Diese Arbeit wurde durch NIH R01 GM50322, um PM Kane unterstützt. Die Autoren danken Dr. Rajini Rao, Johns Hopkins University für die Bereitstellung der Hefe-pHluorin Plasmiden und für Erfahrungsberichte zu ratiometrische pH-Messungen, und Dr. Gloria A. Martinez Munoz für das Arbeiten out diese Protokolle für unsere lab.

Ein. Messung von pH Vacuolar In Vivo Mit BCECF-AM <ol><li> Grow a 50 ml Flüssigkeit Kultur der Hefestamm in der gewünschten Medium gemessen über Nacht werden. Ziel ist es, Zellen in der mittleren logarithmischen Phase haben (OD600 (optische Dichte bei 600 nm) Messung von ca. 0,8 für die Suspension). </li><li> Pellet die Hefezellen durch Zentrifugation. Das Pellet in 0,6 ml Wachstumsmedium und Transfer zu einem Mikrozentrifugenröhrchen, die gewogen hat vorher. Pellet die Zellen wieder in einer Mikrozentrifuge bei 2.000 xg für 60 sec. Entfernen Sie den Überstand so vollständig wie möglich und dann wiegen das Zellpellet. Das Pellet auf eine endgültige Dichte von 0,5 g / ml (w / v), einer Kultur dieses Bandes wird ein Zellpellet von etwa 200 mg und 200 ul Puffer geben würde hinzugefügt, um ein Endvolumen von 400 ul zu ergeben. </li><li> In BCECF-AM zu der Zellsuspension in einer Endkonzentration von 50 mM aus einer 12 mM Stammlösung in DMSO hergestellt. Gut mischen und dann incUbate die Zellen bei 30 ° C für 30 min. auf einem Schaukelstuhl Plattform oder Bandage. </li><li> Während die Zellen Inkubation, bereiten Kalibrierpuffern. Die Kalibrierung Puffer enthält 50 mM MES (2 - (N-Morpholino) ethansulfonsäure), 50 mM HEPES (4 - (2-hydroxyethyl)-1-piperazinethansulfonsäure), 50 mM KCl, 50 mM NaCl, 0,2 M Ammoniumacetat, 10 mM Natriumazid und 10 mM 2-deoxyglucose, und ist der pH-eingestellten Werte für Ihre Kalibrierbereich mit NaOH oder HCl. (Vorsicht: Natriumazid ist sehr giftig und sollte mit Sorgfalt behandelt werden.) Für die Messung des pH-Wertes in Wildtyp-Zellen, würden wir mehrere Mischungen Kalibrierung bei pH 5,5 bis 6,5 zubereiten, aber für Mutanten erwartet, dass eine mehr alkalische pH vacuolar haben ( vma-Mutanten), würden wir uns vorbereiten zusätzliche, höhere pH-Puffer. </li><li> Aliquot 2 ml Puffer Kalibrierung für jede pH in 15 ml konische Röhrchen, und fügen Monensin (15 mM stock) und Nigericin (2 mM Lager), um Endkonzentrationen von 110 pM und 15 pM Monensin Nigericin, r gebenespectively. Mischen Sie gut auf einem Vortex-Mischer. (Achtung: Sowohl Monensin und Nigericin sind giftig und sollten mit Vorsicht behandelt werden.) </li><li> Wenn die BCECF-AM Inkubationszeit abgeschlossen ist, Pellet die Zellsuspension durch Zentrifugation für 30 sec bei 2.000 xg in einer Mikrozentrifuge. Resuspendieren der Zellen 1 ml Wachstumsmedium Glucose mangelt und zentrifugiert wie oben. Wiederholen Sie diesen Waschschritt einmal, und dann das Pellet in 200 ul Wachstumsmedium ohne Glucose. Platz auf dem Eis. </li><li> In 20 ul der Zellsuspension jedem pH Kalibrierungsröhre oben hergestellt. Inkubation bei 30 ° C für 30-60 min. auf einer Bandage. </li><li> Stellen Sie die Fluorimeter abwechselnd bei Anregungswellenlängen 450 nm und 490 nm, jeweils mit einer Emissionswellenlänge von 535 nm zu messen. Stellen Sie den Probenraum Temperatur auf 30 ° C. Alle Messungen unter ständigem Rühren der Mischung in die Küvette. </li><li> In 1,96 ml 1 mM MES (eingestellt auf pH 5 oder 7 in Abhängigkeit vom pH-Wert der Zellen Wachstumsmedium einernd das experimentelle Design). In 20 ul Zellsuspension in der Küvette. Initiieren Fluoreszenzmessungen. Wir sammeln sowohl kontinuierliche kinetische und Zeit-Punkt-Daten in verschiedenen Experimenten. Zur kontinuierlichen