- Email: [email protected]
BAX and BAK at the Gates of Innate Immunity
TICB 1414 No. of Pages 3
Spotlight
BAX and BAK at the Gates of Innate Immunity Lorenzo Galluzzi1,2,3,* and Claire Vanpouille-Box1,* Large BAX/BAK pores that form during apoptosis enable mitochondrial nucleoids to access the cytosol, potentially leading to inflammatory signaling via [69_TD$IF]CGAS. Under physiological conditions, however, BAX/BAK-dependent caspase activation rapidly dismantles dying cells to prevent inflammatory responses. BAX and BAK operate at the interface between apoptotic signaling and innate immunity control. Intrinsic apoptosis and some variants of extrinsic apoptosis rely on the ability of [2_TD$IF]BCL2 associated X, apoptosis regulator (BAX) [70_TD$IF]and BCL2 antagonist/killer [71_TD$IF]1 (BAK1; best known as BAK) to mediate mitochondrial outer membrane permeabilization (MOMP). Widespread MOMP has a variety of detrimental consequences for the cell, including the translocation of cytochrome c from the mitochondrial intermembrane space into the cytosol. Together with deoxyATP and apoptotic peptidase activating factor 1 (APAF1), cytosolic cytochrome c drives the assembly of the so-called ‘apoptosome’, a supramolecular platform for the recruitment and activation of caspase 9 (CASP9), de facto precipitating the proteolytic maturation of executioner caspases including CASP3 and hence sealing the cell fate [1]. Of note, apoptotic cell death is generally (but not always) unable to drive inflammation, at least in part owing to multiple CASP3-dependent processes that promote the emission of anti-inflammatory signals and/or inhibit
the emission of proinflammatory signals [2]. Importantly, MOMP is also associated with the translocation of mitochondrial DNA (mtDNA), often in the form of nucleoids complexed with transcription factor A, mitochondrial (TFAM), from the mitochondrial matrix to the cytosolic compartment [3,4]. This can potently activate [72_TD$IF]cyclic GMP-AMP synthase (CGAS) to initiate a signal transduction cascade that critically relies on transmembrane protein 173 (TMEM[73_TD$IF]173) (best known as STING) and culminates with the synthesis of proinflammatory cytokines such as type I interferon (IFN) [5,6]. Under physiological conditions, however, caspases robustly prevent MOMP-driven CGAS–STING signaling to preserve the anti-inflammatory nature of apoptosis [7,8]. Recent data from McArthur et al. [9] elegantly demonstrate that BAX and BAK are not only responsible for the MOMP-associated efflux of cytochrome c and consequent activation of caspases, but also provide a gateway for the inner mitochondrial membrane to herniate, de facto enabling mitochondrial nucleoids to access the cytosolic compartment. To confirm that mtDNA is released in the cytosol of cells succumbing to intrinsic apoptosis, McArthur et al. harnessed live-cell lattice light-sheet microscopy (LLSM) to image Mcl1 / [68_TD$IF] mouse embryonic fibroblasts (MEFs) stably expressing a fluorescent variant of TFAM plus a form of translocase of outer mitochondrial membrane 20 (TOMM20) tagged with a HaloTag moiety that can be detected on the addition of a cell-permeant fluorescent dye, following the administration of the small molecule ABT-737. In this cellular model, ABT-737 – which operates by simultaneously inhibiting BCL2, apoptosis regulator (BCL2), and BCL2 like 1 (BCL2L1) (best known as BCL-XL) – is sufficient to potently drive BAX/BAKdependent apoptosis. Mcl1 / MEFs rapidly (within 20 min) responded to
