Name: pulling membrane nanotubes from giant unilamellar vesicles
Uploaded: 2017-12-07T12:30:00.000+00:00
Duration: 6 min 26 s
Description: Watch this Scientific Journal Video about Pulling Membrane Nanotubes from Giant Unilamellar Vesicles at JoVE.com

The authors thank Benoit Sorre, Damien Cuvelier, Pierre Nassoy, Fran&#231;ois Quemeneur, and Gil Toombes for their essential contributions to establish the nanotube method in the group. The P.B. group belongs to the CNRS consortium CellTiss, to the Labex CelTisPhyBio (ANR-11-LABX0038), and to Paris Sciences et Lettres (ANR-10-IDEX-0001-02). F.-C. Tsai was funded by the EMBO Long-Term fellowship (ALTF 1527-2014) and Marie Curie actions (H2020-MSCA-IF-2014, project membrane-ezrin-actin). M.S. is a Junior Fellow of the Simons Society of Fellows.

The authors have nothing to disclose.

The method of pulling tubes from GUVs gives rich information on the membrane-protein system, as it is not only the means to measure the fundamental mechanical properties of the membrane, but it helps to shed light on the coupling between proteins and membrane curvature. As discussed in the Introduction, other techniques exist to measure the effects of membrane-curving proteins, either by incubating the proteins with sub-micron liposomes tethered to a passivated surface18,19 or by observing the spontaneous formation of tubules in micropipette-aspirated GUVs upon the injection of a protein20,21. The method described here provides direct geometric connection to endocytosis and is unique in providing the important physical measurements of the system at the same time, such as protein density, membrane tension, and membrane force.
An important limitation of the method is that it is not trivial to construct and calibrate all the components involved in the assay. However, while a combination of confocal microscopy, optical tweezers, and micromanipulation provides maximal information, some components can be substituted as discussed in the Introduction, depending on the information that needs to be learned about the system. The minimal components required to successfully probe the curvature coupling of a protein, or any other potential curvature-coupled molecule, are a micropipette aspirator and a confocal microscope, with the optical tweezers being dispensable. Importantly, many of the calibrations (such as the calibration for membrane tube radius and for determining protein surface density) are typically only needed to be performed once. Another limitation of the method is that it can measure only one GUV at a time, amounting to two to three GUVs per hour (with practice); however each measurement provides a wealth of data.
For higher throughput measurements or if it is essential to study the interaction of proteins with spherical and not tubular geometries, other assays will need to be explored or developed. Future efforts will be devoted to: (1) designing a higher-throughput system in which multiple tubes (from multiple GUVs) could be pulled and measured at the same time, and (2) integrating super-resolution microscopy so to study the details of the protein scaffold that forms on the nanotubes.

