This work is part of the MaxSynBio consortium, which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.

The authors have nothing to disclose.

Successful production of GUVs for phase state observations under symmetric and asymmetric high-salinity conditions
The protocols presented here introduces a strategy to assess the influence of high-salinity buffer and solution asymmetry on the membrane phase state of charged GUVs over a broad range of compositions. One of the major challenges towards achieving this goal was the production of charged GUVs in high-salinity buffers.
We successfully produced GUVs in sucrose solution and high-saline buffer by a simple spontaneous swelling approach, which includes a pre-hydration step of the deposited lipid film and an overnight final hydration step for vesicle growth. It is important to note that the lipid deposition should be done on a roughened PTFE plate to ensure even lipid spreading to yield unilamellar vesicles. Moreover, it is essential to perform every step during vesicle preparation at a temperature where the lipid film is in a homogenous and fluid phase state. Else, the vesicle population may be polydisperse in composition and bias the final population phase state analysis. The specific protocol for the spontaneous swelling of GUVs yields a vesicle clump which on the one hand offers the possibility to re-suspend it in small volumes to obtain highly concentrated vesicle dispersions, and on the other hand provides asymmetric solution conditions across the membrane while minimizing the dilution of vesicles8,28. It is essential that during vesicle dilution or external solution exchange, the inside and outside osmolarities remain matched as morphology changes caused by osmolarity mismatches may induce or prevent Lo+Ld phase separation41 or, in case of hypotonic conditions, may lead to vesicle bursting.
Here, attempts to optimize electroformation protocols in high-salinity solution resulted in the production of no GUVs while PVA-assisted swelling yielded multilamellar vesicles. Even though it requires longer preparation times and results in vesicle batches of lower quality10,17, the successful production of charged vesicles by spontaneous swelling comes with additional advantages. It demands minimal effort while resulting in sufficient yields for statistical batch analyses and, unlike electroformation, no sophisticated equipment or optimization were required. Moreover, no contaminations by lipid oxidation have been observed42,43. According to the literature there are no differences between the lipid compositions of vesicles and the corresponding stocks from which they were grown7,17. Furthermore, the vesicle formation on a PTFE substrate does not prompt the inclusion of any contaminations in contrast to gel-assisted swelling methods where foreign molecules may be introduced by the substrate23. Electroformation comes with further drawbacks related to excessive vesicle dilution when creating asymmetric solution conditions. GUVs produced by electroformation are usually present as a homogeneous dispersion (in contrast to the highly concentrated vesicle suspension in the form of a clump formed during spontaneous swelling). Any dilution of the external solution would substantially dilute the number of vesicles as well. Furthermore, over the course of this work it was observed that DOPG/eSM/Chol GUVs produced by electroformation in sucrose became unstable if diluted in high-salinity buffer. Fluorescence from lipid patches on the microscope slide indicated that vesicles would burst before their visual inspection was possible. Such instability may be ascribed to an elevated membrane tension of vesicles prepared by electroformation as compared to those obtained by spontaneous swelling10.
Although the vesicle dilution is an easy and quick approach to create asymmetric GUV solution conditions, it only accomplishes a partial external solution exchange, albeit to a high fraction (here: 95 %, Figure 2), as upon dilution, traces of the swelling solution will remain. The choice of the degree of the external solution exchange is a trade-off between pipetting up the vesicle clump together with the swelling solution (section 2) and not diluting it too much. Hence, we introduced an alternative microfluidic approach discussed elsewhere in detail37 that allows for a quick and complete external solution exchange during GUV phase state observations to verify the phase state observations made for diluted vesicles. The observations of phase state variations when changing from symmetric to asymmetric solution conditions were indeed observed to be in agreement. Additionally, both methods to generate asymmetric solution conditions shown here are comparably quick (compare Ref.8) and come with no known risks of local composition alterations (compare references30,31), increase in membrane tension (compare reference32), or local heating (compare reference33), as for the alternative methods discussed in the Introduction. During the microfluidic trapping, an osmotic balance between the vesicle interior and exterior is not only essential to avoid phase state artifacts as mentioned above but also deflation caused by hypertonic solution conditions might cause the trapped GUVs to slip through the posts after an external solution exchange.
