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2013 | Buch

Fluorescent Methods to Study Biological Membranes

herausgegeben von: Yves Mély, Guy Duportail

Verlag: Springer Berlin Heidelberg

Buchreihe : Springer Series on Fluorescence

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SUCHEN

Über dieses Buch

Biological membranes play a central role in cell structure, shape and functions. However, investigating the membrane bilayer has proved to be difficult due to its highly dynamic and anisotropic structure, which generates steep gradients at the nanometer scale. Due to the decisive impact of recently developed fluorescence-based techniques, tremendous advances have been made in the last few years in our understanding of membrane characteristics and functions. In this context, the present book illustrates some of these major advances by collecting review articles written by highly respected experts. The book is organized in three parts, the first of which deals with membrane probes and model membranes. The second part describes the use of advanced quantitative and high-resolution techniques to explore the properties of biological membranes, illustrating the key progress made regarding membrane organization, dynamics and interactions. The third part is focused on the investigation of membrane proteins using the same techniques, and notably on the membrane receptors that play a central role in signaling pathways and therapeutic strategies. All chapters provide comprehensive information on membranes and their exploration for beginners in the field and advanced researchers alike.

Inhaltsverzeichnis

Frontmatter
LAURDAN Fluorescence Properties in Membranes: A Journey from the Fluorometer to the Microscope
Abstract
After 32 years since its introduction, the particular fluorescence properties of 6-lauroyl-2-(dimethylamino)-naphtalene (LAURDAN) in model and biological membranes are revisited. This review includes a historical perspective about the design, synthesis and initial description of the probe’s fluorescent properties, a discussion about the proposed mechanism of LAURDAN sensitivity to membrane lateral packing, and a detailed description of the definition of the Generalized Polarization function. This article includes as well examples of the different experimental strategies involving LAURDAN in model and biological membranes, using both bulk fluorescence spectroscopy measurements and spatially resolved information from fluorescence microscopy. The value of this probe in the study of membrane structure and dynamics is reflected in more than 330 papers reported in the existing literature.
L. A. Bagatolli
Application of NBD-Labeled Lipids in Membrane and Cell Biology
Abstract
The fluorescent NBD group has come a long way in terms of biological applications since its discovery a few decades back. Although the field of fluorescently labeled lipids has grown over the years with the introduction of new fluorescent labels, NBD-labeled lipids continue to be a popular choice in membrane and cell biological studies due to desirable fluorescence characteristics of the NBD group. In this chapter, we discuss the application of NBD-labeled lipids in membrane and cell biology taking representative examples with specific focus on the biophysical basis underlying such applications.
Sourav Haldar, Amitabha Chattopadhyay
3-Hydroxychromone Probes Precisely Located and Oriented in Lipid Bilayers: A Toolkit for Biomembrane Research
Abstract
Environment-sensitive dyes due to the sensitivity of their spectra to the physicochemical properties of their environment are unique tools for probing model and biological membranes. Here, we describe a particular class of environment-sensitive dyes based on 3-hydroxychromones. These dyes exhibit excited-state intramolecular proton transfer resulting in dual emission, highly sensitive to environment polarity and hydration. Appropriate molecular design of the new probes allows precise localization and orientation of their fluorophore in the lipid bilayers, which confer high specificity to particular membrane properties. In this respect, interface localization of the probes allows monitoring lipid order, while vertical orientation is required to achieve sensitivity to dipole and transmembrane potentials. Finally, biological applications of these probes for sensing lipid domains (rafts) and apoptosis are shown.
Andrey S. Klymchenko, Guy Duportail, Yves Mély
Lateral Membrane Heterogeneity Probed by FRET Spectroscopy and Microscopy
Abstract
