XB-ART-51074Curr Biol. August 17, 2015; 25 (16): 2177-83.
Zeta-Tubulin Is a Member of a Conserved Tubulin Module and Is a Component of the Centriolar Basal Foot in Multiciliated Cells.
There are six members of the tubulin superfamily in eukaryotes. Alpha- and beta-tubulin form a heterodimer that polymerizes to form microtubules, and gamma-tubulin nucleates microtubules as a component of the gamma-tubulin ring complex. Alpha-, beta-, and gamma-tubulin are conserved in all eukaryotes. In contrast, delta- and epsilon-tubulin are conserved in many, but not all, eukaryotes and are associated with centrioles, although their molecular function is unclear. Zeta-tubulin is the sixth and final member of the tubulin superfamily and is largely uncharacterized. We find that zeta-, epsilon-, and delta-tubulin form an evolutionarily co-conserved module, the ZED module, that has been lost at several junctions in eukaryotic evolution and that zeta- and delta-tubulin are evolutionarily interchangeable. Humans lack zeta-tubulin but have delta-tubulin. In Xenopus multiciliated cells, zeta-tubulin is a component of the basal foot, a centriolar appendage that connects centrioles to the apical cytoskeleton, and co-localizes there with epsilon-tubulin. Depletion of zeta-tubulin results in disorganization of centriole distribution and polarity in multiciliated cells. In contrast with multiciliated cells, zeta-tubulin in cycling cells does not localize to centrioles and is associated with the TRiC/CCT cytoplasmic chaperone complex. We conclude that zeta-tubulin facilitates interactions between the centrioles and the apical cytoskeleton as a component of the basal foot in differentiated cells and propose that the ZED tubulins are important for centriole functionalization and orientation of centrioles with respect to cellular polarity axes.
PubMed ID: 26234217
PMC ID: PMC4546511
Article link: Curr Biol.
Grant support: R01 GM052022 NIGMS NIH HHS , F32 GM103146 NIGMS NIH HHS , T32 CA009302 NCI NIH HHS , T32 CA09302 NCI NIH HHS , 1S10RR026780-01 NCRR NIH HHS , R01HL117164 NHLBI NIH HHS , R01GM052022 NIGMS NIH HHS , R01 HL117164 NHLBI NIH HHS , F32GM103146 NIGMS NIH HHS , S10 RR026780 NCRR NIH HHS
Genes referenced: actb cct2 cetn3 gapdh gnl3 marveld2 pcyt1a picalm.1 tuba4b tubd1 tube1 tubg1 tubz1
Antibodies referenced: Cetn3 Ab1 H3f3a Ab14 Tuba4b Ab2 Tuba4b Ab5 Tubg1 Ab4 Tubz1 Ab1
Morpholinos referenced: Tubz1 MO1 Tubz1 MO2 Tubz1 MO3
Article Images: [+] show captions
|Figure 1. Zeta-Tubulin Is a Conserved Tubulin that Associates with TRiC/CCT (A) Presence or absence of ZED module tubulins in representative organisms. (B) Unrooted phylogenetic tree, based on Clustal Omega alignment. (C–E) Sucrose gradient sedimentation of zeta-tubulin from (C) A6 cells, (D) eggs, and (E) whole oviduct. Lysates were separated on 10%–40% sucrose gradients and fractions probed for zeta-tubulin, the TRiC/CCT component CCT2, and gamma-tubulin. Molecular weights in kilodaltons (left) and increasing sucrose concentration, left to right (above), are indicated. The dividing line in (E) indicates the joint between two membranes, probed and developed identically. See also Table S1 and Figure S1.|
|Figure 2. Zeta-Tubulin Localizes to the Basal Foot in Multiciliated Cells (A) Dissociated Xenopus multiciliated oviduct cells stained for zeta-tubulin (green), acetylated tubulin (red), and DAPI (blue). The scale bar represents 5 μm. (B) Transmission EM of oviduct tissue stained with zeta-tubulin antibody and 10-nm gold-conjugated secondary antibody. Arrowheads indicate the basal body, and arrows indicate labeling of the basal foot. The scale bars represent 100 nm. (C) Confocal image of live tadpole epidermal multiciliated cell expressing GFP-zeta-tubulin (green), CLAMP-RFP (red), and centrin-BFP (blue). The arrow shows the direction of ciliary beating. The scale bar represents 5 μm. (D) Confocal image of live tadpole epidermal multiciliated cell expressing EMTB-3XGFP (green), mCherry-zeta-tubulin (red), and centrin-BFP (blue). The arrow shows the direction of ciliary beating. The scale bar represents 5 μm. See also Figures S2 and S3.|
