Stereospecific Properties and Intracellular Transport of Novel Intrinsically Fluorescent Neurosteroids.

Allopregnanolone (AlloP) is an example of neuroactive steroids (NAS), which is a potent allosteric activator of the γ-aminobutyric acid A (GABA) receptor. The mechanisms underlying the biological activity of AlloP and other NAS are only partially understood. Here, we present intrinsically fluorescent analogs of AlloP (MQ-323) and its 3β-epimer, epi-allopregnanolone (E-AlloP) (YX-11), and show, by a combination of spectroscopic and computational studies, that these analogs mimic the membrane properties of AlloP and E-AlloP very well.

The impact of acyl-CoA:cholesterol transferase (ACAT) inhibitors on biophysical membrane properties depends on membrane lipid composition.

Acyl-coenzyme A: cholesterol acyltransferases are enzymes which are involved in the homeostasis of cholesterol. Impaired enzyme activity is associated with the occurrence of various diseases like Alzheimer's disease, atherosclerosis, and cancers. At present, mitotane is the only inhibitor of this class of enzymes in clinical use for the treatment of adrenocortical carcinoma but associated with common and severe adverse effects.

Ratiometric fluorescence nanoscopy and lifetime imaging of novel Nile Red analogs for analysis of membrane packing in living cells.

Subcellular membranes have complex lipid and protein compositions, which give rise to organelle-specific membrane packing, fluidity, and permeability. Due to its exquisite solvent sensitivity, the lipophilic fluorescence dye Nile Red has been used extensively to study membrane packing and polarity. Further improvement of Nile Red can be achieved by introducing electron-donating or withdrawing functional groups.

Ergosterol promotes 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.

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.

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. 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.

Kinetic modelling of sterol transport between plasma membrane and endo-lysosomes based on quantitative fluorescence and X-ray imaging data.

Niemann Pick type C1 and C2 (NPC1 and NPC2) are two sterol-binding proteins which, together, orchestrate cholesterol transport through late endosomes and lysosomes (LE/LYSs). NPC2 can facilitate sterol exchange between model membranes severalfold, but how this is connected to its function in cells is poorly understood. Using fluorescent analogs of cholesterol and quantitative fluorescence microscopy, we have recently measured the transport kinetics of sterol between plasma membrane (PM), recycling endosomes (REs) and LE/LYSs in control and NPC2 deficient fibroblasts.

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

During starvation in the yeast 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.

Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging.

CRISPR-mediated endogenous tagging of genes provides unique possibilities to explore the function and dynamic subcellular localization of proteins in living cells. Here, we describe experimental strategies for endogenous PCR-tagging of ciliary genes in human RPE1 cells and how image acquisition and analysis of the expressed fluorescently tagged proteins can be utilized to study the dynamic ciliary processes of intraflagellar transport and vesicular trafficking.

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.

Dynamic switching of transcriptional regulators between two distinct low-mobility chromatin states.

How chromatin dynamics relate to transcriptional activity remains poorly understood. Using single-molecule tracking, coupled with machine learning, we show that histone H2B and multiple chromatin-bound transcriptional regulators display two distinct low-mobility states. Ligand activation results in a marked increase in the propensity of steroid receptors to bind in the lowest-mobility state.

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.

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.

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.

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.

Pathways and Mechanisms of Cellular Cholesterol Efflux-Insight From Imaging.

Cholesterol is an essential molecule in cellular membranes, but too much cholesterol can be toxic. Therefore, mammalian cells have developed complex mechanisms to remove excess cholesterol. In this review article, we discuss what is known about such efflux pathways including a discussion of reverse cholesterol transport and formation of high-density lipoprotein, the function of ABC transporters and other sterol efflux proteins, and we highlight their role in human diseases.

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.

Quantitative imaging of membrane contact sites for sterol transfer between endo-lysosomes and mitochondria in living cells.

Mitochondria receive cholesterol from late endosomes and lysosomes (LE/LYSs) or from the plasma membrane for production of oxysterols and steroid hormones. This process depends on the endo-lysosomal sterol transfer protein Niemann Pick C2 (NPC2). Using the intrinsically fluorescent cholesterol analog, cholestatrienol, we directly observe sterol transport to mitochondria in fibroblasts upon treating NPC2 deficient human fibroblasts with NPC2 protein.

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.

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.

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).

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.