A primary microcephaly-associated sas-6 mutation perturbs centrosome duplication, dendrite morphogenesis, and ciliogenesis in Caenorhabditis elegans

Abstract

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 Caenorhabditis elegans. Given that SAS-6 is highly conserved, we modeled this mutation in C. elegans and examined the 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.

Keywords: C. elegans; SAS-6; SASS6; centriole; centrosome; cilia; microcephaly; rare disease.

Fig. 1.

The MCPH-associated sas-6(L69T) mutation was successfully generated in C. elegans by CRISPR/Cas9 editing. a) Schematic of the CRISPR repair template used for inserting the leucine-to-threonine mutation at amino acid 69 of the C. elegans SAS-6 protein (Note: only 10 out of 45 bases of the 5′ and 3′ homology ends of the repair template are shown). The teal highlights represent bases that are identical between the repair template and the sas-6 gene sequence. The magenta highlight indicates the bases that were altered to introduce the leucine 69 to threonine mutation into the endogenous sas-6 gene. The bases highlighted in gray represent silent mutations that were introduced in the repair template to prevent its cutting by the Cas9 enzyme. b) Agarose gel electrophoresis of PCR products digested with EcoRI to screen for homozygous-edited C. elegans carrying the sas-6(L69T) mutation. Black stars: Homozygous-edited worms; het: heterozygote; WT: wild-type.

Fig. 2.

The sas-6(L69T) mutation does not affect C. elegans brood size or embryonic viability. a) Brood sizes of WT and sas-6(L69T) mutant worms were determined at temperatures of 16°C, 20°C, and 25°C. Each circle or triangle represents the brood size of a single adult hermaphrodite. No statistically significant difference in brood size was observed between WT and sas-6(L69T) worms at any of the assessed temperatures. n = number of worms whose brood was analyzed for each strain. n.s: not significant. Error bars represent the standard deviation. The middle bar represents the mean. b) Embryonic viability of WT and sas-6(L69T) worms was analyzed at temperatures of 16°C, 20°C, and 25°C. Each circle or triangle represents the embryonic viability of a single adult hermaphrodite. The middle line represents the median embryonic viability with a 95% confidence interval. No major change in embryonic viability was observed between WT and sas-6(L69T) worms at any of the assessed temperatures. The viability of over 2,000 embryos was analyzed for each strain at every temperature.

Fig. 3.

The sas-6(L69T) mutation increases the centrosome duplication failures and the embryonic lethality of zyg-1(it25) worms. a) Schematic showing the genetic cross that was performed to combine the sas-6(L69T) mutation with the zyg-1(it25) mutation. b) Stills from time-lapse imaging of zyg-1(it25) and zyg-1(it25); sas-6(L69T) strains expressing fluorescently-tagged centrosome and DNA markers. Grayscale: mCherry-SPD-2 (centrosome marker); Green: GFP-histone (DNA marker). In a majority of zyg-1(it25) mutant embryos, centrosome duplication proceeds normally at a semipermissive temperature of 21.8°C, yielding two centrosomes in each cell of a 2-cell C. elegans embryo (Fig. 3b, top panel, white arrows). Scale bar = 10 µm. On the contrary, most of the zyg-1(it25); sas-6(L69T) mutant centrosomes fail to duplicate at 21.8°C resulting in a single centrosome present in each cell of a 2-cell C. elegans embryo (Fig. 3b, bottom panel, white arrows). c) Quantification of b). n = number of centrosomes analyzed. Approximately 83% of the zyg-1(it25) centrosomes duplicate normally at 21.8°C. In contrast, only about 28% of the zyg-1(it25); sas-6(L69T) centrosomes duplicate normally at this temperature. d) Quantification of embryonic viability of zyg-1(it25) single and zyg-1(it25); sas-6(L69T) double mutants at the semipermissive temperature of 21.7°C. Average embryonic viability is reduced from ∼44% to ∼7% in the presence of the sas-6(L69T) mutation. The viability of 1,103 embryos from zyg-1(it25) worms and 912 embryos from zyg-1(it25); sas-6(L69T) worms was analyzed. Embryos from 18 worms were analyzed for each condition. Unpaired two-tailed t-test, P < 0.0001. Error bars represent the standard deviation and the middle bar represents the mean.

Fig. 4.

The L69T mutation does not significantly alter the overall structure of the C. elegans SAS-6 protein. a) Cartoon depicting the location of the L69T mutation within the C. elegans SAS-6 protein oriented with respect to known SAS-6 protein domains and SAS-6 amino acid residues that are important for the interaction of SAS-6 with either itself or with other known interactors (Kitagawa et al. 2011; Lettman et al. 2013). b) Structure of C. elegans SAS-6 protein (PDB accession number: 4G79, Hilbert et al. 2013). c) The C. elegans SAS-6(L69T) protein structure was modeled using AlphaFold2. The coloring for the models is based on the ChimeraX default palette-option where lipophilicity ranges from −20 to 20 going from cyan (least lipophilic) to golden (most lipophilic). Blue arrows: A deep hydrophobic pocket within the mutant SAS-6(L69T) protein is decreased by the L69T mutation.

Fig. 5.

Phasmid cilia length is shorter in sas-6(L69T) mutant worms. a) A schematic of the genetic crossing experiment that was performed to introduce the MCPH-associated sas-6(L69T) mutation into the PY6100 pan-ciliary marker strain. b) Cartoon showing the anatomical positioning of a C. elegans phasmid cilium and dendrite within the tail region of the worm. c) Z-projection confocal images of phasmid neurons from osm-6p::gfp (top panel) and osm-6p::gfp; sas-6(L69T) (bottom panel) worms highlighting the difference in phasmid cilia lengths between the two genotypes. On average, control osm-6p::gfp worms have longer phasmid cilia (top panel, white bracket) than osm-6p::gfp; sas-6(L69T) worms (bottom panel, white bracket). White brackets denote the cilia. Scale bar = 10 µm. d) Quantification of phasmid cilia lengths of control (osm-6p::gfp) and sas-6(L69T) (osm-6p::gfp; sas-6(L69T)) worms. Each circle represents the length of a single phasmid cilium. n = number of phasmid cilia analyzed for each genotype. Phasmid cilia length was reduced from 5.82 µm in control worms (n = 54) to 3.99 µm in osm-6p::gfp; sas-6(L69T) worms (n = 59). P < 0.0001, unpaired two-tailed t-test. The error bars represent the standard deviation and the middle bars represents the mean.

Fig. 6.

Phasmid cilia morphology is defective in a subset of the sas-6(L69T) mutant worms. a) Cartoon showing the organization of a typical phasmid neuron with a ciliary axoneme (A) built on top of the TZ. The dendrite of the phasmid neuron connects to the TZ. b) and c) Representative Z-projection confocal images of phasmid cilia morphology in osm-6p::gfp and osm-6p::gfp; sas-6(L69T) worms, respectively. b) osm-6p::gfp worms have a stereotypical phasmid cilia morphology with two prominent transition zones (TZs) at the base and ciliary axonemes projecting from the TZs. Scale bar = 10 µm. c) A subset of the osm-6p::gfp; sas-6(L69T) worms exhibit deformed phasmid cilia morphologies. Scale bar = 10 µm. d) Quantification of B) and C). While all of the control (osm-6p::gfp) phasmid neurons (n = 61) exhibit a normal phasmid cilia morphology, 15.4% of the analyzed osm-6p::gfp; sas-6(L69T) phasmid neurons show a deformed cilia morphology (n = 65). n = number of phasmid neurons analyzed. P = 0.0014, Fisher's exact test.

Fig. 7.

Dendrite length is shorter in osm-6p::gfp; sas-6(L69T) phasmid neurons. a) Representative Z-projection confocal image of the average phasmid dendrite length in osm-6p::gfp worms. Scale bar = 10 µm. b) Representative Z-projection confocal image of the average phasmid dendrite length in osm-6p::gfp; sas-6(L69T) worms. Scale bar = 10 µm. c) Quantification of A) and B). The average dendrite length of control (osm-6p::GFP) worms is 37.4 µm (n = 38) while that of osm-6p::gfp; sas-6(L69T) worms is 28.1 µm (n = 42). n = number of phasmid dendrites analyzed. P < 0.0001, unpaired two-tailed t-test. The error bars and middle bar represent the standard deviation and the mean, respectively.

Fig. 8.

The sas-6(L69T) mutation reduces C. elegans chemotaxis to 1:500 Butanone. a) Schematic of chemotaxis assay experiments. L4 stage C. elegans were plated onto unseeded NGM plates that had been divided into four quadrants, two with solvent (ethanol) and two with odorant (1:500 butanone in ethanol). The worms were placed in the center of the plate and allowed to move around for 1 hour at 25°C. After 1 h, the number of worms in each quadrant was counted and quantified. The formula CI = (# animals in two odorant quadrants)/(# animals in any of the four quadrants) was used to compute the CI. Worms on any line or within the origin were not counted. Four plates were used for each genotype per experiment. Assays were repeated in triplicate. b) Quantification of data obtained from A). The sas-6(L69T) mutants exhibit a decreased chemotaxis toward 1:500 butanone as compared with control worms. n = Number of worms whose chemotaxis toward 1:500 butanone was analyzed. P < 0.0001, unpaired two-tailed t-test. c) Summary of data from A) The average CI of sas-6(L69T) worms was 0.3307. This CI was less than the average CI of control worms which was 0.4107. CI = Chemotaxis Index.