Abstract
Centromeres are specialized chromosomal domains that direct mitotic kinetochore assembly and are defined by the presence of CENP-A (CID in Drosophila) and CENP-C. While the role of CENP-A appears to be highly conserved, functional studies in different organisms suggest that the precise role of CENP-C in kinetochore assembly is still under debate. Previous studies in vertebrate cells have shown that CENP-C inactivation causes mitotic delay, chromosome missegregation, and apoptosis; however, in Drosophila, the role of CENP-C is not well-defined. We have used RNA interference depletion in S2 cells to address this question and we find that depletion of CENP-C causes a kinetochore null phenotype, and consequently, the spindle checkpoint, kinetochore–microtubule interactions, and spindle size are severely misregulated. Importantly, we show that CENP-C is required for centromere identity as CID, MEI-S332, and chromosomal passenger proteins fail to localize in CENP-C depleted cells, suggesting a tight communication between the inner kinetochore proteins and centromeres. We suggest that CENP-C might fulfill the structural roles of the human centromere-associated proteins not identified in Drosophila.
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Acknowledgments
We would like to thank all the members of the Sunkel lab and Hélder Maiato for comments and suggestions and Terry Orr-Weaver, Christian Lehner, Stefen Heidmann, Mike Goldberg, and Byron Williams for antibodies. We would also like to thank Gohta Goshima for providing the H2B-GFP;mCherry-tubulin stable cell line. BO holds a Doctoral studentship from FCT and this work was supported by a project grant from FCT.
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Supplementary Fig. 1
CENP-C is poorly conserved in Drosophila. Amino acid sequence alignment of CENP-C from various species shows that the Drosophila protein bears very little homology with CENP-C protein in other species. a, b The two most conserved domains of CENP-C fail to show significant homology with the Drosophila protein. Note that the Drosophila CENP-C protein is also significantly larger than in other species (JPEG 844 kb)
Supplementary Fig. 2
CENP-C is required for kinetochore organization. a Quantification of the mean pixel intensity of CID and CENP-meta levels at kinetochores of control and CENP-C depleted cells obtained from cells shown in Fig. 2c. Control levels of CID and CENP-meta have been normalized to 1 (n = more than 200 kinetochores from 15 different cells). b Relative levels of CID versus CENP-meta were plotted to determine the relationship between both proteins 96 h after the addition of the dsRNA, where each dot represents one kinetochore. Note that there is a linear relationship between CENP-meta and CID levels. c Quantification of the relative mean pixel intensity of Nuf2 and Ndc80 levels at kinetochores of both control and CENP-C depleted cells obtained from cells shown in Fig. 2e. Control levels of Nuf2 and Ndc80 have been normalized to 1 (n = more than 150 kinetochores from 15 different cells). Relative levels of CID were plotted against d Ndc80 or e Nuf2 where each dot represents a single kinetochore. Note that there is a population of kinetochores with high levels of CID and low levels of Ndc80 and Nuf2 suggesting that Ndc80 complex mislocalization at kinetochores is specific to CENP-C depletion. All quantifications were performed using Image J software with a previously defined ROI spanning the entire kinetochore region (JPEG 842 kb)
Supplementary Fig. 3
CENP-C depletion causes failure in chromosome congression and aneuploidy. a Control and CENP-C RNAi-treated S2 cells were grown for 96 h and then fixed and stained to reveal phosphohistone H3, α-tubulin, and CID allowing classification into distinct mitotic phases. The quantification of the different mitotic phases shows that in the absence of CENP-C, cells spend little time in prometaphase, none in metaphase but accumulate at telophase. b Analysis of DNA content by fluorescence-activated cell sorting (FACS) at 48 and 96 h after the addition of the dsRNA and results were analyzed using Cell Quest data acquisition software. Note that at 96 h, CENP-C depleted cells exhibit highly variable DNA contents consistent with an S-phase delay and overall aneuploidy (JPEG 1127 kb)
Supplementary Fig. 4
Kinetochore null cells display increased spindle length. a Control and CENP-C depleted cells were incubated in MG132 (2 h) prior to fixation and stained to show DNA (blue), γ-tubulin (green) to reveal spindle poles, and CENP-C (red). Note that CENP-C depleted cells fail to align their chromosomes at the spindle equator and exhibit longer mitotic spindles than those observed in control cells. b Quantification of pole-to-pole distance (length between each γ-tubulin positive signal) in MG132-arrested cells shows that in the absence of CENP-C, spindles elongate 1.5-fold more than they do in control cells (JPEG 563 kb)
Supplementary Fig. 5
Centromere localization of MEI-S332 and CPC proteins is CENP-C dependent. Control and CENP-C depleted cells were incubated in MG132 (2 h) and colchicine for (1 h further) prior to fixation and immunofluorescence staining with specific antibodies. Relative levels of CID were plotted against a MEI-S332 or b INCENP where each dot represents a single centromere/kinetochore pair. Note that there is a population of kinetochores with high levels of CID and low levels of MEI-S332 and INCENP, suggesting that centromeric localization of MEI-S332 and INCENP is CENP-C-dependent rather than dependent on the prior localization of CID. All quantifications were performed using Image J software with a previously defined ROI. c Cells treated with MG132 (2 h) and colchicine (1 h further), then fixed and stained to reveal DNA (gray), Aurora B (green), phosphohistone H3 (PH3; red), and CID (blue). Note that Aurora B mislocalization does not affect PH3 staining (JPEG 686 kb)
Online Resource 1
Monitoring mitotic progression using S2 cells expressing H2B-GFP;mCherry-tubulin. Untreated S2 cells stably expressing H2B-GFP (green);mCherry-tubulin (red). Images were collected using a spinning disk confocal system (see “Materials and methods” section) at intervals of 30 s. NEBD is indicated by the rapid entry of mCherry-tubulin into the nuclear space and anaphase onset takes place when chromatid separation is observed (MPG 1184 kb)
Online Resource 2
CENP-C depletion causes accelerated mitotic exit and chromosome missegregation. S2 cells stably expressing H2B-GFP (green);mCherry-tubulin (red) previously treated for 96 h with specific dsRNA against CENP-C. Images were collected using a spinning disk confocal system (see “Materials and methods” section) at intervals of 30 s. Note that in the absence of CENP-C, the time from NEBD to anaphase onset is severely shortened and overall chromosome missegregation is observed (MPG 679 kb)
Online Resource 3
CENP-C depletion causes accelerated mitotic exit and chromosome missegregation. S2 cells stably expressing H2B-GFP (green);mCherry-tubulin (red) previously treated for 96 h with specific dsRNA against CENP-C. Images were collected using a spinning disk confocal system (see “Materials and methods” section) at intervals of 30 s. Note that in the absence of CENP-C, the time from NEBD to anaphase onset is severely shortened and overall chromosome missegregation is observed (MPG 727 kb)
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Orr, B., Sunkel, C.E. Drosophila CENP-C is essential for centromere identity. Chromosoma 120, 83–96 (2011). https://doi.org/10.1007/s00412-010-0293-6
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DOI: https://doi.org/10.1007/s00412-010-0293-6