Pinocytic loading of cytochrome c into intact cells specifically induces caspase-dependent permeabilization of mitochondria: evidence for a cytochrome c feedback loop.
Previous studies introduced cytochrome c into intact cells via the disruptive techniques of microinjection or electroporation to provide support for the hypothesis that, in whole cells, cytochrome c release from mitochondria triggers caspase activation and other degradative changes. However, the types of measurements that could be undertaken with these techniques was limited. We used the simple and relatively gentle technique of pinocytic loading to demonstrate that, in intact cells, cytosolic cytochrome c specifically induced activation of caspase-3- and -9-like enzymes, and a loss of mitochondrial polarization coincident with an increase in mitochondrial permeability. Our results support the prediction from in vitro studies that activation of caspases-3 and -9 is downstream of cytochrome c release and provide the first direct evidence that, in whole cells, cytochrome c-dependent caspase-activation can exert a feedback effect to elicit mitochondrial permeabilization and collapse of the mitochondrial trans-membrane potential.
Gilmore
K J
KJ
Department of Biological Sciences, University of Wollongong, Northfields Avenue, Wollongong. NWS. 2522. Australia.
Quinn
H E
HE
Wilson
M R
MR
D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D003574:Cytochrome c Group / Q000378:metabolism*; D004789:Enzyme Activation; D005246:Feedback; D005434:Flow Cytometry; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism*; D019169:Jurkat Cells; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D010539:Permeability / Q000187:drug effects; D010873:Pinocytosis / Q000187:drug effects; D013997:Time Factors; D020298:U937 Cells
D003574:Cytochrome c Group; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases