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

Wüstner, Daniel

2022 Volume 23, Issue 1, Page(s) 334

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Animals ; Caenorhabditis elegans ; Fluorescent Dyes ; Kinetics ; Microscopy, Fluorescence/methods ; Photobleaching
Chemical Substances	Fluorescent Dyes
Language	English
Publishing date	2022-08-12
Publishing country	England
Document type	Journal Article
ZDB-ID	2041484-5
ISSN	1471-2105 ; 1471-2105
ISSN (online)	1471-2105
ISSN	1471-2105
DOI	10.1186/s12859-022-04881-x
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Article ; Online: Dynamic Mode Decomposition of Fluorescence Loss in Photobleaching Microscopy Data for Model-Free Analysis of Protein Transport and Aggregation in Living Cells.

Wüstner, Daniel

Sensors (Basel, Switzerland)

2022 Volume 22, Issue 13

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Fluorescence Recovery After Photobleaching/methods ; Humans ; Microscopy, Confocal ; Microscopy, Fluorescence/methods ; Neurodegenerative Diseases ; Photobleaching ; Protein Transport ; Proteins
Chemical Substances	Proteins
Language	English
Publishing date	2022-06-23
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2052857-7
ISSN	1424-8220 ; 1424-8220
ISSN (online)	1424-8220
ISSN	1424-8220
DOI	10.3390/s22134731
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Article ; Online: Steady state analysis of influx and transbilayer distribution of ergosterol in the yeast plasma membrane.

Wüstner, Daniel

Theoretical biology & medical modelling

2019 Volume 16, Issue 1, Page(s) 13

Abstract: 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 ...

Abstract	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.
MeSH term(s)	Anaerobiosis ; Biological Transport ; Cell Membrane/metabolism ; Endoplasmic Reticulum/metabolism ; Ergosterol/chemistry ; Ergosterol/metabolism ; Lipid Bilayers/metabolism ; Models, Biological ; Phospholipids/metabolism ; Saccharomyces cerevisiae/growth & development ; Saccharomyces cerevisiae/metabolism
Chemical Substances	Lipid Bilayers ; Phospholipids ; Ergosterol (Z30RAY509F)
Language	English
Publishing date	2019-08-15
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2156462-0
ISSN	1742-4682 ; 1742-4682
ISSN (online)	1742-4682
ISSN	1742-4682
DOI	10.1186/s12976-019-0108-2
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Article ; Online: Dynamic Mode Decomposition of Multiphoton and Stimulated Emission Depletion Microscopy Data for Analysis of Fluorescent Probes in Cellular Membranes.

Wüstner, Daniel / Egebjerg, Jacob Marcus / Lauritsen, Line

Sensors (Basel, Switzerland)

2024 Volume 24, Issue 7

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Fluorescent Dyes ; Microscopy ; Cell Membrane ; Cholesterol ; Lipids
Chemical Substances	Fluorescent Dyes ; Cholesterol (97C5T2UQ7J) ; Lipids
Language	English
Publishing date	2024-03-25
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2052857-7
ISSN	1424-8220 ; 1424-8220
ISSN (online)	1424-8220
ISSN	1424-8220
DOI	10.3390/s24072096
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Article: Pathways and Mechanisms of Cellular Cholesterol Efflux-Insight From Imaging.

Juhl, Alice Dupont / Wüstner, Daniel

Frontiers in cell and developmental biology

2022 Volume 10, Page(s) 834408

Abstract: 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 ... ...

Abstract	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. Attention is paid to the biophysical principles governing efflux of sterols from cells. We also discuss recent evidence for cholesterol efflux by the release of exosomes, microvesicles, and migrasomes. The role of the endo-lysosomal network, lipophagy, and selected lysosomal transporters, such as Niemann Pick type C proteins in cholesterol export from cells is elucidated. Since oxysterols are important regulators of cellular cholesterol efflux, their formation, trafficking, and secretion are described briefly. In addition to discussing results obtained with traditional biochemical methods, focus is on studies that use established and novel bioimaging approaches to obtain insight into cholesterol efflux pathways, including fluorescence and electron microscopy, atomic force microscopy, X-ray tomography as well as mass spectrometry imaging.
Language	English
Publishing date	2022-03-01
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2022.834408
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Article ; Online: Endogenous Tagging of Ciliary Genes in Human RPE1 Cells for Live-Cell Imaging.

Kuhns, Stefanie / Juhl, Alice Dupont / Anvarian, Zeinab / Wüstner, Daniel / Pedersen, Lotte B / Andersen, Jens S

Methods in molecular biology (Clifton, N.J.)

2023 Volume 2725, Page(s) 147–166

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Humans ; Protein Transport/genetics ; Cilia/metabolism ; Proteins/metabolism ; Biological Transport
Chemical Substances	Proteins
Language	English
Publishing date	2023-10-19
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-3507-0_9
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Article ; Online: Conformational changes in the Niemann-Pick type C1 protein NCR1 drive sterol translocation.

Frain, Kelly M / Dedic, Emil / Nel, Lynette / Bohush, Anastasiia / Olesen, Esben / Thaysen, Katja / Wüstner, Daniel / Stokes, David L / Pedersen, Bjørn Panyella

Proceedings of the National Academy of Sciences of the United States of America

2024 Volume 121, Issue 15, Page(s) e2315575121

Abstract: The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes. ...

