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BBA - Biomembranes (v.1798, #7)
The liquid-ordered state comes of age by Ole G. Mouritsen (pp. 1286-1288).
Biomembranes are unique states of soft matter that share some of their materials properties with the mesophases of liquid crystals. Although of genuinely fluid character, membranes can display ordered states under physiological conditions, and it appears that their lateral organization and the related functional properties are intimately coupled to states in-between order and disorder. Hence, the liquid-ordered state of membranes, which owes its existence to the unique ability of cholesterol to mediate between order and disorder, has moved center stage in the characterization of membranes in terms of domains or rafts.
Keywords: Lipid bilayer; Liquid-ordered phase; Cholesterol; Lipid domains
Taking another look with fluorescence microscopy: Image processing techniques in Langmuir monolayers for the twenty-first century by Benjamin L. Stottrup; Andrew H. Nguyen; Tuzel Erkan Tüzel (pp. 1289-1300).
Fluorescence microscopy has become a powerful and standard complementary technique in the study of amphiphilic films at the air–water interface. For nearly three decades the coupling of traditional thermodynamic measurements with direct visualization has provided a better understanding of self-assembled Langmuir monolayers and their application in the study of the physical properties of membranes and interfaces. As an introduction we provide a brief overview of this established technique and demonstrate its continued utility in the recent observation of novel phase behavior in monolayers of 25-hydroxycholesterol (25-OH) and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC). We then focus our review on new analysis techniques which take advantage of the ability to store, process, and analyze large sets of images. We pay particular attention to efforts measuring the line tension between coexisting two dimensional fluid phases in the Langmuir monolayer. Using non-perturbative methods, we can measure fundamental mechanical properties of these two dimensional systems. Finally, we highlight the use of Model Convolution Microscopy as a new tool to provide insight on the experimental limits in these studies.
Keywords: Fluorescence microscopy; Langmuir film balance; Line tension; Model Convolution Microscopy; Lipid; Cholesterol; Image processing
Multiphoton excitation fluorescence microscopy in planar membrane systems by Jonathan Brewer; Jorge Bernardino de la Serna; Kerstin Wagner; Luis A. Bagatolli (pp. 1301-1308).
The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air–water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when “classical” fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26±2mN/m and 28±3mN/m for DOPC and DPPC, respectively.
Keywords: Langmuir films; LAURDAN; Two-photon excitation microscopy; Planar supported membranes
The action of sphingomyelinase in lipid monolayers as revealed by microscopic image analysis by Maria Laura Fanani; Steffen Hartel; Bruno Maggio; Luisina De Tullio; Jorge Jara; Felipe Olmos; Rafael Gustavo Oliveira (pp. 1309-1323).
In recent years, new evidence in biomembrane research brought about a holistic, supramolecular view on membrane-mediated signal transduction. The consequences of sphingomyelinase (SMase)-driven formation of ceramide (Cer) at the membrane interface involves reorganization of the lateral membrane structure of lipids and proteins from the nm to the μm level. In this review, we present recent insights about mechanisms and features of the SMase-mediated formation of Cer-enriched domains in model membranes, which have been elucidated through a combination of microscopic techniques with advanced image processing algorithms. This approach extracts subtle morphological and pattern information beyond the visual perception: since domain patterns are the consequences of subjacent biophysical properties, a reliable quantitative description of the supramolecular structure of the membrane domains yields a direct readout of biophysical properties which are difficult to determine otherwise. Most of the information about SMase action on simple lipid interfaces has arisen from monolayer studies, but the correspondence to lipid bilayer systems will also be discussed. Furthermore, the structural changes induced by sphingomyelinase action are not fully explained just by the presence of ceramide but by out-of equilibrium surface dynamics forcing the lipid domains to adopt transient supramolecular pattern with explicit interaction potentials. This rearrangement responds to a few basic physical properties like lipid mixing/demixing kinetics, electrostatic repulsion and line tension. The possible implications of such transient codes for signal transduction are discussed for SMase controlled action on lipid interfaces.
