Unsere Arbeit wird durch Zuschüsse von NIH / NIGMS, NSF, USDA / NIFA und BARD zu VC unterstützt.

Hintergrund Symplastisch Transport von Makromolekülen durch Anlage interzellulären Verbindungen, die Plasmodesmen, ist von Interesse für viele Pflanzen-Pathologen und Biologen. Zum Beispiel sind mehrere virale Proteine ​​bekannt, plasmodesmal Größenausschlusschromatographie Grenzen zu regulieren, damit virale Bewegung 1-3. Auch sind einige endogene Proteine, darunter wichtige Entwicklungs-Regulatoren, angenommen von Zelle zu Zelle bewegen, vermutlich durch Plasmodesmen, nicht-Zell-autonome 4 Funktionstasten. So ist eine zuverlässige Methode zur Identifizierung und Visualisierung von makromolekularen Transport zwischen Pflanzenzellen sehr gefragt. 1) wachsen Zielpflanzen Für hohe Transformationseffizienz, sollten gesunde, robuste Pflanzen verwendet werden. 1. Arabidopsis-Pflanzen Wachsen ein Arabidopsis-Pflanze auf Pro-Mix BX in einem Topf (10 cm x 10 cm x 10 cm) in einer Umgebung, kontrollierten Kammer mit einer kurzen Photoperiode (8 Std. von 130-150 uE m -2 s -1 Licht bei 23 ° C/16 h Dunkelheit bei 20 ° C) und 40-65% relativer Luftfeuchtigkeit für 6 bis 8 Wochen 5. Düngen Sie sie gelegentlich mit handelsüblichen Produkten wie beschrieben 5. Blätter mit der Größe von mehr als 15 mm x 35 mm (der Längenmessung umfasst Blattstiel) sind für die Experimente ausgewählt. 2. N. benthamiana und N. tabacum Wachsen einer Pflanze auf Pro-Mix BX in einem Topf (20 cm x 20 cm x 20 cm) in einer Umgebung, kontrollierten Kammer mit einer langen Photoperiode (16 hr von 130-150 uE m -2 s -1 Licht bei 23 ° C / 8 h Dunkelheit bei 20 ° C) und 40-65% relativer Luftfeuchtigkeit für 7 bis 10 Wochen. Düngen Sie sie gelegentlich mit handelsüblichen Produkten wie beschreiben. Blätter mit einer Größe größer als 50 mm x 70 mm für N. benthamiana oder 100 mm x 125 mm für N. tabacum (diese Länge Messungen umfassen nicht Blattstiel) sind für die Experimente ausgewählt. 2) Herstellung der Gene Gun Cartridge mit DNA-beschichteten Gold-Mikropartikel Das Protokoll für diesen experimentellen Schritt wurde im Detail bereits 6 beschrieben. Es ist sehr wichtig, gut gereinigte Plasmid-DNA bei hohen Konzentrationen (~ 1 ug / ul) auf den höchsten Wirkungsgrad bei der die Leichtigkeit der konfokalen Mikroskopie Analyse in den späteren Phasen des Experiments zu erhalten verwenden. Für diesen Test ist es wichtig festzustellen, dass die vorbereitete Kartusche nicht verwandeln zwei oder mehr benachbarte Zellen gleichzeitig mit hoher Frequenz, weil die Zahl der Zellen assoziieren mit einem Fluoreszenz-Signal-Cluster als ein Indikator für das Ausmaß der symplastisch Verkehr (dh verwendet wird , eine einzelne Zelle mit dem Signal zeigt an, keine Bewegung, während ein multicell Signal-Cluster zeigt Bewegung). Die Qualität der einzelnen Patronen kann durch die Analyse der Expression des Proteins 16-20 Stunden post Beschuss geprüft werden. Unser Protokoll 6 erzeugt Patronen, die multicell Ausdruck Cluster nur in &lt;3% aller Ausdruck Ereignisse in diesem Zeitraum, so geeignet für dieses Experiment ergeben. 