Name: using fluorescent proteins to monitor glycosome dynamics in the african trypanosome
Uploaded: 2014-08-19T13:00:00.000+00:00
Duration: 10 min 4 s
Description: Watch this Scientific Journal Video about Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome at JoVE.com

This work was funded by the Creative Inquiry Program for Undergraduate Research and the Calhoun Honors College at Clemson University.

1. Generale Tripanosoma Zootecnica <ol><li> Pesare solidi per SDM79 preparazione supporti (Tabella 1). </li><li> Conservare a 4 ° C in 50 ml conica o un sacchetto a chiusura ermetica. NOTA: I reagenti sono stabili per almeno 6 mesi. </li><li> Scongelare siero fetale bovino (FBS) in un bagnomaria a 37 ° C, e mescolare periodicamente per inversione. NOTA: FBS viene ricevuto dal fornitore come una soluzione sterile. Filtro sterilizzante FBS riduce la sua capacità di sostenere la crescita del parassita. </li><li> Una volta che l&#39;intera bottiglia è scongelato, il calore inattivare il siero per 30 minuti in un bagnomaria a 56 ° C, mescolando periodicamente per ridurre al minimo la precipitazione di componenti del siero. </li><li> Thaw penicillina / streptomicina soluzione (pen / strep), emina (2 mg / ml in NaOH 50 mM), e la soluzione a medio basale aquila vitamina a temperatura ambiente. </li><li> Per 1.000 ml cilindro graduato, aggiungere 20,2 g di solidi SDM79 (Tabella 1) ai componenti liquidi (Tabella 2) e mescolare bene su un piatto mescolare. </li><li> Regolare il pH a 7,35, e portare il volume a 850 ml con acqua. </li><li> Filtro sterilizzare supporti collegando un tubo di aspirazione sul fondo di una bottiglia filtro. Applicare vuoto finché la soluzione viene filtrata. </li><li> Rimuovere il filtro in alto nell&#39;armadio biosicurezza e aggiungere 150 ml di FBS inattivato al calore. Rimuovere rivestimento in plastica dal tappo sterile e bottiglia mezzi di tenuta. </li><li> Preparare supporti SDM80 nello stesso modo come SDM79 con le seguenti eccezioni; non aggiungere il glucosio o glucosamina e utilizzare MEM, senza glutammina. Al posto di 150 ml di FBS inattivato al calore, aggiungere 135 ml di dializzato, FBS inattivato al calore, e 15 ml di FBS inattivato al calore. </li><li> Cultura PCF parassiti a 25 centimetri pallone 2 con 10 ml di mezzo appropriato a 27 ° C e 5% di CO 2. NOTA: Le cellule devono essere conteggiati tutti i giorni con un emocitometro o un citofluorimetro (vedi paragrafo 6) e le colture devono essere mantenute a densità tra 1 x 10 5 e 5 x 10 6 cellule / ml. </li></ol> 2 Transfection di PCF parassiti con il Reporter Construct fluorescente NOTA: Per seguire glycosome dinamica, una proteina reporter contenente la sequenza di targeting dei perossisomi (PTS2) di aldolasi fusa alla proteina fluorescente gialla maggiore è espressa nei parassiti. La sequenza codificante la proteina di fusione viene clonato in pXS2bla 12, che contiene il promotore procyclin e le regioni intergeniche tubulina, che ricombinazione omologa diretto nel genoma e il gene di resistenza blasticidin per la selezione. Linee cellulari prociclico che ospitano i geni che codificano la polimerasi e tetraciclina repressore T7 (PF29-13) vengono trasformate con il costrutto di targeting, pXS2: PTS2eYFP. <ol><li> Preparare cytomix miscelando i componenti elencati nella Tabella 3. Filtro sterilizzare e conservare a temperatura ambiente. </li><li> Linearizza DNA plasmidico con MluI pipettando 10 ml di DNA (1 mg / mL), 5 ml di buffer enzima di restrizione, acqua 33 ml, e 2 μ; L di MluI enzima in una provetta. Incubare a 37 ° C per 1 ora. </li><li> Purificare la restrizione digerire con l&#39;aggiunta di 1 volume di tampone di legame per l&#39;enzima di restrizione