kinetischen Daten wir Messungen alle 6 Sekunden für 5 min, dann addieren Glucose zu einer endgültigen Konzentration von Glucose 50 mM und weiterhin Messung für 5-10 min. Für einzelne Zeit-Punkt-Messungen, nehmen wir grundsätzlich Messungen bei 1 und 5 min. nach Zugabe von Zellen in die Küvette, fügen Glucose als in den kinetischen Messungen, und dann eine weitere Messung 5 min. nach Glukose hinaus. Diese Zusätze können variiert werden, und zusätzliche Komponenten (Inhibitoren usw.) können ebenfalls zugegeben werden. </li><li> Nachdem die experimentellen Messungen abgeschlossen sind, entfernen Sie die Kalibrierung Rohre aus dem 30 ° C Inkubation und übertragen das gesamte Volumen von 2 ml für jede der Fluorimeters Küvette. Messen der Fluoreszenz bei den gleichen Einstellungen (siehe 1.8) alle 5 Sek. über insgesamt 30 sec für jede Probe. </li><li>Exportieren Fluoreszenz Daten in Microsoft Excel. (Für unsere Fluorimeters erfordert dies den Export von Daten als Text in durch Tabulatoren getrennte Form und Import in Excel.) Gewinne eine Eichkurve, indem das Verhältnis der Fluoreszenz bei 490 nm bis 450 nm für jede Kalibrierung Mischung. Die Fluoreszenz-Verhältnis wird dann gegen pH aufgetragen, um eine Eichkurve zu erhalten. </li><li> Konvertieren Sie die experimentellen Daten zur Fluoreszenz-Verhältnis und pH berechnen anhand der Standardkurve bestimmt. Vacuolar pH-Wert kann dann gegen die Zeit aufgetragen (Kinetik der pH-Änderungen) oder im Vergleich unter verschiedenen Bedingungen (Abbildung 1) oder in verschiedenen Mutanten. </li></ol> 2. Die Messung der cytosolischen pH In Vivo Mit Hefe pHluorin <ol><li> Verwandeln Sie den gewünschten Hefestamm mit der Hefe pHluorin Plasmid durch Standardprotokolle, und wählen Sie für die Transformanten auf Minimalmedium ergänzt ohne Uracil (SC-Uracil). </li><li> Grow a 50 ml Flüssigkeit Kultur der transformierten Zellen in SC-Uracil bis zur mittleren logarithmischen Phase (OD <sub&gt; 600 = 0,8 oder niedriger). </li><li> Kalibrierstandards für vacuolar pH, sondern Puffer auf einen pH-Bereich geeignet cytosolischen pH-Messungen, in der Regel pH 6-8 beschrieben. Aliquot 2 ml Puffer Kalibrierung auf den gewünschten pH-Wert in mehrere 15 ml konischen Röhrchen eingestellt. In Monensin und Nigericin wie oben (1.4) beschrieben und gut mischen. </li><li> Ernten der Zellen durch Zentrifugation wie oben beschrieben, und das Pellet in 600 ul des Wachstumsmediums. Transfer zu einem gewogen Mikrozentrifugenröhrchens und Pellet-Zellen durch Zentrifugation bei 5.000 rpm für 30 sec. Resuspendieren des Pellets in 1 ml Wachstumsmedium ohne Glucose (SC-Uracil-glucose), Pellet-Zellen und wiederholen. Nach der letzten Zentrifugation den Überstand so gründlich wie möglich und wiegen das Zellpellet. Die Zellen auf eine endgültige Dichte von 0,5 g / ml in SC-Uracil ohne Glucose. </li><li> In 20 ul Zellsuspension in jedes Röhrchen der Kalibrierung Puffer und gut mischen auf einem Vortex-Mischer. Inkubation bei 30 ° C auf einer sich drehenden Trommel Rotor für 60 min. </li><li> Richten Sie die Fluorimeters zur Anregung bei Wellenlängen 405 und 485 nm und einer Emissionswellenlänge von 508 nm auf. Fahren Sie mit dem einzigen Zeitpunkt oder kinetische Messungen und Zugabe von Glucose, wie oben für BCECF Messung beschrieben. </li><li> Messen Fluoreszenz Kalibrierproben und kann eine Kalibrationskurve für BCECF (siehe 1,10-1,11). Eine Auftragung der Fluoreszenz gegen pH Verhältnis linear über den Bereich der am cytosolischen pH-Messungen (pH 6,0-8,0) 9 sind so experimentellen Fluoreszenzverhältnis einfache Messung pH umgewandelt. </li></ol>

