Diese Arbeit wurde in Teilen durch die NIH gewährt GM073197 und RR025336 finanziert.

Eine typische Gliederung eines in meso Kristallisation Experiment ist in Abb. 1 1,2 dargestellt. Pre-Kristallisation LCP-FRAP-Assays sind optional, aber sie können erheblich beschleunigen den Prozess der Suche nach anfänglichen Kristallisation Bedingungen, insbesondere im Fall der schwer Membran Proteinen 3. 1. Protein Rekonstitution in LCP <ol><li> Purify ein Membranprotein von Interesse in einer Reinigungslösung und konzentrieren das Protein / Detergenz-Komplexen zu ~ 10 bis 20 mg / mL, dabei nicht über-Konzentrat das Waschmittel 1,4. </li><li> Transfer ~ 25 mg eines LCP-Host-Lipid (typischerweise Monoolein) oder einer Lipid-Mischung in eine 1,5 ml Plastikröhrchen und Inkubieren bei 40 ° C für wenige Minuten, bis die Lipid-schmilzt. </li><li> Bringen Sie eine Spritze Kupplung zu 100 ul gasdichte Spritze. </li><li> Legen Sie die Spritze mit dem geschmolzenen Lipid mit einem Volumen einstellbare Pipette. Notieren Sie die Lautstärke des Lipids in der Spritze. </li><li> Legen Sie weitere 100 ul Spritze mit dem Protein-Lösung bei einer Protein-Lösung-to-Lipid-Verhältnis 2 / 3 v / v. </li><li> Verbinden Sie beide Spritzen gemeinsam durch die Spritze Koppler. </li><li> Drücken Sie den Spritzenkolben abwechselnd an die Lipid-und Protein durch die innere Nadel des Kopplers bewegen, hin und her, bis die Lipid-Mesophase homogen wird. LCP bildet sich spontan auf ein mechanisches Mischen, und das Protein wird rekonstituiert in die Lipid-Doppelschicht der LCP. Die Bildung von LCP kann durch seine transparente und gelartige Konsistenz und durch das Fehlen der Doppelbrechung überprüft werden, wenn sie unter einem Mikroskop mit cross-Polarisatoren ausgestattet angesehen, oder, wenn möglich, durch kleine Winkel Röntgenbeugung 1. </li></ol> 2. LCP-FRAP Pre-Kristallisation Assays LCP-FRAP-Assays wurden entwickelt, um die Diffusion Eigenschaften von Membranproteinen rekonstituiert in LCP in einer Vielzahl von Screening-Bedingungen 3 zu messen. Die Langstrecken-Diffusion von Membranproteinen in LCP ist von wesentlicher Bedeutung für eine erfolgreiche Kristallisation, allerdings schränkt die Mikrostruktur von LCP Verbreitung von großen Proteinen oder oligomeren Protein-Aggregaten. Eine häufige Ursache für Ausfall eines in meso Kristallisation Experiment ist eine schnelle Protein-Aggregation führt zu einem Verlust der Diffusion. Es hat sich gezeigt, dass das Aggregationsverhalten des Proteins auf das jeweilige Protein zu konstruieren, die Host-Lipid-und die Zusammensetzung der Screening-Lösung 3 abhängt. <ol><li> Beschriften Sie die Protein mit einem fluoreszierenden Farbstoff (Cy3 oder ähnlich) an ein Protein / Farbstoff-Verhältnis von ~ 100 / 1, entfernen Sie das nicht umgesetzte Farbstoff und konzentrieren das Protein ~ 1 mg / mL. Label entweder freie Amine oder freie Thiole. Bei der Kennzeichnung freie Amine, verwenden Sie pH-Wert zwischen 7 und 7,5 bis überwiegend Etikett der freien N-Terminus. Seien Sie sich bewusst, dass Aminosäuren Kennzeichnung können auch Label Lipiden mit dem Protein 2,3 co-gereinigt. </li><li> Lösen Sie das markierte Protein in LCP, wie in Abschnitt 1 beschrieben). </li><li> Set up Testplatten, wie in Abschnitt 3 beschrieben) mit LCP-FRAP Screening-Lösungen anstelle von Kristallisation Bildschirme 2. </li><li> Die Inkubation bei 20 ° C im Dunkeln für mindestens 12 Stunden, um einen Gleichgewichtszustand zu erreichen. </li><li> Legen Sie eine der Platten auf dem LCP-FRAP-Station und konzentrieren sich auf die erste Bohrung mit einem 10x-Objektiv. </li><li> Erwerben 5 Leuchtfarben Bilder auf den ersten Pre-gebleichten Zustand zu erfassen. </li><li> Auslöser des Lasers. 