title: Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint.
abstract: The spindle assembly checkpoint (SAC) restrains anaphase until all chromosomes become bi-oriented on the mitotic spindle. The SAC protein Mad2 can fold into two distinct conformers, open (O) and closed (C), and can asymmetrically dimerize. 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 Mad1 to adopt the closed conformation and that the activity of the Mad1:C-Mad2 complex undergoes regulation by p31comet-dependent 'capping'. Furthermore, C-Mad2 antibody microinjection caused an abrupt termination of the SAC and accelerated mitotic progression. Remarkably, microinjection of a Mad1-neutralizing antibody triggered a comparable mitotic acceleration. Our study provides direct in vivo evidence for the model that a kinetochore complex of Mad1:C-Mad2 acts as a template to sustain the SAC and it challenges the distinction between SAC and mitotic timer.
authors.affiliation: Growth and Development, Biozentrum, University of Basel, Basel, Switzerland.
authors.forename: Luca L
authors.initials: LL
authors.lastname: Fava
authors.forename: Manuel
authors.initials: M
authors.lastname: Kaulich
authors.forename: Erich A
authors.initials: EA
authors.lastname: Nigg
authors.forename: Anna
authors.initials: A
authors.lastname: Santamaria
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000276:immunology / Q000502:physiology*; D000705:Anaphase / Q000502:physiology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D019281:Dimerization; D006801:Humans; D018386:Kinetochores / Q000378:metabolism; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008938:Mitosis / Q000187:drug effects / Q000502:physiology; D022022:Nuclear Pore / Q000378:metabolism; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D011487:Protein Conformation; D017510:Protein Folding; D025941:Protein Interaction Mapping; D034741:RNA, Small Interfering / Q000494:pharmacology; D011817:Rabbits; D012097:Repressor Proteins / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D008941:Spindle Apparatus / Q000502:physiology*; D013997:Time Factors
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; C470309:MAD2L1BP protein, human; D046911:Macromolecular Substances; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; D012097:Repressor Proteins; C577488:CDC20 protein, human