<ol>
	<li>Casey, J. R., Grinstein, S., Orlowski, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensors+and+regulators+of+intracellular+pH.">Sensors and regulators of intracellular pH.</a> Nat. Rev. Mol. Cell Biol. 11, 50-61 (2010).</li><li>Orij, R., Brul, S., Smits, G. J. <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+is+a+tightly+controlled+signal+in+yeast.">Intracellular pH is a tightly controlled signal in yeast.</a> Biochim. Biophys. Acta. 1810, 933-944 (2011).</li><li>Diakov, T. T., Kane, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+vacuolar+proton-translocating+ATPase+activity+and+assembly+by+extracellular+pH.">Regulation of vacuolar proton-translocating ATPase activity and assembly by extracellular pH.</a> J. Biol. Chem. 285, 23771-23778 (2010).</li><li>Martinez-Munoz, G. A., Kane, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vacuolar+and+plasma+membrane+proton+pumps+collaborate+to+achieve+cytosolic+pH+homeostasis+in+yeast.">Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast.</a> J. Biol. Chem. 283, 20309-20319 (2008).</li><li>Tarsio, M., Zheng, H., Smardon, A. M., Martinez-Munoz, G. A., Kane, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Consequences+of+loss+of+Vph1+protein-containing+vacuolar+ATPases+(V-ATPases)+for+overall+cellular+pH+homeostasis.">Consequences of loss of Vph1 protein-containing vacuolar ATPases (V-ATPases) for overall cellular pH homeostasis.</a> J. Biol. Chem. 286, 28089-28096 (2011).</li><li>Plant, P. J., Manolson, M. F., Grinstein, S., Demaurex, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alternative+mechanisms+of+vacuolar+acidification+in+H(+)-ATPase-deficient+yeast.">Alternative mechanisms of vacuolar acidification in H(+)-ATPase-deficient yeast.</a> J. Biol. Chem. 274, 37270-37279 (1999).</li><li>Ali, R., Brett, C. L., Mukherjee, S., Rao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+sodium/proton+exchange+by+a+Rab-GTPase-activating+protein+regulates+endosomal+traffic+in+yeast.">Inhibition of sodium/proton exchange by a Rab-GTPase-activating protein regulates endosomal traffic in yeast.</a> J. Biol. Chem. 279, 4498-4506 (2004).</li><li>Miesenbock, G., De Angelis, D. A., Rothman, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+secretion+and+synaptic+transmission+with+pH-sensitive+green+fluorescent+proteins.">Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins.</a> Nature. 394, 192-195 (1998).</li><li>Brett, C. L., Tukaye, D. N., Mukherjee, S., Rao, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+endosomal+Na+K+/H++exchanger+Nhx1+regulates+cellular+pH+to+control+vesicle+trafficking.">The yeast endosomal Na+K+/H+ exchanger Nhx1 regulates cellular pH to control vesicle trafficking.</a> Mol. Biol. Cell. 16, 1396-1405 (2005).</li><li>Owen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+spectrum-shifting+intracellular+pH+probes+5'(and+6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7,+1'(3'H)-isobenzofuran]-3'-one+and+2',7'-biscarboxyethyl-5(and+6)-carboxyfluorescein.">Comparison of spectrum-shifting intracellular pH probes 5'(and 6')-carboxy-10-dimethylamino-3-hydroxyspiro[7H-benzo[c]xanthene-7, 1'(3'H)-isobenzofuran]-3'-one and 2',7'-biscarboxyethyl-5(and 6)-carboxyfluorescein.</a> Anal. Biochem. 204, 65-71 (1992).</li><li>Dechant, R., 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=Cytosolic+pH+is+a+second+messenger+for+glucose+and+regulates+the+PKA+pathway+through+V-ATPase.">Cytosolic pH is a second messenger for glucose and regulates the PKA pathway through V-ATPase.</a> Embo J. 29, 2515-2526 (2010).</li><li>Gustavsson, M., Barmark, G., Larsson, J., Muren, E., Ronne, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+genomics+of+monensin+sensitivity+in+yeast:+implications+for+post-Golgi+traffic+and+vacuolar+H+-ATPase+function.">Functional genomics of monensin sensitivity in yeast: implications for post-Golgi traffic and vacuolar H+-ATPase function.</a> Mol. Genet. Genomics. 280, 233-248 (2008).</li><li>Kovac, L., Bohmerova, E., Butko, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionophores+and+intact+cells.+I.+Valinomycin+and+nigericin+act+preferentially+on+mitochondria+and+not+on+the+plasma+membrane+of+Saccharomyces+cerevisiae.">Ionophores and intact cells. I. Valinomycin and nigericin act preferentially on mitochondria and not on the plasma membrane of Saccharomyces cerevisiae.</a> Biochim. Biophys. Acta. 721, 341-348 (1982).</li><li>Braun, N. A., Morgan, B., Dick, T. P., Schwappach, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+yeast+CLC+protein+counteracts+vesicular+acidification+during+iron+starvation.">The yeast CLC protein counteracts vesicular acidification during iron starvation.</a> J. Cell Sci. 123, 2342-2350 (2010).</li><li>Orij, R., Postmus, J., Beek, T. e. r., Brul, A., S, G. J., Smits, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+measurement+of+cytosolic+and+mitochondrial+pH+using+a+pH-sensitive+GFP+derivative+in+Saccharomyces+cerevisiae+reveals+a+relation+between+intracellular+pH+and+growth.">In vivo measurement of cytosolic and mitochondrial pH using a pH-sensitive GFP derivative in Saccharomyces cerevisiae reveals a relation between intracellular pH and growth.</a> Microbiology. 155, 268-278 (2009).</li><li>Zhang, Y. Q., 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=Requirement+for+ergosterol+in+V-ATPase+function+underlies+antifungal+activity+of+azole+drugs.">Requirement for ergosterol in V-ATPase function underlies antifungal activity of azole drugs.</a> PLoS Pathog. 6, e1000939 (2010).</li><li>Brett, C. 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=Genome-wide+analysis+reveals+the+vacuolar+pH-stat+of+Saccharomyces+cerevisiae.">Genome-wide analysis reveals the vacuolar pH-stat of Saccharomyces cerevisiae.</a> PLoS One. 6, e17619 (2011).</li><li>Roberts, C. J., Raymond, C. K., Yamashiro, C. T., Stevens, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+the+yeast+vacuole.">Methods for studying the yeast vacuole.</a> Methods Enzymol. 194, 644-661 (1991).</li><li>Chan, C. Y., 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=Inhibitors+of+V-ATPase+proton+transport+reveal+uncoupling+functions+of+tether+linking+cytosolic+and+membrane+domains+of+V0+subunit+a+(Vph1p).">Inhibitors of V-ATPase proton transport reveal uncoupling functions of tether linking cytosolic and membrane domains of V0 subunit a (Vph1p).</a> J. Biol. Chem. 287, 10236-10250 (2012).</li><li>Johnson, R. 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=Identification+of+inhibitors+of+vacuolar+proton-translocating+ATPase+pumps+in+yeast+by+high-throughput+screening+flow+cytometry.">Identification of inhibitors of vacuolar proton-translocating ATPase pumps in yeast by high-throughput screening flow cytometry.</a> Anal. Biochem. 398, 203-211 (2010).</li></ol>

