In Vitro FRAP Identifies the Minimal Requirements for Mad2 Kinetochore Dynamics

Martin Vink, Marco Simonetta, Pietro Transidico, Karin Ferrari, Marina Mapelli, Anna De Antoni, Lucia Massimiliano, Andrea Ciliberto, Mario Faretta, Edward D. Salmon, Andrea Musacchio

Research output: Contribution to journalArticle

72 Citations (Scopus)

Abstract

Background: Mad1 and Mad2 are constituents of the spindle-assembly checkpoint, a device coupling the loss of sister-chromatid cohesion at anaphase to the completion of microtubule attachment of the sister chromatids at metaphase. Fluorescence recovery after photobleaching (FRAP) revealed that the interaction of cytosolic Mad2 with kinetochores is highly dynamic, suggesting a mechanism of catalytic activation of Mad2 at kinetochores followed by its release in a complex with Cdc20. The recruitment of cytosolic Mad2 to kinetochores has been attributed to a stable receptor composed of a distinct pool of Mad2 tightly bound to Mad1. Whether specifically this interaction accounts for the kinetochore dynamics of Mad2 is currently unknown. Results: To gain a precise molecular understanding of the interaction of Mad2 with kinetochores, we reconstituted the putative Mad2 kinetochore receptor and developed a kinetochore recruitment assay with purified components. When analyzed by FRAP in vitro, this system faithfully reproduced the previously described in vivo dynamics of Mad2, providing an unequivocal molecular account of the interaction of Mad2 with kinetochores. Using the same approach, we dissected the mechanism of action of p31comet, a spindle-assembly checkpoint inhibitor. Conclusions: In vitro FRAP is a widely applicable approach to dissecting the molecular bases of the interaction of a macromolecule with an insoluble cellular scaffold. The combination of in vitro fluorescence recovery after photobleaching with additional fluorescence-based assays in vitro can be used to unveil mechanism, stoichiometry, and kinetic parameters of a macromolecular interaction, all of which are important for modeling protein interaction networks.

Original languageEnglish
Pages (from-to)755-766
Number of pages12
JournalCurrent Biology
Volume16
Issue number8
DOIs
Publication statusPublished - Apr 18 2006

Fingerprint

Fluorescence Recovery After Photobleaching
Kinetochores
Photobleaching
kinetochores
Fluorescence
Recovery
M Phase Cell Cycle Checkpoints
Assays
Chromatids
chromatids
Macromolecules
Kinetic parameters
Scaffolds
Stoichiometry
Protein Interaction Maps
Anaphase
receptors
Chemical activation
In Vitro Techniques
fluorescence recovery after photobleaching

Keywords

  • CELLBIO

ASJC Scopus subject areas

  • Agricultural and Biological Sciences(all)

Cite this

In Vitro FRAP Identifies the Minimal Requirements for Mad2 Kinetochore Dynamics. / Vink, Martin; Simonetta, Marco; Transidico, Pietro; Ferrari, Karin; Mapelli, Marina; De Antoni, Anna; Massimiliano, Lucia; Ciliberto, Andrea; Faretta, Mario; Salmon, Edward D.; Musacchio, Andrea.

In: Current Biology, Vol. 16, No. 8, 18.04.2006, p. 755-766.

Research output: Contribution to journalArticle

Vink, M, Simonetta, M, Transidico, P, Ferrari, K, Mapelli, M, De Antoni, A, Massimiliano, L, Ciliberto, A, Faretta, M, Salmon, ED & Musacchio, A 2006, 'In Vitro FRAP Identifies the Minimal Requirements for Mad2 Kinetochore Dynamics', Current Biology, vol. 16, no. 8, pp. 755-766. https://doi.org/10.1016/j.cub.2006.03.057
Vink, Martin ; Simonetta, Marco ; Transidico, Pietro ; Ferrari, Karin ; Mapelli, Marina ; De Antoni, Anna ; Massimiliano, Lucia ; Ciliberto, Andrea ; Faretta, Mario ; Salmon, Edward D. ; Musacchio, Andrea. / In Vitro FRAP Identifies the Minimal Requirements for Mad2 Kinetochore Dynamics. In: Current Biology. 2006 ; Vol. 16, No. 8. pp. 755-766.
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AU - De Antoni, Anna

AU - Massimiliano, Lucia

AU - Ciliberto, Andrea

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AU - Salmon, Edward D.

AU - Musacchio, Andrea

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