70% des markierten Proteins in der Mitte des gebleichten Fleck - Die Laserleistung und Anzahl der Impulse sollten zu bleichen ~ 30 eingestellt werden. </li><li> Unmittelbar nach dem Auslösen des Lasers, starten Sie die Aufnahme einer schnellen post-Bleichsequenz von ~ 200 Bilder mit der schnellstmöglichen Geschwindigkeit. </li><li> Folgen Sie mit der Aufnahme eines langsamen post-bleach Sequenz von ~ 50 Bilder, die Auswahl der Verzögerung zwischen den Bildern als 1-20 s, abhängig von der Diffusionsrate des Proteins. </li><li> Integrieren Sie die Intensität in der Bleich-Spot in allen Frames und korrigieren Sie zum Bleichen und Lichtintensitätsschwankungen bei der Akquisition, indem die Intensität im gebleichten vor Ort durch die gemittelte Intensität einer Referenz vor Ort außerhalb des Lasers gebleichten Bereich. </li><li> Normalisieren der korrigierten Intensität der Vor-gebleichtem Intensität gleich 1 und die anfängliche gebleichtem Intensität gleich 0 zu machen. </li><li> Fit der Kurve der normierten Intensität gegen die Zeit, F (t), mit der folgenden Gleichung 5: F (t) = M x exp (-2T / t) x (I 0 (2T / t) + I 1 (2T / t)), (Eq.1) wo M das mobile Bruchteil der diffundierenden Moleküle ist, T die charakteristische Diffusionszeit ist, t die Echtzeit von jedem aufgenommenen Bild, I 0 und I 1 sind die 0-te und 1. Ordnung modifizierten Bessel-Funktionen. </li><li> Berechnen Sie die Diffusionskoeffizienten, D, wie: D = R 2 / 4T, (Gleichung 2) wobei R der Radius des gebleichten Stelle. </li><li> Bewegen to den nächsten gut und wiederholen Sie die Schritte 2,5) - 2,13). </li><li> Vergleichen Sie die mobilen Fraktionen und Diffusionskoeffizienten für die verschiedenen Screening-Bedingungen erhalten. Design neuer Kristallisationspunkt Bildschirme auf Basis der Komponenten, Protein Diffusion und ohne Bedingungen, für die Protein-Diffusion nicht beobachtet wurde erleichtert. Wenn das Protein nicht diffuse in einem der abgeschirmten Bedingungen Erwägung einer Ausweitung des Screening-Raum oder versuchen ein neues Protein zu konstruieren. </li></ol> 3. Einrichten LCP Kristallisation Trials <ol><li> Lösen Sie das Protein in LCP, wie in Abschnitt 1 beschrieben). </li><li> Übertragen Sie die Protein-beladenen LCP in ein 10 l gasdichte Spritze an einem sich wiederholenden Spritzenspender. </li><li> Bringen Sie eine kurze austauschbare Nadel (Spurweite 26, 10 mm Länge), um die 10 l Spritze. </li><li> Geben Sie 200 nL Boli von LCP auf der Oberfläche von vier benachbarten Vertiefungen bilden ein 2x2-Quadrat. </li><li> Overlay jeder der LCP Boli mit 1 ul der entsprechenden Kristallisation Bildschirm-Lösung. </li><li> Cap vier beladene Brunnen mit einem 18 mm im Quadrat Deckglas. Tragen Sie einen sanften Druck auf das Deckglas in die Vertiefungen zu versiegeln. </li><li> Wiederholen Sie die Schritte 3.4) -3,6) mit dem nächsten Satz von 4 Brunnen, bis die ganze Platte gefüllt ist. </li><li> Die Platte mit einer konstanten Temperatur, regelmäßige Überprüfung zur Kristallbildung und Wachstum. </li></ol> 4. Harvesting Kristalle aus LCP <ol><li> Legen Sie eine Platte mit Proteinkristallen unter einem Stereomikroskop mit variablem Zoom, mit einem linearen rotierenden Polarisator und Analysator ausgestattet. </li><li> Konzentrieren Sie sich auf das Wohl von Interesse mit einem Low-Power-Zoom, so dass das ganze auch im Blickfeld platziert. </li><li> Ergebnis der Deckglas Glas in vier Schläge, einen Platz innerhalb der Grenzen und mit einem scharfen Ecke eines Keramikkapillare Schneiden von Stein. </li><li> Drücken Sie rund um die erzielte Umfang mit starken scharfen Punkt einer Pinzette die Kratzer, die durch die Dicke des Deckglases Glas ausbreiten. </li><li> Punch zwei kleine Löcher an gegenüberliegenden Ecken des Platzes erzielt. </li><li> Inject wenigen ul Fällungslösung durch eines der Löcher, um eine Dehydratation während der nachfolgenden Schritte zu reduzieren. </li><li> Mit einer abgewinkelten spitzen