Diese Arbeit wurde durch das Stipendium Nr. 2016/23/D/NZ3/01314 des National Science Centre (NCN), Krakau, Polen, unterstützt.

1. Erzeugung von Expressionsplasmiden
<ol><li>Untersuchen Sie die Membrantopologie der interessierenden Proteine mit einem Topologievorhersagewerkzeug.</li>
	<li>Entwerfen Sie die Klonierungsstrategie so, dass die größeren und kleineren Fragmente dem Zytoplasma gegenüberstehen, sobald die Fusionsproteine in die Golgi-Membranen eingefügt wurden. Wenn, wie im hier dargestellten Fall, sowohl N- als auch C-Termini der interessierenden Proteine zytoplasmatisch orientiert sind, markieren Sie die Proteine auf acht mögliche Arten (siehe Abbildung 1B). Wenn der N- oder C-Terminus eines oder beider Proteine von Interesse luminal orientiert ist, schließen Sie ihn vom Tagging aus.</li>
	<li>Subklonen Sie die interessierenden Gene in geeignete Expressionsvektoren (siehe Materialtabelle), indem Sie die Standard-Klonierungsprotokolle befolgen. HINWEIS: Es wird empfohlen, alle möglichen Ausrichtungen zu testen, da einige Tagging-Optionen aufgrund unzureichender Nähe, suboptimaler Ausrichtung oder räumlicher Einschränkungen möglicherweise nicht funktionieren.</li>
</ol>2. Transiente Transfektion der Expressionsplasmide in die Zellen
<ol><li>Ernten Sie die adhärente HEK293T-Zellkultur durch Trypsinisierung und resuspendieren Sie die Zellen in einem speziellen vollständigen Wachstumsmedium. Platten Sie die Zellen (2 x 104/100 μL/Well) auf eine klare untere, weiße 96-Well-Platte. Passen Sie die Gesamtzahl der Bohrlöcher an, um alle getesteten Kombinationen und Kontrollen einschließlich Replikationen aufzunehmen. HINWEIS: Versuchen Sie, nur die inneren 60 Vertiefungen der Platte zu verwenden, um thermische Verschiebungen zu minimieren und eine Verdunstung über Nacht zu vermeiden. Die Verwendung von Poly-D-Lysin-beschichteten Platten, wie sie in der Materialtabelle angegeben sind, wird dringend empfohlen, da sich sonst Zellen während der nachfolgenden Waschschritte ablösen können.</li>
	<li>Kultivieren Sie die Zellen über Nacht unter Standardbedingungen (37 °C, 5% CO2).</li>
	<li>Am nächsten Tag werden die Zellen mit den gewünschten Kombinationen von Expressionsplasmiden transfiziert, die in Nummer 1.1 erhalten wurden.<ol>
<li>Verdünnte Expressionsplasmide in einem serumfreien Medium (siehe Materialtabelle) auf 6,25 ng/μL für jedes Konstrukt.</li>
		<li>Fügen Sie das lipidbasierte Transfektionsreagenz in einem geeigneten Lipid-zu-DNA-Verhältnis hinzu und inkubieren Sie es gemäß den Anweisungen des Herstellers.</li>
		<li>8 μL Lipid:DNA-Gemisch in die dafür vorgesehenen Vertiefungen geben. Mischen Sie den Inhalt der Platte durch sanfte Rotation. Dies führt zu einer Transfektion beider Ausdruckskonstrukte bei 50 ng/well.</li>
	</ol>
</li>
	<li>Kultivieren Sie die Zellen für 20-24 h unter Standardbedingungen (37 °C, 5% CO2). HINWEIS: Die Kultivierung der Zellen über einen längeren Zeitraum kann zu einer höheren Expression von Fusionsproteinen führen, was eine unspezifische Assoziation zwischen den Fragmenten fördern kann.</li>
</ol>3. Mittlerer Austausch
<ol><li>Ersetzen Sie am nächsten Tag das konditionierte Medium durch 100 μL eines serumfreien Mediums in jedem Bohrloch. Stellen Sie sicher, dass sich die Zellen beim Mediumsaustausch nicht gelöst haben. HINWEIS: Dieser Schritt sollte 2-3 Stunden vor der Zugabe der Furimazin-Arbeitslösung durchgeführt werden. Der Serumentzug minimiert den Hintergrund, der durch die Autolumineszenz von Furimazin verursacht wird.</li>
</ol>4. Vorbereitung der Furimazin-Arbeitslösung
<ol><li>Mischen Sie kurz vor der Messung 1 Volumen Furimazin mit 19 Volumina eines Verdünnungspuffers (eine 20-fache Verdünnung). HINWEIS: Das Gesamtvolumen der herzustellenden Furimazin-Arbeitslösung hängt von der Anzahl der einzelnen zu analysierenden Vertiefungen ab (die Furimazin-Arbeitslösung wird dem Zellkulturmedium im Verhältnis 1:5 zugesetzt, daher sollte jedem zuvor mit 100 μL gefüllten Bohrloch eines serumfreien Mediums 25 μL der Furimazin-Arbeitslösung zugegeben werden).</li>
	<li>Fügen Sie die Furimazin-Arbeitslösung zu den vorgesehenen Vertiefungen hinzu (25 μL/Well). Mischen Sie die Platte vorsichtig von Hand oder mit einem Orbitalschüttler (z. B. 15 s bei 300-500 U / min).</li>
</ol>5. Messung der Lumineszenz
<ol><li>Setzen Sie die Platte in einen Lumineszenz-Mikroplattenleser ein.<ol>
<li>Für Experimente, die bei 37 °C durchgeführt werden sollen, gleicht die Platte für 10-15 min bei der angegebenen Temperatur aus.</li>
	</ol>
</li>
	<li>Wählen Sie die zu analysierenden Vertiefungen aus.</li>
	<li>Leselumineszenz mit einer Integrationszeit von 0,3 s. Überwachen Sie bei Bedarf bis zu 2 Stunden lang die Lumineszenz.</li>
</ol>6. Datenanalyse
<ol><li>Berechnen Sie Mittelwerte und Standardabweichungen für alle geprüften und Regelkombinationen.</li>
	<li>Analysieren Sie Daten mit unidirektionaler ANOVA mit mehreren Vergleichen.</li>
	<li>Berechnen Sie Faltungsänderungswerte, indem Sie eine mittlere Lumineszenz, die für Kombinationen von Interesse erhalten wird, durch eine mittlere Lumineszenz dividieren, die für die entsprechenden Negativkontrollen erhalten wird. Bewerten Sie die Ergebnisse. ANMERKUNG: Der hier vorgeschlagene Ansatz zur Datenanalyse geht davon aus, dass die Wechselwirkung beansprucht werden kann, wenn die für die getestete Kombination erhaltene Lumineszenz statistisch signifikant höher ist als die für die entsprechende Kontrollkombination erhaltene Lumineszenz und gleichzeitig das Verhältnis dieser beiden Werte 10 übersteigt.</li>
</ol>

