Abstract
因申請專利緣故，資料延後公開;Abstract Bax is predominantly a cytosolic monomer in healthy cells. Upon receiving apoptotic signal, it performs a series of biological processes, which include conformational changes, translocation to mitochondria as well as oligomerization, consequently leading to the event of mitochondrial outer membrane permeabilization (MOMP) and then cell death. However, little is known about the molecular basis of the driving forces that direct the translocation of Bax to bring about the MOMP. Localization of Bax to the mitochondrial outer membrane is a key step to the activation ability of Bax to modulate mitochondrial membrane integrity. In this thesis, we used giant unilamellar vesicle (GUV) along with confocal fluorescence microscope to investigate how this Bax translocation, upon receipt of apoptotic signaling, depend on the lipid compositions and properties. The activation of Bax was induced with the addition of BimBH3 peptides, which is known to be useful to promote the oligomerization of Bax (o-Bax). This study prepared GUVs of several different lipid compositions using PC (POPC), PE (POPE), PG (POPG), and CL (Cardiolipin). Quantitative analyses regarding the membrane permeability and pore size were obtained by the time-dependent fluorescence intensity observed within GUVs from three fluorescence probes of different molecular size. In PC-only GUVs, some leakages due to the associations of Bax with membrane surface could be observed. The leakages were found to be mostly caused by small pores (> 0.45 nm in radius), although there existed small number of large pores (> 6 nm) that appeared transiently during the observations. In GUVs composed of PC/PG, both the membrane surface association of Bax and the pore permeability were largely abolished. The charged property of PG, though similar to CL, does not seem to be important. In the PC-based GUVs containing of PE or CL, stable large pores (> 6 nm) could only be II observed in the presence of o-Bax. The membrane permeability and the stability of the large pores were found to be higher in the PC/CL than in the PC/PE vesicles, indicating that the greater the negative curvature of the doped lipid is, the greater the number of the large pores is observed. Collectively, our study indicates that the formation and stability of large pores, which are essential to the MOMP process, depend critically on the lipid compositions. Inactive Bax monomers could easily cause disturbances to membrane surface. The disturbances result in some accidental breaks for small probes to flow in and, therefore, are considered as a measure of the membrane surface association of Bax monomers. O-Bax was found to effectively induce large pores on PC-based GUVs containing either CL or PE but rather than PG. Nevertheless, only in the presence of CL could the large pores remain stable over a long time. Our studies demonstrate the importance of the curvature property of lipids in the MOMP process.