Nadel Fühlerbruch das Glas entlang einer oder zwei Seiten, die cut-out Platz frei. </li><li> Heben Sie das Glas Platz gerade für die kubische Phase Bolus. Wenn der Bolus den Deckglas geklebt wird, dann drehen Sie das Glas Platz über und auf dem Grund des Brunnens. </li><li> Fügen Sie ein zusätzliches paar ul Fällungslösung, ergänzt mit einem Kryo-Schutzmittel, falls erforderlich, auf der exponierten kubischen Phase Bolus in den Brunnen. </li><li> Erhöhung des Mikroskops und konzentrieren sich auf einen Kristall. </li><li> Passen Sie den Winkel zwischen dem Polarisator und den Analysator, um den Kontrast zwischen dem doppelbrechenden Kristall und dem Hintergrund zu erhöhen, und dabei genug Licht, um die Ernte Schleife zu sehen. </li><li> Wählen Sie einen MiTeGen MicroMount mit einem Durchmesser passenden Größe des Kristalls und dann ernten den Kristall direkt aus dem LCP mit einer Schöpfkelle in die MicroMount. </li><li> Flash-Freeze der MicroMount mit den geernteten Kristalle in flüssigem Stickstoff, und verschicken Sie es zu einem Synchrotron Source Beamline für X-ray-Daten Sammlung 6. </li></ol> 5. Repräsentative Ergebnisse: Eine manipulierte menschliche beta 2-adrenerge G-Protein-gekoppelten Rezeptor (β 2 AR-T4L) wurde in Baculovirus infizierten Sf9-Insektenzellen und gereinigt in Dodecylmaltosid (DDM) / Cholesteryl hemisuccinat (CHS) Reinigungslösung gebunden an eine partielle inverse Agonisten Carazolol 7 ausgedrückt. Das Protein wurde mit Cy3-NHS-Ester markiert und in LCP-FRAP Vorkristallisation Assays (Abbildung 2). Grobes Raster-Bildschirme von mehreren Bedingungen aus den Ergebnissen der LCP-FRAP-Assays ausgewählt wurden produziert erste Kristall-like hits (Abbildung 3). Weitere Optimierung der Fällungsmittel Bedingungen ergab Beugung Qualität Kristalle (Abbildung 4). <img src="/files/ftp_upload/2501/2501fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Flow-Diagramm eines typischen LCP Kristallisation Experiment. Schritte in den grauen Kästen sind nicht in der aktuellen Protokollen beschrieben. <img src="/files/ftp_upload/2501/2501fig2.jpg" alt="Abbildung 2" /> Abbildung 2. LCP-FRAP-Assay mit β 2 AR-T4L/carazolol in Monoolein Basis LCP. A) Ergebnisse einer LCP-FRAP-Assay in einer automatischen Hochdurchsatz-Modus durchgeführt, in denen jede Probe einer 96-Well-Platte sequentiell und gebleicht Fluoreszenz Erholung ist nach einer 30 min Inkubation gemessen. Die erhaltenen Fluoreszenz Erholungen, die den mobilen Teil in jeder Probe stellen, sind für alle 96 Proben aufgetragen. Die Screening-Lösungen enthalten 0,1 M Tris pH 8, 30% v / v PEG 400 mit 48 verschiedene Salze in zwei verschiedenen Konzentrationen kombiniert. B) Fluorescence Recovery-Profile für mehrere reptreter Bedingungen. Die durchgezogene Linie Kurven stellen passt durch Gl. 1.Die mobilen Fraktionen und die Diffusionskoeffizienten werden anhand Gl. 1 und 2. Schnelle Wiederherstellung von weniger als 10% in der Probe mit Na-Chlorid ist darauf zurückzuführen, fluoreszenzmarkierten Lipiden mit dem Protein co-gereinigt. <img src="/files/ftp_upload/2501/2501fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Initial Kristall Hits von β 2 AR-T4L/carazolol durch ein grobes Raster-Screening rund vielversprechendsten Bedingungen durch LCP-FRAP identifiziert erhalten, mit Na-Sulfat (Panel A) und Na Formate (Panel B). Das Protein wird mit Cy3-NHS-Ester markiert und die Fluoreszenz-Bilder sind aufgenommen mit einer Anregung bei 543 nm und Emission bei 605 nm. <img src="/files/ftp_upload/2501/2501fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Optimized Kristalle von β 2 AR-T4L/carazolol. Die Bilder der Kristalle in Gegenwart von Na-Sulfat gewachsen (Abb. A und B) und K Formate (Panels C und D) sind in der Hellfeld-Modus (Abb. A und C) entnommen und mittels Cross-Polarisatoren (Platten B und D).

