Supplemental Information. Supernumerary Centrosomes. Nucleate Extra Cilia and Compromise. Primary Cilium Signaling. Current Biology, Volume 22

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1 Current Biology, Volume 22 Supplemental Information Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary Cilium Signaling Moe R. Mahjoub and Tim Stearns Supplemental Inventory 1. Supplemental Figures and Tables Figure S1, related to Figure 1 Figure S2, related to Figure 1 Tables S1A and S1B, related to Figure 1A 2. Supplemental Experimental Procedures 3. Supplemental References

3 Figure S1. Related to Figure 1 (A) Immunofluorescence images of stably-expressed Htr6-GFP (green) in mono- and bi-ciliated IMCD-3 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 5 µm. Graph (right panel) shows ratio of Htr6-GFP/glutamylated tubulin levels in cilia of mono-, and bi-ciliated cells. (B) Immunofluorescence images of ciliary PKHD-GFP (green) in mono-and bi-ciliated IMCD-3 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) shows ratio of PKHD-GFP/glutamylated tubulin levels in cilia of mono-, and biciliated cells. (C) Immunofluorescence images of PKHD-GFP (green) in mono-ciliated IMCD-3 cells, showing total cellular levels of the fusion protein. Cells were co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) shows distribution of total PKHD1-GFP signal intensity in cells with one and two cilia. (D) Immunofluorescence images of Htr6-GFP (green) in mono-ciliated IMCD-3 cells, showing total cellular levels of the fusion protein. Cells were co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) showing distribution of total Htr6- GFP signal intensity in cells with one and two cilia. (E) Distribution of ciliary levels of PKHD1-GFP (per unit length cilium) compared with total (whole-cell) PKHD1-GFP signal intensity in cells. (F) Distribution of ciliary levels of HTr6-GFP (per unit length cilium) compared with total (whole-cell) HTr6-GFP signal intensity in cells. For all graphs, N = 50 for each sample (from 2 independent experiments; *** indicates P < ).

5 Figure S2. Related to Figure 1 (A) Immunofluorescence images of endogenous Arl13b (green) in mono- and bi-ciliated RPE-1 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) shows ratio of endogenous Arl13b/glutamylated tubulin levels in cilia of mono- and bi-ciliated cells. (B) Immunofluorescence images of endogenous Arl13b (green) in mono-ciliated RPE-1 cells, containing either 2 or >4 centrioles. Cells were co-stained with glutamylated tubulin (G.T., red). Scale bar, 2 µm. Graph (right panel) shows levels of endogenous Arl13b per unit length, in mono-ciliated wild-type and super-centrosomal cells (N = 25 for each sample). (C) Immunofluorescence images of endogenous IFT88 (green) in mono- and bi-ciliated RPE-1 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 5 µm. Graph (right panel) shows ratio of IFT88/glutamylated tubulin levels in cilia of mono- and bi-ciliated cells. (D) Immunofluorescence images of endogenous Smo in mono-ciliated cells, compared to biciliated bi-nucleate MEFs generated by blocking cytokinesis. Cells were co-stained with glutamylated tubulin (G.T.) and DAPI. Scale bar, 10 µm. Graph (right panel) shows the ratio of Smo/glutamylated tubulin levels in these cells. For all graphs, N = 50 for each sample (from 2 independent experiments; *** indicates P < ).

6 Tables S1A and S1B. Related to Figure 1A (A) Frequency of ciliogenesis over time. Centriole amplification was achieved by briefly inducing expression of Plk4 in RPE-1 cells as described in the Methods section. Cells were then grown for 1, 2 or 3 additional days to allow centriole maturation. To induce ciliogenesis, cells were incubated with low-serum (0.5%) medium for 24 h, and the number of cilia per cell was determined for each time point (N = 200 for each sample, from 2 independent experiments). (B) Fraction of centrioles forming cilia over time. Super-ciliated cells were also analyzed on the indicated days for the number of centrioles per cell, the fraction of centrioles that form cilia per cell, and the fraction of mature centrioles per cell. (N = 50, from 2 independent experiments).

7 Supplemental Results Generality of the Ciliary Dilution Phenotype in Superciliated Cells We addressed the generality and nature of the dilution phenotype in super-ciliated cells by examining the ciliary concentrations of two additional receptors that localize to cilia. The serotonin 6 (Htr6) protein is a G-protein-coupled receptor that localizes to the ciliary membrane in neurons [1], and in mouse kidney epithelial (IMCD) cells expressing it from an introduced construct [2]. IMCD-3 cells stably-expressing Htr6-GFP were transfected with Plk4 to produce super-ciliated cells, and the amount of Htr6-GFP per unit length of cilium was determined. As for Smo, the ciliary concentration of Htr6-GFP was reduced in bi-ciliated cells compared to mono-ciliated cells (Supplementary Figure S1A). The same dilution phenotype was also observed in IMCD-3 cells stably-expressing the ciliary-targeted cytoplasmic tail of the fibrocystin protein ( CTS PKHD1-GFP; Supplementary Figure S1B; [3]). The difference in ciliary concentration in mono- and bi-ciliated cells is not due to a difference in the expression level of the introduced protein, as quantification of whole-cell fluorescence values showed a similar range in both types of cells (Supplementary Figure S1C-D). Because there was substantial variation in expression, we could also address whether the ciliary dilution phenotype might be due simply to limiting amounts of the transported receptor protein. For both CTS PKHD1-GFP and Htr6-GFP, the amount of cilium-localized protein in mono-ciliated cells became saturated relative to the whole cell protein, and remained constant over a three-fold concentration range in the whole cell (Supplementary Figure S1E-F). Thus, the dilution of membrane-associated proteins in super-ciliated cells is true for several membrane proteins, and is not due to limitation of the total concentration of the transported protein. We next tested whether the super-ciliated dilution phenotype was restricted to membraneassociated proteins, or was common to other components of cilia. We examined the ciliary concentration of Arl13b, a small GTPase that localizes to the ciliary matrix (the compartment between the membrane and the axoneme) and is mutated in patients with ciliopathies [4]. Expression of Plk4 was induced in human retinal pigment epithelial (RPE-1) cells to generate super-ciliated cells, and the ciliary concentration of endogenous Arl13b was assessed. Similar to the membrane proteins, the ciliary concentration of Arl13b was reduced in bi-ciliated cells compared to mono-ciliated cells (Supplementary Figure S2A), demonstrating that proteins localized within the ciliary matrix may also be diluted in super-ciliated cells. We note that for each of the above proteins, Smo, Htr6, PKHD and Arl13b, the total amount of ciliary protein was similar in mono- and bi-ciliated cells, and that the dilution phenotype is due to partitioning of that protein over two cilia. To determine whether the ciliary dilution phenotype was due to the presence of extra cilia, rather than extra centrioles, we compared normal cells (containing 2 centrioles and one cilium) with cells bearing extra centrioles, but only one cilium. The ciliary concentration of endogenous Arl13b was identical in both cases (Supplementary Figure S2B), indicating that the dilution phenotype is due specifically to the presence of extra cilia. Finally, since super-ciliated cells assembled cilia of similar length to those in monociliated cells, we reasoned that components of the ciliary machinery might not display the dilution phenotype we have described for molecules involved in ciliary signaling. We examined the ciliary concentration of IFT88, a conserved component of the IFT machinery that is essential for the assembly of cilia from Chlamydomonas to mammals [5]. Consistent with this hypothesis,

