Sphinganine Induces Osmotic Instability and Increased Plasma Membrane Permeability in Neurospora crassa.

Jennifer A. Gerlash, Sara L. Schecter, Cassandra Teeter, Kenneth M. Bart, Lawrence Aaronson

 

Abstract: Sphinganine is an 18-carbon straight chain lipid that is concentrated in the superficial layers of mammalian epidermis and it is thought to provide a natural antifungal barrier in the skin. The lipid is inhibitory to the growth and viability of the nonpathogenic mold, Neurospora crassa. Exogenous sphinganine causes the loss of multiple metabolite transport systems in Neurospora, suggesting disruption of the transmembrane proton gradient. Sphinganine-traeted conidia exhibit abnormal ultrastructure, most notably the presence of swollen organelles. Germinating conidia treated with sphinganine in hypertonic medium show not only the absence of organellar swelling, but shrinkage and condensation of organelles, supporting the hypothesis that sphinganine induces osmotic instability in fungi. Sphinganine treatment of Neurospora not only allows osmotic movement across the fungal plasma membrane, but also induces leakage of cytoplasmic constituents. Taken together, these data suggest that exogenous sphinganine perturbs the function and integrity of the fungal plasma membrane.

A- Control; B- 15 minutes in 25uM Sphinganine; C- 15 minutes in 100uM Sphinganine; D- 60 minutes in 100uM Sphinganine


 

 

 

Mitochondria Reconstruction

Click to see a Quick Time animation (3.5MB) of an in vivo mitochondria from Neurospora crassa, treated with sphinganine. These images were formed using Electron Tomographic Imaging in collaboration with Carmen Mannella, Biological Microscopy Reconstruction Resource, Wadsworth Center, NYS Department of Health, Albany, New York.

 

Click here for Quicktime Movie (3.6MB)

 


 

 

Mitochondrial Activity and Morphology Are Affected by Sphinganine in Neurospora Crassa

J. A. Gerlash, K.M. Bart and L. R. Aaronson

 

Abstract: Sphinganine, a sphingoid base found concentrated in mammalian epidermis, may serve as a natural antifungal barrier, preventing infection by pathogenic fungi. Neurospora crassa is being used as a model to study the effects of sphinganine in fungal pathogens. In Neurospora, exogenous sphinganine is metabolized into complex sphingolipids, such as ceramides and cerebrosides over a 1 hour period, suggesting that the lipid is being internalized. However, only sphinganine is associated with mitochondrial fractions during this time. Sphinganine appears to have a dramatic effect on mitochondrial structure and function. Transmission electron microscopy demonstrates that the disruption of ultrastructure is both a time and concentration dependent phenomenon. Exposure to 100 mM sphinganine for 1 hour completely disrupts the outer membranes of most mitochondria. Analysis also shows that short-term exposure to subinhibitory concentrations of sphinganine causes swelling of mitochondria, while exposure to increasing concentrations of the lipid results in the complete disruption of outer membranes. The presence of cytoplasmic lamellar and mesosome-like structures near the plasma membrane, and enlarged vacuolar bodies, further indicate the deterioration of cellular activity. The changes observed in mitochondrial ultrastructure appear to coincide with a loss of mitochondrial function. Using the fluorescent dye Rhodamine 123, it was observed that germinating cells treated with to 100 mM sphinganine exhibit a severe decline in electrogenic mitochondrial activity. These results demonstrate that the loss of cell growth and viability observed in sphinganine-treated Neurospora may be due in part to the disruption of mitichondrial morphology and activity.

 

 

 

Left Panel: Rhodamine 123 Results and Right Panel: Effect of Sphinganine on Ultrastructure

 

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