Name: pulling membrane nanotubes from giant unilamellar vesicles
Uploaded: 2017-12-07T12:30:00
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.

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 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.

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

Research

JoVE Journal

Biology

Measuring Peptide Translocation into Large Unilamellar Vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1. Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors1-3. Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms4,5, particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes6,7. In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides1,8. A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)9-13. LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies14,15. Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II16,17. In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties18, to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.

This protocol details a method for the quantitative measure of peptide translocation into large unilamellar lipid vesicles. This method also provides information about the rate of membrane translocation and can be used to identify peptides that efficiently and spontaneously cross lipid bilayers.

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors1.  Many of these are referred ...

Examining BCL-2 Family Function with Large Unilamellar Vesicles

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate apoptosis1. Following cellular stress (e.g., DNA damage), the pro-apoptotic BCL-2 family effectors BAK (BCL-2 antagonistic killer 1) and/or BAX (BCL-2 associated X protein) become activated and compromise the integrity of the outer mitochondrial membrane (OMM), though the process referred to as mitochondrial outer membrane permeabilization (MOMP)1. After MOMP occurs, pro-apoptotic proteins (e.g., cytochrome c) gain access to the cytoplasm, promote caspase activation, and apoptosis rapidly ensues2.
In order for BAK/BAX to induce MOMP, they require transient interactions with members of another pro-apoptotic subset of the BCL-2 family, the BCL-2 homology domain 3 (BH3)-only proteins, such as BID (BH3-interacting domain agonist)3-6. Anti-apoptotic BCL-2 family proteins (e.g., BCL-2 related gene, long isoform, BCL-xL; myeloid cell leukemia 1, MCL-1) regulate cellular survival by tightly controlling the interactions between BAK/BAX and the BH3-only proteins capable of directly inducing BAK/BAX activation7,8. In addition, anti-apoptotic BCL-2 protein availability is also dictated by sensitizer/de-repressor BH3-only proteins, such as BAD (BCL-2 antagonist of cell death) or PUMA (p53 upregulated modulator of apoptosis), which bind and inhibit anti-apoptotic members7,9. As most of the anti-apoptotic BCL-2 repertoire is localized to the OMM, the cellular decision to maintain survival or induce MOMP is dictated by multiple BCL-2 family interactions at this membrane.
 Large unilamellar vesicles (LUVs) are a biochemical model to explore relationships between BCL-2 family interactions and membrane permeabilization10. LUVs are comprised of defined lipids that are assembled in ratios identified in lipid composition studies from solvent extracted Xenopus mitochondria (46.5% phosphatidylcholine, 28.5% phosphatidylethanoloamine, 9% phosphatidylinositol, 9% phosphatidylserine, and 7% cardiolipin)10. This is a convenient model system to directly explore BCL-2 family function because the protein and lipid components are completely defined and tractable, which is not always the case with primary mitochondria. While cardiolipin is not usually this high throughout the OMM, this model does faithfully mimic the OMM to promote BCL-2 family function. Furthermore, a more recent modification of the above protocol allows for kinetic analyses of protein interactions and real-time measurements of membrane permeabilization, which is based on LUVs containing a polyanionic dye (ANTS: 8-aminonaphthalene-1,3,6-trisulfonic acid) and cationic quencher (DPX: p-xylene-bis-pyridinium bromide)11. As the LUVs permeabilize, ANTS and DPX diffuse apart, and a gain in fluorescence is detected. Here, commonly used recombinant BCL-2 family protein combinations and controls using the LUVs containing ANTS/DPX are described.

Biochemically-defined large unilamellar vesicles (LUVs) are a convenient model system to analyze BCL-2 family interactions with immediate implications in better understanding the mitochondrial pathway of apoptosis. A method to produce LUVs, along with standard BCL-2 family protein combinations and controls to examine LUV permeabilization, are presented.

The BCL-2 (B cell CLL/Lymphoma) family is comprised of approximately twenty proteins that collaborate to either maintain cell survival or initiate ...

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 ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular mechanisms regulating vascular permeability in cultured endothelial cell monolayers and the functional exchange properties of whole microvascular beds. A method to cannulate and perfuse venular microvessels of rat mesentery and measure the hydraulic conductivity of the microvessel wall is described. The main equipment needed includes an intravital microscope with a large modified stage that supports micromanipulators to position three different microtools: (1) a beveled glass micropipette to cannulate and perfuse the microvessel; (2) a glass micro-occluder to transiently block perfusion and enable measurement of transvascular water flow movement at a measured hydrostatic pressure, and (3) a blunt glass rod to stabilize the mesenteric tissue at the site of cannulation. The modified Landis micro-occlusion technique uses red cells suspended in the artificial perfusate as markers of transvascular fluid movement, and also enables repeated measurements of these flows as experimental conditions are changed and hydrostatic and colloid osmotic pressure difference across the microvessels are carefully controlled. Measurements of hydraulic conductivity first using a control perfusate, then after re-cannulation of the same microvessel with the test perfusates enable paired comparisons of the microvessel response under these well-controlled conditions. Attempts to extend the method to microvessels in the mesentery of mice with genetic modifications expected to modify vascular permeability were severely limited because of the absence of long straight and unbranched microvessels in the mouse mesentery, but the recent availability of the rats with similar genetic modifications using the CRISPR/Cas9 technology is expected to open new areas of investigation where the methods described herein can be applied.

The modified Landis technique enables paired measurement of the hydraulic conductivity of individual microvessels in the mesentery of normal and genetically modified rats under control and test conditions using microperfusion techniques. It provides a convenient method to evaluate mechanisms that regulate microvessel permeability and transvascular exchange under physiological conditions.

Experiments to measure the permeability properties of individually perfused microvessels provide a bridge between investigation of molecular and cellular ...

Ground State Depletion Super-resolution Imaging in Mammalian Cells

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to dramatic leaps in our understanding of how cells function. The development and availability of super-resolution microscopy has considerably extended the limits of optical resolution from ~250-20 nm. Biologists are no longer limited to describing molecular interactions in terms of colocalization within a diffraction limited area, rather it is now possible to visualize the dynamic interactions of individual molecules. Here, we outline a protocol for the visualization and quantification of cellular proteins by ground-state depletion microscopy&#160;for fixed cell imaging. We provide examples from two different membrane proteins, an element of the endoplasmic reticulum translocon, sec61&#946;, and a plasma membrane-localized voltage-gated L-type Ca2+ channel (CaV1.2). Discussed are the specific microscope parameters, fixation methods, photo-switching buffer formulation, and pitfalls and challenges of image processing.

This article outlines a protocol for the detection of one or more plasma and/or intracellular membrane proteins using Ground State Depletion (GSD) super-resolution microscopy in mammalian cells. Here, we discuss the benefits and considerations of using such approaches for the visualization and quantification of cellular proteins.

Advances in fluorescent microscopy and cell biology are intimately correlated, with the enhanced ability to visualize cellular events often leading to ...

Flow Cytometric Analysis of Extracellular Vesicles from Cell-conditioned Media

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for phenotypic analyses of extracellular vesicles (EV) including exosomes (Exo) in the peripheral blood and other body fluids. The small size of EV mandates the use of dedicated instruments having a detection threshold around 50-100 nm. Alternatively, EV can be bound to latex microbeads that can be detected by FC. Microbeads, conjugated with antibodies that recognize EV-associated markers/Cluster of Differentiation CD63, CD9, and CD81 can be used for EV capture. Exo isolated from CM can be analyzed with or without pre-enrichment by ultracentrifugation. This approach is suitable for EV analyses using conventional FC instruments. Our results demonstrate a linear correlation between Mean Fluorescence Intensity (MFI) values and EV concentration. Disrupting EV through sonication dramatically decreased MFI, indicating that the method does not detect membrane debris. We report an accurate and reliable method for the analysis of EV surface antigens, which can be easily implemented in any laboratory.

The protocol describes a reproducible method designed for use with cell culture supernatants to detect surface epitopes on small extracellular vesicles (EV). It utilizes specific EV immunoprecipitation using beads coupled with antibodies that recognize surface antigen CD9, CD63, and CD81. The method is optimized for downstream flow cytometry analysis.

Flow cytometry (FC) is the method of choice for semi-quantitative measurement of cell-surface antigen markers. Recently, this technique has been used for ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Detection and Quantification of Tunneling Nanotubes Using 3D Volume View Images

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely &#34;tunneling nanotubes&#34; (TNTs). TNTs are defined as open-ended, lipid bilayer-encircled membrane extensions that mediate continuity between neighboring cells of diameters ranging between 50 nm and 1 &#181;m. TNTs were demonstrated initially in neuronal cells, but successive studies have revealed the existence of TNTs in several cell types and diseases, such as neurodegenerative diseases, viral infections, and cancer. Several studies have referred to close-ended, electrically coupled membrane nanostructures between neighboring cells as TNTs or TNT-like structures.
The elucidation of ultrastructure in terms of membrane continuity at the endpoint is technically challenging. In addition, studies on cell-cell communication are challenging in terms of the characterization of TNTs using conventional methods due to the lack of specific markers. TNTs are primarily defined as F-actin-based, open-ended membrane protrusions. However, one major limitation is that F-actin is present in all types of protrusions, making it challenging to differentiate TNTs from other protrusions. One of the notable characteristics of F-actin-based TNTs is that these structures hover between two cells without touching the substratum. Therefore, distinct F-actin-stained TNTs can conveniently be distinguished from other protrusions such as filopodia and neurites based on their hovering between cells.
We have recently shown that the internalization of oligomeric amyloid-&#946;1-42 (oA&#946;) via actin-dependent endocytosis stimulates activated p21-activated kinase-1 (PAK1), which mediates the formation of F-actin-containing TNTs coexpressed with phospho-PAK1 between SH-SY5Y neuronal cells. This protocol outlines a 3D volume analysis method to identify and characterize TNTs from the captured z-stack images of F-actin- and phospho-PAK1-immunostained membrane protrusions in oA&#946;-treated neuronal cells. Further, TNTs are distinguished from developing neurites and neuronal outgrowths based on F-actin- and &#946;-III tubulin-immunostained membrane conduits.

Tunneling nanotubes (TNTs) are primarily open-ended F-actin membrane nanotubes that connect neighboring cells, facilitating intercellular communication. The notable characteristic that distinguishes TNTs from other cell protrusions is the hovering nature of the nanotubes between cells. Here, we characterize TNTs by constructing a 3D volume view of confocal z-stack images.

Recent discoveries have revealed that cells perform direct, long-range, intercellular transfer via nano-scale, actin-membrane conduits, namely ...

Production and Imaging of Recombinant Protein-Filled Vesicles from &lt;em&gt;E. coli&lt;/em&gt;

Optimized Production and Analysis of Recombinant Protein-Filled Vesicles from E. coli

This innovative system, using a short peptide tag, that exports multiple recombinant proteins in membrane bound vesicles from E. coli, provides an effective solution to a range of problems associated with bacterial recombinant protein expression. These recombinant vesicles compartmentalise proteins within a micro-environment that facilitates the production of otherwise challenging, toxic, insoluble, or disulfide-bond containing proteins from bacteria. Protein yield is increased considerably when compared to typical bacterial expression in the absence of the vesicle-nucleating peptide tag. The release of vesicle-packaged proteins supports isolation from the culture medium&#160;and permits long-term active protein storage. This technology gives rise to increased yields of vesicle-packaged, functional proteins for simplified downstream processing for a diverse range of applications from applied biotechnology to discovery science and medicine. In the present article and the associated video, a detailed protocol of the method is provided, which highlights key steps in the methodology to maximize recombinant protein-filled vesicle production.

Author Spotlight: The Production of Recombinant Proteins 

The present protocol describes a detailed method for the bacterial production of recombinant proteins, including typically insoluble or disulfide-bond containing proteins, packaged inside extracellular membrane-bound vesicles. This has the potential to be applied to versatile areas of scientific research, including applied biotechnology and medicine.