Förster resonance energy transfer (FRET) is a photophysical process highly dependent on interchromophore distance. Due to this feature, it is very sensitive to membrane lateral heterogeneity, as the donor and acceptor fluorophores involved in FRET tend to have different preference for distinct types of lipid bilayer domains. In this chapter, the basic formalisms of FRET in situations of increasing complexity (from a single donor-acceptor pair at a fixed distance to non-random probe distribution) are presented and illustrated with selected examples from the literature. The importance of time-resolved fluorescence data is emphasized. It is shown that FRET can be used to study the occurrence of domain formation, allowing their detection as well as size estimation. Lateral lipid distribution heterogeneity may also result from peptide- or protein-lipid interaction. Formalisms that apply to these situations are also presented, as well as selected examples of their use. Applications of FRET under the microscope have recently come to the fore, and representative studies are mentioned.
Luís M. S. Loura, Manuel Prieto
FRET Analysis of Protein-Lipid Interactions
Abstract
Förster resonance energy transfer (FRET) is an old but constantly developing spectroscopic tool possessing enormous potential for studies on structure and dynamics of biological macromolecules and their assemblies. One of the main advantages of FRET technique is the possibility of measuring the nanometer-scale distances between donor and acceptor fluorophores. This chapter highlights some aspects of FRET-based monitoring of intermolecular interactions in membrane systems. Analytical model of energy transfer between membrane-associated donors and acceptors randomly distributed over parallel planes separated by a fixed distance is presented. The factors determining the efficiency of energy transfer are considered with special attention to orientational behavior of the donor emission and acceptor absorption transition dipoles. It is demonstrated that FRET can provide proof for specific orientation of the protein molecule relative to lipid-water interface. The applications of FRET to quantification of protein-lipid binding parameters and membrane position of protein fluorophores are exemplified. It is illustrated how FRET may help in obtaining evidence for protein aggregation in a membrane environment and domain formation.
Galyna Gorbenko, Paavo K. J. Kinnunen
Hydration and Mobility in Lipid Bilayers Probed by Time-Dependent Fluorescence Shift
Abstract
Biological membranes as an indispensable part of living organisms are permanently surrounded by the molecules of water. The presence of water is essential for maintaining their structure and functionality. Therefore, lipid bilayer hydration, mobility of the hydrated lipids, and their changes upon perturbations are appealing characteristics in the lipid membrane research. Time-dependent fluorescent shift (TDFS) measurements enable probing these properties in biologically relevant fully hydrated liquid crystalline lipid bilayers with a simple instrumentation and easy data treatment. Since the native lipid molecules do not fluoresce naturally, the extrinsic probing with a suitable fluorescent dye is necessary. There are a number of fluorescent membrane polarity probes designed for this purpose with different spectral properties and locations within the lipid bilayer. The basics of the technique are explained together with some useful additional considerations. The convenience of the TDFS method is demonstrated with examples from recent research on the study of the interactions of ions with lipid bilayers, and the monitoring of mobility and hydration changes along the bilayer normal upon addition of the oxidized phospholipids.
Sarka Pokorna, Agnieszka Olżyńska, Piotr Jurkiewicz, Martin Hof
Visual Discrimination of Membrane Domains in Live Cells by Widefield Microscopy
Abstract
Membrane dynamics is a fast-evolving field with the many new methods and probes being developed each year affording ever increased insights into how membranes behave in the laboratory. Typically, these developments are first tested in model membranes using high-cost, bespoke microscopes which often employ confocal and two-photon systems and which give little consideration to preservation of cellular integrity and homeostasis during experiments. This chapter addresses the clear need to rapidly apply and deploy this work into mainstream biological laboratories by development of economical, four-dimensional imaging on user-friendly low-cost systems using widefield optics and simultaneous capture of multiple fluorescent markers. Such systems are enabling biologists to consider the coordinated processes triggered from signalling platforms during cellular interaction with the environment. In this chapter, we describe the progress made to date and in particular we focus on the Laurdan family of fluorescent probes, which are being used to image whole cells and tissues using widefield epifluorescence microscopy and which can be usefully combined with simultaneous capture at longer wavelengths (yellow through far red) for imaging of cell morphology or for following expressed markers such as fluorescent adaptor proteins.