Die Autoren haben keine Interessenkonflikte offen zu legen.

Wir haben diese Protokolle verwendet, um eine Reihe von Aspekten der pH-Homöostase adressieren. Zum Beispiel haben wir cytosolischen pH und Antworten von Wildtyp-und V-ATPase-defizienten mutierten Zellen 4,5 verglichen. Wir haben auch die Auswirkungen von Veränderungen der Wachstumsbedingungen, insbesondere extrazellulären pH auf pH vacuolar Reaktion auf Glukose 3 untersucht. Wichtig ist, dass die Antworten, die wir beobachten, sowohl im Einklang mit anderen Methoden der quantitativen pH-Messung...

pH-Homöostase ist ein dynamisches und stark regulierten Prozess in allen Organismen 1,2. Biochemische Prozesse sind eng durch pH-reguliert und intrazellulären Umgebungen zu engen pH-Bereich abgestimmt, um eine optimale Wirkung der Enzyme Bewohner zu ermöglichen. Allerdings kann intrazelluläre pH-Homöostase durch schnelle Änderungen der Umweltbedingungen pH-, Stoffwechsel-Verschiebungen und bestimmte Signalwege angefochten werden. Darüber hinaus können intrazelluläre pH selbst als ein wichtiges Signal dienen. Schließlich führen viele Organellen luminalen pH-Werten, die sich von der umgebenden Cytosol und wichtig für organellspezifisch Funktionen sind. Die Hefe Saccharomyces cerevisiae Aktien eine Reihe von pH-Homöostase Mechanismen mit höheren Eukaryoten 2. In den sauren Organellen der endozytischen / lysosomalen Weg wird vor allem durch den pH-Wert hoch konserviert vacuolar Protonen-Translokation ATPase (V-ATPase) gesteuert wird, in Abstimmung mit den vielen exchangers abhängig vom pH-Gradienten. Alle eukaryotischen Zellen haben auch Protonen-Export-Mechanismen. In Pilzen und Pflanzen, eine zweite, deutlich Protonenpumpenhemmer an der Plasmamembran, PMA1 Exporte metabolische Protonen und ist vermutlich die wichtigste Determinante der cytosolischen pH-und Plasma-Membran Potenzial. Die genetische Flexibilität S. cerevisiae und seine wirtschaftliche Bedeutung, haben es ein sehr interessantes und wichtiges Modell für die Untersuchung pH 2 Homöostase. Abgesehen davon, dass die primären Treiber der Organellen Versauerung, sind V-ATPasen hoch Enzyme reguliert und wird in unserem Labor zu verstehen Mechanismen der V-ATPase Regulierung interessiert. Mit diesem Ziel haben wir in vivo Messungen von pH Vakuole und cytosolischen pH wurde mit: 1), um Antworten auf veränderte extrazelluläre Bedingungen, wie Glukose Deprivation und Neuansatz, 2) zu überwachen, um die Auswirkungen von Mutationen, dass ein Kompromiss V-ATPase-Aktivität zu untersuchen, und 3) zur Erkundung der Koorrung der Organellen und Plasmamembran Protonenpumpen 3-5. Diese Experimente wurden erst durch die Entwicklung von robusten ratiometrischen pH-Indikatoren zugänglich in Hefezellen nutzen möglich. . Pflanze et al zeigten erstmals, dass BCECF (2&#39;7&#39;-Bis-(2-Carboxyethyl) -5 - (und 6)-Carboxyfluorescein), das verwendet wurde, weitgehend auf cytosolischen pH in Säugetierzellen zu messen, in der Hefe Vakuole akkumuliert anstatt das Cytosol 6. Dieser Unterschied in der BCECF Lokalisierung hat der vielen hydrolytischen Enzymen in die Vakuole, die geeignet sind, die für die Spaltung der Acetoxy-Methylester aus BCECF-AM (Acetoxymethylester BCECF) und Vakuolen Retention 6 zurückgeführt. Ali et al. 7 weiterentwickelt vacuolar pH-Messung mit BCECF und angepasst diese Messungen mit einem Fluoreszenz-Reader Format. Brett et al. Eingeführt Hefe pHluorin als Mittel zur Messung der cytosolischen pH in Hefe durch Expression einer Plasmid-borne ratiometric pH-sensitive GFP 8 unter der Kontrolle eines Hefe-Promotors 9. Die Anregungsspektren sowohl BCECF und Hefe pHluorin sind pH-empfindlich, so dass sie als ratiometrische pH-Indikatoren, wobei das Verhältnis der Fluoreszenz bei zwei Anregungswellenlängen, in einer einzigen Emissionswellenlänge gemessen, ein Maß von pH 8,10 verwendet. Diese Hefe Vakuole und cytosolischen pH-Sensoren sind sowohl für Single-Cell-und bevölkerungsbezogenen Messungen verwendet worden. Einzel-Zell-Messungen 6,11 werden durch Fluoreszenz-Mikroskopie und Bildanalyse durchgeführt. Vacuolar oder cytosolische Fluoreszenz bei den beiden Wellenlängen für jede Zelle gemessen. Die Bevölkerung-basierten Messungen werden entweder in einem Mikroplatten-Reader mit entsprechenden Fluoreszenz-Funktionen oder in einem Fluorimeter durchgeführt. Wir haben unsere Messungen im Allgemeinen in einem Fluorimeter durchgeführt, weil sie einen einfachen Zugang für die Zugabe von Komponenten, wie Glucose während con bietetkontinuierlichen kinetischen Messungen. Unsere aktuellen Labor-Protokolle für die Messung der Vakuole und cytosolischen pH sind unten aufgeführt, beide sind auch leicht zu Mikrotiterplatten-Assays angepasst.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>Spectrofluorometer</td>
 <td>Horiba Jobin Yvon</td>
 <td>Model Fluoromax-4</td>
 <td>Temperature control and stirring capability are desirable.</td>
 </tr>
 <tr>
 <td>BCECF-AM</td>
 <td>Invitrogen/Molecular Probes</td>
 <td>B1150</td>
 <td>Prepare a 12 mM stock in dry DMSO, store as aliquots at -20 &deg;C </td>
 </tr>
 <tr>
 <td>monensin</td>
 <td>Sigma</td>
 <td>M5273</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>nigericin</td>
 <td>Sigma</td>
 <td>N7143</td>
 <td>Toxic. </td>
 </tr>
 <tr>
 <td>MES</td>
 <td>Sigma</td>
 <td>M8250</td>
 <td></td>
 </tr>
 </tbody>
</table>

