1. TTLL3 Is a Tubulin Glycine Ligase that Regulates the Assembly of Cilia
Dorota Wloga, Danielle M. Webster, Krzysztof Rogowski, Marie-Hélène Bré, Nicolette Levilliers, Maria Jerka-Dziadosz, Carsten Janke, Scott T. Dougan, Jacek Gaertig 
Developmental Cell 
Volume 16, Issue 6, Pages (June 2009)
DOI: /j.devcel
Copyright © 2009 Elsevier Inc. Terms and Conditions

2. Figure 1 TTLL3 Enzymes Increase Tubulin Monoglycylation in Tetrahymena
(A and B) A cell expressing GFP-Ttll3Ap before (A) and after (B) induction with 2.5 μg/ml CdCl2 for 5 hr, labeled by TAP952.
(C) The GFP fluorescence in GFP-Ttll3Ap-expressing cells. Scale bar, 10 μm. c, cilia; oc, oral cilia.
(D) A fluorogram of an SDS-PAGE gel with brain microtubule proteins after in vitro glycylation with fractions enriched in overexpressed TTLL3 proteins of Tetrahymena.
(E) A graph showing levels of incorporation of 3H-glycine (in disintegrations per min) into brain α- or β-tubulin in in vitro glycylation assays.
(F) Western blots of ciliary proteins from wild-type and cells overproducing either GFP-Ttll3Ap or GFP-DN-Ttll3Ap.
(G) A Western blot of cells expressing either GFP-Ttll3Ap or GFP-DN-Ttll3Ap before (−) and after (+) cadmium induction, probed either with anti-GFP or anti-α-tubulin antibodies.
Developmental Cell  , DOI: ( /j.devcel )
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3. Figure 2
Disruption of TTLL3 Genes Reduces Tubulin Glycylation and Increases Tubulin Glutamylation
(A–D) WT (fed with India ink) and 3AB-KO cells (arrows) imaged side by side. (A) and (C) show immunofluorescence with TAP952 and AXO49 respectively, while (B) and (D) show the corresponding phase contrast images. Insets show higher magnifications (2.5×) of boxed areas of cilia.
(E–E′) Growth curves of WT and TTLL3 knockout strains grown without (E) or with (E′) 35 μM paclitaxel. Note that in (E), WT and 3AB-KO curves completely overlap.
(F) Western blots of ciliary proteins from WT (loaded at multiple dilutions) and TTLL3 knockout strains.
(G) A graph representing the length of cilia in WT and TTLL3 knockout cells. Error bars represent SEM. The differences between WT and 3AB-KO, 3ABCD-KO, and 3ABCDEF-KO cells were statistically significant (p < ).
(G′–G″) Immunofluorescence images of cilia in WT and sextuple knockout cells labeled with anti-tubulin antibodies. Images of the entire cells are shown in Figures S3K and S3L.
(H–K) Immunofluorescence with anti-total tubulin antibodies of Tetrahymena cells that are either WT (H and J) or 3AB-KO (I and K), and were either untreated (H–I) or treated with 25 μM paclitaxel for 4 hr (J and K).
Developmental Cell  , DOI: ( /j.devcel )
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4. Figure 3
TTLL3 Proteins and Tubulin Glycylation Are Restricted to Outer Microtubules and Dominant-Negative TTLL3 Disrupts the Axoneme Structure
(A–G) TEM cross-sections of axonemes of WT (A) and GFP-DN-Ttll3Ap-overproducing cells (B–G). The arrow in (G) marks an abnormal triplet. Scale bar, 0.2 μm.
(H–J) Immunogold localization of either GFP-Ttll3Ap (H) or GFP-DN-Ttlll3Ap (I and J) with anti-GFP antibodies. Scale bar, 0.2 μm.
(K–L) Longitudinal TEM sections of cilia of GFP-DN-Ttll3Ap overproducing cells. Scale bar, 0.2 μm.
(M and N) TEM cross-sections of axonemes of 3AB-KO (M) and 3ABCDEF-KO (N) cells. Scale bar, 0.2 μm.
(O and P) Immunogold localization of monoglycylated (O) or polyglycylated tubulin (P) in WT cells using TAP952 and AXO49 mAb, respectively. Scale bar, 0.2 μm.
Developmental Cell  , DOI: ( /j.devcel )
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5. Figure 4
Expression of a Dominant-Negative TTLL3 Affects Ciliary Length
(A–C) Cells expressing GFP-DN-Ttll3Ap induced with 2.5 μg/ml CdCl2 for 6 hr. The GFP fluorescence is shown in (A), AXO49 immunofluorescence in (B), and a merged image in (C). GFP-DN-Ttlll3Ap is enriched in short, newly formed cilia (arrowheads) and in the distal segments of long preexisting cilia (arrows).
