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.

The human SASS6(I62T) missense mutation has been linked with the incidence of primary microcephaly in a Pakistani family, although the mechanisms by which this mutation causes disease remain unclear. The SASS6(I62T) mutation corresponds to SAS-6(L69T) in C. elegans. Given that SAS-6 is highly conserved, we modeled this mutation in C. elegans and examined sas-6(L69T) effect on centrosome duplication, ciliogenesis and dendrite morphogenesis. Our studies revealed that all the above processes are perturbed by the sas-6(L69T) mutation. Specifically, C. elegans carrying the sas-6(L69T) mutation exhibit an increased failure of centrosome duplication in a sensitized genetic background. Further, worms carrying this mutation also display shortened phasmid cilia, an abnormal phasmid cilia morphology, shorter phasmid dendrites and chemotaxis defects. Our data show that the centrosome duplication defects caused by this mutation are only uncovered in a sensitized genetic background, indicating that these defects are mild. However, the ciliogenesis and dendritic defects caused by this mutation are evident in an otherwise wild-type background, indicating that they are stronger defects. Thus, our studies shed light on the novel mechanisms by which the sas-6(L69T) mutation could contribute to the incidence of primary microcephaly in humans.

A missense mutation within the humanSASS6gene has been linked with the incidence of primary microcephaly in a Pakistani family. However, the mechanism by which this mutation causes disease is still unclear. SASS6 protein function is conserved between humans andC. elegans. Therefore, in the present study, we usedC. elegansto model this primary microcephaly-associated mutation to determine which molecular pathways are affected by this mutation inC. elegans. The human primary microcephaly-associated mutation corresponds to SAS-6(L69T) inC. elegans. Our studies revealed that both centrosome duplication and ciliogenesis are perturbed by thesas-6(L69T)mutation. Specifically,C. eleganscarrying thesas-6(L69T)mutation exhibit an increased failure of centrosome duplication in a sensitized genetic background. Further, worms carrying this mutation also display shortened phasmid cilia, an abnormal phasmid cilia morphology, defective phasmid dendrite morphology and chemotaxis defects. Our data show that the centrosome duplication defects caused by this mutation are only uncovered in a sensitized genetic background, indicating that these defects are mild. However, the ciliogenesis defects caused by this mutation are evident in an otherwise wild-type background, indicating that they are stronger defects. Based on our findings, we propose that the ciliogenesis defects induced by this mutation are likely a more important contributing factor to the development of primary microcephaly in humans carrying this mutation than centrosome duplication defects. Thus, our studies shed light on a novel mechanism by which thesas-6(L69T)mutation could lead to the incidence of primary microcephaly in humans.

The mammalian retromer is comprised of subunits VPS26, VPS29 and VPS35, and a more loosely-associated sorting nexin (SNX) heterodimer. Despite known roles for the retromer in multiple trafficking events in yeast and mammalian cells, its role in development is poorly understood, and its potential function in primary ciliogenesis remains unknown. Using CRISPR-Cas9 editing, we demonstrated that vps-26 homozygous knockout C. elegans have reduced brood sizes and impaired vulval development, as well as decreased body length which has been linked to defects in primary ciliogenesis. Since many endocytic proteins are implicated in the generation of primary cilia, we addressed whether the retromer regulates ciliogenesis in mammalian cells. We observed VPS35 localized to the primary cilium, and depletion of VPS26, VPS35 or SNX1/SNX5 led to decreased ciliogenesis. Retromer also coimmunoprecipitated with the capping 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, and suggest a novel role for the retromer in CP110 removal from the mother centriole.