Name: using fluorescent proteins to monitor glycosome dynamics in the african trypanosome
Uploaded: 2014-08-19T13:00:00
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. Generelt trypanosominfektion Dyrehold <ol><li> Faststoffer til SDM79 medier forberedelse (tabel 1) afvejes. </li><li> Opbevares ved 4 ° C i 50 ml konisk eller en Ziploc pose. BEMÆRK: Reagenserne er stabile i mindst 6 måneder. </li><li> Optø føtalt bovint serum (FBS) i en 37 ° C vandbad og blandes periodisk ved inversion. BEMÆRK: FBS modtages fra leverandøren som en steril opløsning. Filtersterilisere FBS reducerer dens evne til at understøtte parasit vækst. </li><li> Når hele flasken er...

<ol>
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Glycosomes er vigtige, dynamiske, parasit-specifikke organeller. De processer, der regulerer Biogenese, vedligeholdelse, spredning og ombygning af disse organeller sandsynligvis omfatte lægemiddelvirkninger, der kunne udnyttes til terapeutiske formål. På trods af den potentielt høj overflod af disse lægemiddelkandidater, har inden for glycosome biogenese haltet bag studiet af tilsvarende processer i andre organismer, overvejende på grund af manglen på et medgørligt, high-throughput-system ved at overvåge hurtig...

Trypanosoma brucei forårsager afrikansk sovesyge hos mennesker, og en wasting disease, nagana i kvæg. Lægemidler, der anvendes i behandlingen af disse sygdomme, er forældede og ekstremt giftigt, vacciner ikke er tilgængelige, og potentialet for udvikling af resistens nødvendiggør søgen efter nye lægemiddelkandidater 1. I løbet af sin livscyklus, T. brucei, veksler mellem et insekt vektor og pattedyrværten; to værter, der præsenterer meget forskellige miljøer, hvor parasitten skal overleve. En række metaboliske og morfologiske ændringer forekomme som parasitten er udsat for forskellige miljøforhold. Nogle af de mest dramatiske ændringer observeres i single-membran-afgrænset parasit specifikke microbodies, kaldet glycosomes 13. Glucose niveauer er relativt høj (~ 5 mm) i blodbanen og blodbanen parasitter (BSF) generere ATP udelukkende via glycolyse while mitokondrie stofskifte er undertrykt 14. I modsætning til andre eukaryoter, hvor glycolyse forekommer i cytoplasmaet, T. brucei compartmentalizes fleste af de glycolytiske enzymer i glycosomes 14,15. Parasitterne er taget op af tsetsefluen under en blodmel og opleve et fald i glukose, som falder til påviselige niveauer inden for 15 minutter for at blive indtaget af fluen. Metabolismen af insekt, procyklisk formular (PCF), parasitter er mere fleksibel og glucose, samt aminosyrer, såsom prolin, kan anvendes i syntesen af ATP 16-18. Sammenlignende proteom undersøgelser viser livscyklus afhængige ændringer i glycosomal og mitokondrielle proteiner med glycolytiske proteiner steg i blodbanen parasitter og mitokondrielle proteiner involveret i TCA cyklus og respiratoriske kæde 13,19. Mens mange undersøgelser har fokuseret på forskellene mellem BSF og PCF glycosomes vides der kun lidt om ændringerne i PCF glycosomes, der opstår som reaktion på ENVjøområdet ændringer. I stortarmen af fluen, blodsukkerniveauet er lavt med forbigående stigninger i løbet af en fodring 20. I de fleste in vitro-undersøgelser, der PCF parasitter dyrkes i medier indeholdende glucose. Imidlertid har nyere undersøgelser vist, at PCF metabolisme ændringer betydeligt som reaktion på glucose tilgængelighed 17. I fravær af glucose, prolin optagelse og prolin dehydrogenase-aktivitet stigning på 18. Denne ændring i mitochondriel stofskifte sandsynligvis ledsaget af en ændring i glycosome sammensætning og morfologi, men dette er ikke blevet foretaget en direkte vurdering. Elektron og fluorescens mikroskopi er almindelige teknikker, der anvendes til at studere glycosome dynamik i T. brucei 2,21-24. Disse protokoller er tid og arbejdskrævende, dyrt og vanskeligt at tilpasse sig til real-time undersøgelser og high throughput protokoller. For at overvinde denne begrænsning, en fluorescerende organel reportersystem used at studere organeller i mammale og gær-systemer er blevet modificeret til brug i T. brucei 12. Fluorescerende-organel reportersystemer har været udbredt i højere eukaryoter, såsom gær, plante og mammale celler 25-27. I sådanne systemer er et fluorescerende protein, fusioneret til en aminosyresekvens, som målretter proteinet til specifikke organeller. Nedbrydningen eller syntese af de målrettede proteiner måles via fluorescens og ændringer i organel sammensætning afspejles af ændringer i celle fluorescens. Når den åbne læseramme af et forstærket gult fluorescerende protein (EYFP) er fusioneret til en type II peroxisomal målretning sekvens (PTS2) 12 er PTS2eYFP proteinet importeres til modne, import-kompetent glycosomes og fluorescens kan overvåges via flowcytometri. Variationer i glycosome sammensætning afspejles af ændringer i cellulær fluorescens. Dette system kan hjælpe med resolVing de mekanismer, der regulerer miljømæssigt inducerede ændringer i glycosome sammensætning. Dette håndskrift beskriver frembringelsen af ​​et glycosome reportersystem i PCF parasitter i forbindelse med flowcytometri til at overvåge realtid glycosome dynamik i levende parasitter, og er et eksempel på, hvordan den er blevet anvendt til at følge ændringer i glycosome præparat som respons på forskellige miljøer. Sammenfattende er glycosome sammensætning påvirkes af ekstracellulære glukosekoncentrationer og passage af log-fase kulturer i frisk medier udløser ændringer i glycosome sammensætning. Dette system kan modificeres til at studere dynamiske adfærd andre organeller i trypanosomer og andre parasitter.

