Palmitoylation of acetylated tubulin and association with ceramide-rich platforms is critical for ciliogenesis

Abstract

Microtubules are polymers composed of αβ-tubulin subunits that provide structure to cells and play a crucial role in in the development and function of neuronal processes and cilia, microtubule-driven extensions of the plasma membrane that have sensory (primary cilia) or motor (motile cilia) functions. To stabilize microtubules in neuronal processes and cilia, α tubulin is modified by the posttranslational addition of an acetyl group, or acetylation. We discovered that acetylated tubulin in microtubules interacts with the membrane sphingolipid, ceramide. However, the molecular mechanism and function of this interaction are not understood. Here, we show that in human induced pluripotent stem cell-derived neurons, ceramide stabilizes microtubules, which indicates a similar function in cilia. Using proximity ligation assays, we detected complex formation of ceramide with acetylated tubulin in Chlamydomonas reinhardtii flagella and cilia of human embryonic kidney (HEK293T) cells, primary cultured mouse astrocytes, and ependymal cells. Using incorporation of palmitic azide and click chemistry-mediated addition of fluorophores, we show that a portion of acetylated tubulin is S-palmitoylated. S-palmitoylated acetylated tubulin is colocalized with ceramide-rich platforms in the ciliary membrane, and it is coimmunoprecipitated with Arl13b, a GTPase that mediates transport of proteins into cilia. Inhibition of S-palmitoylation with 2-bromo palmitic acid or inhibition of ceramide biosynthesis with fumonisin B1 reduces formation of the Arl13b-acetylated tubulin complex and its transport into cilia, concurrent with impairment of ciliogenesis. Together, these data show, for the first time, that ceramide-rich platforms mediate membrane anchoring and interaction of S-palmitoylated proteins that are critical for cilium formation, stabilization, and function.

Keywords: Arl13b; acetylation; cell biology; ceramide; cilia; lipid rafts; lipids; microtubules; palmitoylation; tubulin.

Fig. 1

Ceramide colocalizes with acetylated tubulin and stabilizes microtubules in human iPS cell–derived neuroprogenitors and neurons. Human iPS cells were differentiated to neural progenitors (A–C) and then to neurons (D, E). A–C: Immunolabeling with anti-ceramide (green) and anti-acetylated tubulin antibodies (red). In addition, labeling of the neural progenitor marker nestin is shown in (A) and (C) (blue). D: Immunolabeling shows juxtaposition of ceramide (red) to microtubules (acetylated tubulin in green). E, F: Incubation with 10 μM FB1 (middle panel) or 10 μM FB1, and 2 μM C24:1 ceramide (right panel) shows that inhibition of ceramide biosynthesis disrupts neuronal process formation (middle panel), whereas addition of C24:1 ceramide rescues neuronal processes (right panel, the left panel is control). Acetylated tubulin is shown in green. Differentiation is not likely to be affected as shown by expression of the astrocyte marker glial fibrillary acidic protein (red). N = 5. P < 0.001. FB1, fumonisin B1; iPS, induced pluripotent stem.

Fig. 2

Ceramide forms complexes with acetylated tubulin at microtubules in neuronal processes and cilia. A: PLA for CAPs forming a complex with ceramide at CRPs, the principle of the method. Anti-ceramide rabbit IgG and anti-CAP mouse IgG (here acetylated tubulin) mouse IgG are used for the PLA reaction (red in A–H). Additional immunolabeling with secondary antibodies then shows where the complex is localized within the distribution of ceramide (blue) and acetylated tubulin (green). B: Immunolabeling and PLA using human iPS cell–derived neurons. C–E: The same as in (B) for Chlamydomonas reinhardtii. F: Immunolabeling of ceramide and its complex with acetylated tubulin in primary cilia of HEK293T cells. G: The same as in (F) for primary cultured mouse astrocytes. H: The same as in G for primary cultured mouse ependymal cells.

Fig. 3

Acetylated tubulin is S-palmitoylated in Chlamydomonas reinhardtii. A: Metabolic labeling of S-palmitoylated proteins with palmitic azide (PAz), the principle of the method. Cells (here C. reinhardtii) are metabolically labeled with PAz and then subjected to click reaction–mediated addition of a fluorophore (here Cy5) to the S-palmitoylated protein. 2-Bromo palmitic acid (2BPA) is used to specifically inhibit S-palmitoylation. B–D: SDS-PAGE and immunoblot of protein from C. reinhardtii metabolically labeled with PAz and subjected to click reaction as described in (A). B: Ponceau staining; (C) Cy5 fluorescence of S-palmitoylated protein (pseudocolored in red); (D) immunoblot for acetylated tubulin (pseudocolored in green). −PAz is the negative control without PAz. E–J: Immunoprecipitation of S-palmitoylated protein using anti-acetylated tubulin rabbit IgG for the precipitation reaction and anti-acetylated tubulin mouse IgG for immunoblotting. −PAz is the negative control without S-palmitoylation. E–G: Show immunoblot of the precipitate with metabolic PAz labeling, but without inhibition of S-palmitoylation. H–J: The same as in (E–G), but with inhibition of S-palmitoylation (2BPA). αaT, precipitation reaction with anti-acetylated tubulin rabbit IgG; IgG, precipitation control with nonspecific rabbit IgG; In, input.

