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Exploring plasma membrane heterogeneity in saccharomyces cerevisiae with a focus on sphingolipid hydroxylation
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Resumo(s)
Amidst a growing global trend of fungal infections and diminishing antifungal drug efficacy,
the fungal plasma membrane emerges as a potential target for enhanced antifungal treatments. By
employing innovative experimental approaches, the concomitant use of fluorescent membrane probes
1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivatives, cationic TMA-DPH and anionic PA-DPH, this
study aimed to i) develop a new methodology to study sphingolipid-enrich domains (SLEDs) in gel
phase in Saccharomyces cerevisiae plasma membrane and ii) further characterize the biophysical
properties and organization of the plasma membrane of Saccharomyces cerevisiae cells. All probes were
shown to not compromise cellular viability. In terms of cellular distribution, DPH was confirmed to
distribute across both the plasma and internal membranes, while TMA-DPH and PA-DPH localized
predominantly in the plasma membrane. Notably, fluorescence anisotropy assays over time revealed
that, at short incubation times, TMA-DPH and PA-DPH label different regions of the membrane, most
likely due to their opposite charges. It is hypothesized that TMA-DPH, owing to its positive charge, will
firstly incorporate into SLEDs due to electrostatic interactions with negative charges present in
sphingolipids polar head group. This trend was observed in both wild-type and sur2∆ cells lacking C4 -
OH group in the sphingoid base.
Trans-parinaric acid (t-PnA) was also employed to assess the properties of gel SLEDs, which
were identified in sur2∆ cells for the first time. Considering the long lifetime component of t-PnA in
these cells (~44 ns), SLEDs are more compact compared to wt cells. This is in line with previous results
obtained in other yeast strains with altered sphingolipid profile, namely scs7∆ and ipt1∆, where SLEDs
also exhibited alterations in their compactness. This study reinforces that lipid composition, especially
the hydroxylation pattern of sphingolipids, plays a pivotal role in defining membrane properties, thus
SLEDs may constitute an explored target for antifungal therapies.
Descrição
Tese de mestrado, Bioquímica e Biomedicina , 2023, Universidade de Lisboa, Faculdade de Ciências
Palavras-chave
Membrana plasmática fúngica Saccharomyces cerevisiae Domínios ricos em esfingolípidos (SLEDs) Hidroxilação de esfingolípidos Sonda fluorescente de membrana Teses de mestrado - 2024