JCS004812 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. TAC-1 and ZYG-9 protein levels in mutant embryonic extracts. Western-blot analysis of TAC-1 (A, top) and ZYG-9 (B, top) protein levels in mixed-stage embryonic extracts from the indicated genotypes. The blots were reprobed with α-tubulin antibodies (A and B, bottom). The α−tubulin signal was used to normalize loading between lanes; numbers below each lane indicate the relative amount of TAC-1 or ZYG-9 protein compared to the wild type.

• Supplemental Figure S2 -

  Fig. S2. Sequence alignment of ZYG-9 and TAC-1 related proteins. (A) Multiple sequence alignment of ZYG-9 with C-terminal fragments of its homologues D. melanogaster Msps1 (starting at aa 557), X. laevis XMAP215 (starting at aa 562) and H. sapiens Ch-TOG (starting at aa 565). Yellow box: TOG domains with HEAT repeats HR 0-6 (Al-Bassam et al., 2007); Underlined sequences: strong (thick) and weak (thin) TAC-1 binding region (Bellanger and Gönczy, 2003). (B) Multiple sequence alignment of TAC-1 with C-terminal fragments of its homologues D. melanogaster D-TACC (starting at aa 933), X. laevis Maskin (starting at aa 622), as well as H. sapiens Hs-TACC-1 (starting at aa 555), Hs-TACC-2 (starting at aa 778) and Hs-TACC-3 (starting at aa 596). Cyan box: TACC domain. Alignments were realized at the Multalin web interface (http://bioinfo.genopole-toulouse.prd.fr/multalin/multalin.html). Red: consensus >90%; blue: consensus >50%. Numbering is relative to ZYG-9 in (A) and TAC-1 in (B);. The mutations identified in the novel zyg-9 and tac-1 alleles are indicated in (A) and (B) by a red star close to the name of the allele and the nature of the amino acid substitution.

• Supplemental Figure S3 -

  Fig. S3. Rapid inactivation of tac-1 or zyg-9 during mitosis can phenocopy zyg-8 mutant embryos. (A-C) Merged immunostainings of anaphase one-cell stage embryos from indicated genotypes after a 7 minutes shift to the restrictive temperature prior to fixation. α-tubulin: green, SPD-5: red, DNA: blue. C is a maximum intensity projection of two ∼1-μm-thick confocal sections; A and B are single 1-μm-thick confocal sections.

• Supplemental Figure S4 -

  Fig. S4. TAC-1 and ZYG-8 do not stabilize each other. (A) Embryonic extracts of the genotypes indicated above the lanes analyzed by Western-blot using antibodies against ZYG-8, α-tubulin (as a loading control) or TAC-1, as indicated on the left. Note that the left-most two lanes are from one experiment, the remaining lanes from another experiment. (B-E) Wild-type (B, two-cells stage; D, one-cell stage), tac-1(or455) (C) or zyg-8(t1650) (E) one-cell-stage embryos during mitosis stained with antibodies against ZYG-8 (B, C) or TAC-1 (D, E) and α-tubulin. Left panels show ZYG-8 or TAC-1 staining alone, right panels the merge of ZYG-8 or TAC-1 (red), α-tubulin (green) and DNA (blue). Identical results were obtained with zyg-8(or436), zyg-8(or450), zyg-8(or478), zyg-8(or484) and zyg-8(or490) mutant embryos, as well as with tac-1(RNAi) mutant embryos (data not shown). Note that the DNA derived from the female pronucleus has remained outside the spindle in this particular zyg-8(t1650) mutant embryos, as is occasionally the case (Gönczy et al., 2001).

• Supplemental Figure S5 -

  Fig. S5. zyg-8(or484) encodes a protein that interacts with TAC-1 but does not localize to microtubules. (A,B) COS-7 cells transfected with full-length HA-ZYG-8wild-type (A) or HA-ZYG-8or484 (B) were stained with antibodies against HA and α-tubulin. Left panels show HA staining alone, right panels the merge of HA (red), α-tubulin (green) and DNA (blue). (C) Yeast two-hybrid experiment measuring the interaction of amino acids 1-464 of ZYG-8wild-type versus ZYG-8or484 with TAC-1. Plates containing 50 mM 3-aminotriazol were grown for 3 days at 37°C before being photographed. The yeast two-hybrid assay was carried out essentially as described (Vidal, 1997).