Many cellular processes, such as endocytosis, trafficking, the formation of filopodia, infection, etc., are accompanied by a dramatic change in the shape of cell membranes1,2. In the cell, a number of proteins participate in these processes by binding to the membrane and altering their shape. The most notable examples are members of the Bin/Amphiphysin/Rvs (BAR) protein family, containing a characteristic intrinsically curved BAR domain3,4,5,6,7. Typically, they interact with the membrane by adhering the BAR domain to the surface and, in many cases, also shallowly inserting amphipathic helices into the bilayer. The shape, size, and charge of the BAR domain together with the number of amphipathic helices determines: (1) the direction of membrane curvature (i.e., whether they will induce invaginations or protrusions), and (2) the magnitude of membrane curvature5,8. Of note, here positive curvature is defined as the convex side of the curved membrane, i.e., the bulge toward the interacting particle, and negative otherwise. Moreover, quantitative studies of BAR proteins revealed that their effect on the membrane depends on a set of physical parameters: surface density of proteins, membrane tension, and membrane shape (flat versus tubular versus spherical shape)7. Depending on these parameters BAR proteins can: (1) act as sensors of membrane curvature, (2) bend membranes, or (3) induce membrane scission7.
Due to the sheer number of components involved in membrane reshaping in the cell, studying the quantitative aspects of the phenomena, such as endocytosis, in vivo is extremely challenging. In vitro reconstitution of minimal components mimicking curved membranes in the cell provides means to gain a mechanistic understanding of how membrane-curving proteins operate. This article describes a protocol to reconstitute a membrane nanotube in vitro using micromanipulation, confocal microscopy, and optical tweezers. The approach can be used to study, in a quantitative way, how proteins, lipids, or small molecules interact with curved membranes. Lipid GUVs are used as models of a cell membrane, whose curvature is negligible compared to the size of interacting membrane-curving molecules. They are prepared using the electroformation method9 in which the vesicles are formed by hydrating a lipid film and swelling it into GUVs under an alternating current (AC)10. Most common substrates on which GUVs are grown are either semi-conductive plates coated with indium tin oxide (ITO) or platinum wires (Pt-wires)11. In this work, GUVs are grown on Pt-wires as this method has been shown to work much better than the alternative in making GUVs in the presence of salts in the buffer12. Although the electroformation protocol is described here in sufficient detail to reproduce it, we refer the reader to previous articles in which similar and other methods of making GUVs have been described in detail13,14. In our hands, electroformation on Pt-wires has successfully yielded GUVs from a mix of synthetic lipids or from natural lipid extracts in a buffer containing ~100 mM NaCl. Furthermore, it was also possible to encapsulate proteins inside GUVs during growth. An example electroformation chamber is shown in Figure 1A; it comprises two ~10-cm-long Pt-wires inserted into a holder made from polytetrafluoroethylene (PTFE) that can be sealed on both sides with glass coverslips ~1 - 2 cm apart (Figure 1A).
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56086/56086fig1.jpg" /> 
Figure 1: Experimental setup. (A) The GUV electroformation chamber with electrical connectors attached to Pt-wires. (B) Left: the experimental system showing the microscope, the experimental chamber above the objective and two micropipettes (left and right) attached to the micromanipulators and inserted into the experimental chamber for tube pulling and protein injection. Right: a close-up view of the experimental chamber mounted above the objective showing the tips of the aspiration and the injection micropipettes inserted. (C) A syringe equipped with a thin dispenser inserted into a micropipette at its back end. The bottom is a close-up view of the dispenser inside the micropipette with the blue dotted line outlining the micropipette. This system is used to fill the micropipette with casein to passivate the glass surface and also to back fill with mineral oil when needed. (D) A system used to aspirate &#181;L quantities of the protein solution. The needle is connected to a syringe and to tubing which is connected to the injection micropipette. The micropipette tip is carefully immersed into the protein solution and aspirated so to fill the micropipette tip. The micropipette is then back filled with mineral oil using the system shown in panel C. <a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56086/56086fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A membrane nanotube, ranging in radius from 7 nm to several