<table border="0" cellpadding="0" cellspacing="0" width="1257">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:396px;">Adenosine</td>
			<td style="width:425px;">Avocado Research Chemicals Ltd</td>
			<td style="width:180px;">A10781</td>
			<td style="width:256px;">SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-Alanine</td>
			<td>Avocado Research Chemicals Ltd</td>
			<td>A15804</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-arginine</td>
			<td>CalBiochem</td>
			<td>1820</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">p-aminobenzoic acid</td>
			<td>ICN Biomedicals</td>
			<td>102569</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Basal Medium Eagle Vitamin Solution (100X)</td>
			<td>Sigma</td>
			<td>B6891</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotin</td>
			<td>Fisher</td>
			<td>BP232-1</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium Chloride</td>
			<td>VWR</td>
			<td>BDH0224</td>
			<td>Cytomix</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td>Fisher</td>
			<td>S311-100</td>
			<td>Cytomix ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EZNA Gel Extraction kit</td>
			<td>Omega Biotek</td>
			<td>D2500-01</td>
			<td>DNA purifiation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Research grade Serum</td>
			<td>Fisher</td>
			<td>03-600-511</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Folic acid</td>
			<td>ICN Biomedicals</td>
			<td>101725</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucosamine HCl</td>
			<td>ICN Biomedicals</td>
			<td>194671</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>GIBCO</td>
			<td>15023-021</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-glutamine</td>
			<td>CalBiochem</td>
			<td>3520</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>Acros Organics</td>
			<td>Ac15892-0010</td>
			<td>Freezing media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Graces insect cell media powder</td>
			<td>GIBCO</td>
			<td>11300-043</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemin</td>
			<td>MP Biomedicals</td>
			<td>194025</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Guanosine</td>
			<td>Avocado Research Chemicals Ltd</td>
			<td>A11328</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HEPES</td>
			<td>MP Biomedicals</td>
			<td>194025</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium Chloride</td>
			<td>Fisher</td>
			<td>BP214-500</td>
			<td>Cytomix ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-methionine</td>
			<td>Fisher</td>
			<td>BP388-100</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MEM Amino Acids (50X)</td>
			<td>Cellgro</td>
			<td>25-030-CI</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NEAA Mixture (100X)</td>
			<td>Lonza</td>
			<td>13-114E</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimal Essential Medium (1X) with L-glutamine</td>
			<td>Cellgro</td>
			<td>10-010-CM</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MOPS</td>
			<td>Fisher</td>
			<td>BP308-500</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Biocarbonate</td>
			<td>Fisher</td>
			<td>S233-500</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin Solution</td>
			<td>Cellgro</td>
			<td>30-002-CI</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-phenylalanine</td>
			<td>ICN Biomedicals</td>
			<td>102623</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Fisher</td>
			<td>P217-500</td>
			<td>Cytomix ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Phosphate Dibasic Anhydrous</td>
			<td>Fisheer</td>
			<td>P290-212</td>
			<td>Cytomix ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-proline</td>
			<td>Fisher</td>
			<td>BP392-100</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-serine</td>
			<td>Acros Organics</td>
			<td>56-45-1</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pyruvic acid, sodium salt</td>
			<td>Acros Organics</td>
			<td>113-24-6</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-taurine</td>
			<td>TCI America</td>
			<td>A0295</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-threonine</td>
			<td>Acros Organics</td>
			<td>72-19-5</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-tyrosine</td>
			<td>ICN Biomedicals</td>
			<td>103183</td>
			<td>SDM79 Ingredient</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">E.Z.N.A.Cycle Pure kit</td>
			<td>Omega Biotek</td>
			<td>D6492-02</td>
			<td>DNA purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Binding buffer</td>
			<td>Omega Biotek</td>
			<td>PDR041</td>
			<td>DNA purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPW wash buffer&#160;</td>
			<td>Omega Biotek</td>
			<td>PDR045</td>
			<td>DNA purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gene Pulser Xcell&#160;</td>
			<td>Biorad</td>
			<td>165-2660</td>
			<td>Trypanosome transformation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 er en kinetoplastid parasit, der forårsager menneskelig afrikansk trypanosomiasis (HAT), eller sovesyge, og en wasting disease, nagana i kvæg 1. Parasitten veksler mellem blodbane pattedyrsværten og tsetsefluen vektor. Sammensætningen af mange cellulære organeller ændringer som reaktion på disse forskellige ekstracellulære forhold 2-5. Glycosomes er højt specialiserede peroxisomer, hvor mange af de enzymer, der er involveret i glykolysen er opdelte. Glycosome sammensætning ændringer i en udviklingsmæssig og miljømæssigt reguleret måde 4-11. I øjeblikket er de mest almindelige teknikker, der anvendes til at studere glycosome dynamik er elektron og fluorescens mikroskopi; teknikker, der er dyre, tid og arbejdskrævende, og ikke let tilpasses til højt gennemløb analyser. For at overvinde disse begrænsninger, en fluorescerende-glycosome reporter systemsom forbedret gul fluorescerende protein (EYFP) er fusioneret til en peroxisome targetingsekvens (PTS2), som dirigerer fusionsproteinet glycosomes 12, er blevet etableret. Ved import af det PTS2eYFP fusionsproteinet glycosomes bliver fluorescerende. Organel nedbrydning og genanvendelse medfører tab af fluorescens, der kan måles ved flowcytometri. Store antal celler (5000 celler / sek) kan analyseres i realtid uden omfattende prøveforberedelse såsom fiksering og montering. Denne metode giver en hurtig måde at detektere ændringer i organel sammensætning som reaktion på svingende miljøforhold.

