Dynamics of outer mitochondrial membrane permeabilization during apoptosis

Abstract

Individual cells within a population undergo apoptosis at distinct, apparently random time points. By analyzing cellular mitotic history, we identified that sibling HeLa cell pairs, in contrast to random cell pairs, underwent apoptosis synchronously. This allowed us to use high-speed cellular imaging to investigate mitochondrial outer membrane permeabilization (MOMP), a highly coordinated, rapid process during apoptosis, at a temporal resolution approximately 100 times higher than possible previously. We obtained new functional and mechanistic insight into the process of MOMP: We were able to determine the kinetics of pore formation in the outer mitochondrial membrane from the initiation phase of cytochrome-c-GFP redistribution, and showed differential pore formation kinetics in response to intrinsic or extrinsic apoptotic stimuli (staurosporine, tumor necrosis factorrelated apoptosis-inducing ligand (TRAIL)). We also detected that the onset of mitochondrial permeabilization frequently proceeded as a wave through the cytosol, and that the frequency of wave occurrence in response to TRAIL was reduced by inhibition of protein kinase CK2. Computational analysis by a partial differential equation model suggested that the spread of permeabilization signals could sufficiently be explained by diffusion-adsorption velocities of locally generated permeabilization inducers. Taken together, our study yielded the first comprehensive analysis of clonal cell-to-cell variability in apoptosis execution and allowed to visualize and explain the dynamics of MOMP in cells undergoing apoptosis. Apoptosis is an evolutionary conserved cell death process that is fundamental to remove superfluous and damaged cells from the bodies of multicellular organisms. Impaired or excessive apoptosis has repeatedly been implicated as a major contributor to proliferative and degenerative diseases. Initiated by BH-3-only proteins of the Bcl-2 protein family, mitochondrial outer membrane permeabilization (MOMP) and the subsequent activation of effector caspases are rapid key processes crucial for the efficient execution of apoptotic cell death.Previous single-cell imaging studies using fluorescently tagged proteins showed that MOMP results in the coordinate release of mitochondrial intermembrane space proteins, including cytochrome-c (cyt-c) and Smac/Diablo, into the cytosol. The release was suggested to occur synchronously throughout the cytosol and with very fast kinetics, which seemed independent of the type of the apoptotic stimulus used, and also independent of downstream effector caspase activity. [1][2][3][4][5] Cytosolic cyt-c induces the rapid formation of the large multiprotein apoptosome complex, caspase-9 activation and subsequent activation of effector caspases-3 and -7. 6,7 In parallel, cytosolic Smac neutralizes x-linked inhibitor of apoptosis protein, the key inhibitor of caspases-9, -3 and -7. 8 As long as all key components of the execution network are present, effector caspase activation proceeds as a rapid and kinetically largely i...

Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation

“…7 Furthermore, in recent work we also provided evidence that MOMP inducers, once activated, will translocate within seconds to mitochondria. 15,29 Nevertheless, even at optimised sensitivity at nonphototoxic conditions, we still detected mitochondrial depolarisation, indicative of MOMP, before the onset of mitochondrial Bax accumulation in YFP-Bax-expressing DU-145 cells. As this was much less frequently observed in cells in which bak gene expression was silenced, it is possible that such cell-to-cell variability arose from different endogenous levels of Bak protein expression, or from different protein expression levels of other Bcl-2 family proteins.…”

“…14 Single-cell imaging studies using Cyt-C-GFP or other GFP-or fluorescently labelled mitochondrial intermembrane proteins have demonstrated that MOMP is a rapid process, completed in cells within minutes if not seconds. 10,12,15 Although these studies suggested a high efficiency of the permeabilisation process, quantitative insights into the initiation of MOMP and the respective roles of Bax and Bak in this process have so far primarily been obtained from studies using artificial membranes and liposomes 5,7,8 but are still lacking from experiments in living cells within an intact biological environment. We here performed a quantitative confocal microscopy and mathematical modelling study of the temporal and spatial dynamics of Bax translocation and oligomerisation relative to MOMP initiation in single living cells.…”

Intramitochondrial recruitment of endolysosomes mediates Smac degradation and constitutes a novel intrinsic apoptosis antagonizing function of XIAP E3 ligase

“…[15][16][17][18] While targeting of damaged mitochondria suggests that mitophagy may counter apoptotic mitochondria, mitophagy occurs progressively over days, 12,14,16,17,19 a rate that is likely insufficient to alter intracellular propagation of mitochondrial apoptosis, which can occur within minutes. 20,21 Indeed, Bnip3-and Fundc1-induced mitophagy have no direct effect on apoptosis, 14,18 and we determined that Bnip3-mediated mitophagy was cytoprotective if activated before apoptosis. 17 While MDV delivery of mitochondria to lysosomes operates at a higher rate, minutes to hours, 10 this process is regulated by Parkin and restricted to specific mitochondrial components.…”

“…Mitochondrial apoptosis transmits within a cell over minutes, 20,21 yet mitophagy can require transcriptional upregulation, 14,16,17,19 with mitochondrial elimination occurring progressively over days. [15][16][17] In contrast, here we demonstrate that, concurrent with reduced Smac levels, cytochrome c release was delayed in XIAP-and Bax-targeted mitochondria, indicating that XIAP can uncouple MOMP from apoptosis signaling, at least initially.…”

Temporal Heterogeneity Metrics in Apoptosis Induced by Anticancer Drugs

“…Apoptosis is a form of programmed cell death that occurs in a largely asynchronous manner in cell populations (Kroemer et al 1995;Messam and Pittman 1998;Rehm et al 1992;Green 2005;Albeck et al 2008;Hellwig et al 2008;Bhola and Simon 2009;Rehm et al 2009;Spencer et al 2009). Indeed, heterogeneity is an intrinsic feature of cell populations and is the basis of cell fate regulation in a variety of processes (Spiller et al 2010).…”