The conserved AAA-ATPase PCH-2 TRIP13 regulates spindle checkpoint strength.

Spindle checkpoint strength is dictated by the number of unattached kinetochores, cell volume, and cell fate. We show that the conserved AAA-ATPase PCH-2/TRIP13, which remodels the checkpoint effector Mad2 from an active conformation to an inactive one, controls checkpoint strength in Caenorhabditis elegans. Having previously established that this function is required for spindle checkpoint activation, we demonstrate that in cells genetically manipulated to decrease in cell volume, PCH-2 is no longer required for the spindle checkpoint or recruitment of Mad2 at unattached kinetochores. This role is not limited to large cells: the stronger checkpoint in germline precursor cells also depends on PCH-2. PCH-2 is enriched in germline precursor cells, and this enrichment relies on conserved factors that induce asymmetry in the early embryo. Finally, the stronger checkpoint in germline precursor cells is regulated by CMT-1, the ortholog of p31comet, which is required for both PCH-2's localization to unattached kinetochores and its enrichment in germline precursor cells. Thus, PCH-2, likely by regulating the availability of inactive Mad2 at and near unattached kinetochores, governs checkpoint strength. This requirement may be particularly relevant in oocytes and early embryos enlarged for developmental competence, cells that divide in syncytial tissues, and immortal germline cells.

RZZ and Mad1 dynamics in Drosophila mitosis.

The presence or absence of Mad1 at kinetochores is a major determinant of spindle assembly checkpoint (SAC) activity, the surveillance mechanism that delays anaphase onset if one or more kinetochores remain unattached to spindle fibers. Among the factors regulating the levels of Mad1 at kinetochores is the Rod, Zw10, and Zwilch (RZZ) complex, which is required for Mad1 recruitment through a mechanism that remains unknown. The relative dynamics and interactions of Mad1 and RZZ at kinetochores have not been extensively investigated, although Mad1 has been reported to be stably recruited to unattached kinetochores. In this study, we directly compare Mad1-green fluorescent protein (GFP) turnover dynamics on unattached Drosophila kinetochores with that of RZZ, tagged either with GFP-Rod or GFP-Zw10. We find that nearly 40 % of kinetochore-bound Mad1 has a significant dynamic component, turning over with a half-life of 12 s. RZZ in contrast is essentially stable on unattached kinetochores. In addition, we report that a fraction of RZZ and Mad1 can co-immunoprecipitate, indicating that the genetically determined recruitment hierarchy (in which Mad1 depends on RZZ) may reflect a physical association of the two complexes.

A maternal effect rough deal mutation suggests that multiple pathways regulate Drosophila RZZ kinetochore recruitment.

Proper kinetochore recruitment and regulation of dynein and the Mad1-Mad2 complex requires the Rod-Zw10-Zwilch (RZZ) complex. Here, we describe rod(Z3), a maternal-effect Drosophila mutation changing a single residue in the Rough Deal (Rod) subunit of RZZ. Although the RZZ complex containing this altered subunit (denoted R(Z3)ZZ) is present in early syncytial stage embryos laid by homozygous rod(Z3) mothers, it is not recruited to kinetochores. Consequently, the embryos have no spindle assembly checkpoint (SAC), and syncytial mitoses are profoundly perturbed. The polar body (residual meiotic products) cannot remain in its SAC-dependent metaphase-like state, and decondenses into chromatin. In neuroblasts of homozygous rod(Z3) larvae, R(Z3)ZZ recruitment is only partially reduced, the SAC is functional and mitosis is relatively normal. R(Z3)ZZ nevertheless behaves abnormally: it does not further accumulate on kinetochores when microtubules are depolymerized; it reduces the rate of Mad1 recruitment; and it dominantly interferes with the dynein-mediated streaming of RZZ from attached kinetochores. These results suggest that the mutated residue of rod(Z3) is required for normal RZZ kinetochore recruitment and function and, moreover, that the RZZ recruitment pathway might differ in syncytial stage embryos and post-embryonic somatic cells.