Keywords: Abbreviations; SMase; Sphingomyelinase; PLA; 2; phospholipase A; 2; Cer; N-acylsphingosine; SM; sphingomyelin; chol; cholesterol; GUVs; giant unilamellar vesicles; ROIs; Regions of Interest; hgd; Highest Gap Distance; GGVF; generalized gradient vector flow; LE; liquid-expanded; LC; liquid-condensed; ld; liquid-disordered; lo; liquid-ordered; P; 2; /A; perimeter; 2; /area; AFM; Atomic Force MicroscopySphingomyelin; Ceramide; Ceramide-enriched domain; Epifluorescence microscopy; Microscopic image processing; Active contours
GUV preparation and imaging: Minimizing artifacts by Nelson F. Morales-Penningston; Jing Wu; Elaine R. Farkas; Shih Lin Goh; Tatyana M. Konyakhina; Judy Y. Zheng; Watt W. Webb; Gerald W. Feigenson (pp. 1324-1332).
The components of biological membranes are present in a physical mixture. The nonrandom ways that the molecules of lipids and proteins mix together can strongly influence the association of proteins with each other, and the chemical reactions that occur in the membrane, or that are mediated by the membrane. A particular type of nonrandom mixing is the separation of compositionally distinct phases. Any such phase separation would result in preferential partition of some proteins and lipids between the coexisting phases, and thus would influence which proteins could be in contact, and whether a protein could find its target. Phase separation in a plasma membrane would also influence the binding of molecules from outside the cell to the membrane, including recognition proteins on viruses, bacteria, and other cells. The concept of these and other events associated with membrane phase separation are sometimes grouped together as the “raft model” of biological membranes. Several types of experiments are aimed at detecting and characterizing membrane phase separation. Visualizing phase separation has special value, both because the immiscibility is so decisively determined, and also because the type of phase can often be identified. The fluorescence microscope has proven uniquely useful for yielding images of separated phases, both in certain cell preparations, and especially in models of cell membranes. Here we discuss ways to prepare useful model membranes for image studies, and how to avoid some of the artifacts that can plague these studies.
Keywords: Abbreviations; GUV; giant unilamellar vesicle; PG; phosphatidylglycerol; PS; phosphatidylserine; PC; phosphatidylcholine; DSPC; 1,2-Distearoyl-; sn; -Glycero-3-Phosphocholine; SM; sphingomyelin; POPC; 1-Palmitoyl-2-Oleoyl-; sn; -Glycero-3-Phosphocholine; EDTA; ethylenediaminetetraacetic acid; ITO; indium tin oxide; DOPC; 1,2-Dioleoyl-; sn; -Glycero-3-Phosphocholine; L; d; liquid-disordered; L; o; liquid-ordered; SOPC; 1-Stearoyl-2-Oleoyl-; sn; -Glycero-3-Phosphocholine; chol; cholesterol; TLC; Thin layer chromatography; C12:0-DiI; 1,1'-didodecanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; C18:0-DiI; 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; C20:0-DiI; 1,1'-dieicosanyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate; (16:0,Bodipy-PC); 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-; s; -indacene-3-pentanoyl)-1-hexadecanoyl -; sn; -glycero-3-phosphocholine; TR-DHPE; Texas Red 1,2-dihexadecanoyl-sn-glycerol-3-phosphoethanolamine; PAH; polycyclic aromatic hydrocarbons; LR-DPPE; Lissamine Rhodamine 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine; NPG; n-propyl gallate; b-SM; brain sphingomyelinLight-induced; Lipid peroxidation; Phase boundary shift; Giant unilamellar vesicle; Gentle hydration; Electroformation; Electrode; ITO; Complementary dye; Budding vesicle
Impact of membrane-anchored fluorescent probes on the mechanical properties of lipid bilayers by Hélène Bouvrais; Tanja Pott; Luis A. Bagatolli; John H. Ipsen; Meleard Philippe Méléard (pp. 1333-1337).
