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

During starvation in the yeast Saccharomyces cerevisiae vacuolar vesicles fuse and lipid droplets (LDs) can become internalized into the vacuole in an autophagic process named lipophagy. There is a lack of tools to quantitatively assess starvation-induced vacuole fusion and 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 these processes in yeast. We focus on yeast homologs of mammalian NPC1 (NPC intracellular cholesterol transporter 1; Ncr1 in yeast) and NPC2 proteins, whose dysfunction leads to Niemann Pick type C (NPC) disease in humans. We developed a convolutional neural network (CNN) model which classifies fully fused versus partially fused vacuoles based on fluorescence images of stained cells. This CNN, named Deep Yeast Fusion Network (DYFNet), revealed that cells lacking Ncr1 (ncr1∆ cells) or Npc2 (npc2∆ cells) have a reduced capacity for vacuole fusion. Using a second CNN model, we implemented a pipeline named LipoSeg to perform automated instance segmentation of LDs and vacuoles from high-resolution reconstructions of X-ray tomograms. From that, we obtained 3D renderings of LDs inside and outside of the vacuole in a fully automated manner and additionally measured droplet volume, number, and distribution. We find that ncr1∆ and npc2∆ cells could ingest LDs into vacuoles normally but showed compromised degradation of LDs and accumulation of lipid vesicles inside vacuoles. Our new method is versatile and allows for analysis of vacuole fusion, droplet size and lipophagy in intact cells.Abbreviations: BODIPY493/503: 4,4-difluoro-1,3,5,7,8-pentamethyl-4-bora-3a,4a-diaza-s-Indacene; BPS: bathophenanthrolinedisulfonic acid disodium salt hydrate; CNN: convolutional neural network; DHE; dehydroergosterol; npc2∆, yeast deficient in Npc2; DSC, Dice similarity coefficient; EM, electron microscopy; EVs, extracellular vesicles; FIB-SEM, focused ion beam milling-scanning electron microscopy; FM 4-64, N-(3-triethylammoniumpropyl)-4-(6-[4-{diethylamino} phenyl] hexatrienyl)-pyridinium dibromide; LDs, lipid droplets; Ncr1, yeast homolog of human NPC1 protein; ncr1∆, yeast deficient in Ncr1; NPC, Niemann Pick type C; NPC2, Niemann Pick type C homolog; OD600, optical density at 600 nm; ReLU, rectifier linear unit; PPV, positive predictive value; NPV, negative predictive value; MCC, Matthews correlation coefficient; SXT, soft X-ray tomography; UV, ultraviolet; YPD, yeast extract peptone dextrose.

Natamycin interferes with ergosterol-dependent lipid phases in model membranes.

Natamycin is an antifungal polyene macrolide that is used as a food preservative but also to treat fungal keratitis and other yeast infections. In contrast to other polyene antimycotics, natamycin does not form ion pores in the plasma membrane, but its mode of action is poorly understood. Using nuclear magnetic resonance (NMR) spectroscopy of deuterated sterols, we find that natamycin slows the mobility of ergosterol and cholesterol in liquid-ordered (Lo) membranes to a similar extent. This is supported by molecular dynamics (MD) simulations, which additionally reveal a strong impact of natamycin dimers on sterol dynamics and water permeability. Interference with sterol-dependent lipid packing is also reflected in a natamycin-mediated increase in membrane accessibility for dithionite, particularly in bilayers containing ergosterol. NMR experiments with deuterated sphingomyelin (SM) in sterol-containing membranes reveal that natamycin reduces phase separation and increases lipid exchange in bilayers with ergosterol. In ternary lipid mixtures containing monounsaturated phosphatidylcholine, saturated SM, and either ergosterol or cholesterol, natamycin interferes with phase separation into Lo and liquid-disordered (Ld) domains, as shown by NMR spectroscopy. Employing the intrinsic fluorescence of natamycin in ultraviolet-sensitive microscopy, we can visualize the binding of natamycin to giant unilamellar vesicles (GUVs) and find that it has the highest affinity for the Lo phase in GUVs containing ergosterol. Our results suggest that natamycin specifically interacts with the sterol-induced ordered phase, in which it disrupts lipid packing and increases solvent accessibility. This property is particularly pronounced in ergosterol containing membranes, which could underlie the selective antifungal activity of natamycin.

