Kinetochore-Microtubule Attachment and the Spindle Checkpoint

The kinetochore is a remarkable protein machine that links chromosomes to mitotic spindle microtubules. A kinetochore acts principally as a force-coupling device that transduces chemical energy released through microtubule polymerization and depolymerization into mechanical forces to move and organize its associated chromosome. The mechanical properties of yeast kinetochores depend on microtubule-binding subcomplexes, including the Dam1 complex, which are required for the initial capture of sister chromosomes by microtubules following DNA replication, their retrieval and biorientation on the spindle, and their eventual segregation. But kinetochores also incorporate signaling proteins that continuously monitor kinetochore-microtubule interactions and the organization of chromosomes on the spindle throughout mitosis. One key group of such components is a conserved biochemical surveillance network called the spindle checkpoint. As a critical line of defense against aneuploidy, the spindle checkpoint is sensitive to even very subtle defects in kinetochore-microtubule interactions. Checkpoint activation serves to generate signals that inhibit the activities of cell cycle components that otherwise promote anaphase entry, in order to prevent the initiation of chromosome segregation until all the chromosomes are properly organized in the cell. In my research, I have used a variety of cellular, genetic, bioinformatic, biochemical, and biophysical approaches to investigate important questions regarding kinetochore- microtubule attachments and the spindle checkpoint.Three key questions motivated my studies of the spindle checkpoint: (1) what factors control the recruitment of the spindle checkpoint component Mad1 to kinetochores and its non-kinetochore binding sites at nuclear pore complexes (NPCs), (2) what mechanisms control the sensitivity of the spindle checkpoint to kinetochore-microtubule attachment defects, and (3) what cellular components and/or activities contribute to spindle checkpoint inactivation after all the chromosomes are properly organized in the cell. As my primary approach, I used a centromere reactivation assay to observe the dynamic localization of a fluorescent derivative of the spindle checkpoint protein Mad1 to pairs of detached sister kinetochores in budding yeast. Using this assay, I characterized the recruitment of Mad1 upon de novo kinetochore assembly, and I observed the events preceding Mad1 disappearance from kinetochores following capture by microtubules in a variety of strains. I also created analytical tools to measure the spatial distribution of detached kinetochores and to quantify the relative abundance of Mad1 at kinetochores following kinetochore assembly. Using these tools, I was able to exclude the possibility that Mad1 binding sites at NPCs tether detached kinetochores to the nuclear periphery, suggesting that individual Mad1 molecules do not bind to both structures simultaneously. I also found that perturbations that alter the number of high-affinity Mad1 binding sites in the nucleus significantly impacted the amount of Mad1 recruited to pairs of detached kinetochores. Together, these results suggested that competition between alternative binding sites for Mad1 limits the amount of free Mad1, so that the amount of Mad1 available is less than saturating for even one pair of detached sister kinetochores. Thus, Mad1's localization to kinetochores responds disproportionately in the regime of one or only a few detachments, which may help to explain why even a one detached kinetochore can arrest the cell cycle. I also discovered that half of the Mad1 at sister kinetochores is released following their lateral attachment to a microtubule, implying that the budding yeast spindle checkpoint is satisfied by lateral attachments, and the checkpoint monitors the attachment status of each sister kinetochore independently of the other. These results are presented in Chapter 2-3. In Chapter 3, I also present experiments directed at identifying the Mad1 receptor at detached kinetochores, which pointed to the Bub1/3 complex as a potential target for future investigation, and I describe my efforts to establish a genetic screen to identify genes involved in silencing the checkpoint after all the chromosomes are bioriented.I also initiated work, presented in Chapter 4, to address critical questions regarding the biophysics of kinetochore-microtubule attachment, motivated by observations that the Dam1 complex forms microtubule-encircling rings and tracks the ends of dynamic microtubules in vitro. I used a biochemical approach to begin to investigate whether Dam1-binding proteins discriminate between Dam1 rings and non-ring forms of Dam1. I found that although a component of the microtubule-binding Ndc80 kinetochore complex, Spc25, could bind to recombinant Dam1 complex in yeast extract, it exhibited no preference for Dam1 alone or Dam1 bound to microtubules. I also propose experiments directed at testing several biophysical models that seek to explain how Dam1 can maintain a stable association to the ends of a depolymerizing or polymerizing microtubule. Toward this end, I generated and purified recombinant Dam1HaloTag, Dam1mEos2, and Dam1sfGFP* complexes and, in collaboration with others from my lab, purified native tubulin from budding yeast, tools which will enable quantitative fluorescence analysis and super-resolution imaging of Dam1 in vitro in order to distinguish between these models. Finally, an important question raised by many investigators is whether the Dam1 complex actually forms rings at kinetochores in vivo. Here, I present simulations of super-resolution fluorescence microscopy and real experimental data that suggest that subdiffraction imaging might allow us to directly observe the spatial organization of the Dam1 complex at individual budding yeast kinetochores