Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

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).

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