<p>Bid is a member of the Bcl-2 family of proteins, which are known as the regula- tors of apoptosis. Bid recruits Bax, another Bcl-2 family protein, which forms large oligomers that permeabilize the mitochonrdial outer membrane during apoptosis. In this thesis, Bid complexes embedded in a mitochondria-like supported lipid bilayer were investigated using single molecule fluorescence techniques. The bilayer, con- taining a lipophilic tracer, was formed on a mica surface and ATTO647 labelled Bid was added to it. For experiments where the effect of Bax on Bid complexes was investigated, a wild type Bax or a HiLyte488 labelled Bax was added as well. The protein-bilayer sample was imaged using total internal reflection fluorescence (TIRF). The formation of a fluid bilayer was confirmed by the observation of the lateral diffusion of DiD. Single particle tracking of the lipid molecules was used to measure the diffusion coefficent of DiD which was determined to be 2.2μm2 /s. The TIRF images also revealed two populations of Bid complexes, immobile and mobile. The diffusion coefficient of the observed Bid complexes was determined to be about three times slower than that of DiD (0.8±0.5μm2 /s). This provides evidence that mobile Bid is embedded in the bilayer. Image analysis of immobile Bid complexes showed a step-wise decrease in the fluorescence intensity due to photobleaching. The oligomeric distribution of the immobile Bid complexes was determined from the num- ber of steps, which corresponds to the number of particles in each complex. From these distributions it was concluded that the imaged immobile Bid existed mainly as monomers. However dimer and trimer complexes of Bid were also observed. The detected oligomeric distribution was not affected by the presence of either wild type Bax or Hilyte488 Bax. However Bid was imaged for the first time participating in Bax complexes. The acquired results somewhat differ from what had been observed in confocal imaging of the same samples, where mostly larger Bid complexes (dimers and up) were detected. We attribute the difference to the superior sensitivity of the TIRF method presented here.</p> / Master of Science (MSc)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/15349 |
| Date | 24 April 2015 |
| Creators | Hirmiz, Nehad |
| Contributors | Fradin, Cécile, Physics and Astronomy |
| Source Sets | McMaster University |
| Detected Language | English |
| Type | thesis |
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