Name: in vitro investigation of the mexab efflux pump from pseudomonas aeruginosa
Uploaded: 2014-02-17T21:30:00
Duration: 13 min 40 s
Description: Watch this Scientific Journal Video about In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa at JoVE.com

This study was supported by the Agence Nationale de la Recherche (project ANR-2010-BLAN-1535) and by a grant from R&#233;gion Ile-de-France (DIM-Malinf 110058).

1. Protein Production and Purification
<ol>
	<li>MexA, MexB from Pseudomonas aeruginosa
	<ol>
		<li>Transform the plasmids harboring the MexA and MexB genes in an E. coli production strain (e.g. C43 DE3). Plate on LB Agar medium supplemented with the appropriate antibiotic. Pick a single colony to inoculate an O/N&#160;preculture. The next morning, inoculate the preculture in 1&#160;L of LB and grow at 37 &#176;C under agitation (240 rpm).</li>
		<li>Once the cells have reached the exponential phase (OD600&#160;= 0.6-0.8) induce with 1 mM IPTG. Perform induction for 2-3 hr&#160;at 30 &#176;C under agitation.</li>
		<li>Centrifuge cells (9,000 x g for 20 min) and resuspend&#160;in 30 ml Tris-HCl 20 mM, pH 8, NaCl 150 mM.</li>
		<li>Disrupt cells using a French press (10,000 psi, 4 pass). Discard unbroken cells and inclusion bodies by centrifuging at 9,000 x g for 20 min.</li>
		<li>Pellet membranes 1 hr at 100,000 x g and resuspend each pellet in 30 ml Bis-Tris 10 mM pH 7.4, glycerol 20% (w/v), imidazole 10 mM, NaCl 500 mM.</li>
		<li>Determine the total membrane protein concentration using the BCA colorimetric assay and solubilize the membranes O/N&#160;with 2% dodecyl maltoside (DDM) in a final volume determined so that detergent: protein is 1:1.5 (w/w).</li>
		<li>Ultracentrifuge at 100,000 x g for 1 hr to discard non solubilized and aggregated material.</li>
		<li>Incubate the supernatant&#160;for 2 hr at 4 &#176;C with a nickel resin (MexA purification) or a cobalt resin (MexB purification) equilibrated with Bis-Tris 10 mM pH 7.4, glycerol 5% (w/v), imidazole 10 mM, NaCl 500 mM, DDM 0.2%.</li>
		<li>Pour the resin in an empty column. Collect the Flow Through.</li>
		<li>Wash the resin with 50 ml of Bis-Tris 10 mM pH 7.4, glycerol 20% (w/v), imidazole 10 mM, NaCl 500 mM, DDM 0.2%, then with 25 ml of Bis-Tris 10 mM pH 6, glycerol 5% (w/v), imidazole 10 mM, NaCl 500 mM, DDM 0.2%.</li>
		<li>Elute the protein with 20 ml Bis-Tris 10 mM pH 7.4, glycerol 5% (w/v), imidazole 300 mM, NaCl 500 mM, DDM 0.2%.</li>
		<li>Concentrate the protein with an ultrafiltration device down to 3 ml and load on a desalting column equilibrated with an imidazole-free buffer. Just before reconstitution (see below), ultracentrifuge the solubilized proteins (100,000 x g for 20 min) to get rid of lipids and unsolubilized proteins. Then concentrate the protein again to 0.3 ml.</li>
	</ol>
	</li>
	<li>Bacteriorhodopsin from Halobacter salinarium
	<ol>
		<li>Grow Halobacter salinarium cells under illumination at 37 &#176;C for 10 days in a liquid growth medium containing NaCl 4 M, MgSO4 150 mM, citrate 10&#160;mM, KCl 30 mM, yeast extract 5 g/L, and peptone 5 g/L.</li>
		<li>Disrupt cells by dialyzing against deionized water.</li>
		<li>Purify the purple membrane on a 30-40% (w/v) sucrose gradient (100,000 x g for 17 hr).</li>
		<li>Solubilize the membrane suspension with 2% octylglucoside for 24 hr at 37 &#176;C (volume for the solubilization in order to resuspend the membrane at a 2:1 (w/w) detergent : protein ratio). Estimate the concentration of solubilized BR using &#949; (570 nm) = 54,000 M-1&#160;cm-1. Just before reconstitution (see below), ultracentrifuge the solubilized BR (100,000 x g for 20 min) to get rid of lipids and unsolubilized proteins. 
		Note: The native membranes are naturally enriched in BR so no further purification is needed.</li>
	</ol>
	</li>
</ol>
2. Preparation of Proteoliposomes
<ol>
	<li>Liposome formation: Put 400&#160;&#956;l&#160;of DOPC dissolved in chloroform (25 mg/ml) in a glass beaker and add 1.5 mg of cholesterol stored as a powder. Dry them just before manipulation under a steam of nitrogen or vacuum for at least 1&#160;hr in a glass beaker. The DOPC:cholesterol molar ratio is 3.3:1.
	<ol>