Even though spontaneous swelling has been successfully applied to grow uncharged vesicles from a DOPC/eSM/Chol system, in other cases, the absence of charges may impair GUV swelling due to the resulting lack of repulsion between the individual bilayers44. Extending the pre-swelling period or introducing bulky lipid headgroups may counter this issue45. Furthermore, the vesicle stability after their dilution in solutions different from the one used for swelling may differ for different lipid compositions and dilution media, which we have not investigated here. We also have not explored the possibility of tuning the average GUV diameter with the preparation method presented here. But parameters such as vesicle composition and swelling solution are likely to influence the results. The application of alternative methods46 may yield larger vesicles for the lipids and solutions used here, however, they may come with other drawbacks associated with the method. The above-described approach to produce GUVs in symmetric and asymmetric high-salinity conditions provides a potential tool for further studies of vesicles made up of different lipid compositions and dispersed in different media. As we have not explored those possibilities, future trials will show how generally the GUV preparation and dilution methods may be applied.
Observing phase separation at varying temperatures
Different experimental setups suitable to study GUVs at varying temperatures exist. While these setups are not commonly described in detail within the literature, the current work presents a basic assembly applicable for such studies.
Control measurements show that the temperature of this in-house designed and built chamber is precisely controlled by a thermostat and temperature gradients inside the chamber are within the experimental temperature resolution. It is ensured that the experimental thermal conditions are consistent with the reading of the thermostat.
During the assessment of GUV phase states over a broad temperature range, it is important that the observed vesicles are well equilibrated after the temperature has been changed. One possible way to ensure this is to check for hysteresis. If hysteresis is present, the temperature steps should be decreased and/or equilibration times increased. As temperature control in this work is established by a water-based thermostat, the range of working temperatures is ideally limited to 0 - 100 &#176;C. This range can be expanded by using other temperature control liquids such as oil or by employing other setups, e.g. a Peltier device. In practice, the working temperature is also limited by possible condensation or evaporation. In addition, for temperatures far away from room temperature, the occurrence of a steady state temperature gradient across the observation chamber becomes more likely. Also, the imaging equipment may be harmed at extreme temperatures. For typical temperature ranges appropriate for lipid vesicle studies (~10 - 50 &#176;C7,9) damage to the observation equipment should be considered but is typically not expected.
Vesicle domain observation artifacts
There are a number of sources for observation artifacts using wide-field fluorescence microscopy. First of all, one should be aware that the maximum resolution r of the object applied for the visual inspection of vesicle phase states determines the detection limit of lipid domains according to: 
<img alt="Equation 2" src="/files/ftp_upload/56034/56034eq2v2.jpg" /> 
where &#955; is the emission wavelength, and NA is the numeric aperture of the objective. A typical objective with a 40x magnification and an NA of 0.6 which detects green emission light around 560 nm would reach an optical resolution of ~0.6 &#181;m. Hence, studies that compare phase states of vesicles made from particular lipid mixtures among different conditions should use the same objective for the same lipid mixture.
Another artifact is the occurrence of lipid domains as a consequence of lipid photo-oxidation due to an extended exposure to excitation light41. Photo-damage occurs preferentially on unsaturated hydrocarbon lipid moieties. In fact, for some lipid compositions, such domain formation of initially homogeneous vesicles was observed here after an extended period of excitation light exposure (~30 s). To counter that issue, the excitation light was kept focused at one field of view for only a few seconds for the phase state assessment. Hence, DiIC18 was suitable for our purposes. Other dyes, however, may be much more sensitive and may need to be handled at lower excitation intensities and with shorter excitation light exposure times.
Mechanical shear stress from the pipette transfer of the vesicles potentially mixes domains temporarily, thereby distorting the apparent vesicle phase behavior. For some batches, different vesicles showed different phase behaviors 0 min and 5 min after pipette transfer onto the microscope coverslip. Also the shear stress induced by the fluid flow in the microfluidic device has been shown to result in domain mixing47. Vesicles should be left undisturbed for a sufficient amount of time for equilibration before observation. Within this study, vesicles trapped on the microfluidic device were left undisturbed for 1 h after vesicle loading and solution exchanges before observation.
To avoid some of the above-mentioned difficulties as well as the restriction imposed by the light diffraction limit, alternative methods such as nuclear magnetic resonance spectroscopy48 or super resolution microscopy techniques49 could be employed.
Conclusion and outlook
The presented work demonstrates a set of methods that allows for the analysis of the influence of high-salinity symmetric and asymmetric solution conditions on membrane phase separation. All of the presented methods are suitable for other applications. The microfluidic device for example provides a platform to study the kinetics of domain formation and disappearance upon the induction of solution asymmetry. Also, domain appearance as a function of salt concentration could be examined that way. All methods could also be used to look at the influence on the phase behavior using any other solutions of interest.

