Das Plasmid pCG001 Ausdruck UBP1 wurde freundlicherweise von Rohan Baker an der John Curtin School of Medical Research. Diese Arbeit wurde von March of Dimes Research Grant 1-FY2011, March of Dimes Basil O&#39;Connor Starter Scholar Research Award # 5-FY20 und NSF CAREER Award 0.746.796 finanziert.

Ein. Belastung Engineering Workflow <ol><li> Legen Sequenzen kodiert (a) eine Membran-Lokalisierungssequenz, (b) eine schnell reifenden fluoreszierendes Protein und (c) eine Protease-Erkennungssequenz N-terminal zu und im Leserahmen mit einem Protein von Interesse (zB ein Transkriptionsfaktor). Wir verwendeten die Membran gezielt Tsr-Venus Reporter 2 und fusioniert an die Protease Erkennungssequenz Ubiquitin (Ub), die Anzahl der Bakteriophage λ ausgedrückt Repressor CI Proteinmoleküle zählen. Einzelheiten über konstruierten wir das Fusionsprotein Tsr-Venus-Ub-CI, Ersetzen des Wildtyp-CI Kodierregion und Einbringen des Konstrukts in die E. coli-Chromosom mit λ Red Rekombination 3, sind im Detail in ref beschrieben. Ein. </li><li> Überprüfen Sie alle Änderungen durch Sequenzierung. </li><li> Transformieren eines Plasmids, das eine Protease spezifisch für die Erkennungssequenz oben in einen Stamm, der die fluoreszierende Reporter und Protein von Interesse verwendetin translationale Fusion. Wir verwendeten die Protease UBP1, die sofort spaltet nach der C-terminale Rest 4 Ubiquitin. </li></ol> 2. Kultur Cells und Probe vorbereiten <ol><li> Wählen Sie ein E. coli-Kolonie von Interesse aus einer frisch ausgestrichenen Agarplatte in 1 ml M9 Minimalmedium 5 mit 1X MEM Aminosäuren und geeigneten Antibiotika ergänzt. Inkubieren in einem Schüttler bei gewünschte Temperatur lange genug, um zu erreichen OD 600&gt; 1,0 (optische Dichte bei 600 nm). </li><li> Verdünnen Sie die Kultur in 1 ml frischem M9-Medium mit geeigneten Antibiotika zur OD 600 = 0,02. Inkubieren in einem Schüttler bei geeigneter Temperatur. Initial Zelldichte erhöht werden, wenn für die Tieftemperatur-Wachstum benötigt werden. </li><li> Wenn die OD 600 = 0,2-0,3 (bei ​​37 ° C mit einem anfänglichen Zellkultur bei OD 600 = 0,02, dies wird 3-4 Stunden dauern kann), beginnen die Vorbereitung der Agarosegel-Pad (Schritt 3). </li><li> Die Zellen sind zur Bildgebung bereit, wenn OD <sub&gt; 600 = 0,3-0,4. </li><li> Übertragen 1 ml inkubierten Kultur in einem 1,5 ml Mikrozentrifugenröhrchen Zentrifuge bei 10.000 xg für 1 min in einem Tischgerät Mikrozentrifuge. </li><li> Überstand verwerfen und 1 ml frisches Medium M9 und das Pellet vorsichtig. </li><li> Zentrifugieren bei 10.000 × g für 1 Minute. </li><li> Wiederholen Sie Schritt 1,6 und 1,7. Überstand verwerfen. </li><li> Vorsichtig in 1 ml M9-Medien resuspendieren. Bis 100-fache zu geringen Zelldichten für timelapse Bildgebung sicherzustellen - Zellen können direkt für Mikroskopabbildanordnung oder verdünntem 10 verwendet werden. Beachten Sie, dass geringen Zelldichten wichtig sind für längere timelapse Bildgebung. Die Abbildungskammer (Schritt 4) während des Experiments abgedichtet. Durch exponentielle Wachstum der Zellen, können hohe anfängliche Zellkonzentration signifikant zum Abbau Sauerstoff in die Kammer nach längerer Wachstum in der Gel-Pad, wodurch fluoreszierende Protein Reifung und beeinflussen Zellwachstum. </li></ol> 3. Bereiten Sie die Agarose-Lösung <ol><li> Wiegen 10-20 mglow-Schmelztemperatur Agarose in einem 1,5 ml Mikrozentrifugenröhrchen. </li><li> Fügen Sie entsprechende Volumen M9 Minimalmedium ohne Antibiotika, um eine 3% igen Lösung herzustellen. </li><li> Erwärmen der Agarose-Lösung für 30 