Quantitative and Mechanistic Studies on the Spindle Assembly Checkpoint in Human Cells Reveal the Origin of Its Sensitivity

Abstract

Equal distribution of replicated genetic materials packed in chromosomes from a dividing parent cell to two daughter cells is the hallmark of mitosis. This task is carried out by the spindle, whose microtubules attach to chromosomes through adaptors named kinetochores. Failure to achieve faithful chromosome segregation is often associated with cancers and many pathological syndromes.

My thesis investigates a surveillance mechanism named the spindle assembly checkpoint (SAC) that safeguards faithful chromosome segregation. The SAC is activated at kinetochores lacking end-on spindle microtubule attachment to stall the progression of mitosis. The SAC can robustly arrest a mammalian cell in mitosis in the presence of either many unattached kinetochores or merely one. Although the biochemical events associated with the SAC have been mostly elucidated, how the SAC reaches such "sensitivity" to effectively arrest a cell in mitosis in the presence of a single unattached kinetochore remains unclear. Here, I will describe how multiple proteins and reactions cooperate at various layers to enable this sensitivity.

First, to study the origin of the sensitivity of the SAC, we need a quantitative readout of the input (the phosphorylation of the scaffold protein KNL1 which recruits SAC proteins and activates the SAC at signaling kinetochores) and the output (the duration of the mitotic arrest caused by the SAC). In Chapter 2, we engineer a cytosolic probe that ectopically activates the SAC, which solves the technical challenge to gauge the phosphorylation of KNL1 in live cells. Dose-response analyses using the probe reveal that under certain conditions, the SAC signaling activity is stronger with a smaller number of phosphorylated KNL1 proteins. This striking observation strongly indicates positive cooperativity in the SAC, which may underlie the sensitivity of the SAC.

Next, I validate some of the preconditions of this cooperativity model above in the context of the endogenous kinetochore-based SAC in Chapter 3. We demonstrate that the numbers of SAC proteins recruited per signaling kinetochore are inversely correlated with the total number of signaling kinetochores in the cell. Additionally, I show that the localization at signaling kinetochores of BUBR1, an important constituent of the mitotic checkpoint complex (MCC, the effector molecule that stalls the progression of mitosis made up of BUBR1, BUB3, CDC20, and MAD2), strengthens the SAC activity. These observations reinforce the idea that cooperative signaling contributes to the sensitivity of the SAC.

Finally, in Chapter 4, I explore the catalytic mechanism of the rate-limiting step in the assembly of the MCC - the formation of CDC20-MAD2. Unveiling this catalytic mechanism is fundamental to understanding how an unattached kinetochore can produce an adequate signal to stall mitosis. Given that both CDC20 and MAD2 bind to the catalyst MAD1, I hypothesize that the structural flexibility of MAD1 helps to position CDC20 and MAD2 closely, thereby coordinating the formation of CDC20-MAD2. Our data show that disrupting the structural flexibility of MAD1 impairs the SAC signaling activity, based on which I propose a model for the catalytic mechanism of the formation of CDC20-MAD2 scaffolded by MAD1.

My thesis research reveals the role of cooperative synergy in shaping the sensitivity of the SAC. It also establishes the foundation for future studies aiming for a complete understanding of the origin of its sensitivity.