measurement of vacuolar and cytosolic ph in vivo in yeast cell suspensions

Vacuolar und cytosolischen pH sind hoch in Hefezellen reguliert und nehmen eine zentrale Rolle in der Gesamtstrategie pH-Homöostase. Wir beschreiben Protokolle für ratiometrischen Messung von pH in vivo mit pH-sensitiven Fluorophoren lokalisiert zur Vakuole oder Cytosol. Vacuolar pH wird unter Verwendung BCECF, die in der Vakuole lokalisiert, wenn sie in Hefe-Zellen in ihrer Acetoxymethylester Form eingeführt. Cytosolischen pH-Wert mit einem pH-sensitive GFP unter Kontrolle eines Hefe-Promotors, Hefe pHluorin ausgedrückt gemessen. Verfahren zur Messung der Fluoreszenz-Verhältnisse in Hefe Zellsuspensionen in einem Fluorimeter beschrieben. Durch diese Protokolle haben einzigen Zeitpunkt Messungen des pH unter verschiedenen Bedingungen oder in verschiedenen Hefe-Mutanten verglichen und Veränderungen im pH im Laufe der Zeit wurden überwacht. Diese Methoden haben auch zu einem Fluoreszenz-Reader-Format für High-Throughput-Experimente angepasst. Vorteile von ratiometrischen pH-Messungen im Vergleich zu anderen apAnsätze derzeit im Einsatz, mögliche experimentelle Probleme und Lösungen und Perspektiven für die zukünftige Verwendung dieser Techniken werden ebenfalls beschrieben.

Abbildung 1 zeigt vacuolar pH Daten auf Wildtyp-Hefezellen in reiche Medium (Hefeextrakt-Pepton-Dextran; YEPD) erhalten, gepuffert auf pH 5 mit 50 mM MES. Oft werden die Zellen in einer gepufferten Medium, weil der pH-Wert des Mediums dramatisch ändern kann während dem Wachstum über Nacht, insbesondere für Minimalmedium, und wir haben festgestellt, dass der pH-Wert des Nährmediums kann sich vacuolar pH 3 Reaktionen. Es ist jedoch auch annehmbar, für viele Experimente, um die Zellen in u...

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Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

Vacuolar und cytosolischen pH-Wert kann in lebende Hefe gemessen werden (<em&gt; S. cerevisiae</em&gt;)-Zellen unter Verwendung ratiometrische Fluoreszenzfarbstoffe lokalisiert spezifischen zellulären Kompartimenten. Wir beschreiben Methoden zur Messung des pH-Wertes mit Vakuolen BCECF-AM, die der Vakuole lokalisiert in Hefe und cytosolischen pH mit einer zytosolischen ratiometrische pH-sensitive GFP (Hefe pHluorin).

Messung von pH Vacuolar und Cytosolic<em&gt; In Vivo</em&gt; In Hefe Zellsuspensionen

Vacuolar and  cytosolic pH are highly regulated in yeast cells and occupy a central role in  overall pH homeostasis. We describe  protocols for ...

measurement-vacuolar-cytosolic-ph-vivo-yeast-cell

Research

JoVE Journal

Biology

Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

19.8K Aufrufe.  SUNY Upstate Medical University. Vacuolar und cytosolischen pH-Wert kann in lebende Hefe gemessen werden ( S. cerevisiae)-Zellen unter Verwendung ratiometrische Fluoreszenzfarbstoffe lokalisiert spezifischen zellulären Kompartimenten. Wir beschreiben Methoden zur Messung des pH-Wertes mit Vakuolen BCECF-AM, die der Vakuole lokalisiert in Hefe und cytosolischen pH mit einer zytosolischen ratiometrische pH-sensitive GFP (Hefe pHluorin).