ABT-737 by undergoing mitochondrial rearrangement followed by the release of TFAM-associated mtDNA into the cytosol, irrespective of the presence of the pan-caspase inhibitor QVD-QPh, which was required to avoid rapid detachment of the cells from the coverslip. 3D surface reconstruction and live 3D structured illumination microscopy (3D-SIM) revealed that TFAM-containing nucleoids: (i) are released from a single site on fragmented mitochondria; and (ii) generally remain in the proximity of the specific mitochondrion of origin. In this setting, cytochrome c efflux preceded mitochondrial rearrangements and the release of TFAM-associated mtDNA. Consistent with previous findings, Mcl1 / MEFs maintained in the presence of QVD-QPh secreted measurable amounts of interferon beta 1 (IFNB1) 4–6 h after the administration of ABT-737, a time at which various proteins of the mitochondrial matrix including TFAM could be detected in the cytosolic fraction by immunoblotting [9]. Next, the authors set to investigate the influence of mitochondrial dynamics on the release of TFAM-containing nucleoids from MEFs succumbing to apoptosis. To this aim, they generated Opa1 / [74_TD$IF]Mcl1 / and Mfn1 / Mfn2 / Mcl1 / MEFs, both of which are defective in mitochondrial fusion, as well as Dnm1l / Mcl1 / MEFs, which are defective in mitochondrial fission. Opa1 / Mcl1 / and Mfn1 / Mfn2 / Mcl1 / MEFs exhibited hyperfragmented mitochondria at baseline and were slightly more sensitive to ABT-737-driven cytochrome c release and nucleoid efflux than Mcl1 / MEFs. MEFs from Dnm1l / Mcl1 / mice displayed hyperfused mitochondria under baseline conditions and responded to ABT-737 with incomplete mitochondrial rearrangement coupled to the formation of ‘beads-on-string’ structures and limited release of TFAM-associated mtDNA.
Trends in Cell Biology, Month Year, Vol. xx, No. yy
1
RelaƟve degree
TICB 1414 No. of Pages 3
ApoptoƟc sƟmulus
Cytochrome c release
Mitochondrial fragmentaƟon
mtDNA release
ApoptoƟc RCD
CGAS–STING signaling
Figure 1. K [6_TD$IF] inetics of Intrinsic Apoptosis Impacts on Immunogenicity. On induction of intrinsic apoptosis, mitochondria rapidly release cytochrome c via small BAX/BAK pores. This enables prompt caspase activation and hence generates a cellular microenvironment that is poorly permissive for CGAS–STING signaling. As mitochondrial fragments and large BAX/BAK pores form, mitochondrial DNA (mtDNA) accessing the cytosol can no longer drive the synthesis of type I interferon, and cells succumb to apoptosis in the absence of inflammatory signaling. RCD, regulated cell death.
These latter findings, however, could not be reproduced by deleting Dnm1l from Mcl1 / MEFs with the CRISPR–Cas9 system. In this latter Dnm1l / Mcl1 / cellular model, mitochondrial responses to ABT-737 were similar to those observed in Mcl1 / MEFs. Despite such an unresolved discrepancy, all Dnm1l / cells tested produced normal amounts of IFNB1 on treatment with ABT-737 in the presence of QVD-QPh, demonstrating that fission is not involved in mtDNAdriven CGAS activation [9].
Trends in Cell Biology, Month Year, Vol. xx, No. yy
In summary, McArthur et al. [7_TD$IF]provided an elegant demonstration that BAX and BAK enable the efflux of mitochondrial nucleoids in the course of intrinsic apoptosis as the inner mitochondrial membrane herniates. BAX and BAK are also responsible for the apoptotic release of cytochrome c and consequent activation of caspases, which (at least in the model employed by these authors) occur before the efflux of TFAM-associated mtDNA. It is tempting to speculate, but remains to be formally elucidated, that the kinetics of process may be critical for multiple instances of apoptosis to remain immunologically silent [10] (Figure 1). Irrespective of this untested possibility, the findings by McArthur et al. add to an abundant literature that delineates [78_TD$IF]an intimate crosstalk between cell death control and the regulation of innate immunity [3,4].
cannot be detected by LLSM. Conversely, the release of TFAM-associated mtDNA was observed only after the formation of large BAX/BAK pores, which encircled the point of mtDNA exit from fragmented mitochondria, as elegantly demonstrated by 3D-SIM. A fluorophore targeted to the mitochondrial matrix exited the mitochondria of ABT-737treated Mcl1 / [75_TD$IF] MEFs in a manner similar to TFAM-containing nucleoids, strongly suggesting the existence of a physical barrier to cytosolic diffusion. Expression of a fluorescent variant of distal membrane arm assembly complex 1 (DMAC1) coupled to LLSM and 3D-SIM confirmed that such a physical barrier is provided by the inner mitochondrial membrane, which herniates across BAX/BAK macropores to form cytosolic bulges staining positively for TFAM. Immunogold labeling for fluoAcknowledgments rescent TFAM and transmission electron C.V-B. is supported by the 2017 Kellen Junior Faculty microscopy provided additional data in Fellowship from the Anna-Maria and Stephen Kellen support of this mechanism [9]. Foundation (New York, NY, USA). L.G. is supported
ABT-737 induced rapid dissipation of the mitochondrial transmembrane potential (Dcm) in Mcl1 / MEFs, which was not etiologically involved in the release of TFAM-containing nucleoids, as well as the formation of large mitochondrial foci containing BAX or BAK (as demonstrated by reconstituting Bax / Bak1 / Mcl1 / MEFs with fluorescent variants of either protein), which appeared 4–8 min after cytochrome c loss. This suggests that cytochrome c exits the intermembrane Of note, CGAS did not relocalize to space by small BAX/BAK pores that mtDNA-containing cytoplasmic bulges
2
and manifested no discernible change in subcellular distribution for up to 4–6 h after ABT-737 administration, when the supernatant of Mcl1 / MEFs exposed to ABT-737 in the presence of QVD-QPh contained detectable amounts of IFNB1. Correlative light and electron microscopy (CLEM) revealed that a minority of hernias forming in this setting lose their integrity starting at 90 min after ABT-767 administration, potentially constituting the source of mtDNA for CGAS activation. In a very few instances, single-[76_TD$IF] membraned TFAM-containing structures were also observed. The significance of such entities, resembling (at least to some degree) the CGAS-activating micronuclei that form in response to DNA damage [6], remains to be elucidated [9].
by intramural funds from the Department of Radiation Oncology of Weill Cornell Medical College (New York, NY, USA) and by donations from Phosplatin
TICB 1414 No. of Pages 3
Therapeutics (New York, NY, USA), the Luke Heller
References
TECPR2 Foundation (Boston, NY, USA), and Sotio a.
1. Galluzzi, L. et al. (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 25, 486–541
s. (Prague, Czech Republic). 1
Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA 2 Sandra and Edward Meyer Cancer Center, New York, NY, USA 3 Université Paris Descartes/Paris V, Paris, France *Correspondence: [email protected] (L. Galluzzi) and [email protected] (C. Vanpouille-Box).
2. Galluzzi, L. et al. (2016) Caspases connect cell-death signaling to organismal homeostasis. Immunity 44, 221–231 3. Mehta, M.M. et al. (2017) Mitochondrial control of immunity: beyond ATP. Nat. Rev. Immunol. 17, 608–620 4. Galluzzi, L. et al. (2012) Mitochondria: master regulators of danger signalling. Nat. Rev. Mol. Cell Biol. 13, 780–788 5. Chen, Q. et al. (2016) Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142–1149
6. Galluzzi, L. et al. Snapshot: CGAS–STING signaling. Cell (in press) 7. Rongvaux, A. et al. (2014) Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 159, 1563–1577 8. White, M.J. et al. (2014) Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 159, 1549–1562 9. McArthur, K. et al. (2018) BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science 359, eaao6047 10. Galluzzi, L. et al. (2017) Immunogenic cell death in cancer and infectious disease. Nat. Rev. Immunol. 17, 97–111
https://doi.org/10.1016/j.tcb.2018.02.010
Trends in Cell Biology, Month Year, Vol. xx, No. yy
3
Spotlight
BAX and BAK at the Gates of Innate Immunity Lorenzo Galluzzi1,2,3,* and Claire Vanpouille-Box1,* Large BAX/BAK pores that form during apoptosis enable mitochondrial nucleoids to access the cytosol, potentially leading to inflammatory signaling via [69_TD$IF]CGAS. Under physiological conditions, however, BAX/BAK-dependent caspase activation rapidly dismantles dying cells to prevent inflammatory responses. BAX and BAK operate at the interface between apoptotic signaling and innate immunity control. Intrinsic apoptosis and some variants of extrinsic apoptosis rely on the ability of [2_TD$IF]BCL2 associated X, apoptosis regulator (BAX) [70_TD$IF]and BCL2 antagonist/killer [71_TD$IF]1 (BAK1; best known as BAK) to mediate mitochondrial outer membrane permeabilization (MOMP). Widespread MOMP has a variety of detrimental consequences for the cell, including the translocation of cytochrome c from the mitochondrial intermembrane space into the cytosol. Together with deoxyATP and apoptotic peptidase activating factor 1 (APAF1), cytosolic cytochrome c drives the assembly of the so-called ‘apoptosome’, a supramolecular platform for the recruitment and activation of caspase 9 (CASP9), de facto precipitating the proteolytic maturation of executioner caspases including CASP3 and hence sealing the cell fate [1]. Of note, apoptotic cell death is generally (but not always) unable to drive inflammation, at least in part owing to multiple CASP3-dependent processes that promote the emission of anti-inflammatory signals and/or inhibit
the emission of proinflammatory signals [2]. Importantly, MOMP is also associated with the translocation of mitochondrial DNA (mtDNA), often in the form of nucleoids complexed with transcription factor A, mitochondrial (TFAM), from the mitochondrial matrix to the cytosolic compartment [3,4]. This can potently activate [72_TD$IF]cyclic GMP-AMP synthase (CGAS) to initiate a signal transduction cascade that critically relies on transmembrane protein 173 (TMEM[73_TD$IF]173) (best known as STING) and culminates with the synthesis of proinflammatory cytokines such as type I interferon (IFN) [5,6]. Under physiological conditions, however, caspases robustly prevent MOMP-driven CGAS–STING signaling to preserve the anti-inflammatory nature of apoptosis [7,8]. Recent data from McArthur et al. [9] elegantly demonstrate that BAX and BAK are not only responsible for the MOMP-associated efflux of cytochrome c and consequent activation of caspases, but also provide a gateway for the inner mitochondrial membrane to herniate, de facto enabling mitochondrial nucleoids to access the cytosolic compartment. To confirm that mtDNA is released in the cytosol of cells succumbing to intrinsic apoptosis, McArthur et al. harnessed live-cell lattice light-sheet microscopy (LLSM) to image Mcl1 / [68_TD$IF] mouse embryonic fibroblasts (MEFs) stably expressing a fluorescent variant of TFAM plus a form of translocase of outer mitochondrial membrane 20 (TOMM20) tagged with a HaloTag moiety that can be detected on the addition of a cell-permeant fluorescent dye, following the administration of the small molecule ABT-737. In this cellular model, ABT-737 – which operates by simultaneously inhibiting BCL2, apoptosis regulator (BCL2), and BCL2 like 1 (BCL2L1) (best known as BCL-XL) – is sufficient to potently drive BAX/BAKdependent apoptosis. Mcl1 / MEFs rapidly (within 20 min) responded to
ABT-737 by undergoing mitochondrial rearrangement followed by the release of TFAM-associated mtDNA into the cytosol, irrespective of the presence of the pan-caspase inhibitor QVD-QPh, which was required to avoid rapid detachment of the cells from the coverslip. 3D surface reconstruction and live 3D structured illumination microscopy (3D-SIM) revealed that TFAM-containing nucleoids: (i) are released from a single site on fragmented mitochondria; and (ii) generally remain in the proximity of the specific mitochondrion of origin. In this setting, cytochrome c efflux preceded mitochondrial rearrangements and the release of TFAM-associated mtDNA. Consistent with previous findings, Mcl1 / MEFs maintained in the presence of QVD-QPh secreted measurable amounts of interferon beta 1 (IFNB1) 4–6 h after the administration of ABT-737, a time at which various proteins of the mitochondrial matrix including TFAM could be detected in the cytosolic fraction by immunoblotting [9]. Next, the authors set to investigate the influence of mitochondrial dynamics on the release of TFAM-containing nucleoids from MEFs succumbing to apoptosis. To this aim, they generated Opa1 / [74_TD$IF]Mcl1 / and Mfn1 / Mfn2 / Mcl1 / MEFs, both of which are defective in mitochondrial fusion, as well as Dnm1l / Mcl1 / MEFs, which are defective in mitochondrial fission. Opa1 / Mcl1 / and Mfn1 / Mfn2 / Mcl1 / MEFs exhibited hyperfragmented mitochondria at baseline and were slightly more sensitive to ABT-737-driven cytochrome c release and nucleoid efflux than Mcl1 / MEFs. MEFs from Dnm1l / Mcl1 / mice displayed hyperfused mitochondria under baseline conditions and responded to ABT-737 with incomplete mitochondrial rearrangement coupled to the formation of ‘beads-on-string’ structures and limited release of TFAM-associated mtDNA.