hundred nm, can be pulled from a GUV by an external force. This method was initially designed to measure the elastic properties of cell membranes and vesicles, such as the bending rigidity15,16. In most recent works, the method was extended to study the interaction of proteins with curved membranes by microinjecting the proteins near the pulled nanotube7,17. Other methods have been developed for studying membrane-curving proteins. In one method, proteins are incubated with differently sized liposomes tethered to a passivated surface. Confocal microscopy is used to measure the protein binding as a function of liposome diameter, which can indicate curvature-induced sorting18,19. In another method, proteins are injected near a micro-aspirated GUV to measure their ability to spontaneously induce tubules20,21. The method described in this protocol is uniquely suited to study membrane-curving proteins involved in endocytosis, where most proteins typically encounter preformed membrane nanotubes connecting the cargo-containing membrane invagination with the underlying flat plasma membrane. Furthermore, in this method, unlike in the assay with tethered small liposomes, the membrane nanotube is continuously connected to the membrane; therefore, it is in mechanical equilibrium with the GUV, a situation expected in vivo. Hence, fundamental membrane physics applies and we can infer a plethora of mechanical properties from our measurements22,23,24.
For a full implementation of this method, the necessary equipment includes a confocal microscope, optical tweezers, and one or two micropipettes connected to a water tank (Figure 1B). By combining all three, it is possible to simultaneously measure membrane tension, membrane curvature, surface density of proteins, and tube force25. Micropipette aspiration is essential and it is easily constructed by inserting a glass micropipette into a holder connected to a water tank, which, via hydrostatic pressure, controls the aspiration pressure26. The micropipette and the holder are controlled by a micromanipulator and, ideally, in one direction by a piezo-actuator for precision movement. To pull a nanotube, the microaspirated GUV is briefly stuck to a micron-sized bead then pulled away creating a nanotube. In this implementation, the bead is held by optical tweezers, which can be constructed by following a published protocol27. It is possible to dispense of the optical tweezers and pull nanotubes in different ways, although at the cost of accurate force measurements. If it is too challenging to build an optical trap or if force measurements are not essential, such as if one simply wants to check the preference of proteins for curved membranes, a tube can be pulled using a bead aspirated at the tip of a second micropipette28. It is also possible to pull tubes using gravitational force29 or flow30,31. Furthermore, confocal microscopy is not essential either; however, it is preferred so to measure the surface density of proteins. It also allows measuring the nanotube radius from fluorescence intensity of lipids in the tube, thus independently of membrane force and tension. Inferring tube radius from fluorescence is particularly important if the relationship between these quantities deviates from well-established equations due to the presence of membrane-adhered proteins25. Importantly, one cannot dispense of both the optical trap and confocal microscopy, as it will not be possible to measure the tube curvature.
The method as described in this protocol has been used to study the curvature-induced sorting of various peripheral membrane proteins on nanotubes, mostly those from the BAR family25,32,33,34. It was also shown that the conically shaped transmembrane potassium channel KvAP is enriched on curved nanotubes in the same way as BAR proteins35. By optimizing the method to encapsulate proteins inside GUVs, the interaction of proteins with negative curvature has been recently investigated as well36. Furthermore, this method has been used to elucidate the formation of protein scaffolds25,37 and to study the mechanism of membrane scission by either line tension38, protein dynamin39, or by BAR proteins40,41. In addition to proteins, small molecules or ions can also induce curvature. Using this method, calcium ions were shown to induce positive curvature under salt-free conditions42. Interestingly, it has also been shown that lipids can undergo curvature sorting, although only for compositions that are near a demixing point43,44. In sum, the method can be used by researchers interested in investigating how either integral membrane components (e.g., lipids or transmembrane proteins) or peripherally binding molecules (either inside or outside GUVs) interact with cylindrically curved membranes, from mechanical and quantitative points of view. It is also intended for those interested in measuring the mechanical properties of the membrane itself22,23,45.

