The authors are deeply indebted to all the members of the scientific community working on pH and ion transport, who have originated and improved the measurements described here. They particularly thank Dr. Jacques Pouyss&#233;gur who originated the H+-killing selection technique used here. They acknowledge the University of Nice-Sophia Antipolis, the CNRS, the ANR (JCJC SVSE1 NHEint) and the ICST Labex for support.

1. H+ Killing Selection
<ol>
	<li>Cell lines
	<ol>
		<li>Stably transfect NHE-deficient cells (e.g., the CCL39-derived PS120 cell line16) using any combination of mammalian expression vector and transfection method that will produce efficient transfection yields and selection in a fibroblast cell line. 
		NOTE: For many years Calcium Phosphate precipitation19 was used with good transfection yields. This has been replaced more recently by commercial reagents such as Lipofectamine2000, the transfections being performed following the manufacturer&#8217;s instructions.</li>
	</ol>
	</li>
	<li>Solutions
	<ol>
		<li>Prepare three sterile solutions described as below. Sterilize these solutions by filtration (0.22 &#181;m).
		<ol>
			<li>Prepare a NH4+ Loading solution (pH 7.4) comprised of 50 mM NH4Cl, 70 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM MOPS at pH 7.4.</li>
			<li>Prepare a rinse Solution (pH 7.0) comprised of 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.0.</li>
			<li>Prepare a Recovery solution (pH 7.4) comprised of 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Before starting the selection, obtain a cell culture incubator that can be used at 37 &#176;C with no additional CO2 from external tank (termed CO2-&#8220;free&#8221; incubator), as 5% CO2 will result in buffering that may impair the acidification. Amplify the cell line expressing the NHE of interest up to about 2 x 108 cells (typically about 20 confluent 100 mm plates).</li>
	<li>H+ killing procedure:
	<ol>
		<li>Incubate the cells expressing the intracellular NHE of interest for 1 hour in the Loading Solution, at 37 &#176;C in the CO2-free incubator.</li>
		<li>Aspirate the loading solution and then rinse them twice in the above described-rinse solution. Perform this step efficiently to eliminate the residual extracellular NH4Cl that would impair the creation of a steep NH4+ gradient that will generate the acidification.</li>
		<li>Eliminate the rinse medium by aspiration and incubate the cells for one hour in recovery solution again at 37 &#176;C in the CO2 free incubator.</li>
		<li>Replace the recovery medium with regular culture medium (typically DMEM with 7.5% FCS) and grow the cells in standard culture conditions (37 &#176;C in a humidified atmosphere of 5% CO2 and 95% air).</li>
	</ol>
	</li>
	<li>Repeat this cycle of selection twice a week until stable clones emerge. 
	NOTE: Take special care concerning the cell culture and selection conditions. Months of work can be ruined by any contamination that is more prone to occur after a long period of culture and repeated cell manipulations.
	<ol>
		<li>Here, choose whether to amplify the clones and characterize them individually as further described in sections 2-3, or pool them together to generate a cellular population before characterization.</li>
	</ol>
	</li>
	<li>Apply occasional selection (about once every two weeks) to retain the acidification-resistant cells by counter-selecting those that may have reverted to the parental acid-sensitive phenotype (Figure 1B).</li>
</ol>
2. Intracellular pH Measurements
2.1) Fluorescence pH Imaging
<ol>
	<li>Use the ratiometric pH-sensitive fluorescent dye BCECF/AM, which is pH sensitive when excited at 490 nm and possesses a 445 nm isosbestic point, following manufacturer&#8217;s protocols.
	<ol>
		<li>Alternatively, use other pH&#8211;sensitive probes, following the manufacturer&#8217;s protocols.</li>
	</ol>
	</li>
	<li>Use an imaging set consisting of an inverted microscope coupled to a high sensitivity video camera. Equip this set with 450 nm and 490 nm narrow band interference filters paired with appropriate quartz neutral-density filters for excitation.
	<ol>
		<li>If possible, use the appropriate set of filters and illumination conditions to setup comparable fluorescence values for &#955;1 and &#955;2 so that the ratio approaches 1. Also avoid basal fluorescence values that are set either saturating or too low, both for &#955;1 and/or &#955;2. This may yield important artifacts as then the ratio may not been detectable although intracellular pH is changing. 
		NOTE: This system must enable rapid perfusion, time-lapse excitation at the two above-mentioned wavelengths and acquisition at the proper emission wavelength (535 nm for BCECF). Equip the system with software enabling automatic data acquisition and storage.</li>
	</ol>
	</li>
</ol>
2.2) Measurement of NHE Forward Activity (Extrusion of Protons from the Cytosol)
<ol>
	<li>Acidify cells by a 1 hr incubation in the NH4+ Loading solution (see step 1.4) in the absence of CO2.</li>
	<li>Add BCECF-AM (5 &#181;M final concentration) to the solution for the last five minutes and then rinse it with NH4+ loading solution to eliminate the extracellular probe.</li>
	<li>Mount the cells on the microscope and take several images to record a stable baseline. Perfuse the rinse solution (see above). This results in a drop of fluorescence corresponding to the acidification.</li>
	<li>After pH stabilization, perfuse any of the solutions given below to measure pH recovery rates mediated by Li+/H+, Na+/H+ or K+/H+ exchange. Each solution contains a potential coupling cation to be tested for H+ exchange. If one of those is transported, this will result in a fluorescence increase at 495 nm that will correspond to a raise in intracellular pH: 
	120 mM LiCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4 
	120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4 
	125 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4</li>
</ol>
2.3) Measurement of NHE Reverse Activity
NOTE: The principle here is to invert the transmembrane ionic gradients.
<ol>
	<li>Prior to measurement, load the cells with the intracellular cation of interest (see below).</li>
	<li>Incubate them for 5 min with BCECF/AM (see step 2.2.2); rinse them, mount them on the video microscope.</li>
	<li>Perfuse the loading solution and take several images to record a stable baseline.</li>
	<li>After the baseline stabilization, image the pH variations when cells are perfused with an extracellular acidic medium containing 120 mM choline chloride, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM MES at pH 6.5.</li>
	<li>For LiCl loading, incubate cells for 2 hr in a solution composed of 120 mM LiCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4. Intracellular lithium measured by atomic absorption spectroscopy yields a value of about 60 mM.</li>
	<li>For Na+ loading, incubate cells for two hours in a solution composed of 120 mM NaCl, 5 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 5 mM glucose and 15 mM HEPES at pH 7.4 in the presence of 1 mM ouabain to block the Na/K ATPase. In these conditions, intracellular Na+ concentration is in the range of 40 mM. 
	NOTE: K+ loading is not necessary as cytosolic K+ concentration is in the range of 140 mM and an outwardly-directed K+ gradient is therefore easy to achieve.</li>
</ol>
2.4) Data Treatment and Calibration:
NOTE: This step applies both to the forward and reverse measurements.
<ol>
	<li>At the end of each experiment, perfuse cells with a solution of 140 mM KCl, 20 mM HEPES and 5 &#181;M nigericin adjusted to pH values between 6.5 and 7.4.</li>
	<li>Collect data as fluorescence measurements from the images (grey levels representing intensity levels) and export under text format and treat as explained below using a spreadsheet program.</li>
	<li>Calculate intracellular pH values for each individual cell or region of interest using the following equation: 
	pH = pKa + (Log (R-Rmin)/(Rmax-R))x Fmin(&#955;2)/Fmax(&#955;2)</li>
	<li>Use the Fmin(&#955;2)/Fmax(&#955;2) ratio if there is probe bleaching or leakage, resulting in a decrease in the 450 nm fluorescence throughout the experiments. This is however very rarely observed in the conditions used in the described experiments.</li>
	<li>As the calibration data are present at the end of each pH measurement, check whether the corresponding calculated values differ from the pH values used for calibration. If it is the case, then adjust all experimentally-determined pH values to the calibration using the following procedure:
	<ol>
		<li>Calculate the following quotient (Q, the difference between the measured values/Difference between the calibration values). Multiply the whole dataset by Q. Make the final adjustment by addition or subtraction so that the calculated values will equal the corresponding calibration values.</li>
	</ol>
	</li>
</ol>
3. Measurements of Initial Rates of NHE7 by Fast Li+ Uptake
<ol>
	<li>Seed cells on multi well plates (6 to 24-well) and acidify them using either the NH4+ loading technique described above or alternately the nigericin/bovine serum albumin (BSA) acidification described in20.</li>
	<li>Rinse plates twice rapidly using the above-described rinse solution and then incubate the cells in uptake solutions containing 1-10 mM lithium (slightly below Km values) and the desired concentrations of the cations and/or inhibitors of interest for the desired amount of time.</li>
	<li>Maintain short and consistent uptake durations (typically one minute or below) to ensure that transport operates in initial rates conditions. Also make sure that all the solutions used for uptake are isotonic.</li>
	<li>At the end of the uptake time, carefully eliminate the uptake medium and wash the cells four times with ice-cold phosphate buffered saline (PBS). Perform these washes as fast as possible (less than 10 sec for the four of them is ideal) to prevent lithium efflux.</li>
	<li>Lyse the cells in 25% Nitric acid. Apply 250 &#181;l per well of 25% Nitric acid and let it sit for at least 1 h, then scrap each well with the end of the pipette tip and transfer the whole well suspension in a new microcentrifuge tube.</li>
	<li>Transfer the lysate in 1.5 ml microcentrifuge tubes, centrifuge them for 5 minutes at 15,000 x g (room temperature) to remove the cellular debris.</li>
	<li>Measure the lithium content of the supernatants by atomic absorption spectroscopy21. 
	NOTE: Different companies provide atomic absorption spectrometers, which have to be equipped with a Lithium hollow cathode lamp. Follow the manufacturer&#8217;s instructions as these machines are very precise but also quite delicate.</li>
</ol>

<ol>
	<li>Marshansky, V., Futai, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+V-type+H+-ATPase+in+vesicular+trafficking:+targeting,+regulation+and+function.">The V-type H+-ATPase in vesicular trafficking: targeting, regulation and function.</a> Curr Opin Cell Biol. 20 (4), 415-426 (2008).</li><li>Jentsch, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride+and+the+endosomal-lysosomal+pathway:+emerging+roles+of+CLC+chloride+transporters.">Chloride and the endosomal-lysosomal pathway: emerging roles of CLC chloride transporters.</a> J Physiol. 578 (3), 633-640 (2007).</li><li>Kornak, U., 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=Mutations+in+the+a3+subunit+of+the+vacuolar+H(+)-ATPase+cause+infantile+malignant+osteopetrosis.">Mutations in the a3 subunit of the vacuolar H(+)-ATPase cause infantile malignant osteopetrosis.</a> Hum Mol Genet. 9 (13), 2059-2063 (2000).</li><li>Gunther, W., Piwon, N., Jentsch, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ClC-5+chloride+channel+knock-out+mouse+-+an+animal+model+for+Dent's+disease.">The ClC-5 chloride channel knock-out mouse - an animal model for Dent's disease.</a> Pflugers Arch. 445 (4), 456-462 (2003).</li><li>Kasper, D., 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=Loss+of+the+chloride+channel+ClC-7+leads+to+lysosomal+storage+disease+and+neurodegeneration.">Loss of the chloride channel ClC-7 leads to lysosomal storage disease and neurodegeneration.</a> Embo J. 24 (5), 1079-1091 (2005).</li><li>Poet, M., 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=Lysosomal+storage+disease+upon+disruption+of+the+neuronal+chloride+transport+protein+ClC-6.">Lysosomal storage disease upon disruption of the neuronal chloride transport protein ClC-6.</a> Proc Natl Acad Sci U S A. 103 (37), 13854-13859 (2006).</li><li>Nakamura, N., Tanaka, S., Teko, Y., Mitsui, K., Kanazawa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Four+Na+/H++exchanger+isoforms+are+distributed+to+Golgi+and+post-Golgi+compartments+and+are+involved+in+organelle+pH+regulation.">Four Na+/H+ exchanger isoforms are distributed to Golgi and post-Golgi compartments and are involved in organelle pH regulation.</a> J Biol Chem. 280 (2), 1561-1572 (2005).</li><li>Gilfillan, G. D., 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=SLC9A6+mutations+cause+X-linked+mental+retardation,+microcephaly,+epilepsy,+and+ataxia,+a+phenotype+mimicking+Angelman+syndrome.">SLC9A6 mutations cause X-linked mental retardation, microcephaly, epilepsy, and ataxia, a phenotype mimicking Angelman syndrome.</a> Am J Hum Genet. 82 (4), 1003-1010 (2008).</li><li>Mignot, 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=Novel+mutation+in+SLC9A6+gene+in+a+patient+with+Christianson+syndrome+and+retinitis+pigmentosum.">Novel mutation in SLC9A6 gene in a patient with Christianson syndrome and retinitis pigmentosum.</a> Brain &amp; Development. 35 (2), 172-176 (2013).</li><li>Morrow, E. M., 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=Identifying+autism+loci+and+genes+by+tracing+recent+shared+ancestry.">Identifying autism loci and genes by tracing recent shared ancestry.</a> Science. 321 (5886), 218-223 (2008).</li><li>Lasky-Su, J., 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=Genome-wide+association+scan+of+the+time+to+onset+of+attention+deficit+hyperactivity+disorder.">Genome-wide association scan of the time to onset of attention deficit hyperactivity disorder.</a> Am J Med Genet B Neuropsychiatr Genet. 147B (8), 1355-1358 (2008).</li><li>Franke, B., Neale, B. M., Faraone, S. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome-wide+association+studies+in+ADHD.">Genome-wide association studies in ADHD.</a> Hum Genet. 126 (1), 13-50 (2009).</li><li>Meda, S. A., 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=A+large+scale+multivariate+parallel+ICA+method+reveals+novel+imaging-genetic+relationships+for+Alzheimer's+disease+in+the+ADNI+cohort.">A large scale multivariate parallel ICA method reveals novel imaging-genetic relationships for Alzheimer's disease in the ADNI cohort.</a> Neuroimage. 60 (3), 1608-1621 (2012).