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Liposomes, Exosomes, and Virosomes: From Modeling Complex
Membrane Processes to Medical Diagnostics and Drug Delivery
Poster Abstracts
71
58-POS
Board 29
Lipid-Gel Domains in Living Cells: Tackling the Biological Significance of Atypically
Ordered Ceramide-Domains
Ana E. Ventura
1,2,3
, Sandra Pinto
2
, Ana R. Varela
1,2,3
, Giovanni D’Angelo
4
, Anthony Futerman
3
,
Manuel Prieto
2
,
Liana C. Silva
1
.
1
Faculdade de Farmácia, Universidade de Lisboa, Lisboa, Portugal,
2
Instituto Superior Técnico,
Universidade de Lisboa, Lisboa, Portugal,
4
Institute of Protein Biochemistry, National Research
Council of Italy, Naples, Italy.
3
Weizmann Institute of Science, Rehovot, Israel,
Ceramides are bioactive sphingolipids with important roles in cell physiology and pathology.
Ceramides activity have been related to their unique biophysical properties, namely to their
ability to form tightly packed membrane domains [1]. Nevertheless, the biophysical impact of
ceramides in living cells is still poorly characterized. To tackle this issue we employed multiple
methodologies, including steady-state and time-resolved fluorescence spectroscopy, confocal and
2-photon microscopy and fluorescence lifetime imaging (FLIM). Our results show that ceramide
formation upon stimulation with TNF-α resulted in an increase in the bulk membrane order and
in the formation of intracellular vesicles. Surprisingly, these vesicles displayed biophysical
features typical of the gel phase, as shown by the very high lifetime of trans-parinaric acid and
Laurdan generalized polarization suggesting that ceramide enriched domains accumulate in these
structures. Inhibition of neutral sphingomyelinase blocked the formation of those intracellular
vesicles and the increase in membrane order, further showing that the observed alterations are
dependent on ceramide formation. Colocalization imaging with different markers suggests that
ceramide-derived vesicles are involved in endo-lysosomal trafficking. Overall our results
highlight the existence of ceramide-derived biologically-relevant gel domains in cellular
membranes in response to physiological stress stimulation.
Acknowledgments: This work was supported by Fundação para a Ciência e Tecnologia (FCT),
Portugal: PTDC/BBB-BQB/0506/2012, PTDC/BBB-BQB/3710/2014, SFRH/BD/110093/2015
to AEV and Investigador FCT2014 to LCS
[1]. Castro, B. M., Prieto, M. & Silva, L. C. Ceramide: A simple sphingolipid with unique
biophysical properties. Prog. Lipid Res. 54, 53–67 (2014).