Meiosis-specific functions of kinetochore protein SPC105R required for chromosome segregation in Drosophila oocytes

Abstract

The reductional division of meiosis I requires the separation of chromosome pairs towards opposite poles. We have previously implicated the outer kinetochore protein SPC105R/KNL1 in driving meiosis I chromosome segregation through lateral attachments to microtubules and co-orientation of sister centromeres. To identify the domains of SPC105R that are critical for meiotic chromosome segregation, an RNAi-resistant gene expression system was developed. We found that SPC105R's C-terminal domain (aa 1284-1960) is necessary and sufficient for recruiting NDC80 to the kinetochore and building the outer kinetochore. Furthermore, the C-terminal domain recruits BUBR1, which in turn recruits the cohesion protection proteins MEI-S332 and PP2A. Of the remaining 1283 amino acids, we found the first 473 are most important for meiosis. The first 123 amino acids of the N-terminal half of SPC105R contain the conserved SLRK and RISF motifs that are targets of PP1 and Aurora B kinase and are most important for regulating the stability of microtubule attachments and maintaining metaphase I arrest. The region between amino acids 124 and 473 are required for two activities that are critical for accurate chromosome segregation in meiosis I, lateral microtubule attachments and bi-orientation of homologs.

Figure 1:

Spc105R^B can rescue most wild-type SPC105R functions. (A) A schematic of the two known Drosophila Spc105R isoforms and mutants used in this study. The first 9 amino acids in SPC105R^B are changed from MNANKRRSS to MVDLLFLQ in SPC105R ^A. The coordinates on the schematic represent the first amino acid of each domain. The N-terminal includes the SLRK and RISF motifs and is where the two isoforms differ. Following this is a domain with three MELT-like motifs, a region that contains two KI-like repeats, a central domain containing repeats with the consensus ExxEED, and the C-terminal region containing coiled-coil motifs. All transgenes included missense mutations to make them resistant to the shRNA GL00392. (B) Viability of Spc105R mutants, all in a Spc105R^RNAi background. Data shows the relative amounts of progeny expressing the Spc105R^RNAi and mutants (Tub-Gal4) to siblings that did not (no Gal4) (n> 200). (C) Confocal images of wild-type, Spc105R^RNAi, and Spc105R^B, Spc105R^RNAi oocytes. Merged images show DNA (blue), Tubulin (green), centromeres (white), and SPC105R (red). Centromeres and SPC105R are shown in separate channels. Scale bars are 5 μm.

Figure 2:

SPC105R domains required for homologous chromosome bi-orientation. A) Confocal images of Spc105R^RNAi oocytes expressing the indicated transgene. Merged images show DNA (blue), Tubulin (green), the X-Chromosome (Yellow), the second chromosome (Red), and the third chromosome (white). DNA and FISH probes are shown in a separate channel. Scale bars are 5 μm. B) Quantification for percent of chromosome mono-orientation: Spc105R ^B, Spc105R^RNAi oocytes (n=93), Spc105R ^ΔN, Spc105R^RNAi oocytes (n=117), Spc105R ^ΔExxEED, Spc105R^RNAi oocytes (n=65), Spc105R ^ΔM, Spc105R^RNAi oocytes (n=81), Spc105R ^ΔMELT-KI, Spc105R^RNAi oocytes (n=123), Spc105R ^C, Spc105R^RNAi oocytes (n=63). Significance in frequency of mono-orientation om oocytes determined by Fisher’s exact test, with * = p-values < 0.01, **** = p-value < 0.0001.

Figure 3:

Spc105R’s C-Terminal Domain recruits NDC80. The N-terminal 1284 amino acids of SPC105R is deleted in Spc105R ^C, retaining only the last 676 amino acids (Figure 1). A) Confocal images of Spc105R^RNAi oocytes expressing the indicated transgene, with DNA (blue), Tubulin (green), centromeres (white), and the kinetochore protein NDC80 (red). Centromeres and NDC80 are shown in separate channels. Scale bars are 5 μm. B) Quantification of NDC80 intensity at the centromeres in wild-type (n=68), Spc105R^RNAi (n=77), Spc105R^B, Spc105R^RNAi (n=131), and Spc105R^C, Spc105R^RNAi (n=107) oocytes. Error bars indicate standard deviation and **** = p-value < 0.0001 by a one-sided unpaired t-test. C) Confocal images of Spc105R^RNAi oocytes expressing the indicated transgene, with DNA (blue), Tubulin (green), and centromeres (white). DNA is shown in separate channels. Scale bars are 5 μm. D) Quantification of end-to-end chromosome length of wild-type (n=34), Spc105R^RNAi (n=12), Spc105R^C, Spc105R^RNA (n=22), and Spc105R ^C, Spc105R^RNAi, Ndc80^RNAi (n=30) oocytes. Error bars indicate standard deviation, and *** = p-value < 0.001, **** = p-value < 0.0001, ns = not significant by a one-sided unpaired t-test.