digest. Mescolare binding buffer e DNA digerito con il vortex. Brevemente centrifugare per raccogliere il campione al fondo della provetta. </li><li> Inserire una colonna vincolante in un tubo di raccolta da 2 ml DNA, e il trasferimento di DNA digerito / soluzione tampone a colonna vincolante. </li><li> Centrifugare per 10.000 xg per 1 minuto. Dopo centrifugazione, scartare filtrato dal tubo di raccolta e riportare la colonna al tubo di raccolta. </li><li> Aggiungere 700 ml di SPW tampone di lavaggio e centrifugare a 10.000 xg per 1 min. </li><li> Scarta filtrato dal tubo di raccolta, riportare la colonna al tubo di raccolta e ripetere SPW fase di lavaggio e centrifugazione. </li><li> Eliminare filtrato, il ritorno colonna di tubo di raccolta, e centrifugare colonna vuota per 3 min a 10.000 xg per rimuovere l&#39;etanolo residuo. </li><li> In un armadio biosicurezza, buttare via coltubo colta e colonna posto in una provetta sterile. </li><li> Per eluire il DNA, aggiungere 25 ml di acqua sterile per colonna e incubare a temperatura ambiente per 2 min. Centrifugare a 10.000 xg per 1 min. </li><li> Aggiungere altri 25 ml di acqua sterile in cappa di biosicurezza a colonna e incubare a temperatura ambiente per 2 min. </li><li> Centrifugare a 10.000 xg velocità per 1 min. </li><li> In un armadio biosicurezza trasferire intero volume di DNA in una nuova provetta sterile. NOTA: Questo passaggio impedisce la contaminazione dal coperchio aperto durante la centrifugazione. </li><li> Aggiungere la sterile, purificato, DNA linearizzato (10 mg) per 400 ml di cytomix sterilizzata per filtrazione. </li><li> Contare le celle come descritto nella sezione 6 e raccogliere 5 x10 7 -10 8 cellule per centrifugazione a 800 xg per 15 minuti a temperatura ambiente. </li><li> Eliminare il surnatante e risospendere il pellet cellulare in 450 ml di DNA + cytomix con una pipetta 1 ml sierologica. </li><li> Trasferire la soluzione contenente ce<html> LLS, DNA, e cytomix ad una sterile 4 millimetri divario cuvetta e posto cuvetta nella camera di elettroporazione. </li><li> Selezionare &quot;decadimento esponenziale&quot; e immettere manualmente le seguenti impostazioni: Tensione: 1.5 kV, Capacità: 25 mF, Resistance: Ω, e Cuvette: 4 mm. Premere impulso. Una volta che il polso, rimuovere cuvetta dalla camera di elettroporazione e tornare al mobile biosicurezza. </li><li> In un nuovo beuta sterile, pipetta 10 ml di SDM79. Trasferire le cellule trasformate al pallone con 10 ml di SDM79. </li><li> Incubare senza selezione della droga per 24 ore a 27 ° C, 5% di CO 2. Dopo 24 ore, aggiungere G418 (15 mg / ml), igromicina (50 mg / ml), e blasticidin (10 mg / ml). Passare 1 ml di cellule trasformate con la droga in 9 ml di SDM79 con la droga. </li><li> Analizzare le cellule quotidianamente come descritto nelle sezioni 6 e 7 Quando le cellule sono fluorescenti, e hanno raggiunto una densità cellulare di 1 x 10 7 / ml, fare stabilates per la conservazione a lungo termine (3.1-3.3). </li></ol>ve_title &quot;&gt; 3. Fare Stabilates <ol><li> Per rendere congelamento supporti, aggiungere un volume equivalente di 100% glicerolo a cytomix e filtro sterilizzare. </li><li> Aggiungere 200 ml di mezzi di congelamento a 800 ml di cellule (1 x 10 7) in una provetta Microbank ™, mescolare delicatamente per inversione e posto tra due rack di polistirolo e congelare a -80 ° C. </li><li> Una volta congelato (~ 24 ore), le cellule di trasferimento per l&#39;azoto liquido. NOTA: E &#39;importante spostare le celle in LN 2 dopo 24 ore, come una conservazione più lunga a -80 ° C porta ad una diminuzione della vitalità cellulare. </li></ol> 4. Azioni Scongelamento congelati <ol><li> Rimuovere flacone