		<li>After they have dried, hydrate the lipids with 1 ml&#160;of HEPES 25 mM pH 7, K2SO4 100 mM, MgCl2 2 mM, pyranine 2 mM. The suspension should appear turbid at this stage.</li>
		<li>Heat the solution for 10 min&#160;at 37 &#176;C. Sonicate for 10 min at 40 W with 30 sec pulse, 30 sec pause cycles at RT. The suspension should appear clear at this stage.</li>
		<li>To obtain a monodisperse population of liposomes, perform two cycles of extrusion and for each cycle, pass the liposome suspension through the filter at least 11x. For the first cycle, use a membrane pore size of 200&#160;nm and for the second cycle, use a membrane pore size of 100 nm. Dynamic Light scattering measurements can be performed at this step to check the homogeneity of the suspension.</li>
	</ol>
	</li>
	<li>Protein incorporation 
	Principle: membrane proteins can be reconstituted in liposomes thanks to the solubilizing effect of detergents. Detergent-solubilized liposomes are incubated with the detergent-solubilized membrane protein and formation of the proteoliposomes is triggered by rapid elimination of the detergent with polystyrene beads9.
	<ol>
		<li>Add 28 mg of Triton X-100 (0.56% final) and incubate O/N&#160;at 4 &#176;C (the solubilization temperature can be adapted to the protein being studied).</li>
		<li>Add the detergent-solubilized protein (BR, MexB and MexA) to the solubilized liposomes and incubate 15 min&#160;at 4 &#176;C. Add the proteins to the solubilized liposome suspension at the following ratios&#160;(w/w): lipids/MexB = 20, MexB/MexA = 2.5, and lipids/BR = 30.</li>
		<li>Add polystyrene beads at a 30:1 beads:detergent ratio (w/w, considering the total weight of detergent, including detergent added with the purified proteins) and incubate 5 hr, in the dark (because of the BR) at RT&#160;under gentle stirring. Prior to this procedure, activate the biobeads with methanol and ethanol incubation and extensively wash with water.</li>
		<li>Purify the proteoliposomes protein and free substrates using a PD-10 desalting column equilibrated with HEPES 25 mM pH 7, K2SO4 100 mM and MgCl2 2 mM.</li>
		<li>Store the proteoliposomes at 18 &#176;C in the dark for up to 2-3 weeks.</li>
	</ol>
	</li>
	<li>Assessment of the reconstitution efficacy on sucrose gradient. 
	To check that both the MexB and the BR have been reconstituted in liposomes, purify the proteoliposome suspension on a discontinuous sucrose gradient.
	<ol>
		<li>Gently settle the proteoliposomes on a five-layer gradient of sucrose (60%, 20%, 10%, 5%, and 2.5%, w/v).</li>
		<li>Ultracentrifuge for 17 hr at 100,000 x g (with minimal acceleration and deceleration rates).</li>
		<li>Carefully collect the various gradient fractions (especially the interfaces between each layer of sucrose) and analyze them on SDS-PAGE (10%) using Coomassie staining or Western blotting. Aggregated proteins are found at the bottom of the tube, while nonincorporated, detergent-solubilized proteins are found at the top of the tube. Proteoliposomes and liposomes are found at sucrose interfaces that correspond to their intrinsic density. Empty liposomes are recovered farther up in the gradient than the proteoliposomes.</li>
	</ol>
	</li>
</ol>
3. Fluorescence Measurement
<ol>
	<li>Perform fluorescence measurements at 25.0 &#176;C using a spectrofluorometer allowing for dual-wavelength measurements (Xe lamp, 150 W). Alternate illumination periods (to activate BR with excitation&#160;/&#160;emission wavelengths of 550 nm / 550 nm) and fluorescence measurements with excitation / emission wavelengths of 455 nm / 509 nm for 2 sec&#160;to measure the pyranine fluorescence. Set the excitation and emission bandwidths to 5 nm. Perform the measurements in the presence of 50 nM valinomycin to prevent the formation of a reverse membrane potential &#916;&#936;. 
	Note: Indeed, because the BR pumps protons, there are more positive charges inside of the liposome than outside. This charge gradient can be discarded using valinomycin, which is a K+&#160;selective ionophore. Once added to the liposomes suspension valinomycin, as a hydrophobic molecule, will insert into the liposome membrane, passively transport K+, and therefore dissipate the membrane potential &#916;&#936;.</li>
</ol>

