TTLL12 is required for primary ciliary axoneme formation in polarized epithelial cells

Abstract

The primary cilium is a critical sensory organelle that is built of axonemal microtubules ensheathed by a ciliary membrane. In polarized epithelial cells, primary cilia reside on the apical surface and must extend these microtubules directly into the extracellular space and remain a stable structure. However, the factors regulating cross-talk between ciliation and cell polarization, as well as, axonemal microtubule growth and stabilization in polarized epithelia are not fully understood. In this study, we find TTLL12, a previously uncharacterized member of the Tubulin Tyrosine Ligase-Like (TTLL) family, localizes to the base of primary cilia and is required for cilia formation in polarized renal epithelial cells. We also show that TTLL12 directly binds to the α/β-tubulin heterodimer in vitro and regulates microtubule dynamics, stability, and post-translational modifications (PTMs). While all other TTLLs catalyze the addition of glutamate or glycine to microtubule C-terminal tails, TTLL12 uniquely affects tubulin PTMs by promoting both microtubule lysine acetylation and arginine methylation. Together, this work identifies a novel microtubule regulator and provides insight into the requirements for apical extracellular axoneme formation.

Figure 1:. Identification of TTLL12 as a Rab19 binding partner

A. Schematic of the TTLL family. Blue indicates TTL is a tyrosinase. *Indicates a glutamylase that functions in a complex. **Indicates a glutamylase that is classified only by sequence similarity. B. Identification of TTLL12 binding partners. Spectral counts of candidate proteins identified through co-immunoprecipitation/mass spectrometry on GFP-TTLL12. The full list of proteins identified is in Table S1. C. TTLL12 binding to tubulin. Immunoprecipitation of GPF-TTLL12 followed by western blot for α-tubulin. Left column shows lysates (input) probed for GFP and α-tubulin. Right column shows immunoprecipitates probed for GFP and α-tubulin.

Figure 2:. TTLL12 directly binds to the α/β-tubulin heterodimer

A. Microtubule and TTLL12 co-sedimentation assay. Taxol-stabilized porcine brain microtubules were mixed with recombinant 6His-TTLL12 and pelleted by centrifugation. The supernatant and pellet were separated and run on an SDS/PAGE gel followed by Coomassie blue staining. B. Quantification of TTLL12 in the pellet with or without microtubules from A. Graph shows mean ± SD. C. TTLL12 binding to the tubulin heterodimer. Binding assay with recombinant 6His-TTLL12 and porcine brain tubulin. TTLL12 and tubulin were mixed O/N at 4° C to prevent microtubule polymerization followed by the addition of nickel beads. Bead elutates were run on an SDS/PAGE gel followed by Coomassie blue staining. D. Quantification of tubulin band intensity in C. Graph shows mean ± SD. E. TTLL12 N-terminus binding to tubulin. Binding assay with recombinant GST-TTLL12 (aa 1-260) and porcine brain tubulin. TTLL12 and tubulin were mixed O/N at 4° C followed by the addition of glutathione beads and western blot probing for GST and α-tubulin. F. Quantification of α-tubulin band intensity in E. Graph shows mean ± SD. G. Competitive binding experiment between recombinant 6His-TTLL12, GST-Rab19, and untagged porcine brain tubulin. Increasing concentrations of GST-Rab19 were added to 6His-TTLL12 and tubulin followed by the addition of nickel beads. The presence of TTLL12 and Rab19 was confirmed by Coomassie and western blot was used to probe for α-tubulin. H. Quantification of α-tubulin band intensity in G. Graph shows mean ± SD.

Figure 3:. TTLL12 regulates microtubule stability and dynamics

A. Representative western blot and quantification of acetylated α-tubulin in MDCK WT, TTLL12 KO, and TTLL12 KO +GFP-TTLL12 cells. N=4. Graph shows mean ± SD. B. Microtubule cold-stability assay. Representative images of MDCK WT and TTLL12 KO cells placed at 4° C for 30 minutes to depolymerize microtubules and stained for DNA and tubulin. Scalebar = 5 µm. C. Quantification of tubulin intensity from A. Data from three independent experiments (N=3, n=30 for WT, n=30 for KO). Graph shows median and quartiles. D. Representative images of MDCK WT and TTLL12 KO cells treated with 1µM Taxol before 4° C for 30 minutes and stained for DNA and tubulin. Scalebar = 5 µm. E. Quantification of tubulin intensity from Taxol-treated cells in C. Data from three independent experiments (N=3, n=19 for WT, n=20 for KO). Graph shows median and quartiles. F. Representative western blot and quantification of acetylated α-tubulin in RPE1 WT, TTLL12 KO, and TTLL12 KO 2 cells. N=3. Graph shows mean ± SD. G. Example image of WT RPE1 cell expressing GFP-MACF18 used for live imaging of microtubule polymerization. Scalebar = 5 µm. H. Quantification of the number of microtubule polymerization events that occur in each cell over the course of 4 minutes. Images were obtained from three independent experiments. n=13 cells for both WT and TTLL12 KO. Graph shows mean ± SD. I. Quantification of microtubule polymerization rates measured from GFP-MACF18 comets. Violin plot represents all microtubules measured (n=20,427 for WT, n=19,525 for KO). Black circles represent the average polymerization rate from each independent experiment (N=3). T-test was performed on the means from the experiment. J. Quantification of MACF18 comet lifetime. Plot represents all microtubules measured (n=20,427 for WT, n=19,525 for KO). Black circles represent the average polymerization rate from each independent experiment (N=3). T-test was performed on the means from the experiment.