3) Biolistic Lieferung von DNA-beschichteten Mikropartikeln <ol><li> Entfernen Sie die Blätter der gleichen Entwicklungsstufe (dh mit gleicher Größe und gleichem Alter, siehe Schritt 1) ​​mit einer scharfen Rasierklinge und sofort legen Sie sie mit der abaxialen Seiten nach oben auf eine flache Oberfläche aus Styropor. Die abaxialen Seite des Blattes ist ein besseres Substrat für Bombardements aufgrund seiner geringeren Dichte Trichom und dünner Cuticula. Arabidopsis Blättern, die aufgrund ihrer relativ geringen Größe, mit einem Stück Fenster Siebgewebe sollte abgedeckt werden, und das Netz dann mit Reißzwecken befestigt die Styropor-Oberfläche. Die Aufrechterhaltung Blätter flach erhöht die Effizienz des Teilchens Lieferung und minimiert die Schädigung des Gewebes während microbombardment. </li><li> Setzen Sie die Patrone mit DNA-beschichteten Mikropartikeln (hergestellt in Schritt 1) ​​in der Pistole. Für Arabidopsis, ist der Shooting bei einem Druck von 80-110 psi für 1-um Mikropartikel, und 140-160 psi für 0,6-um Mikropartikel durchgeführt. Für N. benthamiana und Tabak, ist der Shooting bei einem Druck von 100-120 bar für 1-um Mikropartikel, und 160-180 psi für 0,6-um Mikropartikel durchgeführt. Für Arabidopsis, wir verwenden normalerweise eine Patrone pro Blatt, weil jedes Blatt hat genug Fläche nur für eine Aufnahme, die Mikropartikel erstreckt sich über eine Fläche von 10-12 mm im Durchmesser. Bei größeren N. benthamiana und Tabakblättern, mehrere Bombardierungen der selben Blatt sind möglich, jedoch müssen sie bei der symmetrischen Positionen auf jeder Seite der Mittelrippe, die Blattflächen auf derselben Entwicklungsstufe Ziel (siehe Diskussion) durchgeführt werden. </li><li> Entfernen Sie die Blätter aus dem Styropor Oberfläche und sie inicht eine Petrischale auf 3 Schichten nassen Whatman Filterpapier, versiegeln Sie die Petrischale mit Parafilm, und inkubieren Sie bei Raumtemperatur für 36-48 Stunden, um die Expression der Transgene geliefert und das Potenzial von Zelle zu Zelle Bewegung ihrer Eiweiss-Produkte ermöglichen . </li></ol> 4) Imaging of Protein Expression Das Fluoreszenzsignal des transient exprimiert markierte Proteine ​​visualisiert durch konfokale Mikroskopie. Es sollte darauf geachtet, um eine optimale Mikroskopie Einstellungen für die Erkennung der jeweils getesteten Protein zu finden. Zum Beispiel Proteine, die begrenzte intrazelluläre Akkumulation Show mit schwachen Signalintensitäten, wie die plasmodesmal Lokalisation des Tabak-Mosaik-Virus Bewegung Protein (TMV MP) 1-3,6, sollten unter einem 40x Objektiv, das eine höhere Auflösung und bietet beobachtet werden Empfindlichkeit, während Proteine, die zytoplasmatische Verteilung zeigen, mit starken Signalintensitäten, wie freie YFP, kann unter