Il pCG001 plasmide che esprime Ubp1 è stato gentilmente fornito da Rohan Baker presso la John Curtin School of Medical Research. Questo lavoro è stato finanziato dal March of Dimes Research Grant 1-FY2011, March of Dimes Basil O&#39;Connor Research Scholar Award Starter N ° 5-FY20 e di aggiudicazione CARRIERA NSF 0746796.

1. Flusso di lavoro Strain Ingegneria <ol><li> Inserire codifica sequenze (a) una membrana-localizzazione sequenza, (b) una rapida maturazione proteina fluorescente e (c) una sequenza di riconoscimento proteasi N-terminale e in frame con una proteina di interesse (ad esempio, un fattore di trascrizione). Abbiamo usato la membrana mirata Tsr-Venus 2 giornalista e fuso alla sequenza di riconoscimento proteasi ubiquitina (Ub) per contare il numero di espresse batteriofago λ repressore molecole proteiche CI. I dettagli su come abbiamo costruito la proteina di fusione Tsr-Venere-Ub-CI, che sostituisce il wild-type CI regione codificante e incorporando il costrutto in E. cromosoma coli usando λ ricombinazione Red 3, sono descritte in dettaglio in rif. 1. </li><li> Verificare che tutte le modifiche mediante sequenziamento. </li><li> Trasformare un plasmide codificante una proteasi specifica per la sequenza di riconoscimento utilizzato sopra in un ceppo che esprime il reporter fluorescente e la proteina di interessein fusione traslazionale. Abbiamo usato la proteasi Ubp1, che scinde subito dopo il residuo C-terminale Ubiquitina 4. </li></ol> 2. Cellule Cultura e preparare campioni <ol><li> Scegli uno E. colonia coli di interesse da una piastra di agar appena striato in 1 ml di M9 minima media 5 integrato con 1X acidi MEM amminoacidi e antibiotici appropriati. Incubare in un agitatore a temperatura voluta abbastanza a lungo per raggiungere OD 600&gt; 1,0 (densità ottica a 600 nm). </li><li> Diluire la cultura in 1 ml di terreno fresco M9 con antibiotici appropriati per OD 600 = 0,02. Incubare in un agitatore a temperatura adeguata. Densità cellulare iniziale può essere aumentato se necessario per la bassa temperatura di crescita. </li><li> Quando la OD 600 = 0,2-0,3 (a 37 ° C con una coltura cellulare iniziale OD 600 = 0,02, ciò richiederà hr 3-4), iniziare a preparare il gel di agarosio pad (passo 3). </li><li> Le cellule sono pronte per l&#39;imaging quando OD <sub&gt; 600 = 0,3-0,4. </li><li> Trasferire 1 ml di coltura incubata in una provetta da microcentrifuga da 1,5 ml, centrifugare a 10.000 xg per 1 min in microcentrifuga. </li><li> Eliminare il supernatante e aggiungere 1 ml di terreno fresco M9 e risospendere il pellet delicatamente. </li><li> Centrifugare a 10.000 xg per 1 min. </li><li> Ripetere il punto 1.6 e 1.7. Eliminare il surnatante. </li><li> Risospendere delicatamente in 1 ml di M9 media. Cellule può essere direttamente utilizzata per l&#39;imaging microscopio o diluito 10 - a 100 volte per garantire densità cellulari bassi per l&#39;imaging timelapse. Notare che basse densità cellulari sono importanti per l&#39;imaging timelapse esteso. La camera di imaging (fase 4) è sigillata durante l&#39;esperimento. A causa della crescita esponenziale di cellule, elevate concentrazioni iniziali di cellule può significativamente esaurire l&#39;ossigeno all&#39;interno della camera di crescita dopo prolungata nel cuscinetto di gel, riducendo maturazione proteina fluorescente e influenzando la crescita cellulare. </li></ol> 3. Preparare la soluzione di agarosio <ol><li> Pesare 10-20 mgbassa temperatura di fusione agarosio in una provetta da microcentrifuga 1,5 ml. </li><li> Aggiungere mezzo adeguato volume minimo M9 senza antibiotici per ottenere una soluzione al 3% di agarosio. </li><li> Riscaldare la soluzione di agarosio per 30 minuti a 70 ° C per fondere, invertendo la provetta per garantire che la soluzione è completamente fusa e omogenea. Il cuscinetto di gel può essere versato a questo punto (passo 4) o la temperatura può essere ridotta a 50 ° C e mantenuta per diverse ore per un uso successivo. </li></ol> 4. Preparare Pad Gel sulla Camera <ol><li> Circa 50 minuti prima di imaging, lavare uno scivolo Microaqueduct con acqua. </li><li> Risciacquo uno pulito vetro di copertura (utilizzando un metodo rigoroso di pulizia come quello descritto in 6) e Microaqueduct scivolo con acqua sterile e asciugare soffiando con aria compressa. </li><li> Posizionare la guarnizione di gomma sul vetrino Microaqueduct modo che copra le entrate e le uscite sul lato del vetro scorrevole Microaqueduct (questo può essere modificato per applicazioni inquale supporto è perfuso attraverso il campione). Applicare 50 ul di soluzione di agarosio (step 3) al centro della slitta Microaqueduct. </li><li> Top la soluzione di agarosio con la pulizia, vetro di copertura a secco. </li><li> Lasciare la soluzione di agarosio riposare a temperatura ambiente per 30 min. </li><li> Nel frattempo, preparare un campione di cellule (fase 2). </li><li> Attentamente staccare il vetro di copertura dalla parte superiore del cuscinetto di gel. Aggiungere 1,0 microlitri coltura cellulare lavato per la parte superiore del cuscinetto di gel. Attendere ~ 2 min per la coltura di essere assorbita dal cuscinetto di gel. E &#39;importante non aspettare così a lungo che le overdries gel pad, ma abbastanza a lungo che le cellule siano correttamente rispettate il cuscinetto di gel. Il tempo ideale di attesa varia a seconda della temperatura e dell&#39;umidità. </li><li> In attesa, asciugare un altro bicchiere predepurata copertura. </li><li> Coprire il campione con il vetro di copertura nuova assicurare che il vetro e il coperchio scorrevole Microaqueduct sono ben allineati. </li><li> Montare la camera di crescita della temperatura controllata seguendo le istruzioni del produttore.Si può ora essere utilizzato per l&#39;imaging su microscopio (passo 5). </li></ol> 5. Acquisizione di immagini e filmati Timelapse <ol><li> Accendere il microscopio laser e seguendo le istruzioni del produttore (ad esempio, il laser potrebbe essere necessario riscaldato per ~ 0,5 ore prima dell&#39;esperimento). </li><li> Bloccare la camera montata per l&#39;inserto sul palco microscopio. Impostare la temperatura della camera di crescita e il riscaldatore obiettivo a 37 ° C o altra temperatura di crescita desiderata. </li><li> Se l&#39;imaging sopra della temperatura ambiente, la messa a fuoco o cuscinetto di gel di solito deviare significativamente per ~ 15 minuti dopo il turno temperatura iniziale. In pratica, utilizzare questo tempo per trovare le regioni di interesse e modificare gli script di imaging, se necessario, per un esperimento. </li><li> Trova celle del microscopio e memorizzare la posizione di ciascuna cella dopo centrandolo all&#39;interno di una regione imaging predefiniti e allineati. Più di una cella può essere ripreso in ciascuna finestra di tempo di acquisizione dell&#39;immagine. Per timelapse di imaging su mtutte le generazioni, in modo che le cellule sotto forma di immagini sono inizialmente separati da altre cellule di almeno qualche centinaio di micron in modo che altre colonie non entrerà nella regione di imaging durante la crescita. </li><li> Regolare intensità del laser di eccitazione in modo che quasi tutti fotosbiancante molecole fluorescenti entro 6 esposizioni (Figura 5). </li><li> Utilizzo di uno script automatico di imaging / rivista, acquisire i dati timelapse utilizzando l&#39;algoritmo descritto nel flusso di lavoro sperimentale in Figura 3. </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>

