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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 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.
