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.

1. Preparation of GUVs by Electroformation on Pt-wires
<ol>
	<li>Clean the electroformation chamber (see Introduction and Figure 1A) and the Pt-wires with an organic solvent such as ethanol or acetone to wash away the lipids and with water to wash away the salts. 
	NOTE: We suggest thoroughly wiping away the residue with ethanol-soaked tissue then sonicating in acetone, ethanol, then water, each for 5 min.</li>
	<li>Prepare a lipid mix of a desired lipid composition a...

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,<sup class="xre...

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/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-&#945;-phosphatidylcholine (eggPC)</td>
			<td>Avanti</td>
			<td>840051</td>
			<td>used for calibrating the tube radius constant</td>
		</tr>
		<tr>
			<td>&#946;-casein from bovine milk (&#62;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 (&#955; = 488 nm and 543 nm); optical tweezers induced by a 5 W ytterbium fiber continuous wave laser (&#955; &#62; 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 &#181;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/5...

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 ...

The overall goal of this nanotube pulling methodology is to quantitatively study the interactions between specific proteins of interest and highly curved membranes. This method can help answer key question in the biology of cell membranes, such as endocytosis and intracellular trafficking. This technique allow us to directly quantify how proteins bind to curved membranes and reciprocally deform them.Initially, this method was used to measure the basic mechanical properties of lipid membranes. Recently, it has been applied to elucidate important mechanisms in protein membrane interactions, particularly protein-shaping membranes. Begin by collecting the giant unilamellar vesicles directly from the platinum wires of the electroformation chamber.To construct the experimental chamber, place two rectangular glass cover slips one millimeter apart on a metallic base, leaving openings along the long edges large enough to allow a glass micropipette tip to reach to at least the center of the chamber. Fill both the experimental chamber and a glass micropipette with five milligrams per milliliter of a highly pure beta-casein solution, taking care to avoid bubbles. While the chamber is being passivated, mount it onto a light microscope centered above the objective, and insert the tip of the aspiration micropipette through the opening along the long edge until the tip is above the objective.Adjust the water tank level so that the aspiration pressure is near zero, and fill an injection micropipette with the molecule of interest dissolved in experimental buffer at the appropriate experimental concentration. Mount the injection micropipette inside a micromanipulator, and insert the micropipette through the other side of the experimental chamber. At the end of the incubation, remove the beta-casein solution from the chamber, and rinse the chamber several times with experimental buffer.Then, add a few microliters of vesicle solution to the chamber, followed by a few microliters of streptavidin-coated beads, to a final concentration of 0.1 times 10 to the negative 3%bead rate per volume or less. After the vesicles and beads have settled to the bottom of the chamber, allow the experimental buffer to evaporate for about 15 minutes, at which point the vesicles should visibly undulate under a light microscope. When the vesicles are appropriately floppy, aspirate individual vesicles into the micropipette until the lengths of the membranes within the pipette are equal to or larger than the pipette radius.When an appropriately sized aspiration tongue has been identified, seal the open edges of the chamber with mineral oil. To set the zero position of the aspiration pressure, position the end of the aspiration pipette near a bead, and adjust the height of the water tank so that the bead is neither sucked in nor blown away by the pipette. Adjusting the height of the water is critical to control the membrane tension of the vesicle that is aspirated by the micropipette.Aspirate a vesicle, and move the micropipette up and out of focus without breaking the micropipette. Using optical tweezers, trap a bead approximately 20 micrometers away from the bottom of the chamber. Bring the vesicle back into focus away from the bead and aligned with the optical trap, and record the movement of the bead for one to two minutes to measure the equilibrium position.Reduce the pressure inside the micropipette as much as possible without losing the vesicle to decrease the membrane tension, and carefully bring the vesicle in contact with the bead for around a second to establish the streptavidin-biotin bonds. Then, using a piezo actuator, gently pull the vesicle back to create a nanotube. Increase the aspiration pressure to recreate the aspiration tongue and align the tube within the axis of the aspiration pipette.Bring the tube and the vesicle fully into focus, and record the movement of the bead at a 30-hertz acquisition speed and the height of the water tank with respect to the zero position for a few minutes under bright-field microscopy. When all of the images have been obtained, record the movement of the bead again at different aspiration pressures and membrane tensions. To inject proteins or molecules of interest, bring the injection micropipette near the nanotube, taking care that the bead in the optical trap is not perturbed, and gently inject the proteins at one to two pascals of pressure.After the protein binding has equilibrated, repeat the stepwise measurements in the presence of the added proteins, as demonstrated for the bare membrane. Force and radius measurements on nanotubes pulled from 100%DOPC vesicles are independent, as the force is measured from the displacement of the bead in the optical trap and the radius from fluorescence intensity, providing two ways of calculating the membrane bending rigidity. The relative enrichment of BAR or potassium channel proteins on membrane tubes over the underlying vesicles indicates that the proteins are more likely to bind to curved membranes.Indeed, binding of the N-BAR protein endophilin A2 to the membrane gradually reduces the tube force to zero, indicating the formation of a three-dimensional scaffold that keeps the tube stable. Following this procedure, other techniques can be combined to answer additional questions, for instance, high-resolution microscopy to detect protein organization on membrane tubes or single particle tracking to measure protein mobility.

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10.8K Görüntüleme.  PSL Research University, CNRS UMR168. The overall goal of this nanotube pulling methodology is to quantitatively study the interactions between specific proteins of interest and highly curved membranes. This method can help answer key question in the biology of cell membranes, such as endocytosis and intracellular trafficking. This technique allow us to directly quantify how proteins bind to curved membranes and reciprocally deform them.Initially, this method was used to measure the basic mechanical properties of lipid mem...