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.

Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. Saccharomyces cerevisiae NCR1 is localized to the vacuolar membrane, where it is suggested to carry sterols across the protective glycocalyx and deposit them into the vacuolar membrane. However, documentation of a vacuolar glycocalyx in fungi is lacking, and the mechanism for sterol translocation has remained unclear. Here, we provide evidence supporting the presence of a glycocalyx in isolated S. cerevisiae vacuoles and report four cryo-EM structures of NCR1 in two distinct conformations, named tense and relaxed. These two conformations illustrate the movement of sterols through a tunnel formed by the luminal domains, thus bypassing the barrier presented by the glycocalyx. Based on these structures and on comparison with other members of the Resistance-Nodulation-Division (RND) superfamily, we propose a transport model that links changes in the luminal domains with a cycle of protonation and deprotonation within the transmembrane region of the protein. Our model suggests that NPC proteins work by a generalized RND mechanism where the proton motive force drives conformational changes in the transmembrane domains that are allosterically coupled to luminal/extracellular domains to promote sterol transport.

Transient accumulation and bidirectional movement of KIF13B in primary cilia.

Primary cilia are microtubule-based sensory organelles whose assembly and function rely on the conserved bidirectional intraflagellar transport (IFT) system, which is powered by anterograde kinesin-2 and retrograde cytoplasmic dynein-2 motors. Nematodes additionally employ a cell-type-specific kinesin-3 motor, KLP-6, which moves within cilia independently of IFT and regulates ciliary content and function. Here, we provide evidence that a KLP-6 homolog, KIF13B, undergoes bursts of bidirectional movement within primary cilia of cultured immortalized human retinal pigment epithelial (hTERT-RPE1) cells. Anterograde and retrograde intraciliary velocities of KIF13B were similar to those of IFT (as assayed using IFT172-eGFP), but intraciliary movement of KIF13B required its own motor domain and appeared to be cell-type specific. Our work provides the first demonstration of motor-driven, intraciliary movement by a vertebrate kinesin other than kinesin-2 motors.

Dynamic Mode Decomposition of Multiphoton and Stimulated Emission Depletion Microscopy Data for Analysis of Fluorescent Probes in Cellular Membranes.

An analysis of the membrane organization and intracellular trafficking of lipids often relies on multiphoton (MP) and super-resolution microscopy of fluorescent lipid probes. A disadvantage of particularly intrinsically fluorescent lipid probes, such as the cholesterol and ergosterol analogue, dehydroergosterol (DHE), is their low MP absorption cross-section, resulting in a low signal-to-noise ratio (SNR) in live-cell imaging. Stimulated emission depletion (STED) microscopy of membrane probes like Nile Red enables one to resolve membrane features beyond the diffraction limit but exposes the sample to a lot of excitation light and suffers from a low SNR and photobleaching. Here, dynamic mode decomposition (DMD) and its variant, higher-order DMD (HoDMD), are applied to efficiently reconstruct and denoise the MP and STED microscopy data of lipid probes, allowing for an improved visualization of the membranes in cells. HoDMD also allows us to decompose and reconstruct two-photon polarimetry images of TopFluor-cholesterol in model and cellular membranes. Finally, DMD is shown to not only reconstruct and denoise 3D-STED image stacks of Nile Red-labeled cells but also to predict unseen image frames, thereby allowing for interpolation images along the optical axis. This important feature of DMD can be used to reduce the number of image acquisitions, thereby minimizing the light exposure of biological samples without compromising image quality. Thus, DMD as a computational tool enables gentler live-cell imaging of fluorescent probes in cellular membranes by MP and STED microscopy.

Image segmentation and separation of spectrally similar dyes in fluorescence microscopy by dynamic mode decomposition of photobleaching kinetics.

