Deciphering the dynamics and molecular mechanisms .. (APCACTIVATION)
Deciphering the dynamics and molecular mechanisms of APC/C activity in vitro and in live cells
Start date: Jan 1, 2014,
End date: Jan 31, 2016
"The spindle assembly checkpoint (SAC) inhibits chromosome segregation and mitotic exit prior to biorientation of all chromosomes on the mitotic spindle by restraining the activity of the anaphase promoting complex or cyclosome (APC/C) towards Cyclin B1 and Securin. In contrast, other APC/C substrates like Cyclin A2 bypass this inhibition and are degraded in the presence of an active SAC. Despite its central role in mitotic progression, the molecular mechanisms activating the APC/C towards different substrates are poorly defined. Here, I present a research plan that aims to decipher the dynamics and molecular mechanisms of APC/C activity in live cells and in vitro. Specifically, I will establish an assay to quantify the spatial and temporal dynamics of APC/C-dependent ubiquitylation in live human cells by Foerster resonance energy transfer (FRET) between endogenously tagged Cyclin B1 and ubiquitin. In parallel, I will reconstitute the APC/C in vitro from recombinant proteins and quantify ubiquitylation activity of the reconstituted complex using fluorescently labelled substrates to assess the function of individual APC/C subunits, cofactors and posttranslational modifications. Together, the results obtained in live cells and in vitro will substantially advance our understanding of the molecular mechanisms that govern mitotic progression in human cells."
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