Abstract	The membrane protein Niemann-Pick type C1 (NPC1, named NCR1 in yeast) is central to sterol homeostasis in eukaryotes.
MeSH term(s)	Sterols/metabolism ; Saccharomyces cerevisiae/genetics ; Saccharomyces cerevisiae/metabolism ; Carrier Proteins/metabolism ; Natural Cytotoxicity Triggering Receptor 1/metabolism ; Niemann-Pick C1 Protein/metabolism ; Membrane Glycoproteins/metabolism
Chemical Substances	Sterols ; Carrier Proteins ; Natural Cytotoxicity Triggering Receptor 1 ; Niemann-Pick C1 Protein ; Membrane Glycoproteins
Language	English
Publishing date	2024-04-03
Publishing country	United States
Document type	Journal Article
ZDB-ID	209104-5
ISSN	1091-6490 ; 0027-8424
ISSN (online)	1091-6490
ISSN	0027-8424
DOI	10.1073/pnas.2315575121
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Zs.A 1148: Show issues

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Jg. 1995 - 2021: Lesesall (1.OG)
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Article ; Online: Cholesterol binding to the sterol-sensing region of Niemann Pick C1 protein confines dynamics of its N-terminal domain.

Dubey, Vikas / Bozorg, Behruz / Wüstner, Daniel / Khandelia, Himanshu

PLoS computational biology

2020 Volume 16, Issue 10, Page(s) e1007554

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Cholesterol/chemistry ; Cholesterol/metabolism ; Humans ; Intracellular Signaling Peptides and Proteins/chemistry ; Intracellular Signaling Peptides and Proteins/metabolism ; Lysosomes/metabolism ; Models, Molecular ; Niemann-Pick Disease, Type C ; Protein Binding ; Protein Domains
Chemical Substances	Intracellular Signaling Peptides and Proteins ; NPC1 protein, human ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2020-10-06
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2193340-6
ISSN	1553-7358 ; 1553-734X
ISSN (online)	1553-7358
ISSN	1553-734X
DOI	10.1371/journal.pcbi.1007554
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Article ; Online: Modeling the Sterol-Binding Domain of Aster-A Provides Insight into Its Multiligand Specificity.

Moesgaard, Laust / Reinholdt, Peter / Wüstner, Daniel / Kongsted, Jacob

Journal of chemical information and modeling

2020 Volume 60, Issue 4, Page(s) 2268–2281

Abstract: 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 ... ...

Abstract	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.
MeSH term(s)	Cell Membrane/metabolism ; Cholesterol/metabolism ; Endoplasmic Reticulum/metabolism ; Protein Binding ; Sterols/metabolism
Chemical Substances	Sterols ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2020-04-10
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	190019-5
ISSN	1549-960X ; 0095-2338
ISSN (online)	1549-960X
ISSN	0095-2338
DOI	10.1021/acs.jcim.0c00086
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Zs.A 1230: Show issues

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Article ; Online: Following intracellular cholesterol transport by linear and non-linear optical microscopy of intrinsically fluorescent sterols.

Wüstner, Daniel

Current pharmaceutical biotechnology

2010 Volume 13, Issue 2, Page(s) 303–318

Abstract: Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the ... ...

Abstract	Elucidation of intracellular cholesterol transport is important for understanding the molecular basis of several metabolic and neuronal diseases, like atheroclerosis or lysosomal storage disorders. Progress in this field depends crucially on the development of new technical approaches to follow the cellular movement of this essential lipid molecule. In this article, a survey of the various methods being used for analysis of sterol trafficking is given. Various classical biochemical methods are presented and their suitability for analysis of sterol trafficking is assessed. Special emphasis is on recent developments in imaging technology to follow the intracellular fate of intrinsically fluorescent sterols as faithful cholesterol markers. In particular, UV-sensitive wide field and multiphoton microscopy of the sterol dehydroergosterol, DHE, is explained and new methods of quantitative image analysis like pixel-wise bleach rate fitting and multiphoton image correlation spectroscopy are introduced. Several applications of the new technology including observation of vectorial sterol trafficking in polarized human hepatoma cells for investigation of reverse cholesterol transport are presented.
MeSH term(s)	Animals ; Biological Transport/physiology ; Carcinoma, Hepatocellular/chemistry ; Carcinoma, Hepatocellular/metabolism ; Cholesterol/metabolism ; Fluorescent Dyes/chemistry ; Fluorescent Dyes/metabolism ; Humans ; Liver Neoplasms/chemistry ; Liver Neoplasms/metabolism ; Microscopy, Fluorescence/methods ; Sterols/chemistry ; Sterols/metabolism
Chemical Substances	Fluorescent Dyes ; Sterols ; Cholesterol (97C5T2UQ7J)
Language	English
Publishing date	2010-11-25
Publishing country	Netherlands
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	2132197-8
ISSN	1873-4316 ; 1389-2010
ISSN (online)	1873-4316
ISSN	1389-2010
DOI	10.2174/138920112799095301
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Zs.A 6212: Show issues

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