BACKGROUND: Image segmentation in fluorescence microscopy is often based on spectral separation of fluorescent probes (color-based segmentation) or on significant intensity differences in individual image regions (intensity-based segmentation). These approaches fail, if dye fluorescence shows large spectral overlap with other employed probes or with strong cellular autofluorescence. RESULTS: Here, a novel model-free approach is presented which determines bleaching characteristics based on dynamic mode decomposition (DMD) and uses the inferred photobleaching kinetics to distinguish different probes or dye molecules from autofluorescence. DMD is a data-driven computational method for detecting and quantifying dynamic events in complex spatiotemporal data. Here, DMD is first used on synthetic image data and thereafter used to determine photobleaching characteristics of a fluorescent sterol probe, dehydroergosterol (DHE), compared to that of cellular autofluorescence in the nematode Caenorhabditis elegans. It is shown that decomposition of those dynamic modes allows for separating probe from autofluorescence without invoking a particular model for the bleaching process. In a second application, DMD of dye-specific photobleaching is used to separate two green-fluorescent dyes, an NBD-tagged sphingolipid and Alexa488-transferrin, thereby assigning them to different cellular compartments. CONCLUSIONS: Data-based decomposition of dynamic modes can be employed to analyze spatially varying photobleaching of fluorescent probes in cells and tissues for spatial and temporal image segmentation, discrimination of probe from autofluorescence and image denoising. The new method should find wide application in analysis of dynamic fluorescence imaging data.

Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.

The phase separation and aggregation of proteins are hallmarks of many neurodegenerative diseases. These processes can be studied in living cells using fluorescent protein constructs and quantitative live-cell imaging techniques, such as fluorescence recovery after photobleaching (FRAP) or the related fluorescence loss in photobleaching (FLIP). While the acquisition of FLIP images is straightforward on most commercial confocal microscope systems, the analysis and computational modeling of such data is challenging. Here, a novel model-free method is presented, which resolves complex spatiotemporal fluorescence-loss kinetics based on dynamic-mode decomposition (DMD) of FLIP live-cell image sequences. It is shown that the DMD of synthetic and experimental FLIP image series (DMD-FLIP) allows for the unequivocal discrimination of subcellular compartments, such as nuclei, cytoplasm, and protein condensates based on their differing transport and therefore fluorescence loss kinetics. By decomposing fluorescence-loss kinetics into distinct dynamic modes, DMD-FLIP will enable researchers to study protein dynamics at each time scale individually. Furthermore, it is shown that DMD-FLIP is very efficient in denoising confocal time series data. Thus, DMD-FLIP is an easy-to-use method for the model-free detection of barriers to protein diffusion, of phase-separated protein assemblies, and of insoluble protein aggregates. It should, therefore, find wide application in the analysis of protein transport and aggregation, in particular in relation to neurodegenerative diseases and the formation of protein condensates in living cells.

Cholesterol binding to the sterol-sensing region of Niemann Pick C1 protein confines dynamics of its N-terminal domain.

Lysosomal accumulation of cholesterol is a hallmark of Niemann Pick type C (NPC) disease caused by mutations primarily in the lysosomal membrane protein NPC1. NPC1 contains a transmembrane sterol-sensing domain (SSD), which is supposed to regulate protein activity upon cholesterol binding, but the mechanisms underlying this process are poorly understood. Using atomistic simulations, we show that in the absence of cholesterol in the SSD, the luminal domains of NPC1 are highly dynamic, resulting in the disengagement of the NTD from the rest of the protein. The disengaged NPC1 adopts a flexed conformation that approaches the lipid bilayer, and could represent a conformational state primed to receive a sterol molecule from the soluble lysosomal cholesterol carrier NPC2. The binding of cholesterol to the SSD of NPC1 allosterically suppresses the conformational dynamics of the luminal domains resulting in an upright NTD conformation. The presence of an additional 20% cholesterol in the membrane has negligible impact on this process. The additional presence of an NTD-bound cholesterol suppresses the flexing of the NTD. We propose that cholesterol acts as an allosteric effector, and the modulation of NTD dynamics by the SSD-bound cholesterol constitutes an allosteric feedback mechanism in NPC1 that controls cholesterol abundance in the lysosomal membrane.

