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

1. General Trypanosome Husbandry
<ol>
	<li>Weigh solids for SDM79 media preparation (Table 1).</li>
	<li>Store at 4 &#176;C in 50 ml conical or a Ziploc&#160;bag. NOTE: Reagents are stable for at least 6 months.</li>
	<li>Thaw fetal bovine serum (FBS) in a 37 &#176;C waterbath, and mix periodically by inversion. NOTE: FBS is received from the supplier as a sterile solution. Filter sterilizing FBS reduces its ability to support parasite growth.</li>
	<li>Once the entire bottle is thawed, heat inactivate serum for 30 min in a 56 &#176;C waterbath, mixing periodically to minimize precipitation of serum components.</li>
	<li>Thaw penicillin/streptomycin solution (pen/strep), hemin (2 mg/ml in 50 mM NaOH), and basal medium eagle vitamin solution at room temperature.</li>
	<li>To a 1,000 ml graduated cylinder, add 20.2 g of SDM79 solids (Table 1) to the liquid components (Table 2) and mix well on a stir plate.</li>
	<li>Adjust pH to 7.35, and bring volume to 850 ml with water.</li>
	<li>Filter sterilize media by attaching a vacuum hose to the bottom of a filter bottle. Apply vacuum until the solution is filtered.</li>
	<li>Remove filter top in the biosafety cabinet and add 150 ml of heat inactivated FBS. Remove plastic covering from sterile cap and seal media bottle.</li>
	<li>Prepare SDM80 media in the same way as SDM79 with the following exceptions; do not add glucose or glucosamine and use MEM without glutamine. In place of 150 ml of heat inactivated FBS, add 135 ml of dialyzed, heat inactivated FBS, and 15 ml of heat inactivated FBS.</li>
	<li>Culture PCF parasites in 25 cm2 flask with 10 ml appropriate medium at 27 &#176;C and 5% CO2. NOTE: Cells should be counted daily using a hemocytometer or a flow cytometer (see section 6) and cultures should be maintained at densities between 1 x 105 and 5 x 106 cells/ml.</li>
</ol>
2. Transfection of PCF Parasites with the Fluorescent Reporter Construct
NOTE: To follow glycosome dynamics, a reporter protein containing the peroxisomal targeting sequence (PTS2) of aldolase fused to enhanced yellow fluorescent protein is expressed in the parasites. The sequence encoding the fusion protein is cloned into pXS2bla12, which contains the procyclin promoter and the tubulin intergenic regions, which direct homologous recombination into the genome and the blasticidin resistance gene for selection. Procyclic cell lines harboring the genes encoding the T7 polymerase and tetracycline repressor (PF29-13) are transformed with the targeting construct, pXS2:PTS2eYFP.
<ol>
	<li>Prepare cytomix by mixing the components listed in Table 3. Filter sterilize and store at RT.</li>
	<li>Linearize plasmid DNA with MluI by pipetting 10 &#956;l of DNA (1 &#956;g/&#956;l), 5 &#956;l restriction enzyme buffer, 33 &#956;l water, and 2 &#956;l of MluI enzyme into a microcentrifuge tube. Incubate at 37 &#176;C for 1 hr.</li>
	<li>Purify the restriction digest by adding 1 volume of binding buffer to the restriction enzyme digest. Mix binding buffer and digested DNA by vortexing. Briefly centrifuge to collect the sample at the bottom of the tube.</li>
	<li>Insert a DNA binding column into a 2 ml collection tube, and transfer digested DNA/binding buffer solution to column.</li>
	<li>Centrifuge for 10,000 x g for 1 min. After centrifugation, discard filtrate from collection tube and return the column to the collection tube.</li>
	<li>Add 700 &#181;l of SPW wash buffer and centrifuge at 10,000 x g for 1 min.</li>
	<li>Discard filtrate from collection tube, return the column to the collection tube and repeat SPW wash step and centrifugation.</li>