Serie: Measurement of Vacuolar and Cytosolic pH In Vivo in Yeast Cell Suspensions

In vivo Micro-circulation Measurement in Skeletal Muscle by Intra-vital Microscopy

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of imaging had invented. While many macroscopic parameters of blood flow reflect flow velocity, changes in blood flow velocity and red blood cell (RBC) flux does not hold linear relationship in the microscopic observations. There are reports of discrepancy between RBC velocity and RBC flux, RBC flux and plasma flow volume, and of spatial and temporal heterogeneity of flow regulation in the peripheral tissues in microscopic observations, a scientific basis for the requirement of more detailed studies in microcirculatory regulation using intravital microscopy. METHODS: We modified Jeff Lichtman's method of in vivo microscopic observation of mouse sternomastoid muscles. Mice are anesthetized,
ventilated, and injected with PKH26L-fluorescently labeled RBCs for microscopic observation.RESULT &amp; CONCLUSIONS: Fluorescently labeled RBCs are detected and distinguished well by a wide-field microscope. Muscle contraction evoked by electrical stimulation induced increase in RBC flux. Quantification of other parameters including RBC velocity and capillary density were feasible. Mice tolerated well the surgery, injection of stained RBCs, microscopic observation, and electrical stimulation. No muscle or blood vessel damage was observed, suggesting that our method is relatively less invasive and suited for long-term observations.

A new versatile method for observation of microcirculation is presented. It is considered suitable for long-term observation, and for combination with pharmacophysiological or molecular biological interventions.

In-vivo-Mikro-Zirkulation Measurement im Skelettmuskel von Intra-vital-Mikroskopie

BACKGROUND: Regulatory factors and detailed physiology of in vivo microcirculation have remained not fully clarified after many different modalities of ...

Forschung

Biologie

Single-cell Suction Recordings from Mouse Cone Photoreceptors

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment are open and allow cations to flow steadily inwards across the membrane, depolarizing the cell. Light exposure triggers the closure of the CNG channels, blocks the inward cation current flow, and thus results in cell hyperpolarization. Based on the polarity of photoreceptors, a suction recording method was developed in 1970s that, unlike the classic patch-clamp technique, does not require penetrating the plasma membrane 1. Drawing the outer segment into a tightly-fitting glass pipette filled with extracellular solution allows recording the current changes in individual cells upon test-flash exposure. However, this well-established "outer-segment-in (OS-in)" suction recording is not suitable for mouse cone recordings, because of the low percentage of cones in the mouse retina (3%) and the difficulties in identifying the cone outer segments. Recently, an inner-segment-in (IS-in) recording configuration was developed to draw the inner segment/nuclear region of the photoreceptor into the recording pipette 2,3. In this video, we will show how to record from individual mouse cone photoresponses using single-cell suction electrode.

We will show how to record flash responses from single mouse cones using a suction electrode.

Einzel-Zell-Saug-Recordings aus Maus Zapfen-Photorezeptoren

Rod and cone photoreceptors in the retina are responsible for light detection. In darkness, cyclic nucleotide-gated (CNG) channels in the outer segment ...

Quantitative Measurement of Invadopodia-mediated Extracellular Matrix Proteolysis in Single and Multicellular Contexts

Cellular invasion into local tissues is a process important in development and homeostasis. Malregulated invasion and subsequent cell movement is characteristic of multiple pathological processes, including inflammation, cardiovascular disease and tumor cell metastasis1. Focalized proteolytic degradation of extracellular matrix (ECM) components in the epithelial or endothelial basement membrane is a critical step in initiating cellular invasion. In tumor cells, extensive in vitro analysis has determined that ECM degradation is accomplished by ventral actin-rich membrane protrusive structures termed invadopodia2,3. Invadopodia form in close apposition to the ECM, where they moderate ECM breakdown through the action of matrix metalloproteinases (MMPs). The ability of tumor cells to form invadopodia directly correlates with the ability to invade into local stroma and associated vascular components3. 
Visualization of invadopodia-mediated ECM degradation of cells by fluorescent microscopy using dye-labeled matrix proteins coated onto glass coverslips has emerged as the most prevalent technique for evaluating the degree of matrix proteolysis and cellular invasive potential4,5. Here we describe a version of the standard method for generating fluorescently-labeled glass coverslips utilizing a commercially available Oregon Green-488 gelatin conjugate. This method is easily scaled to rapidly produce large numbers of coated coverslips. We show some of the common microscopic artifacts that are often encountered during this procedure and how these can be avoided. Finally, we describe standardized methods using readily available computer software to allow quantification of labeled gelatin matrix degradation mediated by individual cells and by entire cellular populations. The described procedures provide the ability to accurately and reproducibly monitor invadopodia activity, and can also serve as a platform for evaluating the efficacy of modulating protein expression or testing of anti-invasive compounds on extracellular matrix degradation in single and multicellular settings.

We describe the prototypical method for producing microscope coverslips coated with fluorescent gelatin for visualizing invadopodia-mediated matrix degradation. Computational techniques using available software are presented for quantifying the resultant levels of matrix proteolysis by single cells within a mixed population and for multicellular groups encompassing entire microscopic fields.

Quantitative Messung der Invadopodia-vermittelte extrazellulären Matrix Proteolyse in Einzel-und Multizelluläre Contexts

Cellular invasion into local tissues is a process important in development and homeostasis.  Malregulated invasion and subsequent cell movement is ...

