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 + Sélection Tuer <ol><li> Les lignées cellulaires <ol><li> Transfecter des cellules de manière stable NHE-déficients (par exemple, le PS120 lignée de cellules dérivées 16 CCL39) à l&#39;aide d&#39;une combinaison du vecteur d&#39;expression de mammifère et un procédé de transfection qui produira des rendements de transfection efficace et sélection dans une lignée cellulaire de fibroblastes. REMARQUE: Pendant de nombreuses années phosphate de calcium précipitations 19 a été utilisé avec de bons rendements de transfection. Cela a été remplacé par plus récemment réactifs commerciaux tels que Lipofectamine2000, les transfections étant effectuées suivant les instructions du fabricant. </li></ol></li><li> Solutions <ol><li> Préparer trois solutions stériles décrites ci-dessous. Stériliser ces solutions par filtration (0,22 um). <ol><li> Préparer une solution de 4 + NH chargement (pH 7,4) composé de 50 mM de NH 4 Cl, 70 mM de chlorure de choline, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, 5 mM glucose et 15 mM MOPS à pH 7,4. </li><li> Préparer une solution de rinçage (pH 7,0), composé de 120 mM de chlorure de choline, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, 5 mM de glucose et 15 mM de HEPES à pH 7,0. </li><li> Préparer une solution de récupération (pH 7,4) constitué de NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, glucose 5 mM et HEPES 15 mM à pH 7,4. </li></ol></li></ol></li><li> Avant de commencer la sélection, l&#39;obtention d&#39;un incubateur de culture de cellules qui peut être utilisé à 37 ° C sans CO supplémentaire 2 du réservoir externe (appelé CO 2 - incubateur «libre»), tel que 5% de CO 2 entraînera tampon qui peut nuire à la acidification. Amplifier la lignée cellulaire exprimant le NHE d&#39;intérêt jusqu&#39;à environ 2 x 10 8 cellules (typiquement d&#39;environ 20 100 plaques confluentes mm). </li><li> H + tuant procédure: <ol><li> Incuber les cellules exprimant le NHE intracellulaire d&#39;intérêt pendant 1 heure dans la solution de charge, à 37 ° C dans l&#39;incubateur à CO2 exempt. </li><li> Aspirer la solution de chargement, puis les rincer deux fois dans la solution décrite ci-dessus rinçage. Effectuez cette étape efficace pour éliminer le résidu extracellulaire NH 4 Cl qui porterait atteinte à la création d&#39;un NH 4 + forte pente qui va générer l&#39;acidification. </li><li> Eliminer le milieu de rinçage par aspiration et on incube les cellules pendant une heure dans une solution de récupération de nouveau à 37 ° C dans l&#39;incubateur CO 2 libre. </li><li> Remplacer le moyen de récupération du milieu de culture ordinaire (typiquement du DMEM avec 7,5% de FCS) et faire croître les cellules dans des conditions standard de culture (37 ° C dans une atmosphère humidifiée de 5% de CO 2 et 95% d&#39;air). </li></ol></li><li> Répétez ce cycle de la sélection deux fois par semaine jusqu&#39;à ce que des clones stables émergent. NOTE: Faites attention concernant la culture de cellules et les conditions de sélection. Mois de travail peuvent être ruinés par toute contamination qui est plus enclin à oCCur après une longue période de culture et de manipulations répétées de cellules. <ol><li> Ici, choisir d&#39;amplifier les clones et de caractériser individuellement comme décrit dans la dans les sections 2-3, ou les réunir pour générer une population cellulaire avant caractérisation. </li></ol></li><li> Appliquer la sélection occasionnelle (environ une fois toutes les deux semaines) pour conserver les cellules d&#39;acidification résistant par contre-sélection de ceux qui peuvent être revenu au phénotype sensible à l&#39;acide parentale (figure 1B). </li></ol> 2. Mesures pH intracellulaire 2.1) Fluorescence Imaging pH <ol><li> Utilisez le ratiométrique sensible au pH colorant fluorescent BCECF / AM, qui est sensible au pH lorsqu&#39;il est excité à 490 nm et possède un point isosbestique nm 445, en suivant les protocoles du fabricant. <ol><li> Vous pouvez également utiliser d&#39;autres sondes