Polyribosome Fractionation Cells were incubated with cycloheximide and cytoplasmic lysates were fractionated by centrifugation through 1050%

April 27, 2017

understood. The MCC might bind to the APC as a pseudosubstrate due to a KEN-box motif in BubR1. This indicates that the MCC needs to disassemble from the APC at metaphase to elicit anaphase. Bub1 and Aurora-B kinase contribute directly to the formation of a complex of the MCC with the APC ). Unattached AZD 1152 custom synthesis kinetochores might sensitize the APC for inhibition by the MCC ). In addition to kinetochore attachment, tension is important for MSAC inactivation: if both sister kinetochores attach to microtubules from the same pole, not enough tension 23863710 is generated and microtubules kinetochore attachment is destabilized to correct the problem. This destabilization depends on Aurora-B kinase. Again, these effects are subsumed by the switching parameter u. For complex dissociation we consider two model variants: In the ��Dissociation variant”, we assume that MCC binds to APC and that this binding is reversible ). Free Cdc20 has to bind reversibly to APC ), effectively competing with MCC. In the ��Convey variant”, we do not assume that the APC:MCC complex simply dissociates into APC and MCC, but that the MCC complex falls apart so that the Cdc20 contained in the MCC complex can bind to the APC ). Methods Biochemical background Our model incorporates three MSAC-related mechanisms: the Template Model, the MCC formation, and the APC inhibition. Their biochemical details will be explained in the following. Mad2 Template Model DeAntoni et al. proposed the ��Template Model��explaining the mechanism of Mad2 recruitment to the kinetochore during checkpoint activation and subsequent transfer to sequester Cdc20. Recent work by Vink et al. and Mapelli et al. provide additional support for the Template Model. Moreover, this model has been confirmed by Nezi et al., and is entirely consistent with recent Fluorescence Recovery After 20522545 Photobleaching data. The Template Model is superior and more solid than the Exchange Model, which we confirmed in a recent in silico study . The Mad2 Template Model is described by the reaction equations Eqs. . It is assumed that Mad1 and C-Mad2 form a stable core complex Mad1:C-Mad2 at unattached kinetochores. In our nonlinear ordinary differential equations model, we assume that this process has already been completed. Therefore, there is no free Mad1. Equation describes how the Mad1:C-Mad2 core complex binds additional molecules of O-Mad2 through formation of conformational heterodimers between the C- Mad2 subunit of the Mad1:C-Mad2 complex and O-Mad2. Upon Mad1:CMad2 binding, O-Mad2 adopts an intermediate conformation, which can quickly and efficiently bind Cdc20 and switch to the C-conformation. This process is documented by Eq.: Cdc20 binding to the complex Mad1:C-Mad2:O-Mad2 leads to Control by attachment Several reactions in the reaction scheme are controlled by the attachment of microtubules to the kinetochore which is realized by the factor u present in several reaction equations. Factor u represents the function of proteins like p31comet, UbcH10, and Dynein. p31comet prevents further Spindle Assembly Checkpoint Mad2 turnover on Mad1 and neutralizes the inhibitory activity of Cdc20-bound Mad2. Catalytically active UbcH10 can promote the release of checkpoint proteins from APC. Dynein removes the Mad1:C-Mad2 2:2 complex from the kinetochore site after microtubule attachment. Thus, p31comet, UbcH10, and Dynein work in concert during checkpoint inactivation. Also the MCC:APC complex dissociation might be attachment controlled. We there