min bei 70 ° C zu schmelzen, Invertieren des Rohres zu gewährleisten, dass die Lösung vollständig geschmolzen und homogen ist. Das Gelkissen kann an diesem Punkt (Schritt 4) gegossen werden, oder die Temperatur kann auf 50 ° C gesenkt werden und dort für mehrere Stunden zur späteren Verwendung. </li></ol> 4. Bereiten Gel Pad auf der Kammer <ol><li> Etwa 50 min vor der Bildgebung, spülen ein Microaqueduct Rutsche mit Wasser. </li><li> Lidspalt ein Deckglas reinigen (mit einem strengen Reinigungsverfahren wie das in 6 beschrieben) und Microaqueduct Schlitten mit sterilem Wasser und trocken durch Abblasen mit Druckluft. </li><li> Platzieren Sie die Gummidichtung auf den Schieber Microaqueduct so dass er die Ein-und Auslässe auf der Glasseite der Microaqueduct Schieber (deckt diese für Anwendungen im modifiziert werdenwelche Medien durch die Probe perfundiert). Anwenden 50 ul Agaroselösung (Schritt 3) in der Mitte des Schiebers Microaqueduct. </li><li> Ganz oben auf der Agarose-Lösung mit dem gereinigten, trockenen Deckglas. </li><li> Lassen Sie die Agarose-Lösung bei Raumtemperatur stehen für 30 min. </li><li> Unterdessen bereiten eine Zellprobe (Schritt 2). </li><li> Ziehen Sie vorsichtig die Glasabdeckung von der Spitze des Gel-Polster. Fügen Sie 1,0 ul gewaschene Zellkultur an die Spitze des Gel-Polster. Warten für ~ 2 Minuten für die Kultur von der Gel-Polster absorbiert werden. Es ist wichtig, nicht so lange zu warten, dass die Gel-Polster overdries, aber lang genug, dass die Zellen richtig auf das Gel Pad eingehalten werden. Das Ideal Wartezeit wird mit Temperatur und Luftfeuchtigkeit variieren. </li><li> Während des Wartens, trocknen anderen vorgereinigte Deckglas. </li><li> Decken Sie die Probe mit dem neuen Deckglas sicherzustellen, dass das Deckglas und Microaqueduct Objektträgers ausgerichtet sind. </li><li> Montieren Sie den temperierten Klimakammer nach den Anweisungen des Herstellers.Es kann nun zur Abbildung auf Mikroskop (Schritt 5) verwendet werden. </li></ol> 5. Imaging und Erwerb von Timelapse Filme <ol><li> Einschalten des Lasers und Mikroskop nach Herstellerangaben (zB der Laser muss möglicherweise aufgewärmt für ~ 0,5 Stunden vor dem Experiment). </li><li> Verriegeln Sie den zusammengesetzten Kammer auf die Bühne Einsatz am Mikroskop. Stellen Sie die Temperatur der Wachstumskammer und des Ziels Heizeinrichtung bei 37 ° C oder eine andere gewünschte Wachstumstemperatur. </li><li> Wenn Bildgebung oberhalb der Raumtemperatur, wird der Fokus oder die Gel-Polster in der Regel deutlich driften für ~ 15 min nach der anfänglichen Temperatur-Verschiebung. In der Praxis diese Zeit nutzen, um Bereiche von Interesse zu finden und zu ändern Bildgebung Script als notwendig für ein Experiment. </li><li> Finden Zellen auf dem Mikroskop und Speichern der Position einer jeden Zelle nach Zentrierung er innerhalb eines vordefinierten und ausgerichtet Bildgebungsregion. Mehr als eine Zelle in jeder Bildaufnahmezeit Fenster abgebildet werden. Für timelapse Abbildung über mirgendwelche Generationen, damit abgebildeten Zellen zunächst von anderen Zellen durch mindestens ein paar hundert um getrennt, so dass andere Kolonien nicht betreten wird die Bildgebungsregion während des Wachstums. </li><li> Passen Laseranregung Intensität, so dass fast alle fluoreszierende Moleküle Photobleichmittels innerhalb von 6 Aufnahmen (Abbildung 5). </li><li> Mit Hilfe eines automatisierten Imaging-Skript / journal erwerben timelapse Daten mit Hilfe des Algorithmus in der experimentellen Workflow in Abbildung 3 beschrieben. </li></ol>