<table><tbody><tr><td>1,2-Dioleoyl-sn-glycero-3-phosphatidylcholine (DOPC)</td><td>Avanti</td><td>850375</td><td>Example lipid used in data for Figure 3</td></tr><tr><td>1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[biotinyl(polyethyleneglycol)-2000] [DSPE-PEG(2000)-biotin]</td><td>Avanti</td><td>880129</td><td>biotinylated lipid</td></tr><tr><td>BODIPY-TR-C5-ceramide</td><td>Molecular Probes (ThermoFisher)</td><td>D7540</td><td>fluorescent lipid</td></tr><tr><td>BODIPY- FLC5-hexadecanoyl phosphatidylcholine (HPC*)</td><td>Molecular Probes (ThermoFisher)</td><td></td><td>fluorescent lipid for protein density calibration</td></tr><tr><td>egg L-&amp;alpha;-phosphatidylcholine (eggPC)</td><td>Avanti</td><td>840051</td><td>used for calibrating the tube radius constant</td></tr><tr><td>&amp;beta;-casein from bovine milk (&gt;99%)</td><td>Sigma Aldrich</td><td>C6905</td><td>used for passivating the micropipette and the experimental chamber</td></tr><tr><td>Sucrose</td><td>Sigma Aldrich</td><td>S7903</td><td></td></tr><tr><td>D(+) glucose</td><td>Sigma Aldrich</td><td>G7021</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>Tris</td><td>Sigma Aldrich</td><td>10708976001</td><td></td></tr><tr><td>osmometer</td><td>Loser</td><td>n/a</td><td></td></tr><tr><td>Pt-wires, 0.5 mm diameter, 99.99% pure</td><td>Goodfellow USA</td><td>PT005139</td><td>used for GUV electroformation</td></tr><tr><td>function generator</td><td></td><td>n/a</td><td>used to create current for GUV electroformation</td></tr><tr><td>putty sealant</td><td>Vitrex (from CML France)</td><td>CRIT 140013</td><td>used to seal the electroformation chamber</td></tr><tr><td>bath sonicator</td><td></td><td>n/a</td><td>useful to clean the electroformation chamber, but not crucial</td></tr><tr><td>Nikon TE2000 inverted microscope, eC1 confocal system (Nikon), with two laser lines (&amp;lambda; = 488 nm and 543 nm); optical tweezers induced by a 5 W ytterbium fiber continuous wave laser (&amp;lambda; &gt; 1070 nm; IPG GmBH Germany)</td><td></td><td></td><td>an example of a confocal microscopy system equipped with optical tweezers</td></tr><tr><td>micromanipulators</td><td></td><td>n/a</td><td></td></tr><tr><td>borosilicate capillaries (with internal and external radii of 0.78 mm and 1 mm, respectively)</td><td>Harvard apparatus</td><td>30-0036</td><td></td></tr><tr><td>micropipette puller</td><td>Sutter Instrument</td><td>P-2100</td><td></td></tr><tr><td>microforge</td><td>Narishige</td><td>MF-800</td><td></td></tr><tr><td>piezoelectric actuator</td><td>Physik Instrumente</td><td>n/a</td><td></td></tr><tr><td>polystyrene streptavidin coated beads (diameter 3.2 &amp;micro;m)</td><td>Spherotech</td><td>SVP-30-5</td><td></td></tr></tbody></table>

