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

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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.

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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

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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).

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Authors and Affiliations

  1. MOE Key Laboratory of Laser Life Science and College of Biophotonics, South China Normal University, Guangzhou, China

    Fangfang Yang, Wenfeng Qu, Mengyan Du, Zihao Mai, Bin Wang, Yunyun Ma & Tongsheng Chen

  2. Department of Pain Management, The First Affiliated Hospital of Jinan University, Guangzhou, China

    Xiaoping Wang

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  1. Fangfang Yang
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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.

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Correspondence to Xiaoping Wang or Tongsheng Chen.

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Yang, F., Qu, W., Du, M. et al. Stoichiometry and regulation network of Bcl-2 family complexes quantified by live-cell FRET assay. Cell. Mol. Life Sci. 77, 2387–2406 (2020). https://doi.org/10.1007/s00018-019-03286-z

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