Supplemental Information. Glutamylation Regulates Transport, Specializes. Function, and Sculpts the Structure of Cilia

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1 Current Biology, Volume 27 Supplemental Information Glutamylation Regulates Transport, Specializes Function, and Sculpts the Structure of Cilia Robert O'Hagan, Malan Silva, Ken C.Q. Nguyen, Winnie Zhang, Sebastian Bellotti, Yasmin H. Ramadan, David H. Hall, and Maureen M. Barr

2 A Length of PKD-2::GFP Accum. (µm) wild type (35) (19) (11) ; (23) * b (26) B Length of PKD-2::GFP Accum. (µm) R3B b (13) ; (9) (3) (10) wild type (14) Figure S1 mutants were PKD-2::GFP ciliary localization (Cil) defective; related to Figure 1). We measured the length of PKD-2::GFP accumulations in ciliary bases and dendrites of and R3B neurons. Length of patches was more variable in neurons in the head, but abnormal PKD-2::GFP patches in the tail were clearly expanded in all mutant genotypes. (Mean ± sem; indicates p<0.0001, * indicates p=0.013 by ANOVA and post-hoc Tukey tests; number in parentheses indicates n neurons scored.)

3 OLQ or PKD-2::GFP polye / 10 µm b * * * ; ;b Figure S2 Detection of long glutamate side chains by indirect immunofluorescence (related to Figure 2). The IN105 polye antibody recognizes linear side-chains of 3 or more glutamates S1]. Results were similar to those in Figure 2, in which GT335 was used to detect the branch point of glutamate side-chains S2]. We stained males on the first day of adulthood and found that, in wild type males, the channel ciliary middle segments and OLQ or cilia were brightly stained. Loss of both TTLL-11 isoforms in (tm4059) mutants abolished polyglutamylation side-chains from neuronal sensory cilia. However, in b(gk482) mutants, polye detected ciliary middle segments, although possibly less abundantly than in wild type. In mutants, polye staining was seen in additional neuronal cilia in the nose. In, polye staining abnormally localized posterior to middle segment regions in a diffuse dendritic trail marked by speckles (*). In ; double mutants, polye staining of sensory cilia was virtually abolished but the speckled trail of staining posterior to the middle segment was still visible (*). In ;b mutants, ciliary middle segments and other cilia were illuminated by polye staining. The trailing region of speckled polye staining was also visible (*).

4 OSM-3::GFP B ; 2.45µm 3.78µm 3.4µm C KAP-1::GFP 3.7µm 2.9µm ; 3.2µm 2.8µm 2.4µm duration: 20s Cilium Length duration: 40s duration: 40s Anterograde Retrograde 4.5µm 3.1µm ; GFP::KLP-6 A 3.2µm 2.8µm Figure S3 Images of GFP-tagged kinesin markers in cilia and kymographs of motile puncta (related to Table 1). cilia are indicated by orange brackets, scale bar= 3 µm for still images of each kinesin marker (anterior is right, to match kymographs). Kymographs were created using Kymograph Direct S3] in genotypes indicated above each image for (A) Kinesin-3 GFP::KLP-6; (B) Kinesin-2 OSM3::GFP; and (C) Kinesin II non-motor subunit KAP-1::GFP. Analysis of velocities shown in Table 1.

5 Fig. S3. PKD-2::GFP-labeled EVs A C Adult Nose Molting L4 Tail Adult Tail Wild Type PKD-2::GFP 10 µm 20 µm ; 20 µm D B L4 Male Tail (19) (10) ** Adult Male Tail ; b (11) (10) (4) (29) ; b (18) (15) (9) (9) Figure S4 PKD-2::GFP-labeled EVs in L4 and adult males (related to Figure 4). (A) In late wild-type L4 males, abundant EVs released from RnB ciliated neurons were trapped in the molting L4 cuticle in the tail S4].,, and ; had few EVs in molting L4 cuticles. Yellow dotted lines indicate outline of molting L4 cuticle. (B) Quantification of EVs trapped in L4 cuticles. (C) Abundant EVs were also shed by adult males ciliated sensory neurons into the environment surrounding the nose and tail in wild type.,, and ; adult males had few EVs surrounding the nose and tail. Note that nose images for wild type and here are the same as in Figure 4. (D) Quantification of PKD-2::GFP-labeled EVs released by the adult male tail. The same animals were used here for scoring EVs released from the adult male tail here and EVs released from the adult male nose in Figure 4 (Mean ± sem; **p<0.001, p< by ANOVA and post-hoc Tukey tests. N animals scored in parentheses for each genotype.)

6 wild type 200 nm ; Figure S5 EVs shed into the glial lumen surrounding cephalic neuron cilia were visible in TEM sections (related to Figure 4). Representative images near the transition zone were selected for each genotype. For all images, the dotted line indicates the border of the lumen formed by glial cells surrounding the and neurons. Abnormally abundant EVs were present in and mutants. Images sizes were adjusted so that scale bar applies to all four images. Magnification settings used to capture images were: 46,000X (wild type and ;); 25,000X ( and ). Ciliary defects occur rarely in wild type s. In this case, although a doublet MT was displaced in the wild type (and a vesicle was present in ), we did not exclude this sensillum from our examination of EV counts.

7 Mutant Glutamylation of /RnB Cilia (GT335) PKD-2::GFP Localization GFP::KLP-6 Localization KLP-6 Vel. OSM-3 Vel. + or "* - - " "! - +! + MT Structure Reduced # singlets Stabilized doublets Released EVs Lumenal EVs! "! " ;! Stabilized doublets! + Table S1: Summary of mutants and phenotypes examined (related to Figures 1, 2, 3, 4, 5, 6, and Table 1) Wild-type phenotype indicated by +, mutant phenotype indicated by -, reduction in intensity, velocity or amount indicated by!, increase indicated by ". *indicates that under experimental conditions used here, GT335 staining was not increased relative to, however previous experiments detected an increase in GT335 staining in neurons in mutants relative to wild type. Supplemental References S1. van Dijk, J., Rogowski, K., Miro, J., Lacroix, B., Edde, B., and Janke, C. (2007). A targeted multienzyme mechanism for selective microtubule polyglutamylation. Molecular cell 26, S2. Wolff, A., de Nechaud, B., Chillet, D., Mazarguil, H., Desbruyeres, E., Audebert, S., Edde, B., Gros, F., and Denoulet, P. (1992). Distribution of glutamylated alpha and betatubulin in mouse tissues using a specific monoclonal antibody, GT335. European journal of cell biology 59, S3. Mangeol, P., Prevo, B., and Peterman, E.J. (2016). KymographClear and KymographDirect: two tools for the automated quantitative analysis of molecular and cellular dynamics using kymographs. Mol Biol Cell 27, S4. Wang, J., Silva, M., Haas, L.A., Morsci, N.S., Nguyen, K.C., Hall, D.H., and Barr, M.M. (2014). C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr Biol 24, !