sensibles au pH, en suivant les protocoles du fabricant. </li></ol></li><li> Utilisez une imagerie mis conune superficie d &#39;un microscope inversé couplé à une caméra vidéo à haute sensibilité. Équipé cet ensemble avec 450 nm et 490 filtres d&#39;interférence à bande étroite nm jumelé avec quartz appropriée des filtres de densité neutre pour l&#39;excitation. <ol><li> Si possible, utilisez l&#39;ensemble approprié des filtres et des conditions d&#39;éclairage de configuration valeurs de fluorescence comparables pour λ1 et λ2 de sorte que le ratio se rapproche de 1. Evitez également les valeurs de fluorescence de base qui sont définies soit saturer ou trop faible, tant pour λ1 et / ou λ2. Cela peut donner des artefacts importants que le ratio peut pas détectable bien pH intracellulaire est en train de changer. NOTE: Ce système doit permettre la perfusion rapide, time-lapse excitation à deux longueurs d&#39;onde mentionnées ci-dessus et l&#39;acquisition à la longueur d&#39;onde d&#39;émission appropriée (535 nm pour BCECF). Équipé du système avec un logiciel permettant l&#39;acquisition automatique des données et le stockage. </li></ol></li></ol> 2.2) Mesure de l&#39;activité NHE Forward (Extrusion duLes protons du cytosol) <ol><li> Acidifier cellules par une incubation de 1 h dans la solution NH 4 + chargement (voir l&#39;étape 1.4) en l&#39;absence de CO 2. </li><li> Ajouter BCECF-AM (5 uM de concentration finale) à la solution pour les cinq dernières minutes, puis rincer avec de NH 4 + solution de charge pour éliminer la sonde extracellulaire. </li><li> Monter les cellules sur le microscope et prendre plusieurs images pour enregistrer une ligne de base stable. Perfuser la solution de rinçage (voir ci-dessus). Il en résulte une chute de la fluorescence correspondant à l&#39;acidification. </li><li> Après stabilisation du pH, perfuser l&#39;une des solutions ci-dessous pour mesurer les taux de récupération de pH médiées par Li + / H +, Na + / H + ou K + / H + échange. Chaque solution contient un cation de couplage pourrait être testé pour l&#39;échange de H +. Si l&#39;un de ceux qui est transporté, cela se traduira par une augmentation de la fluorescence à 495 nm qui correspond à une augmentation du pH intracellulaire: 120 mM de LiCl, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, glucose 5 mM et HEPES 15 mM à pH 7,4 NaCl 120 mM, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, glucose 5 mM et HEPES 15 mM à pH 7,4 125 mM de KCl, 1 mM de MgCl2, CaCl2 2 mM, glucose 5 mM et HEPES 15 mM à pH 7,4 </li></ol> 2.3) Mesure de NHE inverse Activité NOTE: Le principe ici est d&#39;inverser les gradients ioniques transmembranaires. <ol><li> Avant la mesure, charger les cellules avec le cation intracellulaire d&#39;intérêt (voir ci-dessous). </li><li> Incuber pendant 5 min avec BCECF / AM (voir l&#39;étape 2.2.2); rincez-les, montez-les sur le microscope vidéo. </li><li> Perfuser la solution de chargement et de prendre plusieurs images pour enregistrer une ligne de base stable. </li><li> Après la stabilisation de base, l&#39;image les variations de pH lorsque les cellules sont perfusées avec un milieu acide extracellulaire contenant 120 mM de chlorure de choline, 5 mM de KCl, 1 mM de MgCl2, CaCl2 2 mM, glucose 5 mM et 15 mM de MES à pH 6,5. </li><li> Pour LiCl chargement, incuber les cellules pendant 2 heures dans une solution composée de 120 mM de LiCl, KCl 5 mM, MgCl2 1 mM, CaCl2 2 mM, glucose 5 mM et HEPES 15 mM à pH 7,4. Lithium intracellulaire mesuré par spectroscopie d&#39;absorption atomique donne une valeur d&#39;environ 60 mM. </li><li> Pour le chargement Na +, les cellules incuber pendant deux heures dans une solution composée de 120 mM de NaCl, 5 mM de KCl, 1 mM de MgCl2, 2 mM de CaCl2, 5 mM de glucose et 15 mM de HEPES à pH 7,4 en présence de 1 mM d&#39;ouabaïne de bloquer la Na / K ATPase. Dans ces conditions, la concentration intracellulaire de Na + est de l&#39;ordre de 40 mM. REMARQUE: K + chargement ne est pas nécessaire que K + cytosolique concentration est dans la gamme de 