<ol>
	<li>Granseth, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+protein+structural+biology+-+How+far+can+the+bugs+take+us?+(Review).">Membrane protein structural biology - How far can the bugs take us? (Review).</a> Molecular Membrane Biology. 24 (5-6), 329 (2007).</li><li>Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidrug+resistance+in+bacteria.">Multidrug resistance in bacteria.</a> Annu Rev Biochem. 78, 119 (2009).</li><li>Pos, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+transport+mechanism+of+the+AcrB+efflux+pump.">Drug transport mechanism of the AcrB efflux pump.</a> Biochim Biophys Acta. 1794 (5), 782 (2009).</li><li>Murakami, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structures+of+a+multidrug+transporter+reveal+a+functionally+rotating+mechanism.">Crystal structures of a multidrug transporter reveal a functionally rotating mechanism.</a> Nature. 443 (7108), 173 (2006).</li><li>Seeger, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Asymmetry+of+AcrB+Trimer+Suggests+a+Peristaltic+Pump+Mechanism.">Structural Asymmetry of AcrB Trimer Suggests a Peristaltic Pump Mechanism.</a> Science. 313 (5791), 1295 (2006).</li><li>Sennhauser, G., Bukowska, M. A., Briand, C., Grutter, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+multidrug+exporter+MexB+from+Pseudomonas+aeruginosa.">Crystal structure of the multidrug exporter MexB from Pseudomonas aeruginosa.</a> J Mol Biol. 389 (1), 134 (2009).</li><li>Su, C. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+the+CusBA+heavy-metal+efflux+complex+of+Escherichia+coli.">Crystal structure of the CusBA heavy-metal efflux complex of Escherichia coli.</a> Nature. 470 (7335), (2011).</li><li>Nikaido, H., Takatsuka, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+RND+multidrug+efflux+pumps.">Mechanisms of RND multidrug efflux pumps.</a> Biochim Biophys Acta. 1794 (5), 769 (2009).</li><li>Rigaud, J. L., Levy, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+membrane+proteins+into+liposomes.">Reconstitution of membrane proteins into liposomes.</a> Methods in enzymology. 372, 65 (2003).</li><li>Aires, J. R., Nikaido, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Aminoglycosides+are+captured+from+both+periplasm+and+cytoplasm+by+the+AcrD+multidrug+efflux+transporter+of+Escherichia+coli.">Aminoglycosides are captured from both periplasm and cytoplasm by the AcrD multidrug efflux transporter of Escherichia coli.</a> J Bacteriol. 187 (6), 1923 (2005).</li><li>Picard, M., Verchere, A., Broutin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+the+active+transport+of+efflux+pumps+after+their+reconstitution+into+proteoliposomes:+caveats+and+keys.">Monitoring the active transport of efflux pumps after their reconstitution into proteoliposomes: caveats and keys.</a> Anal Biochem. 420 (2), 194 (2012).</li><li>Verchere, A., Dezi, M., Broutin, I., Picard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Basic+Methods+in+Protein+Purification+and+Analysis,+edited+by+iConcept+Press.">Basic Methods in Protein Purification and Analysis, edited by iConcept Press.</a> , (2012).</li><li>Verchere, A., Broutin, I., Picard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photo-induced+proton+gradients+for+the+in+vitro+investigation+of+bacterial+efflux+pumps.">Photo-induced proton gradients for the in vitro investigation of bacterial efflux pumps.</a> Sci Rep. 2, 306 (2012).</li></ol>

There is nothing to disclose.

