Spindle Checkpoint at Kinetochores




Kim, Soonjoung

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The kinetochore—a large protein assembly on centromeric chromatin—functions as the docking site for spindle microtubules and as a signaling hub for the spindle checkpoint. The Constitutive Centromere-Associated Network (CCAN) at the inner kinetochore nucleates the formation of the mature outer kinetochore during mitosis, including the recruitment of the KMN network that consists of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C). The KMN is a critical receptor for microtubules, and provides a landing pad for various spindle checkpoint proteins and regulatory factors. The spindle checkpoint protein Mad2 has multiple conformations, including the inactive open Mad2 (O-Mad2) and the active closed Mad2 (C-Mad2). The kinetochore-bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of O-Mad2 and serves as a catalytic engine of checkpoint signaling. The activated C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Deficient spindle checkpoint signaling leads to premature sister-chromatid separation and aneuploidy. Research in this thesis has provided several key insights into spindle checkpoint signaling at kinetochores. First, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2S195D mutant and the phospho-S195 Mad2 protein do not form C-Mad2 on their own. Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20. Our results establish for the first time that the conformational change of Mad2 is regulated by posttranslational mechanisms. Second, we have studied how Mad1 is targeted to kinetochores. We have determined the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. The structure reveals unexpected fold similarity between Mad1 CTD and known kinetochore-binding modules. Functional studies then validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Interestingly, deletion of the CTD does not abolish Mad1 kinetochore localization. Non-overlapping Mad1 fragments retain detectable kinetochore targeting. Our results indicate that the CTD–Bub1 connection is one of several mechanisms of targeting Mad1 to kinetochores. Finally, we show that the proper assembly of KMN is required for generating the spindle checkpoint signal at kinetochores. We have developed several strategies to inactivate KMN at kinetochores in human cells, and demonstrate its requirement for the spindle checkpoint in the absence of microtubules. We further show that two quasi-independent pathways mediate the mitosis-specific assembly of KMN at kinetochores. In one pathway, the centromeric kinase Aurora B phosphorylates the Mis12C component Dsn1, and strengthens Mis12C binding to the CCAN component CENP-C. In the second pathway, CENP-T anchors the CENP-H/I/K sub-complex at kinetochores, which in turn recruits Ndc80C. Inactivation of both pathways abolishes KMN at kinetochores and causes gross spindle checkpoint defects. In conclusion, combining cell biology and structural biology methods, our studies have defined a new posttranslational mechanism of Mad2 regulation, uncovered a critical way for targeting Mad1 to kinetochores, and dissected assembly pathways of the KMN checkpoint sensor at kinetochores.

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