Doctor of Philosophy
Faculty of Science
Smail, Alison, Investigations into the role of mitochandrial permeability transition in apoptosis, Doctor of Philosophy thesis, Faculty of Science, University of Wollongong, 2002. http://ro.uow.edu.au/theses/1866
The role of mitochondrial permeability transition (MPT) in apoptosis has been a hard issue to resolve, mainly due to the lack of adequate techniques to measure M P T in intact cells. Most previous research has measured mitochondrial depolarisation and assumed that this reflected the occurrence of MPT, which is not necessarily true, as mitochondrial depolarisation can be caused by other events. The measurement of M P T directly in intact cells would help to establish the role of MPT in apoptosis. The aims of this thesis were to establish a system to study low conductance (LC-PT) and high conductance P T (HC-PT) in isolated mitochondria, perform time course experiments on Jurkat, U937 and H L 6 0 cells to establish the timing of M P T , mitochondrial depolarisation, release of cytochrome c and activation of caspase-3-like enzymes during apoptosis, and establish techniques to measure changes in MPT and mitochondria during apoptosis.
Conditions to induce both LC- and HC-PT were established in isolated mitochondria; mitochondria induced to undergo LC-PT with 40 uM PPIX released proteins including cytochrome c which activated caspase-3 in cytosol fractions of Jurkat cells. This raises the possibility that in vivo, LC-PT (and not just HC-PT) may release proteins to induce apoptotic degradative events. Experiments measuring M P T in intact cells and comparing the timing of induction with mitochondrial depolarisation, cytochrome c release and activation of caspase-3-like enzymes showed that MPT did have a controlling role in inducing apoptotic degradative events in U937 cells induced to undergo apoptosis with TNFa and cycloheximide. In Jurkat cells induced to undergo apoptosis with a-Fas antibody, MPT did not have a controlling role, while in HL60 cells induced to undergo apoptosis with etoposide the role of M P T needs further clarification. Co-staining cells to measure both mitochondrial depolarisation and HC-PT in the time courses mentioned above showed that MPT and mitochondrial depolarisation can occur independently in individual cells undergoing apoptosis. Lastly, the development of two new methods for measuring changes in mitochondria during apoptosis was attempted. A new confocal microscopy-based method to measure MPT in intact cells was developed. This method uses pinocytic loading to load cells with calcein and visualises mitochondria as darkened voids within a bright green cytosol. a-AIF (Apoptosis Inducing Factor) antibodies were successfully produced which recognised the AIF peptides used for immunisation. However these antibodies were unsuitable for immunofluorescence studies because they poorly recognised AIF in fixed, permeabilised cells and appeared to cross-react with other proteins in the cell.
This thesis provides new information about the role that MPT plays in apoptosis and also describes a new technique developed to measure MPT in intact cells.