Advertisement

Stoichiometry and regulation network of Bcl-2 family complexes quantified by live-cell FRET assay

  • Fangfang Yang
  • Wenfeng Qu
  • Mengyan Du
  • Zihao Mai
  • Bin Wang
  • Yunyun Ma
  • Xiaoping WangEmail author
  • Tongsheng ChenEmail author
Original Article

Abstract

The stoichiometry and affinity of Bcl-2 family complexes are essential information for understanding how their interactome network is orchestrated to regulate mitochondrial permeabilization and apoptosis. Based on over-expression model system, FRET analysis was used to quantify the protein–protein interactions among Bax, Bcl-xL, Bad and tBid in healthy and apoptotic cells. Our data indicate that the stoichiometry and affinity of Bcl-2 complexes are dependent on their membrane environment. Bcl-xL, Bad and tBid can form hetero-trimers in mitochondria. Bcl-xL binds preferentially to Bad, then to tBid and Bax in mitochondria, whilst Bcl-xL displays higher affinity to Bad or tBid than to itself. Strikingly, Bax can bind to Bcl-xL in cytosol. In cytosol of apoptotic cells, Bcl-xL associates with Bax to form hetero-trimer with 1:2 stoichiometry, while Bcl-xL associates with Bad to form hetero-trimer with 2:1 stoichiometry and Bcl-xL associates with tBid to form hetero-dimer. In mitochondria, Bcl-xL associates with Bax/Bad to form hetero-dimer in healthy cells, while Bcl-xL associates with Bad to form hetero-tetramer with 3:1 stoichiometry in apoptotic cells.

Keywords

Affinity Bcl-2 proteins FRET Living cells Stoichiometry 

Abbreviations

FRET

Fluorescence resonance energy transfer

BH

Bcl-2 homology domains

Bax

Bcl-2-associated X protein

Bak

Bcl-2 antagonist killer

MOM

Mitochondrial outer membrane

Bcl-2

B-cell lymphoma 2

Bcl-xL

B-cell lymphoma, long isoform

Bcl-w

Bcl2L2, Bcl-2-like protein 2

A1

Bcl-2-related protein A1

Mcl-1

Myeloid cell leukemia sequence 1

BH3

Bcl-2 homology 3

Bid

BH3-interacting domain death agonist

tBid

Truncated Bid protein

Bim

Bcl-2-protein 11

Puma

p53 upregulated modulator of apoptosis

Bad

Bcl-2 antagonist of cell death

Noxa

PMAIP1, Phorbol-12-myristate-13-acetate-induced protein 1

FCCS

Fluorescence cross-correlation spectroscopy

DMEM

Dulbecco’s modified Eagle’s medium

FBS

Fetal bovine serum

STS

Staurosporine

DiIC1

1,1′,3,3,3′,3′-Hexamethylin-dodicarbocyanine iodide

PbFRET

Partial acceptor photobleaching-based FRET

ROIs

Regions of interest

Notes

Acknowledgements

We thank David W. Andrews for providing mCherry-Bad and mCherry-tBid plasmids and Andrew P. Gilmore for providing CFP-Bax plasmid. This work was supported by grants from the National Natural Science Foundation of China (NSFC), (Grant Nos. 61527825, 61875056 and 81572184).

Author contributions

FY designed and performed experiments, analyzed data, and wrote the manuscript. WQ, MD, ZM, BW, and YM performed experiments. XW and TC designed the study, planned experiments, and wrote the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

18_2019_3286_MOESM1_ESM.docx (7 mb)
Supplementary material 1 (DOCX 7122 kb)