Ever since the observation of micron-sized domains in liquid-liquid phase-separated giant unilamellar vesicles (GUVs) by fluorescence microscopy, GUVs have been&#160;used as a model system to investigate the lipid raft hypothesis1,2,3. As the area of their free-standing bilayer lies in the range of that from biological cells, they are suitable mimics of plasma membranes featuring the hypothesized rafts. Numerous studies on such GUVs have been performed with vesicles dispersed in pure water, sucrose or low-salinity solutions4,5,6,7,8. These conditions, however, do not reflect physiologically relevant exposure of biomembranes to high-salinity environments and trans-membrane solution asymmetry as are the conditions for cells.
In this work and in a previous publication from our group9, the phase states of charged multicomponent GUVs were examined as a function of the presence of salt and solution asymmetry across the membrane. GUVs were prepared from mixtures of different ratios of dioleoylphosphatidylglycerol (DOPG), egg sphingomyelin (eSM), and cholesterol (Chol) in either sucrose solution (with osmolarity of 210 mOsm/kg) or high-salinity buffer (100 mM NaCl, 10 mM Tris, pH 7.5, 210 mOsm/kg). The lipid choice was justified by the data already obtained on the phase diagram of this mixture6,8.
A number of methods for the preparation of GUVs are available in the literature10,11,12 (note that here, we will not consider those involving transfer of lipids from an oil-based to an aqueous phase13,14 because of the inherent danger of the remaining oil residues in the membrane which may affect the phase behavior). The preparation of GUVs in high-salinity buffer is associated with specific challenges. For solutions of low ionic strength, the electroformation method15,16 presents a quick way to prepare GUVs in high yields with little defects10,17. The method is based on depositing a layer of lipids on a conductive surface (the electrode), drying them, and hydrating them with an aqueous solution while applying an AC field. However, this method requires adjustments if salt is present in the aqueous solution18,19. It is assumed that the driving force for vesicle growth by electroformation is electroosmosis16 which is hindered at high conductivities20. Hence, electroswelling of GUVs in high-salinity solutions is not a straightforward approach as it requires optimization for different salt concentrations present in the swelling solution. Gel-assisted vesicle swelling21,22 is a potential alternative to electroformation with even faster formation times. This approach builds on the enhanced lipid film hydration when a gel (agarose or polyvinyl alcohol (PVA)) is used as a substrate. These approaches, however, come with the risk of membrane contamination in the case of agarose-based swelling23 and/or temperature limits as in the case of PVA-based swelling. Similarly, a protocol to grow GUVs on a cellulose paper substrate has recently been established24. General issues of this method are the lack of control over the substrate purity as well as the usage of large amounts of lipid. In this work, we will introduce and present the advantages of the most traditional method for GUV preparation, namely the spontaneous swelling method25,26. It consists of drying a lipid layer on a lipophobic substrate, hydrating it in water-vapor atmosphere, and subsequent swelling in the desired swelling solution (see Figure 1 and details in the Protocol section). This method does not offer control over the vesicle size distribution and results in overall smaller vesicles as compared to methods where the production is assisted by electric field, polymer substrate or microfluidic means. However, the vesicle quality and size is appropriate for examining the membrane phase state as explored here.
Creating asymmetry between the solutions across the vesicle membrane is associated with certain challenges as well. One commonly used approach is the direct dilution of the vesicle suspension in the desired external solution27,28. However, this also decreases the vesicle distribution density. Another strategy is to slowly exchange the external solution around GUVs settled at the bottom of a flow-cell that allows for solution in- and outflux. To avoid disturbing or even losing the vesicles with the flow, low flow rates are applied8, which renders this approach time-inefficient. Moreover, neither of these approaches guarantees full external solution exchange. An obvious solution is to immobilize vesicles in order to avoid losing them during an external solution exchange. For instance, biotinylated GUVs can be tethered onto a streptavidin-coated surface29. However, this approach may lead to compositional variations at the adhered and hence the non-adhered membrane segments30,31.The application of magnetic or electric fields to trap vesicles results in imposing membrane tension32. Employing optical tweezers to trap a vesicle requires having a handle attached (i.e. a bead), while the use of optical stretchers may involve local heating33. Trapping of GUVs can also be accomplished by growing them on platinum wires without final detachment34. This however yields vesicles which are not isolated and which are usually connected to the wires or other vesicles by thin lipid tubes (tethers).
The presented work highlights strategies to overcome the aforementioned limitations. We first present a detailed description of the spontaneous swelling method adapted and optimized for the production of GUVs in high-salinity buffers. We then introduce two approaches to efficiently create asymmetric solution conditions by simple dilution or the utilization of a microfluidic device. Because our goal is the analysis of the membrane phase state of GUVs in different solution conditions, the subsequent sections describe criteria for successful statistical analysis and present hints for avoiding false categorization.
The analyses were performed under isothermal conditions as well as under varying temperatures. While temperature control is commonly employed, details about experimental temperature-control chambers are rarely described. Here, an in-house built setup to observe GUVs at various temperature conditions is presented.