congelato da -80 ° C e scongelare a temperatura ambiente per circa 10 min. </li><li> Pipettare 9 ml di SDM79 con la droga in una nuova boccetta sterile. Aggiungi cellule scongelati a questo fiasco. Passare cellule 1:10 aggiungendo 1 ml di questa coltura a 9 ml di SDM79 in un&#39;altra beuta (concentrazione finale di 1 x 10 5 / ml). </li><li> Prima che le cellule raggiungono una densità di 10 7 / ml, iniziare citometro sete saggi di diluizione. </li></ol> 5. citometro Setup <ol><li> Accendere il citofluorimetro e aprire il programma CFLOW più. NOTA: Prima di eseguire i campioni, i fluidica dovrebbero essere backflush e risciacquati in base alle fasi 5,2-5,7. </li><li> Sostituire il tubo di acqua Nanopure in cui il SIP viene memorizzato con un tubo vuoto. </li><li> Selezionare il pulsante &quot;controlavaggio&quot;. </li><li> Una volta backflush, rimuovere provetta da sorseggiare e scarto. </li><li> Posizionare una nuova provetta contenente 1 ml di acqua nuova Nanopure sul sorso. </li><li> Selezionare un nuovo pozzo in programma CFLOW. Sotto &quot;Limiti Run&quot; impostare il tempo per 2 min. Sotto &quot;Fluidics&quot; selezionare &quot;veloce&quot; e &quot;Run&quot;. </li><li> Una volta che il limite di tempo di 2 minuti, fare clic sul pulsante &quot;Elimina dati di esempio&quot;. </li></ol> 6. conteggio cellulare utilizzando il citometro di flusso <ol><li> Sostituire l&#39;acqua con il campione da contare. Impostare &quot;Limiti Esegui &quot;per 30 secondi,&quot; pneumatica &quot;a&quot; veloce &quot;e quindi selezionare&quot; Esegui &quot;. </li><li> Dopo il termine di 30 sec è completa, CFLOW oltre fornirà gli eventi / ml sotto &quot;Ultima esecuzione.&quot; Numero di ripetizioni per ogni cultura. NOTA: Prima di cellule raggiungono 1 x 10 7 cellule / ml, procedere con test di diluizione. Le cellule devono essere interrotti dopo un test di diluizione viene completata. Le cellule in coltura per periodi più lunghi perdono la loro capacità di rispondere alle condizioni ambientali. </li></ol> 7 fluorescenza di misura utilizzando il citometro di flusso <ol><li> Per valutare la fluorescenza, impostare &quot;Limiti Run&quot; a 10.000 eventi, e &quot;pneumatica&quot; per &quot;rallentare&quot;. Selezionare &quot;Imposta soglia&quot;. Sotto &quot;Threshold Primaria&quot;, selezionare &quot;Permanentemente eliminare eventi su&quot;, &quot;FSC-H&quot; (nella casella a discesa) e immettere meno di 30.000. Fare clic su &quot;Applica&quot;, quindi su &quot;Chiudi&quot;. </li><li> Selezionare il220, &quot;pulsante in una delle nuove finestre trama e selezionare&quot; Istogramma FSC-A &quot;Dal menu a tendina, selezionare&quot; FL1-A &quot;. NOTA: misure FL1-A fluorescenza a lunghezza d&#39;onda 530 nm. </li><li> Selezionare &quot;Esegui&quot; e ripetere per tutte le culture. </li><li> Eseguire pulizia soluzione attraverso la linea fluidica per 2 min e acqua per 2 min. </li></ol> 8. diluizione saggi <ol><li> Passare cellule ad una densità di 1 x 10 5 cellule / ml in 3 ml di SDM79 in un pallone di 25 centimetri 2 cultura e misurare fioritura immediatamente dopo passaggio e poi alle 3 ore e 24 ore. </li></ol> Analisi dei dati 9. <ol><li> Selezionare &quot;Analizza&quot; linguetta CFLOW più. </li><li> Fare clic apparirà il pulsante &quot;Istogramma&quot; sotto &quot;Crea una nuova trama.&quot; Select &quot;FSC-A&quot; e un elenco a discesa. Selezionare il canale &quot;FL1-A.&quot; </li><li> Evidenziare un bene da analizzare. Selezionare il pulsante &quot;Gate&quot;, e disegnare a mano un cancelloper la popolazione di interesse. </li><li> Flusso Inoltre fornirà cella &quot;conte&quot; all&#39;interno di questa porta e &quot;% di questo complotto&quot;. </li></ol>

