Biology subjects

Thaysen, K.

Publications and source records attributed to Thaysen, K..

3 recordsLinked to original sources

bioRxiv2024

Structural and biochemical analysis of ligand binding in yeast Niemann-Pick type C1-related protein

In eukaryotes, integration of sterols into the vacuolar/lysosomal membrane is critically dependent on the Niemann-Pick type C (NPC) system. The system consists of an integral membrane protein, called NCR1 in yeast, and NPC2, a luminal soluble protein that transfers sterols to the N-terminal domain (NTD) of NCR1 before membrane integration. Both proteins have been implicated in sterol homeostasis of yeast and humans. Here, we investigate sterol and lipid binding of the NCR1/NPC2 transport system and determine crystal structures of the sterol-binding NTD. The NTD binds both ergosterol and cholesterol, with nearly identical conformations of the binding pocket. Apart from sterols, the NTD can also bind fluorescent analogs of phosphatidylinositol, phosphatidylcholine and phosphatidylserine as well as sphingosine and ceramide. We confirm the multi-lipid scope of the NCR1/NPC2 system using photo-crosslinkable and clickable lipid analogs, namely pac-cholesterol, pac-sphingosine and pac-ceramide. Finally, we reconstitute the transfer of pac-sphingosine from NPC2 to the NTD in vitro. Collectively, our results support that the yeast NPC system can work as versatile machinery for vacuolar homeostasis of structurally diverse lipids, besides ergosterol. Summary blurbResults of X-ray crystallography and binding assays with different lipids expand our knowledge of the substrate scope of the Niemann-Pick type C1-related proteins NCR1 and NPC2 in yeast. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/598172v2_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@102579borg.highwire.dtl.DTLVardef@c5be27org.highwire.dtl.DTLVardef@471096org.highwire.dtl.DTLVardef@191ee26_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Ergosterol mediates aggregation of natamycin in the yeast plasma membrane

Polyene macrolides are antifungal substances, which interact with cells in a sterol-dependent manner. While being widely used, their mode of action is poorly understood. Here, we employ ultraviolet-sensitive (UV) microscopy to show that the antifungal polyene natamycin binds to the yeast plasma membrane (PM) and causes permeation of propidium iodide into cells. Right before membrane permeability becomes compromised, we observed clustering of natamycin in the PM that was independent of PM protein domains. Aggregation of natamycin was paralleled by cell deformation and membrane blebbing as revealed by soft X-ray microscopy. Substituting ergosterol for cholesterol decreased natamycin binding and resulted in reduced clustering of natamycin in the PM. Blocking of ergosterol synthesis necessitates sterol import via the ABC transporters Aus1/Pdr11 to ensure natamycin binding. Quantitative imaging of dehydroergosterol (DHE) and cholestatrienol (CTL), two analogs of ergosterol and cholesterol, respectively, revealed a largely homogeneous lateral sterol distribution in the PM, ruling out that natamycin binds to pre-assembled sterol domains. Depletion of sphingolipids using myriocin increased natamycin binding to yeast cells, likely by increasing the ergosterol fraction in the outer PM leaflet. We conclude that ergosterol-specific aggregation of natamycin in the yeast PM underlies its antifungal activity, which can be synergistically enhanced by inhibitors of sphingolipid synthesis. SignificanceErgosterol is the major sterol in the membranes of fungi and a major target for antifungal treatments. Polyene macrolides, such as natamycin, are known to target ergosterol but the underlying mechanisms for their preference for this yeast sterol compared to mammalian cholesterol is not understood. This study shows that natamycin forms aggregates when associated with yeast S. cerevisiae in an ergosterol-dependent manner. Cholesterol can only partially substitute for ergosterol with respect to natamycin binding and aggregation. Membrane-associated aggregation of natamycin is not the result of pre-formed sterol domains in the cell membrane, as we show by direct visualization of minimally modified ergosterol and cholesterol analogs. Inhibiting sphingolipid synthesis increased membrane association and antifungal activity of natamycin, suggesting that targeting sphingolipids in combination with polyene macrolides could lead to novel drug treatment approaches against fungal infections.

biophysics↗

bioRxiv2023

Automated quantification of lipophagy in Saccharomyces cerevisiae from fluorescence and cryo-soft X-ray microscopy data using deep learning

Lipophagy is a form of autophagy by which lipid droplets (LDs) become digested to provide nutrients as a cellular response to starvation. Lipophagy is often studied in yeast, Saccharomyces cerevisiae, in which LDs become internalized into the vacuole. There is a lack of tools to quantitatively assess lipophagy in intact cells with high resolution and throughput. Here, we combine soft X-ray tomography (SXT) with fluorescence microscopy and use a deep learning computational approach to visualize and quantify lipophagy in yeast. We focus on yeast homologs of mammalian Niemann Pick type C proteins, whose dysfunction leads to Niemann Pick type C disease in humans, i.e., NPC1 (named NCR1 in yeast) and NPC2. We developed a convolutional neural network (CNN) model which classifies ring-shaped versus lipid-filled or fragmented vacuoles containing ingested LDs in fluorescence images from wild-type yeast and from cells lacking NCR1 ({Delta}ncr1 cells) or NPC2 ({Delta}npc2 cells). Using a second CNN model, which performs automated segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms, we can obtain 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measure droplet volume, number, and distribution. We find that cells lacking functional NPC proteins can ingest LDs into vacuoles normally but show compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. This phenotype is most severe in{Delta} npc2 cells. Our new method is versatile and allows for automated high-throughput 3D visualization and quantification of lipophagy in intact cells.

bioinformatics↗