Analysis of Bcl-2 family protein interactions in live cells by fluorescence recovery after photobleaching.

  • Ricardo Rodriguez-Enriquez

Student thesis: Phd

Abstract

The Bcl-2 family of proteins strictly regulates the intrinsic pathway of apoptosis.Direct physical interactions between Bcl-2 proteins regulate mitochondrial outerpermeabilisation (MOMP), which occurs in response to various cell stresses andapoptotic stimuli. How changes in Bcl-2 protein activity regulate apoptosiscommitment is still unclear, especially with regard to how they interact with eachother within the context of the mitochondrial membrane. Recent studies haveshown that Bcl-2 proteins exist in a dynamic equilibrium between the mitochondriaand the cytosol. In this thesis, by using FRAP, I have measured changes in Bcl-XLand Mcl-1 dynamics in single cells. Surprisingly, individual cells within a populationshow widely differing Bcl-XL and Mcl-1 dynamics. There is a corelation betweenBcl-XL and Mcl-1 dynamics with BH3-only protein expression. Anti-apoptotic andpro-apoptotic Bcl-2 proteins stabilise each other on the OMM. Together, theseresults indicate that cells constantly fine tune mitochondrial priming and thatanalysing anti-apoptotic Bcl-2 proteins by FRAP allows this to be measured at asingle cell level in real time before MOMP.
Date of Award31 Dec 2014
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorAndrew Gilmore (Supervisor) & Patrick Caswell (Supervisor)

Keywords

  • Bcl-2 family protein, FRAP

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