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.

Design, Synthesis, and Evaluation of a Luminescent Cholesterol Mimic.

The development of new chemical tools with improved properties is essential to chemical and cell biology. Of particular interest is the development of mimics of small molecules with important cellular function that allow the direct observation of their trafficking in a cell. To this end, a novel 15-azasterol has been designed and synthesized as a luminescent cholesterol mimic for the monitoring of cholesterol trafficking. The brightness of this probe, which is ∼32-times greater than the widely used dehydroergosterol probe, is combined with resistance to photobleaching in solution and in human fibroblasts and an exceptionally large Stokes-like shift of ∼150-200 nm. The photophysical properties of the probe have been studied experimentally and computationally, suggesting an intersystem crossing to the triplet excited state with subsequent phosphorescent decay. Molecular dynamics simulations show a similar binding mode of cholesterol and the azasterol probe to NPC proteins, demonstrating the structural similarity of the probe to cholesterol.

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.

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.

One- and two-photon solvatochromism of the fluorescent dye Nile Red and its CF3, F and Br-substituted analogues.

The solvatochromic fluorophore Nile Red, 9-diethylamino-5H-benzo[a]phenoxazine-5-one, is one of the most commonly used stains to enhance contrast of lipid-rich areas of microscopic biosamples. Quite surprisingly, relatively little is known about the spectrally-resolved two-photon absorption (2PA) properties of this dye despite its promising features for two-photon microscopy of biological matter. For this reason, the two-photon solvatochromism of Nile Red still remains an uncharted territory as well. Also, no study has yet reported on how electron-withdrawing substituents attached to the Nile Red backbone affect its solvatochromic properties and two-photon brightness. In this paper, we demonstrate how solvent polarity influences the one- and two-photon absorption spectra of Nile Red as well as its fluorescence parameters, and we present new analogues that contain -CF3, -F and -Br substituents on its eastern side. Two-photon excited fluorescence experiments in a broad spectral range (780-1240 nm) and electronic structure calculations show that both the nature and location of the substituent have particular influence on the strength of 2PA, peaking in all cases at approx. 860 and 1050 nm. 2PA cross sections are higher at 1050 nm than at 860 nm, which suggests that Nile Red and its analogues are best suited for two-photon imaging employing excitation in the NIR-II optical transparency window of biological tissues.

Modeling the Sterol-Binding Domain of Aster-A Provides Insight into Its Multiligand Specificity.

Intracellular transport of cholesterol and related sterols relies to a large degree on nonvesicular mechanisms, which are only partly understood at the molecular level. Aster proteins belonging to the Lam family of sterol transfer proteins have recently been identified as important catalysts of nonvesicular sterol exchange between the plasma membrane (PM) and endoplasmic reticulum (ER). Here, we used a range of computational tools to study the molecular mechanisms underlying sterol binding as well as multisterol ligand specificity of Aster-A. Our study focused primarily on gaining atomistic insight into the bound ligand-protein complex and was, on this basis, performed in the absence of any membrane. Molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations provide a rationale for the experimentally found ranking of binding affinities of various sterols to Aster-A. In particular, the polarity of the sterols and the length of their alkyl chain could be identified as being critical determinants of ligand affinity. A Gibbs free energy decomposition identified a charged residue, Glu444, at the base of the binding pose as an important control point for sterol binding. Removing its net charge via protonation was found to cause significant changes to the environment surrounding this residue. In addition, the protonation of Glu444 was found to be paralleled by a large redistribution of molecular flexibility in the Aster domain. This finding was supplemented by multiple branched adaptive steered molecular dynamics (MB-ASMD) simulations by which we defined a possible molecular path for sterol release and demonstrated the importance of Glu444 in this process.

