Characterization of mitotic checkpoint complexes

In eukaryotes, chromosome segregation critically depends on the establishment of productive contacts between kinetochores (KTs), specialized chromosomal structures, and the spindle microtubules (MTs). In mitosis, the spindle assembly checkpoint (SAC) is the major surveillance mechanism that restrains anaphase onset until all KTs become bi-oriented by spindle MTs. Several SAC proteins act in concert to relay the presence of unattached KTs to the cell cycle machinery in the cytoplasm. The SAC protein Mad2 plays a pivotal role in this signal transduction cascade, contributing both to the KT sensor and to the SAC cytoplasmic effector. Mad2 can fold into two distinct conformers, Open (O) and Closed (C), and can asymmetrically dimerize. Biophysical and structural work had demonstrated that the conformational dynamics of Mad2 is crucial for its activation in vitro, but models arising from this work could not be exhaustively tested in cells. Here, we describe a monoclonal antibody that specifically recognizes the dimerization interface of C-Mad2. This antibody revealed several conformation specific features of Mad2 in human cells. Notably, we show that Mad2 requires association with its KT-receptor Mad1 to adopt the Closed conformation. Furthermore, C-Mad2 antibody microinjection interfered with Mad2 asymmetric dimerization and abrogated the SAC, accelerating mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Finally, we show that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent ‘capping’. We also suggest that p31comet capping is negatively regulated by the SAC kinase Mps1 and the SAC regulator Tpr. Collectively, this work provides direct in vivo evidence for the model that a KT complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer.