ABSTRACT
Protocol 10.1: Isolation of cytosolic extracts 265
Protocol 10.2: Isolation of soluble membrane extracts 265
Protocol 10.3: Isolation of mouse liver mitochondria 266
Protocol 10.4: Isolation of mitochondria from cultured cells 267
Protocol 10.5: Induction of permeability transition (PT) in isolated mitochondria
Protocol 10.6: Induction of mitochondrial outer membrane permeabilisation
10.3 Detecting the translocation of proteins to and from mitochondria in cells
Protocol 10.7: Cellular fractionation to detect cytochrome c release
Protocol 10.8: Detection of apoptosis inducing factor 273
Protocol 10.9: Detecting adenylate kinase 274
Protocol 10.10: Measurement of protein translocation by immunocytochemistry
Protocol 10.11: Quantitative analysis of cytochrome c-GFP redistribution in cells by flow cytometry
10.4 The impact of mitochondrial outer membrane permeabilisation on mitochondrial function
Protocol 10.12: Measurement of ∆Ψm in live cells 280
Protocol 10.13: Measurement of ∆Ψm in cells that are to be fixed 280
Protocol 10.14: ATP measurements in cells 281
Protocol 10.15: Measurement of oxygen consumption by isolated mitochondria
Protocol 10.16: Measurement of oxygen consumption in permeabilised cells
Protocol 10.17: Measurement of reactive oxygen species in intact cells
10.5 Physical changes in mitochondria 286
Protocol 10.18: Measurement of complex III accessibility 287
Protocol 10.19: Measurement of complex IV accessibility 287
Protocol 10.20: Measurement of mitochondrial swelling 288
Protocol 10.21: Detection of PT in isolated mitochondria using calcein-AM fluorescence
Protocol 10.22: Detection of PT in cells using calcein-AM fluorescence
Protocol 10.23: Determination of mitochondrial mass 290
10.6 References 290
10.1 Introduction
Mitochondria are complex energy-producing organelles enclosed by an inner and an outer membrane. During apoptosis, the outer membrane becomes permeable and proteins contained within the mitochondrial intermembrane space are released into the cytosol. These include cytochrome c, AIF, SMAC (also known as DIABLO) and htrA2, all of which have been shown to exert pro-apoptotic activities. Oncogenes such as Bcl-2, which prevents permeabilisation of the outer membrane and the release of intermembrane space proteins, block apoptotic cell death and
Cell Proliferation and Apoptosis, David Hughes and Huseyin Mehmet (Eds) © 2003 BIOS Scientific Publishers Ltd, Oxford
maintain the clonogenic potential of the cell. In contrast, inhibition of apoptosis downstream of mitochondrial outer membrane permeabilisation only delays eventual cell death. Mitochondrial outer membrane permeabilisation has therefore received much interest as a critical point of regulation in the apoptotic process. In this chapter we will discuss some of the studies that have established a role for mitochondria in apoptosis and the methods that have been used to dissect some of the complex issues surrounding mitochondrial outer membrane permeabilisation.