</li><li>Zhang, L., 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=A+microdeletion+in+Xp11.3+accounts+for+co-segregation+of+retinitis+pigmentosa+and+mental+retardation+in+a+large+kindred.">A microdeletion in Xp11.3 accounts for co-segregation of retinitis pigmentosa and mental retardation in a large kindred.</a> Am J Med Genet A. 140 (4), 349-357 (2006).</li><li>Sardet, C., Franchi, A., Pouyss&#233;gur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning,+primary+structure,+and+expression+of+the+human+growth+factor-activatable+Na+/H++antiporter.">Molecular cloning, primary structure, and expression of the human growth factor-activatable Na+/H+ antiporter.</a> Cell. 56 (2), 271-280 (1989).</li><li>Pouyssegur, J., Sardet, C., Franchi, A., L'Allemain, G., Paris, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+specific+mutation+abolishing+Na+/H++antiport+activity+in+hamster+fibroblasts+precludes+growth+at+neutral+and+acidic+pH.">A specific mutation abolishing Na+/H+ antiport activity in hamster fibroblasts precludes growth at neutral and acidic pH.</a> Proc Natl Acad Sci U S A. 81 (15), 4833-4837 (1984).</li><li>Franchi, A., Cragoe, E., Pouyss&#233;gur, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+properties+of+fibroblast+mutants+overexpressing+an+altered+Na+/H++antiporter.">Isolation and properties of fibroblast mutants overexpressing an altered Na+/H+ antiporter.</a> J Biol Chem. 261 (31), 14614-14620 (1986).</li><li>Milosavljevic, N., 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=The+Intracellular+Na+/H++Exchanger+NHE7+effects+a+Na++coupled,+but+not+K++coupled+proton-loading+mechanism+in+endocytosis.">The Intracellular Na+/H+ Exchanger NHE7 effects a Na+ coupled, but not K+ coupled proton-loading mechanism in endocytosis.</a> Cell Reports. 7 (3), 1-8 (2014).</li><li>Wigler, M., 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=Transformation+of+mammalian+cells+with+genes+from+procaryotes+and+eucaryotes.">Transformation of mammalian cells with genes from procaryotes and eucaryotes.</a> Cell. 16 (4), 777-785 (1979).</li><li>Lacroix, J., Po&#235;t, M., Maherel, C., Counillon, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanism+for+the+activation+of+the+Na/H+exchanger+NHE-1+by+cytoplasmic+acidification+and+mitogens.">A mechanism for the activation of the Na/H exchanger NHE-1 by cytoplasmic acidification and mitogens.</a> EMBO Reports. 5 (1), 91-96 (2004).</li><li>Milosavljevic, N., 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=Nongenomic+Effects+of+Cisplatin:+Acute+Inhibition+of+Mechanosensitive+Transporters+and+Channels+without+Actin+Remodeling.">Nongenomic Effects of Cisplatin: Acute Inhibition of Mechanosensitive Transporters and Channels without Actin Remodeling.</a> Cancer Res. 70 (19), 7514-7522 (2010).</li><li>Boron, W. F., De Weer, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+pH+transients+in+squid+giant+axons+caused+by+CO2,+NH3,+and+metabolic+inhibitors.">Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors.</a> J Gen Physiol. 67 (1), 91-112 (1976).</li><li>Paradiso, A. M., Tsien, R. Y., Machen, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Na+-H++exchange+in+gastric+glands+as+measured+with+a+cytoplasmic-trapped,+fluorescent+pH+indicator.">Na+-H+ exchange in gastric glands as measured with a cytoplasmic-trapped, fluorescent pH indicator.</a> Proc Natl Acad Sci U S A. 81 (23), 7436-7440 (1984).</li><li>Paradiso, A. M., Tsien, R. Y., Machen, T. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Digital+image+processing+of+intracellular+pH+in+gastric+oxyntic+and+chief+cells.">Digital image processing of intracellular pH in gastric oxyntic and chief cells.</a> Nature. 325, 447-450 (1987).</li><li>Quentin, F., 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=RhBG+and+RhCG,+the+putative+ammonia+transporters,+are+expressed+in+the+same+cells+in+the+distal+nephron.">RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.</a> J Am Soc Nephrol. 14 (3), 545-554 (2003).</li><li>Geyer, R. R., Musa-Aziz, R., Enkavi, G., Mahinthichaichan, P., Tajkhorshid, E., Boron, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Movement+of+NH3+through+the+human+urea+transporter+B:+a+new+gas+channel.">Movement of NH3 through the human urea transporter B: a new gas channel.</a> Am J Physiol Renal Physiol. 304 (12), F1447-F1457 (2013).</li></ol>