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A., Coincon, M., Drew, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+the+delivery+of+activated+sialic+acid+into+Golgi+for+sialyation.">Structural basis for the delivery of activated sialic acid into Golgi for sialyation.</a> Nature Structural and Molecular Biology. 26 (6), 415-423 (2019).</li><li>Parker, J. L., Corey, R. A., Stansfeld, P. J., Newstead, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+basis+for+substrate+specificity+and+regulation+of+nucleotide+sugar+transporters+in+the+lipid+bilayer.">Structural basis for substrate specificity and regulation of nucleotide sugar transporters in the lipid bilayer.</a> Nature Communications. 10 (1), 4657 (2019).</li><li>Wiertelak, W., Sosicka, P., Olczak, M., Maszczak-Seneczko, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+homologous+and+heterologous+interactions+between+UDP-galactose+transporter+and+beta-1,4-galactosyltransferase+1+using+NanoBiT.">Analysis of homologous and heterologous interactions between UDP-galactose transporter and beta-1,4-galactosyltransferase 1 using NanoBiT.</a> Analytical Biochemistry. 593, 113599 (2020).</li><li>Hong, K., Ma, D., Beverley, S. M., Turco, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Leishmania+GDP-mannose+transporter+is+an+autonomous,+multi-specific,+hexameric+complex+of+LPG2+subunits.">The Leishmania GDP-mannose transporter is an autonomous, multi-specific, hexameric complex of LPG2 subunits.</a> Biochemistry. 39 (8), 2013-2022 (2000).</li><li>Sosicka, P., 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=An+insight+into+the+orphan+nucleotide+sugar+transporter+SLC35A4.">An insight into the orphan nucleotide sugar transporter SLC35A4.</a> Biochimica et Biophysica Acta. 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S., 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=NanoLuc+Complementation+Reporter+Optimized+for+Accurate+Measurement+of+Protein+Interactions+in+Cells.">NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells.</a> ACS Chemical Biology. 11 (2), 400-408 (2016).</li><li>Tung, J. K., Berglund, K., Gutekunst, C. -. A., Hochgeschwender, U., Gross, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+imaging+in+live+cells+and+animals.">Bioluminescence imaging in live cells and animals.</a> Neurophotonics. 3 (2), 025001 (2016).</li><li>Hu, C. D., Chinenov, Y., Kerppola, T. 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Hier stellen wir ein detailliertes Protokoll zur Verfügung, das den Nachweis heterologer Komplexe ermöglicht, die zwischen Golgi-residenten Typ-III-Membranproteinen wie NSTs gebildet werden, indem wir den Split-Luciferase-Komplementierungsassay verwenden. Der vorgeschlagene Ansatz zur Datenanalyse und -interpretation besteht darin, die für die interessierende Proteinkombination erhaltene Lumineszenz mit der für die entsprechende Kontrollkombination erhaltenen Lumineszenz in Beziehung zu setzen, die aus einem der inte...