einem 10x Objektiv mit konfokaler Zoom-Funktion für eine schnellere Bildgebung (siehe Abbildung 1) visualisiert. Symplastisch Transport aus dem Auftreten von mehrzelligen Cluster, dass die Fluoreszenz-Signal enthalten abgeleitet. Die Zahl solcher Cluster und die Anzahl der Zellen in jedem Cluster ist bezeichnend für das Ausmaß der Zelle zu Zelle zu transportieren. Um zuverlässige Daten, sollte mindestens 100 Ausdruck Cluster pro experimentelles System erfasst werden. Zum Beispiel sollte, wenn die Zelle zu Zelle Bewegung eines Proteins ist in zwei unterschiedlichen genetischen Hintergründen (zB Wildtyp und transgenen Pflanzen), mit einem Gesamtwert von 200 Expressions-Clustern verglichen aufgezeichnet werden. Wichtig ist, dass Versuche, sollten die Ergebnisse, die miteinander verglichen werden, sind gleichzeitig durchgeführt werden. 5. Repräsentative Ergebnisse Die folgende Abbildung zeigt repräsentative Experimente zum Nachweis von symplastisch Transport von fluoreszenzmarkierten Proteinen. Die Abbildungen A und B zeigen die typische konfokale Bilder, die folgenden microbombardment einer N. gewonnen werden benthamiana Blatt mit einem TMV MP-YFP-exprimierenden konstruieren. In Panel A, symplastisch Bewegung der TMV MP-YFP wird basierend auf dem Aussehen der multicell Cluster der YFP-Signal beobachtet. Nicht alle transient exprimiert TMV MP-YFP ist in der Lage sich zu bewegen, wie Single-Cell-Signal in einigen microbombardments (Panel B) zeigt. Statistisch gesehen, in &lt;40% der gezählten Signal-Cluster ist TMV MP-YFP nicht zwischen den Zellen während bewegen sich in&gt; 60% der Cluster, bewegt sich das Protein zwischen 2-5 Zellen, wobei die beiden Zellen verteilt die häufigste (ca. ~ 50% der Fälle) (Abbildung 1C). Die Proteine ​​mit relativ kleinen Molekülgröße, ohne angeborene Bewegung Aktivität kann durch PD diffuse, um von Zelle zu Zelle bewegen. Zum Beispiel, breitet sich kostenlos YFP oder 1xYFP (ca. 27 kDa, Panel D), zwischen mehreren Zellen in 30% der gezählten Cluster (Panels C). Diese unspezifische Diffusion nicht für eine translationale YFP Dimmer oder 2xYFP (54 kDa, Panel E), die in der Größe vergleichbar ist, TMV MP-YFP-Fusionsprotein (57 kDa), und die vollständig zellautonome auftreten (Abb. 1C). <img src="/files/ftp_upload/2208/2208fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Typische Ergebnisse aus der symplastisch Transport-Assay in N. erhalten benthamiana Blattgewebe. (A, B) Visualisierung von TMV MP-YFP. (C) Quantifizierung der Signal-Cluster. 1xYFP und 2xYFP, frei YFP und translationale YFP Dimer bzw.. (D) Visualisierung von 1xYFP. (E) Visualisierung von 2xYFP. In Aufnahmen zeigen die linken Panels der YFP-Signal und das Recht Tafeln zeigen fusionierten Bilder von YFP (in grün) und Chloroplasten-Autofluoreszenz (in weiß) Signale. Die Bilder werden einzelne konfokale Schnitte. Sternchen in den Aufnahmen zeigen die Epidermiszellen zeigt YFP-Signal. Bars = 50 um.