Fluorescent probes are used in membrane biophysics studies to provide information about physical properties such as lipid packing, polarity and lipid diffusion or to visualize membrane domains. However, our understanding of the effects the dyes themselves may induce on the membrane structure and properties are sparse. As mechanical properties like bending elasticity were already shown to be highly sensitive to the addition of “impurities” into the membranes, we have investigated the impact of six different commonly used fluorescent membrane probes (LAURDAN, TR-DPPE, Rh-DPPE, DiIC18, Bodipy-PC and NBD-PC) on the bending elasticity of dye containing POPC GUVs as compared to single component POPC GUVs. Small changes in the membrane bending elasticity compared to single POPC bilayers are observed when 2mol% of Rh-DPPE, Bodipy-PC or NBD-PC are added in POPC membranes. These binary membranes are showing non reproducible mechanical properties attributed to a photo-induced peroxidation processes that may be controlled by a reduction of the fluorescent dye concentration. For TR-DPPE, a measurable decrease of the bending elasticity is detected with reproducible bending elasticity measurements. This is a direct indication that this dye, when exposed to illumination by a microscope lamp and contrary to Rh-DPPE, does not induce chemical degradation. At last, LAURDAN and DiIC18 probes mixed with POPC do not significantly affect the bending elasticity of pure POPC bilayers, even at 2mol%, suggesting these latter probes do not induce major perturbations on the structure of POPC bilayers.
Keywords: Lipid bilayer; Vesicle fluctuations analysis; Membrane mechanics; Fluorescent probes; Bending elasticity; GUV
Vesicles with charged domains by Cíntia C. Vequi-Suplicy; Karin A. Riske; Roland L. Knorr; Rumiana Dimova (pp. 1338-1347).
This work summarizes results obtained on membranes composed of the ternary mixture dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM) and cholesterol (Chol). The membrane phase state as a function of composition is characterized from data collected with fluorescence microscopy on giant unilamellar vesicles. The results suggest that the presence of the charged DOPG significantly decreases the composition region of coexistence of liquid ordered and liquid disordered phases as compared to that in the ternary mixture of dioleoylphosphatidycholine, sphingomyelin and cholesterol. The addition of calcium chloride to DOPG:eSM:Chol vesicles, and to a lesser extent the addition of sodium chloride, leads to the stabilization of the two-phase coexistence region, which is expressed in an increase in the miscibility temperature. On the other hand, addition of the chelating agent EDTA has the opposite effect, suggesting that impurities of divalent cations in preparations of giant vesicles contribute to the stabilization of charged domains. We also explore the behavior of these membranes in the presence of extruded unilamellar vesicles made of the positively charged lipid dioleoyltrimethylammoniumpropane (DOTAP). The latter can induce domain formation in DOPG:eSM:Chol vesicles with initial composition in the one-phase region.
Keywords: Giant vesicle; Confocal microscopy; Phase separation; Lipid raft; Membrane domain; Charged lipid; DOPG; Egg sphingomyelin; DOTAP
Ceramide: From lateral segregation to mechanical stress by Lopez-Montero Iván López-Montero; Francisco Monroy; Velez Marisela Vélez; Philippe F. Devaux (pp. 1348-1356).
Ceramide is a sphingolipid present in eukaryotic cells that laterally segregates into solid domains in model lipid membranes. Imaging has provided a wealth of structural information useful to understand some of the physical properties of these domains. In biological membranes, ceramide is formed on one of the membrane leaflets by enzymatic cleavage of sphyngomyelin. Ceramide, with a smaller head size than its parent compound sphyngomyelin, induces an asymmetric membrane tension and segregates into highly ordered domains that have a much high shear viscosity than that of the surrounding lipids. These physical properties, together with the rapid transmembrane flip-flop of the locally produced ceramide, trigger a sequence of membrane perturbations that could explain the molecular mechanism by which ceramide mediates different cell responses. In this review we will try to establish a connection between the physical membrane transformations in model systems known to occur upon ceramide formation and some physiologically relevant process in which ceramide is known to participate.