The authors have nothing to disclose.

This protocol describes how to select cells expressing intracellular Na+/H+ exchangers at the plasma membrane without altering their primary sequence by site-directed mutagenesis. These exchangers can now be characterized.
This method is based on the cellular toxicity of intracellular protons to select cell lines that will express vesicular Na+/H+ exchangers at the plasma membrane. It might be possible, in principle, to use other cations that may be ...

Most intracellular compartments display an acidic luminal pH, which is a key parameter for maturation, trafficking, recycling of proteins or hormones and neurotransmitters loading. It has been shown that the pH gradient between cytosol and vesicular content is generated by vacuolar H+ ATPases1, coupled to vesicular ClC chloride transporters2. Both in knock-out (KO) mice and human patients, the importance of these transporters has been highlighted by the heavy phenotypes caused by mutations in their genes3-6.
The members of the Sodium-Hydrogen exchangers SLC9A family, also termed NHEs for Na+/H+ exchangers, have been shown to be key effectors in intracellular pH and cell volume regulation, as well as in vectorial transport of acid-base equivalents across epithelia. Besides the plasma membrane NHEs, three highly conserved Na+/H+ exchangers, NHE 6, 7 and 9 are expressed in trans-Golgi network and in early endosomes7. Mutations in their genes have been linked with Angelman-like or Christianson Syndromes8-9, family-based autism10 and Attention Deficit Hyperactivity Disorder11-12. These exchangers have also been involved in neurodegenerative problems such as Alzheimer disease susceptibility13 and X-linked mental retardation contiguous genes syndromes14. Taken together, these studies highlight the importance of these intracellular NHEs in brain development and/or function.
The intracellular localization of these exchangers prevents accurate measurements of their ion selectivity, transport direction, kinetic parameters, and regulation. As is the case for all transporters expressed in intracellular compartments, it is extremely difficult to assess their biochemical activities and hence to fully understand their physiological roles and the mechanisms underlying their pathological implications. Based on the high cytosolic K+ concentration, the most-commonly accepted hypothesis was that they were working as K+ coupled proton efflux transporters. The existence of such a proton leak had been hypothesized, as it may counterbalance proton pumping by the V-ATPases in order to maintain a steady state vesicular pH. The aim of this visual article is (i) to demonstrate a method that allows the genetic selection of cell lines that express such vesicular transporters at their plasma membrane, and (ii) to show two independent approaches to measure the functions of these transporters.
Three decades ago, Pouyss&#233;gur and Franchi have pioneered a genetic approach that enabled the molecular cloning and characterization of the members of the NHE family15. This was based on the toxicity of intracellular protons as a screening method. The first step was to obtain cell lines deficient in any Na+/H+ exchange expressed at the plasma membrane, using the reversibility of this transporter. Fibroblasts (CCL39 cell line) were preloaded with Na+ or Li+ and then placed in an acidic extracellular medium (pH 6.5) for 2 hr. This led to the death of cells expressing a functional Na+/H+ exchange and to the selection of antiporter-deficient cells (PS120 cell line)16. When cultivated in bicarbonate-free medium, these cells are very sensitive to acute intracellular acidification. Consequently, the expression of any functional proton efflux mechanism at the plasma membrane will be positively selected (see17) if such cells are submitted to acute intracellular acidifications. Such acidification techniques can be used to isolate cell lines with trafficking defects enabling the forced expression of WT intracellular NHEs at the plasma membrane.
As eukaryotic Na+/H+ exchangers are electroneutral, they are not measurable by the electrophysiological approaches that have been used with great success to measure channels. This manuscript therefore demonstrates how to measure the activity of this exchanger by intracellular pH measurements and rapid kinetics of lithium uptake. As the underlying concepts are the same, it is interesting to notice that many of the processes developed for the selection section are also used directly for functional measurements.
Interestingly, we have observed that the trafficking defect present in the cell lines selected using the approach described in this manuscript leads to a greater expression of other vesicular proteins at the plasma membrane such as the vesicular potassium channel TWIK118. This points out toward the selection of a general retention defect mechanism for vesicular transmembrane proteins. Hence this selection procedure and the cells that it generates may constitute a promising tool for the scientific community working on the membrane proteins of intracellular compartments. As well the measurement techniques presented here might be applicable for studying other non-electrogenic transporters.

<table border="0" cellpadding="0" cellspacing="0" width="621">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Standard cell culture equipement</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td>Used for H+ killing selection composed of many devices with different catalog numbers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubator with CO2</td>
			<td style="width:119px;">Sanyo</td>
			<td style="width:119px;">MCO15A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Incubator without CO2</td>
			<td style="width:119px;">Heraeus instrument</td>
			<td style="width:119px;">BB6220</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Laminar flow hood PSM1200NF</td>
			<td style="width:119px;">Fisher&#160;</td>
			<td style="width:119px;">52010120</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM medium</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">D5796</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS gold</td>
			<td style="width:119px;">GE Healthcare</td>
			<td style="width:119px;">A15-151</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicilin/streptomycin</td>
			<td style="width:119px;">PAA</td>
			<td>P11-010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin 10X</td>
			<td style="width:119px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Atomic Absorption spectrometer with Zeeman furnace system</td>
			<td style="width:119px;">Thermo Scientific</td>
			<td style="width:119px;">ICE 3500 GFZ</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LiCl</td>
			<td style="width:119px;">sigma</td>
			<td style="width:119px;">L4408</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nitric Acid</td>
			<td style="width:119px;">sigma</td>
			<td align="right" style="width:119px;">438073</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fluorescence videomicroscopy set</td>
			<td style="width:119px;">Leica</td>
			<td style="width:119px;"></td>
			<td>Composed of many devices with different catalog numbers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Inverted automated microscope</td>
			<td style="width:119px;">Leica</td>
			<td style="width:119px;">DMI6000B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">microscope stand</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888906</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11505180</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888377</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">incident fluorescence</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888901</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11504166</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">motor bracket</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888379</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11505234</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521505</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11522106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LED transmission light&#160;</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">8097321</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">8102034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521580</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">motorized plate</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11522068</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11531172</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521734</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11521719</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888423</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888424</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">camera output</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888373</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11507807</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888393</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888259</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11888258</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11541510</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">images acquisition/analysis software</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11888375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">optics</td>
			<td style="width:119px;">leica</td>
			<td align="right" style="width:119px;">11506507</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11506243</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11506203</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">fluorescence Xenon lamp</td>
			<td style="width:119px;">leica</td>
			<td style="width:119px;">DMI6000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">camera</td>
			<td style="width:119px;">hamamatsu</td>
			<td align="right" style="width:119px;">8100601</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">metafluor/Mmfluor software</td>
			<td style="width:119px;"></td>
			<td align="right" style="width:119px;">11640905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">pH sensitive probe, BCECF-AM</td>
			<td style="width:119px;">life technologies</td>
			<td style="width:119px;">B1170</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nigericin</td>
			<td style="width:119px;">Sigma</td>
			<td style="width:119px;">N7143</td>
			<td></td>
		</tr>
	</tbody>
</table>