<ol>
	<li>Hensel, Z., Feng, H. D., Han, B., Hatem, C., Wang, J., Xiao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stochastic+expression+dynamics+of+transcription+factor+revealed+by+single-molecule+noise+analysis.">Stochastic expression dynamics of transcription factor revealed by single-molecule noise analysis.</a> Nat. Struct. Mol. Biol. 19, 797-802 (2012).</li><li>Yu, J., Xiao, J., Ren, X., Lao, K., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+Gene+Expression+in+Live+Cells,+One+Protein+Molecule+at+a+Time.">Probing Gene Expression in Live Cells, One Protein Molecule at a Time.</a> Science. 311, 1600-1603 (2006).</li><li>Datsenko, K. A., Wanner, 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=One-step+inactivation+of+chromosomal+genes+in+Escherichia+coli+K-12+using+PCR+products.">One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.</a> Proc. Nat. Acad. Sci. U.S.A. 97, 6640-6645 (2000).</li><li>Tobias, J. W., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+and+Functional+Analysis+of+the+Ubiquitin-specific+Protease+Gene+UBP1+of+Saccharomyces+cerevisiae.">Cloning and Functional Analysis of the Ubiquitin-specific Protease Gene UBP1 of Saccharomyces cerevisiae.</a> J. Biol. Chem. 266, 12021-12028 (1991).</li><li>Sambrook, J., Russell, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular Cloning: A Laboratory Manual. , A2.2 (2001).</li><li>Xiao, J., Elf, J., Li, G., Yu, J., Xie, X. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+gene+expression+in+living+cells+at+the+single-molecule+level.">Imaging gene expression in living cells at the single-molecule level.</a> Single Molecules: a laboratory manual. , 149-169 (2007).</li><li>Nagai, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+variant+of+yellow+fluorescent+protein+with+fast+and+efficient+maturation+for+cell-biological+applications.">A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications.</a> Nat. Biotechnol. 20, 87-90 (2002).</li><li>Stagaman, G., Forsyth, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright-field+microscopy+of+semitransparent+objects.">Bright-field microscopy of semitransparent objects.</a> J. Opt. Soc. Am. A. 5, 648-659 (1988).</li><li>Tobias, J. W., Shrader, T. E., Rocap, G., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+N-end+rule+in+bacteria.">The N-end rule in bacteria.</a> Science. 254, 1374-137 (1991).</li></ol>

Wir haben keine Angaben.

Die CoTrAC Verfahren kann verallgemeinert werden, um die Produktion von anderen Proteinen, wo herkömmliche N-oder C-terminale Fusionen fluoreszierendes Protein-Protein-Aktivität zerstören kann messen. Die CoTrAC Strategie hat drei einzigartige Vorteile gegenüber den derzeitigen Methoden. Erstens sorgt co-translationale Fusion, daß ein Molekül des fluoreszierenden Reporter für jedes Molekül des Proteins von Interesse hergestellt wird, was eine genaue Zählung der Proteinproduktion in Echtzeit. Zweitens die Membra...

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare (optional) </td></tr><tr><td> Agarose (Low Schmelztemperatur) </td><td> Lonza </td><td> 50100 </td><td></td></tr><tr><td> Milli-Q-H 2 O </td><td></td><td></td><td></td></tr><tr><td> 5 × M9-Salze </td><td></td><td></td><td> Folgende Rezept in 9 beschrieben </td></tr><tr><td> 20% Glucose </td><td></td><td></td><td></td></tr><tr><td> MgSO 4 </td><td></td><td></td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> 50 × MEM Aminosäurelösung </td><td> Invitrogen </td><td> 11130-051 </td><td></td></tr><tr><td> Temperatur-Controlled GrowthKammer Tischadapter </td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td> Ziel Heater </td><td> Bioptechs </td><td> Modell hängt Mikroskopobjektiv </td><td></td></tr><tr><td> Microaqueduct Slide </td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td> Micro Deckgläser </td><td> VWR </td><td> 40CIR-1 </td><td> Kann schwierig sein Quelle; ebenfalls erhältlich von Bioptechs </td></tr><tr><td> Deckglas / slide Dichtung </td><td> Bioptechs </td><td> FCS2 0,75 mm </td><td></td></tr><tr><td> Fluoreszenzmikroskop </td><td> Verschiedene </td><td> Beispiel setup: Coherent Innova 308C Argon-Ionen-Laser, Olympus IX-81 Mikroskop Olympus PlanApo 100X NA 1,45 Ziel, Metamorph-Software </td><td> Muss Laseranregung, automatisierte xyz Bühne, Automatisierungs-Software in der Lage ist scripted Bildgebung und Autofokus, Optik in der Lage Resolvin habeng einzelner fluoreszierender Proteine </td></tr><tr><td> EM-CCD-Kamera </td><td> Verschiedene </td><td> Beispiel setup: Andor Ixon DU-898 </td><td> Muss niedrig genug Lärm haben, einzelne fluoreszierende Proteine ​​über den Hintergrund erkennen </td></tr></tbody></table>

single-molecule imaging of gene regulation in vivo using cotranslational activation by cleavage (cotrac)