We describe a reconstitution procedure designed for assaying membrane protein transport. Once established, the protein reconstitution procedure leads to measurements that are very reproducible. However, the exact conditions for reconstituting the protein will vary from one protein to the other. Much care must be taken in optimizing the following parameters: i) quality of the purification (purity and absence of aggregated material); ii) efficiency of the detergent solubilizing step (when possible, low cmc detergent should be preferred because they are easier to get rid of); iii) desorption of the detergent (it is for instance possible to combine the use of polystyrene beads with a dialysis step but this may affect the rate of desorption, a critical parameter for the subsequent activity of the protein, see ref [9]); iv) lipid reconstitution (the chemical nature of the lipid, the lipid to protein ratio, the presence of additional amphiphiles are critical parameters that must be varied and optimized). In addition temperature and incubation time affect all these issues.
Quality control is thus primordial at each step of the reconstitution procedure. From this point of view, light scattering is a very convenient technique because it is rapid, nondestructive, and it only requires 20 &#956;l of sample at a concentration in the micromolar range. Once the proteoliposomes are formed, sucrose gradient is also of great help because it opens the way towards systematic verification of the reconstitution: qualitative (is the protein indeed embedded into the liposome membrane) as well as quantitative (how many protein in one liposome). The latter would be addressed by precisely measuring the protein and lipid concentrations.
Our assay does not rely on the titration of the substrate itself and this avoids uneasy considerations regarding possible artifactual passive diffusion of the molecule due to hydrophobic partitioning in the membranes. The assay exploits properties of pyranine as a reliable and quantitative probe for monitoring pH changes. Our results are in agreement with results obtained with another procedure where proton gradient formation was performed using valinomycin10, but it allows a more robust and reproducible gradient11. In addition, the proton gradient can be precisely tuned, simply by varying the concentration of the buffer (for further detail see Verchere et al.12).
In the procedure a control measurement is first performed in the absence of substrate (this gives access to the passive activity of the protein). The actual membrane protein transporter activity is then measured after the substrate has been added in the same cuvette. This is truly an asset because the experiment can be considered as a genuine, non ambiguous, substrate-induced outcome.
The protocol could be adapted to high-medium throughput (for instance 96-wells measurements) automatization because illuminating the sample triggers the reaction. Automation and parallelization would consist in preparing a large batch of proteoliposome, and in dispensing aliquots in a 96-wells plate. Substrates (and also possible inhibitors) would then be added and after incubation in the dark for 45 min the plate would be subjected to illumination / pyranine fluorescence measurement cycles on a microplate reader.
For additional information on the protocol, readers are invited to read Verchere et al.12,13

Integral membrane proteins represent up to 30% of the genes encoded in eukaryotic genomes. They share pivotal roles such as gate keeping (receptors), transporting nutrients, ions or noxious compounds (transporters) or maintaining of the permeability across membrane bilayers (channels) amongst all living cells1. Efflux pumps have a central role in resistance against antibiotic therapy2. In the Gram-negative bacteria Pseudomonas aeruginosa, which is protected by an outer membrane, efflux transporters are organized as tripartite systems where MexB, the efflux pump located in the inner membrane, works in conjunction with MexA, a periplasmic protein, and OprM, an outer membrane channel. The cytoplasmic inner membrane protein acts as an energy-dependent pump with broad substrate specificity. The outer membrane protein acts as a porin whereas the third one is located in the periplasmic space and is thought to stabilize the whole complex3. In the following, we focus on the design of a functional assay for the MexA MexB assembly.
Structural information has accumulated over the last 5 years thanks to the X-ray determination of the homologous protein AcrB from Escherichia coli4-7. Although it is clearly important to couple such information with dynamic and kinetic data, designing a functional assay for this transporter is a real challenge8. Indeed, the membrane proteins must be maintained in a membranous environment and a closed compartment must be maintained for a vectorial transport of substrate to be achieved.
Reconstitution of membrane proteins into proteoliposomes has been extensively presented and reviewed (see Rigaud and Levy9). Such protocols allow for the monitoring of membrane proteins activity, for instance by following the substrates across the liposome membrane. In this case limitations arise from the availability of titratable substrates (fluorescent, radioactive, etc.) and from the fact that very often these substrates are hydrophobic and have a tendency to cross membranes irrespective of the presence of the transporter. As an alternative, one can follow the spectroscopic variations of a reporting dye sensitive to chemical changes that occur as a consequence of transport. As stated above, protons energize transport via MexB. Hence, a relevant option is to monitor the spectroscopic variations of a pH-sensitive reporting dye to follow pH variations due to proton-driven substrate transport. The choice of a fluorescent dye avoids any artifactual effects due to the hydrophobic nature of the substrate.
An additional difficulty is the generation of a proton gradient. Several protocols can be found in the literature. Among them, the use of valinomycin technique is widespread but we have shown that it is tedious to perform10-11. Moreover it is not reproducible and it is not reversible. We have resorted to the use of bacteriorhodopsin (BR), a light-activated proton pump from Halobacter salinarium, a halophilic marine Gram-negative obligate aerobic archaeon. This protein is coreconstituted into liposomes with MexB and, upon illumination, BR pumps protons inside of the liposomes and hence creates the &#8710;pH (acidic inside) needed by the protein to transport the substrate12-13.