Steady state analysis of influx and transbilayer distribution of ergosterol in the yeast plasma membrane.

BACKGROUND: The transbilayer sterol distribution between both plasma membrane (PM) leaflets has long been debated. Recent studies in mammalian cells and in yeast show that the majority of sterol resides in the inner PM leaflet. Since sterol flip-flop in model membranes is rapid and energy-independent, a mechanistic understanding for net enrichment of sterol in one leaflet is lacking. Import of ergosterol in yeast can take place via the ABC transporters Aus1/Pdr11 under anaerobic growth conditions, eventually followed by rapid non-vesicular sterol transport to the endoplasmic reticulum (ER). Little is known about how these transport steps are dynamically coordinated. METHODS: Here, a kinetic steady state model is presented which considers sterol import via Aus1/Pdr11, sterol flip-flop across the PM, bi-molecular complex formation and intracellular sterol release followed by eventual transport to and esterification of sterol in the ER. The steady state flux is calculated, and a thermodynamic analysis of feasibility is presented. RESULTS: It is shown that the steady state sterol flux across the PM can be entirely controlled by irreversible sterol import via Aus1/Pdr11. The transbilayer sterol flux at steady state is a non-linear function of the chemical potential difference of sterol between both leaflets. Non-vesicular release of sterol on the cytoplasmic side of the PM lowers the attainable sterol enrichment in the inner leaflet. Including complex formation of sterol with phospholipids or proteins can explain several puzzling experimental observations; 1) rapid sterol flip-flop across the PM despite net sterol enrichment in one leaflet, 2) a pronounced steady state sterol gradient between PM and ER despite fast non-vesicular sterol exchange between both compartments and 3) a non-linear dependence of ER sterol on ergosterol abundance in the PM. CONCLUSIONS: A steady state model is presented that can account for the observed sterol asymmetry in the yeast PM, the strong sterol gradient between PM and ER and threshold-like expansion of ER sterol for increasing sterol influx into the PM. The model also provides new insight into selective uptake of cholesterol and its homeostasis in mammalian cells, and it provides testable predictions for future experiments.

Photophysical investigation of two emissive nucleosides exhibiting gigantic stokes shifts.

We report spectroscopic characterization of two emissive 2'-deoxycytidine analogues: 5-(5-phenylfuran-2-yl)-2'-deoxycytidine and 5-(1-phenyl-1H-pyrazol-3-yl)-2'-deoxycytidine. Their fluorescent properties were examined using a combined experimental and theory/simulation approach, where the latter was based on Born-Oppenheimer molecular dynamics and time-dependent density functional theory. The analogues were found to exhibit unusually large Stokes shifts in polar media (>100 nm), moderate fluorescence quantum yields, and their emissions were found to be very sensitive to the local dielectric environment. These two analogues of 2'-deoxycytidine thus hold a promising potential as probes in chemical biology. In addition, the accuracy of the theoretical models for determining the optical properties is validated, which opens up for a convenient way of assessing the potential of future probes.

Rational design of novel fluorescent analogues of cholesterol: a "step-by-step" computational study.

In this paper we show a theoretical rational design approach on a series of intrinsically fluorescent analogues of cholesterol (FLACs), called polyene-sterols (P-sterols), followed by a step-by-step selection of potential candidates, employing, sequentially, state-of-the-art quantum mechanical (QM) computations of the optical properties (single- and multiphoton absorption electronic spectroscopies and emission), molecular dynamics (MD) simulations in model membranes, and multiscale approaches (polarizable embedding). This selection converged to a promising candidate that shows simultaneously interesting single- and multiphoton absorption properties as well as emitting properties and good abilities to mimic cholesterol order effects in model membranes.

SpatTrack: an imaging toolbox for analysis of vesicle motility and distribution in living cells.

