Abstract
Plasma membrane lipid microdomains have been considered as a sort of ‘closed chamber’, where several subcellular activities, including CD95/Fas-mediated proapoptotic signaling, take place. In this work we detected GD3 and GM3 gangliosides in isolated mitochondria from lymphoblastoid CEM cells. Moreover, we demonstrated the presence of microdomains in mitochondria by immunogold transmission electron microscopy. We also showed that GD3, the voltage-dependent anion channel-1 (VDAC-1) and the fission protein hFis1 are structural components of a multimolecular signaling complex, in which Bcl-2 family proteins (t-Bid and Bax) are recruited. The disruption of lipid microdomains in isolated mitochondria by methyl-β-cyclodextrin prevented mitochondria depolarization induced by GD3 or t-Bid. Thus, mitochondrion appears as a subcompartmentalized organelle, in which microdomains may act as controllers of their apoptogenic programs, including fission-associated morphogenetic changes, megapore formation and function. These results disclose a new scenario in which mitochondria-associated lipid microdomains can act as regulators and catalysts of cell fate.
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Abbreviations
- BSA:
-
bovine serum albumin
- COX-4:
-
cytochrome c oxidase
- Cyt c:
-
cytochrome c
- ΔΨ:
-
mitochondrial membrane potential
- DISC:
-
death-inducing signaling complex
- EGTA:
-
ethylene glycol-bis(-aminoethyl ether) N,N′,N″-tetraacetic acid
- ER:
-
endoplasmic reticulum
- FCS:
-
fetal calf serum
- FITC:
-
fluorescein isothiocyanate
- GEM:
-
glycosphingolipid enriched microdomains
- GM-130:
-
Golgi marker 130
- HPTLC:
-
high-performance thin layer chromatography
- HRP:
-
horseradish peroxidase
- LAMP-1:
-
lysosome-associated membrane glycoprotein
- MβCD:
-
methyl-β-cyclodextrin
- MoAb:
-
monoclonal antibody
- MOPS:
-
3[N-Morpholino] butane-sulfonic acid
- PBS:
-
phosphate-buffered saline
- PKC:
-
protein kinase C
- PMSF:
-
phenylmethylsulfonyl fluoride
- PVDF:
-
polyvinilidene difluoride
- SB:
-
swelling buffer
- SDS-PAGE:
-
sodium-dodecyl sulphate polyacrilamide gel electrophoresis
- TEM:
-
transmission electron microscopy
- TMRM:
-
tetramethylrhodamine-methyl-ester-perchlorate
- VDAC-1:
-
voltage-dependent anion channel-1
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Acknowledgements
We thank Dr. Mauro Degli Esposti, University of Manchester, UK; Dr. Stefano Rufini, University of Rome ‘Tor Vergata’, Italy; and Dr. Paola Matarrese, Istituto Superiore di Sanità, Rome, Italy, for helpful suggestions and criticisms. This work was supported by grants from Ministero della Salute, Italy to MS AP and WM.
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Garofalo, T., Giammarioli, A., Misasi, R. et al. Lipid microdomains contribute to apoptosis-associated modifications of mitochondria in T cells. Cell Death Differ 12, 1378–1389 (2005). https://doi.org/10.1038/sj.cdd.4401672
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DOI: https://doi.org/10.1038/sj.cdd.4401672
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