Abnormal chromosome segregation during mitosis in Spc105 mutants. (A) Embryos were collected from Spc105¹/TM3, Ubx-lacZ flies and fixed at the stage of the 16th round of mitosis. Embryos were labelled with a DNA stain (DNA), an antibody against Cenp-A/Cid (Cenp-A) and an antibody against β-galactosidase for genotype determination (data not shown). In Spc105⁺ sibling control embryos (Spc105⁺, left panel), centromeres form a tight cluster during anaphase/telophase on the poleward side of the chromosomes (see arrows). In Spc105 mutants (Spc105⁻; right panel), centromeres are rarely clustered in anaphase/telophase and lagging chromosomes are frequent (see arrows). Moreover, more severe perturbations during anaphase/telophase were also apparent (dashed circles). The bar diagram indicates the percentage of abnormal anaphase/telophase figures in wild-type and Spc105 mutant embryos. The total number of the observed anaphase/telophase figures (n>100) in the analysed epidermal regions from at least six different embryos per genotype was set to 100%. Genotypes are indicated below the bars. Bar=2 μm. (B) Time-lapse in vivo imaging of mitoses in Spc105⁺ or Spc105⁻ embryos expressing Cenp-A/Cid–EGFP and histone H2AvD–mRFP1. The time (min:s) indicated in each frame is given relative to the first anaphase frame, which was set to zero in the top three rows, or relative to the start of chromosome condensation in the bottom row. The top two rows illustrate progression through mitosis 15, in which chromosome segregation during anaphase is already less synchronous in Spc105 mutants (second row). The bottom two rows illustrate progression through mitosis 16 in Spc105 mutants, in which chromosome congression was either no longer observed (class I, fourth row) or in which it was followed by an abnormally delayed anaphase (class II, third row). Bar=3 μm.

High-resolution localization of Spc105 within the kinetochore. (A) The localization of Spc105 was compared with that of Cenp-A/Cid. Native chromosomes released from syncytial embryos co-expressing EGFP–Spc105 (EGFP-Spc105) and a red fluorescent Cenp-A/Cid (Cenp-A–mRFP) were labelled with a DNA stain (DNA). The green and red signal intensities were quantified along the inter-kinetochore axis (white line). The spatial separation between the red and green fluorescent proteins was determined as described by Schittenhelm et al (2007) by averaging data obtained from 100 chromosomes. Bar=100 nm. (B) The average spatial separation observed in experiments with Spc105 and other kinetochore proteins is indicated in the context of a kinetochore map. The position of Cenp-A/Cid, N- and C-terminus of Cenp-C, Spc25, Mis12 and Nuf2 along the inner–outer kinetochore axis is given in nm as determined previously (Schittenhelm et al, 2007). Double arrows and numbers below indicate the spacing (nm) observed in additional experiments using Spc105, Nsl1 and Ndc80.

The C-terminal domain of Spc105 partially rescues the Spc105 mutant phenotypes. (A) For an analysis of the different Spc105 domains, UAS transgenes, expressing either the N-terminal (N), middle (M) or the C-terminal (C) region fused to EGFP (green boxes), were generated with the indicated fragments. Numbers indicate amino-acid positions. The subcellular localization of these domains was analysed after zygotic expression of paternally inherited UAS transgenes driven by maternal α4tub-GAL4-VP16. Embryos at the stage of mitosis 16 were double labelled with an antibody against Cenp-A/Cid (Cenp-A) and a DNA stain (DNA). Representative metaphase figures illustrate that the C-terminal domain localizes to the kinetochore, as full-length Spc105 (FL), whereas the middle and the N-terminal domain do not show kinetochore localization. Bar=4 μm. (B) Spc105¹ embryos expressing the UAS transgenes (see panel A) were fixed at the stage of mitosis 16 and double labelled with an antibody against Cenp-A/Cid (Cenp-A) and a DNA stain (DNA). Spc105¹ embryos without a UAS transgene served as controls (−). Expression of UAS-Spc105(N)-EGFP and UAS-EGFP-Spc105(M) in Spc105¹ embryos does not prevent the mitotic defects during mitosis 16. Centromeres are rarely clustered in anaphase and lagging chromosomes are frequent (dashed circles). In contrast, in Spc105¹ embryos expressing UAS-Spc105(FL)-EGFP or UAS-EGFP-Spc105(C), centromeres form a tight cluster during anaphase on the poleward side of the chromosomes (arrows). Also see (C). Bar=6 μm. (C) The bar diagram indicates the percentage of abnormal anaphase/telophase figures in Spc105¹ mutant embryos at the stage of mitosis 16 after α4tub-GAL4-VP16-mediated expression of Spc105(FL)–EGFP (FL), Spc105(N)–EGFP (N), EGFP–Spc105(M) (M) or EGFP–Spc105(C) (C). Spc105¹ embryos without a UAS transgene served as control (−). (D) To evaluate SAC function, 10–12 h embryos were incubated for 45 min in medium without (− colchicine) or with colchicine (+ colchicine) before fixation and labelling with a DNA stain (DNA) and antibodies against phospho-histone H3 (PH3) and against β-galactosidase for genotype identification (data not shown). High magnification views of CNS regions are shown from Spc105⁺ sibling embryos (Spc105⁺), from Spc105¹ mutant embryos with transgenes driving the expression of either full-length (Spc105¹+FL) or the C-terminal region of Spc105 (Spc105¹+C), as well as of mad2 mutant embryos (mad2^P). Bar=20 μm. (E) Bar diagram represents quantification of the number of PH3-positive cells in the CNS of embryos treated as described and illustrated in (D). The average density of PH3 cells in mock-treated Spc105⁺ sibling embryos was set to 1. Ten embryos were analysed for each of the genotypes indicated below the bars.

