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. 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...

<ol>
	<li>Stuart, K., 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=Kinetoplastids:+related+protozoan+pathogens,+different+diseases.">Kinetoplastids: related protozoan pathogens, different diseases.</a> J Clin Invest. 118, 1301-1310 (2008).</li><li>Herman, M., Perez-Morga, D., Schtickzelle, N., Michels, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Turnover+of+glycosomes+during+life-cycle+differentiation+of+Trypanosoma+brucei.">Turnover of glycosomes during life-cycle differentiation of Trypanosoma brucei.</a> Autophagy. 4, 294-308 (2008).</li><li>Herman, M., Gillies, S., Michels, P. A., Rigden, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autophagy+and+related+processes+in+trypanosomatids:+insights+from+genomic+and+bioinformatic+analyses.">Autophagy and related processes in trypanosomatids: insights from genomic and bioinformatic analyses.</a> Autophagy. 2, 107-118 (2006).</li><li>Michels, P. A., Bringaud, F., Herman, M., Hannaert, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+functions+of+glycosomes+in+trypanosomatids.">Metabolic functions of glycosomes in trypanosomatids.</a> Biochim Biophys Acta. 1763, 1463-1477 (2006).</li><li>Michels, P. A., 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=Peroxisomes,+glyoxysomes+and+glycosomes+(review).">Peroxisomes, glyoxysomes and glycosomes (review).</a> Mol Membr Biol. 22, 133-145 (2005).</li><li>Moyersoen, J., Choe, J., Fan, E., Hol, W. G., Michels, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+peroxisomes+and+glycosomes:+trypanosomatid+glycosome+assembly+is+a+promising+new+drug+target.">Biogenesis of peroxisomes and glycosomes: trypanosomatid glycosome assembly is a promising new drug target.</a> FEMS Microbiol Rev. 28, 603-643 (2004).</li><li>Parsons, M., Furuya, T., Pal, S., Kessler, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+and+function+of+peroxisomes+and+glycosomes.">Biogenesis and function of peroxisomes and glycosomes.</a> Molecular and biochemical parasitology. 115, 19-28 (2001).</li><li>Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosomes:+parasites+and+the+divergence+of+peroxisomal+purpose.">Glycosomes: parasites and the divergence of peroxisomal purpose.</a> Mol Microbiol. 53, 717-724 (2004).</li><li>Michels, P. A., Hannaert, V., Bringaud, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabolic+aspects+of+glycosomes+in+trypanosomatidae+-+new+data+and+views.">Metabolic aspects of glycosomes in trypanosomatidae - new data and views.</a> Parasitol Today. 16, 482-489 (2000).</li><li>Michels, P. A., Hannaert, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+evolution+of+kinetoplastid+glycosomes.">The evolution of kinetoplastid glycosomes.</a> J Bioenerg Biomembr. 26, 213-219 (1994).</li><li>Hannaert, V., Michels, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+function,+and+biogenesis+of+glycosomes+in+kinetoplastida.">Structure function, and biogenesis of glycosomes in kinetoplastida.</a> J Bioenerg Biomembr. 26, 205-212 (1994).</li><li>Bauer, S., Morris, J. C., Morris, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmentally+regulated+glycosome+protein+composition+in+the+african+trypanosome.">Environmentally regulated glycosome protein composition in the african trypanosome.</a> Eukaryot Cell. 12, 1072-1079 (2013).</li><li>Colasante, C., Ellis, M., Ruppert, T., Voncken, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+proteomics+of+glycosomes+from+bloodstream+form+and+procyclic+culture+form+Trypanosoma+brucei+brucei.">Comparative proteomics of glycosomes from bloodstream form and procyclic culture form Trypanosoma brucei brucei.</a> Proteomics. 6, 3275-3293 (2006).</li><li>Opperdoes, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Compartmentation+of+carbohydrate+metabolism+in+trypanosomes.">Compartmentation of carbohydrate metabolism in trypanosomes.</a> Annu Rev Microbiol. 41, 127-151 (1987).</li><li>Kessler, P. S., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+the+role+of+compartmentation+of+glycolysis+in+procyclic+form+Trypanosoma+brucei:+RNA+interference+studies+of+PEX14,+hexokinase,+and+phosphofructokinase.">Probing the role of compartmentation of glycolysis in procyclic form Trypanosoma brucei: RNA interference studies of PEX14, hexokinase, and phosphofructokinase.</a> The Journal of biological chemistry. 280, 9030-9036 (2005).</li><li>Bringaud, F., Riviere, L., Coustou, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Energy+metabolism+of+trypanosomatids:+adaptation+to+available+carbon+sources.">Energy metabolism of trypanosomatids: adaptation to available carbon sources.