<ol>
	<li>Waigmann, E., Ueki, S., Trutnyeva, K., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ins+and+outs+of+non-destructive+cell-to-cell+and+systemic+movement+of+plant+viruses.+.">The ins and outs of non-destructive cell-to-cell and systemic movement of plant viruses. .</a> Crit Rev Plant Sci. 23, 195-250 (2004).</li><li>Lucas, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viral+movement+proteins:+agents+for+cell-to-cell+trafficking+of+viral+genomes.">Plant viral movement proteins: agents for cell-to-cell trafficking of viral genomes.</a> Virology. 344, 169-184 (2006).</li><li>Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+viruses+spread+by+diffusion+on+ER-associated+movement-protein-rafts+through+plasmodesmata+gated+by+viral+induced+host+beta-1,3-glucanases.">Plant viruses spread by diffusion on ER-associated movement-protein-rafts through plasmodesmata gated by viral induced host beta-1,3-glucanases.</a> Semin Cell Dev Biol. 20, 1074-1081 (2009).</li><li>Zambryski, P. C., Crawford, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmodesmata:+gatekeepers+for+cell-to-cell+transport+of+developmental+signals+in+plants.">Plasmodesmata: gatekeepers for cell-to-cell transport of developmental signals in plants.</a> Annu Rev Cell Dev Biol. 16, 393-421 (2000).</li><li>Rivero-Lepinckas, L., Crist, D., Scholl, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+plants+and+presercation+of+seeds.">Growth of plants and presercation of seeds.</a> Methods Mol Biol. 323, 3-12 (2006).</li><li>Ueki, S., Lacroix, B., Krichevsky, A., Lazarowitz, S. G., Citovsky, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+transient+genetic+transformation+of+Arabidopsis+leaves+by+biolistic+bombardment.">Functional transient genetic transformation of Arabidopsis leaves by biolistic bombardment.</a> Nat Protoc. 4, 71-77 (2009).</li><li>Oparka, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple,+but+not+branched,+plasmodesmata+allow+the+nonspecific+trafficking+of+proteins+in+developing+tobacco+leaves.">Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.</a> Cell. 97, 743-754 (1999).</li><li>Imlau, A., Truernit, E., Sauer, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+and+long-distance+trafficking+of+the+green+fluorescent+protein+in+the+phloem+and+symplastic+unloading+of+the+protein+into+sink+tissues.">Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues.</a> Plant Cell. 11, 309-322 (1999).</li><li>Gisel, A., Barella, S., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+and+spatial+regulation+of+symplastic+trafficking+during+development+in+Arabidopsis+thaliana+apices.">Temporal and spatial regulation of symplastic trafficking during development in Arabidopsis thaliana apices.</a> Development. 126, 1879-1889 (1999).</li><li>Kim, I., Cho, E., Crawford, K. M., Hempel, F. D., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-to-cell+movement+of+GFP+during+embryogenesis+and+early+seedling+development+in+Arabidopsis.">Cell-to-cell movement of GFP during embryogenesis and early seedling development in Arabidopsis.</a> Proc Natl Acad Sci USA. 102, 2227-2231 (2005).</li><li>Crawford, K. M., Zambryski, P. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-targeted+and+targeted+protein+movement+through+plasmodesmata+in+leaves+in+different+developmental+and+physiological+states.">Non-targeted and targeted protein movement through plasmodesmata in leaves in different developmental and physiological states.</a> Plant Physiol. 125, 1802-1812 (2001).</li><li>Roberts, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phloem+unloading+in+sink+leaves+of+Nicotiana+benthamiana:+comparison+of+a+fluorescent+solute+with+a+fluorescent+virus.">Phloem unloading in sink leaves of Nicotiana benthamiana: comparison of a fluorescent solute with a fluorescent virus.</a> Plant Cell. 9, 1381-1396 (1997).</li><li>Guenoune-Gelbart, D., Elbaum, M., Sagi, G., Levy, A., Epel, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tobacco+mosaic+virus+(TMV)+replicase+and+movement+protein+function+synergistically+in+facilitating+TMV+spread+by+lateral+diffusion+in+the+plasmodesmal+desmotubule+of+Nicotiana+benthamiana.">Tobacco mosaic virus (TMV) replicase and movement protein function synergistically in facilitating TMV spread by lateral diffusion in the plasmodesmal desmotubule of Nicotiana benthamiana.</a> Mol Plant Microbe Interact. 21, 335-345 (2008).</li></ol>