References

  1. 1.
    Youle RJ, Strasser A (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nat Rev Mol Cell Biol 9:47–59CrossRefGoogle Scholar
  2. 2.
    Garcia-Saez AJ (2012) The secrets of the Bcl-2 family. Cell Death Differ 19:1733–1740CrossRefGoogle Scholar
  3. 3.
    Pena-Blanco A, Garcia-Saez AJ (2018) Bax, Bak and beyond—mitochondrial performance in apoptosis. FEBS J 285:416–431CrossRefGoogle Scholar
  4. 4.
    Ku B, Liang C, Jung JU, Oh B-H (2011) Evidence that inhibition of BAX activation by BCL-2 involves its tight and preferential interaction with the BH3 domain of BAX. Cell Res 21:627–641CrossRefGoogle Scholar
  5. 5.
    Cartron PF, Gallenne T, Bougras G, Gautier F, Manero F, Vusio P, Meflah K, Vallette FM, Juin P (2004) The first alpha helix of Bax plays a necessary role in its ligand-induced activation by the BH3-only proteins bid and PUMA. Mol Cell 16:807–818CrossRefGoogle Scholar
  6. 6.
    Billen LP, Shamas-Din A, Andrews DW (2008) Bid: a Bax-like BH3 protein. Oncogene 27:S93–S104CrossRefGoogle Scholar
  7. 7.
    Merino D, Giam M, Hughes PD, Siggs OM, Heger K, O’Reilly LA, Adams JM, Strasser A, Lee EF, Fairlie WD, Bouillet P (2009) The role of BH3-only protein Bim extends beyond inhibiting Bcl-2-like prosurvival proteins. J Cell Biol 186:355–362CrossRefGoogle Scholar
  8. 8.
    Chipuk JE, Moldoveanu T, Llambi F, Parsons MJ, Green DR (2010) The BCL-2 family reunion. Mol Cell 37:299–310CrossRefGoogle Scholar
  9. 9.
    Yan J, Zhang H, Xiang J, Zhao Y, Yuan X, Sun B, Lin A (2018) The BH3-only protein BAD mediates TNFα cytotoxicity despite concurrent activation of IKK and NF-κB in septic shock. Cell Res 28:701–718CrossRefGoogle Scholar
  10. 10.
    Hsu YT, Wolter KG, Youle RJ (1997) Cytosol-to-membrane redistribution of Bax and Bcl-X(L) during apoptosis. Proc Natl Acad Sci USA 94:3668–3672CrossRefGoogle Scholar
  11. 11.
    Hausmann G, O’Reilly LA, van Driel R, Beaumont JC, Strasser A, Adams JM, Huang DC (2000) Pro-apoptotic apoptosis protease-activating factor 1 (Apaf-1) has a cytoplasmic localization distinct from Bcl-2 or Bcl-x(L). J Cell Biol 149:623–634CrossRefGoogle Scholar
  12. 12.
    Jeong SY, Gaume B, Lee YJ, Hsu YT, Ryu SW, Yoon SH, Youle RJ (2004) Bcl-x(L) sequesters its C-terminal membrane anchor in soluble, cytosolic homodimers. EMBO J 23:2146–2155CrossRefGoogle Scholar
  13. 13.
    Bleicken S, Hantusch A, Das KK, Frickey T, Garcia-Saez AJ (2017) Quantitative interactome of a membrane Bcl-2 network identifies a hierarchy of complexes for apoptosis regulation. Nat Commun 8:73CrossRefGoogle Scholar
  14. 14.
    Edlich F, Banerjee S, Suzuki M, Cleland MM, Arnoult D, Wang C, Neutzner A, Tjandra N, Youle RJ (2011) Bcl-x(L) retrotranslocates Bax from the mitochondria into the cytosol. Cell 145:104–116CrossRefGoogle Scholar
  15. 15.
    Schellenberg B, Wang P, Keeble JA, Rodriguez-Enriquez R, Walker S, Owens TW, Foster F, Tanianis-Hughes J, Brennan K, Streuli CH, Gilmore AP (2013) Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming. Mol Cell 49:959–971CrossRefGoogle Scholar
  16. 16.
    Aranovich A, Liu Q, Collins T, Geng F, Dixit S, Leber B, Andrews DW (2012) Differences in the mechanisms of proapoptotic BH3 proteins binding to Bcl-XL and Bcl-2 quantified in Live MCF-7 cells. Mol Cell 45:754–763CrossRefGoogle Scholar
  17. 17.
    Du MY, Yang FF, Mai ZH, Qu WF, Lin FR, Wei LC, Chen TS (2018) FRET two-hybrid assay by linearly fitting FRET efficiency to concentration ratio between acceptor and donor. Appl Phys Lett 112:153702CrossRefGoogle Scholar
  18. 18.
    Li HL, Zhu H, Xu CJ, Yuan JY (1998) Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 94:491–501CrossRefGoogle Scholar
  19. 19.
    Chou JJ, Li HL, Salvesen GS, Yuan JY, Wagner G (1999) Solution structure of BID, an intracellular amplifier of apoptotic signaling. Cell 96:615–624CrossRefGoogle Scholar
  20. 20.
    Eskes R, Desagher S, Antonsson B, Martinou JC (2000) Bid induces the oligomerization and insertion of Bax into the outer mitochondrial membrane. Mol Cell Biol 20:929–935CrossRefGoogle Scholar
  21. 21.