Simple and Efficient Technique for the Preparation of Testicular Cell Suspensions

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline cells. This heterogeneity is an important drawback concerning the study of the bases of mammalian spermatogenesis, as pure or enriched cell populations in certain stages of sperm development are needed for most molecular analyses1.
 Various strategies such as Staput2,3, centrifugal elutriation1, and flow cytometry (FC)4,5 have been employed to obtain enriched or purified testicular cell populations in order to enable differential gene expression studies. 
It is required that cells are in suspension for most enrichment/ purification approaches. Ideally, the cell suspension will be representative of the original tissue, have a high proportion of viable cells and few multinucleates - which tend to form because of the syncytial nature of the seminiferous epithelium6,7 - and lack cell clumps1 . Previous reports had evidenced that testicular cell suspensions prepared by an exclusively mechanical method clumped more easily than trypsinized ones1 . On the other hand, enzymatic treatments with RNAses and/or disaggregating enzymes like trypsin and collagenase lead to specific macromolecules degradation, which is undesirable for certain downstream applications. The ideal process should be as short as possible and involve minimal manipulation, so as to achieve a good preservation of macromolecules of interest such as mRNAs. Current protocols for the preparation of cell suspensions from solid tissues are usually time-consuming, highly operator-dependent, and may selectively damage certain cell types1,8 . 
	The protocol presented here combines the advantages of a highly reproducible and extremely brief mechanical disaggregation with the absence of enzymatic treatment, leading to good quality cell suspensions that can be used for flow cytometric analysis and sorting4, and ulterior gene expression studies9 .

A novel protocol for the mechanical preparation of testicular cell suspensions from rodent material, avoiding enzymes and detergents, is described. The method is very simple, fast, reproducible, and renders good quality cell suspensions, which are suitable for flow sorting and RNA extraction.

Einfache und effiziente Technik zur Herstellung von Hodenkrebs Zellsuspensionen

Mammalian testes are very complex organs that contain over 30 different cell types, including somatic testicular cells and different stages of germline ...

Simultaneous pH Measurement in Endocytic and Cytosolic Compartments in Living Cells using Confocal Microscopy

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial for homeostatic control of physiological processes that include: protein, DNA and RNA synthesis, vesicular trafficking, cell growth and cell division. Alterations in cellular pH homeostasis can lead to detrimental functional changes and promote progression of various diseases2. Various methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of pH in the cytoplasm and in organelles. Here, we describe in detail a rapid and accurate method for the simultaneous measurement of cytoplasmic and organellar pH by using confocal microscopy on living cells3. This goal is achieved with the use of two pH-sensing ratiometric dyes that possess selective cellular compartment partitioning. For instance, SNARF-1 is compartmentalized inside the cytoplasm whereas HPTS is compartmentalized inside endosomal/lysosomal organelles. Although HPTS is commonly used as a cytoplasmic pH indicator, this dye can specifically label vesicles along the endosomal-lysosomal pathway after being taken up by pinocytosis3,4. Using these pH-sensing probes, it is possible to simultaneously measure pH within the endocytic and cytoplasmic compartments. The optimal excitation wavelength of HPTS varies depending on the pH while for SNARF-1, it is the optimal emission wavelength that varies. Following loading with SNARF-1 and HPTS, cells are cultured in different pH-calibrated solutions to construct a pH standard curve for each probe. Cell imaging by confocal microscopy allows elimination of artifacts and background noise. Because of the spectral properties of HPTS, this probe is better suited for measurement of the mildly acidic endosomal compartment or to demonstrate alkalinization of the endosomal/lysosomal organelles. This method simplifies data analysis, improves accuracy of pH measurements and can be used to address fundamental questions related to pH modulation during cell responses to external challenges.

Several methods are available for measuring intracellular pH but very few of these allow simultaneous measurement of cytoplasmic and organellar pH. Here, we describe in detail a rapid and accurate methodology to simultaneously measure cytoplasmic and vesicular pH by ratiometric imaging of living cells.

Gleichzeitige pH-Messung in endozytischen und Cytosolic Fächer in lebenden Zellen mittels konfokaler Mikroskopie

Intracellular pH is tightly regulated and differences in pH between the cytoplasm and organelles have been reported1. Regulation of cellular pH is crucial ...

Measurement of Heme Synthesis Levels in Mammalian Cells

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in various molecular and cellular processes such as gene transcription, translation, cell differentiation and cell proliferation. The biosynthesis levels of heme vary across different tissues and cell types and is altered in diseased conditions such as anemia, neuropathy and cancer. This technique uses [4-14C] 5-aminolevulinic acid ([14C] 5-ALA), one of the early precursors in the heme biosynthesis pathway to measure the levels of heme synthesis in mammalian cells. This assay involves incubation of cells with [14C] 5-ALA followed by extraction of heme and measurement of the radioactivity incorporated into heme. This procedure is accurate and quick. This method measures the relative levels of heme biosynthesis rather than the total heme content. To demonstrate the use of this technique the levels of heme biosynthesis were measured in several mammalian cell lines.

Altered intracellular heme levels are associated with common diseases such as cancer. Thus, there is a need to measure heme biosynthesis levels in diverse cells. The goal of this protocol is to provide a fast and sensitive method to measure and compare the levels of heme synthesis in different cells.