<table border="0" cellpadding="0" cellspacing="0" width="1051">
	<tbody>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">DOPC (1,2-Dioleoyl-sn-glycero-3-phosphocholine)</td>
			<td style="width:344px;">Avanti Polar Lipids</td>
			<td style="width:344px;">850375C</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Cholesterol</td>
			<td style="width:344px;">Sigma Aldrich</td>
			<td style="width:344px;">C8667</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Hoechst 33342</td>
			<td style="width:344px;">Sigma Aldrich</td>
			<td style="width:344px;">B2261</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Triton X-100</td>
			<td style="width:344px;">Sigma Aldrich</td>
			<td style="width:344px;">93427</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Valinomycin</td>
			<td style="width:344px;">Invitrogen</td>
			<td style="width:344px;">V-1644</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Pyranine (8-hydroxypyrene-1,3,6-trisulphonic acid)</td>
			<td style="width:344px;">Invitrogen</td>
			<td style="width:344px;">H-348</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">DDM (n-Dodecyl-&#946;-D-maltopyranoside)</td>
			<td style="width:344px;">Affymetrix</td>
			<td style="width:344px;">D310LA</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Extruder kit (extruder, Hamilton gas-tight syringes)</td>
			<td style="width:344px;">Avanti Polar Lipids</td>
			<td style="width:344px;">610000</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Biobeads SM-2 Adsorbents</td>
			<td style="width:344px;">Bio-Rad</td>
			<td style="width:344px;">152-3920</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Econo-Pac Chromatography empty column</td>
			<td style="width:344px;">Biorad</td>
			<td style="width:344px;">732-1010</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:363px;">Desalting columns</td>
			<td style="width:344px;">Bio-Rad</td>
			<td style="width:344px;">54805</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:363px;">Dynamic light scattering instrument</td>
			<td style="width:344px;">Wyatt Technology</td>
			<td style="width:344px;">DynaPro NanoStar</td>
		</tr>
		<tr height="31">
			<td height="31" style="height:31px;width:363px;">Fluorometer JASCO FP-6200</td>
			<td style="width:344px;">JASCO</td>
			<td style="width:344px;">6816-J002A</td>
		</tr>
	</tbody>
</table>

in vitro investigation of the mexab efflux pump from pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution of efflux pumps from the Gram-negative bacteria Pseudomonas aeruginosa in a biomimetic environment that allows for an accurate investigation of their activity of transport. Three prerequisites are fulfilled: compartmentation in a lipidic environment, use of a relevant index for transport, and generation of a proton gradient. The membrane protein transporter is reconstituted into liposomes together with bacteriorhodopsin, a light-activated proton pump that generates a proton gradient that is robust as well as reversible and tunable. The activity of the protein is deduced from the pH variations occurring within the liposome, using pyranin, a pH-dependent fluorescent probe. We describe a step-by-step procedure where membrane protein purification, liposome formation, protein reconstitution, and transport analysis are addressed. Although they were specifically designed for an RND transporter, the described methods could potentially be adapted for use with any other membrane protein transporter energized by a proton gradient.

