Defining the relationship between kinetochore structure and spindle checkpoint signaling

In this dissertation, I use the early embryo of the nematode Caenorhabditis elegans to probe the relationships between kinetochore formation and spindle checkpoint signaling. The spindle checkpoint is a conserved signaling pathway that delays cell cycle progression in mitosis until all chromosomes have properly attached to microtubules emanating from opposing spindle poles. To trigger checkpoint activation, I undertook a strategy of inhibiting centriole duplication, using RNAi specific for components of the centriole duplication machinery. Such depletions result in bipolar spindles in the first mitotic division (serving as useful internal controls), while leading to monopolar spindle formation in the second and third divisions. Monopolar spindles are known to trigger checkpoint activation, as kinetochores lack both full attachment and tension. By performing live imaging video microscopy on these embryos, I was able to quantitatively assess checkpoint status by timing cell cycle progression. I found that cells with monopolar spindles exhibited cell cycle delays that were dependent on each of the conserved checkpoint proteins in C. elegans. Kinetochores are proteinaceus organelles that connect DNA to microtubules, and are essential for chromosome segregation in mitosis. Using the timing assay described above, I set out to comprehensively determine the contribution of kinetochore proteins for checkpoint signaling, and found that these proteins could be placed into one of three categories based on their phenotypes in the timing assay; required for checkpoint signaling, not required, or those that trigger checkpoint activation when depleted. Checkpoint activation is correlated with enrichment of specific components of the pathway on unattached kinetochores. To correlate checkpoint protein recruitment with the functional analysis of checkpoint signaling, I generated worm strains stably expressing fluorescently tagged checkpoint proteins Mad2MDF⁻², Mad3SAN⁻¹, and BUB-3, and systematically determined the requirements for their localization and enrichment at kinetochores. I also used these assays to characterize the roles of the recently discovered kinetochore protein SPDL-1 in checkpoint activation and signaling. Taken together, my results indicate that checkpoint activation is coordinately directed by three key kinetochore components - the NDC-80 complex, the Rod/Zwilch/Zw10 complex, and BUB -1 - that are targeted independently of one another by the outer kinetochore scaffold protein KNL-1. Surprisingly, I found that Mad3SAN⁻¹, unlike other checkpoint proteins, does not enrich at unattached kinetochores, and that subtly elevating Mad2MDF⁻², levels bypasses the requirement for BUB-3 and Mad3SAN⁻¹ in kinetochore-dependent checkpoint activation. I propose that a core kinetochore-generated Mad1MDF⁻¹/super>/ Mad2MDF⁻² signal is integrated with a largely independent cytoplasmic Mad3SAN⁻¹/BUB-3-based signal to coordinately regulate checkpoint activation, possibly in response to different checkpoint-activating states. I discuss the rationale for the partitioning of the checkpoint signaling pathway into two branches, and the newly elucidated relationships between kinetochore assembly and checkpoint signalin