<ol>
	<li>Caffrey, M., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallizing+membrane+proteins+using+lipidic+mesophases.">Crystallizing membrane proteins using lipidic mesophases.</a> Nat. Protoc. 4, 706-731 (2009).</li><li>Cherezov, V., Abola, E., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+progress+in+the+structure+determination+of+GPCRs,+a+membrane+protein+family+with+high+potential+as+pharmaceutical+targets.">Recent progress in the structure determination of GPCRs, a membrane protein family with high potential as pharmaceutical targets.</a> Methods Mol. Biol. 654, 141-170 (2010).</li><li>Cherezov, V., Liu, J., Hanson, M. A., Griffith, M. T., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LCP-FRAP+assay+for+pre-screening+membrane+proteins+for+in+meso+crystallization.">LCP-FRAP assay for pre-screening membrane proteins for in meso crystallization.</a> J. Cryst. Growth Design. 8, 4307-4315 (2008).</li><li>Misquitta, Y., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detergents+destabilize+the+cubic+phase+of+monoolein:+implications+for+membrane+protein+crystallization.">Detergents destabilize the cubic phase of monoolein: implications for membrane protein crystallization.</a> Biophys. J. 79, 394-405 (2003).</li><li>Soumpasis, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+fluorescence+photobleaching+recovery+experiments.">Theoretical analysis of fluorescence photobleaching recovery experiments.</a> Biophys. J. 41, 95-97 (1983).</li><li>Cherezov, V., Hanson, M. A., Griffith, M. T., Hilgart, M. C., Sanishvili, R., Nagarajan, V., Stepanov, S., Fischetti, R. F., Kuhn, P., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rastering+strategy+for+screening+and+centering+of+microcrystal+samples+of+human+membrane+proteins+with+a+sub-10+micrometer+size+X-ray+synchrotron+beam.">Rastering strategy for screening and centering of microcrystal samples of human membrane proteins with a sub-10 micrometer size X-ray synchrotron beam.</a> J. R. Soc. Interface. 6, S587-S597 (2009).</li><li>Cherezov, V., Rosenbaum, D. M., Hanson, M. A., Rasmussen, S. G., Thian, F. S., Kobilka, T. S., Choi, H. J., Kuhn, P., Weis, W. I., Kobilka, B. K., Stevens, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+crystal+structure+of+an+engineered+human+beta2-adrenergic+G+protein-coupled+receptor.">High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor.</a> Science. 318, 1258-1265 (2007).</li><li>Cherezov, V., Peddi, A., Muthusubramaniam, L., Zheng, Y. F., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+robotic+system+for+crystallizing+membrane+and+soluble+proteins+in+lipidic+mesophases.">A robotic system for crystallizing membrane and soluble proteins in lipidic mesophases.</a> Acta Crystallogr. D. 60, 1795-1807 (2004).</li><li>Kissick, D. J., Gualtieri, E. J., Simpson, G. J., Cherezov, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonlinear+optical+imaging+of+integral+membrane+protein+crystals+in+lipidic+mesophases.">Nonlinear optical imaging of integral membrane protein crystals in lipidic mesophases.</a> Anal. Chem. 82, 491-497 (2010).</li><li>Cheng, A., Hummel, B., Qiu, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+mechanical+mixer+for+small+viscous+lipid-containing+samples.">A simple mechanical mixer for small viscous lipid-containing samples.</a> Chem. Phys. Lipids. 95, 11-21 (1998).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+inexpensive+nanoliter-volume+dispenser+for+highly+viscous+materials+used+in+membrane+protein+crystallization.">A simple and inexpensive nanoliter-volume dispenser for highly viscous materials used in membrane protein crystallization.</a> J. Appl. Cryst. 38, 398-400 (2005).</li><li>Cherezov, V., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nano-volume+plates+with+excellent+optical+properties+for+fast,+inexpensive+crystallization+screening+of+membrane+proteins.">Nano-volume plates with excellent optical properties for fast, inexpensive crystallization screening of membrane proteins.</a> J. Appl. Cryst. 36, 1372-1377 (2003).</li><li>Cherezov, V., Fersi, H., Caffrey, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystallization+screens:+compatibility+with+the+lipidic+cubic+phase+for+in+meso+crystallization+of+membrane+proteins.">Crystallization screens: compatibility with the lipidic cubic phase for in meso crystallization of membrane proteins.</a> Biophys J. 81, 225-242 (2001).</li></ol>