Der Schlüssel für den Erfolg der symplastisch Transport-Assay ist eine hohe Transformationseffizienz, die Produktion von statistisch signifikant und leicht nachweisbaren Signal-Cluster ermöglicht zu erhalten. Dies kann erreicht werden mit den Blättern von gesunden, robusten Pflanzen geerntet, und die Vorbereitung Goldpartikel durch eine reine und konzentrierte DNA-Präparation beschichtet werden. Mit den Blättern an der gleichen Wachstumsphase ist auch entscheidend für den Test Zuverl?...

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<td>Gold microparticles, 1.0 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2262</td>
<td>&nbsp;</td>
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<td>Gold microparticles, 0.6 &mu;m in diameter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2263</td>
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<td>Spermidine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>S0266-1G</td>
<td>&nbsp;</td>
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<td>Tefzel tubing</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2441</td>
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<td>Helios cartridge preparatory station</td>
<td>&nbsp;</td>
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<td>Tubing cutter</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2422</td>
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<td>Helios gene gun</td>
<td>&nbsp;</td>
<td>Bio-Rad</td>
<td>165-2432</td>
<td>&nbsp;</td>
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<td>Helium gas regulator</td>
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a cell-to-cell macromolecular transport assay in planta utilizing biolistic bombardment

Hier stellen wir ein einfaches und schnelles Protokoll zu erkennen und zu beurteilen, das Ausmaß der Zell-Zell-makromolekularen Transport in planta. In diesem Protokoll wird ein fluoreszenzmarkierten-Protein von Interesse vorübergehend in Pflanzengewebe folgenden biolistischen Auslieferung der kodierenden DNA-Konstrukt zum Ausdruck gebracht. Die intra-und interzelluläre Verteilung der markierte Protein wird dann durch konfokale Mikroskopie analysiert. Wir beschreiben diese Technik im Detail und bietet Schritt-für-Schritt-Protokolle Assay und der Beurteilung von symplastisch Transport von Proteinen in drei Pflanzenarten, Arabidopsis thaliana, Nicotiana benthamiana und N. tabacum (Tabak).

Watch this Scientific Journal Video about A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment at JoVE.com

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Makromolekulare Handel zwischen Pflanzenzellen können durch transiente Expression eines Fluoreszenz-markierten Proteins von Interesse und die Analyse ihrer intra-und interzelluläre Verteilung durch konfokale Mikroskopie beurteilt werden.

Ein Cell-to-cell Makromolekulare Transport Assay In Planta Verwendung Biolistic Bombardment

Here, we present a simple and rapid protocol to detect and assess the extent of cell-to-cell macromolecular transport in planta. In this protocol, a ...

a-cell-to-cell-macromolecular-transport-assay-planta-utilizing

Research

JoVE Journal

Biology

A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

11.8K Aufrufe.  State University of New York at Stony Brook. Makromolekulare Handel zwischen Pflanzenzellen können durch transiente Expression eines Fluoreszenz-markierten Proteins von Interesse und die Analyse ihrer intra-und interzelluläre Verteilung durch konfokale Mikroskopie beurteilt werden.

Serie: A Cell-to-cell Macromolecular Transport Assay in Planta Utilizing Biolistic Bombardment

Generation of Transgenic C. elegans by Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid generation time and short life span, easy and inexpensive maintenance, fully sequenced genome, and array of RNAi resources and mutant animals. Additionally, analysis of the C. elegans genome revealed a great similarity between worms and higher vertebrates, which suggests that research in worms could be an important adjunct to studies performed in whole mice or cultured cells. A powerful and important part of worm research is the ability to use transgenic animals to study gene localization and function. Transgenic animals can be created either via microinjection of the worm germline or through the use of biolistic bombardment. Bombardment is a newer technique and is less familiar to a number of labs. Here we describe a simple protocol to generate transgenic worms by biolistic bombardment with gold particles using the Bio-Rad PDS-1000 system. Compared with DNA microinjection into hermaphrodite germline, this protocol has the advantage of not requiring special skills from the operator with regards to identifying worm anatomy or performing microinjection. Further multiple transgenic lines are usually obtained from a single bombardment. Also in contrast to microinjection, biolistic bombardment produces transgenic animals with both extrachromosomal arrays and integrated transgenes. The ability to obtain integrated transgenic lines can avoid the use of mutagenic protocols to integrate foreign DNA. In conclusion, biolistic bombardment can be an attractive method for the generation of transgenic animals, especially for investigators not interested in investing the time and effort needed to become skilled at microinjection.

Generation of Transgenic C. elegans by Biolistic Transformation

Transgenic worms are commonly used in C. elegans research. Described is a simple, yet effective, protocol to introduce transgenes into worms using biolistic bombardment with DNA-coated gold particles. The effort involved and results of bombardment compare favorably with microinjection for the generation of transgenic animals.

Erzeugung von transgenen C. elegans Nach Biolistic Transformation

The number of laboratories using the free living nematode C. elegans is rapidly growing. The popularity of this biological model is attributed to a rapid ...