Dieses Manuskript beschreibt ein Schritt-für-Schritt-Protokoll zur Überprüfung auf das Vorhandensein heterologer Interaktionen zwischen Golgi-residenten Typ-III-Membranproteinen in transient transfizierten menschlichen Zellen unter Verwendung der neuesten Variante des Split-Lucifererase-Komplementierungsassays. Das Verfahren wurde am umfangreichsten gegen Nukleotid-Zuckertransporter (NSTs) getestet, aber wir konnten auch positive Ergebnisse für andere Golgi-residente Typ-III-Membranproteine erzielen, deren N- und/oder C-Termini dem Zytoplasma zugewandt sind.
Unsere Forschungsgruppe untersucht die Rolle von NSTs bei der Glykosylierung von Makromolekülen. NSTs sind Golgi- und/oder ER-residente Typ-III-Membranproteine mit N- und C-Termini, die der zytoplasmatischen Seite der Organellamembran zugewandt sind1. Es wird angenommen, dass NSTs nukleotidaktivierte Zucker über Organellenmembranen transportieren, um Glykosyltransferasen mit ihren Substraten zu versorgen. NSTs bilden Dimere und/oder höhere Oligomere2,3,4,5,6,7,8,9,10. Darüber hinaus wurden auch heterologe Wechselwirkungen zwischen verschiedenen NSTs berichtet6,11. Es wurde auch gezeigt, dass NSTs Komplexe mit funktionell verwandten Glykosylierungsenzymen bilden12,13,14. Wir suchten nach einer Alternative zur derzeit verwendeten Technik, dem FLIM-basierten FRET-Ansatz (Fluorescence Lifetime Imaging), um die Wechselwirkungen von NSTs und funktionell verwandten Golgi-residenten Proteinen zu untersuchen, also beschlossen wir, den Split-Luciferase-Komplementierungsassay zu testen. Es ermöglichte uns, eine neuartige Wechselwirkung zwischen einem NST und einem funktionell verwandten Glykosylierungsenzym zu identifizieren9.
Die jüngste Modifikation des Split-Luciferase-Komplementierungsassays NanoBiT wird in dem hier vorgestellten Protokoll verwendet15. Es beruht auf der Rekonstitution des Luciferase-Enzyms (z. B. NanoLuc) aus den beiden Fragmenten - dem großen, das als großes BiT oder LgBiT bezeichnet wird, einem 17,6-kDa-Protein, und dem kleinen, das aus nur 11 Aminosäuren besteht, das als kleines BiT oder SmBiT bezeichnet wird. Die beiden interessierenden Proteine werden mit den komplementären Fragmenten verschmolzen und vorübergehend in einer menschlichen Zelllinie exprimiert. Wenn die beiden Fusionsproteine interagieren, entsteht in situ durch Zugabe eines zelldurchlässigen Substrats eine Lumineszenz. Diese beiden Fragmente wurden so optimiert, dass sie sich mit minimaler Affinität assoziieren, es sei denn, sie werden durch eine Wechselwirkung zwischen den Proteinen von Interesse, mit denen sie verschmolzen sind, zusammengebracht.
Im Allgemeinen haben biolumineszenzbasierte Methoden einige Vorteile gegenüber denen, die auf Fluoreszenz basieren. Biolumineszenzsignale haben ein höheres Signal-Rausch-Verhältnis, da die Hintergrundlumineszenz im Vergleich zum Luciferase-abgeleiteten Signal16 vernachlässigbar ist. Im Gegensatz dazu leiden fluoreszenzbasierte Ansätze in der Regel unter einem relativ hohen Hintergrund, der durch das Phänomen der Autofluoreszenz verursacht wird. Außerdem ist die Biolumineszenz für die analysierten Zellen weniger schädlich als die Fluoreszenz, da im ersten Fall keine Notwendigkeit besteht, die Probe anzuregen. Aus diesen Gründen übertreffen biolumineszierende Ansätze zur Untersuchung von PPIs in vivo die häufig verwendeten fluoreszierenden Methoden wie Förster Resonance Energy Transfer (FRET) oder Bimolecular Fluorescence Complementation (BiFC).
Unser Protokoll beruht auf der Bezugnahme der Lumineszenz, die für die Proteinkombination von Interesse ist, auf die Lumineszenz, die für die Kontrollkombination erhalten wird. Letzteres umfasst eines der getesteten Proteine, das mit einem größeren Fragment verschmolzen ist, und ein Kontrollprotein (z. B. HaloTag), das mit einem kleineren Fragment verschmolzen ist. Letzteres ist ein Protein bakteriellen Ursprungs, von dem nicht erwartet wird, dass es mit einem der Säugetierproteine interagiert. Die Verwendung dieses Proteins als Kontrolle schränkt die Topologie der zu analysierenden Golgi-residenten Proteinpaare ein. Da dieses Protein in Säugetierzellen im Zytoplasma synthetisiert wird, sollten beide interessierenden Proteine mindestens einen zytoplasmatischen Schwanz haben.
Dieser Ansatz kann besonders nützlich für das anfängliche Screening von PPIs sein. Es kann die Methode der Wahl werden, wenn die interessierenden Fusionsproteine in Konzentrationen ausgedrückt werden, die für die Anwendung anderer Ansätze einfach nicht ausreichen. In ähnlicher Weise kann der Split-Luciferase-Komplementierungsassay die beste Option sein, wenn die interessierenden Proteine in hohen Konzentrationen exprimiert werden, was sich jedoch nachteilig auf ihre subzelluläre Lokalisation auswirkt oder dafür bekannt ist, unspezifische Wechselwirkungen zu erzwingen. Da das kleinere Fragment nur 11 Aminosäuren enthält, kann der Split-Luciferase-Komplementierungsassay angewendet werden, wenn die Verwendung größerer Tags unmöglich ist. Schließlich kann es verwendet werden, um Daten, die mit anderen Techniken erhalten wurden, wie in dem hier vorgestellten Fall, weiter zu bestätigen.