Figure 4:. TTLL12 regulates ciliation timing and length in RPE1 cells

A. Representative images of RPE1 WT and TTLL12 KO cells grown on collagen-coated glass coverslips, serum starved for 48 hours, and stained for DNA, Arl13b, and acetylated tubulin (Ac. Tub). Scalebar = 5 µm. B. Representative images of RPE1 WT and TTLL12 KO cells serum starved for 8 or 48 hours and stained for DNA, Arl13b, and GT335 (Glut. Tub.). Scalebar = 5 µm. Yellow arrowhead indicates a shorter than average cilium and yellow arrow indicates a longer than average cilium. C. Quantification of ciliation frequency through a serum starvation time course using Arl13b and GT335 as markers for cilia. Graph shows mean ± SD. D. Quantification of the rate of ciliation from the time course in C. Graph shows mean ± SD. E. Quantification of primary cilium length after 48 hours of serum starvation. Violin plot represents all primary cilia measured. Black circles represent average cilium length from three independent experiments, on which a t-test was performed (N=3, n=1438 for WT, n=1352 for KO). F. Quantification of the length variance from three independent experiments. Graph shows mean ± SD. G. Representative images of TTLL12 localization in ciliated RPE1 WT and TTLL12 KO cells. Scalebar = 5 µm. Scalebar = 1 µm in inset. H. Quantification of TTLL12 at the base of the primary cilium from G. N=1 and 35 cells were measured in each condition. Graph shows mean ± SD.

Figure 5:. TTLL12 is required for axoneme formation in polarized epithelial cells

A. Representative images of MDCK WT, TTLL12 KO, and TTLL12 KO cells stably expressing GFP-TTLL12 grown on transwell filters, polarized and stained for actin, gamma-tubulin, and acetylated tubulin (Ac. Tub). Scalebar = 5 µm. B. Inset from A (white boxes). Scalebar = 5 µm. C. Quantification of cells with a primary cilium marked by acetylated tubulin in A. Graph shows mean ± SD from three independent experiments. D. Representative images of MDCK WT and TTLL12 KO cells stained for actin, Arl13b, and GT335. Scalebar = 5 µm. E. Quantification of cells with a primary cilium marked by glutamylated tubulin (GT335). Graph shows mean ± SD from three independent experiments. F. Representative images of MDCK cells stained for actin, gamma-tubulin, and Arl13b. Scalebar = 5 µm. G. Quantification of cells with Arl13b at the centrosome (marked by gamma tub.). Graph shows mean ± SD from three independent experiments. H. Quantification of Arl13b intensity at each gamma tubulin puncta. Black circles represent average Arl13b intensity from three independent experiments (N=3, n=1658 for WT, n=1616 for KO). Graph shows mean ± SD. I. SEM images of the apical surface of MDCK WT and TTLL12 KO polarized on transwell filters. Images are at x4000 magnification with 5 µm scale bar. White arrows indicate primary cilia-like structures. Boxes mark the location of higher magnification insets.

Figure 6:. TTLL12 regulates tubulin methylation

A. Alpha-fold structure of TTLL12. SET domain is purple. TTL domain is blue. Amino acids not in defined domains are green. B. Fluorescence-based assay to measure methyltransferase activity. 1µM 6His-TTLL12 was incubated with methyl donor SAM and 6µM porcine brain tubulin and fluorescence was measured over time (N=3). Mass spectrometry identifying methylation of tubulin alone and TTLL12 + tubulin is in Table S2. C. Methyltransferase activity was calculated using the slopes of the curves from C (inside the dotted box). D. Immunoprecipitation of mono-methyl arginine from RPE1 cells followed by western blot for α-tubulin. Left columns show lysates (input) probed for Rme1 and α-tubulin. Right columns show immunoprecipitates probed for Rme1 and α-tubulin. E. Quantification of α-tubulin band from D. IgG control was subtracted as background. Graph shows mean ± SD from four independent experiments. F. Immunoprecipitation of di-methyl arginine from RPE1 cells followed by western blot for α-tubulin. Left column shows lysates (input) probed for Rme2 and α-tubulin. Right columns show immunoprecipitates probed for Rme2 and α-tubulin. G. Quantification of α-tubulin band from F. IgG control was subtracted as background. Graph shows mean ± SD from three independent experiments.

Figure 7:. Model Figure

In polarized epithelial cells, the centrosome migrates to the apical surface, and Rab19 is required for apical actin clearing and initial ciliary membrane recruitment. TTLL12 also resides at the centrosome and interacts with the α/β-tubulin heterodimer to promote tubulin arginine methylation and lysine acetylation, which is required for axoneme growth and stability.