Centromere function requires the coordination of many processes including kinetochore assembly, sister chromatid cohesion, spindle attachment and chromosome movement. Here we show that CID, the Drosophila homologue of the CENP-A centromere-specific H3-like proteins, colocalizes with molecular-genetically defined functional centromeres in minichromosomes. Injection of CID antibodies into early embryos, as well as RNA interference in tissue-culture cells, showed that CID is required for several mitotic processes. Deconvolution fluorescence microscopy showed that CID chromatin is physically separate from proteins involved in sister cohesion (MEI-S332), centric condensation (PROD), kinetochore function (ROD, ZW10 and BUB1) and heterochromatin structure (HP1). CID localization is unaffected by mutations in mei-S332, Su(var)2-5 (HP1), prod or polo. Furthermore, the localization of POLO, CENP-meta, ROD, BUB1 and MEI-S332, but not PROD or HP1, depends on the presence of functional CID. We conclude that the centromere and flanking heterochromatin are physically and functionally separable protein domains that are required for different inheritance functions, and that CID is required for normal kinetochore formation and function, as well as cell-cycle progression.
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The authors thank K. Hari, J. Cordeiro and W. Sullivan for help with antibody injection, and K. Maggert, K. Sullivan and members of the Karpen lab for guidance and critical comments on the manuscript. We are grateful to B. Sullivan, B. Sullivan, S. Henikoff, C. Sunkel, T. Orr-Weaver, T. Torok, R. Karess and B. Williams for reagents, and M. Baker and The Salk Institute Sequencing Facility for analyses. M.B. is supported by an CMG training grant, and this project was funded by a grant from the NIH.
Figure S1 Structures and transmission frequencies of minichromosome derivatives used in these studies. (PDF 26 kb)
Movie 1 Anti-CID TMR: embryo injected with tetramethyl-rhodamine labelled anti-CID. Interphase injection: the antibody does not enter the nucleus until nuclear envelope breakdown at mitosis. The antibody specifically binds centromeres during all stages of the cell cycle, and binds in a gradient with decreasing antibody bound with increasing distance from the site of injection. (MOV 749 kb)
Movie 2 Anti-CID injection: embryo injected with anti-CID show a series of phenotypes. Embryos injected in interphase show phenotypes two cycles after injection. (MOV 2823 kb)
Movie 3 Anti-CID control: embryos injected with heat-killed anti-CID show few mitotic defects. (MOV 1247 kb)
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