Keywords: Abbreviations; Chol; cholesterol; Cer; ceramide; C2Cer; acetoyl-Cer; C6Cer; hexanoyl-Cer; C8Cer; octanoyl-Cer; C10Cer; decanoyl-Cer; C12Cer; lauroyl-Cer; C14Cer; myristoyl-Cer; C16Cer; palmitoyl-Cer; C18Cer; stearoyl-Cer; C20Cer; arachidoyl-Cer; C24Cer; lignoceroyl-Cer; C24:1Cer; nervonoyl-Cer; ECer; Egg-Cer; PC; phosphatidylcholine; DMPC; 1,2-dimyristoyl-PC; DPPC; dipalmitoyl-PC; POPC; 1-palmitoyl-2-oleoyl-PC; DOPC; 1,2-dioleoyl-PC; SOPC; 1-stearoyl-1-oleyl-PC; EPC; Egg-PC; SM; sphingomyelin; C16SM; palmitoyl-SM; C18SM; stearoyl-SM; ESM; Egg-SM; BSM; brain-SM; PE; phosphatidyl ethanolamine; DPPE; dipalmitoyl-PE; DOPE; 1,2-dioleoyl-PE; EPE; Egg-PE; L; o; liquid-ordered phase; L; d; liquid-disordered phase; L; β; solid phase; Smase; sphingomyelinase; AFM; atomic force microscopy; FCS; fluorescence correlation spectroscopy; GUV; giant unilamellar vesicle; FITC; fluorescein isothiocyanate; Cy3; cyanine 3Ceramide; Sphingomyelinase; Mechanical stress; Lipid domains
Imaging cerebroside-rich domains for phase and shape characterization in binary and ternary mixtures by Marjorie L. Longo; Craig D. Blanchette (pp. 1357-1367).
The objective of this paper is to review phase behavior and shape characterization of cerebroside-rich domains in binary and ternary lipid bilayers, as obtained by microscopy techniques. These lipid mixtures provide a format to examine molecular (e.g. headgroup, tail unsaturation, and tail hydroxylation) and thermodynamic (e.g. temperature and mole percentages) factors that determine phase behavior, molecular partitioning, crystal/atomic scale structure, and microstructure/shape (particularly of phase-separated domains). Microscopy can provide excellent spatial (often with high resolution) characterization of cerebroside-rich domains (and their surroundings) to identify, describe or infer with high certainty these characteristics. In the introduction to this review we review briefly the molecular structure, phase behavior, and intermolecular interactions of cerebrosides, in comparison to ceramides and sphingomyelins and in some binary and biological systems. The bulk of the review is then devoted to microscopy investigations of cerebroside-rich domain microstructure and shape dynamics in binary and ternary (one component is cholesterol) systems. Quantitative and/or high-resolution microscopy techniques have been used to interrogate cerebroside-rich domains such as freeze-fracture electron microscopy, atomic force microscopy, imaging elipsometry, two-photon fluorescence microscopy, and LAURDAN generalized polarization in addition to the laboratory workhorse technique of epifluorescence microscopy that allows a quick often qualitative assessment of microstructure and dynamics. We particularly focus on the information these microscopy investigations have revealed with respect to phase behavior, cholesterol partitioning, domain shape, and determinants of domain shape.
Keywords: Membrane raft; AFM; GUV; Liquid-ordered; Myelin; HIV
Dynamics of lipid domain formation: Fluctuation analysis by Anna Celli; Enrico Gratton (pp. 1368-1376).
Scanning-fluctuation correlation spectroscopy was used to detect subresolution organizational fluctuations in the lipid liquid-crystalline phase for single lipid model systems. We used the fluorescent probe Laurdan which is sensitive to the amount of water in the membrane to show that there is a spatial heterogeneity on the scale of few pixels (the size of the pixel is 50 nm). We calculated the pixel variance of the GP function and we found that the variance has a peak at the phase transition for 3 different samples made of pure lipids. The pixel variance has an abrupt change at the phase transition of the membrane and then it slowly decreases at higher temperature. The relatively large variance of the GP indicates that the liquid phase of the membrane is quite heterogeneous even several degrees higher than the phase transition temperature. We interpreted this result as evidence of an underlying microscale structure of the membrane in which water is not uniformly distributed at the micron scale. Imaging of these microstructures shows that the pixels with different GP tend to concentrate in specific domains in the membrane. In the case of single lipid membrane, the statistical and fluctuation analysis of the GP data shows that even such simple lipid systems are capable of generating and maintaining stable structural and organizational heterogeneities.