Identification of Drosophila Spc105. (A) The Spc105¹ mutation was identified as a dominant enhancer of a rough eye phenotype caused by GMR-thrΔC, which results in eye-specific expression of a dominant-negative Separase regulatory subunit. GMR-thrΔC was expressed in either Spc105^+/+ (GMR-thrΔC; +/+) or Spc105^−/+ (GMR-thrΔC; Spc105¹/+) flies. The increased eye abnormalities observed in Spc105¹ heterozygous flies were reverted by the presence of the gSpc105 transgene driving expression of wild-type Spc105 (GMR-thrΔC; Spc105¹/+; gSpc105). (B) The structure of wild-type and mutant Spc105/CG11451 alleles, as well as the gSpc105 transgene is illustrated schematically. The triangles indicate the insertion sites of an incomplete DOC element in Spc105¹ and of a P element (P01511LE) in Spc105². The genomic fragment used for the construction of the gSpc105 transgene is indicated by the thin black line below the gene model. Arrows above the gene models indicate the direction of transcription. Boxes represent exons with UTR regions (white) and coding regions (black). (C) The intracellular localization of Drosophila Spc105 was analysed in syncytial embryos expressing an Spc105–EGFP fusion (Spc105–EGFP) after double labelling with an antibody against Cenp-A/Cid (Cenp-A) and a DNA stain (DNA). EGFP signals were detected at centromeres only during mitosis (lower row) and not during interphase (upper row). Bar=5 μm. (D) Y2H experiments were used for an analysis of protein–protein interactions of Spc105, Mis12/MIND complex subunits (Mis12, Nsl1, Nnf1a) and the N-terminal region of Bub1. Spc105 was divided into an N-terminal (N), middle (M) and C-terminal region (C). Interactions represented by single arrows were only observed when the protein next to the arrowhead was fused to the transcriptional activation domain, and the protein at the base of the arrow to the DNA-binding domain of GAL4, but not with the reciprocal fusion protein configuration. Interactions indicated by double arrows were observed with both configurations.

Requirements for kinetochore localization of Spc105 and other kinetochore proteins. (A) Localization of EGFP fusions of Cenp-C, Mis12, Nsl1, Spc25, Ndc80, Nuf2 and BubR1 in Spc105¹ (Spc105⁻) and Spc105⁺ sibling control embryos (Spc105⁺) during mitosis 16. Representative anaphase figures are shown (except for prometaphases in case of BubR1) with kinetochore proteins in green and DNA staining in red. Bar=3 μm. (B) Localization of Spc105–EGFP (shown in green) in Cenp-C^prl41 (Cenp-C⁻) and Mis12^f03756 (Mis12⁻) mutant embryos in comparison with sibling control embryos (Cenp-C⁺ and Mis12⁺, respectively). Representative anaphases with DNA staining in red are shown at the stage in which mitotic abnormalities start in the mutant embryos, that is during mitosis 16 in Cenp-C mutants and during the later mitotic divisions in the CNS in Mis12 mutants. Bar=3 μm.

The rapidly evolving repeat region of Spc105 does not provide essential functions. (A) The repetitive middle region of Spc105 evolves rapidly by repeat loss and expansions. The repeat motifs in the middle region of DmSpc105 (red boxes) and DwSpc105 (blue boxes) are illustrated. Most of the D. willistoni (Dw) repeats seem to have arisen recently from an expansion of a period composed of four repeats. These 5.5 periods are indicated on the line below the blue boxes. Reflecting their recent expansion, the D. willistoni repeats are more similar to each other than the D. melanogaster (Dm) repeats, as illustrated by the logo representation (Crooks et al, 2004) of the repeat consensus sequences. The numbers (amino-acid positions) and dashed lines illustrate the repeat domain swap constructs (also see panel B). (B) For a functional characterization of the repetitive middle region, the transgenes gSpc105^wr30 (wr30) gSpc105^wr18 (wr18) and gSpc105^wr1 (wr1) expressing DmSpc105 with a variable number of D. willistoni repeats instead the D. melanogaster repeats were generated. These transgenes provide all essential Spc105 functions in D. melanogaster according to complementation tests.