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The authors have nothing to disclose.

Glycosomes sono,, organelli specifici del parassita dinamiche essenziali. I processi che regolano la biogenesi, la manutenzione, la proliferazione e rimodellamento di questi organelli probabilmente sono bersagli farmacologici che potrebbero essere sfruttati per scopi terapeutici. Nonostante l&#39;elevato potenziale abbondanza di tali bersagli farmacologici, il campo di glycosome biogenesi è rimasta indietro lo studio dei processi simili in altri organismi, prevalentemente a causa della mancanza di un sistema high-throu...

Trypanosoma brucei causa la malattia africana sonno negli esseri umani e una malattia devastante, nagana, nei bovini. Farmaci utilizzati nel trattamento di queste malattie sono antiquate ed estremamente tossico, i vaccini non sono disponibili, e il potenziale di sviluppo di resistenza ai farmaci richieda l&#39;ricerca di nuovi bersagli farmacologici 1. Durante il suo ciclo di vita, T. brucei, si alterna tra un insetto vettore e ospite mammiferi; due host che presentano molto diversi ambienti in cui il parassita deve sopravvivere. Una serie di cambiamenti metabolici e morfologici si verificano come il parassita è esposto a diverse condizioni ambientali. Alcuni dei cambiamenti più drammatici sono stati osservati in parassiti microcorpi specifici-single-membrana delimitata, chiamato glycosomes 13. I livelli di glucosio sono relativamente alti (~ 5 mm) nel sangue e parassiti del sangue (BSF) generano ATP esclusivamente attraverso wh glicolisimetabolismo mitocondriale ile è represso 14. A differenza di altri eucarioti in cui glicolisi avviene nel citoplasma, T. brucei compartimentalizza maggior parte degli enzimi glicolitici in glycosomes 14,15. I parassiti sono prese dalla mosca tse-tse durante un farine e sperimentare un calo di glucosio, che scende a livelli non rilevabili entro 15 min di essere ingerito dalla mosca. Il metabolismo di insetti, forma prociclico (PCF), parassiti è più flessibile e glucosio, nonché aminoacidi come prolina, possono essere utilizzati nella sintesi di ATP 16-18. Studi di proteomica comparativi rivelano ciclo di vita variazioni dipendenti in glycosomal e proteine ​​mitocondriali con le proteine ​​glicolitici aumentato in parassiti del sangue e proteine ​​mitocondriali coinvolte nel ciclo TCA e catena respiratoria 13,19. Mentre molti studi si sono concentrati sulle differenze tra BSF e glycosomes PCF, poco si sa circa i cambiamenti in glycosomes PCF che si verificano in risposta a envmodifiche ironmental. Nel hindgut della mosca, i livelli di glucosio sono bassi con aumenti transitori nel corso di una alimentazione 20. In più studi in vitro, i parassiti PCF vengono coltivate in terreni contenenti glucosio. Tuttavia, studi recenti hanno dimostrato che i cambiamenti del metabolismo PCF significativamente in risposta al glucosio disponibilità 17. In assenza di glucosio, prolina captazione e prolina aumento deidrogenasi 18. Questo cambiamento nel metabolismo mitocondriale è probabilmente accompagnato da un cambiamento nella composizione glycosome e morfologia, tuttavia, questo non è stato valutato direttamente. Elettroni e microscopia a fluorescenza sono comuni tecniche utilizzate per studiare le dinamiche glycosome in T. brucei 2,21-24. Questi protocolli sono tempo e lavoro, costoso e difficile adattarsi agli studi in tempo reale e protocolli ad alto rendimento. Per superare questa limitazione, un sistema reporter fluorescente-organello used per studiare organelli nei sistemi di mammiferi e di lievito è stata modificata per l&#39;uso in T. brucei 12. Sistemi reporter fluorescente-organelli sono stati ampiamente utilizzati in eucarioti superiori come il lievito, piante e cellule di mammifero 25-27. In tali sistemi, una proteina fluorescente è fuso ad una sequenza di amminoacidi che ha come bersaglio la proteina di organelli specifici. La degradazione o la sintesi delle proteine ​​target viene misurata tramite fluorescenza e cambiamenti nella composizione organelli sono riflesse dai cambiamenti nella fluorescenza delle cellule. Quando la cornice di lettura aperta della proteina fluorescente gialla maggiore (EYFP) è fusa ad un perossisomica sequenza di targeting di tipo II (PTS2) 12, la proteina PTS2eYFP viene importato in, glycosomes e fluorescenza di importazione-competente maturi può essere monitorato tramite citometria a flusso. Variazioni nella composizione glycosome sono riflesse dai cambiamenti nella fluorescenza cellulare. Questo sistema può aiutare a risolVing i meccanismi che regolano i cambiamenti provocati dall&#39;ambiente in glycosome composizione. Questo manoscritto descrive la generazione di un sistema glycosome giornalista in parassiti PCF in collaborazione con citometria a flusso per monitorare le dinamiche glycosome in tempo reale in parassiti vivi e fornisce un esempio di come è stato utilizzato per seguire cambiamenti nella composizione glycosome in risposta a diversi ambienti. In sintesi, glycosome composizione è influenzata dalla concentrazione di glucosio extracellulare e passaggio di culture log-fase in mezzi freschi innesca cambiamenti nella glycosome composizione. Questo sistema può essere modificato per studiare il comportamento dinamico di altri organelli in tripanosomi e altri parassiti.