|Figure 3. Zeta-Tubulin Depletion Disrupts Basal Body Orientation and Spacing (A) Tadpole epidermal multiciliated cells in morphant and control embryos co-expressing CLAMP-GFP (green) and centrin-(RFP or BFP) (red). Clumps of basal bodies are indicated by arrowheads and insets. The scale bars represent 5 μm. (B) Quantification of mean rootlet angle from experiments as in (A). Each arrow represents one cell, where length indicates uniformity of rootlet angles in that cell. Mean cellular rootlet orientation was statistically similar between morphants and controls (mean vector angle −64.4° in controls and −74.8° in morphants; Watson-Williams). The number of cells counted is as in (C). (C) Vector length of plots shown in (B) is significantly reduced in morphants (∗∗∗p < 0.0001; Mann-Whitney). n indicates number of cells counted, with total number of embryos in parentheses. Error bars represent the SEM. (D and E) Quantification of basal body clumping phenotype from experiments as in (A). n indicates the number of cells counted, with total number of embryos in parentheses (shown in E). (D) The distance between each basal body and its nearest neighbor is shown as average percentage of binned nearest neighbor distances for each condition. (E) Mean distance between basal bodies is shown; this is significantly reduced in morphants (∗∗∗p < 0.0001; Mann-Whitney). In (E), error bars represent the SEM. See also Figure S4.|
|Figure 4. Zeta-Tubulin Function and ZED Tubulin Localization (A) Microtubule organization in tadpole epidermal multiciliated cells visualized by co-expressing EMTB-3XGFP (green) and centrin-RFP (red). The scale bars represent 5 μm. (B) Tadpole epidermal multiciliated cells expressing centrin-BFP (white) fixed and stained with Alexa Fluor 568 phalloidin (red). Apical actin (apical inset) and subapical foci (subapical slice; ∼1.2 μm below the apical section) are shown. The scale bars represent 5 μm. (C) Tadpole epidermal multiciliated cell expressing GFP-epsilon-tubulin (green), mCherry-zeta-tubulin (red), and centrin-BFP (blue). The scale bar represents 5 μm. (D) Mouse tracheal epithelial cell cultures infected with GFP-zeta-tubulin-expressing lentivirus and stained for GFP (green), centrin (red), and DAPI (blue). x-z projections (below) show a slice through the area marked by the white rectangle. The scale bar represents 5 μm. See also Figures S3 and S4|
|Figure S1. Zeta-tubulin is expressed and can be specifically detected via immunoblot and immunofluorescence. Related to Figure 1. A) Zeta-tubulin is expressed in adult tissues of X. tropicalis, with highest expression in the testis. The number of transcripts of zeta-tubulin per million reads for two biological replicates are shown for each tissue. B) Protein samples were probed for zeta-tubulin, with gamma-tubulin as a loading control. Zeta-tubulin antibody recognizes a single band in both A6 cell lysate and egg extract that is of equal size to in vitro translated zeta-tubulin. Molecular weights in kilodaltons are indicated on the left. Dividing line indicates joint between lanes on the same membrane. C) Identically loaded protein samples were probed with zeta-tubulin antibody incubated with the peptide used for antibody production (+ peptide) or without (- peptide). All bands recognized are specific to the antigen. Sample entitled “peak fraction” represents the most concentrated fraction of zeta-tubulin from a 10-40% sucrose gradient (as in Figure 1D). Molecular weights in kilodaltons are indicated on the left. D) U2OS cells were transfected with GFP -zeta-tubulin and stained for GFP (green), zeta-tubulin (red), and DAPI (blue). Prior to staining, zeta-tubulin antibody was incubated with the peptide used for antibody production (+ peptide) or without (- peptide). Zeta-tubulin antibody not incubated with peptide recognizes GFP-zeta-tubulin, whereas peptide-blocked zeta-tubulin antibody no longer recognizes GFP-zeta-tubulin. Scale bar, 5 µm.|
|Figure S3. Zeta-tubulin precipitation reveals TRiC/CCT and GFP-zetatubulin is cytoplasmic in frog and mouse tissue culture cells. Related to Figures 2 and 4D. A) GFP nanobody-conjugated beads were used to affinity purify GFP-zetatubulin from GFP-zeta-tubulin stable cells, and GFP nanobody-conjugated beads were incubated separately with A6 cell lysate as a control. The most abundant co-purifying proteins as identified by mass spectrometry are shown, relative to their respective peptide counts. Hits include zeta-tubulin (blue), all 8 subunits of TRiC/CCT (green), heat shock or protein folding factors (pink), and proteasome associated proteins (yellow). B) Wild-type A6 cells and the GFP-zeta-tubulin stable line were fixed in methanol and stained for GFP (green), gamma-tubulin (red), and DAPI (blue). GFP was only detected in the stable cells, and GFP-zetatubulin labels cytoplasm and does not co-localize with centrosomes. Scale bar, 5 µm. C) 3T3 mouse fibroblasts were infected with GFP-zeta-tubulin lentiviruses and fixed in methanol. Cells were stained for GFP (green), poly-glutamylated tubulin (red), and DAPI (blue). Inset shows a magnified image of the cilium (3x). Zeta-tubulin does not localize to microtubule-based structures in mouse cycling cells. Scale bar, 5 µm.|