<table>
	<tbody>
		<tr>
			<td>0.25% trypsin-EDTA solution</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Adherent mammalian cell line</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BioCoat Poly-D-Lysine 96-well White/Clear Flat Bottom TC-treated Microplate, with Lid</td>
			<td>Corning</td>
			<td>356651</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture supplements (heat-inactivated fetal bovine serum, L-glutamine, penicillin, streptamycin)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Expression plasmids encoding protein(s) of interest not tagged with NanoBiT fragments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FuGENE HD Transfection Reagent</td>
			<td>Promega</td>
			<td>E2311</td>
			<td></td>
		</tr>
		<tr>
			<td>GloMax Discover Microplate Reader (or a different luminescence microplate reader)</td>
			<td>Promega</td>
			<td>GM3000</td>
			<td></td>
		</tr>
		<tr>
			<td>Growth medium dedicated to the cell line used</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Materials and reagents for standard molecular cloning (bacteria, thermostable polymerase, restriction enzymes, DNA ligase, materials and reagents for nucleic acid purification)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NanoBiT MCS Starter System</td>
			<td>Promega</td>
			<td>N2014</td>
			<td>This kit contains vectors enabling tagging of the proteins of interest with NanoBiT fragments at different orientations as well as the control plasmid encoding HaloTag protein fused with SmBiT and a positive control plasmid pair.</td>
		</tr>
		<tr>
			<td>Nano-Glo Live Cell Assay System</td>
			<td>Promega</td>
			<td>N2011</td>
			<td>This kit contains furimazine, which is a substrate enabling detection of the NanoLuc activity in living cells, and a dedicated dilution buffer.</td>
		</tr>
		<tr>
			<td>Opti-MEM I Reduced Serum Medium, no phenol red</td>
			<td>Gibco</td>
			<td>11058021</td>
			<td></td>
		</tr>
		<tr>
			<td>Oribital shaker</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Software for data analysis (e.g. GraphPad Prism)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Thermocycler</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