<table><tbody><tr><td>Adenosine</td><td>Avocado Research Chemicals Ltd</td><td>A10781</td><td>SDM79 Ingredient</td></tr><tr><td>L-Alanine</td><td>Avocado Research Chemicals Ltd</td><td>A15804</td><td>SDM79 Ingredient</td></tr><tr><td>L-Arginine</td><td>CalBiochem</td><td>1820</td><td>SDM79 Ingredient</td></tr><tr><td>p-Aminobenzoic acid</td><td>ICN Biomedicals</td><td>102569</td><td>SDM79 Ingredient</td></tr><tr><td>Basal Medium Eagle Vitamin Solution (100x)</td><td>Sigma</td><td>B6891</td><td>SDM79 Ingredient</td></tr><tr><td>Biotin</td><td>Fisher</td><td>BP232-1</td><td>SDM79 Ingredient</td></tr><tr><td>Calcium chloride</td><td>VWR</td><td>BDH0224</td><td>Cytomix</td></tr><tr><td>EDTA</td><td>Fisher</td><td>S311-100</td><td>Cytomix ingredient</td></tr><tr><td>EZNA Gel Extraction kit</td><td>Omega Biotek</td><td>D2500-01</td><td>DNA purifiation</td></tr><tr><td>Research grade Serum</td><td>Fisher</td><td>03-600-511</td><td>SDM79 Ingredient</td></tr><tr><td>Folic acid</td><td>ICN Biomedicals</td><td>101725</td><td>SDM79 Ingredient</td></tr><tr><td>Glucosamine HCl</td><td>ICN Biomedicals</td><td>194671</td><td>SDM79 Ingredient</td></tr><tr><td>Glucose</td><td>GIBCO</td><td>15023-021</td><td>SDM79 Ingredient</td></tr><tr><td>L-Glutamine</td><td>CalBiochem</td><td>3520</td><td>SDM79 Ingredient</td></tr><tr><td>Glycerol</td><td>Acros Organics</td><td>Ac15892-0010</td><td>Freezing media</td></tr><tr><td>Graces insect cell media powder</td><td>GIBCO</td><td>11300-043</td><td>SDM79 Ingredient</td></tr><tr><td>Hemin</td><td>MP Biomedicals</td><td>194025</td><td>SDM79 Ingredient</td></tr><tr><td>Guanosine</td><td>Avocado Research Chemicals Ltd</td><td>A11328</td><td>SDM79 Ingredient</td></tr><tr><td>HEPES</td><td>MP Biomedicals</td><td>194025</td><td>SDM79 Ingredient</td></tr><tr><td>Magnesium chloride</td><td>Fisher</td><td>BP214-500</td><td>Cytomix ingredient</td></tr><tr><td>L-Methionine</td><td>Fisher</td><td>BP388-100</td><td>SDM79 Ingredient</td></tr><tr><td>MEM Amino Acids (50x)</td><td>Cellgro</td><td>25-030-CI</td><td>SDM79 Ingredient</td></tr><tr><td>NEAA Mixture (100x)</td><td>Lonza</td><td>13-114E</td><td>SDM79 Ingredient</td></tr><tr><td>Minimal Essential Medium (1x) with L-glutamine</td><td>Cellgro</td><td>10-010-CM</td><td>SDM79 Ingredient</td></tr><tr><td>MOPS</td><td>Fisher</td><td>BP308-500</td><td>SDM79 Ingredient</td></tr><tr><td>Sodium biocarbonate</td><td>Fisher</td><td>S233-500</td><td>SDM79 Ingredient</td></tr><tr><td>Penicillin-streptomycin Solution</td><td>Cellgro</td><td>30-002-CI</td><td>SDM79 Ingredient</td></tr><tr><td>L-Phenylalanine</td><td>ICN Biomedicals</td><td>102623</td><td>SDM79 Ingredient</td></tr><tr><td>Potassium chloride</td><td>Fisher</td><td>P217-500</td><td>Cytomix ingredient</td></tr><tr><td>Potassium phosphate dibasic anhydrous</td><td>Fisheer</td><td>P290-212</td><td>Cytomix ingredient</td></tr><tr><td>L-Proline</td><td>Fisher</td><td>BP392-100</td><td>SDM79 Ingredient</td></tr><tr><td>L-Serine</td><td>Acros Organics</td><td>56-45-1</td><td>SDM79 Ingredient</td></tr><tr><td>Pyruvic acid, sodium salt</td><td>Acros Organics</td><td>113-24-6</td><td>SDM79 Ingredient</td></tr><tr><td>L-Taurine</td><td>TCI America</td><td>A0295</td><td>SDM79 Ingredient</td></tr><tr><td>L-Threonine</td><td>Acros Organics</td><td>72-19-5</td><td>SDM79 Ingredient</td></tr><tr><td>L-Tyrosine</td><td>ICN Biomedicals</td><td>103183</td><td>SDM79 Ingredient</td></tr><tr><td>E.Z.N.A. Cycle Pure kit</td><td>Omega Biotek</td><td>D6492-02</td><td>DNA purification</td></tr><tr><td>Binding buffer</td><td>Omega Biotek</td><td>PDR041</td><td>DNA purification</td></tr><tr><td>SPW wash buffer</td><td>Omega Biotek</td><td>PDR045</td><td>DNA purification</td></tr><tr><td>Gene Pulser Xcell</td><td>Biorad</td><td>165-2660</td><td>Trypanosome transformation</td></tr><tr><td>4 mm Electroporation cuvettes</td><td>VWR</td><td></td><td>Trypanosome transformation</td></tr></tbody></table>

using fluorescent proteins to monitor glycosome dynamics in the african trypanosome