<table><tbody><tr><td>1,2-dioleoyl-&lt;em&gt;sn&lt;/em&gt;-glycero-3-phospho-(1&#039;-rac-glycerol), sodium salt</td><td>Avanti Polar Lipids</td><td>840475C</td><td>abbreviated as DOPG in the text</td></tr><tr><td>chicken egg sphingomyelin</td><td>Avanti Polar Lipids</td><td>860061</td><td>abbreviated as eSM</td></tr><tr><td>cholesterol (ovine wool, &gt; 98 %)</td><td>Avanti Polar Lipids</td><td>700000</td><td>abbreviated as Chol</td></tr><tr><td>1,1&#039;-dioctadecyl-3,3,3&#039;,3&#039;-tetramethylindocarbocyanine perchlorate</td><td>Molecular Probes</td><td>D-282</td><td>abbreviated as DiIC&lt;sub&gt;18&lt;/sub&gt;</td></tr><tr><td>Chloroform, HPLC grade (&amp;ge; 99.8 %)</td><td>Merck</td><td></td><td></td></tr><tr><td>NaCl (&gt; 99.8 %)</td><td>Roth</td><td></td><td></td></tr><tr><td>HCl (37 %)</td><td>Roth</td><td></td><td></td></tr><tr><td>Tris (&amp;ge; 99.9 %)</td><td>Roth</td><td></td><td></td></tr><tr><td>Sucrose (&amp;ge; 99.5 %)</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Parafilm</td><td></td><td></td><td></td></tr><tr><td>Threaded vial 45x27 mm, 15 mL</td><td>Kimble</td><td></td><td>Soda flat bottom, white screw cap</td></tr><tr><td>pH meter</td><td>Mettler Toledo</td><td>MP220</td><td></td></tr><tr><td>Osmometer</td><td>Gonotec</td><td>Osmomat030</td><td></td></tr><tr><td>Epi-fluorescence microscope</td><td>Zeiss</td><td>Axio Observer D1</td><td></td></tr><tr><td>Confocal laser scanning microscope</td><td>Leica</td><td>TCS SP5</td><td></td></tr><tr><td>Objective 40x, 0.6 NA</td><td>Zeiss</td><td>LD Achroplan</td><td></td></tr><tr><td>Objective 40x, 0.75 NA</td><td>Leica</td><td>506174</td><td></td></tr><tr><td>Objective 63x, 0.9 NA</td><td>Leica</td><td>506148</td><td></td></tr><tr><td>Microscope slide, 56x26 mm, 0.17 &amp;plusmn; 0.01 mm</td><td>Menzel-Gl&amp;auml;ser</td><td></td><td></td></tr><tr><td>Cover slip, 22x22 mm, 0.17 &amp;plusmn; 0.01 mm</td><td>Menzel-Gl&amp;auml;aser</td><td></td><td></td></tr><tr><td>Parafilm &quot;M&quot;</td><td>Bremix Flexible Packaging</td><td></td><td></td></tr><tr><td>Syringes, 5 mL, 10 mL</td><td>Braun</td><td></td><td></td></tr><tr><td>0.45 &amp;micro;m syringe filter</td><td>GVS North America</td><td>Cameo 25AS, 1213723</td><td>Acetate, sterile</td></tr></tbody></table>

phase behavior of charged vesicles under symmetric and asymmetric solution conditions monitored with fluorescence microscopy