functional characterization of na+/h+ exchangers of intracellular compartments using proton-killing selection to express them at the plasma membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and neurotransmitter loading in synaptic vesicles. This acidification is described to be mediated by proton ATPases, coupled to ClC chloride transporters. Highly-conserved electroneutral protons transporters, the Na+/H+ exchangers (NHE) 6, 7 and 9 are also expressed in these compartments. Mutations in their genes have been linked with human cognitive and neurodegenerative diseases. Paradoxically, their roles remain elusive, as their intracellular localization has prevented detailed functional characterization. This manuscript shows a method to solve this problem. This consists of the selection of mutant cell lines, capable of surviving acute cytosolic acidification by retaining intracellular NHEs at the plasma membrane. It then depicts two complementary protocols to measure the ion selectivity and activity of these exchangers: (i) one based on intracellular pH measurements using fluorescence video microscopy, and (ii) one based on the fast kinetics of lithium uptake. Such protocols can be extrapolated to measure other non-electrogenic transporters. Furthermore, the selection procedure presented here generates cells with an intracellular retention defective phenotype. Therefore these cells will also express other vesicular membrane proteins at the plasma membrane. The experimental strategy depicted here may therefore constitute a potentially powerful tool to study other intracellular proteins that will be then expressed at the plasma membrane together with the vesicular Na+/H+ exchangers used for the selection.

Selection:
The H+ killing selection is based on the diffusion of the ammonium weak base, as depicted in Figure 1A. The effect of weak bases and acids diffusion on intracellular pH has been pioneered by Walter Boron and collaborators22. The elegant idea to use this phenomenon to produce a lethal acidification for positive genetic selection was then developed by Jacques Pouyss&#233;gur17. Under such a protocol, the intracellular pH drops to about...

Watch this Scientific Journal Video about Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane at JoVE.c

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers.

Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Endosomal acidification is critical for a wide range of processes, such as protein recycling and degradation, receptor desensitization, and ...

functional-characterization-na-h-exchangers-intracellular

Research

JoVE Journal

Biology

9.2K Views.  Université Nice-Sophia Antipolis, Laboratoire de Physiomédecine Moléculaire, CNRS UMR7370, and Laboratories of Excellence Ion Channel Science and Therapeutics. The first part of this article shows how to select mutant cell lines expressing vesicular Na+/H+ exchangers at their plasma membrane. The second part provides protocols based on intracellular pH measurements and fast ion uptake, which are used to determine the ion selectivity and the kinetic parameters of these exchangers. 

Video: Functional Characterization of Na+/H+ Exchangers of Intracellular Compartments Using Proton-killing Selection to Express Them at the Plasma Membrane

Measuring Near Plasma Membrane and Global Intracellular Calcium Dynamics in Astrocytes

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, increasing evidence suggests that astrocytes may also actively participate in brain function through functional interactions with neurons.  However, many fundamental aspects of astrocyte biology remain controversial, unclear and/or experimentally unexplored. One important issue is the dynamics of intracellular calcium transients in astrocytes. This is relevant because calcium is well established as an important second messenger and because it has been proposed that astrocyte calcium elevations can trigger the release of transmitters from astrocytes. However, there has not been any detailed or satisfying description of near plasma membrane calcium signaling in astrocytes. Total internal reflection fluorescence (TIRF) microscopy is a powerful tool to analyze physiologically relevant signaling events within about 100 nm of the plasma membrane of live cells. Here, we use TIRF microscopy and describe how to monitor near plasma membrane and global intracellular calcium dynamics almost simultaneously. The further refinement and systematic application of this approach has the potential to inform about the precise details of astrocyte calcium signaling. A detailed understanding of astrocyte calcium dynamics may provide a basis to understand if, how, when and why astrocytes and neurons undergo calcium-dependent functional interactions.

We describe how to measure near membrane and global intracellular calcium dynamics in cultured astrocytes using total internal reflection and epifluorescence microscopy.

The brain contains glial cells.  Astrocytes, a type of glial cell, have long been known to provide a passive supportive role to neurons. However, ...

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the lipidic cubic phase or in meso method. The method has been shown to be quite versatile in that it has been used to solve X-ray crystallographic structures of prokaryotic and eukaryotic proteins, proteins that are monomeric, homo- and hetero-multimeric, chromophore-containing and chromophore-free, and alpha-helical and beta-barrel proteins. Recent successes using in meso crystallization are the human engineered beta2-adrenergic and adenosine A2a G protein-coupled receptors. Protocols are presented for reconstituting the membrane protein into the monoolein-based mesophase, and for setting up crystallizations in the manual mode. Additional steps in the overall process, such as crystal harvesting, are to be addressed in future video articles. The time required to prepare the protein-loaded mesophase and to set up a crystallization plate manually is about one hour.

Herein is described the procedure implemented in the Caffrey Membrane Structural and Functional Biology Group to set up manually crystallization trials of membrane proteins in lipidic mesophases.

A detailed protocol for crystallizing membrane proteins by using lipidic mesophases is described. This method has variously been referred to as the ...