Natamycin sequesters ergosterol and interferes with substrate transport by the lysine transporter Lyp1 from yeast.

Natamycin is a polyene macrolide, widely employed to treat fungal keratitis and other yeast infections as well as to protect food products against fungal molds. In contrast to other polyene macrolides, such as nystatin or amphotericin B, natamycin does not form pores in yeast membranes, and its mode of action is not well understood. Here, we have employed a variety of spectroscopic methods, computational modeling, and membrane reconstitution to study the molecular interactions of natamycin underlying its antifungal activity. We find that natamycin forms aggregates in an aqueous solution with strongly altered optical properties compared to monomeric natamycin. Interaction of natamycin with model membranes results in a concentration-dependent fluorescence increase which is more pronounced for ergosterol- compared to cholesterol-containing membranes up to 20 mol% sterol. Evidence for formation of specific ergosterol-natamycin complexes in the bilayer is provided. Using nuclear magnetic resonance (NMR) and electron spin resonance (ESR) spectroscopy, we find that natamycin sequesters sterols, thereby interfering with their well-known ability to order acyl chains in lipid bilayers. This effect is more pronounced for membranes containing the sterol of fungi, ergosterol, compared to those containing mammalian cholesterol. Natamycin interferes with ergosterol-dependent transport of lysine by the yeast transporter Lyp1, which we propose to be due to the sequestering of ergosterol, a mechanism that also affects other plasma membrane proteins. Our results provide a mechanistic explanation for the selective antifungal activity of natamycin, which can set the stage for rational design of novel polyenes in the future.

Novel methods to characterise spatial distribution and enantiomeric composition of usnic acids in four Icelandic lichens.

Usnic acid is an antibiotic metabolite produced by a wide variety of lichenized fungal lineages. The enantiomers of usnic acid have been shown to display contrasting bioactivities, and hence it is important to determine their spatial distribution, amounts and enantiomeric ratios in lichens to understand their roles in nature and grasp their pharmaceutical potential. The overall aim of the study was to characterise the spatial distribution of the predominant usnic acid enantiomer in lichens by combining spatial imaging and chiral chromatography. Specifically, separation and quantification of usnic acid enantiomers in four common lichens in Iceland was performed using a validated chiral chromatographic method. Molecular dynamics simulation was carried out to rationalize the chiral separation mechanism. Spatial distribution of usnic acid in the lichen thallus cross-sections were analysed using Desorption Electrospray Ionization-Imaging Mass Spectrometry (DESI-IMS) and fluorescence microscopy. DESI-IMS confirmed usnic acid as a cortical compound, and revealed that usnic acid can be more concentrated around the algal vicinity. Fluorescence microscopy complemented DESI-IMS by providing more detailed distribution information. By combining results from spatial imaging and chiral separation, we were able to visualize the distribution of the predominant usnic acid enantiomer in lichen cross-sections: (+)-usnic acid in Cladonia arbuscula and Ramalina siliquosa, and (-)-usnic acid in Alectoria ochroleuca and Flavocetraria nivalis. This study provides an analytical foundation for future environmental and functional studies of usnic acid enantiomers in lichens.

Photophysical and Structural Characterization of Intrinsically Fluorescent Sterol Aggregates.

Self-association of cholesterol into aggregates and crystals is a hallmark of developing atherosclerosis. Intrinsically fluorescent sterols, such as dehydroergosterol (DHE), can be used to study sterol aggregation by fluorescence spectroscopy and microscopy, but a thorough understanding of DHE's photophysical and structural properties in the aggregated state is missing. Here, we show that DHE forms submicron fluorescent aggregates when evaporated from an ethanol solution. Using atomic force microscopy, we find that DHE, like cholesterol, forms compact oblate-shape aggregates of <100 nm in diameter. DHE's fluorescence is lowered in the aggregate compared to the monomeric form, and characteristic spectral changes accompany the aggregation process. Electronic structure calculations of DHE dimers in water indicate that Frenkel-type exciton coupling contributes to the lowered DHE fluorescence in the aggregates. Using molecular dynamics (MD) simulations, we show that DHE forms compact aggregates on the nanosecond scale and with strong intermolecular attraction, in which a broad range of orientations, and therefore electronic couplings, will take place. Tight packing of DHE in aggregates also lowers the apparent absorption cross section, further reducing the molecular brightness of the aggregates. Our results pave the way for systematic solubility studies of intrinsically fluorescent analogues of biologically relevant sterols.

Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles.

The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2's known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface.

Substituted 9-Diethylaminobenzo[a]phenoxazin-5-ones (Nile Red Analogues): Synthesis and Photophysical Properties.

Nile Red is a benzo[a]phenoxazone dye containing a diethylamino substituent at the 9-position. In recent years, it has become a popular histological stain for cellular membranes and lipid droplets due to its unrivaled fluorescent properties in lipophilic environments. This makes it an attractive lead for chemical decoration to tweak its attributes and optimize it for more specialized microscopy techniques, e.g., fluorescence lifetime imaging or two-photon excited fluorescence microscopy, to which Nile Red has never been optimized. Herein, we present synthesis approaches to a series of monosubstituted Nile Red derivatives (9-diethylbenzo[a]phenoxazin-5-ones) starting from 1-naphthols or 1,3-naphthalenediols. The solvatochromic responsiveness of these fluorophores is reported with focus on how the substituents affect the absorption and emission spectra, luminosity, fluorescence lifetimes, and two-photon absorptivity. Several of the analogues emerge as strong candidates for reporting the polarity of their local environment. Specifically, the one- and two-photon excited fluorescence of Nile Red turns out to be very responsive to substitution, and the spectroscopic features can be finely tuned by judiciously introducing substituents of distinct electronic character at specific positions. This new toolkit of 9-diethylbenzo[a]phenoxazine-5-ones constitutes a step toward the next generation of optical molecular probes for advancing the understanding of lipid structures and cellular processes.

Direct observation of nystatin binding to the plasma membrane of living cells.

Nystatin is an antifungal polyene macrolide which is widely applied to treat yeast infections. Nystatin has also been used as a laboratory tool to inhibit endocytic processes in mammalian cells. The interaction of nystatin with model membranes has been studied thoroughly by various spectroscopic methods, making use of its weak fluorescence in the ultraviolet (UV). Studying its interaction with cells would require direct imaging, which, so far, required attachment of a fluorophore to nystatin. Using UV-sensitive microscopy, we show here how to visualize the interaction of nystatin with the plasma membrane (PM) directly. We find that nystatin forms micron-sized aggregates in buffer, and molecular dynamics simulations confirm that nystatin rapidly self-assembles into aggregates in aqueous solution. Using UV-sensitive microscopy, we find that large nystatin aggregates adhere to the surface of Chinese Hamster Ovarian (CHO) cells, causing slow spreading of nystatin fluorescence into the PM. Binding of nystatin to CHO cells does not interfere with cellular uptake or lateral membrane diffusion of the cholesterol analogue TopFluor-cholesterol (TF-Chol). Nystatin binds extensively to the PM of yeast cells as inferred from a strong UV signal in this membrane. Loading a yeast mutant unable to synthesize ergosterol with cholesterol gave much less nystatin membrane staining compared to loading such cells with ergosterol. These results explain the selective fungicidal effect of nystatin by differential interaction of nystatin with yeast membranes containing ergosterol compared to the mammalian cholesterol. Our combined experimental and computational approach provides a toolset for future design of new polyene macrolides.

Membrane organization and intracellular transport of a fluorescent analogue of 27-hydroxycholesterol.