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to investigate the lipid raft hypothesis. Numerous studies, however, neglect the impact of physiological solution conditions. On that account, the current work presents the effect of high-salinity buffer and trans-membrane solution asymmetry on liquid-liquid phase separation in charged GUVs grown from dioleylphosphatidylglycerol, egg sphingomyelin, and cholesterol. The effects were studied under isothermal and varying temperature conditions.
We describe equipment and experimental strategies applicable for monitoring the stability of coexisting liquid domains in charged vesicles under symmetric and asymmetric high-salinity solution conditions. This includes an approach to prepare charged multicomponent GUVs in high-salinity buffer at high temperatures. The protocol entails the option to perform a partial exchange of the external solution by a simple dilution step while minimizing the vesicle dilution. An alternative approach is presented utilizing a microfluidic device that allows for a complete external solution exchange. The solution effects on phase separation were also studied under varying temperatures. To this end, we present the basic design and utility of an in-house built temperature control chamber. Furthermore, we reflect on the assessment of the GUV phase state, pitfalls associated with it and how to circumvent them.

Watch this Scientific Journal Video about Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy at JoVE.com

Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

Experiments on phase separated giant unilamellar vesicles (GUVs) frequently neglect physiological solution conditions. This work presents approaches to study the effect of high-salinity buffer on liquid-liquid phase separation in charged multicomponent GUVs as a function of trans-membrane solution asymmetry and temperature.

Phase-separated giant unilamellar vesicles (GUVs) exhibiting coexisting liquid-ordered and liquid-disordered domains are a common biophysical tool to ...

phase-behavior-charged-vesicles-under-symmetric-asymmetric-solution

Universidad San Sebastian

Research

JoVE Journal

Chemistry

9.1K Views.  Max Planck Institute of Colloids and Interfaces. Experiments on phase separated giant unilamellar vesicles (GUVs) frequently neglect physiological solution conditions. This work presents approaches to study the effect of high-salinity buffer on liquid-liquid phase separation in charged multicomponent GUVs as a function of trans-membrane solution asymmetry and temperature.

Video: Phase Behavior of Charged Vesicles Under Symmetric and Asymmetric Solution Conditions Monitored with Fluorescence Microscopy

Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used for ATP production. The amphiphilic nature of membrane proteins requires particular attention to their handling, and reconstitution into the natural lipid environment is indispensable when studying membrane transport processes like proton translocation. Here, we detail a method that has been used for the investigation of the proton-pumping mechanism of membrane redox enzymes, taking cytochrome bo3 from Escherichia coli as an example. A combination of electrochemistry and fluorescence microscopy is used to control the redox state of the quinone pool and monitor pH changes in the lumen. Due to the spatial resolution of fluorescent microscopy, hundreds of liposomes can be measured simultaneously while the enzyme content can be scaled down to a single enzyme or transporter per liposome. The respective single enzyme analysis can reveal patterns in the enzyme functional dynamics that might be otherwise hidden by the behavior of the whole population. We include a description of a script for automated image analysis.

Here, we present a protocol to study the molecular mechanism of proton translocation across lipid membranes of single liposomes, using cytochrome bo3 as an example. Combining electrochemistry and fluorescence microscopy, pH changes in the lumen of single vesicles, containing single or multiple enzyme, can be detected and analyzed individually.

Proton-pumping enzymes of electron transfer chains couple redox reactions to proton translocation across the membrane, creating a proton-motive force used ...

Biochemistry

Membrane Remodeling of Giant Vesicles in Response to Localized Calcium Ion Gradients

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local variations in calcium ion concentration. The main molecular components involved in these processes have been identified; however, the specific interplay between calcium ion gradients and the lipids within the cell membrane is far less known, mainly due to the complex nature of biological cells and the difficultly of observation schemes. To bridge this gap, a synthetic approach is successfully implemented to reveal the localized effect of calcium ions on cell membrane mimics. Establishing a mimic to resemble the conditions within a cell is a severalfold problem. First, an adequate biomimetic model with appropriate dimensions and membrane composition is required to capture the physical properties of cells. Second, a micromanipulation setup is needed to deliver a small amount of calcium ions to a particular membrane location. Finally, an observation scheme is required to detect and record the response of the lipid membrane to the external stimulation. This article offers a detailed biomimetic approach for studying the calcium ion-membrane interaction, where a lipid vesicle system, consisting of a giant unilamellar vesicle (GUV) connected to a multilamellar vesicle (MLV), is exposed to a localized calcium gradient formed using a microinjection system. The dynamics of the ionic influence on the membrane were observed using fluorescence microscopy and recorded at video frame rates. As a result of the membrane stimulation, highly curved membrane tubular protrusions (MTPs) formed inside the GUV, oriented away from the membrane. The described approach induces the remodeling of the lipid membrane and MTP production in an entirely contactless and controlled manner. This approach introduces a means to address the details of calcium ion-membrane interactions, providing new avenues to study the mechanisms of cell membrane reshaping.