Forschung

Biologie

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 ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

In-vitro-Analyse der PDZ-abhängigen CFTR Makromolekulare Signalkomplexe

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media (mCeHR). Additionally, the steps for exposing and acclimatizing C. elegans grown on E. coli to axenic liquid media are described. Finally, downstream experiments that utilize axenic C. elegans illustrate the benefits of this procedure. The ability to analyze and determine C. elegans&#160;nutrient requirement was illustrated by growing N2 wild type worms in axenic liquid media with varying heme concentrations. This procedure can be replicated with other nutrients to determine the optimal concentration for worm growth and development or, to determine the toxicological effects of drug treatments. The effects of varied heme concentrations on the growth of wild type worms were determined through qualitative microscopic observation and by quantitating the number of worms that grew in each heme concentration. In addition, the effect of varied nutrient concentrations can be assayed by utilizing worms that express fluorescent sensors that respond to changes in the nutrient of interest. Furthermore, a large number of worms were easily produced for the generation of transgenic C. elegans using microparticle bombardment.

Culturing Caenorhabditis elegans in Axenic Liquid Media and Creation of Transgenic Worms by Microparticle Bombardment

C. elegans is usually grown on solid agar plates or in liquid cultures seeded with E. coli. To prevent bacterial byproducts from confounding toxicological and nutritional studies, we utilized an axenic liquid medium, CeHR, to grow and synchronize a large number of worms for a range of downstream applications.

Die Züchtung<em&gt; Caenorhabditis elegans</em &gt; In Axenic Flüssige Medien und Erstellung von transgenen Worms durch Mikropartikelbeschuss

In this protocol, we present the required materials, and the procedure for making modified&#160;C. elegans&#160;Habituation and Reproduction media ...

Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in &#181;Manager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.

We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using F&#246;rster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in &#181;Manager and data analysis is undertaken in FLIMfit.

Open Source High Content Analyse Unter Verwendung Automatische Fluorescence Lifetime Imaging Mikroskopie

We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions ...

Optimization of Crystal Growth for Neutron Macromolecular Crystallography

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX beamlines having been built and increased availability of structure refinement software. However, the neutron sources currently available for NMX are significantly weaker than equivalent sources for X-ray crystallography. Despite advances in this field, significantly larger crystals will always be required for neutron diffraction studies, particularly with the tendency to study ever-larger macromolecules and complexes. Further improvements in methods and instrumentation suited to growing larger crystals are therefore necessary for the use of NMX to expand.
In this work, we introduce rational strategies and a crystal growth bench (OptiCrys) developed in our laboratory that combines real-time observation through a microscope-mounted video camera with precise automated control of crystallization solutions (e.g., precipitant concentration, pH, additive, temperature). We then demonstrate how this control of temperature and chemical composition facilitates the search for optimal crystallization conditions using model soluble proteins. Thorough knowledge of the crystallization phase diagram is crucial for selecting the starting position and the kinetic path for any crystallization experiment. We show how a rational approach can control the size and number of crystals generated based on knowledge of multidimensional phase diagrams.

Structural studies of biomacromolecules by crystallography require high-quality crystals. Here we demonstrate a protocol that can be used by OptiCrys (a fully automated instrument developed in our lab) and/or microdialysis buttons for growing large high-quality crystals based on knowledge of the crystallization phase diagram.

Optimierung des Kristallwachstums für die Neutronenmakromolekulare Kristallographie

The use of neutron macromolecular crystallography (NMX) is expanding rapidly with most structures determined in the last decade thanks to new NMX ...