140 mM et un gradient K + dirigée vers l&#39;extérieur est donc facile à réaliser. </li></ol> 2.4) Le traitement des données et d&#39;étalonnage: NOTE: Cette étape se applique à la fois pour les mesures avant et arrière. <ol><li> A la fin de chaque expérience, perfuser les cellules avec une solution de KCl 140 mM, HEPES 20 mM et 5 uM nigéricine ajustée à des valeurs de pH entre 6,5 et 7,4. </li><li> Recueillir des données que des mesures de fluorescence à partir des images (niveaux de gris représentant niveaux d&#39;intensité) et l&#39;exportation sous format texte et traiter comme il est expliqué ci-dessous à l&#39;aide d&#39;un tableur. </li><li> Calculer les valeurs de pH intracellulaire pour chaque cellule ou d&#39;une région d&#39;intérêt en utilisant l&#39;équation suivante individuelle: pH = pKa + (Log (R-Rmin) / (R max -R)) x F min (λ2) / F max (λ2) </li><li> Utiliser le F min (de λ2) / F x MA (λ2) se il existe rapport blanchiment de sonde ou de fuite, ce qui entraîne une diminution de la fluorescence de 450 nm tout au long des expériences. Ce ne est cependant très rarement observée dans les conditions utilisées dans le DEScribed expériences. </li><li> Comme les données d&#39;étalonnage sont présents à la fin de chaque mesure de pH, de vérifier si les valeurs calculées correspondantes diffèrent des valeurs de pH utilisées pour l&#39;étalonnage. Si ce est le cas, puis réglez toutes les valeurs de pH déterminé de manière expérimentale à l&#39;étalonnage en utilisant la procédure suivante: <ol><li> Calculer le quotient suivante (Q, la différence entre la valeurs mesurées / Différence entre les valeurs d&#39;étalonnage). Multipliez l&#39;ensemble des données par Q. Faire l&#39;ajustement final par addition ou soustraction de sorte que les valeurs calculées seront égales les valeurs d&#39;étalonnage correspondantes. </li></ol></li></ol> 3. Mesures de taux initiaux de NHE7 par Fast Li + Absorption <ol><li> cellules de semences sur des plaques à puits multiples (6 à 24 puits) et acidifier les utiliser soit la technique NH 4 + de chargement décrit ci-dessus ou alternativement le nigéricine / sérum albumine bovine (BSA) acidification décrite dans 20. </li><li&gt; Rincer les plaques deux fois rapidement en utilisant la solution de rinçage décrit ci-dessus et ensuite incuber les cellules dans des solutions contenant de 1 à 10 mM absorption lithium (légèrement en dessous de valeurs de Km) et les concentrations désirées de cations et / ou des inhibiteurs d&#39;intérêt pour la quantité souhaitée de temps. </li><li> Maintenir courtes et cohérentes durées d&#39;absorption (généralement une minute ou ci-dessous) afin de se assurer que le transport fonctionne dans des conditions de taux initiaux. Assurez-vous également que toutes les solutions utilisées pour l&#39;absorption sont isotoniques. </li><li> A la fin du temps d&#39;absorption, éliminer soigneusement le milieu d&#39;absorption et laver les cellules à quatre reprises avec une solution saline phosphate glacée tamponnée (PBS). Effectuez ces lavages aussi vite que possible (moins de 10 secondes pour les quatre d&#39;entre eux est idéal) pour empêcher lithium efflux. </li><li> Lyse des cellules dans 25% d&#39;acide nitrique. Appliquer 250 pi par puits de 25% d&#39;acide nitrique et laisser reposer pendant au moins 1 h, puis la ferraille chaque puits avec l&#39;extrémité de la pointe de la pipette et transférer la suspension de l&#39;ensemble ainsi dans une nouvelle kmTube crocentrifuge. </li><li> Transférer le lysat dans 1,5 ml microtubes, centrifuger les pendant 5 minutes à 15 000 xg (à température ambiante) pour éliminer les débris cellulaires. </li><li> Mesurer la teneur en lithium des surnageants par spectroscopie d&#39;absorption atomique 21. NOTE: Différentes entreprises offrent spectromètres d&#39;absorption atomique, qui doivent être équipé d&#39;une lampe à cathode creuse au lithium. Suivez les instructions du fabricant, car ces machines sont très précises mais aussi très délicate. </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.