We present a technique for contactless micromanipulation of vesicles, using localized calcium ion gradients. Microinjection of a calcium ion solution, in the vicinity of a giant lipid vesicle, is utilized to remodel the lipid membrane, resulting in the production of membrane tubular protrusions.

In a wide variety of fundamental cell processes, such as membrane trafficking and apoptosis, cell membrane shape transitions occur concurrently with local ...

Atomic Force Microscopy Imaging and Force Spectroscopy of Supported Lipid Bilayers

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient magnification to examine membrane substructures and even individual molecules. AFM can act as a force probe to measure interactions and mechanical properties of membranes. Supported lipid bilayers are conventionally used as membrane models in AFM studies. In this protocol, we demonstrate how to prepare supported bilayers and characterize their structure and mechanical properties using AFM. These include bilayer thickness and breakthrough force. 
The information provided by AFM imaging and force spectroscopy help define mechanical and chemical properties of membranes. These properties play an important role in cellular processes such as maintaining cell hemostasis from environmental stress, bringing membrane proteins together, and stabilizing protein complexes.

We describe a protocol for preparation of supported lipid bilayers and its characterization using atomic force microscopy and force spectroscopy.

Atomic force microscopy (AFM) is a versatile, high-resolution imaging technique that allows visualization of biological membranes. It has sufficient ...

Bioengineering

Single Molecule Fluorescence Microscopy on Planar Supported Bilayers

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background in optics and laser physics to a discipline that is now enjoying vivid attention by life-scientists of all venues 1. This is because single molecule imaging has the unique potential to reveal protein behavior in situ in living cells and uncover cellular organization with unprecedented resolution below the diffraction limit of visible light 2. Glass-supported planar lipid bilayers (SLBs) are a powerful tool to bring cells otherwise growing in suspension in close enough proximity to the glass slide so that they can be readily imaged in noise-reduced Total Internal Reflection illumination mode 3,4. They are very useful to study the protein dynamics in plasma membrane-associated events as diverse as cell-cell contact formation, endocytosis, exocytosis and immune recognition. Simple procedures are presented how to generate highly mobile protein-functionalized SLBs in a reproducible manner, how to determine protein mobility within and how to measure protein densities with the use of single molecule detection. It is shown how to construct a cost-efficient single molecule microscopy system with TIRF illumination capabilities and how to operate it in the experiment.

Preparation of protein-functionalized planar glass-supported lipid bilayers, determination of protein mobility within and measurement of protein densities is shown here. A roadmap to building a noise-reduced Total Internal Reflection microscope is outlined, which allows visualizing single bilayer-resident fluorochromes with high spatiotemporal resolution.

In the course of a single decade single molecule microscopy has changed from being a secluded domain shared merely by physicists with a strong background ...

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main common features required for this activity resulted from the interactions of CPPs with the plasma membrane (lipids) and more particularly with components of the extracellular matrix of the membrane itself (heparan sulphate). Indeed, independent of the direct translocation or the endocytosis-dependent internalization, lipid bilayers are involved in the internalization process both at the level of the plasma membrane and at the level of intracellular traffic (endosomal vesicles). In this article, we present a detailed protocol describing the different steps of a large unilamellar vesicles (LUVs) formulation, purification, characterization, and application in fluorescence leakage assay in order to detect possible CPP-membrane destabilization/interaction and to address their role in the internalization mechanism. LUVs with a lipid composition reflecting the plasma membrane content are generated in order to encapsulate both a fluorescent dye and a quencher. The addition of peptides in the extravesicular medium and the induction of peptide-membrane interactions on the LUVs might thus induce in a dose-dependent manner a significant increase in fluorescence revealing a leakage. Examples are provided here with the recently developed tryptophan (W)- and arginine (R)-rich Amphipathic Peptides (WRAPs), which showed a rapid and efficient siRNA delivery in various cell lines. Finally, the nature of these interactions and the affinity for lipids are discussed to understand and to improve the membrane translocation and/or the endosomal escape.