Quantitative Measurement of GLUT4 Translocation to the Plasma Membrane by Flow Cytometry

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces glucose uptake by insulin-sensitive tissues, most notably the brain, skeletal muscle, and adipocytes. Patients suffering from type-2 diabetes and/or obesity often develop insulin resistance and are unable to control their glucose homeostasis. New insights into the mechanisms of insulin resistance may provide new treatment strategies for type-2 diabetes.
The GLUT family of glucose transporters consists of thirteen members distributed on different tissues throughout the body1. Glucose transporter type 4 (GLUT4) is the major transporter that mediates glucose uptake by insulin sensitive tissues, such as the skeletal muscle. Upon binding of insulin to its receptor, vesicles containing GLUT4 translocate from the cytoplasm to the plasma membrane, inducing glucose uptake. Reduced GLUT4 translocation is one of the causes of insulin resistance in type-2 diabetes2,3.
The translocation of GLUT4 from the cytoplasm to the plasma membrane can be visualized by immunocytochemistry, using fluorophore-conjugated GLUT4-specific antibodies.
Here, we describe a technique to quantify total amounts of GLUT4 translocation to the plasma membrane of cells during a chosen duration, using flow cytometry. This protocol is rapid (less than 4 hours, including incubation with insulin) and allows the analysis of as few as 3,000 cells or as many as 1 million cells per condition in a single experiment. It relies on anti-GLUT4 antibodies directed to an external epitope of the transporter that bind to it as soon as it is exposed to the extracellular medium after translocation to the plasma membrane.

This protocol describes a rapid technique to quantify the translocation of GLUT4 from the cytoplasm to the plasma membrane of cells by flow cytometry.

Glucose is the main source of energy for the body, requiring constant regulation of its blood concentration. Insulin release by the pancreas induces ...

Identifying the Effects of BRCA1 Mutations on Homologous Recombination using Cells that Express Endogenous Wild-type BRCA1

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the BRCA1 have been complicated by the lack of a robust assay for the full length BRCA1 protein and the difficulties inherent in working with cell lines that express hypomorphic BRCA1 protein1,2,3,4,5. We developed a system whereby the endogenous BRCA1 protein in a cell was acutely depleted by RNAi targeting the 3'-UTR of the BRCA1 mRNA and replaced by co-transfecting a plasmid expressing a BRCA1 variant. One advantage of this procedure is that the acute silencing of BRCA1 and simultaneous replacement allow the cells to grow without secondary mutations or adaptations that might arise over time to compensate for the loss of BRCA1 function. This depletion and add-back procedure was done in a HeLa-derived cell line that was readily assayed for homologous recombination activity. The homologous recombination assay is based on a previously published method whereby a recombination substrate is integrated into the genome (Figure 1)6,7,8,9. This recombination substrate has the rare-cutting I-SceI restriction enzyme site inside an inactive GFP allele, and downstream is a second inactive GFP allele. Transfection of the plasmid that expresses I-SceI results in a double-stranded break, which may be repaired by homologous recombination, and if homologous recombination does repair the break it creates an active GFP allele that is readily scored by flow cytometry for GFP protein expression. Depletion of endogenous BRCA1 resulted in an 8-10-fold reduction in homologous recombination activity, and add-back of wild-type plasmid fully restored homologous recombination function. When specific point mutants of full length BRCA1 were expressed from co-transfected plasmids, the effect of the specific missense mutant could be scored. As an example, the expression of the BRCA1(M18T) protein, a variant of unknown clinical significance10, was expressed in these cells, it failed to restore BRCA1-dependent homologous recombination. By contrast, expression of another variant, also of unknown significance, BRCA1(I21V) fully restored BRCA1-dependent homologous recombination function. This strategy of testing the function of BRCA1 missense mutations has been applied to another biological system assaying for centrosome function (Kais et al, unpublished observations). Overall, this approach is suitable for the analysis of missense mutants in any gene that must be analyzed recessively.

We provide a method for testing BRCA1 variants in a tissue culture based assay for homologous recombination repair of DNA damage by depleting endogenous BRCA1 protein from a cell using RNAi and replacing it with a BRCA1 point mutant that contains a coding change.

The functional analysis of missense mutations can be complicated by the presence in the cell of the endogenous protein. Structure-function analyses of the ...

Use of a Robot for High-throughput Crystallization of Membrane Proteins in Lipidic Mesophases

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through macromolecular X-ray crystallography (MX). An essential ingredient of MX is a steady supply of ideally diffraction-quality crystals. The in meso or lipidic cubic phase (LCP) method for crystallizing membrane proteins is one of several methods available for crystallizing membrane proteins. It makes use of a bicontinuous mesophase in which to grow crystals. As a method, it has had some spectacular successes of late and has attracted much attention with many research groups now interested in using it. One of the challenges associated with the method is that the hosting mesophase is extremely viscous and sticky, reminiscent of a thick toothpaste. Thus, dispensing it manually in a reproducible manner in small volumes into crystallization wells requires skill, patience and a steady hand. A protocol for doing just that was developed in the Membrane Structural &amp; Functional Biology (MS&amp;FB) Group1-3. JoVE video articles describing the method are available1,4. 
The manual approach for setting up in meso trials has distinct advantages with specialty applications, such as crystal optimization and derivatization. It does however suffer from being a low throughput method. Here, we demonstrate a protocol for performing in meso crystallization trials robotically. A robot offers the advantages of speed, accuracy, precision, miniaturization and being able to work continuously for extended periods under what could be regarded as hostile conditions such as in the dark, in a reducing atmosphere or at low or high temperatures. An in meso robot, when used properly, can greatly improve the productivity of membrane protein structure and function research by facilitating crystallization which is one of the slow steps in the overall structure determination pipeline. 
In this video article, we demonstrate the use of three commercially available robots that can dispense the viscous and sticky mesophase integral to in meso crystallogenesis. The first robot was developed in the MS&amp;FB Group5,6. The other two have recently become available and are included here for completeness.	
An overview of the protocol covered in this article is presented in Figure 1. All manipulations were performed at room temperature (~20 &deg;C) under ambient conditions.