Keywords: Abbreviations; DMPC; 1,2 di-myristoyl phosphatidyl choline; POPC; 1-palmitoyl-2-oleylphosphatidylcholine; DLPC; 1,2 di-lauroyl phosphatidyl choline; DOPC; 1,2 di-oleylphosphatidylcholine; DPPC; 1,2-dipalmitoylphatidylcholine; PSF; point spread functionLipid phase; Fluctuation spectroscopy; Laurdan GP; GUVs
Lipid diffusion in planar membranes investigated by fluorescence correlation spectroscopy by Machaň Radek Macháň; Martin Hof (pp. 1377-1391).
Investigation of lipid lateral mobility in biological membranes and their artificial models provides information on membrane dynamics and structure; methods based on optical microscopy are very convenient for such investigations. We focus on fluorescence correlation spectroscopy (FCS), explain its principles and review its state of the art versions such as 2-focus, Z-scan or scanning FCS, which overcome most artefacts of standard FCS (especially those resulting from the need for an external calibration) making it a reliable and versatile method. FCS is also compared to single particle tracking and fluorescence photobleaching recovery and the applicability and the limitations of the methods are briefly reviewed. We discuss several key questions of lateral mobility investigation in planar lipid membranes, namely the influence which membrane and aqueous phase composition (ionic strength and sugar content), choice of a fluorescent tracer molecule, frictional coupling between the two membrane leaflets and between membrane and solid support (in the case of supported membranes) or presence of membrane inhomogeneities has on the lateral mobility of lipids. The recent FCS studies addressing those questions are reviewed and possible explanations of eventual discrepancies are mentioned.
Keywords: Supported lipid bilayer; Giant unilamellar vesicle; Fluorescence recovery after photobleaching; Single particle tracking; Membrane domain
Protein–protein and protein–lipid interactions in domain-assembly: Lessons from giant unilamellar vesicles by Nicoletta Kahya (pp. 1392-1398).
Giant Unilamellar Vesicles (GUVs) provide a key model membrane system to study lipid–lipid and lipid–protein interactions, which are relevant to vital cellular processes, by (single-molecule) optical microscopy. Here, we review the work on reconstitution techniques for membrane proteins and other preparation methods for developing GUVs towards most suitable close-to-native membrane systems. Next, we present a few applications of protein-containing GUVs to study domain assembly and protein partitioning into raft-like domains.
Keywords: Abbreviations; SPT; single particle tracking; FCS; fluorescence correlation spectroscopy; FRET; Föster resonance energy transfer; GUVs; giant unilamellar vesicles; BLMs; black lipid membranes; BR; bacteriorhodopsin; DOPC; dioleoyl-phosphatidylcholine; SM; sphingomyelin (C18:0); APP; amyloid precursor protein; BACE; beta-site-amyloid cleaving enzyme; LUVs; large unilamellar vesicles; DRMs; detergent resistant membranes; PLAP; placental alkaline phosphataseGiant unilamellar vesicle; Lipid raft; Membrane protein reconstitution; Optical microscopy
Lipid packing determines protein–membrane interactions: Challenges for apolipoprotein A-I and high density lipoproteins by Sanchez Susana A. Sánchez; M. Alejandra Tricerri; Giulia Ossato; Enrico Gratton (pp. 1399-1408).
Protein and protein–lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid–protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.
Keywords: Abbreviations; DMPC; 1,2 di-myristoyl phosphatidyl choline; POPC; 1-palmitoyl-2-oleylphosphatidylcholine; DSPC; 1,2 di-stearoyl phosphatidyl choline; DOPC; 1,2 di oleylphosphatidylcholine; DPPC 1; 2-dipalmitoylphatidylcholine; SM; sphingomylin; MβCD; methyl beta cyclo dextrine; HDL; high density lipoproteins; SUVs, MLVs, LUVs; small unilamellar, multilamellar, and large unilamellar vesicles, respectively; PCH; Photon Counting Histogram; FC; free cholesterolrHDL; apoA-I; Fluorescence microscopy; Laurdan GP; GUV
Visualizing association of lipidated signaling proteins in heterogeneous membranes−Partitioning into subdomains, lipid sorting, interfacial adsorption, and protein association by Katrin Weise; Gemma Triola; Sascha Janosch; Herbert Waldmann; Roland Winter (pp. 1409-1417).