8 the ciliary concentration of IFT88 was equal in mono- and bi-ciliated cells (Supplementary Figure S2C). In the above experiments the number of cilia per cell was manipulated while keeping ploidy constant. We considered the possibility that the ratio of cilia to genome might determine the amount of transported signaling molecules, and thus examined cells in which both cilium number and ploidy were increased two-fold by blocking cytokinesis. NIH3T3 cells were enriched in S-phase by a single thymidine block for 16 h, released and allowed to progress to mitosis. The cells were treated with cytochalasin B for 1.5 h to block cytokinesis, and released into low-serum medium to induce ciliogenesis, then treated with Shh and assessed for ciliary Smo as described above. In this case, bi-nucleate, bi-ciliated cells were compared with mononucleate, mono-ciliated cells in the same population, which had presumably not undergone mitosis during the cytochalasin treatment. Remarkably, the bi-nucleate bi-ciliated cells displayed the same Smo dilution phenotype with respect to the mono-nucleate mono-ciliated cells (Supplementary Figure S2D). This suggests that the mechanism determining ciliary protein levels assesses the number of cilia per cell, rather than cilia per unit genome. Supplemental Experimental Procedures Cell Culture and Media Cultured mammalian cells including MEFs, NIH3T3, NIH3T3::Gli-GFP (gift from James Chen, Stanford, CA), RPE-1::Tet-Plk4 (gift from Bryan Tsou, Sloan-Kettering, NY), Tsc2 +/+ and Tsc2 - /- MEFs (gift from Elizabeth Henske, Harvard Medical School, MA) were grown in DMEM with 10% FBS (Invitrogen). IMCD-3, IMCD-3::Htr6-GFP and IMCD-3:: CTSPKHD1-GFP (gift from Max Nachury, Stanford, CA) cells were grown in DMEM/F12 medium with 10% FBS. Cilium formation was induced by incubating cells in low-serum medium (0.5% serum) for 24 h. All media were supplemented with 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). For spheroid formation assays, IMCD-3 cells were trypsinized, washed with PBS and resuspended in DMEM/F12 supplemented with 2% FBS and 2% growth factor-deleted Matrigel (BD Bioscience). Lab-Tek chamber slides (8-well) were treated with 40 µl of 100% Matrigel and incubated at 37 C for 15 min to allow the Matrigel to solidify. Approximately 5,000 cells/well were layered over the bed of Matrigel and grown at 37 C for 4 days to form spheroids. Electron Microscopy For TEM analysis, RPE-1::Tet-Plk4 cells were grown on Aclar strips, and centriole amplification/ciliogenesis was induced as described above. Cells were fixed with PBS containing 4% paraformaldehyde and 2% glutaraldehyde for 5 h, post fixed in 1% osmium tetroxide (Electron Microscopy Systems) for 1 h at room temperature, washed 3X with filtered water, then en-bloc stained for 2 h at room temperature (or incubated at 4 o C overnight). The cells were then dehydrated in a series of 50%, 70%, and 95% ethanol washes for 5 minutes each at 4 o C, allowed to rise to room temperature, washed twice with 100% ethanol, followed by incubation in acetonitrile for 15 min. Samples were infiltrated with a 1:1 mixture of acetonitrile:embed-812 resin (Electron Microscopy Systems), followed by 1:2 mixture, then EMbed-812, with each incubation lasting 2 h. Polymerization was achieved by incubating the samples at 65 o C overnight. 90 nm thin-sections were collected on formvar/carbon coated slot grids or 100 mesh

9 Cu grids (Electron Microscopy Systems), contrast stained for 30 seconds in 1:1 saturated uranyl acetate (~7.7%) to 100% acetone followed by 30 seconds of staining in 0.2% lead citrate. Grids were imaged in a JEOL JEM-1400 TEM at 120 kv and images taken using a Gatan Orius digital camera. Supplemental References 1. Hamon, M., Doucet, E., Lefevre, K., Miquel, M.C., Lanfumey, L., Insausti, R., Frechilla, D., Del Rio, J., and Verge, D. (1999). Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors. Neuropsychopharmacology 21, 68S-76S. 2. Berbari, N.F., Johnson, A.D., Lewis, J.S., Askwith, C.C., and Mykytyn, K. (2008). Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors. Mol Biol Cell 19, Follit, J.A., Li, L., Vucica, Y., and Pazour, G.J. (2010). The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence. J Cell Biol 188, Lim, Y.S., Chua, C.E., and Tang, B.L. (2011). Rabs and other small GTPases in ciliary transport. Biol Cell 103, Pazour, G.J., Dickert, B.L., Vucica, Y., Seeley, E.S., Rosenbaum, J.L., Witman, G.B., and Cole, D.G. (2000). Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 151,

Report. Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary Cilium Signaling

Report. Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary Cilium Signaling Current Biology 22, 1628 1634, September 11, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.06.057 Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary