Sphingolipid domains in the plasma membrane of fungal cells : Interplay with membrane proteins and antifungal resistance

Santos, Filipa C.

http://hdl.handle.net/10451/57798

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Authors

Santos, Filipa C.

Advisor(s)

Almeida, Rodrigo F. M. de

Abstract(s)

Plasma membrane (PM) carries out multiple functions a tightly regulated dynamic organization into specialized domains of different size, stability, and composition. Sphingolipids are a major class of lipids of PM being crucial for its structure and function. A specific type of ergosterol-depleted PM domains in fungi, where lipids are tightly packed in a very rigid gel phase, comprises the sphingolipid-enriched domains (SLEDs). In this work, the presence of SLEDs in a mold, Neurospora crassa, was disclosed, as well as their involvement in the response to the antifungal staurosporine. A comparison with the budding yeast Saccharomyces cerevisiae was performed, taking into consideration the known differences between their PM sphingolipidomes. The studies with N. crassa were performed in conidial suspensions along different time points of conidial germination, using several fluorescence spectroscopy techniques and a multiprobe approach. S. cerevisiae living cells were studied in mi-exponential phase. To evaluate the impact of changing sphingolipid composition in the organization of the two main yeast PM compartments, MCC (arginine/ H+ symporter Can1p) and MCP (H+ ATPase Pma1p), the time-resolved fluorescence properties of Can1p tagged with GFP and Pma1p tagged with mRFP, respectively, were studied by FLIM in wt and a mutant strain with a different sphingolipid profile, ipt1∆. IPT1 deletion strongly affects the rigidity of gel domains and Pma1p PM distribution, whereas no significant alterations could be perceived neither in ergosterol-enriched domains nor Can1p distribution. Thus, this work strongly suggests that proper SLEDs hydrophobic chain packing is required for an adequate organization of the MCP, but not MCC. Finally, considering that the PM is crucial for antifungal action, a biophysical connection between SLEDs and antifungal activity was explored in S. cerevisiae.