Wir beschreiben ein Verfahren der Fluoreszenzmikroskopie, Co-translationale Aktivierung durch Spaltung (CoTrAC) zur Abbildung der Herstellung von Protein-Molekülen in lebende Zellen mit Einzel-Molekül-Präzision ohne Stören des Proteins Funktionalität. Dieses Verfahren macht es möglich, die Zahl der Proteinmoleküle in einer Zelle während sequentiell, fünfminütigen Zeitfenster produziert zählen. Es erfordert ein Fluoreszenzmikroskop mit Laseranregung Leistungsdichte von ~ 0,5 bis 1 kW / cm 2, die empfindlich genug, um einzelne Moleküle fluoreszierende Protein in lebenden Zellen nachzuweisen ist. Das fluoreszierende Reporter in diesem Verfahren verwendet wird, besteht aus drei Teilen: eine Membran-Targeting-Sequenz, eine Fast-Reifung, gelb fluoreszierendes Protein und ein Protease-Erkennungssequenz. Der Reporter translatorisch an den N-Terminus eines Proteins von Interesse fusioniert ist. Zellen werden auf einer Temperatur-gesteuerten Mikroskoptisch gewachsen. Alle fünf Minuten werden fluoreszierende Moleküle in Zellen abgebildet (und später coUNTED durch die Analyse von Fluoreszenz-Bildern) und anschließend Lichtgeschädigte so dass nur neu übersetzt Proteine ​​in der nächsten Messung gezählt. Fluoreszenzbilder aus diesem Verfahren kann durch Erfassen Fluoreszenzpunkte in jedem Bild, ihre Zuordnung zu einzelnen Zellen und dann Zuweisen Zellen Zelllinien analysiert werden. Die Anzahl von Proteinen innerhalb eines Zeitfensters in einer gegebenen Zelle erzeugt wird durch Dividieren der integrierten Intensität der Fluoreszenz von Flecken durch die durchschnittliche Intensität der einzelnen fluoreszierenden Moleküle berechnet. Wir verwendeten diese Methode zum Expressionslevel in dem Bereich von 0-45 Molekülen in einzelnen Zeitfenstern 5 min zu messen. Dieses Verfahren ermöglichte es uns, Rauschen in dem die Expression des λ-Repressor CI messen, und hat viele andere potentielle Anwendungen in Systembiologie.

Typische Ergebnisse aus einem Versuch CoTrAC Verfolgen der Herstellung des λ-Repressor CI sind in den 4 und 5 gezeigt. In diesem Experiment wurden 12 Kolonien in 5 min Intervallen abgebildet. Zu jedem Zeitpunkt wurde die erste Kolonie autofocused und zentralisiert innerhalb der Bildgebungsregion. Als nächstes wurde die mittig / fokussierte Position gespeichert und ein Hellfeld Bild erworben wurde. Die Stufe wurde dann von ~ 0,5 um entlang der z-Achse von der translozi...

Watch this Scientific Journal Video about Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC at JoVE.com

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Wir beschreiben ein Verfahren der Fluoreszenzmikroskopie, Co-translationale Aktivierung durch Spaltung (CoTrAC), zur Abbildung der Herstellung von Protein-Molekülen in lebende Zellen mit Einzel-Molekül-Präzision ohne Stören des Proteins Funktionalität. Dieses Verfahren wurde verwendet, um die Expression stochastische Dynamik eines Transkriptionsfaktors, dem λ-Repressor CI folgen<sup&gt; 1</sup&gt;.