Die Protokolle bieten eine grundlegende visuelle Orientierung für die wichtigsten Schritte bei der Durchführung in meso Kristallisation Experimenten beteiligt. Weitere ausführliche Informationen im Zusammenhang mit diesen Protokollen, betont mögliche Fallstricke sind Mängel oder alternative Routen an anderer Stelle 1,2. Optional LCP-FRAP-Untersuchungen können in früheren Phasen helfen, die vielversprechendsten Protein zu konstruieren, LCP Host Lipid-und Lipid-Additive sowie das Spektrum der möglichen Fällungs-und Pufferbedingungen 3 zu wählen. Nach einer ersten Kristallisation Hit gefunden wird, sollte sie optimiert, um bessere Qualität Kristalle zu erhalten. Optimierung in meso Kristallisationsbedingungen ist im Wesentlichen ähnlich Optimierung der Bedingungen für lösliche Proteine ​​mit der Zugabe von zusätzlichen Parametern mit der Zusammensetzung des LCP 1 zugeordnet. Membrane Protein-Kristalle in Lipid-Mesophase gewachsen sind typischerweise kleiner als Kristalle in Wasch-Lösung erhalten, aber mehr bestellt, so profitiert stark von der Nutzung Mikrofokus Strahlrohre am modernen Synchrotron-Quellen 6 zur Verfügung. Viele der Verfahren im Zusammenhang mit in meso Kristallisation, einschließlich der Einrichtung Kristallisation oder Testplatten, die Durchführung von LCP-FRAP-Assays und Detektion Kristalle, wurden halb-oder vollautomatische 1,2,8,9, so dass Screening einer großen Reihe von Bedingungen und verbrauchen dabei kleine Mengen von Protein-und Lipid. Auf der anderen Seite bleiben Protein Rekonstruktionen in LCP und Kristall Ernte manuelle und langwierige Operationen und damit eine Notwendigkeit für Verbesserungen.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>100 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7656-01</td>
<td>2 syringes are required</td>
</tr>
<tr>
<td>10 &mu;L gas-tight syringe</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7653-01</td>
<td>&nbsp;</td>
<tr>
<td>Syringe coupler</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7770-02 30902</td>
<td>The coupler can be made using available parts as described in refs. 10</td>
</tr>
<tr>
<td>Repetitive syringe dispenser</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>83700</td>
<td>The repetitive syringe dispenser can be modified to reduce dispensing volume by ~3 times11</td>
</tr>
<tr>
<td>Short (0.375&#x201D;) flat-tipped removable needle (point style 3, gauge 26)</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>7804-03</td>
<td>&nbsp;</td>
<tr>
<td>96-well glass sandwich plate</td>
<td>&nbsp;</td>
<td>Marienfeld</td>
<td>08 900 03</td>
<td>For manual operations it is more convenient to assemble the glass sandwich plate using a standard microscope glass slides and a double sticky tape with punched holes1,2,12.</td>
</tr>
<tr>
<td>Glass cover slip</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>63787-01</td>
<td>&nbsp;</td>
<tr>
<td>monoolein</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M7765</td>
<td>&nbsp;</td>
<tr>
<td>Crystallization screens</td>
<td>&nbsp;</td>
<td>Hampton Research, Molecular Dimensions, Emerald Biosystems, Jena Bioscience</td>
<td>&nbsp;</td>
<td>Most of available commercial screens can be used for initial screening. Conditions that consistently disrupt LCP can be diluted 2x for better compatibility13.</td>
</tr>
<tr>
<td>Capillary cutting stone</td>
<td>&nbsp;</td>
<td>Hampton Research</td>
<td>HR4-334</td>
<td>&nbsp;</td>
<tr>
<td>Fine point tweezers</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>510</td>
<td>&nbsp;</td>
<tr>
<td>Angled sharp probe</td>
<td>&nbsp;</td>
<td>Ted Pella</td>
<td>13650</td>
<td>&nbsp;</td>
<tr>
<td>MicroMounts</td>
<td>&nbsp;</td>
<td>MiTeGen</td>
<td>M1-Lxx-xx</td>
<td>Select MicroMount diameter to match the crystal size</td>
</tr>		</tbody>
		</table>
		</div>

