Abstract
Neurospora crassa is a non-pathogenic filamentous fungus widely used as a multicellular eukaryotic model. Recently, the biophysical properties of the plasma membrane of N. crassa conidia were thoroughly characterized. They evolve during conidial germination at a speed that depends on culture conditions, suggesting an important association between membrane remodeling and the intense membrane biogenesis that takes place during the germinative process. Staurosporine (STS) is a drug used to induce programmed cell death in various organisms. In N. crassa, STS up-regulates the expression of the ABC transporter ABC-3, which localizes at the plasma membrane and pumps STS out. To understand the role of plasma membrane biophysical properties in the fungal drug response, N. crassa was subjected to STS treatment during early and late conidial development stages. Following 1 h treatment with STS, there is an increase in the abundance of the more ordered, sphingolipid-enriched, domains in the plasma membrane of conidia. This leads to higher fluidity in other membrane regions. The global order of the membrane remains thus practically unchanged. Significant changes in sphingolipid-enriched domains were also observed after 15 min challenge with STS, but they were essentially opposite to those verified for the 1 h treatment, suggesting different types of drug responses. STS effects on membrane properties that are more dependent on ergosterol levels also depend on the developmental stage. There were no alterations on 2 h-grown cells, clearly contrasting to what happens at longer growth times. In this case, the differences were more marked for longer STS treatment, and rationalized considering that the drug prevents the increase in the ergosterol/glycerophospholipid ratio that normally takes place at the late conidial stage/transition to the mycelial stage. This could be perceived as a drug-induced development arrest after 5 h growth, involving ergosterol, and pointing to a role of lipid rafts possibly related with an up-regulated expression of the ABC-3 transporter. Overall, our results suggest the involvement of membrane ordered domains in the response mechanisms to STS in N. crassa.
Highlights
Antifungal drug resistance is a major society concern, since the mortality rate associated with resistant fungal infections has increased dramatically, in hospital environments and in patients with decreased immunological response (Gulshan and Moye-Rowley, 2007; Shahi and Moye-Rowley, 2009; Prevention CFDCA., 2017).The emerging role of apoptosis as key regulator of fungal development suggests that it might be possible to develop new means of controlling fungal infections through the manipulation of some key components/organelles involved in the apoptotic cascade
We studied the biophysical effects of STS on the plasma membrane lipid domains of N. crassa and evaluated if the drug interacts with lipids of the plasma membrane
The fluorescence intensity decay allows computing the amplitude-weighted and intensity-weighted mean fluorescence lifetime, after analysis using equation 3 with a bi-exponential model (n = 2) and equations 4 and 5. Those two parameters reflect the microenvironment of the probe: the contribution of each of them to the total decay can change with the solvent polarity and specific interactions, or with the pathways the excited fluorophore follows to return to the ground state, which depend on collisions with other molecules, vibrations and torsions (Berezin and Achilefu, 2010)
Summary
Antifungal drug resistance is a major society concern, since the mortality rate associated with resistant fungal infections has increased dramatically, in hospital environments and in patients with decreased immunological response (Gulshan and Moye-Rowley, 2007; Shahi and Moye-Rowley, 2009; Prevention CFDCA., 2017).The emerging role of apoptosis as key regulator of fungal development suggests that it might be possible to develop new means of controlling fungal infections through the manipulation of some key components/organelles involved in the apoptotic cascade. STS-induced programmed cell death in filamentous fungus (Fernandes et al, 2011) is one of the many examples of important fungal physiological activities, such as cell proliferation or differentiation, sensing and signaling, that are usually found to be closely related to membrane composition and biophysical features, through their involvement with membrane microdomain organization and lipid homeostasis (Malinsky and Operakova, 2016). The plasma membrane protein for which mRNA levels determined in the transcriptional profiling study are increased by a larger amount, attaining a 30-fold increase is ABC3 (Fernandes et al, 2011). This protein is responsible for most of the energy-dependent efflux of STS and a null-mutant of this ABC transporter (abc3) is extremely sensitive to STS and accumulates more STS than the wild type strain (Fernandes et al, 2011)
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