demonstration of heterologous complexes formed by golgi-resident type iii membrane proteins using split luciferase complementation assay

Das Ziel dieses Protokolls ist es, die Anwendbarkeit der neuesten Variante der gespaltenen Luciferase-Komplementierung für den Nachweis heterologer Komplexe, die von Nukleotid-Zuckertransportern (NSTs) gebildet werden, zu untersuchen. Diese ER- und Golgi-residenten Multitransmembranproteine transportieren die zytoplasmatisch synthetisierten Nukleotidzucker über Organellenmembranen, um Enzyme zu liefern, die die Glykosylierung mit ihren Substraten vermitteln. NSTs existieren als Dimere und/oder höhere Oligomere. Heterologe Wechselwirkungen zwischen verschiedenen NSTs wurden ebenfalls berichtet. Um zu überprüfen, ob die Technik für die Untersuchung des Phänomens der NST-Heteromerisierung geeignet ist, haben wir sie gegen eine Kombination der beiden Golgi-ansässigen NSTs getestet, von denen zuvor gezeigt wurde, dass sie sich auf verschiedene andere Weise assoziieren. Der Luciferase-Komplementierungsassay scheint besonders geeignet zu sein, um Wechselwirkungen zwischen Golgi-residenten Membranproteinen zu untersuchen, da er keine hohen Expressionsniveaus erfordert, die oft eine Proteinfehllokalisation auslösen und das Risiko von falsch positiven Ergebnissen erhöhen.

Um die zuverlässigsten Daten in diesem Ansatz zu erhalten, sollten alle möglichen Kombinationen getestet werden (siehe Abbildung 1). Parallel dazu sollten Positiv- und Negativkontrollen einbezogen werden. Die Positivkontrolle sollte aus den beiden Proteinen bestehen, von denen bekannt ist, dass sie interagieren, von denen eines mit dem größeren Fragment und das andere mit dem kleineren Fragment verschmolzen ist. Die Negativkontrolle sollte idealerweise aus den beiden nicht interagierende...