Simultaneous Live Imaging of Multiple Insect Embryos in Sample Chamber-Based Light Sheet Fluorescence Microscopes

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample chamber-based setups, which are specifically designed to preserve the three-dimensional integrity of the specimen and usually feature sample rotation, are the best choice in developmental biology. For instance, they have been used to document the entire embryonic morphogenesis of the fruit fly Drosophila melanogaster and the red flour beetle Tribolium castaneum. However, many available live imaging protocols provide only experimental frameworks for single embryos. Especially for comparative studies, such approaches are inconvenient, since sequentially imaged specimens are affected by ambient variance. Further, this limits the number of specimens that can be assayed within a given time. We provide an experimental framework for simultaneous live imaging that increases the throughput in sample chamber-based setups and thus ensures&#160;similar ambient conditions for all specimens. Firstly, we provide a calibration guideline for light sheet fluorescence microscopes. Secondly, we propose a mounting method for multiple embryos that is compatible with sample rotation. Thirdly, we provide exemplary three-dimensional live imaging datasets of Drosophila, for which we juxtapose three transgenic lines with fluorescently labeled nuclei, as well as of Tribolium, for which we compare the performance of three transgenic sublines that carry the same transgene, but at different genomic locations. Our protocol is specifically designed for comparative studies as it pro-actively addresses ambient variance, which is always present in sequential live imaging. This is especially important for quantitative analyses and characterization of aberrational phenotypes, which result e.g., from knockout experiments. Further, it increases the overall throughput, which is highly convenient when access to light sheet&#160;fluorescence microscopes is limited. Finally, the proposed mounting method can be adapted for other insect species and further model organisms, e.g., zebrafish, with basically no optimization effort.

Light sheet-based fluorescence microscopy is the most valuable tool in developmental biology. A major issue in comparative studies is ambient variance. Our protocol describes an experimental framework for simultaneous live imaging of multiple specimens and, therefore, addresses this issue pro-actively.

Gleichzeitige Live-Bildgebung mehrerer Insektenembryonen in probenkammerbasierten Lichtbogenfluoreszenzmikroskopen

Light sheet-based fluorescence microscopy offers efficient solutions to study complex processes on multiple biologically relevant scales. Sample ...

Untersuchung des Zinktransportmechanismus in Säugetierzellen mit dem FluoZin-3-Assay

Characterizing Mammalian Zinc Transporters Using an In Vitro Zinc Transport Assay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured indirectly by determining the expression level of the transporter under different concentrations of Zn2+. This was done by utilizing immunohistochemistry, measuring mRNA in the tissue, or determining the cellular Zn2+ levels. With the development of intracellular Zn2+ sensors, the activities of zinc transporters are currently primarily determined by correlating changes in intracellular Zn2+, detected using fluorescent probes, with the expression of the Zn2+ transporters. However, even today, only a few labs monitor dynamic changes in intracellular Zn2+ and use it to measure the activity of zinc transporters directly. Part of the problem is that out of the 10 zinc transporters of the ZnT family, except for ZnT10 (transports manganese), only zinc transporter 1 (ZnT1) is localized at the plasma membrane. Therefore, linking the transport activity to changes in the intracellular Zn2+ concentration is hard. This article describes a direct way to determine the zinc transport kinetics using an assay based on a zinc-specific fluorescent dye, FluoZin-3. This dye is loaded into mammalian cells in its ester form and then trapped in the cytosol due to cellular di-esterase activity. The cells are loaded with Zn2+ by utilizing the Zn2+ ionophore pyrithione. The ZnT1 activity is assessed from the linear part of the reduction in fluorescence following the cell washout. The fluorescence measured at an excitation of 470 nm and emission of 520 nm is proportional to the free intracellular Zn2+. Selecting the cells expressing ZnT1 tagged with the mCherry fluorophore allows for monitoring only the cells expressing the transporter. This assay is used to investigate the contribution of different domains of ZnT1 protein to the transport mechanism of human ZnT1, a eukaryotic transmembrane protein that extrudes excess zinc from the cell.

Autor im Rampenlicht: Erforschung der zellulären Zinkregulation durch ZnT1-Funktionalität 

Zinc transport has proven challenging to measure due to the weak causal links to protein function and the low temporal resolution. This protocol describes a method for monitoring, with high temporal resolution, Zn2+ extrusion from living cells by utilizing a Zn2+ sensitive fluorescent dye, thus providing a direct measure of Zn2+ efflux.

Charakterisierung von Zinktransportern in Säugetieren mit einem In-vitro-Zinktransportassay

Transition metals such as Zn2+ ions must be tightly regulated due to their cellular toxicity. Previously, the activity of Zn2+ transporters was measured ...