Trypanosoma brucei è un parassita che causa kinetoplastidi tripanosomiasi umana africana (HAT), o malattia del sonno, e una malattia devastante, nagana, nei bovini 1. I supplenti parassita tra il sangue del mammifero ospite e la mosca tse-tse vettore. La composizione di molti organelli cellulari cambia in risposta a queste differenti condizioni extracellulari 2-5. Glycosomes perossisomi sono altamente specializzati in cui molti degli enzimi coinvolti nella glicolisi sono compartimenti stagni. Composizione cambia Glycosome in maniera evolutiva e regolamentato l&#39;ambiente 4-11. Attualmente, le tecniche più comuni utilizzate per studiare glycosome dinamiche sono elettroni e microscopia a fluorescenza; tecniche che sono costosi, tempo e lavoro, e non facilmente adattato alle analisi di throughput elevato. Per superare queste limitazioni, un sistema reporter fluorescente-glycosome inche migliorato proteina fluorescente gialla (EYFP) si fonde a una sequenza di targeting dei perossisomi (PTS2), che dirige la proteina di fusione per glycosomes 12, è stato istituito. Con l&#39;importazione della proteina di fusione PTS2eYFP, glycosomes diventano fluorescenti. Degrado organello e risultati riciclaggio nella perdita di fluorescenza che può essere misurato mediante citometria a flusso. Un gran numero di cellule (5.000 cellule / sec) possono essere analizzati in tempo reale senza una preparazione del campione come la fissazione e il montaggio. Questo metodo offre un modo rapido di rilevare cambiamenti nella composizione organello in risposta alle mutevoli condizioni ambientali.

In questo sistema, è stato osservato un cambiamento glucosio-dipendente glycosome composizione. Quando le cellule sono coltivate in terreni contenenti glucosio, si osservano due popolazioni; una luminosa e una dim (Figura 2A). Cellule Dim porto glycosomes immaturi, che non hanno importato la PTS2eYFP mentre le cellule luminose ospitano un mix di maturi e immaturi glycosomes 12. Quando il glucosio è presente nei media, mislocalization di glycosome proteine ​​è letale 15,28 e ...

Watch this Scientific Journal Video about Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome at JoVE.com

Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Trypanosoma brucei is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in ...

using-fluorescent-proteins-to-monitor-glycosome-dynamics-african

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

8.9K Views.  Clemson University Eukaryotic Pathogens Innovation Center. Glycosome dynamics in African trypanosomes are difficult to study by traditional cell biology techniques such as electron and fluorescence microscopy. As a means of observing dynamic organelle behavior, a fluorescent-organelle reporter system has been used in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites.

Video: Using Fluorescent Proteins to Monitor Glycosome Dynamics in the African Trypanosome

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3. CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. Thus, when a CFSE-labeled cell divides, its progeny are endowed with half the number of carboxyfluorescein-tagged molecules and thus each cell division can be assessed by measuring the corresponding decrease in cell fluorescence via Flow cytometry. The capacity of CFSE to label lymphocyte populations with a high fluorescent intensity of exceptionally low variance, coupled with its low cell toxicity, make it an ideal dye to measure cell division. Since it is a fluorescein-based dye it is also compatible with a broad range of other fluorochromes making it applicable to multi-color flow cytometry. This article describes the procedures typically used for labeling mouse lymphocytes for the purpose of monitoring up to 8 cell divisions. These labeled cells can be used both for in vitro and in vivo studies.

The Use of Carboxyfluorescein Diacetate Succinimidyl Ester CFSE to Monitor Lymphocyte Proliferation

CFSE covalently labels long-lived intracellular molecules with the fluorescent dye, carboxyfluorescein. As such, when a CFSE-labeled cell divides, its progeny have half the amount of fluorescence, which can thereby be used to assess cell division. This article describes the procedures typically used for labeling mouse lymphocytes with CFSE.

L&#39;uso di diacetato Carboxyfluorescein succinimidile Ester (CFSE) per monitorare la proliferazione dei linfociti

Carboxyfluorescein succinimidyl ester (CFSE) is an effective and popular means to monitor lymphocyte division1-3.  CFSE covalently labels long-lived ...