The assay consists of monitoring pyranine fluorescence as a function of time while a proton gradient is generated. To that purpose, samples are subjected alternatively to illumination (hence proton pumping by the BR is triggered) and then to fluorescence measurement using the excitation and emission wavelengths of pyranine (&#955;ex = 455 nm and &#955;em = 509 nm).
Figure 1 shows a representative control obtained with the assay, in the absence of MexA/MexB, verifying that the proton gradient generated by the BR is reversible. After one cycle of acidification, proteoliposomes are kept in the dark for 45 min in order for the BR to stop pumping protons (protons diffuse slowly and passively across lipid bilayers following their concentration gradient). After this recovery time, activation of BR is possible, simply by illuminating the same suspension again.
Figure 2 corresponds to an actual transport measurement. A negative control with protein free liposomes shows that, as expected, the pH is constant upon illumination (Figures 2A and 2B, orange circles). However, proteoliposomes containing BR in their membranes do pump proton, thus the pH inside the liposome decreases and a stable gradient is built (Figures 2A and 2B, purple squares). Grey triangles and red diamonds (Figure 2A) represent pH inside of proteoliposomes containing BR and MexB, in the presence or in the absence of Hoechst 33342, respectively. We observe a substrate-independent activity where the proton gradient is only partially dissipated by MexB counter-transport. We attribute this observation to a basal activity of MexB.
In order to test the effect of MexA on the activity of MexB, MexA was added to the reconstitution, first in the absence of any substrate (Figure 2B, blue diamonds). Again, the proton gradient generated by the BR is only partially dissipated. The actual transport measurement is realized on the very same sample. Beforehand, the proton gradient must be reinitialized and the substrate must be added in the suspension in order to reach its binding site in the protein. To that purpose the suspension is incubated in the dark in the presence of the substrate for 45 min. Upon subsequent illumination, one can see that the proton gradient generated by the BR is now totally dissipated by MexAB (Figure 2B, green triangles), as a consequence of substrate transport by the pump.
<img alt="Figure 1" fo:content-width="5in" fo:src="/files/ftp_upload/50894/50894fig1highres.jpg" src="/files/ftp_upload/50894/50894fig1.jpg" /> 
Figure 1. Reversibility of the proton gradient built by BR illumination: Pyranine fluorescence of BR proteoliposomes measured as a function of time. From 0-15 min, BR pumps protons as a result of light activation. From 15-60 min, proteoliposomes are incubated in the dark; during this time, protons passively move out of the liposomes until the intravesicular pH and extravesicular pH are equal. From 60-75 min, BR is still functional and pumps protons upon illumination.
<img alt="Figure 2" fo:content-width="5in" fo:src="/files/ftp_upload/50894/50894fig2highres.jpg" src="/files/ftp_upload/50894/50894fig2.jpg" /> 
Figure 2. Transport assay: pyranine fluorescence, converted to the corresponding pH variations, as a function of time. A) pH inside of control liposomes (orange circles); pH inside of proteoliposomes containing BR in the membrane (purple squares); pH inside of proteoliposomes containing BR, and MexB in the membrane without Hoechst 33342 (red diamonds); pH inside of proteoliposomes containing BR and MexB in the membrane with Hoechst 33342 (grey triangles). B) pH inside of control liposomes (orange circles); pH inside of proteoliposomes containing BR in the membrane (purple squares); pH inside of proteoliposomes containing BR, MexB and MexA in the membrane without Hoechst 33342 (blue diamonds); pH inside of proteoliposomes containing BR, MexB and MexA in the membrane with Hoechst 33342 (green triangles).

Watch this Scientific Journal Video about In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa at JoVE.com

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

We present a protocol for the in vitro investigation of efflux pumps from Pseudomonas aeruginosa. This protocol allows for the generation of a robust, reversible, and tunable proton gradient within the liposome membrane and hence should be adaptable to any membrane protein energized by the protomotive force.

In vitro Investigation of the MexAB Efflux Pump From Pseudomonas aeruginosa

There is an emerging scientific need for reliable tools for monitoring membrane protein transport. We present a methodology leading to the reconstitution ...