pulling membrane nanotubes from giant unilamellar vesicles

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many different proteins associate with curved membranes because of their ability to sense or induce membrane curvature. Typically, these processes involve a multitude of proteins making them too complex to study quantitatively in the cell. We describe a protocol to reconstitute a curved membrane in vitro, mimicking a curved cellular structure, such as the endocytic neck. A giant unilamellar vesicle (GUV) is used as a model of a cell membrane, whose internal pressure and surface tension are controlled with micropipette aspiration. Applying a point pulling force on the GUV using optical tweezers creates a nanotube of high curvature connected to a flat membrane. This method has traditionally been used to measure the fundamental mechanical properties of lipid membranes, such as bending rigidity. In recent years, it has been expanded to study how proteins interact with membrane curvature and the way they affect the shape and the mechanics of membranes. A system combining micromanipulation, microinjection, optical tweezers, and confocal microscopy allows measurement of membrane curvature, membrane tension, and the surface density of proteins, concurrently. From these measurements, many important mechanical and morphological properties of the protein-membrane system can be inferred. In addition, we lay out a protocol of creating GUVs in the presence of physiological salt concentration, and a method of quantifying the surface density of proteins on the membrane from fluorescence intensities of labeled proteins and lipids.

The tube-pulling experiment can give vital mechanical information about the membrane. In the absence of proteins or other molecules that couple with membrane curvature, the membrane force and tube radius can be related with membrane tension by applying the Canham-Helfrich Hamiltonian equation to a tube pulled from a GUV50,51
<img alt="Equation 3" src="/files/ftp_upload/56086/56086eq3v6.jpg" /> (Equation 1)
where <img alt="Equation 4" src="/files/ftp_upload/56086/56086eq4v3.jpg" /> is the membrane free energy of the tube, K membrane bending rigidity, r and R0 are, respectively, the mean and the spontaneous radii of curvature, &#963; membrane tension, A the area of the tube, L the length of the tube, and f the point force exerted by the tube on the bead. At equilibrium, r is the tube radius, denoted as R, and f becomes the tube-retraction force, F, i.e., the equilibrium force by which the tube is pulling the bead in the optical trap. To compute F and R, we minimize <img alt="Equation 4" src="/files/ftp_upload/56086/56086eq4v3.jpg" /> with respect to L and r, yielding22,23
<img alt="Equation 5" src="/files/ftp_upload/56086/56086eq5v4.jpg" /> (Equation 2)
<img alt="Equation 6" src="/files/ftp_upload/56086/56086eq6v5.jpg" /> (Equation 3)
R0 is neglected if both leaflets of the membrane have the same composition, although it is speculated that other factors, such as charge repulsions can induce some spontaneous curvature42. Force and radius measurements on tubes pulled from 100% DOPC GUVs are in excellent agreement with theoretical predictions in Equation 2 and Equation 3 (Figure 4). The two measurements are independent as the force is measured from the displacement of the bead in the optical trap and the radius from fluorescence intensity, therefore providing two ways of calculating the membrane bending rigidity. Fitting Equation 2 and Equation 3 to the shown data yields: K = (25.1 &#177; 1.4) (mean &#177; SD) kBT and (22.1 &#177; 1.5) kBT, respectively, as previously measured42.
If experimenting with curvature-coupling proteins, it is suggested to use the more sophisticated Hamiltonian&#39;s equation than the one in Equation 1, as Equation 1 only accounts for an effective spontaneous curvature and does not account for the effect of proteins on the membrane&#39;s mechanical properties or possible protein-protein interactions. See, e.g.,25,35,36,52,53. Although advanced theoretical models are essential for an in-depth understanding of a specific protein-membrane system, without resorting to them, it is still possible to obtain very useful information from the tube-pulling method.
For example, to test if proteins sense curvature, the relative intensity of proteins on the tube is measured compared to the GUV, which is considered flat. The interaction with positive curvature is studied by injecting the proteins near the tube with the second micropipette25. The interaction with negative curvature or the behavior of transmembrane proteins is studied by encapsulating or reconstituting proteins during GUV formation35,36. The relative enrichment of BAR and KvAP proteins on membrane tubes over the underlying GUVs indicates that they prefer curved membranes (Figure 5). To be more quantitative, this enrichment may be calculated as a sorting coefficient, S, according to:
<img alt="Equation 7" src="/files/ftp_upload/56086/56086eq7v4.jpg" /> (Equation 4)
where Iprot,tub and Iprot,GUV are the fluorescence intensities of proteins on the tube and on the GUV, respectively, and Ilip,tub and Ilip,GUV are the fluorescence intensities of lipids on the tube and on the GUV, respectively.
Another example is measuring the tube force as the protein binds to the membrane. Figure 5B,C shows that an N-BAR protein endophilin A2 gradually reduces the tube force, decreasing it to zero, indicating that it has formed a three-dimensional structure that keeps the tube stable, which is termed a scaffold. Such structures play important roles in endocytosis, by fixing the tube radius but also in enabling scission7,41. Measurement of S, as well as the mechanical and morphological effects that proteins impose on membranes provides important insights into how they operate in the membrane-bending phenomena in the cell.

<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56086/56086fig4.jpg" /> 
Figure 4: Representative results of mechanical measurements in the absence of proteins. Shown are linearized force and tube radius dependence on membrane tension for a 100% DOPC membrane, from three independent measurements, published previously42. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56086/56086fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56086/56086fig5.jpg" /> 
Figure 5: Representative results of curvature sensing and scaffold formation by BAR proteins. (A) Curvature sensing by the I-BAR protein IRSp53. The protein senses negative membrane curvature and is bound on the inside of the GUV, with negligible amount of protein present on outer membrane leaflet. Scale bar = 5 &#181;m. Reprinted from36, under the Creative Commons CC-BY license, Copyright 2015, Nature Publishing Group. (B) Forming a protein scaffold by the N-BAR protein endophilin. Proteins are injected near the tube and are therefore bound on the outer membrane leaflet. As described in37, endophilin binds to the tube&#39;s base and forms a scaffold that continuously grows along the tube from the GUV toward the optical trap (OT, white circle). (C) A kymogram of endophilin scaffold growth from the GUV to the bead, with the lipid and protein channels overlaid. The plot shows tube force, F, as a function of time, t. The timescales in the kymogram and the plot are the same (x-axis is shared). In B and C, Scale bar = 2 &#181;m. Reprinted with permission from reference37, Copyright (2016) National Academy of Sciences. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56086/56086fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Pulling Membrane Nanotubes from Giant Unilamellar Vesicles at JoVE.com

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

Many proteins in the cell sense and induce membrane curvature. We describe a method to pull membrane nanotubes from lipid vesicles to study the interaction of proteins or any curvature-active molecule with curved membranes in vitro.

