Completion of BAX recruitment facilitates mitochondrial fragmentation during apoptosis
Maes, ME1,2, Schlamp, CL1, Nickells, RW1.
1Department of Ophthalmology and Visual Sciences, University of Wisconsin-Madison, Madison, WI, USA; 2Institute of Science and Technology Austria, Klosterneuburg, Austria
BAX controls the committed step of the intrinsic apoptotic program. Excess cellular stress stimulates BAX recruitment to the mitochondrial outer membrane (MOM), propelling the cell beyond the 'point of no return.' One hallmark of the apoptotic program is mitochondrial fragmentation. BAX is consistently associated with mitochondrial fission, however its role in mitochondrial fragmentation during apoptosis remains undefined.
BAX recruitment is best described by a sigmoid curve where each phase of the curve (baseline, logarithmic growth, and plateau) is separated by key cellular events. Cytochrome c release identifies the transition from baseline and logarithmic phases of BAX recruitment. Here, we show that mitochondrial fragmentation identifies the second transition from logarithmic to plateau phase of BAX recruitment. Time-lapse imaging showed a significant decrease in mitochondria volume in the plateau phase of BAX recruitment when compared to either baseline or logarithmic phases (*p < 0.05 for two cell lines).
A P168A BAX mutant showed that BAX recruitment must proceed beyond the baseline phase for mitochondrial fragmentation to occur. P168A BAX lacks MOM-integration, which prevents the logarithmic transition, however this mutant maintains mitochondrial fusion capabilities. In a BAX-/-/BAK-/- cells, P168A BAX does not induce mitochondrial fragmentation after STS addition when compared to WT BAX (p = 0.44). We further tested BAX's role by preventing mitochondrial fission using cells that lack functional DRP1. HeLaDRP1-/- cells or HeLa WT cells expressing a dominant negative R247E DRP1 showed no differences in the time of BAX recruitment initiation (p=0.21, 0.44) or in the rate of BAX recruitment (p=0.63, 0.29). However, in cell conditions where mitochondria did not fragment, there was a significant reduction in total recruited BAX (HeLaDRP1-/-, p < 0.01; HeLa+R247E DRP1, p < 0.001). These data suggest that the transition to the plateau phase, or the completion of BAX recruitment, facilitates mitochondrial fragmentation during apoptosis.