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Biology

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

The isolated microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of factors that control vessel diameter, and thus, perfusion resistance1-5. This is a classic experimental preparation that was, in large measure, initially described by Uchida et al.15 several decades ago. This initial description provided the basis for the techniques that was extensively modified and enhanced, primarily in the laboratory of Dr. Brian Duling at the University of Virginia6-8, and we present a current approach in the following pages. This preparation will specifically refer to the gracilis arteriole in a rat as the microvessel of choice, but the basic preparation can readily be applied to vessels isolated from nearly any other tissue or organ across species9-13. Mechanical (i.e., dimensional) changes in the isolated microvessels can easily be evaluated in response to a broad array of physiological (e.g., hypoxia, intravascular pressure, or shear) or pharmacological challenges, and can provide insight into mechanistic elements comprising integrated responses in an intact, although ex vivo, tissue. The significance of this method is that it allows for facile manipulation of the influences on the integrated regulation of microvessel diameter, while also allowing for the control of many of the contributions from other sources, including intravascular pressure (myogenic), autonomic innervation, hemodynamic (e.g., shear stress), endothelial dependent or independent stimuli, hormonal, and parenchymal influences, to provide a partial list. Under appropriate experimental conditions and with appropriate goals, this can serve as an advantage over in vivo or in situ tissue/organ preparations, which do not readily allow for the facile control of broader systemic variables. 
The major limitation of this preparation is essentially the consequence of its strengths. By definition, the behavior of these vessels is being studied under conditions where many of the most significant contributors to the regulation of vascular resistance have been removed, including neural, humoral, metabolic, etc. As such, the investigator is cautioned to avoid over-interpretation and extrapolation of the data that are collected utilizing this preparation. The other significant area of concern with regard to this preparation is that it can be very easy to damage cellular components such as the endothelial lining or the vascular smooth muscle, such that variable source of error can be introduced. It is strongly recommended that the individual investigator utilize appropriate measurements to ensure the quality of the preparation, both at the initiation of the experiment and periodically throughout the course of a protocol.

The ex vivo Isolated Skeletal Microvessel Preparation for Investigation of Vascular Reactivity

An ex vivo preparation is described for isolation of the largest gracilis muscle resistance arterioles for interrogation of both vascular responses to vasoactive stimuli and the assessment of basic structural properties via passive wall mechanics.

The isolated  microvessel preparation is an ex vivo preparation that allows for examination of the different contributions of  factors that control vessel ...

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures of polypeptides fulfill many purposes, it is now recognized that most proteins can adopt an alternative assembly of beta-sheet rich amyloid. Insoluble amyloid fibrils are initially associated with multiple human ailments, but they are increasingly shown as functional players participating in various important cellular processes. In addition, amyloid deposited in patient tissues contains nonproteinaceous components, such as nucleic acids and glycosaminoglycans (GAGs). These cofactors can facilitate the formation of amyloid, resulting in the generation of different types of insoluble precipitates. By taking advantage of our understanding how proteins misfold via an intermediate stage of soluble amyloid precursor, we have devised a method to convert native proteins to amyloid fibrils in vitro. This approach allows one to prepare amyloid in large quantities, examine the properties of amyloid generated from specific proteins, and evaluate the structural changes accompanying the conversion.

Rapid Generation of Amyloid from Native Proteins In vitro

Proteins can either adopt a native structure or misfold into insoluble amyloid. Conditions that favor the misfolding pathway lead to the formation of different types of amyloid fibrils. The methods described here allow rapid conversion of native proteins into amyloid in vitro.

Proteins carry out crucial tasks in organisms by exerting functions elicited from their specific three dimensional folds. Although the native structures ...

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the homogenization of the tissue of interest that could potentially cause changes in the metabolic pathways being studied1. Mass spectrometric imaging (MSI) is a powerful analytical tool that can provide spatial information of analytes within intact slices of biological tissue samples1-5. This technique has been used extensively to study various types of compounds including proteins, peptides, lipids, and small molecules such as endogenous metabolites. With matrix-assisted laser desorption/ionization (MALDI)-MSI, spatial distributions of multiple metabolites can be simultaneously detected. Herein, a method developed specifically for conducting untargeted metabolomics MSI experiments on legume roots and root nodules is presented which could reveal insights into the biological processes taking place. The method presented here shows a typical MSI workflow, from sample preparation to image acquisition, and focuses on the matrix application step, demonstrating several matrix application techniques that are useful for detecting small molecules. Once the MS images are generated, the analysis and identification of metabolites of interest is discussed and demonstrated. The standard workflow presented here can be easily modified for different tissue types, molecular species, and instrumentation.

MALDI-Mass Spectrometric Imaging for the Investigation of Metabolites in Medicago truncatula Root Nodules

Mass spectrometric imaging (MSI) is a powerful tool that can be used to discover and identify various chemical species in intact tissues, preserving the compounds in their native environments, which can provide new insights into biological processes. Herein a MSI method developed for the analysis of small molecules is described.&#160;

Most techniques used to study small molecules, such as pharmaceutical drugs or endogenous metabolites, employ tissue extracts which require the ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

The cell cycle is important for growth, genome replication, and development in all cells. In bacteria, studies of the cell cycle have focused largely on unsynchronized cells making it difficult to order the temporal events required for cell cycle progression, genome replication, and division. Caulobacter crescentus provides an excellent model system for the bacterial cell cycle whereby cells can be rapidly synchronized in a G0 state by density centrifugation. Cell cycle synchronization experiments have been used to establish the molecular events governing chromosome replication and segregation, to map a genetic regulatory network controlling cell cycle progression, and to identify the establishment of polar signaling complexes required for asymmetric cell division. Here we provide a detailed protocol for the rapid synchronization of Caulobacter NA1000 cells. Synchronization can be performed in a large-scale format for gene expression profiling and western blot assays, as well as a small-scale format for microscopy or FACS assays. The rapid synchronizability and high cell yields of Caulobacter make this organism a powerful model system for studies of the bacterial cell cycle.

