JCS028621 Supplementary Material

Files in this Data Supplement:

• Supplemental Table S1 -

  Table S1. Distribution of B9-domain-containing proteins in ciliated organisms and absence from non-ciliated species. Individual proteins are grouped into their respective MKS or MKSR clades based on results of neighbor-joining ClustalW algorithm (not shown) and Bayesian analysis (Fig. 1A).

• Supplemental Figure S1 -

  Fig. S1. V5-tagged human MKS1, MKSR1 or MKSR2 transiently expressed in non-ciliated IMCD3 cells (stained red) colocalizes with γ-tubulin, a centriolar and centrosomal marker (green), showing overlap in signals (yellow) in the merged image. Arrows indicate centrosomes.

• Supplemental Figure S2 -

  Fig. S2. Mutations in the C. elegans mks-1, mksr-1 and mksr-2 genes and analysis of the corresponding transcripts. (A) C. elegans strains used in this study harbor the indicated mutations in the mks-1 (xbx-7/R148.1), mksr-1 (tza-2/K03E6.4) and mksr-2 (tza-1/Y38F2AL.2) genes. The positions and sequences of X-box regulatory regions are indicated, exons are shown as gray boxes, and the deleted regions are highlighted by dashed boxes along with their allele designations (tm2705; tm3083; tm2452). (B) RT-PCR analysis of wild-type (N2) and mks/mksr mutant transcripts by sequencing. Coding regions for the indicated proteins are shown as lines and the respective B9 domains as a blue box. The predicted MKS-1 protein in the mks-1(tm2705) mutant is missing residues 68-141, which results in a truncated N-terminal region but does not appear to affect the B9 domain. The predicted MKSR-1 mutant protein (in the tm3083 strain) has residues 78-94 of the wild-type protein replaced by 18 distinct amino acids, resulting from improper splicing of intron 2 with exon 3 between nucleotides 1032-1057 and 1276. This produces a B9 domain replaced with an entirely different sequence containing an extra residue. The predicted MKSR-2 mutant protein (in the tm2452 allele) is encoded by a transcript that results from the fusion of exon 1 with the 5′ of the truncated exon 3, resulting in a peptide that is missing amino acids 30-78, all of which fall within the predicted B9 domain (the deletion in the B9 domain is denoted by a squiggle).

• Supplemental Figure S3 -

  Fig. S3. mks/mksr mutant animals do not display dye-filling defects indicative of abrogated ciliary structures, or Nile Red (lipid content) phenotypes. (A) Wild-type and mks/mksr single, double and triple mutant animals incubated with DiI take up the fluorescent dye equally well, based on the number of sensory neurons that are stained and their relative staining intensity. (B) mks-1, mksr-2 and the mks-1;mksr-2 double mutants diplay equivalent Nile Red staining compared with wild-type (N2) animals based on relative fluorescence intensities of the dye in the intestine.

• Supplemental Figure S4 -

  Fig. S4. mks/mksr mutant animals display wild-type localization of intraflagellar transport (IFT) marker proteins and normal IFT rates along the middle and distal segments of cilia. (A) Fluorescence images of GFP-tagged XBX-2 (dynein component), OSM-3 (homodimeric kinesin) or KAP-1 (component of heterotrimeric Kinesin-II) in the amphid (head) or phasmid (tail) sensory neurons of wild-type (N2) or mutant backgrounds, as indicated. Arrows point to the relative positions of transition zones (TZ) and ciliary axonemes are indicated with brackets. Scale bar: 5 µm. (B) IFT rate measurements of IFT markers in N2 or mutant animals, as indicated. n, number of particles measured; n/a, not applicable.

• Supplemental Figure S5 -

  Fig. S5. Transmission electron microscopy (TEM) analyses of wild-type (N2) and mks-1;mksr-1 animals cross-sectioned through their amphid sensory neurons, showing their ciliary transition zones, cilia middle segment (doublet microtubules) and cilia distal segment (singlet microtubules), as indicated. No anomalies in ciliary ultrastructure were seen in the sections of the mutants compared to N2, of which only representative samples for the N2 and mks-1;mksr-2 double mutants are shown. Insets show close-ups of representative (individual) ciliary axonemes. Scale bars: 200 nm (white) and 500 nm (black).