Ce protocole décrit la manière de sélectionner les cellules exprimant intracellulaire de Na + / H + échangeurs à la membrane plasmique sans modification de leur séquence primaire par mutagenèse dirigée. Ces échangeurs peuvent maintenant être caractérisés. Cette méthode est basée sur la toxicité cellulaire des protons intracellulaires pour sélectionner des lignées cellulaires qui expriment vésiculeuse Na + / H + échangeurs à la m...

La plupart des compartiments intracellulaires afficher un luminale pH acide, qui est un paramètre clé pour la maturation, le trafic, le recyclage des protéines ou des hormones et des neurotransmetteurs chargement. Il a été montré que le gradient de pH entre le cytosol et vésiculaire contenu est généré par vacuolaire H + ATPases 1, couplé transporteurs vésiculaires de chlorure ClC 2. Tant dans knock-out (KO) chez la souris et des patients humains, l&#39;importance de ces transporteurs a été soulignée par les phénotypes lourds causés par des mutations dans leurs gènes 3-6. Les membres de la famille de SLC9A échangeurs sodium-hydrogène, aussi appelés NHE pour Na + / H + échangeurs, ont été montré pour être effecteurs clés du pH intracellulaire et la régulation du volume cellulaire, ainsi que dans le transport vectoriel d&#39;équivalents acide-base à travers l&#39;épithélium . Outre les NHE de la membrane plasmique, trois hautement conservée Na + / H + échangeurs NHE 6, 7et 9 sont exprimés dans le réseau trans-Golgi et les endosomes précoces 7. Des mutations dans leurs gènes ont été associés à Angelman-like ou Christianson Syndromes 8-9, l&#39;autisme en milieu familial et 10 Attention Deficit Hyperactivity Disorder 11-12. Ces échangeurs ont également été impliqués dans des problèmes neurodégénératives telles que la maladie d&#39;Alzheimer 13 et la susceptibilité des gènes contigus retard mental lié à l&#39;X syndromes 14. Pris ensemble, ces études mettent en évidence l&#39;importance de ces NHE intracellulaires dans le développement et / ou le fonctionnement du cerveau. La localisation intracellulaire de ces échangeurs empêche des mesures précises de leur sélectivité d&#39;ions, la direction de transport, les paramètres cinétiques et la réglementation. Comme ce est le cas pour tous les transporteurs exprimées dans des compartiments intracellulaires, il est extrêmement difficile d&#39;évaluer leurs activités biochimiques et donc de comprendre pleinement leurs rôles physiologiques et l&#39;Mechanismes qui sous-tend leurs implications pathologiques. Basé sur la concentration élevée K + cytosolique, l&#39;hypothèse la plus communément admise était-qu&#39;ils travaillaient comme K + couplés protons transporteurs d&#39;efflux. L&#39;existence d&#39;une telle fuite de protons a été émis l&#39;hypothèse, car il peut contrebalancer protons pompage par les V-ATPases afin de maintenir un pH vésiculaire état stable. Le but de cet article visuelle est (i) de démontrer une méthode qui permet la sélection génétique de lignées cellulaires qui expriment ces transporteurs vésiculaires à leur membrane plasmique, et (ii) montrent deux approches indépendantes pour mesurer les fonctions de ces transporteurs. Il ya trois décennies, Pouyssegur et Franchi ont mis au point une approche génétique qui a permis le clonage moléculaire et la caractérisation des membres de la famille NHE 15. Cela était basé sur la toxicité des protons intracellulaires en tant que procédé de criblage. La première étape a consisté à obtenir des lignées cellulaires déficientesdans ne importe quel / H échange de Na + + exprimé à la membrane de plasma, en utilisant la réversibilité de ce transporteur. Les fibroblastes (lignée cellulaire de CCL39) ont été préchargées avec Na + ou Li + et ensuite placées dans un milieu extracellulaire acide (pH 6,5) pendant 2 heures. Cela a conduit à la mort des cellules exprimant un Na + / H + échange fonctionnel et à la sélection des cellules déficientes en antiport (lignée cellulaire de 16 PS120). Lorsque cultivées dans un milieu sans bicarbonate, ces cellules sont très sensibles à l&#39;acidification intracellulaire aiguë. Par conséquent, l&#39;expression de tout mécanisme d&#39;efflux de protons fonctionnelle à la membrane plasmique sera sélectionné positive (voir 17) si ces cellules sont soumises à acidifications intracellulaires aigus. De telles techniques d&#39;acidification peuvent être utilisées pour isoler des lignées de cellules de trafic de défauts permettant l&#39;expression forcée de NHE intracellulaires WT à la membrane plasmique. Comme eucaryote Na + / H+ Échangeurs sont électroneutre, ils ne sont pas mesurables par les approches électrophysiologiques qui ont été utilisés avec succès pour mesurer canaux. Ce manuscrit montre donc comment mesurer l&#39;activité de cet échangeur par des mesures de pH intracellulaires et une cinétique rapide de l&#39;absorption de lithium. Comme les concepts sous-jacents sont les mêmes, il est intéressant de remarquer que la plupart des procédés mis au point pour la section de sélection sont également utilisés directement pour les mesures fonctionnelles. Chose intéressante, nous avons observé que le défaut de traite présent dans les lignées cellulaires sélectionnées en utilisant l&#39;approche décrite dans ce manuscrit conduit à une plus grande expression d&#39;autres protéines vésiculaires à la membrane plasmique, comme le canal potassique 18 TWIK1 vésiculaire. Ceci souligne vers la sélection d&#39;un mécanisme général de défauts de conservation des protéines transmembranaires vésiculaires. Ce est pourquoi cette procédure de sélection et les cellules qu&#39;elle génère maiconstituent un outil prometteur pour la communauté scientifique travaillant sur les protéines membranaires de compartiments intracellulaires. Ainsi les techniques de mesure présentées ici pourraient être applicables pour l&#39;étude d&#39;autres transporteurs non-électrogènes.

