In vitro FRAP identifies the minimal requirements for Mad2 kinetochore dynamics.
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 p31(comet), 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.
Vink
Martin
M
Department of Experimental Oncology, European Institute of Oncology, Milan, Italy.
Simonetta
Marco
M
Transidico
Pietro
P
Ferrari
Karin
K
Mapelli
Marina
M
De Antoni
Anna
A
Massimiliano
Lucia
L
Ciliberto
Andrea
A
Faretta
Mario
M
Salmon
Edward D
ED
Musacchio
Andrea
A
D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology; D036681:Fluorescence Recovery After Photobleaching; D018386:Kinetochores / Q000378:metabolism*; D064246:Mad2 Proteins; D009687:Nuclear Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000378:metabolism*; D008941:Spindle Apparatus / Q000378:metabolism
D002135:Calcium-Binding Proteins; D064199:Cdc20 Proteins; D018797:Cell Cycle Proteins; C501384:MAD1L1 protein, human; C577697:MAD2L1 protein, human; D064246:Mad2 Proteins; D009687:Nuclear Proteins; D012097:Repressor Proteins; C577488:CDC20 protein, human