Watch this Scientific Journal Video about Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay at JoVE.com

Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Das hier vorgestellte Protokoll soll das Auftreten heterologer Interaktionen zwischen Golgi-residenten Typ-III-Membranproteinen mit zytoplasmatisch exponierten N- und/oder C-Termini in lebenden Säugetierzellen unter Verwendung der neuesten Variante des Split-Lucifererase-Komplementierungsassays demonstrieren.

Nachweis heterologer Komplexe, die von Golgi-residenten Typ-III-Membranproteinen gebildet werden, mittels Split-Luciferase-Komplementierungsassay

The goal of this protocol is to explore the applicability of the most recent variant of split luciferase complementation for demonstrating heterologous ...

demonstration-heterologous-complexes-formed-golgi-resident-type-iii

Research

JoVE Journal

Biology

2.2K Aufrufe.  University of Wroclaw. Das hier vorgestellte Protokoll soll das Auftreten heterologer Interaktionen zwischen Golgi-residenten Typ-III-Membranproteinen mit zytoplasmatisch exponierten N- und/oder C-Termini in lebenden Säugetierzellen unter Verwendung der neuesten Variante des Split-Lucifererase-Komplementierungsassays demonstrieren.

Serie: Demonstration of Heterologous Complexes formed by Golgi-Resident Type III Membrane Proteins using Split Luciferase Complementation Assay

Studying Membrane Biogenesis with a Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane biogenesis occurs rapidly and in an inducible manner. We have found that phagocytosis of latex beads is practical for these purposes as cells rapidly synthesize membrane lipids to replenish membrane pools lost as  wrapping material  during particle engulfment. Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-Glo  Luciferase Assay System from Promega.

Here, we describe procedures for studying changes in phagocytosis-induced gene expression with a luciferase-based reporter gene approach using the Dual-GloTM Luciferase Assay System from Promega.

Studieren Membrane Biogenesis mit einem Luciferase-Based Reporter Gene Assay

To study the coordination of different lipid synthesis pathways during membrane biogenesis it is useful to work with an experimental system where membrane ...

Forschung

Biologie

Crystallizing Membrane Proteins for Structure Determination using Lipidic Mesophases

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

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

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 ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

Identification of Growth Inhibition Phänotypen durch Expression von bakteriellen Typ III Effektoren in Hefe Induced

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Crystallization of Membrane Proteins in Lipidic Mesophases

Membrane proteins perform critical functions in living cells related to signal transduction, transport and energy transformations, and, as such, are implicated in a multitude of malfunctions and diseases. However, a structural and functional understanding of membrane proteins is strongly lagging behind that of their soluble partners, mainly, due to difficulties associated with their solubilization and generation of diffraction quality crystals. Crystallization in lipidic mesophases (also known as in meso or LCP crystallization) is a promising technique which was successfully applied to obtain high resolution structures of microbial rhodopsins, photosynthetic proteins, outer membrane beta barrels and G protein-coupled receptors. In meso crystallization takes advantage of a native-like membrane environment and typically produces crystals with lower solvent content and better ordering as compared to traditional crystallization from detergent solutions. The method is not difficult, but requires an understanding of lipid phase behavior and practice in handling viscous mesophase materials. Here we demonstrate a simple and efficient way of making LCP and reconstituting a membrane protein in the lipid bilayer of LCP using a syringe mixer, followed by dispensing nanoliter portions of LCP into an assay or crystallization plate, conducting pre-crystallization assays and harvesting crystals from the LCP matrix. These protocols provide a basic guide for approaching in meso crystallization trials; however, as with any crystallization experiment, extensive screening and optimization are required, and a successful outcome is not necessarily guaranteed.

The protocols describe the essential steps for obtaining diffraction quality crystals of a membrane protein starting from reconstitution of the protein in a lipidic cubic phase (LCP), finding initial conditions with LCP-FRAP pre-crystallization assays, setting up LCP crystallization trials and harvesting crystals.

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 ...