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

L&#39;acidification des endosomes est essentiel pour un large éventail de processus, tels que le recyclage des protéines et de la dégradation, désensibilisation des récepteurs, et neurotransmetteur chargement dans les vésicules synaptiques. Cette acidification est décrite être médiée par ATPases de protons, couplés à des transporteurs de chlorure ClC. Hautement conservé protons électroneutres transporteurs, le Na + / H + (NHE) échangeurs 6, 7 et 9 sont également exprimés dans ces compartiments. Des mutations dans leurs gènes ont été liés à des maladies cognitives et neurodégénératives humaines. Paradoxalement, leurs rôles demeurent insaisissables, que leur localisation intracellulaire a empêché la caractérisation fonctionnelle détaillée. Ce manuscrit montre une méthode pour résoudre ce problème. Il se agit de la sélection de lignées cellulaires mutantes, capables de survivre acidification cytosolique aigu en conservant NHE intracellulaires à la membrane plasmique. Il décrit ensuite deux protocoles complémentaires pour mesurer la sélectivité et l&#39;activité d&#39;ionsde ces échangeurs: (i) une basés sur des mesures de pH intracellulaire par microscopie de fluorescence vidéo, et (ii) une base sur la cinétique de l&#39;absorption rapide de lithium. Ces protocoles peuvent être extrapolés à mesurer d&#39;autres transporteurs non-électrogènes. De plus, la procédure de sélection présentés ici génère des cellules avec un phénotype défectueux rétention intracellulaire. Par conséquent, ces cellules vont aussi exprimer d&#39;autres protéines de la membrane vésiculaire dans la membrane plasmique. La stratégie expérimentale représenté ici peut donc constituer un outil potentiellement puissant pour étudier d&#39;autres protéines intracellulaires qui seront ensuite exprimés à la membrane plasmique avec le vésiculaire Na + / H + échangeurs utilisés pour la sélection.