(D–F) WT (D), GFP-Ttll3Ap- (E), and GFP-DN-Ttll3Ap (F)-overexpressing cells were labeled with 12G10 anti-α-tubulin (red) and polyG antibodies (blue). Arrows point at short, new cilia, while arrowheads mark long, pre-existing cilia.
(G–I′) WT (G and G′) and either GFP-Ttll3Ap- (H and H′) or GFP-DN-Ttll3Ap (I and I′) -overproducing cells were double labeled with ID5 anti-polyglutamylated tubulin (red) and polyG antibodies (blue). Note that short cilia and distal segments of elongated cilia in GFP-DN-Ttll3Ap cells are hypoglycylated and hyperglutamylated. Scale bar, 10 μm.
(J–L) Graphs representing the distribution of axoneme lengths in cells overexpressing either GFP-Ttll3Ap (J) or GFP-DN-Ttll3Ap (K and L) that were uninduced (black diamonds) or induced with CdCl2 (white circles) for 6 (K) or 11 hr (J and L). Each data point represents a single measured axoneme.
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6. Figure 5
TTLL3 Is Required for Tubulin Glycylation and Cilia Assembly in Zebrafish
(A–F) Images of live control and morphant embryos at 24 (A and B), 48 (E and F), and 55 hpf (C and D). Hydrocephaly is apparent in TTLL3-ATG morphant embryos, as indicated by the enlarged vesicles in the hindbrain (B and F, arrows).
(G) The head of a WT embryo stained by 6-11 B1 antibodies at 72 hpf (the olfactory placode is marked with an arrow).
(H–I″) In olfactory cilia, TTLL3-ATG MOs reduce the levels of tubulin monoglycylation detected by TAP952 mAb at 72 hpf (compare [H] and [I]), but not the levels of tubulin polyglutamylation revealed by polyE antibodies (H′ and I″).
(J and K) The olfactory cilia stained with AXO49 anti-polyglycylated tubulin mAb in a control (J) and a TTLL3-ATG morphant (K) at 72 hpf.
(L and M) Olfactory cilia stained with 6-11 B-1 mAb in a control (L) and a TTLL3-ATG morphant (M) at 72 hpf.
(N–R) SEM images of the zebrafish olfactory placode at 72 hpf in control (N and O) and a TTLL3-ATG morphant (P and R); (O) and (R) are higher magnifications of areas of placodes shown in (N) and (P).
(S) TEM image of a fragment of the olfactory placode in the TTLL3-ATG embryo.
Developmental Cell  , DOI: ( /j.devcel )
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7. Figure 6 ttll3 MOs Reduce Tubulin Monoglycylation in Zebrafish Cilia
(A–B′) Ear in a control (A and A′) and a morphant fish (B and B′) labeled with TAP952 mAb. (A′) and (B′) show higher magnifications (5×) of areas boxed in (A) and (B).
(C–F) The pronephric duct area in control (C and E) and morphant fish (D and F) labeled with TAP952 anti-monoglycylated tubulin mAb (C and D) and GT335 anti-glutamylated tubulin mAb (E and F). Note lack of cilia staining in morphants in corresponding area (all indicated by arrows).
(G–J) The spinal cord and hypochord area in control (G and I) and morphant fish (H and J) labeled with TAP952 (G and H) and GT335 mAb (I and J). Arrows indicate cilia in spinal cord, while arrowheads point to cilia in hypochord in control embryos (G and I). Note lack of cilia staining in morphants in corresponding regions, as indicated by arrows and arrowheads (H and J).
Developmental Cell  , DOI: ( /j.devcel )
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8. Figure 7
Depletion of ttll3 Expression Produces Defects in the LR Asymmetry in Zebrafish
Ventral views of 22 hpf embryos stained for expression of lefty1 (A–D), or 33 hpf embryos stained for expression of cmlc2 (E–H). Insets in (A)–(D) depict 4× magnifications of the heads of embryos. In all cases, the left side of the embryo is to the left of the panel. lefty1 is normally expressed on the left side of the epiphysis (A). ttll3 morphant embryos express lefty1 on the left body side (B), or display bilateral (C) or right-sided expression (D). Arrows mark the epiphysis. In untreated embryos, cmlc2 (E) is expressed in the myocardium, which is normally located in the left lateral plate. The position of the heart field is unaltered in some TTLL3 morphants (F). Other morphants display midline (G) or right-side expression (H). Arrows in (E)–(H) mark the heart field. (I and J) Cilia in KV stained with 6-11 B-1 anti-acetylated K40 α-tubulin mAb in a control (I) and a TTLL3-ATG morphant (J) at 8-10 somite stage at 14 hpf. (K) A graph represents the average length of cilia in KV in control (n = 3) and TTLL3-ATG morphants (n = 3). Data are presented as means ± SEM.
Developmental Cell  , DOI: ( /j.devcel )
Copyright © 2009 Elsevier Inc. Terms and Conditions