    Lovell JF, Billen LP, Bindner S, Shamas-Din A, Fradin C, Leber B, Andrews DW (2008) Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 135:1074–1084CrossRefGoogle Scholar
  22. 22.
    Billen LP, Kokoski CL, Lovell JF, Leber B, Andrews DW (2008) Bcl-XL inhibits membrane permeabilization by competing with Bax. PLoS Biol 6:e147CrossRefGoogle Scholar
  23. 23.
    Llambi F, Moldoveanu T, Tait SWG, Bouchier-Hayes L, Temirov J, McCormick LL, Dillon CP, Green DR (2011) A unified model of mammalian BCL-2 protein family interactions at the mitochondria. Mol Cell 44:517–531CrossRefGoogle Scholar
  24. 24.
    Selvin PR (1995) Fluorescence resonance energy transfer. Method Enzymol 246:300–334CrossRefGoogle Scholar
  25. 25.
    Dussmann H, Rehm M, Concannon CG, Anguissola S, Wurstle M, Kacmar S, Voller P, Huber HJ, Prehn JHM (2010) Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation. Cell Death Differ 17:278–290CrossRefGoogle Scholar
  26. 26.
    Placone J, Hristova K (2012) Direct assessment of the effect of the Gly380Arg achondroplasia mutation on FGFR3 dimerization using quantitative imaging FRET. PLoS One 7:e46678CrossRefGoogle Scholar
  27. 27.
    Erickson MG, Liang HY, Mori MX, Yue DT (2003) FRET two-hybrid mapping reveals function and location of L-type Ca2+ channel CaM preassociation. Neuron 39:97–107CrossRefGoogle Scholar
  28. 28.
    Ben-Johny M, Yue DN, Yue DT (2016) Detecting stoichiometry of macromolecular complexes in live cells using FRET. Nat Commun 7:13709CrossRefGoogle Scholar
  29. 29.
    Valentijn AJ, Metcalfe AD, Kott J, Streuli CH, Gilmore AP (2003) Spatial and temporal changes in Bax subcellular localization during anoikis. J Cell Biol 162:599–612CrossRefGoogle Scholar
  30. 30.
    Butz ES, Ben-Johny M, Shen M, Yang PS, Sang LJ, Biel M, Yue DT, Wahl-Schott C (2016) Quantifying macromolecular interactions in living cells using FRET two-hybrid assays. Nat Protoc 11:2470–2498CrossRefGoogle Scholar
  31. 31.
    Zhang J, Zhang LL, Chai LY, Yang FF, Du MY, Chen TS (2016) Reliable measurement of the FRET sensitized-quenching transition factor for FRET quantification in living cells. Micron 88:7–15CrossRefGoogle Scholar
  32. 32.
    Elder AD, Domin A, Schierle GSK, Lindon C, Pines J, Esposito A, Kaminski CF (2009) A quantitative protocol for dynamic measurements of protein interactions by Forster resonance energy transfer-sensitized fluorescence emission. J R Soc Interface 6:S59–S81CrossRefGoogle Scholar
  33. 33.
    Yu HN, Zhang JW, Li HL, Qu JL, Chen TS (2012) An empirical quantitative fluorescence resonance energy transfer method for multiple acceptors based on partial acceptor photobleaching. Appl Phys Lett 100:253701CrossRefGoogle Scholar
  34. 34.
    Erickson MG, Alseikhan BA, Peterson BZ, Yue DT (2001) Preassociation of calmodulin with voltage-gated Ca2+ channels revealed by FRET in single living cells. Neuron 31:973–985CrossRefGoogle Scholar
  35. 35.
    Hoppe A, Christensen K, Swanson JA (2002) Fluorescence resonance energy transfer-based stoichiometry in living cells. Biophys J 83:3652–3664CrossRefGoogle Scholar
  36. 36.
    Zal T, Gascoigne NRJ (2004) Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J 86:3923–3939CrossRefGoogle Scholar
  37. 37.
    Chen H, Puhl HL, Koushik SV, Vogel SS, Ikeda SR (2006) Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells. Biophys J 91:L39–L41CrossRefGoogle Scholar
  38. 38.
    Zhu W, Cowie A, Wasfy GW, Penn LZ, Leber B, Andrews DW (1996) Bcl-2 mutants with restricted subcellular location reveal spatially distinct pathways for apoptosis in different cell types. EMBO J 15:4130–4141CrossRefGoogle Scholar
  39. 39.
    Chin HS, Li MX, Tan IKL, Ninnis RL, Reljic B, Scicluna K, Dagley LF, Sandow JJ, Kelly GL, Samson AL, Chappaz S, Khaw SL, Chang C, Morokoff A, Brinkmann K, Webb A, Hockings C, Hall CM, Kueh AJ, Ryan MT, Kluck RM, Bouillet P, Herold MJ, Gray DHD, Huang DCS, Van DMF, Dewson G (2018) VDAC2 enables Bax to mediate apoptosis and limit tumor development. Nat Commun 9:4976CrossRefGoogle Scholar
  40. 40.
    Rehm M, Huber HJ, Hellwig CT, Anguissola S, Dussmann H, Prehn JH (2009) Dynamics of outer mitochondrial membrane permeabilization during apoptosis. Cell Death Differ 16:613–623CrossRefGoogle Scholar
  41. 41.
    Grinberg M, Sarig R, Zaltsman Y, Frumkin D, Grammatikakis N, Reuveny E, Gross A (2002) tBID homooligomerizes in the mitochondrial membrane to induce apoptosis. J Biol Chem 277:12237–12245CrossRefGoogle Scholar
  42. 42.
    Szymczak AL, Workman CJ, Wang Y, Vignali KM, Dilioglou S, Vanin EF, Vignali DAA (2004) Correction of multi-gene deficiency in vivo using a single ‘self-cleaving’ 2A peptide-based retroviral vector. Nat Biotechnol 22:589–594CrossRefGoogle Scholar
  43. 43.
    Lopez J, Bessou M, Riley JS, Giampazolias E, Todt F, Rochegue T, Oberst A, Green DR, Edlich F, Ichim G, Tait SWG (2016) Mito-priming as a method to engineer Bcl-2 addiction. Nat Commun 7:10538CrossRefGoogle Scholar
  44. 44.
    Bhola PD, Letai A (2016) Mitochondria-Judges and executioners of cell death sentences. Mol Cell 61:695–704CrossRefGoogle Scholar
  45. 45.
    Letai A, Bassik MC, Walensky L, Sorcinelli MD, Weiler S, Korsmeyer SJ (2002) Distinct BH3 domains either sensitize or activate mitochondrial apoptosis serving as prototype cancer therapeutics. Cancer Cell 2:183–192CrossRefGoogle Scholar
  46. 46.
    Wang K, Yin XM, Chao DT, Milliman CL, Korsmeyer SJ (1996) BID: a novel BH3 domain-only death agonist. Gene Dev 10:2859–2869CrossRefGoogle Scholar
  47. 47.
    Wang Y, Tjandra N (2013) Structural insights of tBid, the caspase-8-activated Bid, and its BH3 domain. J Biol Chem 288:35840–35851CrossRefGoogle Scholar
  48. 48.
    Leber B, Lin JL, Andrews DW (2007) Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes. Apoptosis 12:897–911CrossRefGoogle Scholar
  49. 49.
    Hsu YT, Youle RJ (1998) Bax in murine thymus is a soluble monomeric protein that displays differential detergent-induced conformations. J Biol Chem 273:10777–10783CrossRefGoogle Scholar
  50. 50.
    Ding JZ, Mooers BHM, Zhang Z, Kale J, Falcone D, McNichol J, Huang B, Zhang XJC, Xing CG, Andrews DW, Lin JL (2014) After embedding in membranes antiapoptotic Bcl-XL protein binds both Bcl-2 homology region 3 and helix 1 of proapoptotic Bax protein to inhibit apoptotic mitochondrial permeabilization. J Biol Chem 289:11873–11896CrossRefGoogle Scholar
  51. 51.
    Suzuki M, Youle RJ, Tjandra N (2000) Structure of Bax: coregulation of dimer formation and intracellular localization. Cell 103:645–654CrossRefGoogle Scholar
  52. 52.
    Annis MG, Soucie EL, Dlugosz PJ, Cruz-Aguado JA, Penn LZ, Leber B, Andrews DW (2005) Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J 24:2096–2103CrossRefGoogle Scholar
  53. 53.
    Zhang Z, Subramaniam S, Kale J, Liao C, Huang B, Brahmbhatt H, Condon SGF, Lapolla SM, Hays FA, Ding JZ, He F, Zhang XJC, Li JN, Senes A, Andrews DW, Lin JL (2016) BH3-in-groove dimerization initiates and helix 9 dimerization expands Bax pore assembly in membranes. EMBO J 35:208–236CrossRefGoogle Scholar
  54. 54.
    Puthalakath H, Strasser A (2002) Keeping killers on a tight leash: transcriptional and post-translational control of the pro-apoptotic activity of BH3-only proteins. Cell Death Differ 9:505–512CrossRefGoogle Scholar
  55. 55.
    Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R, Green DR, Newmeyer DD (2002) Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 111:331–342CrossRefGoogle Scholar
  56. 56.
    Adams JM, Cory S (2018) The BCL-2 arbiters of apoptosis and their growing role as cancer targets. Cell Death Differ 25:27–36CrossRefGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Fangfang Yang
    • 1
  • Wenfeng Qu
    • 1
  • Mengyan Du
    • 1
  • Zihao Mai
    • 1
  • Bin Wang
    • 1
  • Yunyun Ma
    • 1
  • Xiaoping Wang
    • 2
    Email author
  • Tongsheng Chen
    • 1
    Email author
  1. 1.MOE Key Laboratory of Laser Life Science and College of BiophotonicsSouth China Normal UniversityGuangzhouChina
  2. 2.Department of Pain ManagementThe First Affiliated Hospital of Jinan UniversityGuangzhouChina

Personalised recommendations