Claire E. Butler, Guy Wheeler, Jeremy Graham, Kevin M. Tyler
Quantitative Fluorescence Studies of Intracellular Sterol Transport and Distribution
Abstract
Unraveling the pathways of intracellular cholesterol transport is of great importance for biomedicine, since disturbed cholesterol trafficking is involved in many metabolic diseases. Most fluorescent probes for cholesterol, however, have physico-chemical properties deviating from the natural sterol. Intrinsically fluorescent sterols like dehydroergosterol (DHE) and the related cholestatrienol (CTL) have great potential for analysis of sterol trafficking due to their close resemblance of ergosterol and cholesterol, respectively. Excitation and emission of both sterols are in the ultraviolet (UV), which, together with high bleaching propensity and low brightness, make fluorescence imaging of DHE and CTL challenging. Here, we present an overview of how UV-sensitive wide field (UV-WF) and multiphoton (MP) microscopy can be applied to image both sterols in living cells and tissues. In addition, we show, for the first time, how applying advanced image denoising can dramatically enhance the signal-to-noise ratio in MP image sequences of DHE. This allowed us to track DHE-containing vesicles and surface protrusions in cells over prolonged time. We also discuss the properties of BODIPY-tagged cholesterol (BChol) compared to DHE and cholesterol and present an overview of fluorescence imaging techniques for analyzing cellular sterol dynamics.
Daniel Wüstner, Frederik W. Lund, Lukasz M. Solanko
Studying Membrane Properties Using Fluorescence Lifetime Imaging Microscopy (FLIM)
Abstract
Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to investigate the structure and composition of biological membranes. A wide variety of fluorescent probes suitable for FLIM experiments have been described. These compounds differ strongly in the details of their incorporation into membranes and in their responses toward changes in the membrane composition. In this chapter, we discuss and compare different classes of fluorescent membranes probes and their applications to studying biological membranes. We devote a section to a detailed description of fluorescent molecular rotors and their application to measuring local viscosity. As Förster resonance energy transfer (FRET) can be directly measured by changes in the donor fluorescence lifetime, FLIM is a very robust method to determine the distances between FRET pairs or the local concentrations of FRET-based membrane probes. Thus, we also discuss advantages and challenges of FRET-FLIM in the context of biological membranes. As biological membranes are considerably dynamic systems, imaging speed is often the limiting factor in biological FLIM experiments. Thus, novel fast imaging approaches and analysis methods to alleviate the issue of low photon statistics are also presented.
Graphical Abstract
Martin T. Stöckl, Ranieri Bizzarri, Vinod Subramaniam
Fluorescence Correlation Spectroscopy to Study Membrane Organization and Interactions
Abstract
This chapter describes the application of fluorescence correlation spectroscopy (FCS) as a powerful technique for the study of membrane organization and interactions. Monitoring the fluorescence signal fluctuations allows resolving concentrations, diffusion coefficients, and binding of several membrane components in experiments in vitro as well as in vivo.
We discuss the basic principles of FCS and explain novel implementations of FCS introduced to overcome the technical difficulties present in the standard version of fluorescence correlation spectroscopy. Finally, we report several examples of studies with the application of FCS on both model and biological membranes to obtain interesting insight in the topic of lateral membrane organization and membrane interactions.
Monika Zelman-Femiak, Yamunadevi Subburaj, Ana J. García-Sáez
Deciphering Cell Membrane Organization Based on Lateral Diffusion Measurements by Fluorescence Correlation Spectroscopy at Different Length Scales
Abstract
