Drosophila CENP-A mutations cause a BubR1-dependent early mitotic delay without normal localization of kinetochore components

Abstract

The centromere/kinetochore complex plays an essential role in cell and organismal viability by ensuring chromosome movements during mitosis and meiosis. The kinetochore also mediates the spindle attachment checkpoint (SAC), which delays anaphase initiation until all chromosomes have achieved bipolar attachment of kinetochores to the mitotic spindle. CENP-A proteins are centromere-specific chromatin components that provide both a structural and a functional foundation for kinetochore formation. Here we show that cells in Drosophila embryos homozygous for null mutations in CENP-A (CID) display an early mitotic delay. This mitotic delay is not suppressed by inactivation of the DNA damage checkpoint and is unlikely to be the result of DNA damage. Surprisingly, mutation of the SAC component BUBR1 partially suppresses this mitotic delay. Furthermore, cid mutants retain an intact SAC response to spindle disruption despite the inability of many kinetochore proteins, including SAC components, to target to kinetochores. We propose that SAC components are able to monitor spindle assembly and inhibit cell cycle progression in the absence of sustained kinetochore localization.

Figure 1. cid Null Embryos Exhibit Multiple Mitotic Phenotypes.

CID, PH3, and DAPI staining of cid/CyO and cid/cid embryos at different stages of development were monitored to evaluate mitotic progression and segregation defects. (A) Heterozygous (cid/CyO) stage 9–10 embryos displayed no mitotic defects and robust CID staining at kinetochores (inset). (B) cid null animals (trans-heterozygous for different cid alleles, see Materials and Methods) exhibited lagging chromosomes during anaphase. Some CID staining was still visible at this stage, demonstrating that these phenotypes resulted from partial loss of CID function, due to the presence of maternal CID protein. (C) cid/CyO stage 14–15 embryos show normal mitotic progression and normal CID staining at kinetochores (inset). (D) cid null animals exhibited an elevated mitotic index, lower nuclear density, and little detectable CID staining in some cells at stage 14–15. The strong depletion of CID staining suggests that these phenotypes are the result of complete loss of zygotic cid function. cid null animals have a large number of presumably polyploidy cells (inset) suggesting high levels of aneuploidy due to repeated failures in cell division. Scale bar indicates 15 μm.

Figure 2. cid Null Mutants Exhibit a G2/Prophase Delay.

Cell cycle progression was monitored in cid/CyO and cid/cid embryos by staining for PH3, cyclin A, cyclin B, and tubulin. (A) Schematic diagram of the appearance and destruction of various cell cycle regulatory factors and markers. ANA, anaphase; META, metaphase; PRO, prophase; PRO-META, prometaphase; TELO, telophase. (B) cid/cid animals displayed an elevated mitotic index, and an increased number of cells in prophase and prometaphase, compared to cid/CyO controls. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase. (C) cid/cid animals had a 2-fold higher number of cyclin A and B positive cells than cid/cid controls. Scale bars indicate 15 μm.

Figure 3. DNA Damage Is Not Responsible for the cid-Mediated Mitotic Delay

The effect of DNA damage on cell cycle progression was determined by treating stage 15 cid null and heterozygous embryos with the topoisomerase II inhibitor doxorubicin. (A–C) cid/CyO cells dramatically decreased entry into mitosis in response to DNA damage, whereas cid/cid cells were unaffected by doxorubicin (Dox.) treatment. (D–F) The MEI-41/ATR kinase was inhibited by treating cid homozygous and heterozygous embryos with 2 mM caffeine. Inactivation of the DNA damage checkpoint did not suppress the cid-mediated mitotic delay, as the mitotic index of cid mutants remained double that of controls, with the majority of the mitotic cells delayed in prophase or prometaphase. A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase. Scale bars indicate 15 μm.

Figure 4. cid Mutants Retain an Intact SAC Response to Microtubule Depolymerization

cid null and heterozygous embryos were treated with colcemid to determine if they have an intact SAC response to spindle disruption. Both cid/CyO and cid/cid cells were able to delay the cell cycle in response to spindle disruption (A), as evidenced by an approximately 2-fold increase in mitotic index and an accumulation of cells in prometaphase (B). A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase. Scale bars indicate 15 μm.

Figure 5. A bubr1 Mutation Partially Suppresses the cid-Mediated Mitotic Delay

(A) cid bubr1 double mutants were examined for mitotic progression by staining for PH3 and DAPI. cid bubr1 mutants show an increased nuclear density and number of anaphases compared to cid single mutants. Scale bar indicates 15 μm. (B) bubr1 suppressed the high mitotic index and high number of cells delayed in prophase and prometaphase observed in cid single mutants (compare to ratios in Figure 2B). A, anaphase; M, metaphase; MI, mitotic index; P, prophase; PM, prometaphase; T, telophase.

Figure 6. Inner and Outer Kinetochore Protein Localizations Are Disrupted in cid Mutant Embryos

Kinetochore localization of CENP-C, ROD, and BUBR1 were determined in stage 15 embryos. In cid/CyO control embryos (left), all three proteins were localized to the centromere/kinetochore during interphase (CENP-C) or the early stages of mitosis (ROD and BUBR1). All three proteins were absent from centromeres/kinetochores in cid/cid animals (right); in some cases, CENP-C was mislocalized in a diffuse pattern. Scale bars indicate 5 μm.

Figure 7. SAC Components Affect Cell Cycle Progression in the Absence of Kinetochore Localization

In normal cells, CENP-A chromatin assembly is followed by the recruitment of inner and outer kinetochore proteins [17]. We propose that until kinetochore assembly is complete, free SAC components may be responsible for cell cycle inhibition (early activation of the SAC). Upon completion of kinetochore assembly, SAC components delay anaphase until all chromosomes have achieved bipolar spindle attachment. In cid null mutants, both inner and outer kinetochore proteins are free, resulting in a SAC-dependent early mitotic delay that does not depend on localization of SAC components to kinetochores.