Non abbiamo informazioni integrative.

Il metodo CoTrAC può essere generalizzato per misurare la produzione di altre proteine ​​con N-convenzionali o C-terminali fusioni proteine ​​fluorescenti possono alterare l&#39;attività della proteina. La strategia CoTrAC ha tre vantaggi unici rispetto ai metodi attuali. First, co-traslazionale fusione assicura che una molecola del reporter fluorescente viene prodotta per ogni molecola di proteina di interesse, permettendo conteggio preciso della produzione di proteina in tempo reale. In secondo luogo, la mem...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti (opzionale) </td></tr><tr><td> Agarosio (bassa temperatura di fusione) </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 × sali M9 </td><td></td><td></td><td> Ricetta seguito descritto in 9 </td></tr><tr><td> 20% di glucosio </td><td></td><td></td><td></td></tr><tr><td> MgSO4 </td><td></td><td></td><td></td></tr><tr><td> CaCl 2 </td><td></td><td></td><td></td></tr><tr><td> 50 × MEM soluzione di aminoacidi </td><td> Invitrogen </td><td> 11130-051 </td><td></td></tr><tr><td> Temperatura controllata di crescitaCamera Fase adattatore </td><td> Bioptechs </td><td> FCS-2 </td><td></td></tr><tr><td> Riscaldatore Obiettivo </td><td> Bioptechs </td><td> Modello dipende obiettivo microscopio </td><td></td></tr><tr><td> Microaqueduct diapositiva </td><td> Bioptechs </td><td> 130119-5 </td><td></td></tr><tr><td> Vetrini coprioggetto Micro </td><td> VWR </td><td> 40CIR-1 </td><td> Può essere difficile da fonte, disponibile anche da Bioptechs </td></tr><tr><td> Copertura in vetro / slide guarnizione </td><td> Bioptechs </td><td> FCS2 0,75 millimetri </td><td></td></tr><tr><td> Microscopio a fluorescenza </td><td> Vario </td><td> Esempio di installazione: Coherent Innova 308C Argon-laser a ioni, Olympus IX-81 microscopio, Olympus Planapo 100X NA 1,45 obiettivo, software Metamorph </td><td> Deve avere eccitazione del laser, automatizzato fase xyz, software di automazione in grado di imaging script e autofocus, ottica in grado di resolving singole proteine ​​fluorescenti </td></tr><tr><td> EM-CCD </td><td> Vario </td><td> Esempio di installazione: Andor Ixon DU-898 </td><td> Deve avere rumore sufficientemente basso per rilevare singole proteine ​​fluorescenti sopra sfondo </td></tr></tbody></table>