Einzel-Molekül-Imaging der Genregulation<em&gt; In vivo</em&gt; Verwenden cotranslationalen Aktivierung durch Spaltung (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

single-molecule-imaging-gene-regulation-vivo-using-cotranslational

Research

JoVE Journal

Biology

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

9.8K Aufrufe.  Johns Hopkins University School of Medicine. Wir beschreiben ein Verfahren der Fluoreszenzmikroskopie, Co-translationale Aktivierung durch Spaltung (CoTrAC), zur Abbildung der Herstellung von Protein-Molekülen in lebende Zellen mit Einzel-Molekül-Präzision ohne Stören des Proteins Funktionalität. Dieses Verfahren wurde verwendet, um die Expression stochastische Dynamik eines Transkriptionsfaktors, dem λ-Repressor CI folgen 1.

Serie: Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

Single-Molecule Imaging of Nuclear Transport

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the last decade. The investigation of molecular interactions with high temporal and spatial resolutions deep within cells has remained challenging due to the inherently weak signals arising from individual molecules. Recent works by Yang et al. demonstrated that narrow-field epifluorescence microscopy allows visualization of nucleocytoplasmic transport at the single molecule level. By the single molecule approach, important kinetics, such as nuclear transport time and efficiency, for signal-dependent and independent cargo molecules have been obtained. Here we described a protocol for the methodological approach with an improved spatiotemporal resolution of 0.4 ms and 12 nm. The improved resolution enabled us to capture transient active transport and passive diffusion events through the nuclear pore complexes (NPC) in semi-intact cells. We expect this method to be used in elucidating other binding and trafficking events within cells.

Single molecule microscopy approch provided novel insights into nuclear transport.

The utility of single molecule fluorescence microscopy approaches has been proven to be of a great avail in understanding biological reactions over the ...

Forschung

Biologie

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1. Apoptosis can be triggered when cells encounter a wide range of cytotoxic stresses. These insults initiate signaling cascades that ultimately cause the release of cytochrome c from the mitochondrial intermembrane space to the cytoplasm2. The release of cytochrome c from mitochondria is a key event that triggers the rapid activation of caspases, the key cellular proteases which ultimately execute cell death3-4.
The pathway of apoptosis is regulated at points upstream and downstream of cytochrome c release from mitochondria5. In order to study the post-mitochondrial regulation of caspase activation, many investigators have turned to direct cytoplasmic microinjection of holocytochrome c (heme-attached) protein into cells6-9. Cytochrome c is normally localized to the mitochondria where attachment of a heme group is necessary to enable it to activate apoptosis10-11. Therefore, to directly activate caspases, it is necessary to inject the holocytochrome c protein instead of its cDNA, because while the expression of cytochrome c from cDNA constructs will result in mitochondrial targeting and heme attachment, it will be sequestered from cytosolic caspases. Thus, the direct cytosolic microinjection of purified heme-attached cytochrome c protein is a useful tool to mimic mitochondrial cytochrome c release and apoptosis without the use of toxic insults which cause cellular and mitochondrial damage.
In this article, we describe a method for the microinjection of cytochrome c protein into cells, using mouse embryonic fibroblasts (MEFs) and primary sympathetic neurons as examples. While this protocol focuses on the injection of cytochrome c for investigations of apoptosis, the techniques shown here can also be easily adapted for microinjection of other proteins of interest.

Activation of Apoptosis by Cytoplasmic Microinjection of Cytochrome c

In this protocol, we describe the direct cytoplasmic microinjection of cytochrome c protein into fibroblasts and primary sympathetic neurons. This technique allows for the introduction of cytochrome c protein into the cytoplasm of cells and mimics the release of cytochrome c from mitochondria, which occurs during apoptosis.

Die Aktivierung der Apoptose durch cytoplasmatische Mikroinjektion von Cytochrom C</em

Apoptosis, or programmed cell death, is a conserved and highly regulated pathway by which cells die1.  Apoptosis can be triggered when cells encounter a ...

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.
Telomerase is a specialized reverse transcriptase1 that adds short DNA repeats, called telomeres, to the 3' end of linear chromosomes2 that serve to protect them from end-to-end fusion and degradation. Following DNA replication, a short segment is lost at the end of the chromosome3 and without telomerase, cells continue dividing until eventually reaching their Hayflick Limit4. Additionally, telomerase is dormant in most somatic cells5 in adults, but is active in cancer cells6 where it promotes cell immortality7.
The minimal telomerase enzyme consists of two core components: the protein subunit (TERT), which comprises the catalytic subunit of the enzyme and an integral RNA component (TER), which contains the template TERT uses to synthesize telomeres8,9. Prior to 2008, only structures for individual telomerase domains had been solved10,11. A major breakthrough in this field came from the determination of the crystal structure of the active12, catalytic subunit of T. castaneum telomerase, TcTERT1.
Here, we present methods for producing large quantities of the active, soluble TcTERT for structural and biochemical studies, and the reconstitution of the telomerase RNP complex in vitro for telomerase activity assays. An overview of the experimental methods used is shown in Figure 1.