Trends in Cell Biology, Month Year, Vol. xx, No. yy
1
RelaƟve degree
TICB 1414 No. of Pages 3
ApoptoƟc sƟmulus
Cytochrome c release
Mitochondrial fragmentaƟon
mtDNA release
ApoptoƟc RCD
CGAS–STING signaling
Figure 1. K [6_TD$IF] inetics of Intrinsic Apoptosis Impacts on Immunogenicity. On induction of intrinsic apoptosis, mitochondria rapidly release cytochrome c via small BAX/BAK pores. This enables prompt caspase activation and hence generates a cellular microenvironment that is poorly permissive for CGAS–STING signaling. As mitochondrial fragments and large BAX/BAK pores form, mitochondrial DNA (mtDNA) accessing the cytosol can no longer drive the synthesis of type I interferon, and cells succumb to apoptosis in the absence of inflammatory signaling. RCD, regulated cell death.
These latter findings, however, could not be reproduced by deleting Dnm1l from Mcl1 / MEFs with the CRISPR–Cas9 system. In this latter Dnm1l / Mcl1 / cellular model, mitochondrial responses to ABT-737 were similar to those observed in Mcl1 / MEFs. Despite such an unresolved discrepancy, all Dnm1l / cells tested produced normal amounts of IFNB1 on treatment with ABT-737 in the presence of QVD-QPh, demonstrating that fission is not involved in mtDNAdriven CGAS activation [9].
Trends in Cell Biology, Month Year, Vol. xx, No. yy
In summary, McArthur et al. [7_TD$IF]provided an elegant demonstration that BAX and BAK enable the efflux of mitochondrial nucleoids in the course of intrinsic apoptosis as the inner mitochondrial membrane herniates. BAX and BAK are also responsible for the apoptotic release of cytochrome c and consequent activation of caspases, which (at least in the model employed by these authors) occur before the efflux of TFAM-associated mtDNA. It is tempting to speculate, but remains to be formally elucidated, that the kinetics of process may be critical for multiple instances of apoptosis to remain immunologically silent [10] (Figure 1). Irrespective of this untested possibility, the findings by McArthur et al. add to an abundant literature that delineates [78_TD$IF]an intimate crosstalk between cell death control and the regulation of innate immunity [3,4].