The endocytic pathway is a complex network of highly dynamic organelles, which has been traditionally studied by quantitative fluorescence microscopy. The data generated by this method can be overwhelming and its analysis, even for the skilled microscopist, is tedious and error-prone. We developed SpatTrack, an open source, platform-independent program collecting a variety of methods for analysis of vesicle dynamics and distribution in living cells. SpatTrack performs 2D particle tracking, trajectory analysis and fitting of diffusion models to the calculated mean square displacement. It allows for spatial analysis of detected vesicle patterns including calculation of the radial distribution function and particle-based colocalization. Importantly, all analysis tools are supported by Monte Carlo simulations of synthetic images. This allows the user to assess the reliability of the analysis and to study alternative scenarios. We demonstrate the functionality of SpatTrack by performing a detailed imaging study of internalized fluorescence-tagged Niemann Pick C2 (NPC2) protein in human disease fibroblasts. Using SpatTrack, we show that NPC2 rescued the cholesterol-storage phenotype from a subpopulation of late endosomes/lysosomes (LE/LYSs). This was paralleled by repositioning and active transport of NPC2-containing vesicles to the cell surface. The potential of SpatTrack for other applications in intracellular transport studies will be discussed.

Computational Analysis of Sterol Ligand Specificity of the Niemann Pick C2 Protein.

Transport of cholesterol derived from hydrolysis of lipoprotein associated cholesteryl esters out of late endosomes depends critically on the function of the Niemann Pick C1 (NPC1) and C2 (NPC2) proteins. Both proteins bind cholesterol but also various other sterols and both with strongly varying affinity. The molecular mechanisms underlying this multiligand specificity are not known. On the basis of the crystal structure of NPC2, we have here investigated structural details of NPC2-sterol interactions using molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) calculations. We found that an aliphatic side chain in the sterol ligand results in strong binding to NPC2, while side-chain oxidized sterols gave weaker binding. Estradiol and the hydrophobic amine U18666A had the lowest affinity of all tested ligands and at the same time showed the highest flexibility within the NPC2 binding pocket. The binding affinity of all ligands correlated highly with their calculated partitioning coefficient (logP) between octanol/water phases and with the potential of sterols to stabilize the protein backbone. From molecular dynamics simulations, we suggest a general mechanism for NPC2 mediated sterol transfer, in which Phe66, Val96, and Tyr100 act as reversible gate keepers. These residues stabilize the sterol in the binding pose via π-π stacking but move transiently apart during sterol release. A computational mutation analysis revealed that the binding of various ligands depends critically on the same specific amino acid residues within the binding pocket providing shape complementary to sterols, but also on residues in distal regions of the protein.

A comparative study on fluorescent cholesterol analogs as versatile cellular reporters.

Cholesterol (Chol) is a crucial component of cellular membranes, but knowledge of its intracellular dynamics is scarce. Thus, it is of utmost interest to develop tools for visualization of Chol organization and dynamics in cells and tissues. For this purpose, many studies make use of fluorescently labeled Chol analogs. Unfortunately, the introduction of the label may influence the characteristics of the analog, such as its localization, interaction, and trafficking in cells; hence, it is important to get knowledge of such bias. In this report, we compared different fluorescent lipid analogs for their performance in cellular assays: 1) plasma membrane incorporation, specifically the preference for more ordered membrane environments in phase-separated giant unilamellar vesicles and giant plasma membrane vesicles; 2) cellular trafficking, specifically subcellular localization in Niemann-Pick type C disease cells; and 3) applicability in fluorescence correlation spectroscopy (FCS)-based and super-resolution stimulated emission depletion-FCS-based measurements of membrane diffusion dynamics. The analogs exhibited strong differences, with some indicating positive performance in the membrane-based experiments and others in the intracellular trafficking assay. However, none showed positive performance in all assays. Our results constitute a concise guide for the careful use of fluorescent Chol analogs in visualizing cellular Chol dynamics.