The fluorescence leakage assay is a simple method that enables the investigation of peptide/membrane interactions in order to understand their involvement in several biological processes and especially the ability of cell-penetrating peptides to disturb phospholipids bilayers during a direct cellular translocation process.

Cell-penetrating peptides (CPPs) are defined as carriers that are able to cross the plasma membrane and to transfer a cargo into cells. One of the main ...

Reconstitution of Septin Assembly at Membranes to Study Biophysical Properties and Functions

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component in coordinating shape changes like cytokinesis, polarized growth, and migration. Septins are filament-forming proteins that assemble to form diverse higher-order structures and, in many cases, are found in different areas of the plasma membrane, most notably in regions of micron-scale positive curvature. Monitoring the process of septin assembly in vivo is hindered by the limitations of light microscopy in cells, as well as the complexity of interactions with both membranes and cytoskeletal elements, making it difficult to quantify septin dynamics in living systems. Fortunately, there has been substantial progress in the past decade in reconstituting the septin cytoskeleton in a cell-free system to dissect the mechanisms controlling septin assembly at high spatial and temporal resolutions. The core steps of septin assembly include septin heterooligomer association and dissociation with the membrane, polymerization into filaments, and the formation of higher-order structures through interactions between filaments. Here, we present three methods to observe septin assembly in different contexts: planar bilayers, spherical supports, and rod supports. These methods can be used to determine the biophysical parameters of septins at different stages of assembly: as single octamers binding the membrane, as filaments, and as assemblies of filaments. We use these parameters paired with measurements of curvature sampling and preferential adsorption to understand how curvature sensing operates at a variety of length and time scales.

Cell-free reconstitution has been a key tool to understand the cytoskeleton assembly, and work in the last decade has established approaches to study septin dynamics in minimal systems. Presented here are three complementary methods to observe septin assembly in different membrane contexts: planar bilayers, spherical supports, and rod supports.

Most cells can sense and change their shape to carry out fundamental cell processes. In many eukaryotes, the septin cytoskeleton is an integral component ...

TIRFM and pH-sensitive GFP-probes to Evaluate Neurotransmitter Vesicle Dynamics in SH-SY5Y Neuroblastoma Cells: Cell Imaging and Data Analysis

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real time and dissecting the different steps of exo-endocytosis at the single-vesicle level are crucial for understanding synaptic functions in health and disease.
Genetically-encoded pH-sensitive probes directly targeted to synaptic vesicles and Total Internal Reflection Fluorescence Microscopy (TIRFM) provide the spatio-temporal resolution necessary to follow vesicle dynamics. The evanescent field generated by total internal reflection can only excite fluorophores placed in a thin layer (&#60;150 nm) above the glass cover on which cells adhere, exactly where the processes of exo-endocytosis take place. The resulting high-contrast images are ideally suited for vesicles tracking and quantitative analysis of fusion events.
In this protocol, SH-SY5Y human neuroblastoma cells are proposed as a valuable model for studying neurotransmitter release at the single-vesicle level by TIRFM, because of their flat surface and the presence of dispersed vesicles. The methods for growing SH-SY5Y as adherent cells and for transfecting them with synapto-pHluorin are provided, as well as the technique&#160;to perform TIRFM and imaging. Finally, a strategy aiming to select, count, and analyze fusion events at whole-cell and single-vesicle levels is presented.
To validate the imaging procedure and data analysis approach, the dynamics of pHluorin-tagged vesicles are&#160;analyzed under resting and stimulated (depolarizing potassium concentrations) conditions. Membrane depolarization increases the frequency of fusion events and causes a parallel raise of the net fluorescence signal recorded in whole cell. Single-vesicle analysis reveals modifications of fusion-event behavior (increased peak height and width). These data suggest that potassium depolarization not only induces a massive neurotransmitter release but also modifies the mechanism of vesicle fusion and recycling.
With the appropriate fluorescent probe, this technique can be employed in different cellular systems to dissect the mechanisms of constitutive and stimulated secretion.

This paper provides a method for investigating neurotransmitter vesicle dynamics in neuroblastoma cells, using a synaptobrevin2-pHluorin construct and Total Internal Reflection Fluorescence Microscopy. The strategy developed for image processing and data analysis is also reported.

Synaptic vesicles release neurotransmitters at chemical synapses through a dynamic cycle of fusion and retrieval. Monitoring synaptic activity in real ...

