer consists of subunits VPS26, VPS29 and VPS35, and a loosely-associated sorting nexin (SNX) heterodimer or a variety of other SNX proteins. Despite involvement in yeast and mammalian cell trafficking, retromer's role in development is poorly understood, and its impact on primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrate that vps-26 knockout worms have reduced brood sizes, impaired vulval development, and decreased body length, which have been linked to ciliogenesis defects. While preliminary studies did not identify worm ciliary defects, and impaired development limited additional ciliogenesis studies, we turned to mammalian cells to investigate the role of retromer in ciliogenesis. VPS35 localized to the primary cilium of mammalian cells, and depletion of VPS26, VPS35, VPS29, SNX1, SNX2, SNX5 or SNX27 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the centriolar protein, CP110, and was required for its removal from the mother centriole. Herein, we characterize new roles for the retromer in C. elegans development and in the regulation of ciliogenesis in mammalian cells, suggesting a novel role for the retromer in CP110 removal from the mother centriole.

A primary microcephaly-associatedsas-6mutation perturbs centrosome duplication, dendrite morphogenesis, and ciliogenesis inCaenorhabditis elegans

“…Thus, the introduction of osm-6p::GFP marks C. elegans ciliated neurons with GFP, allowing for easy visualization of the cilia (Bayer et al . 2020, Xie et al . 2022).…”

“…C. elegans phasmid cilia were imaged as described previously (Xie et al 2022). Briefly, L4 stage C. elegans from IYR026 (oyIs59 Is[osm-6p::gfp] III; sas-6(luv1) IV) and IYR027 (oyIs59 Is[osm-6p::gfp] III; sas-6(luv2) IV) strains were grown at 16C overnight .…”

“…Whole worm extracts for western blotting were prepared as described previously (Xie et al 2022). Briefly, 100 gravid adults from each genotype were washed with M9 buffer twice.…”

“…Western blotting was performed using the Bio-Rad wet-transfer method as described previously (Xie et al 2022). Briefly, for western blotting of whole worm extracts, 10 to 14 µl of extracts were loaded onto each well of a 7.5% SDS-PAGE gel.…”

“…No reuse allowed without permission. All brood count assays and embryonic viability assays were performed as described previously (Xie et al 2022). For both brood count as well as embryonic viability assays, L4 stage C. elegans were plated at the respective indicated temperatures.…”