Sélection: La sélection + H tuant est basé sur la diffusion de l&#39;ammonium base faible, telle que représentée sur la figure 1A. L&#39;effet des bases faibles et acides diffusion sur le pH intracellulaire a été mis au point par Walter bore et ses collaborateurs 22. L&#39;idée élégante d&#39;utiliser ce phénomène pour produire une acidification mortelle pour la sélection génétique positive a été ensuite développé par Jacques Pouysseg...

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.

Caractérisation fonctionnelle de Na<sup&gt; +</sup&gt; / H<sup&gt; +</sup&gt; Echangeurs de intracellulaire Compartiments utilisant la sélection Proton-tuant les exprimer à la membrane plasmique

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

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Research

JoVE Journal

Biology

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

9.2K vues.  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. 

Vidéo: 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.

Mesure Proche membrane plasmique et mondiale dynamique du calcium intracellulaire dans les 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, ...

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Biologie

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.

Cristallisant protéines membranaires pour déterminer la structure à l&#39;aide mésophases lipidique

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.

Mesure quantitative de GLUT4 Translocation à la membrane plasmique par cytométrie en flux

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.

Identifier les effets des mutations du gène BRCA1 sur la recombinaison homologue en utilisant les cellules qui expriment endogène de type sauvage du gène 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 ...

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.

Utilisation d&#39;un robot pour haut-débit cristallisation des protéines membranaires dans des mésophases lipidiques

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.

Cristaux de récolte et de Cryo-refroidissement de protéines membranaires cultivées dans des mésophases lipidiques pour la détermination de la structure par cristallographie macromoléculaire

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.

Microscopie intravitale pour l&#39;imagerie Subcellular ouvrages chez des souris vivantes exprimant des protéines fluorescentes

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.

Imaging Plasma Membrane déformations Avec 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 ...

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure plasma (LPP) discharges contain a broad spectrum of active species, which lead to rapid microbial inactivation. To study the efficiency and mechanisms of sterilization by LPP, we use spores of the test organism Bacillus subtilis because of their extraordinary resistance against conventional sterilization procedures. We describe the production of B. subtilis spore monolayers, the sterilization process by low pressure plasma in a double inductively coupled plasma reactor, the characterization of spore morphology using scanning electron microscopy (SEM), and the analysis of germination and outgrowth of spores by live cell microscopy. A major target of plasma species is genomic material (DNA) and repair of plasma-induced DNA lesions upon spore revival is crucial for survival of the organism. Here, we study the germination capacity of spores and the role of DNA repair during spore germination and outgrowth after treatment with LPP by tracking fluorescently-labelled DNA repair proteins (RecA) with time-resolved confocal fluorescence microscopy. Treated and untreated spore monolayers are activated for germination and visualized with an inverted confocal live cell microscope over time to follow the reaction of individual spores. Our observations reveal that the fraction of germinating and outgrowing spores is dependent on the duration of LPP-treatment reaching a minimum after 120 s. RecA-YFP (yellow fluorescence protein) fluorescence was detected only in few spores and developed in all outgrowing cells with a slight elevation in LPP-treated spores. Moreover, some of the vegetative bacteria derived from LPP-treated spores showed an increase in cytoplasm and tended to lyse. The described methods for analysis of individual spores could be exemplary for the study of other aspects of spore germination and outgrowth.

Investigating the Detrimental Effects of Low Pressure Plasma Sterilization on the Survival of Bacillus subtilis Spores Using Live Cell Microscopy

This protocol illustrates the important consecutive steps required to assess the relevance of monitoring vitality parameter and DNA repair processes in reviving Bacillus subtilis spores after treatment with low pressure plasma by tracking fluorescence-labelled DNA repair proteins via time-resolved confocal microscopy and scanning electron microscopy.

Enquête sur les néfastes effets de basse pression Plasma stérilisation sur la survie de Bacillus subtilis Spores en utilisant Live microscopie des cellules

Plasma sterilization is a promising alternative to conventional sterilization methods for industrial, clinical, and spaceflight purposes. Low pressure ...