In vitro Reconstitution of the Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more than a decade. Here, we present methods for the isolation of the recombinant Tribolium castaneum TERT (TcTERT) and the reconstitution of the active T. castaneum telomerase ribonucleoprotein (RNP) complex in vitro.

In vitro Rekonstitution der Active T. castaneum Telomerase

Efforts to isolate the catalytic subunit of telomerase, TERT, in sufficient quantities for structural studies, have been met with limited success for more ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

Isolierung von Ribosom gebunden Nascent Polypeptide<em&gt; In-vitro-</em&gt; Identifizieren Translational Pause Standorten entlang mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Mit SECM Arrest Sequenz als Werkzeug zum Ribosom gebunden Polypeptide isolieren

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analyse der globalen RNA-Synthese auf der Einzelzellebene folgende Hypoxie

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel (ENaC) by Combining Current Measurements with Detection of Cleavage Fragments

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously expressed in Xenopus laevis oocytes. In combination with site-directed mutagenesis, this approach provides a powerful tool to identify functionally relevant cleavage sites. Proteolytic activation is a characteristic feature of the amiloride-sensitive epithelial sodium channel (ENaC). The final activating step involves cleavage of the channel&#8217;s &#947;-subunit in a critical region potentially targeted by several proteases including chymotrypsin and plasmin. To determine the stimulatory effect of these serine proteases on ENaC, the amiloride-sensitive whole-cell current (&#916;Iami) was measured twice in the same oocyte before and after exposure to the protease using the two-electrode voltage-clamp technique. In parallel to the electrophysiological experiments, a biotinylation approach was used to monitor the appearance of &#947;ENaC cleavage fragments at the cell surface. Using the methods described, it was demonstrated that the time course of proteolytic activation of ENaC-mediated whole-cell currents correlates with the appearance of a &#947;ENaC cleavage product at the cell surface. These results suggest a causal link between channel cleavage and channel activation. Moreover, they confirm the concept that a cleavage event in &#947;ENaC is required as a final step in proteolytic channel activation. The methods described here may well be applicable to address similar questions for other types of ion channels or membrane proteins.

Demonstration of Proteolytic Activation of the Epithelial Sodium Channel ENaC by Combining Current Measurements with Detection of Cleavage Fragments

Proteolytic activation of the epithelial sodium channel (ENaC) heterologously expressed in Xenopus laevis oocytes can be demonstrated by combining current measurements with a biotinylation approach to investigate the appearance of ion channel cleavage products at the cell surface. Functionally important cleavage sites can be identified by using site-directed mutagenesis.

Demonstration der proteolytischen Aktivierung des epithelialen Natriumkanals (ENaC) durch Kombination von Strommessungen mit Nachweis der Spaltfragmente

The described methods can be used to investigate the effect of proteases on ion channels, receptors, and other plasma membrane proteins heterologously ...

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Studieren von DNA-Looping von Einzelmolekül-FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

In-vivo-4-Dimensional-Tracking von hämatopoetischen Stamm-und Vorläuferzellen im erwachsenen Maus Schädeldachknochenmark

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable of measuring SSDC in many single DNA molecules simultaneously. Bead-tethered substrate DNAs, each containing a single copy of the target sequence, are prepared in a microfluidic flow channel. An external magnet applies a weak force to the paramagnetic beads. The integrity of up to 1,000 individual DNAs can be monitored by visualizing the microbeads under darkfield imaging using a wide-field, low magnification objective. Injecting of a restriction endonuclease, NdeI, initiates the cleavage reaction. Video microscopy is used to record the exact moment of each DNA cleavage by observing the frame in which the associated bead moves up and out of the focal plane of the objective. Frame-by-frame bead counting quantifies the reaction, and an exponential fit determines the reaction rate. This method allows collection of quantitative and statistically significant data on single molecule SSDC reactions in a single experiment.

A highly parallel method for measuring the site-specific cleavage of DNA at the single molecule level is described. This protocol demonstrates the technique using the restriction endonuclease NdeI. The method can easily be modified to study any process that results in site-specific DNA cleavage.

Paralleler Hochdurchsatz-Single Molecule Kinetic Assay für standortspezifische DNA-Spaltung

Site-specific DNA cleavage (SSDC) is a key step in many cellular processes, and it is crucial to gene editing. This work describes a kinetic assay capable ...