Fig. 4

S-palmitoylation of acetylated tubulin in flagella is critical for Chlamydomonas reinhardtii motility. A: PLA reaction for S-palmitoylated acetylated tubulin shows signals in the flagella (red, arrows). Acetylated tubulin is shown in green. B, C: Inhibition of S-palmitoylation with 2BPA shows reduction in the number of flagella (acetylated tubulin, green; ceramide, red). D: Motility assay. 2BPA impairs swimming velocity. E–H: Quantitation of (B–D). E: Shows the number of ciliated C. reinhardtii, F shows the average flagella length, (G) shows the proportion of cells that swim toward the direction of the light source in (D) after 20 min, and (H) shows the proportion of swimmers that reach the end of the well after half-time (10 min in D). N = 5. P as indicated in figure.

Fig. 5

Acetylated tubulin in microtubules of mammalian cells is S-palmitoylated. A: Metabolic labeling of HEK293T cells with PAz, with or without inhibition of S-palmitoylation with 2BPA, followed by click chemistry–mediated addition of fluorophore (Cy5) and immunoprecipitation using anti-acetylated tubulin rabbit IgG. Immunoblot developed with anti-acetylated tubulin mouse IgG (pseudocolored in green). Palmitoylation is pseudocolored in red. B, C: PLA reaction for S-palmitoylation of acetylated tubulin shows signal at primary cilia of HEK293T cells (B, arrow) and along microtubules of neuronal processes in human iPS cell–derived neurons (C, arrows). Acetylated tubulin labeling is shown in green and S-palmitoylation in blue (C). αaT, immunoprecipitation using anti-acetylated tubulin rabbit IgG; 2BPA, 2-bromo palmitic acid; IgG, control immunoprecipitation using nonspecific rabbit IgG; In, input; iPS, induced pluripotent stem; PAz, palmitic azide; PLA, proximity ligation assay.

Fig. 6

Acetylated tubulin forms a complex with S-palmitoylated Arl13b in cilia. A–C: Metabolic labeling of HEK293T cells with PAz, followed by click chemistry–mediated addition of fluorophore (Cy5) and immunoprecipitation using anti-Arl13b rabbit IgG. Immunoblot shows coimmunoprecipitated acetylated tubulin, the signal of which is reduced by 2BPA (arrow in B). Quantitation is shown in C. N = 3. P < 0.01. D: PLA for complex of Arl13b with acetylated tubulin (signal in red, arrow) in HEK293T cells. Acetylated tubulin labeling is shown in green and Arl13b in blue. E, F: PLA for S-palmitoylation of Arl13b in HEK cells (E) and ependymal cells (F). Arrows point at S-palmitoylated Arl13b (red) in cilia. Acetylated tubulin labeling is shown in green. αArl, immunoprecipitation using anti-Arl13b rabbit IgG (immunolabeled Arl13b is pseudocolored in green, and palmitoylation is pseudocolored in red); 2BPA, 2-bromo palmitic acid; IgG, control immunoprecipitation using nonspecific rabbit IgG; In, input; PAz, palmitic azide; PLA, proximity ligation assay.

Fig. 7

S-palmitoylation is critical for complex formation of acetylated tubulin with Arl13b and ciliogenesis. A–-D: PLA for complex of Arl13b with acetylated tubulin (signals pseudocolored in red, arrows) in primary culture of mouse ependymal cells, with or without inhibition of S-palmitoylation (2BPA). Labeling of acetylated tubulin is shown in green and Arl13b is in blue. A, C: (detail of A) without 2BPA; B and D (detail of B) with 2BPA showing that inhibition of S-palmitoylation disrupts Arl13b–acetylated tubulin complex formation and ciliogenesis. 2BPA, 2-bromo palmitic acid; PLA, proximity ligation assay.

