Supplemental Materials

This article contains the following supporting material:

• Figure S1 - Morpholino antisense knockdown of zebrafish ttll6. (A) RTPCR of control (Con) and two individual ttll6 morphant larvae (M) with primers flanking exon 10 of zebrafish ttll6 show complete absence of wildtype mRNA in morphants and inclusion intron 10 in morphant ttll6 mRNA (larger RTPCR products). Exon 10 inclusion introduces a stop codon after 10 missense codons. Red shaded sequence represents sequence complementary to the ttll6 exon 10 morpholino oligo. (B) Sequence alignment and annotation of mouse and zebrafish TTLL6 protein. Green shaded sequence highlights the conserved TTLL core domain, Yellow shading represents the C-terminal deleted sequence the mouse TTLL6 N705 construct (van Dijk et al., 2007) and Blue shading represents the predicted C-terminal deleted sequence in zebrafish Ttll6 exon 10 morphant protein.
• Figure S2 - Control data to validate sequential immunofluorescence using two different mouse antibodies. An antibody blocking protocol enabled independent detection of two mouse monoclonal antibodies based on Negoescu et al. (Negoescu et al., 1994) (see Methods). (A) Following initial incubation of permeabilized larvae with primary mouse monoclonal antibody to glutamylated tubulin (mAb GT335) and detection with Alexa 546 labeled anti mouse secondary antibody, larvae were blocked with 10% normal mouse serum (NMS) and anti-mouse IgG Fab fragments at 1:20 dilution. In the absence of a second mouse antibody, subsequent incubation with Alexa 488 anti-mouse secondary (B) did not result in fluorescence signal, indicating that all 6-11-B1 primary antibody was blocked and the signals we detected in our double monoclonal labeling experiments were specific to the two independent antibody reactivities. (D-F) A reduced concentration of anti-mouse IgG Fab fragments (1:50) in the blocking step was also sufficient to block GT335 reactivity to a second Alexa 488 anti-mouse antibody. (G-I) When the concentration of both normal mouse serum (5%) and anti-mouse IgG Fab fragments (1:50) were reduced, some cross-reactivity of Alexa 546 and Alexa 488 anti-mouse antibodies was detected. This provided the lower limits on the blocking reagent concentrations; all subsequent experiments were performed with 10% normal mouse serum and anti-mouse IgG Fab fragments at a dilution of 1:20 (A-C).
• Movie 1 - Wild-type cilia beating in the pronephric duct. Video was acquired at 250 frames per second and replayed here at 15 fps. The cloaca of this larva was obstructed to expand the pronephric lumen and better show individual cilia beating.
• Movie 2 - fleer mutant cilia show a dramatically reduced beat amplitude. Video was acquired at 250 frames per second and replayed here at 15 fps. fleer mutant cilia have similar beat frequency compared to wild-type cilia but beat amplitude is reduced.
• Movie 3 - Confocal 3D movie of GT335/OSM-10 immunofluorescent stained wild type C. elegans hermaphrodite cilia. Red fluorescence identifies GT335-positive outer labial, inner labial and amphid cilia (see figure 9 and text for details). Green fluorescence identifies OSM-10 positive, amphid neuron dendrites.
• Movie 4 - Confocal 3D movie of GT335/OSM-10 immunofluorescent stained dyf-1 mutant C. elegans hermaphrodite cilia. GT335 anti-polyglutamylated tubulin staining (Red) is absent in outer labial cilia while some staining persists in inner labial and amphid cilia. Green fluorescence identifies OSM-10 positive, amphid neuron dendrites.