crystallization of membrane proteins in lipidic mesophases

Membranproteine ​​erfüllen wichtige Funktionen in lebenden Zellen in Bezug auf Transduktion, Verkehr und Energie Transformationen und als solche sind in einer Vielzahl von Störungen und Erkrankungen beteiligt Signal. Jedoch ist eine strukturelle und funktionelle Verständnis von Membranproteinen stark hinterherhinken, dass ihrer löslichen Partnern, vor allem wegen Schwierigkeiten mit ihrer Solubilisierung und Generation der Beugung Qualität Kristalle verbunden. Kristallisation in Lipid-Mesophasen (auch in meso-oder LCP Kristallisation bekannt) ist eine vielversprechende Technik, die erfolgreich angewendet wurde, um hochauflösende Strukturen mikrobieller Rhodopsine, photosynthetischen Proteinen der äußeren Membran beta Fässer und G-Protein-gekoppelten Rezeptoren zu erhalten. In meso Kristallisation nutzt eine native-like-Membran-Umgebung und in der Regel produziert Kristalle mit niedriger Lösemittelgehalt und besseren Ordnung zu herkömmlichen Kristallisation aus Wasch-Lösungen. Die Methode ist nicht schwierig, erfordert aber ein Verständnis der Lipidphase Verhalten und Praxis im Umgang mit viskosen Mesophase Materialien. Hier zeigen wir eine einfache und effiziente Art und Weise zu machen LCP und Wiederherstellung einer Membran-Protein in die Lipid-Doppelschicht der LCP mit einer Spritze Mischer, durch den Verzicht Nanoliter Teile des LCP in einem Test oder Kristallisation Platte, die Durchführung von Vor-Kristallisation Assays und Ernte Kristalle aus, gefolgt die LCP-Matrix. Diese Protokolle stellen ein grundlegender Leitfaden für die Annäherung in meso Kristallisation Studien, aber wie bei jeder Kristallisation Experiment umfangreichen Screening und Optimierung erforderlich sind, und ein erfolgreiches Ergebnis ist nicht unbedingt gewährleistet.

Watch this Scientific Journal Video about Crystallization of Membrane Proteins in Lipidic Mesophases at JoVE.com

Crystallization of Membrane Proteins in Lipidic Mesophases

Die Protokolle beschreiben die wesentlichen Schritte für den Erhalt Beugung Qualität Kristalle von Membranproteinen ab Rekonstitution des Proteins in einer Lipid-kubische Phase (LCP), finden Anfangsbedingungen mit LCP-FRAP Vorkristallisation Assays, die Einrichtung von LCP Kristallisation Studien und Ernte-Kristallen .

Die Kristallisation von Membranproteinen in Lipidische Mesophasen

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are ...

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JoVE Journal

Biology

31.2K Aufrufe.  The Scripps Research Institute. Die Protokolle beschreiben die wesentlichen Schritte für den Erhalt Beugung Qualität Kristalle von Membranproteinen ab Rekonstitution des Proteins in einer Lipid-kubische Phase (LCP), finden Anfangsbedingungen mit LCP-FRAP Vorkristallisation Assays, die Einrichtung von LCP Kristallisation Studien und Ernte-Kristallen .

Serie: Crystallization of Membrane Proteins in Lipidic Mesophases

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.

Crystallizing Membranproteinen zur Strukturaufklärung mittels Lipidische Mesophasen

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

Forschung

Biologie

Assessing Two-dimensional Crystallization Trials of Small Membrane Proteins for Structural Biology Studies by Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray crystallography to study structure-function questions of membrane proteins, as well as soluble proteins. Screening for two-dimensional (2D) crystals by transmission electron microscopy (EM) is the critical step in finding, optimizing, and selecting samples for high-resolution data collection by cryo-EM. Here we describe the fundamental steps in identifying both large and ordered, as well as small 2D arrays, that can potentially supply critical information for optimization of crystallization conditions.
By working with different magnifications at the EM, data on a range of critical parameters is obtained. Lower magnification supplies valuable data on the morphology and membrane size. At higher magnifications, possible order and 2D crystal dimensions are determined. In this context, it is described how CCD cameras and online-Fourier Transforms are used at higher magnifications to assess proteoliposomes for order and size.
While 2D crystals of membrane proteins are most commonly grown by reconstitution by dialysis, the screening technique is equally applicable for crystals produced with the help of monolayers, native 2D crystals, and ordered arrays of soluble proteins. In addition, the methods described here are applicable to the screening for 2D crystals of even smaller as well as larger membrane proteins, where smaller proteins require the same amount of care in identification as our examples and the lattice of larger proteins might be more easily identifiable at earlier stages of the screening.