Ricerca

Immunologia e infezioni

A Visual Assay to Monitor T6SS-mediated Bacterial Competition

Type VI secretion systems (T6SSs) are molecular nanomachines allowing Gram-negative bacteria to transport and inject proteins into a wide variety of target cells1,2. The T6SS is composed of 13 core components and displays structural similarities with the tail-tube of bacteriophages3. The phage uses a tube and a puncturing device to penetrate the cell envelope of target bacteria and inject DNA. It is proposed that the T6SS is an inverted bacteriophage device creating a specific path in the bacterial cell envelope to drive effectors and toxins to the surface. The process could be taken further and the T6SS device could perforate other cells with which the bacterium is in contact, thus injecting the effectors into these targets. The tail tube and puncturing device parts of the T6SS are made with Hcp and VgrG proteins, respectively4,5.
The versatility of the T6SS has been demonstrated through studies using various bacterial pathogens. The Vibrio cholerae T6SS can remodel the cytoskeleton of eukaryotic host cells by injecting an "evolved" VgrG carrying a C-terminal actin cross-linking domain6,7. Another striking example was recently documented using Pseudomonas aeruginosa which is able to target and kill bacteria in a T6SS-dependent manner, therefore promoting the establishment of bacteria in specific microbial niches and competitive environment8,9,10.
In the latter case, three T6SS-secreted proteins, namely Tse1, Tse2 and Tse3 have been identified as the toxins injected in the target bacteria (Figure 1). The donor cell is protected from the deleterious effect of these effectors via an anti-toxin mechanism, mediated by the Tsi1, Tsi2 and Tsi3 immunity proteins8,9,10. This antimicrobial activity can be monitored when T6SS-proficient bacteria are co-cultivated on solid surfaces in competition with other bacterial species or with T6SS-inactive bacteria of the same species8,11,12,13.
The data available emphasized a numerical approach to the bacterial competition assay, including time-consuming CFU counting that depends greatly on antibiotic makers. In the case of antibiotic resistant strains like P. aeruginosa, these methods can be inappropriate. Moreover, with the identification of about 200 different T6SS loci in more than 100 bacterial genomes14, a convenient screening tool is highly desirable. We developed an assay that is easy to use and requires standard laboratory material and reagents. The method offers a rapid and qualitative technique to monitor the T6SS-dependent bactericidal/bacteriostasis activity by using a reporter strain as a prey (in this case Escherichia coli DH5&alpha;) allowing a-complementation of the lacZ gene. Overall, this method is graphic and allows rapid identification of T6SS-related phenotypes on agar plates. This experimental protocol may be adapted to other strains or bacterial species taking into account specific conditions such as growth media, temperature or time of contact.

We describe a qualitative assay to monitor bacterial competition mediated by the Pseudomonas aeruginosa type VI secretion system (T6SS). The assay relies on the survival/killing of Escherichia coli target cells carrying a lacZ-reporter. This technique is adjustable to assess the bactericidal/bacteriostasis activity of T6SS-proficient microorganisms.

Un test per il monitoraggio visivo T6SS-mediata Concorso batterica

Type VI secretion systems (T6SSs) are molecular  nanomachines allowing Gram-negative bacteria to transport and inject proteins  into a wide variety of ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Uso di<em&gt; In vivo</em&gt; Imaging per monitorare la progressione della Sperimentale mouse infezione da citomegalovirus nei neonati

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades the central nervous system (CNS). In vivo study of the late stage has been limited as traditional methodologies require the removal of the brain to determine the presence of the parasites.
Bioluminescence imaging is a non-invasive, highly sensitive form of optical imaging that enables the visualization of a luciferase-transfected pathogen in real-time. By using a transfected trypanosome strain that has the ability to produce late stage disease in mice we are able to study the kinetics of a CNS infection in a single animal throughout the course of infection, as well as observe the movement and dissemination of a systemic infection.
Here we describe a robust protocol to study CNS infections using a bioluminescence model of African trypanosomiasis, providing real time non-invasive observations which can be further analyzed with optional downstream approaches.

This manuscript describes the use of a bioluminescent strain of African trypanosomes to enable the tracking of late stage infection and demonstrates how in vivo live imaging can be used to visualize infections within the central nervous system in real-time.

Bioluminescenza Imaging per rilevare fase avanzata di infezione di africani Tripanosomiasi

Human African trypanosomiasis (HAT) is a multi-stage disease that manifests in two stages; an early blood stage and a late stage when the parasite invades ...

High-throughput Gene Tagging in Trypanosoma brucei

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins can give insights into their function by determining their localization within the cell and enabling interaction partner identification. We recently published a fast and scalable method to generate Trypanosoma brucei cell lines that express a tagged protein from the endogenous locus. The method was based on a plasmid we generated that, when coupled with long primer PCR, can be used to modify a gene to encode a protein tagged at either terminus. This allows the tagging of dozens of trypanosome proteins in parallel, facilitating the large-scale validation of candidate genes of interest. This system can be used to tag proteins for localization (using a fluorescent protein, epitope tag or electron microscopy tag) or biochemistry (using tags for purification, such as the TAP (tandem affinity purification) tag). Here, we describe a protocol to perform the long primer PCR and the electroporation in 96-well plates, with the recovery and selection of transgenic trypanosomes occurring in 24-well plates. With this workflow, hundreds of proteins can be tagged in parallel; this is an order of magnitude improvement to our previous protocol and genome scale tagging is now possible.

High-throughput Gene Tagging in Trypanosoma brucei

Addition of a tag to a protein is a powerful way of gaining insight into its function. Here, we describe a protocol to endogenously tag hundreds of Trypanosoma brucei proteins in parallel such that genome scale tagging is achievable.

High-throughput di Gene Tagging in<em&gt; Trypanosoma brucei</em

Improvements in mass spectrometry, sequencing and bioinformatics have generated large datasets of potentially interesting genes. Tagging these proteins ...