The reshaping of the cell membrane is an integral part of many cellular phenomena, such as endocytosis, trafficking, the formation of filopodia, etc. Many ...

pulling-membrane-nanotubes-from-giant-unilamellar-vesicles

Nanyang Technological University

Research

JoVE Journal

Biology

10.9K Views.  PSL Research University, CNRS UMR168. Veel proteïnen in de cel zin en membraan kromming veroorzaken. Beschrijven we een methode om te halen membraan nanotubes van lipide blaasjes te bestuderen de interactie van proteïnen of elke kromming-actieve molecuul met gebogen membranen in vitro.

Video: Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

Preparation of Giant Vesicles Encapsulating Microspheres by Centrifugation of a Water-in-oil Emulsion

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological materials. Such approaches have received considerable attention in research on the boundary between living and nonliving matter and have been used to construct artificial cells over the past two decades. In particular, Giant Vesicles (GVs) have often been used as artificial cell membranes. In this paper, we describe the preparation of GVs encapsulating highly packed microspheres as a model of cells containing highly condensed biomolecules. The GVs were prepared by means of a simple water-in-oil emulsion centrifugation method. Specifically, a homogenizer was used to emulsify an aqueous solution containing the materials to be encapsulated and an oil containing dissolved phospholipids, and the resulting emulsion was layered carefully on the surface of another aqueous solution. The layered system was then centrifuged to generate the GVs. This powerful method was used to encapsulate materials ranging from small molecules to microspheres.

Giant vesicles containing highly packed micrometer-sized components are useful cell models. The water-in-oil emulsion centrifugation method is a simple, powerful tool for the preparation of giant vesicles with encapsulated materials.

The constructive biology and the synthetic biology approach to creating artificial life involve the bottom-up assembly of biological or nonbiological ...

Biochemistry

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.

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 ...

Chemistry

Lipid Bilayer Vesicle Generation Using Microfluidic Jetting

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This emerging field engages engineers, chemists, biologists, and physicists to design and assemble basic biological components into complex, functioning systems from the bottom up. Such bottom-up systems could lead to the development of artificial cells for fundamental biological inquiries and innovative therapies1,2. Giant unilamellar vesicles (GUVs) can serve as a model platform for synthetic biology due to their cell-like membrane structure and size. Microfluidic jetting, or microjetting, is a technique that allows for the generation of GUVs with controlled size, membrane composition, transmembrane protein incorporation, and encapsulation3. The basic principle of this method is the use of multiple, high-frequency fluid pulses generated by a piezo-actuated inkjet device to deform a suspended lipid bilayer into a GUV. The process is akin to blowing soap bubbles from a soap film. By varying the composition of the jetted solution, the composition of the encompassing solution, and/or the components included in the bilayer, researchers can apply this technique to create customized vesicles. This paper describes the procedure to generate simple vesicles from a droplet interface bilayer by microjetting.

Microfluidic jetting against a droplet interface lipid bilayer provides a reliable way to generate vesicles with control over membrane asymmetry, incorporation of transmembrane proteins, and encapsulation of material. This technique can be applied to study a variety of biological systems where compartmentalized biomolecules are desired.

Bottom-up synthetic biology presents a novel approach for investigating and reconstituting biochemical systems and, potentially, minimal organisms. This ...

Bioengineering

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 ...

Obtention of Giant Unilamellar Hybrid Vesicles by Electroformation and Measurement of their Mechanical Properties by Micropipette Aspiration

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular recognition within biosensors for diagnosis, functional membranes for artificial cells, and development of bioinspired micro/nano-reactors. In all of these applications, the characterization of their membrane properties is of fundamental importance. Among existing characterization techniques, micropipette aspiration, pioneered by E. Evans, allows the measurement of mechanical properties of the membrane such as area compressibility modulus, bending modulus and lysis stress and strain. Here, we present all the methodologies and detailed procedures to obtain giant vesicles from the thin film of a lipid or copolymer (or both), the manufacturing and surface treatment of micropipettes, and the aspiration procedure leading to the measurement of all the parameters previously mentioned.

