The checkpoint protein Zw10 connects CAL1-dependent CENP-A centromeric loading and mitosis duration in Drosophila cells

A defining feature of centromeres is the presence of the histone H3 variant CENP-A that replaces H3 in a subset of centromeric nucleosomes. In Drosophila cultured cells CENP-A deposition at centromeres takes place during the metaphase stage of the cell cycle and strictly depends on the presence of its specific chaperone CAL1. How CENP-A loading is restricted to mitosis is unknown. We found that overexpression of CAL1 is associated with increased CENP-A levels at centromeres and uncouples CENP-A loading from mitosis. Moreover, CENP-A levels inversely correlate with mitosis duration suggesting crosstalk of CENP-A loading with the regulatory machinery of mitosis. Mitosis length is influenced by the spindle assembly checkpoint (SAC), and we found that CAL1 interacts with the SAC protein and RZZ complex component Zw10 and thus constitutes the anchor for the recruitment of RZZ. Therefore, CAL1 controls CENP-A incorporation at centromeres both quantitatively and temporally, connecting it to the SAC to ensure mitotic fidelity. Author summary Segregation of DNA during mitosis is a highly regulated process necessary to ensure the faithful transmission of genetic material to new daughter cells. Centromeric chromatin, which is defined by the presence of the histone H3 variant CENP-A, mediates the interaction of chromosomes with the spindle apparatus. In most organisms, CENP-A incorporation into centromeric chromatin is uncoupled from DNA replication and depends on specialized loading mechanisms and chaperones. In Drosophila cells, CENP-A loading takes place during mitosis and is mediated by its loading factor CAL1. We show that CAL1 controls the amount as well as the timing of CENP-A incorporation into centromeric chromatin. Moreover, CENP-A loading inversely correlates with the duration of mitosis. Mitosis length is influenced by the spindle assembly checkpoint, which ensures that all centromeres are attached correctly to the microtubule spindle. We identified the checkpoint protein and RZZ component Zw10 as a new interactor of CAL1. This interaction establishes a connection between CENP-A loading and mitosis control. We hypothesize that this crosstalk between CENP-A loading and spindle assembly checkpoint ensures that mitosis does not proceed until a sufficient amount of CENP-A is incorporated, thereby safeguarding centromere function and chromosome segregation.