The plasma membrane delineating the cell is a complex multicomponent assembly of lipids and proteins. Deciphering the lateral organization of this supramolecular complex on appropriate length and temporal scales is necessary to unravel its implication in biological function. Here, we show how measurements of diffusion may be taken to shed light on membrane function. We first describe the current methods used to report lateral diffusion of membrane components. We then focus on one fluorescent correlation spectroscopy (FCS)-based method, namely, the spot variation FCS (svFCS), which allows the characterization of the modes of molecular confinement within the plasma membrane of living cells. We next illustrate with different biological systems the progress made toward improving our understanding of cell membrane function. We also discuss the findings with regard to the current view of nanoscale domains/assemblies as a general membrane-organizing principle.
Vincent Rouger, Cyrille Billaudeau, Tomasz Trombik, Sébastien Mailfert, Yannick Hamon, Hai-Tao He, Didier Marguet
STED-FCS Nanoscopy of Membrane Dynamics
Abstract
Lipid-lipid and lipid-protein interactions such as the formation of lipid nanodomains (often denoted “rafts”) are considered to play a functional part in a whole range of membrane-associated processes. However, their direct and noninvasive observation in living cells is impeded by the resolution limit of >200 nm of a conventional far-field optical microscope. With the superior spatial resolution of STED nanoscopy, it is now possible to directly resolve nanoscale membrane organization. While direct imaging of membrane heterogeneities turns challenging due to their fast dynamics, the combination of STED nanoscopy with tools such as fluorescence correlation spectroscopy (FCS) allows the disclosure of complex nanoscopic dynamical processes. By performing FCS measurements in observation spots tuned to a diameter of down to 30 nm, new details of molecular membrane dynamics have been obtained: Unlike fluorescent phosphoglycerolipids, fluorescent sphingolipids are transiently (~10 ms) trapped on the nanoscale in often cholesterol- and cytoskeleton-assisted molecular complexes. These interactions are distinct for different lipids and may play an important role in cellular functionality. Comparison of the trapping characteristics to the organization of the different fluorescent lipid analogs in model membranes reveals details of the role of lipid “rafts”. This chapter reviews how STED-FCS may shed new light on the role of lipid-protein interactions and nanodomains for membrane bioactivity.
Christian Eggeling
Imaging Molecular Order in Cell Membranes by Polarization-Resolved Fluorescence Microscopy
Abstract
The use of light polarization properties in the analysis of fluorescence images has driven a large amount of research toward the measurement of orientational behavior of molecules in cells, in particular in their membranes. This field has been recently revisited to enlarge the possibilities of polarization-resolved fluorescence microscopy. We show that this technique allows retrieving a wealth of information on the constraints that hinder rotational mobility of lipid probes and proteins in membranes, bringing thus new insights on inter-proteins and lipid-protein interactions, on membrane morphology at the sub-diffraction length scale and on local membrane physical properties such as viscosity.
Sophie Brasselet, Patrick Ferrand, Alla Kress, Xiao Wang, Hubert Ranchon, Alicja Gasecka
Near-Field Optical Nanoscopy of Biological Membranes
Abstract
The specific organization and distribution of protein receptors and lipids on the cellular plasma membrane play a crucial role for the spatiotemporal control of many different cellular processes. A great deal of novel knowledge in this area is currently being generated thanks to the advent of modern surperresolution optical techniques combined with single-molecule approaches. In this chapter, we focus on near-field nanoscopy, a technique particularly well suited for the study of biological cell surfaces at the nanometer scale. We first describe the general concept of near-field scanning optical microscopy (NSOM) and specifically focus on how NSOM is being exploited to map the spatiotemporal organization of proteins and lipids. Novel routes toward surperresolution using optical nanoantennas and first applications for cell membrane nanoimaging are discussed. The last part of the chapter describes recent technical breakthroughs that enable the application of NSOM in living cells providing detailed dynamic information on diffusion processes occurring at the nanoscale.
Thomas S. van Zanten, Carlo Manzo, Maria F. Garcia-Parajo
Unveiling Biophysical and Biological Properties of a Hypothetical Membrane Receptor by Exploiting Recent Imaging Advances
Abstract
Fluorescence microscopy is indispensable in the study of biological systems at various length scales. This rapidly evolving field continues to offer researchers cutting-edge techniques that enhance spatial and temporal resolution, especially with the invention of superresolution methodologies. In this chapter, we focus on techniques that have aided in the understanding of various cell biological phenomena. Each technique has certain boundaries of spatial and temporal resolution, and fluorophore density in a particular biological sample may limit the applicability of some techniques. We discuss strengths and weaknesses of many such techniques by considering their use in understanding the biological function of a hypothetical membrane receptor. We conclude that a combination of techniques is required to fully understand any cell biological process.
Pauline Gonnord, Rajat Varma
New Fluorescent Strategies Shine Light on the Evolving Concept of GPCR Oligomerization
Abstract
GPCR oligomerization has been a matter of intense research these last years. FRET and BRET methods have paved the way to a generalized concept of potential GPCR oligomerization in artificial systems (transfected cell lines). More recently, the use of fluorescent ligands compatible with time-resolved FRET studies has opened the possibility of GPCR oligomerization study in their native context and brought evidence of their existence. Furthermore, recent applications of original fluorescence techniques are unveiling new information on the dynamics that govern these complexes and are changing the way we see GPCR oligomeric structures.
Martin Cottet, Orestis Faklaris, Eric Trinquet, Jean-Philippe Pin, Thierry Durroux
Application of Quantitative Fluorescence Microscopic Approaches to Monitor Organization and Dynamics of the Serotonin1A Receptor
Abstract
G protein-coupled receptors (GPCRs) are the largest class of molecules involved in signal transduction across membranes and represent major targets in the development of novel drug candidates in all clinical areas. Recent advances in understanding of the nonrandom distribution of GPCRs, G proteins, and effector molecules have given rise to new challenges and complexities in cellular signaling by GPCRs. In this article, we provide specific examples on the application of quantitative fluorescence microscopic approaches to monitor organization and dynamics of the serotonin1A receptor (a GPCR) in live cells. This assumes broader relevance due to the emerging theme that GPCR function depends on its organization and dynamics. We envisage that with progress in understanding of receptor organization and dynamics, our knowledge of GPCR function would improve considerably, thereby enabling to design better therapeutic strategies to combat diseases related to malfunctioning of GPCRs.
Md. Jafurulla, Amitabha Chattopadhyay
TNF Receptor Membrane Dynamics Studied with Fluorescence Microscopy and Spectroscopy
Abstract
Sensitive fluorescence techniques opened novel opportunities to study the function and interaction of proteins in living cells. Here, we review the contribution of fluorescence correlation spectroscopy (FCS) and single-molecule tracking to study the dynamics of TNF receptor 1 and 2 (TNFR1/2). Although these techniques greatly differ, both report a similar behavior of TNF receptor (TNFR) species in Hela cells under different experimental conditions. FCS as well as single-molecule tracking revealed an increase of the diffusion coefficient of TNFR1 after treating cells with methyl-cyclodextrin.
In addition, FCS studies of the activation of TNFR1 showed that ligand binding hardly affects its diffusion coefficient.
In contrast, unstimulated TNFR2 was observed to diffuse faster than TNFR1, whereas ligand stimulation of TNFR2 decreases the diffusion coefficient.
In conclusion, the results indicate that the two TNFRs compartmentalize in distinct domains of the plasma membrane most likely determined by the respective transmembrane domains and/or transmembrane domain near regions.
Felix Neugart, Darius Widera, Barbara Kaltschmidt, Christian Kaltschmidt, Mike Heilemann
Backmatter
Metadaten
Titel
Fluorescent Methods to Study Biological Membranes
herausgegeben von
Yves Mély
Guy Duportail
Copyright-Jahr
2013
Verlag
Springer Berlin Heidelberg
Electronic ISBN
978-3-642-33128-2
Print ISBN
978-3-642-33127-5
DOI
https://doi.org/10.1007/978-3-642-33128-2

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