In a combined chemical biological and biophysical approach, we studied the partitioning of differently fluorescent-labeled palmitoyl and/or farnesyl lipidated peptides, which represent membrane recognition model systems, as well as the full lipidated N-Ras protein into various model membrane systems including canonical model raft mixtures. To this end, two-photon fluorescence microscopy on giant unilamellar vesicles, complemented by tapping-mode atomic force microscopy (AFM) measurements, was carried out. The measurements were performed over a wide temperature range, ranging from 30 to 80 °C to cover different lipid phase states (solid-ordered (gel), fluid/gel, liquid-ordered/liquid-disordered, all-fluid). The results provide direct evidence that partitioning of the lipidated peptides and N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is liquid-disordered>liquid-ordered≫solid-ordered. Intriguingly, we detect – using the better spatial resolution of AFM – also a large proportion of the lipidated protein located at the liquid-disordered/liquid-ordered phase boundary, thus leading to a favorable decrease in line tension that is associated with the rim of neighboring domains. In an all-liquid-ordered, cholesterol-rich phase, phase separation can be induced by an effective lipid sorting mechanism owing to the high affinity of the lipidated peptides and proteins to a fluid-like lipid environment. At low temperatures, where the overall acyl chain order parameter of the lipid bilayer has markedly increased, such an efficient lipid sorting mechanism is energetically too costly and self-association of the peptide into small clusters takes place. These data reveal the interesting ability of the lipidated peptides and proteins to induce formation of fluid microdomains at physiologically relevant high cholesterol concentrations. Furthermore, our results reveal self-association of the N-Ras protein at the domain boundaries which may serve as an important vehicle for association processes and nanoclustering, which has also been observed in in vivo studies.
Keywords: Fluorescence microscopy; Lipidated protein; Ras signaling; Model biomembrane; AFM
Mechanism of membrane nanotube formation by molecular motors by Cécile Leduc; Otger Campàs; Jean-François Joanny; Jacques Prost; Patricia Bassereau (pp. 1418-1426).
Membrane nanotubes are ubiquitous in eukaryotic cells due to their involvement in the communication between many different membrane compartments. They are very dynamical structures, which are generally extended along the microtubule network. One possible mechanism of tube formation involves the action of molecular motors, which can generate the necessary force to pull the tubes along the cytoskeleton tracks. However, it has not been possible so far to image in living organisms simultaneously both tube formation and the molecular motors involved in the process. The reasons for this are mainly technological. To overcome these limitations and to elucidate in detail the mechanism of tube formation, many experiments have been developed over the last years in cell-free environments. In the present review, we present the results, which have been obtained in vitro either in cell extracts or with purified and artificial components. In particular, we will focus on a biomimetic system, which involves Giant Unilamellar Vesicles, kinesin-1 motors and microtubules in the presence of ATP. We present both theoretical and experimental results based on fluorescence microscopy that elucidate the dynamics of membrane tube formation, growth and stalling.
Keywords: Molecular motor; Membrane tube; Intracellular traffic; Fluorescence microscopy; Numerical simulation
Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles by S.A. Johnson; B.M. Stinson; M.S. Go; L.M. Carmona; J.I. Reminick; X. Fang; T. Baumgart (pp. 1427-1435).