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine ​​Reporter

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Biologia

Visualization of Endoplasmic Reticulum Subdomains in Cultured Cells

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and functions despite the intense interorganelle molecular traffic. The techniques described in this paper are powerful means of studying protein and lipid mobility and trafficking in vivo and in their physiological environment. Fluorescence recovery after photobleaching (FRAP) and fluorescence loss in photobleaching (FLIP) are widely used live-cell imaging techniques for studying intracellular trafficking through the exo-endocytic pathway, the continuity between organelles or subcompartments, the formation of protein complexes, and protein localization in lipid microdomains, all of which can be observed under physiological and pathological conditions. The limitations of these approaches are mainly due to the use of fluorescent fusion proteins, and their potential drawbacks include artifactual over-expression in cells and the possibility of differences in the folding and localization of tagged and native proteins. Finally, as the limit of resolution of optical microscopy (about 200 nm) does not allow investigation of the fine structure of the ER or the specific subcompartments that can originate in cells under stress (i.e. hypoxia, drug administration, the over-expression of transmembrane ER resident proteins) or under pathological conditions, we combine live-cell imaging of cultured transfected cells with ultrastructural analyses based on transmission electron microscopy.

We describe the imaging approaches we use to investigate the distribution and mobility of the transfected fluorescent proteins resident in the endoplasmic reticulum (ER) by means of the confocal imaging of living cells. We also ultrastructurally analyze the effect of their expression on the architecture of this subcellular compartment.

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

The lipids and proteins in eukaryotic cells are continuously exchanged between cell compartments, although these retain their distinctive composition and ...

Sviluppo di un sensore basato su pHluorin2 per il pH glicosomiale in Trypanosoma brucei

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. Little is known about the cellular responses generated when environmental glucose levels change. In both bloodstream and procyclic form (insect stage) parasites, glycosomes house most of glycolysis. These organelles are rapidly acidified in response to glucose deprivation, which likely results in the allosteric regulation of glycolytic enzymes such as hexokinase. In previous work, localizing the chemical probe used to make pH measurements was challenging, limiting its utility in other applications.
This paper describes the development and use of parasites that express glycosomally localized pHluorin2, a heritable protein pH biosensor. pHluorin2 is a ratiometric pHluorin variant that displays a pH (acid)-dependent decrease in excitation at 395 nm while simultaneously yielding an increase in excitation at 475 nm. Transgenic parasites were generated by cloning the pHluorin2 open reading frame into the trypanosome expression vector pLEW100v5, enabling inducible protein expression in either lifecycle stage. Immunofluorescence was used to confirm the glycosomal localization of the pHluorin2 biosensor, comparing the localization of the biosensor to the glycosomal resident protein aldolase. The sensor responsiveness was calibrated at differing pH levels by incubating cells in a series of buffers that ranged in pH from 4 to 8, an approach we have previously used to calibrate a fluorescein-based pH sensor. We then measured pHluorin2 fluorescence at 405 nm and 488 nm using flow cytometry to determine glycosomal pH. We validated the performance of the live transgenic pHluorin2-expressing parasites, monitoring pH over time in response to glucose deprivation, a known trigger of glycosomal acidification in PF parasites. This tool has a range of potential applications, including potentially being used in high-throughput drug screening. Beyond glycosomal pH, the sensor could be adapted to other organelles or used in other trypanosomatids to understand pH dynamics&#160;in the live cell setting.

Autore in primo piano: Progressi nel monitoraggio del pH glicosomiale in Trypanosoma brucei utilizzando il biosensore pHluorin2

We describe a method to study how pH responds to environmental cues in the glycosomes of the bloodstream form of African trypanosomes. This approach involves a pH-sensitive heritable protein sensor in combination with flow cytometry to measure pH dynamics, both as a time-course assay and in a high-throughput screen format.

Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

Glucose metabolism is critical for the African trypanosome, Trypanosoma brucei, as an essential metabolic process and regulator of parasite development. ...

Utilizzo di proteine fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

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Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

Utilizzo di proteine ​​fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Riepilogo

Esplora Altri Video

Capitoli in questo video

L'uso di diacetato Carboxyfluorescein succinimidile Ester (CFSE) per monitorare la proliferazione dei linfociti

Un test per il monitoraggio visivo T6SS-mediata Concorso batterica

Uso di

Bioluminescenza Imaging per rilevare fase avanzata di infezione di africani Tripanosomiasi

High-throughput di Gene Tagging in

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine ​​Reporter

Visualizzazione del reticolo endoplasmatico sottodomini in cellule in coltura

Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

Misurazione delle variazioni dinamiche del pH glisomiale nel Trypanosoma brucei vivente

Utilizzo di proteine fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano

Monitoraggio del sistema ubiquitina-proteasoma attività nelle cellule viventi Utilizzando una degron (dgn)-destabilizzato proteina verde fluorescente (GFP) a base di proteine Reporter