title: Conformation-specific anti-Mad2 monoclonal antibodies for the dissection of checkpoint signaling.
abstract: The spindle assembly checkpoint (SAC) ensures accurate chromosome segregation during mitosis by delaying the activation of the anaphase-promoting complex/cyclosome (APC/C) in response to unattached kinetochores. The Mad2 protein is essential for a functional checkpoint because it binds directly to Cdc20, the mitotic co-activator of the APC/C, thereby inhibiting progression into anaphase. Mad2 exists in at least 2 different conformations, open-Mad2 (O-Mad2) and closed-Mad2 (C-Mad2), with the latter representing the active form that is able to bind Cdc20. Our ability to dissect Mad2 biology in vivo is limited by the absence of monoclonal antibodies (mAbs) useful for recognizing the different conformations of Mad2. Here, we describe and extensively characterize mAbs specific for either O-Mad2 or C-Mad2, as well as a pan-Mad2 antibody, and use these to investigate the different Mad2 complexes present in mitotic cells. Our antibodies validate current Mad2 models but also suggest that O-Mad2 can associate with checkpoint complexes, most likely through dimerization with C-Mad2. Furthermore, we investigate the makeup of checkpoint complexes bound to the APC/C, which indicate the presence of both Cdc20-BubR1-Bub3 and Mad2-Cdc20-BubR1-Bub3 complexes, with Cdc20 being ubiquitinated in both. Thus, our defined mAbs provide insight into checkpoint signaling and provide useful tools for future research on Mad2 function and regulation.
authors.affiliation: a The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Garry G
authors.initials: GG
authors.lastname: Sedgwick
authors.affiliation: a The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Marie Sofie Yoo
authors.initials: MS
authors.lastname: Larsen
authors.affiliation: a The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Lischetti
authors.affiliation: a The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Werner
authors.initials: W
authors.lastname: Streicher
authors.affiliation: a The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Rosa Rakownikow
authors.initials: RR
authors.lastname: Jersie-Christensen
authors.affiliation: a The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Jesper V
authors.initials: JV
authors.lastname: Olsen
authors.affiliation: a The Novo Nordisk Foundation Center for Protein Research, Faculty of Health and Medical Sciences, University of Copenhagen , Copenhagen , Denmark.
authors.forename: Jakob
authors.initials: J
authors.lastname: Nilsson
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D006801:Humans; D059566:M Phase Cell Cycle Checkpoints / Q000502:physiology*; D064246:Mad2 Proteins / Q000737:chemistry / Q000276:immunology*; D011487:Protein Conformation
chemical_list: D000911:Antibodies, Monoclonal; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins
keywords: APC/C; Cdc20; Mad2; SAC; conformational specific; kinetochore