Oxysterols are cholesterol metabolites with multiple functions in controlling cellular homeostasis. In particular, 27-hydroxycholesterol (27-OH-Chol) has been shown to regulate a variety of physiological functions, but little is known about its uptake, intracellular trafficking, and efflux from cells. This is largely due to a lack of suitable analogs of 27-OH-Chol, which mimic this oxysterol closely. Here, we present the intrinsically fluorescent 27-hydroxy-cholestatrienol (27-OH-CTL), which differs from 27-OH-Chol only by having two additional double bonds in the steroid ring system. Based on molecular dynamics (MD) simulations, we show that 27-OH-CTL possesses almost identical membrane properties compared to 27-OH-Chol. By comparative imaging of 27-OH-CTL and of the cholesterol analogue cholestatrienol (CTL) in living cells, we assess the impact of a single hydroxy group on sterol trafficking. We find that human fibroblasts take up more CTL than 27-OH-CTL, but efflux the oxysterol analogue more efficiently. For both sterols, efflux includes shedding of vesicles from the plasma membrane. Intracellular, 27-OH-CTL accumulates primarily in lipid droplets (LDs), while CTL is mostly found in endosomes and lysosomes. Using fluorescence recovery after photobleaching (FRAP), we find for both sterols a rapidly exchanging pool, which moves orders of magnitude faster than sterol containing vesicles and LDs. In summary, by applying a new fluorescent derivative of 27-OH-Chol we demonstrate that human cells can distinguish sterols based on a single hydroxy group in the side chain, resulting in different transport itineraries, dynamics, and efflux kinetics. Both intrinsically fluorescent cholesterol and oxysterol analogues show rapid non-vesicular transport in human fibroblasts.

Mechanistic Insight into Lipid Binding to Yeast Niemann Pick Type C2 Protein.

Niemann Pick type C2 (NPC2) is a small sterol binding protein in the lumen of late endosomes and lysosomes. We showed recently that the yeast homologue of NPC2 together with its binding partner NCR1 mediates integration of ergosterol, the main sterol in yeast, into the vacuolar membrane. Here, we study the binding specificity and the molecular details of lipid binding to yeast NPC2. We find that NPC2 binds fluorescence- and spin-labeled analogues of phosphatidylcholine (PC), phosphatidylserine, phosphatidylinositol (PI), and sphingomyelin. Spectroscopic experiments show that NPC2 binds lipid monomers in solution but can also interact with lipid analogues in membranes. We further identify ergosterol, PC, and PI as endogenous NPC2 ligands. Using molecular dynamics simulations, we show that NPC2's binding pocket can adapt to the ligand shape and closes around bound ergosterol. Hydrophobic interactions stabilize the binding of ergosterol, but binding of phospholipids is additionally stabilized by electrostatic interactions at the mouth of the binding site. Our work identifies key residues that are important in stabilizing the binding of a phospholipid to yeast NPC2, thereby rationalizing future mutagenesis studies. Our results suggest that yeast NPC2 functions as a general "lipid solubilizer" and binds a variety of amphiphilic lipid ligands, possibly to prevent lipid micelle formation inside the vacuole.

Binding and intracellular transport of 25-hydroxycholesterol by Niemann-Pick C2 protein.

Side-chain oxidized cholesterol derivatives, like 25-hydroxycholesterol (25-OH-Chol) are important regulators of cellular cholesterol homeostasis. How transport of oxysterols through the endo-lysosomal pathway contributes to their biological function is not clear. The Niemann-Pick C2 protein (NPC2) is a small lysosomal sterol transfer protein required for export of cholesterol from late endosomes and lysosomes (LE/LYSs). Here, we show that 25-hydroxy-cholestatrienol, (25-OH-CTL), an intrinsically fluorescent analogue of 25-OH-Chol, becomes trapped in LE/LYSs of NPC2-deficient fibroblasts, but can efflux from the cells even in the absence of NPC2 upon removal of the sterol source. Fluorescence recovery after photobleaching (FRAP) of 25-OH-CTL in endo-lysosomes was rapid and extensive and only partially dependent on NPC2 function. Using quenching of NPC2's intrinsic fluorescence, we show that 25-OH-Chol and 25-OH-CTL can bind to NPC2 though with lower affinity compared to cholesterol and its fluorescent analogues, cholestatrienol (CTL) and dehydroergosterol (DHE). This is confirmed by calculations of binding energies which additionally show that 25-OH-CTL can bind in two orientations to NPC2, in stark contrast to cholesterol and its analogues. We conclude that NPC2's affinity for all sterols is energetically favored over their self-aggregation in the lysosomal lumen. Lysosomal export of 25-OH-Chol is not strictly dependent on the NPC2 protein.

Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins.

Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and in vivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50 Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C.

Niemann-Pick C2 protein regulates sterol transport between plasma membrane and late endosomes in human fibroblasts.