</a> Molecular and biochemical parasitology. 149, 1-9 (2006).</li><li>Coustou, V., 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=Glucose-induced+remodeling+of+intermediary+and+energy+metabolism+in+procyclic+Trypanosoma+brucei.">Glucose-induced remodeling of intermediary and energy metabolism in procyclic Trypanosoma brucei.</a> The Journal of biological chemistry. 283, 16342-16354 (2008).</li><li>Lamour, N., 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=Proline+metabolism+in+procyclic+Trypanosoma+brucei+is+down-regulated+in+the+presence+of+glucose.">Proline metabolism in procyclic Trypanosoma brucei is down-regulated in the presence of glucose.</a> The Journal of biological chemistry. 280, 11902-11910 (2005).</li><li>Vertommen, D., 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=Differential+expression+of+glycosomal+and+mitochondrial+proteins+in+the+two+major+life-cycle+stages+of+Trypanosoma+brucei.">Differential expression of glycosomal and mitochondrial proteins in the two major life-cycle stages of Trypanosoma brucei.</a> Molecular and biochemical parasitology. 158, 189-201 (2008).</li><li>Vickerman, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+cycles+and+biology+of+pathogenic+trypanosomes.">Developmental cycles and biology of pathogenic trypanosomes.</a> British medical bulletin. 41, 105-114 (1985).</li><li>Lorenz, P., Maier, A. G., Baumgart, E., Erdmann, R., Clayton, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongation+and+clustering+of+glycosomes+in+Trypanosoma+brucei+overexpressing+the+glycosomal+Pex11p.">Elongation and clustering of glycosomes in Trypanosoma brucei overexpressing the glycosomal Pex11p.</a> The EMBO journal. 17, 3542-3555 (1998).</li><li>Saveria, 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=Conservation+of+PEX19-binding+motifs+required+for+protein+targeting+to+mammalian+peroxisomal+and+trypanosome+glycosomal+membranes.">Conservation of PEX19-binding motifs required for protein targeting to mammalian peroxisomal and trypanosome glycosomal membranes.</a> Eukaryot Cell. 6, 1439-1449 (2007).</li><li>Banerjee, S. K., Kessler, P. S., Saveria, T., Parsons, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+trypanosomatid+PEX19:+functional+characterization+reveals+impact+on+cell+growth+and+glycosome+size+and+number.">Identification of trypanosomatid PEX19: functional characterization reveals impact on cell growth and glycosome size and number.</a> Molecular and biochemical parasitology. 142, 47-55 (2005).</li><li>Milagros Camara Mde, L., Bouvier, L. A., Miranda, M. R., Pereira, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+validation+of+Trypanosoma+cruzi's+glycosomal+adenylate+kinase+containing+a+peroxisomal+targeting+signal.">Identification and validation of Trypanosoma cruzi's glycosomal adenylate kinase containing a peroxisomal targeting signal.</a> Experimental parasitology. 130, 408-411 (2012).</li><li>Wiemer, E. A., Wenzel, T., Deerinck, T. J., Ellisman, M. H., Subramani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+the+peroxisomal+compartment+in+living+mammalian+cells:+dynamic+behavior+and+association+with+microtubules.">Visualization of the peroxisomal compartment in living mammalian cells: dynamic behavior and association with microtubules.</a> The Journal of cell biology. 136, 71-80 (1997).</li><li>Kim, P. K., Mullen, R. T., Schumann, U., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+maintenance+of+mammalian+peroxisomes+involves+a+de+novo+PEX16-dependent+pathway+from+the+ER.">The origin and maintenance of mammalian peroxisomes involves a de novo PEX16-dependent pathway from the ER.</a> The Journal of cell biology. 173, 521-532 (2006).</li><li>Hoepfner, D., Schildknegt, D., Braakman, I., Philippsen, P., Tabak, H. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+endoplasmic+reticulum+to+peroxisome+formation.">Contribution of the endoplasmic reticulum to peroxisome formation.</a> Cell. 122, 85-95 (2005).</li><li>Furuya, 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=Glucose+is+toxic+to+glycosome-deficient+trypanosomes.">Glucose is toxic to glycosome-deficient trypanosomes.</a> Proc Natl Acad Sci U S A. 99, 14177-14182 (2002).</li></ol>

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 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 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 ...

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Immunology and Infection

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

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

Utilizzo di proteine fluorescenti per monitorare Glycosome Dynamics in Tripanosoma africano