I dette system blev en glucose-afhængige ændringer i glycosome sammensætning overholdes. Når celler dyrkes i glucoseholdigt medie, bliver to populationer overholdes; en lyse og én dim (figur 2A). Dim celler havnen umodne glycosomes, der ikke har indført den PTS2eYFP mens lyse celler huser en blanding af modne og umodne glycosomes 12. Når glukose er til stede i medierne, mislocalization af glycosome proteiner er dødelig 15,28 og glycosome proteinekspression er sandsynligvis ...

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.

Brug fluorescerende proteiner til Monitor Glycosome dynamik i det afrikanske trypanosominfektion

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

Research

JoVE Journal

Immunology and Infection

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

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Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

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Brug fluorescerende proteiner til at visualisere og kvantificere<em&gt; Chlamydia</em&gt; Vakuole Vækst Dynamics i levende celler

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of ...

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Bioluminescence Imaging to Detect Late Stage Infection of African Trypanosomiasis

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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 ...

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy (TIRFM)

"Phagosome Closure Assay" to Visualize Phagosome Formation in Three Dimensions Using Total Internal Reflection Fluorescent Microscopy TIRFM

&quot;Fagosom Lukning Assay&quot; til Visualiser fagosom Formation i tre dimensioner Brug total intern refleksion Fluorescent Microscopy (TIRFM)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements ...

Development of a Hepatitis B Virus Reporter System to Monitor the Early Stages of the Replication Cycle

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Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), ...

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

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