Abstract
Multivesicular bodies (MVBs) are endocytic compartments that contain intraluminal vesicles formed by inward budding from the limiting membrane of endosomes. In T lymphocytes, these vesicles contain pro-apoptotic Fas ligand (FasL), which may be secreted as ‘lethal exosomes’ upon fusion of MVBs with the plasma membrane. Diacylglycerol kinase α (DGKα) regulate the secretion of exosomes, but it is unclear how this control is mediated. T-lymphocyte activation increases the number of MVBs that contain FasL. DGKα is recruited to MVBs and to exosomes in which it has a double function. DGKα kinase activity exerts a negative role in the formation of mature MVBs, as we demonstrate by the use of an inhibitor. Downmodulation of DGKα protein resulted in inhibition of both the polarisation of MVBs towards immune synapse and exosome secretion. The subcellular location of DGKα together with its complex role in the formation and polarised traffic of MVBs support the notion that DGKα is a key regulator of the polarised secretion of exosomes.
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Abbreviations
- Ab:
-
antibody
- AICD:
-
activation-induced cell death
- CCh:
-
carbamyl choline (carbachol)
- CTL:
-
cytotoxic T lymphocyte
- DAG:
-
diacylclycerol
- DGK:
-
diacylglycerol kinase
- ESCRT:
-
endosomal sorting complex required for transport
- FACS:
-
fluorescence-activated cell sorter
- FasL:
-
Fas ligand
- HM1R:
-
human muscarinic receptor type 1
- HRP:
-
horseradish peroxidase
- mAb:
-
monoclonal antibody
- ILVs:
-
intraluminal vesicles
- LBPA:
-
lysobisphosphatidic acid
- MTOC:
-
microtubule organising centre
- MVBs:
-
multivesicular bodies
- PM:
-
plasma membrane
- SEE:
-
Staphylococcal Enterotoxin E
- TCR:
-
T-cell receptor
- TGN:
-
trans-golgi network
- WB:
-
western blot
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Acknowledgements
We thank Dr. G Griffiths (University of Oxford, UK), Dr. P Whitley (University of Bath, UK), and Dr. Sanchez-Madrid (Hospital de la Princesa, Madrid, Spain) for generously providing us some of the plasmids used in this study. We thank Alba Garcia and Ana Sanchez for their excellent technical support. This work was supported by grants from the Ministerio de Ciencia e Innovación, BFU2007-61613 and BFU2010-18726. R Alonso was the recipient of a fellowship from the Spanish Ministerio de Ciencia y Tecnología. A Fraile-Ramos is supported by the Ramón y Cajal Program. M Marsh is supported by the UK Medical Research Council. A Avila-Flores was supported by an AECC fellowship.
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Alonso, R., Mazzeo, C., Rodriguez, M. et al. Diacylglycerol kinase α regulates the formation and polarisation of mature multivesicular bodies involved in the secretion of Fas ligand-containing exosomes in T lymphocytes. Cell Death Differ 18, 1161–1173 (2011). https://doi.org/10.1038/cdd.2010.184
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DOI: https://doi.org/10.1038/cdd.2010.184
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