Liquid-ordered (Lo) and liquid-disordered (Ld) phase coexistence has been suggested to partition the plasma membrane of biological cells into lateral compartments, allowing for enrichment or depletion of functionally relevant molecules. This dynamic partitioning might be involved in fine-tuning cellular signaling fidelity through coupling to the plasma membrane protein and lipid composition. In earlier work, giant plasma membrane vesicles, obtained by chemically induced blebbing from cultured cells, were observed to reversibly phase segregate at temperatures significantly below 37°C. In this contribution, we compare the temperature dependence of fluid phase segregation in HeLa and rat basophilic leukemia (RBL) cells. We find an essentially monotonic temperature dependence of the number of phase-separated vesicles in both cell types. We also observe a strikingly broad distribution of phase transition temperatures in both cell types. The binding of peripheral proteins, such as cholera toxin subunit B (CTB), as well as Annexin V, is observed to modulate phase transition temperatures, indicating that peripheral protein binding may be a regulator for lateral heterogeneity in vivo. The partitioning of numerous signal protein anchors and full length proteins is investigated. We find Lo phase partitioning for several proteins assumed in the literature to be membrane raft associated, but observe deviations from this expectation for other proteins, including caveolin-1.
Keywords: Liquid-ordered; Liquid-disordered; Bleb; Giant plasma membrane vesicle; Phase transition; Phase partitioning
Liquid ordered phase in cell membranes evidenced by a hydration-sensitive probe: Effects of cholesterol depletion and apoptosis by Sule Oncul; Andrey S. Klymchenko; Oleksandr A. Kucherak; Alexander P. Demchenko; Sophie Martin; Monique Dontenwill; Youri Arntz; Pascal Didier; Guy Duportail; Mely Yves Mély (pp. 1436-1443).
Herein, using a recently developed hydration-sensitive ratiometric biomembrane probe based on 3-hydroxyflavone (F2N12S) that binds selectively to the outer leaflet of plasma membranes, we compared plasma membranes of living cells and lipid vesicles as model membranes. Through the spectroscopic analysis of the probe response, we characterized the membranes in terms of hydration and polarity (electrostatics). The hydration parameter value in cell membranes was in between the values obtained with liquid ordered (Lo) and liquid disordered (Ld) phases in model membranes, suggesting that cell plasma membranes exhibit a significant fraction of Lo phase in their outer leaflet. Moreover, two-photon fluorescence microscopy experiments show that cell membranes labeled with this probe exhibit a homogeneous lipid distribution, suggesting that the putative domains in Lo phase are distributed all over the membrane and are highly dynamic. Cholesterol depletion affected dramatically the dual emission of the probe suggesting the disappearance of the Lo phase in cell membranes. These conclusions were corroborated with the viscosity sensitive diphenylhexatriene derivative TMA-DPH, showing membrane fluidity in intact cells intermediate between those for Lo and Ld phases in model membranes, as well as a significant increase in fluidity after cholesterol depletion. Moreover, we observed that cell apoptosis results in a similar loss of Lo phase, which could be attributed to a flip of sphingomyelin from the outer to the inner leaflet of the plasma membrane due to apoptosis-driven lipid scrambling. Our data suggest a new methodology for evaluating the Lo phase in membranes of living cells.
Keywords: Cell plasma membrane; Lipid bilayers; Liquid ordered phase; Hydration; Fluorescent probes; Cholesterol depletion
Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy by Stockl M.T. Stöckl; A. Herrmann (pp. 1444-1456).
The discovery that the lipids constituting the plasma membrane are not randomly distributed, but instead are able to form laterally segregated lipid domains with different properties has given hints how the formation of such lipid domains influences and regulates many processes occurring at the plasma membrane. While in model systems these lipid domains can be easily accessed and their properties studied, it is still challenging to determine the properties of cholesterol rich lipid domains, the so called “Rafts”, in the plasma membrane of living cells due to their small size and transient nature. One promising technique to address such issues is fluorescence lifetime imaging (FLIM) microscopy, as spatially resolved images make the visualization of the lateral lipid distribution possible, while at the same time the fluorescence lifetime of a membrane probe yields information about the bilayer structure and organization of the lipids in lipid domains and various properties like preferential protein–protein interactions or the enrichment of membrane probes. This review aims to give an overview of the techniques underlying FLIM probes which can be applied to investigate the formation of lipid domains and their respective properties in model membrane and biological systems. Also a short technical introduction into the techniques of a FLIM microscope is given.
Keywords: Fluorescence lifetime; FLIM; Lipid domain; Raft