Herein is described a robotic approach to high-throughput crystallization of membrane proteins in lipidic mesophases for use in structure determination using macromolecular X-ray crystallography. Three robots capable of handling the viscous and sticky protein-laden mesophase integral to the method are introduced.

Structure-function studies of membrane proteins greatly benefit from having available high-resolution 3-D structures of the type provided through ...

Harvesting and Cryo-cooling Crystals of Membrane Proteins Grown in Lipidic Mesophases for Structure Determination by Macromolecular Crystallography

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at atomic resolution. Presently, there is only one way to capture such information as applied to integral membrane proteins (Figure 1), and the complexes they form, and that method is macromolecular X-ray crystallography (MX). To do MX diffraction quality crystals are needed which, in the case of membrane proteins, do not form readily. A method for crystallizing membrane proteins that involves the use of lipidic mesophases, specifically the cubic and sponge phases1-5, has gained considerable attention of late due to the successes it has had in the G protein-coupled receptor field6-21 (<a href="http://www.mpdb.tcd.ie" target="_blank">www.mpdb.tcd.ie</a>). However, the method, henceforth referred to as the in meso or lipidic cubic phase method, comes with its own technical challenges. These arise, in part, due to the generally viscous and sticky nature of the lipidic mesophase in which the crystals, which are often micro-crystals, grow. Manipulating crystals becomes difficult as a result and particularly so during harvesting22,23. Problems arise too at the step that precedes harvesting which requires that the glass sandwich plates in which the crystals grow (Figure 2)24,25 are opened to expose the mesophase bolus, and the crystals therein, for harvesting, cryo-cooling and eventual X-ray diffraction data collection.
The cubic and sponge mesophase variants (Figure 3) from which crystals must be harvested have profoundly different rheologies4,26. The cubic phase is viscous and sticky akin to a thick toothpaste. By contrast, the sponge phase is more fluid with a distinct tendency to flow. Accordingly, different approaches for opening crystallization wells containing crystals growing in the cubic and the sponge phase are called for as indeed different methods are required for harvesting crystals from the two mesophase types. Protocols for doing just that have been refined and implemented in the Membrane Structural and Functional Biology (MS&amp;FB) Group, and are described in detail in this JoVE article (Figure 4). Examples are given of situations where crystals are successfully harvested and cryo-cooled. We also provide examples of cases where problems arise that lead to the irretrievable loss of crystals and describe how these problems can be avoided. In this article the Viewer is provided with step-by-step instructions for opening glass sandwich crystallization wells, for harvesting and for cryo-cooling crystals of membrane proteins growing in cubic and in sponge phases.

Herein is described procedures implemented in the Caffrey Membrane Structural and Functional Biology Group to harvest and cryo-cool membrane protein crystals grown in lipidic cubic and sponge phases for use in structure determination using macromolecular X-ray crystallography.

An important route to understanding how proteins function at a mechanistic level is to have the structure of the target protein available, ideally at ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

The Cell-based L-Glutathione Protection Assays to Study Endocytosis and Recycling of Plasma Membrane Proteins

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. The cell based L-glutathione protection assays can be used to study endocytosis and recycling of protein receptors, channels, transporters, and adhesion molecules localized at the cell surface. The endocytic assay requires labeling of cell surface proteins with a cell membrane impermeable biotin containing a disulfide bond and the N-hydroxysuccinimide (NHS) ester at 4 &#186;C -&#160;a temperature at which membrane trafficking does not occur. Endocytosis of biotinylated plasma membrane proteins is induced by incubation at 37 &#186;C. Next, the temperature is decreased again to 4 &#186;C to stop endocytic trafficking and the disulfide bond in biotin covalently attached to proteins that have remained at the plasma membrane is reduced with L-glutathione. At this point, only proteins that were endocytosed remain protected from L-glutathione and thus remain biotinylated. After cell lysis, biotinylated proteins are isolated with streptavidin agarose, eluted from agarose, and the biotinylated protein of interest is detected by western blotting. During the recycling assay, after biotinylation cells are incubated at 37 &#176;C to load endocytic vesicles with biotinylated proteins and the disulfide bond in biotin covalently attached to proteins remaining at the plasma membrane is reduced with L-glutathione at 4 &#186;C as in the endocytic assay. Next, cells are incubated again at 37 &#176;C to allow biotinylated proteins from endocytic vesicles to recycle to the plasma membrane. Cells are then incubated at 4 &#186;C, and the disulfide bond in biotin attached to proteins that recycled to the plasma membranes is reduced with L-glutathione. The biotinylated proteins protected from L-glutathione are those that did not recycle to the plasma membrane.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes for degradation or to the plasma membrane for recycling. Methods described in this article are designed to study endocytosis and recycling of plasma membrane proteins.

Membrane trafficking involves transport of proteins from the plasma membrane to the cell interior (i.e. endocytosis) followed by trafficking to lysosomes ...

Imaging Plasma Membrane Deformations With pTIRFM

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

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

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

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...