Neuroscience

SNARE-mediated Fusion of Single Proteoliposomes with Tethered Supported Bilayers in a Microfluidic Flow Cell Monitored by Polarized TIRF Microscopy

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. During neurotransmitter or hormone release via exocytosis, the fusion pore can transiently open and close repeatedly, regulating cargo release kinetics. Pore dynamics also determine the mode of vesicle recycling; irreversible resealing results in transient, &#34;kiss-and-run&#34; fusion, whereas dilation leads to full fusion. To better understand what factors govern pore dynamics, we developed an assay to monitor membrane fusion using polarized total internal reflection fluorescence (TIRF) microscopy with single molecule sensitivity and ~15 msec time resolution in a biochemically well-defined in vitro system. Fusion of fluorescently labeled small unilamellar vesicles containing v-SNARE proteins (v-SUVs) with a planar bilayer bearing t-SNAREs, supported on a soft polymer cushion (t-SBL, t-supported bilayer), is monitored. The assay uses microfluidic flow channels that ensure minimal sample consumption while supplying a constant density of SUVs. Exploiting the rapid signal enhancement upon transfer of lipid labels from the SUV to the SBL during fusion, kinetics of lipid dye transfer is monitored. The sensitivity of TIRF microscopy allows tracking single fluorescent lipid labels, from which lipid diffusivity and SUV size can be deduced for every fusion event. Lipid dye release times can be much longer than expected for unimpeded passage through permanently open pores. Using a model that assumes retardation of lipid release is due to pore flickering, a pore &#34;openness&#34;, the fraction of time the pore remains open during fusion, can be estimated. A soluble marker can be encapsulated in the SUVs for simultaneous monitoring of lipid and soluble cargo release. Such measurements indicate some pores may reseal after losing a fraction of the soluble cargo.

Here, we present a protocol to detect single, SNARE-mediated fusion events between liposomes and supported bilayers in microfluidic channels using polarized TIRFM, with single molecule sensitivity and ~15 msec time resolution. Lipid and soluble cargo release can be detected simultaneously. Liposome size, lipid diffusivity, and fusion pore properties are measured.

In the ubiquitous process of membrane fusion the opening of a fusion pore establishes the first connection between two formerly separate compartments. ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Biology

Imaging Plasma Membrane Deformations With pTIRFM

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during the fusion of vesicle and plasma membranes. These rapid and localized changes are achieved by dynamic interactions between lipids and specialized proteins that control membrane curvature. The absence of such interactions would not only have devastating consequences for vesicle fusion, but a host of other cellular functions that involve control of membrane shape. In recent years, the identity of a number of proteins with membrane-shaping properties has been determined. What remains missing is a roadmap of when, where, and how they act as fusion and content release progress.
Our understanding of the molecular events that enable membrane remodeling has historically been limited by a lack of analytical methods that are sensitive to membrane curvature or have the temporal resolution to track rapid changes. PTIRFM satisfies both of these criteria. We discuss how pTIRFM is implemented to visualize and interpret rapid, submicron changes in the orientation of chromaffin cell membranes during dense core vesicle (DCV) fusion. The chromaffin cells we use are isolated from bovine adrenal glands. The membrane is stained with a lipophilic carbocyanine dye,1,1&#39;-dioctadecyl-3,3,3&#39;,3&#39;-tetramethylindodicarbocyanine, 4-chlorobenzenesulfonate, or&#160;diD. DiD intercalates in the membrane plane with a &#34;fixed&#34; orientation and is therefore sensitive to the polarization of the evanescent field. The diD-stained cell membrane is sequentially excited with orthogonal polarizations of a 561&#160;nm laser (p-pol, s-pol). A 488&#160;nm laser is used to visualize vesicle constituents and time the moment of fusion. Exocytosis is triggered by locally perfusing cells with a depolarizing KCl solution. Analysis is performed offline using custom-written software to understand how diD emission intensity changes relate to fusion pore dilation.

Polarization-based Total Internal Reflection Fluorescence Microscopy (pTIRFM) enables real-time detection of cell membrane dynamics. This article describes the implementation of pTIRFM for the study of membrane remodeling during regulated exocytosis. The technique is generalizable to other processes in cell biology that directly or indirectly involve changes in membrane shape.

To gain novel insights into the dynamics of exocytosis, our group focuses on the changes in lipid bilayer shape that must be precisely regulated during ...