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level. There are several methods, both in vitro and in vivo, to evaluate protein binding, and at least two methods that complement the shortcomings of each other should be conducted to obtain reliable insights. 
For an in vivo assay, the bimolecular fluorescence complementation (BiFC) assay represents the most popular and least invasive approach that enables to detect protein-protein interaction within living cells, as well as identify the intracellular localization of the interacting proteins 1,2. In this assay, non-fluorescent N- and C-terminal halves of GFP or its variants are fused to tested proteins, and when the two fusion proteins are brought together due to the tested proteins&#x2019; interactions, the fluorescent signal is reconstituted3-6. Because its signal is readily detectable by epifluorescence or confocal microscopy, BiFC has emerged as a powerful tool of choice among cell biologists for studying about protein-protein interactions in living cells 3. This assay, however, can sometimes produce false positive results. For example, the fluorescent signal can be reconstituted by two GFP fragments arranged as far as 7 nm from each other due to close packing in a small subcellular compartment, rather that due to specific interactions7.
Due to these limitations, the results obtained from live cell imaging technologies should be confirmed by an independent approach based on a different principle for detecting protein interactions. Co-immunoprecipitation (Co-IP) or glutathione transferase (GST) pull-down assays represent such alternative methods that are commonly used to analyze protein-protein interactions in vitro. However, iIn these assays, however, the tested proteins must be readily soluble in the buffer that supportsused for the binding reaction. Therefore, specific interactions involving an insoluble protein cannot be assessed by these techniques.
 Here, we illustrate the protocol for the protein membrane overlay binding assay, which circumvents this difficulty. In this technique, interaction between soluble and insoluble proteins can be reliably tested because one of the proteins is immobilized on a membrane matrix. This method, in combination with in vivo experiments, such as BiFC, provides a reliable approach to investigate and characterize interactions faithfully between soluble and insoluble proteins. In this article, binding between Tobacco mosaic virus (TMV) movement protein (MP), which exerts multiple functions during viral cell-to-cell transport8-14, and a recently identified plant cellular interactor, tobacco ankyrin repeat-containing protein (ANK) 15, is demonstrated using this technique.

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

Testing protein-protein interaction is indispensable for dissection of protein functionality. Here, we introduce an in vitro protein-protein binding assay to probe a membrane-immobilized protein with a soluble protein. This assay provides a reliable method to test interaction between an insoluble protein and a protein in solution.

Proteinmembran Overlay Assay: Ein Protokoll, die Interaktion zwischen löslichen und unlöslichen Proteinen testen In-vitro-</em

Validating interactions between different proteins is vital for investigation of their biological functions on the molecular level.  There are several ...

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 ...

High-throughput Functional Screening using a Homemade Dual-glow Luciferase Assay

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with Parkinson&#39;s disease. 293T cells are transiently transfected with plasmids from an arrayed ORF expression library, together with luciferase reporter plasmids, in a one-gene-per-well microplate format. Firefly luciferase activity is assayed after 48 hr to determine the effects of each library gene upon alpha-synuclein transcription, normalized to expression from an internal control construct (a hCMV promoter directing Renilla luciferase). This protocol is facilitated by a bench-top robot enclosed in a biosafety cabinet, which performs aseptic liquid handling in 96-well format. Our automated transfection protocol is readily adaptable to high-throughput lentiviral library production or other functional screening protocols requiring triple-transfections of large numbers of unique library plasmids in conjunction with a common set of helper plasmids. We also present an inexpensive and validated alternative to commercially-available, dual luciferase reagents which employs PTC124, EDTA, and pyrophosphate to suppress firefly luciferase activity prior to measurement of Renilla luciferase. Using these methods, we screened 7,670 human genes and identified 68 regulators of alpha-synuclein. This protocol is easily modifiable to target other genes of interest.

We present a rapid and inexpensive screening method for identifying transcriptional regulators using high-throughput robotic transfections and a homemade dual-glow luciferase assay. This protocol rapidly generates direct side-by-side functional data for thousands of genes and is easily modifiable to target any gene of interest.

Hochdurchsatz-Screening mit einem Funktionshomemade Dual-Luciferase Assay-Schein

We present a rapid and inexpensive high-throughput screening protocol to identify transcriptional regulators of alpha-synuclein, a gene associated with ...

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.