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PMID: 21772247 Published · epublish English Journal Article Research Support, Non-U.S. Gov't

Probing the in vivo function of Mad1:C-Mad2 in the spindle assembly checkpoint.

The EMBO journal ·Vol. 30 ·No. 16 ·2011-07-19 ·Pages 3322-36

Fava LL, Kaulich M, Nigg EA, Santamaria A

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.

MeSH Terms
Adaptor Proteins, Signal Transducing/immunology,physiology Anaphase/physiology Animals Antibodies, Monoclonal/immunology Calcium-Binding Proteins/chemistry,immunology,physiology Cdc20 Proteins Cell Cycle Proteins/chemistry,immunology,physiology Dimerization Humans Kinetochores/metabolism Macromolecular Substances Mad2 Proteins Mice Mice, Inbred BALB C Mitosis/drug effects,physiology Nuclear Pore/metabolism Nuclear Proteins/chemistry,immunology,physiology Protein Conformation Protein Folding Protein Interaction Mapping RNA, Small Interfering/pharmacology Rabbits Repressor Proteins/chemistry,immunology,physiology Spindle Apparatus/physiology Time Factors
Chemicals
Adaptor Proteins, Signal Transducing Antibodies, Monoclonal Calcium-Binding Proteins Cdc20 Proteins Cell Cycle Proteins MAD1L1 protein, human MAD2L1 protein, human MAD2L1BP protein, human Macromolecular Substances Mad2 Proteins Nuclear Proteins RNA, Small Interfering Repressor Proteins CDC20 protein, human
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Fava Luca L
Growth and Development, Biozentrum, University of Basel, Basel, Switzerland.
Kaulich Manuel
Nigg Erich A
Santamaria Anna
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Article Info
Journal
The EMBO journal
Abbr.
EMBO J
ISSN
1460-2075
DOI
10.1038/emboj.2011.239
Published
2011-07-19
Epub
2011-00-19
Pages
3322-36
Language
English
Region
England
NLM ID
8208664
PMCID
PMC3160659
Subset
IM
External Links
PubMed source DOI: 10.1038/emboj.2011.239 PMC Full Text View journal details
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