Figure 4:

Spc105R’s C-terminal domain is required for centromeric cohesion protection in meiosis I. A) Confocal images of Spc105R^RNAi oocytes expressing the indicated transgene with DNA (blue), Tubulin (green), centromeres (white), and MEI-S332 (red) and shown as a separate channel. Scale bars are 5 μm. B) Quantification of MEI-S332 intensity at the centromeres for wild-type (n=71), Spc105R^RNAi (n=202), Spc105R^B, Spc105R^RNAi (n=53) and Spc105R^C, Spc105R^RNAi (n=114) oocytes. Error bars indicate standard deviation, and **** = p-value < 0.0001by one-sided unpaired t-test. C) Confocal images of Spc105R^RNAi oocytes expressing the indicated transgene, with DNA (blue), Tubulin (green), centromeres (white), and the PP2A subunit HA-WDB (red), also in a separate channel. D) Quantification of WDB intensity at the centromeres in Spc105R^B, Spc105R^RNAi (n=134), Spc105R^RNAi (n=189) and Spc105R^C Spc105R^RNAi (n=155). Error bars indicate standard deviation and one-sided unpaired t-test show **** = p-value < 0.0001. E) Confocal images of Spc105R^RNAi oocytes expressing the indicated transgene, with DNA (blue), Tubulin (green), centromeres (white), and GFP-BUBR1 (red) and in a separate channel. F) Quantification of BUBR1 intensity at the centromeres, in wild-type (n=247), Spc105R^RNAi (n=153), Spc105R^B, Spc105R^RNA (n=67) Spc105R^C, Spc105R^RNAi (n =149) and Spc105R^MELT-KI, Spc105R^RNAi (n =89) oocytes. Error bars indicate standard deviation and significance is shown by a one-sided unpaired t-test **** = p-value < 0.0001.

Figure 5:

SPC105R N-terminal domain interacts with PP1 to maintain sister centromere co-orientation. Mutants with a deletion of the N-terminal domain (Spc105R^ΔN) or mutations in the SLRK and RISF sequences are shown in Figure S 4A. A) Confocal images of PP1-87B^RNAi or Spc105R^ΔN oocytes. Merged images show DNA (blue), Tubulin (green), and centromeres (white). Centromeres are shown in a separate channel. Scale bars are 5 μm. B) Quantification of centromere foci for images in A. One-sided unpaired t-test showed significance in number of foci. Sample sizes are: 46, 16, 37, 20, 48, 20, 16, in order of the graph. C) Confocal images of oocytes where the RISF site was changed to AAAA, RIAF or RIDF. D) Quantification of centromere foci for images in B. One-sided unpaired t-test showed significance in number of foci between all channels. Sample sizes are: 37, 46, 17, 29, 19, 23, 36, in order of the graph. Error bars indicate standard deviation, **** p < 0.0001, ** p = 0.001.

Figure 6:

SPC105R regulates interactions with microtubules. A) Confocal images of oocytes in a mei-P22 mutant (mei-P22^mut) background. The first image is only Ndc80 RNAi, while the rest express Ndc80^RNAi, Spc105R^RNAi, and a Spc105R transgene. Due to the absence of crossing over, mei-P22^mut mutant oocytes fail to arrest in metaphase, and precociously enter anaphase. B) Oocytes in mei-P22^mut, Spc105R RNAi background. The second image is also expressing a Spc105R-mis12 transgene. Merged images show DNA (blue), Tubulin (green), and centromeres (white). Chromosomes are shown in a separate channel. Scale bars are 5 μm. C) Quantification of the frequency of chromosome separation in oocytes. Sample sizes are: 25, 50, 23, 175, 115, 81, 83, 21, in order of the graph. Significance in frequency of oocytes entering precocious anaphase determined by Fisher’s exact test, with ** = p-value < 0.01, **** = p-value < 0.0001, and ns = not significant.

Figure 7:

The N terminal domain of SPC105R fused to MIS12 A) Confocal images of stage 14 oocytes expressing Spc105R^N-Mis12, either in the presence or absence of Spc105R^RNAi. The fusion protein was detected using an antibody to the Myc tag (red), along with CENP-C (white), and tubulin (green). B) Spc105R^N-MIS12, Spc105R^RNAi oocyte, detected using both an antibody to the Myc tag (red) and SPC105R (yellow), along with CENP-C (white). C) Ndc80 (red) does not localize in Spc105R^N-MIS12, Spc105R^RNAi oocytes. D) Measurement of NDC80 intensity in wild-type (n=137) and Spc105R ^N-MIS12, Spc105R^RNAi (n=160) oocytes. Error bars indicate standard deviation, and one-sided unpaired t-test showed significance **** = p < 0.0001. E) WDB (red) localization in Spc105R ^N-MIS12, Spc105R^RNAi oocytes. F) Measurement of WDB intensity in wild-type (n=154), Spc105R^RNAi (n=195), and Spc105R^N-MIS12, Spc105R^RNAi (n=183) oocytes. Error bars indicate standard deviation and one-sided unpaired t-test showed significance, *** = p < 0.001.