Niemann-Pick disease type C2 is a lipid storage disorder in which mutations in the NPC2 protein cause accumulation of lipoprotein-derived cholesterol in late endosomes and lysosomes (LE/LYSs). Whether cholesterol delivered by other means to NPC2 deficient cells also accumulates in LE/LYSs is currently unknown. We show that the close cholesterol analog dehydroergosterol (DHE), when delivered to the plasma membrane (PM) accumulates in LE/LYSs of human fibroblasts lacking functional NPC2. We measured two different time scales of sterol diffusion; while DHE rich LE/LYSs moved by slow anomalous diffusion in disease cells (D ∼ 4.6∙10-4 µm2/sec; α∼0.76), a small pool of sterol could exchange rapidly with D ∼ 3 µm2/s between LE/LYSs, as shown by fluorescence recovery after photobleaching (FRAP). By quantitative lipid mass spectrometry we found that esterification of 13C-labeled cholesterol but not of DHE is reduced 10-fold in disease fibroblasts compared to control cells. Internalized NPC2 rescued the sterol storage phenotype and strongly expanded the dynamic sterol pool seen in FRAP experiments. Together, our study shows that cholesterol esterification and trafficking of sterols between the PM and LE/LYSs depends on a functional NPC2 protein. NPC2 likely acts inside LE/LYSs from where it increases non-vesicular sterol exchange with other organelles.

Ergosterol is mainly located in the cytoplasmic leaflet of the yeast plasma membrane.

Transbilayer lipid asymmetry is a fundamental characteristic of the eukaryotic cell plasma membrane (PM). While PM phospholipid asymmetry is well documented, the transbilayer distribution of PM sterols such as mammalian cholesterol and yeast ergosterol is not reliably known. We now report that sterols are asymmetrically distributed across the yeast PM, with the majority (~80%) located in the cytoplasmic leaflet. By exploiting the sterol-auxotrophic hem1Δ yeast strain we obtained cells in which endogenous ergosterol was quantitatively replaced with dehydroergosterol (DHE), a closely related fluorescent sterol that functionally and accurately substitutes for ergosterol in vivo. Using fluorescence spectrophotometry and microscopy we found that <20% of DHE fluorescence was quenched when the DHE-containing cells were exposed to membrane-impermeant collisional quenchers (spin-labeled phosphatidylcholine and trinitrobenzene sulfonic acid). Efficient quenching was seen only after the cells were disrupted by glass-bead lysis or repeated freeze-thaw to allow quenchers access to the cell interior. The extent of quenching was unaffected by treatments that deplete cellular ATP levels, collapse the PM electrochemical gradient or affect the actin cytoskeleton. However, alterations in PM phospholipid asymmetry in cells lacking phospholipid flippases resulted in a more symmetric transbilayer distribution of sterol. Similarly, an increase in the quenchable pool of DHE was observed when PM sphingolipid levels were reduced by treating cells with myriocin. We deduce that sterols comprise up to ~45% of all inner leaflet lipids in the PM, a result that necessitates revision of current models of the architecture of the PM lipid bilayer.

Structural design of intrinsically fluorescent oxysterols.

Oxysterols are oxidized derivatives of cholesterol with many important biological functions. Trafficking of oxysterols in and between cells is not well studied, largely due to the lack of appropriate oxysterol analogs. Intrinsically fluorescent oxysterols present a new route towards direct observation of intracellular oxysterol trafficking by fluorescence microscopy. We characterize the fluorescence properties of the existing fluorescent 25-hydroxycholesterol analog 25-hydroxycholestatrienol, and propose a new probe with an extended conjugated system. The location of both probes inside a membrane is analyzed and compared with that of 25-hydroxycholesterol using molecular dynamics simulations. The analogs' one- and two-photon absorption properties inside the membrane are evaluated using electronic structure calculations with polarizable embedding. Due to predicted keto-enol tautomerisation of the new oxysterol analog, we also evaluate the keto form. Both analogs are found to be good probe candidates for 25-hydroxycholesterol, provided that the new analog remains in the enol-form. Only the new analog with extended conjugated system shows significant two-photon absorption, which is strongly enhanced by the presence of the membrane.