Synchronization of Caulobacter Crescentus for Investigation of the Bacterial Cell Cycle

Synchronization of bacterial cells is essential for studies of the bacterial cell cycle and development. Caulobacter crescentus is synchronizable through density centrifugation allowing a rapid and powerful tool for studies of the bacterial cell cycle. Here we provide a detailed protocol for the synchronization of Caulobacter cells.

The cell cycle is important for growth, genome replication, and development in all cells.  In bacteria, studies of the cell cycle have focused largely on ...

Contractility Measurements on Isolated Papillary Muscles for the Investigation of Cardiac Inotropy in Mice

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The contractile characteristics can be evaluated independently of external influences such as vascular tonus or neurohumoral status. It depicts a scientific approach between single cell measurements with isolated cardiac myocytes and in vivo studies like echocardiography. Thus, papillary muscle preparations serve as an excellent model to study cardiac physiology/pathophysiology and can be used for investigations like the modulation by pharmacological agents or the exploration of transgenic animal models. Here, we describe a method of isolating the murine left anterior papillary muscle to investigate cardiac contractility in an organ bath setup. In contrast to a muscle strip preparation isolated from the ventricular wall, the papillary muscle can be prepared in toto without damaging the muscle tissue severely. The organ bath setup consists of several temperature-controlled, gassed and electrode-equipped organ bath chambers. The isolated papillary muscle is fixed in the organ bath chamber and electrically stimulated. The evoked twitch force is recorded using a pressure transducer and parameters such as twitch force amplitude and twitch kinetics are analyzed. Different experimental protocols can be performed to investigate the calcium- and frequency-dependent contractility as well as dose-response curves of contractile agents such as catecholamines or other pharmaceuticals. Additionally, pathologic conditions like acute ischemia can be simulated.

Murine left ventricular papillary muscle can be used to investigate cardiac contractility in vitro. This article describes in detail the isolation and experimental protocols to study cardiac contractile characteristics.

Papillary muscle isolated from adult mouse hearts can be used to study cardiac contractility during different physiological/pathological conditions. The ...

Improving the Accuracy of Flow Cytometric Assessment of Mitochondrial Membrane Potential in Hematopoietic Stem and Progenitor Cells Through the Inhibition of Efflux Pumps

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic homeostasis have been extensively studied by HSC researchers. Mitochondrial activity levels are reflected in their membrane potentials (&#916;&#936;m), which can be measured by cell-permeant cationic dyes such as TMRM (tetramethylrhodamine, methyl ester). The ability of efflux pumps to extrude these dyes from cells can limit their usefulness, however. The resulting measurement bias is particularly critical when assessing HSCs, as xenobiotic transporters exhibit higher levels of expression and activity in HSCs than in differentiated cells. Here, we describe a protocol utilizing Verapamil, an efflux pump inhibitor, to accurately measure &#916;&#936;m across multiple bone marrow populations. The resulting inhibition of pump activity is shown to increase TMRM intensity in hematopoietic stem and progenitor cells (HSPCs), while leaving it relatively unchanged in mature fractions. This highlights the close attention to dye-efflux activity that is required when &#916;&#936;m-dependent dyes are used, and as written and visualized, this protocol can be used to accurately compare either different populations within the bone marrow, or the same population across different experimental models.

Xenobiotic efflux pumps are highly active in hematopoietic stem and progenitor cells (HSPCs) and cause extrusion of TMRM, a mitochondrial membrane potential fluorescent dye. Here, we present a protocol to accurately measure mitochondrial membrane potential in HSPCs by TMRM in the presence of Verapamil, an efflux pump inhibitor.

As cellular metabolism is a key regulator of hematopoietic stem cell (HSC) self-renewal, the various roles played by the mitochondria in hematopoietic ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

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 ...