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First published online 31 July 2007
doi: 10.1242/jcs.004812

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ZYG-9, TAC-1 and ZYG-8 together ensure correct microtubule function throughout the cell cycle of C. elegans embryos

Jean-Michel Bellanger^1,*, J. Clayton Carter², Jennifer B. Phillips^2,, Coralie Canard¹, Bruce Bowerman² and Pierre Gönczy^1,

¹ Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology (EPFL), School of Life Sciences, Lausanne, Switzerland
² Institute of Molecular Biology, University of Oregon, Eugene, OR 97403, USA

View larger version (73K): [in this window] [in a new window]   Fig. 1. Molecular lesions, TAC-1 and ZYG-9 distribution in five new zyg-9 temperature-sensitive mutant alleles. (A) Schematic representation of ZYG-9 (1-1415) showing the position and nature of the mutations identified in the five new zyg-9 alleles. Yellow boxes, TOG domains; line above the protein, TAC-1-binding region TBR (thick line, strong binding; thin line, weak binding) (Bellanger and Gönczy, 2003). (B-M) One-cell-stage embryos during mitosis in the wild-type (B,C) or zyg-9 (D-M) temperature-sensitive mutant alleles shifted at 25°C for >12 hours and stained with antibodies against ZYG-9 (B,D,F,H,J,L) or TAC-1 (C,E,G,I,K,M) and -tubulin. Left panels show ZYG-9 or TAC-1 staining alone, right panels the merge of ZYG-9 or TAC-1 (red), -tubulin (green) and DNA (blue). The histograms on the right of each staining series represent the quantification of ZYG-9 and TAC-1 levels in the cytoplasm (white bars) and the ratio between the levels of ZYG-9 or TAC-1 at centrosomes and in the cytoplasm (black bars) (Materials and Methods). Values are given as the mean ± s.e.m.; a.u., arbitrary units relative to the wild-type values, which have been set to 1 (dashed lines). Numbers of embryos analyzed for TAC-1 and ZYG-9 were: wild type, 17 and 13; zyg-9(or593), 10 and 9; zyg-9(or623), 10 and 13; zyg-9(or628), 10 and 8; zyg-9(or634), 11 and 10; zyg-9(or635), 10 and 10. Anterior is left, posterior is right; bar, 10 µm. Images in panels D, E and K are maximum-intensity projection of two 1-µm-thick confocal sections; the remaining images are single 1-µm-thick confocal sections. We found also that the distribution of ZYG-9 and TAC-1 is comparable with that of the wild type in zyg-9(or623), zyg-9(or634) and zyg-9(or635) embryos at 15°C (data not shown).

View larger version (63K): [in this window] [in a new window]   Fig. 2. Molecular lesions, TAC-1 and ZYG-9 distribution in three new tac-1 temperature-sensitive mutant alleles. (A) Schematic representation of TAC-1 (1-260) showing the position and nature of the mutations identified in the three new tac-1 alleles. Cyan box, TACC domain. (B-I) One-cell-stage embryos during mitosis in the wild-type (B,C) and tac-1 temperature-sensitive mutant alleles shifted at 25°C for >12 hours (D-G) or maintained at 15°C (H,I). B,D,F,H: ZYG-9 and -tubulin staining; C,E,G,I,: TAC-1 and -tubulin staining. Left panels show ZYG-9 or TAC-1 staining alone, right panels the merge of ZYG-9 or TAC-1 (red), -tubulin (green) and DNA (blue). The histograms on the right of each staining series represent the quantification of ZYG-9 and TAC-1 levels in the cytoplasm (white bars) and the ratio between the levels of ZYG-9 or TAC-1 at centrosomes and in the cytoplasm (black bars) (Materials and Methods). Values are given as the mean ± s.e.m.; a.u., arbitrary units relative to the wild-type values, which have been set to 1 (dashed lines). Numbers of embryos analyzed for TAC-1 and ZYG-9 were: wild type, 17 and 13; tac-1(or369) plus tac-1(or402): 19 and 18; tac-1(or455), 10 and 10; tac-1(or455) [15]: 11 and 14. Anterior is left, posterior is right; bars, =10 µm.

View larger version (31K): [in this window] [in a new window]   Fig. 3. Impact of select mutations on the binding between TAC-1 and ZYG-9. (A) GST pull-down experiments with wild-type, M58I or L229F GST-TAC-1 and in vitro translated [35S]-labeled wild-type, I862K or E1317K ZYG-9 TBR (654-1415), as indicated. Top panels show 10% of the input material and 100% of the corresponding pulled-down material detected by autoradiography. The bottom panels show the Coomassie Blue staining of the GST-TAC-1 fusions used in each case, indicating that comparable amounts of proteins had been added. The weaker band detected in the input lanes (denoted by 10%) corresponds to a 60-kDa protein from the in vitro transcription/translation mix. GST alone did not retain significant amounts of ZYG-9 TBR (data not shown). (B) Quantification of TAC-1-ZYG-9 binding, expressed as the percentage of each radioactive product retained on Glutathione-sepharose beads, averaged from three independent experiments, ± s.e.m. 100% corresponds to binding observed with wild-type ZYG-9 TBR and wild-type GST-TAC-1.