Evaluating two-dimensional (2D) crystallization trials for the formation of ordered membrane protein arrays is a highly critical and difficult task in electron crystallography. Here we describe our approach in screening for and identifying 2D crystals of predominantly small membrane proteins in the range of 15 &#x2013; 90kDa.

Die Beurteilung Zweidimensionale Kristallisation Trials of Small Membranproteinen für Strukturbiologie Studien Electron Crystallography

Electron crystallography has evolved as a method that can be used either alternatively or in combination with three-dimensional crystallization and X-ray ...

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane proteins including ion channels2, ion pumps3, and transporters4. Recent developments have combined site-specific fluorophore labeling alongside TEVC to 
concurrently examine the conformational dynamics at specific residues and function of these proteins on the surface of single cells.
We will describe a method to study the conformational dynamics of membrane proteins by simultaneously monitoring fluorescence and current changes using voltage-clamp fluorometry. This approach can be used to examine the molecular motion of membrane proteins site-specifically following cysteine replacement and site-directed fluorophore labeling5,6. Furthermore, this method provides an approach to determine distance constraints between specific residues7,8. 
This is achieved by selectively attaching donor and acceptor fluorophores to two mutated cysteine residues of interest.
In brief, these experiments are performed following functional expression of the desired protein on the surface of Xenopus leavis oocytes. The large surface area of these oocytes enables facile functional measurements and a robust fluorescence signal5. It is also possible to readily change the extracellular conditions such as pH, ligand or cations/anions, which can provide further information on the mechanism of membrane proteins4. Finally, recent developments 
have also enabled the manipulation of select internal ions following co-expression with a second protein9.
Our protocol is described in multiple parts. First, cysteine scanning mutagenesis proceeded by fluorophore labeling is completed at residues located at the interface of the transmembrane and extracellular domains. Subsequent experiments are designed to identify residues which demonstrate large changes in fluorescence intensity (&lt;5%)3 upon a conformational change of the protein. Second, these changes in fluorescence intensity are compared to the kinetic parameters of 
the membrane protein in order to correlate the conformational dynamics to the function of the protein10. This enables a rigorous biophysical analysis of the molecular motion of the target protein. Lastly, two residues of the holoenzyme can be labeled with a donor and acceptor fluorophore in order to determine distance constraints using donor photodestruction methods. It is also possible to monitor the relative movement of protein subunits following labeling with a donor and acceptor fluorophore.

Examining the Conformational Dynamics of Membrane Proteins in situ with Site-directed Fluorescence Labeling

We will describe a method which measures the kinetics of ion transport of membrane proteins alongside site-specific analysis of conformational changes using fluorescence on single cells. This technique is adaptable to ion channels, transporters and ion pumps and can be utilized to determine distance constraints between protein subunits.

Die Untersuchung der Konformationsänderungen Dynamics von Membranproteinen In situ Mit Site-directed Fluoreszenzmarkierung

Two electrode voltage clamp electrophysiology (TEVC) is a powerful tool to investigate the mechanism of ion transport1 for a wide variety of membrane ...

High-throughput Crystallization of Membrane Proteins Using the Lipidic Bicelle Method