The goal of the protocol is to reliably measure membrane mechanical properties of giant vesicles by micropipette aspiration.

Giant vesicles obtained from phospholipids and copolymers can be exploited in different applications: controlled and targeted drug delivery, biomolecular ...

Spontaneous Formation and Rearrangement of Artificial Lipid Nanotube Networks as a Bottom-Up Model for Endoplasmic Reticulum

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense lipidic nanotubes that are, in terms of morphology and dynamics, reminiscent of ER. The networks are derived from phospholipid double bilayer membrane patches adhering to a transparent Al2O3 substrate. The adhesion is mediated by Ca2+ in the ambient buffer. Subsequent depletion of Ca2+ by means of BAPTA/EDTA causes retraction of the membrane, resulting in spontaneous lipid nanotube network formation. The method only comprises phospholipids and microfabricated surfaces for simple formation of an ER model and does not require the addition of proteins or chemical energy (e.g., GTP or ATP). In contrast to the 3D morphology of the cellular endoplasmic reticulum, the model is two-dimensional (albeit the nanotube dimensions, geometry, structure, and dynamics are maintained). This unique in vitro ER model consists of only a few components, is easy to construct, and can be observed under a light microscope. The resulting structure can be further decorated for additional functionality, such as the addition of ER-associated proteins or particles to study transport phenomena among the tubes. The artificial networks described here are suitable structural models for the cellular ER, whose unique characteristic morphology has been shown to be related to its biological function, whereas details regarding formation of the tubular domain and rearrangements within are still not completely understood. We note that this method uses Al2O3 thin-film-coated microscopy coverslips, which are commercially available but require special orders. Therefore, it is advisable to have access to a microfabrication facility for preparation.

Solid-supported, protein-free, double phospholipid bilayer membranes (DLBM) can be transformed into complex and dynamic lipid nanotube networks and can serve as 2D bottom-up models of the endoplasmic reticulum.

We present a convenient method to form a bottom-up structural organelle model for the endoplasmic reticulum (ER). The model consists of highly dense ...

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (&#60;30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

This protocol describes a process for fabricating lipid nanotube networks using gliding kinesin motility in conjunction with giant unilamellar lipid vesicles.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous ...

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 ...

Cell-Free Expression of Membrane Proteins in Giant Unilamellar Vesicles

Reconstitution of the Bacterial Glutamate Receptor Channel by Encapsulation of a Cell-Free Expression System

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy regeneration in synthetic cells. Reconstruction of a wide range of cellular processes, however, requires successful reconstitution of membrane proteins into the membrane of synthetic cells. While the expression of soluble proteins is usually successful in common CFE systems, the reconstitution of membrane proteins in lipid bilayers of synthetic cells has proven to be challenging. Here, a method for reconstitution of a model membrane protein, bacterial glutamate receptor (GluR0), in giant unilamellar vesicles (GUVs) as model synthetic cells based on encapsulation and incubation of the CFE reaction inside synthetic cells is demonstrated. Utilizing this platform, the effect of substituting the N-terminal signal peptide of GluR0 with proteorhodopsin signal peptide on successful cotranslational translocation of GluR0 into membranes of hybrid GUVs is demonstrated. This method provides a robust procedure that will allow cell-free reconstitution of various membrane proteins in synthetic cells.

Author Spotlight: Membrane Protein Reconstitution in Synthetic Cells

This protocol describes the inverted emulsion method used to encapsulate a cell-free expression (CFE) system within a giant unilamellar vesicle (GUV) for the investigation of the synthesis and incorporation of a model membrane protein into the lipid bilayer.

Cell-free expression (CFE) systems are powerful tools in synthetic biology that allow biomimicry of cellular functions like biosensing and energy ...

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...

Pulling Membrane Nanotubes from Giant Unilamellar Vesicles

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