View larger version (66K): [in this window] [in a new window]   Fig. 4. zyg-9 and tac-1 function during mitosis to ensure correct spindle positioning. (A-D) Images from time-lapse DIC microscopy sequences of wild-type (A), zyg-8(or484) (B), zyg-9(or634) (C) or tac-1(or455) (D) embryos shifted to 25°C during centration/rotation; row 1, metaphase; row 2, anaphase; row 3, cytokinesis. Arrowheads point to spindle poles. Last row of panels represent the trajectories of the anterior (red) and posterior (blue) spindle poles during mitosis; spindle-pole position was plotted every 5 seconds for the entire duration of the movies; arrowheads, positions of spindle poles at the onset (black arrowheads correspond to row 1) and the end (white arrowhead correspond to row 3). Elapsed time is indicated in minutes and seconds. Embryos are 50 µm long. Note that in the wild-type, the anterior spindle pole shifts to a sligthly posterior position before moving to a more anterior one. In 9/18 zyg-9(or634) and 5/23 tac-1(or455) embryos, spindle positioning was as depicted here, with the phenotype typically being somewhat less pronounced in tac-1(or455) embryos; 4/18 zyg-9(or634) and 13/23 tac-1(or455) embryos exhibited analogous but weaker spindle positioning defects, like those observed following partial RNAi-mediated inactivation of zyg-8; the spindle snapped in two in 3/18 zyg-9(or634) and 3/23 tac-1(or455) embryos, as is also observed in occasional zyg-8-mutant embryos (Gönczy et al., 2001); in 2/18 zyg-9(or634) and 2/23 tac-1(or455) embryos, spindle positioning was akin to wild type, perhaps because of incomplete or delayed inactivation. In some zyg-9(or634) and tac-1(or455) embryos, centration/rotation was incomplete (see panel D1). Anterior is left, posterior is right.

View larger version (101K): [in this window] [in a new window]   Fig. 5. Synthetic phenotypes in embryos simultaneously compromised for ZYG-9-TAC-1 and ZYG-8. (A-D) Images from time-lapse DIC microscopy sequences of wild-type (A), tac-1(RNAi) (B), zyg-8(or484) (C) and zyg-8(or484) tac-1(RNAi) (D) embryos; Row 1, pseudocleavage stage (interphase); row 2, metaphase; row 3, end of anaphase; row 4, cytokinesis; row 5, merged immunostainings of -tubulin (green), SPD-5 (red) and DNA (blue), documenting the state of microtubules and centrosomes in anaphase one-cell stage embryos of these genotypes. Arrows point to female pronuclei, arrowheads to spindle poles. Elapsed time is indicated in minutes and seconds. Embryos are 50 µm long. See Table 2 for quantifications. Note that the spindle is less elongated and less distant from the posterior cortex in zyg-8(or484) tac-1(RNAi) embryos (D) than in tac-1(RNAi) embryos (B). Note also that the cleavage furrow does not ingress to bisect the spindle in zyg-8(or484) tac-1(RNAi). However, furrowing activity does take place towards the embryo anterior, orthogonal to the A-P axis, like in embryos lacking microtubules (e.g. Gönczy et al., 2001). Notice the numerous small asters present in the cytoplasm of zyg-8(or484) tac-1(RNAi) embryos, which might result from a larger pool of free cytoplasmic tubulin possibly promoting non-centrosomal microtubule nucleation. Anterior is left, posterior is right; bar, 10 µm.

View larger version (35K): [in this window] [in a new window]   Fig. 6. ZYG-8, through its doublecortin domain, interacts with TAC-1, in a ZYG-9-free complex. (A,B) GST-pull-down experiments with (A) in vitro translated [35S]-labeled full-length ZYG-8 (arrowhead) or an unrelated [35S]-labeled control protein (denoted `ctrl', see Materials and Methods), or (B) [35S]-labeled ZYG-8 fragments. Shown are 10% of the input material and 100% of the material pulled-down by either GST-TAC-1 or GST. Ponceau staining indicates that comparable amounts of GST-TAC-1 and GST had been incubated with the radioactive products (data not shown). (C) Co-immunoprecipitation experiments of wild-type embryonic extracts. Extracts were immunoprecipitated with TAC-1, ZYG-8 or ZYG-9 antibodies (indicated above the lanes), and the immunoprecipitated material was analyzed by western blot using antibodies against ZYG-9, ZYG-8 or TAC-1 (indicated on the left).

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