Die Messung der Hämsynthese Levels in Säugerzellen

Heme serves as the prosthetic group for a wide variety of proteins known as hemoproteins, such as hemoglobin, myoglobin and cytochromes. It is involved in ...

Analysis of Cell Suspensions Isolated from Solid Tissues by Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to the analysis of a few fluorochromes, currently there are dozens of different fluorescent dyes, and up to 14-18 different dyes can be combined at a time. However, several limitations still impair the analytical capabilities. Because of the multiplicity of fluorescent probes, data analysis has become increasingly complex due to the need of large, multi-parametric compensation matrices. Moreover, mutant mouse models carrying fluorescent proteins to detect and trace specific cell types in different tissues have become available, so the analysis (by flow cytometry) of auto-fluorescent cell suspensions obtained from solid organs is required. Spectral flow cytometry, which distinguishes the shapes of emission spectra along a wide range of continuous wavelengths, addresses some of these problems. The data is analyzed with an algorithm that replaces compensation matrices and treats auto-fluorescence as an independent parameter. Thus, spectral flow cytometry should be capable of discriminating fluorochromes with similar emission peaks and can provide a multi-parametric analysis without compensation requirements.
This protocol describes the spectral flow cytometry analysis, allowing for a 21-parameter (19 fluorescent probes) characterization and the management of an auto-fluorescent signal, providing high resolution in minor population detection. The results presented here show that spectral flow cytometry presents advantages in the analysis of cell populations from tissues difficult to characterize in conventional flow cytometry, such as the heart and the intestine. Spectral flow cytometry thus demonstrates the multi-parametric analytical capacity of high-performing conventional flow cytometry without the requirement for compensation and enables auto-fluorescence management.

This article describes spectral cytometry, a new approach in flow cytometry that uses the shapes of emission spectra to distinguish fluorochromes. An algorithm replaces compensations and can treat auto-fluorescence as an independent parameter. This new approach allows for the proper analysis of cells isolated from solid organs.

Analyse von Zellsuspensionen isoliert von festen Geweben von Spectral Flow Cytometry

Flow cytometry has been used for the past 40 years to define and analyze the phenotype of lymphoid and other hematopoietic cells. Initially restricted to ...

High-resolution Imaging and Analysis of Individual Astral Microtubule Dynamics in Budding Yeast

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells regulate these processes and how genetic mutations impact regulation. The quantification of microtubule dynamics in metazoan models has a number of associated challenges, including a high microtubule density and limitations on genetic manipulations. In contrast, the budding yeast model offers advantages that overcome these challenges. This protocol describes a method to measure the dynamics of single microtubules in living yeast cells. Cells expressing fluorescently tagged tubulin are adhered to assembled slide chambers, allowing for stable time-lapse image acquisition. A detailed guide for high-speed, four-dimensional image acquisition is also provided, as well as a protocol for quantifying the properties of dynamic microtubules in confocal image stacks. This method, combined with conventional yeast genetics, provides an approach that is uniquely suited for quantitatively assessing the effects of microtubule regulators or mutations that alter the activity of tubulin subunits.

Budding yeast is an advantageous model for studying microtubule dynamics in vivo due to its powerful genetics and the simplicity of its microtubule cytoskeleton. The following protocol describes how to transform and culture yeast cells, acquire confocal microscopy images, and quantitatively analyze microtubule dynamics in living yeast cells.

Hochauflösende Bildgebung und Analyse einzelner Astral Dynamik der Mikrotubuli in Bäckerhefe

Dynamic microtubules are fundamental to many cellular processes, and accurate measurements of microtubule dynamics can provide insight into how cells ...

4D Microscopy of Yeast

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments in yeast are dynamic, and a full understanding of their properties requires time-lapse imaging. Multi-color 4D confocal fluorescence microscopy is a powerful method for tracking the behavior and composition of an intracellular compartment on a time scale of 5-15 min. Rigorous analysis of compartment dynamics requires the capture of thousands of optical sections. To achieve this aim, photobleaching and phototoxicity are minimized by scanning rapidly at very low laser power, and the pixel dimensions and Z-step intervals are set to the largest values that are compatible with sampling the image at full resolution. The resulting 4D data sets are noisy but can be smoothed by deconvolution. Even with high quality data, the analysis phase is challenging because intracellular structures are often numerous, heterogeneous, and mobile. To meet this need, custom ImageJ plugins were written to array 4D data on a computer screen, identify structures of interest, edit the data to isolate individual structures, quantify the fluorescence time courses, and make movies of the projected Z-stacks. 4D movies are particularly useful for distinguishing stable compartments from transient compartments that turn over by maturation. Such movies can also be used to characterize events such as compartment fusion, and to test the effects of specific mutations or other perturbations.

This protocol describes the analysis of fluorescently labeled intracellular compartments in budding yeast using multi-color 4D (time-lapse 3D) confocal microscopy. The imaging parameters are chosen to capture adequate signals while limiting photodamage. Custom ImageJ plugins allow labeled structures to be tracked and quantitatively analyzed.

4D-Mikroskopie der Hefe

The goal of this protocol is to characterize how membrane compartments form and transform in live cells of budding yeast. Many intracellular compartments ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

Wirkung von fluoreszierenden Proteinen auf Fusion Partner mit Trinukleotiderkrankungen Toxizität Assays in Hefe

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...