Fig. 8

Ceramide is critical for complex formation of acetylated tubulin with Arl13b and formation of cilia. A: High-performance thin-layer chromatography (HPTLC) for ceramide isolated from control and FB1-treated HEK293T cells. The arrow points at the ceramide band, the intensity of which is reduced by FB1-mediated inhibition of ceramide biosynthesis. C16, C24, ceramide standards C16:0 ceramide and C24:1 ceramide. B, C: Coimmunoprecipitation assay using anti-Arl13b rabbit IgG for the precipitation reaction and acetylated tubulin mouse IgG for immunoblotting. Incubation with FB1 shows that reduction of ceramide levels as shown in (A) leads to lower amount of coimmunoprecipitated acetylated tubulin (arrow). Quantitation is shown in (C). N = 3. P < 0.0001. D: Immunocytochemistry shows colocalization of acetylated tubulin (green), ceramide (red), and Arl13b (blue) in primary cilia of HEK293T cells. E–H: Immunocytochemistry for acetylated tubulin (green) and ceramide (red) shows that inhibition of ceramide biosynthesis with FB1 disrupts ciliogenesis in primary cultured mouse ependymal cells. FB1, fumonisin B1.

Fig. 9

FB1 and 2BPA prevent complex formation between acetylated tubulin and Arl13b in primary cilia of astrocytes. A: PLA detecting Arl13b-acetylated tubulin complexes (red) and immunocytochemistry for Arl13b (blue) and acetylated tubulin (green) shows that reduction of ceramide levels with FB1 (48 h, 10 μM) or 2BPA (16 h, 10 μM) disrupts complex formation and ciliogenesis in primary cultured mouse astrocytes. Arrows point at cilia. Second panel from the top shows magnified area from the top panel (control). Arrows point at Arl13b-acetylated tubulin complexes that are mainly detected at the top part of primary cilia. B–D: Quantitation of the number of cilia in randomly chosen areas of 50 cells (B), the number of PLA signals/cilium (C), and cilium length (D). N = 5. P as indicated in figure. FB1, fumonisin B1; PLA, proximity ligation assay.

Fig. 10

Ceramide associates with Arl13b, which is critical for complex formation with acetylated tubulin in motile cilia of ependymal cells. A, B: The PLA detecting Arl13b-acetylated tubulin complexes (red) and immunocytochemistry for Arl13b (blue) and acetylated tubulin (green) shows that reduction of ceramide levels with FB1 disrupts complex formation and ciliogenesis in primary cultured mouse ependymal cells (arrows). C: Ependymal cells were metabolically labeled with PAz and S-palmitoylation visualized by click chemistry–mediated addition of fluorophore (Cy5). The PLA was performed using antibodies against ceramide and Arl13b showing that S-palmitoylation (blue) is at least partially colocalized with ceramide-acetylated tubulin complexes (red, arrows). Ceramide immunolabeling is shown in green. FB1, fumonisin B1; PAz, palmitic azide; PLA, proximity ligation assay.

Fig. 11

S-palmitoylated protein cross-links to photoactivatable ceramide in cilia, which is prevented by 2BPA and FB1. A: Simultaneous labeling for S-palmitoylation and cross-linking to photoactivatable ceramide (pacFACer), the principle of the method. Cells were incubated with PAz to metabolically label for S-palmitoylation, and then with pacFACer followed by UV cross-linking to label for ceramide binding of the CAP (here acetylated tubulin). The protein-bound S-palmitoyl azide and pacFACer residues were linked to fluorophores using two different click reactions (copper-free for adding fluorophore [AF488-DBCO] to S-palmitoyl azide residue and copper-catalyzed for adding fluorophore [AF647-azide] to pacFACer residue). B–F: Reaction as described for (A) performed with primary cultured mouse ependymal cells and immunocytochemistry using anti-acetylated tubulin mouse IgG shows colocalization of pacFACer-cross-linked protein with S-palmitoylation in cilia (C and E, arrows), unless S-palmitoylation is inhibited with 2BPA (C) or ceramide biosynthesis with FB1 (D). 2BPA, 2-bromo palmitic acid; CAP, ceramide-associated protein; FB1, fumonisin B1.

Fig. 12

Regulation of ciliary protein complex formation by S-palmitoylation and association with ceramide. In this model, Arl13b and acetylated tubulin are S-palmitoylated and then bound to ceramide in the ciliary membrane at the cilium base. The two proteins form a complex that is transported with CRPs to the cilium tip. Acetylated tubulin is used to extend the growing axoneme, and Arl13b is transported back to the base. Depletion of ceramide with FB1 or inhibition of S-palmitoylation with 2BPA will disrupt binding of Arl13b and acetylated tubulin to the ciliary membrane and subsequently complex formation and transport of the two proteins to the cilium tip. 2BPA, 2-bromo palmitic acid; CRP, ceramide-rich platform; 2FB1, fumonisin B1.