Document type
Academic documents
Document subtype
Master's dissertation
Title
Impact of the sphingolipid profile on the biophysical properties of the yeast plasma membrane
Participants in the publication
Savarino, R. (Author)
de Almeida, R.F.M. (Adviser)
Dep. Química e Bioquímica
CQE
Santucci, A. (Adviser)
Summary
Considering the global rise in fungal infections and the decreasing efficacy of current antifungal drugs, the fungal plasma membrane offers a promising target for developing more effective therapies. The yeast plasma membrane is characterized by two primary compartments: the Can1p-occupied compartment (MCC), which is sterol-rich, and the Pma1p-occupied compartment (MCP), enriched in sphingolipids. A distinctive feature of the yeast PM, absent in mammalian cells, is the presence of highly rigid sphingolipid-enriched domains (SLEDs), which play a crucial role in membrane stability and organization. Understanding the biophysical properties of the yeast PM and its differences from mammalian membranes is essential for identifying potential avenues for selective antifungal agents. \\\\\\\\nThe main objective of this study was to investigate how changes in the sphingolipid profile affect the biophysical properties and organization of the yeast PM, focusing on three mutant strains: sur2Δ (lacking the C4-OH group in the sphingoid base), scs7Δ (lacking the C2-OH group in the acyl chain), and isc1Δ (accumulating complex sphingolipids like mannosyldiinositol phosphorylceramide). These mutants were compared to wild-type cells, with changes in membrane fluidity, order, and domain formation assessed using a combination of fluorescence probes. Specifically, 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to measure global membrane order, 4-[2-[6-(dioctylamino)-2-naphthalenyl]ethenyl]-1-(3-sulfopropyl)-pyridinium (Di-8-ANEPPS) sensible to the membrane dipole potential, and trans-parinaric acid (t-PnA) to assess sphingolipid-enriched domains hydrophobic packing and abundance. Fluorescence intensity decay, steady-state anisotropy, and spectral measurements provided insights into how modifications in sphingolipid structure influenced PM properties. The characterization was carried out in both intact cells and isolated plasma membrane.\\\\\\\\nThe results demonstrated that the sur2Δ strain exhibited the most fluid intracellular membranes, while scs7Δ showed increased rigidity of them. The isc1Δ strain, which accumulates complex sphingolipids, displayed the shortest trans-parinaric fluorescence lifetimes, indicative of severly impaired or even absent SLEDs. The accumulation of sphingolipids in isc1Δ led to significant disruptions in membrane organization, both plasma membrane and intracellular membranes, affecting the stability of lipid domains and correlating with altered drug sensitivity in this strain. These findings highlight the critical role of sphingolipid composition in maintaining the structure of the yeast PM and emphasize that disruptions in sphingolipid metabolism can profoundly impact membrane biophysics. This research provides a foundation for developing antifungal strategies targeting sphingolipid biosynthesis or degradation pathways to destabilize the fungal PM and enhance the effectiveness of existing treatments.\\\\\\\\n
Date of Submisson/Request
2024
Date of Acceptance
2024
Date of Publication
2024
Institution
Università di Siena
Publication Identifiers