Hyperglutamylation of tubulin can either stabilize or destabilize microtubules in the same cell

Abstract

In most eukaryotic cells, tubulin is subjected to posttranslational glutamylation, a conserved modification of unclear function. The glutamyl side chains form as branches of the primary sequence glutamic acids in two biochemically distinct steps: initiation and elongation. The length of the glutamyl side chain is spatially controlled and microtubule type specific. Here, we probe the significance of the glutamyl side chain length regulation in vivo by overexpressing a potent side chain elongase enzyme, Ttll6Ap, in Tetrahymena. Overexpression of Ttll6Ap caused hyperelongation of glutamyl side chains on the tubulin of axonemal, cortical, and cytoplasmic microtubules. Strikingly, in the same cell, hyperelongation of glutamyl side chains stabilized cytoplasmic microtubules and destabilized axonemal microtubules. Our observations suggest that the cellular outcomes of glutamylation are mediated by spatially restricted tubulin interactors of diverse nature.

FIG. 1.

Overproduction of GFP-Ttll6Ap causes elongation of glutamyl side chains on tubulin in vivo. (A) Western blots of total proteins from GFP-Ttll6Ap-expressing cells that are either uninduced (0 h) or induced for 1, 2, or 4 h with either 1 or 2.5 μg of CdCl₂/ml. (B and C) 2D silver-stained protein gels containing cytoskeletons of uninduced (B) or induced (C) GFP-Ttll6Ap cells. The arrows point at strings of highly acidic protein isoforms that appear at the mass level of the main spots of α- and β-tubulin upon induction of GFP-Ttll6Ap. (D to E) Confocal fluorescence images of the GFP signal in growing (D) and cilia regenerating (E) GFP-Ttll6Ap cells induced with 2.5 μg of CdCl₂/ml for 3 h. Note the increase of the signal of GFP-Ttll6Ap in short growing cilia (arrowheads) and on subcortical microtubules (arrows). Bar, 10 μm.

FIG. 2.

Overproduced GFP-Ttll6Ap increases the levels of tubulin polyglutamylation on ciliary and cell-body microtubules in a time- and overexpression strength-dependent manner. (A to D) Confocal fluorescence images of GFP-Ttll6Ap cells that are untreated (A) or induced with 2.5 μg of CdCl₂/ml for 1 (B), 2 (C), or 3 h (D) and subjected to immunofluorescence with a MAb that recognized polyglutamylation (ID5). We reduced the gain levels in B to D to avoid overexposure. Arrows point to short hyperglutamylated cilia, arrowheads points to hyperglutamylated cell body microtubules. Bar, 10 μm. (E to G) Graphs that document the distribution of cells with distinct pattern of tubulin hyperglutamylation as shown in panels A to D, as a function of either cadmium concentration or duration of induction with cadmium.

FIG. 3.

Tubulin hyperglutamylation stabilizes cell body microtubules. (A to I′) Confocal microscopic images of cells stained with anti-α-tubulin MAb 12G10. (A and B) Wild-type cells grown without (A) or with (B) 40 μM paclitaxel. Note the appearance of thick bundles of subcortical microtubules in the drug-treated cells. (C to G). Cells expressing GFP-Ttll6Ap or its truncated variant M241-V929 that are uninduced (C) and induced with cadmium (D to G). Note the appearance of bundles of microtubules in the cell body and macronuclei (arrow) in the overproducing cell. (H to I′) Overexpression of GFP-Ttll6Ap protects subcortical and cytoplasmic microtubules against oryzalin-induced depolymerization. Wild-type (H and H′) and GFP-Ttll6Ap-overexpressing (I and I′) cells grown for 4 h in the presence of 2.5 μg of CdCl₂/ml were treated for 30 min with 10 μM oryzalin. Panels H′ and I′ show the internal sections of cells shown in panels H and I, respectively. Note the nearly complete depolymerization of cell body microtubules in the wild-type cells but not in Ttll6Ap-overproducing cells. Bar, 10 μm. (J) A graph documents the percentages of wild-type and GFP-Ttll6Ap cells with cytoplasmic microtubules during oryzalin treatment.

FIG. 4.

Hyperelongation of glutamyl side chains in the cell body causes accumulation of α-tubulin K40 acetylation. (A) Wild-type and GFP-Ttll6Ap cells (indicated by the asterisk) were grown for 4 h in the presence of 2.5 μg of CdCl₂/ml and labeled side-by-side with antiacetylated tubulin antibodies (6-11 B-1). (A) Projection image of z-section from the top half of the cell; (A′) section showing the middle part of the cell. Bar, 10 μm.

FIG. 5.

Hyperelongation of glutamyl side chains delays cilium regeneration. (A to F) Confocal immunofluorescence images of wild-type (A and B) and GFP-Ttll6Ap (C and D)-, GFP-Ttll6Ap-M241-V929-E662G (E)-, and GFP-Ttll6Ap-M241-V929 (F)-overexpressing cells that regenerated cilia for 1 (A and C), 2 (E and F), or 4 h (B and D) after deciliation. Cells were costained with anti-α-tubulin 12G10 MAb and the anti-polyglutamylation antibody poly(E). Note the shorter size of cilia of GFP-Ttll6Ap cells. Insets show cilia at higher magnifications from the areas indicated by a bracket. (G) A graph shows the average lengths of cilia as a function of time of cilium regeneration. Bar, 10 μm.

FIG. 6.

Hyperglutamylation of ciliary microtubules results in structural defects in axonemes. TEM cross-sections (A, B, and D) and longitudinal sections (C, E, and F) of cilia in wild-type (D) and GFP-Ttll6Ap-overproducing (A to C, E, and F) cells. Note that short cilia (A to C) lack a central pair (9+0, arrows) and that broken microtubules are visible (E and F, arrows). Bar, 200 nm.

FIG. 7.

Paclitaxel partly rescues the destabilizing effect of hyperglutamylation on elongation of assembling cilia in vegetatively growing Tetrahymena. Confocal immunofluorescence images of wild-type (A and B) and GFP-Ttll6Ap-overexpressing (D and E) cells grown without (A and D) or with (B and E) 40 μM paclitaxel. Cells were costained with anti-α-tubulin antibodies and anti-poly(E) antibodies. The arrows in panel E point to hyperglutamylated distal segments. Bar, 10 μm. (C and F) A graph documents the distribution of cilium length in wild-type (C) and GFP-Ttll6Ap-overexpressing (F) cells. Black diamonds indicate the cilium lengths in cells grown without drug, and open triangles represent the cilium lengths in cells grown in the presence of paclitaxel.