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane bilayer that surrounds all cells and organelles. Alterations in the function of MPs result in many human diseases and disorders; thus, an intricate understanding of their structures remains a critical objective for biological research. However, structure determination of MPs remains a significant challenge often stemming from their hydrophobicity. 
MPs have substantial hydrophobic regions embedded within the bilayer. Detergents are frequently used to solubilize these proteins from the bilayer generating a protein-detergent micelle that can then be manipulated in a similar manner as soluble proteins. Traditionally, crystallization trials proceed using a protein-detergent mixture, but they often resist crystallization or produce crystals of poor quality. These problems arise due to the detergent&prime;s inability to adequately mimic the bilayer resulting in poor stability and heterogeneity. In addition, the detergent shields the hydrophobic surface of the MP reducing the surface area available for crystal contacts. To circumvent these drawbacks MPs can be crystallized in lipidic media, which more closely simulates their endogenous environment, and has recently become a de novo technique for MP crystallization.
Lipidic cubic phase (LCP) is a three-dimensional lipid bilayer penetrated by an interconnected system of aqueous channels1. Although monoolein is the lipid of choice, related lipids such as monopalmitolein and monovaccenin have also been used to make LCP2. MPs are incorporated into the LCP where they diffuse in three dimensions and feed crystal nuclei. A great advantage of the LCP is that the protein remains in a more native environment, but the method has a number of technical disadvantages including high viscosity (requiring specialized apparatuses) and difficulties in crystal visualization and manipulation3,4. Because of these technical difficulties, we utilized another lipidic medium for crystallization-bicelles5,6 (Figure 1). Bicelles are lipid/amphiphile mixtures formed by blending a phosphatidylcholine lipid (DMPC) with an amphiphile (CHAPSO) or a short-chain lipid (DHPC). Within each bicelle disc, the lipid molecules generate a bilayer while the amphiphile molecules line the apolar edges providing beneficial properties of both bilayers and detergents. Importantly, below their transition temperature, protein-bicelle mixtures have a reduced viscosity and are manipulated in a similar manner as detergent-solubilized MPs, making bicelles compatible with crystallization robots. 
Bicelles have been successfully used to crystallize several membrane proteins5,7-11 (Table 1). This growing collection of proteins demonstrates the versatility of bicelles for crystallizing both alpha helical and beta sheet MPs from prokaryotic and eukaryotic sources. Because of these successes and the simplicity of high-throughput implementation, bicelles should be part of every membrane protein crystallographer&prime;s arsenal. In this video, we describe the bicelle methodology and provide a step-by-step protocol for setting up high-throughput crystallization trials of purified MPs using standard robotics.

Bicelles are lipid/amphiphile mixtures that maintain membrane proteins (MPs) within a lipid bilayer but have unique phase behavior that facilitates high-throughput screening by crystallization robots. This technique has successfully produced a number of high-resolution structures from both prokaryotic and eukaryotic sources. This video describes protocols for generating the lipidic bicelle mixture, incorporating MPs into the bicelle mixture, setting up crystallizations trials (manually as well as robotically) and harvesting crystals from the medium.

High-Throughput-Kristallisation von Membranproteinen mit dem Lipidische Bicellen Methode

Membrane proteins (MPs) play a critical role in many physiological processes such as pumping specific molecules across the otherwise impermeable membrane ...

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.

Einsatz eines Roboters für High-Throughput-Kristallisation von Membranproteinen in Lipidische Mesophasen

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.

Ernte-und Cryo-Kühlung Kristalle von Membranproteinen in Lipidische Mesophasen zur Strukturaufklärung von Makromolekulare Kristallographie Grown

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.

Intravitalmikroskopie zur Imaging subzellulärer Strukturen in lebenden Mäusen Ausdruck Fluoreszierende Proteine

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, ...

Immunodetection of Outer Membrane Proteins by Flow Cytometry of Isolated Mitochondria

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and pathophysiology. This protocol describes a technique where mitochondria isolated from rodent tissue are immunolabeled and analyzed by flow cytometry. Mitochondria are isolated from rodent spinal cords and subjected to a rapid enrichment step so as to remove myelin, a major contaminant of mitochondrial fractions prepared from nervous tissue. Isolated mitochondria are then labeled with an antibody of choice and a fluorescently conjugated secondary antibody. Analysis by flow cytometry verifies the relative purity of mitochondrial preparations by staining with a mitochondrial specific dye, followed by detection and quantification of immunolabeled protein. This technique is rapid, quantifiable and high-throughput, allowing for the analysis of hundreds of thousands of mitochondria per sample. It is applicable to assess novel proteins at the mitochondrial surface under normal physiological conditions as well as the proteins that may become mislocalized to this organelle during pathology. Importantly, this method can be coupled to fluorescent indicator dyes to report on certain activities of mitochondrial subpopulations and is feasible for mitochondria from the central nervous system (brain and spinal cord) as well as liver.

Described here is a method to detect and quantify mitochondrial outer membrane proteins by immunolabeling of mitochondria isolated from rodent tissue and analysis by flow cytometry. This method can be extended to assess functional aspects of mitochondrial subpopulations.

Immundetektion von Außenmembranproteinen mittels Durchflusszytometrie von isolierten Mitochondrien

Methods to detect and monitor mitochondrial outer membrane protein components in animal tissues are vital to study mitochondrial physiology and ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-basierte Screening Expression von Membranproteinen in<em&gt; Escherichia coli</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Microperfusion Technique to Investigate Regulation of Microvessel Permeability in Rat Mesentery

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

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

Mikroperfusion Technik zur Untersuchung von Mikrogefäßdurchlässigkeit Verordnung in Rattengekröse

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