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

Descriviamo un metodo di microscopia a fluorescenza, Co-traslazionale di attivazione da Cleavage (CoTrAC) per l&#39;immagine della produzione di molecole di proteine ​​in cellule vive con singola molecola di precisione senza perturbare la funzionalità della proteina. Questo metodo permette di contare il numero di molecole di proteine ​​prodotte in una cella durante sequenziali, cinque minuti finestre temporali. Si richiede un microscopio a fluorescenza con densità di potenza laser di eccitazione di ~ 0,5 a 1 kW / cm 2, che è sufficientemente sensibile per rivelare singole molecole di proteine ​​fluorescenti in cellule vive. Il reporter fluorescente utilizzata in questo metodo si compone di tre parti: una sequenza di targeting membrana, una rapida maturazione, proteina fluorescente gialla e una sequenza di riconoscimento proteasi. Il reporter è traduzionalmente fusa all&#39;N-terminale di una proteina di interesse. Le cellule vengono coltivate su un temperatura controllata portaoggetti. Ogni cinque minuti, le molecole fluorescenti all&#39;interno delle cellule vengono esposte (e poi counted analizzando le immagini di fluorescenza) e, successivamente, in modo che le proteine ​​photobleached solo appena tradotti sono conteggiati nella misura successiva. Immagini di fluorescenza risultanti da tale metodo può essere analizzato rilevando macchie fluorescenti in ogni immagine, assegnando loro singole celle e quindi assegnare cellule per linee cellulari. Il numero di proteine ​​prodotte entro una finestra temporale in una data cella è calcolato dividendo l&#39;intensità di fluorescenza delle macchie integrato per l&#39;intensità media di singole molecole fluorescenti. Abbiamo usato questo metodo per misurare i livelli di espressione nella gamma di 0-45 molecole in finestre singole 5 min di tempo. Questo metodo consente di misurare il rumore nell&#39;espressione del repressore λ CI, e ha molte altre applicazioni potenziali in biologia dei sistemi.

Risultati tipici di un esperimento CoTrAC tracciamento della produzione del repressore λ CI sono mostrati nelle Figure 4 e 5. In questo esperimento, sono stati ripresi 12 colonie a intervalli di 5 min. Ad ogni punto di tempo, la colonia fu autofocused e centralizzato all&#39;interno della regione di imaging. Successivamente, l&#39;centrato / focalizzato posizione è stata memorizzata e l&#39;immagine è stata acquisita campo chiaro. Lo stadio è stato poi traslocato da ~ 0,5 micron lun...

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

Descriviamo un metodo di microscopia a fluorescenza, Co-traslazionale di attivazione da Cleavage (CoTrAC), per l&#39;immagine della produzione di molecole di proteine ​​in cellule vive con singola molecola di precisione senza perturbare la funzionalità della proteina. Questo metodo è stato utilizzato per seguire la dinamica stocastica espressione di un fattore di trascrizione, il repressore λ CI<sup&gt; 1</sup&gt;.

Singola molecola di imaging del regolamento Gene<em&gt; In vivo</em&gt; Utilizzo di attivazione Cotranslational da Cleavage (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 Views.  Johns Hopkins University School of Medicine. Descriviamo un metodo di microscopia a fluorescenza, Co-traslazionale di attivazione da Cleavage (CoTrAC), per l'immagine della produzione di molecole di proteine ​​in cellule vive con singola molecola di precisione senza perturbare la funzionalità della proteina. Questo metodo è stato utilizzato per seguire la dinamica stocastica espressione di un fattore di trascrizione, il repressore λ CI 1.

Video: 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.

Singola molecola Imaging dei trasporti nucleari

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

Ricerca

Biologia

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.

Attivazione di apoptosi da Microiniezione citoplasmatici del Citocromo 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.

Ricostituzione in vitro di Active T. castaneum telomerasi

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.

Isolamento dei ribosomi Polipeptidi Bound nascenti<em&gt; In vitro</em&gt; Per identificare i siti di pausa traslazione lungo 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.

Utilizzando Sequence SECM arresto come uno strumento per isolare ribosoma Polipeptidi Bound

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.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

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.

Dimostrazione di Proteolytic attivazione della epiteliale di sodio Channel (ENAC), combinando misure di corrente con rilevamento di Decolleté Frammenti

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.

Studiare Looping DNA a singola molecola 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 inseguimento 4-dimensionale di emopoietiche staminali e progenitrici celle di topo adulto cranica di Midollo Osseo

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.

Analisi cinetica ad alta velocità ad alto rendimento parallela per cleavage del DNA specifico del sito

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 ...