cannot be detected by LLSM. Conversely, the release of TFAM-associated mtDNA was observed only after the formation of large BAX/BAK pores, which encircled the point of mtDNA exit from fragmented mitochondria, as elegantly demonstrated by 3D-SIM. A fluorophore targeted to the mitochondrial matrix exited the mitochondria of ABT-737treated Mcl1 / [75_TD$IF] MEFs in a manner similar to TFAM-containing nucleoids, strongly suggesting the existence of a physical barrier to cytosolic diffusion. Expression of a fluorescent variant of distal membrane arm assembly complex 1 (DMAC1) coupled to LLSM and 3D-SIM confirmed that such a physical barrier is provided by the inner mitochondrial membrane, which herniates across BAX/BAK macropores to form cytosolic bulges staining positively for TFAM. Immunogold labeling for fluoAcknowledgments rescent TFAM and transmission electron C.V-B. is supported by the 2017 Kellen Junior Faculty microscopy provided additional data in Fellowship from the Anna-Maria and Stephen Kellen support of this mechanism [9]. Foundation (New York, NY, USA). L.G. is supported
ABT-737 induced rapid dissipation of the mitochondrial transmembrane potential (Dcm) in Mcl1 / MEFs, which was not etiologically involved in the release of TFAM-containing nucleoids, as well as the formation of large mitochondrial foci containing BAX or BAK (as demonstrated by reconstituting Bax / Bak1 / Mcl1 / MEFs with fluorescent variants of either protein), which appeared 4–8 min after cytochrome c loss. This suggests that cytochrome c exits the intermembrane Of note, CGAS did not relocalize to space by small BAX/BAK pores that mtDNA-containing cytoplasmic bulges
2
and manifested no discernible change in subcellular distribution for up to 4–6 h after ABT-737 administration, when the supernatant of Mcl1 / MEFs exposed to ABT-737 in the presence of QVD-QPh contained detectable amounts of IFNB1. Correlative light and electron microscopy (CLEM) revealed that a minority of hernias forming in this setting lose their integrity starting at 90 min after ABT-767 administration, potentially constituting the source of mtDNA for CGAS activation. In a very few instances, single-[76_TD$IF] membraned TFAM-containing structures were also observed. The significance of such entities, resembling (at least to some degree) the CGAS-activating micronuclei that form in response to DNA damage [6], remains to be elucidated [9].
by intramural funds from the Department of Radiation Oncology of Weill Cornell Medical College (New York, NY, USA) and by donations from Phosplatin
TICB 1414 No. of Pages 3
Therapeutics (New York, NY, USA), the Luke Heller
References
TECPR2 Foundation (Boston, NY, USA), and Sotio a.
1. Galluzzi, L. et al. (2018) Molecular mechanisms of cell death: recommendations of the Nomenclature Committee on Cell Death 2018. Cell Death Differ. 25, 486–541
s. (Prague, Czech Republic). 1
Department of Radiation Oncology, Weill Cornell Medical College, New York, NY, USA 2 Sandra and Edward Meyer Cancer Center, New York, NY, USA 3 Université Paris Descartes/Paris V, Paris, France *Correspondence: [email protected] (L. Galluzzi) and [email protected] (C. Vanpouille-Box).
2. Galluzzi, L. et al. (2016) Caspases connect cell-death signaling to organismal homeostasis. Immunity 44, 221–231 3. Mehta, M.M. et al. (2017) Mitochondrial control of immunity: beyond ATP. Nat. Rev. Immunol. 17, 608–620 4. Galluzzi, L. et al. (2012) Mitochondria: master regulators of danger signalling. Nat. Rev. Mol. Cell Biol. 13, 780–788 5. Chen, Q. et al. (2016) Regulation and function of the cGAS–STING pathway of cytosolic DNA sensing. Nat. Immunol. 17, 1142–1149
6. Galluzzi, L. et al. Snapshot: CGAS–STING signaling. Cell (in press) 7. Rongvaux, A. et al. (2014) Apoptotic caspases prevent the induction of type I interferons by mitochondrial DNA. Cell 159, 1563–1577 8. White, M.J. et al. (2014) Apoptotic caspases suppress mtDNA-induced STING-mediated type I IFN production. Cell 159, 1549–1562 9. McArthur, K. et al. (2018) BAK/BAX macropores facilitate mitochondrial herniation and mtDNA efflux during apoptosis. Science 359, eaao6047 10. Galluzzi, L. et al. (2017) Immunogenic cell death in cancer and infectious disease. Nat. Rev. Immunol. 17, 97–111
https://doi.org/10.1016/j.tcb.2018.02.010
Trends in Cell Biology, Month Year, Vol. xx, No. yy
3