	<li>Discard filtrate, return column to collection tube, and centrifuge empty column for 3 min at 10,000 x g to remove residual ethanol.</li>
	<li>In a biosafety cabinet, throw away collection tube and place column in a sterile microcentrifuge tube.</li>
	<li>To elute the DNA, add 25 &#181;l of sterile water to column and incubate at room temperature for 2 min. Centrifuge at 10,000 x g for 1 min.</li>
	<li>Add another 25 &#181;l of sterile water in biosafety hood to column and incubate at room temperature for 2 min.</li>
	<li>Centrifuge at 10,000 x g speed for 1 min.</li>
	<li>In a biosafety cabinet transfer entire volume of DNA to a new sterile microcentrifuge tube. NOTE: This step prevents contamination from the open lid during centrifugation.</li>
	<li>Add the sterile, purified, linearized DNA (10 &#181;g) to 400 &#181;l of filter-sterilized cytomix.</li>
	<li>Count cells as described in section 6 and harvest 5 x107-108 cells by centrifugation at 800 x g for 15 min&#160;at RT.</li>
	<li>Pour off supernatant and resuspend the cell pellet in 450 &#181;l of DNA + cytomix using a 1 ml serological pipette.</li>
	<li>Transfer solution containing cells, DNA, and cytomix to a sterile 4 mm gap cuvette and place cuvette in the electroporation chamber.</li>
	<li>Select &#8220;Exponential decay&#8221; and manually enter the following settings: Voltage: 1.5 kV, Capacitance: 25 mF, Resistance: &#937;, and Cuvette: 4 mm. Press pulse. Once the pulse is complete, remove cuvette from the electroporation chamber and return to the biosafety cabinet.</li>
	<li>Into a new sterile flask, pipette 10 ml of SDM79. Transfer the transformed cells to the flask with 10 ml of SDM79.</li>
	<li>Incubate WITHOUT drug selection for 24 hr at 27 &#176;C, 5% CO2. After 24 hr, add G418 (15 &#181;g/ml), hygromycin (50 &#181;g/ml), and blasticidin (10 &#181;g/ml). Pass 1 ml of transformed cells with drug into 9 ml of SDM79 with drug.</li>
	<li>Analyze cells daily as described in sections 6 and 7. When cells are fluorescent, and have reached a cell density of 1 x 107/ml, make stabilates for long-term storage (3.1-3.3).</li>
</ol>
3. Making Stabilates
<ol>
	<li>To make freezing media, add an equal volume of 100% glycerol to cytomix and filter sterilize.</li>
	<li>Add 200 &#181;l of freezing media to 800 &#181;l of cells (1 x 107) in a cryovial, mix gently by inversion and place between two Styrofoam racks and freeze at -80 &#176;C.</li>
	<li>Once frozen (~24 hr), transfer cells to liquid nitrogen. NOTE: It is important to move cells into LN2 after 24 hr, as longer storage at -80 &#176;C leads to a decrease in cell viability.</li>
</ol>
4. Thawing Frozen Stocks
<ol>
	<li>Remove frozen vial from -80 &#176;C and thaw at RT for approximately 10 min.</li>
	<li>Pipette 9 ml of SDM79 with drug into a new sterile flask. Add thawed cells to this flask. Pass cells 1:10 by adding 1 ml of this culture to 9 ml of SDM79 in a new flask (final concentration of 1 x 105/ml).</li>
	<li>Before the cells reach a density of 107/ml, begin cytometer setup and dilution assays.</li>
</ol>
5. Cytometer Setup
<ol>
	<li>Turn on the flow cytometer and open CFlow Plus program. NOTE: Before running any samples, the fluidics should be backflushed and rinsed according to steps 5.2-5.7.</li>
	<li>Replace the tube of nanopure water in which the sip is stored with an empty tube.</li>
	<li>Select the &#8220;backflush&#8221; button.</li>
	<li>Once backflush is complete, remove microcentrifuge tube from sip and discard.</li>
	<li>Place a new microcentrifuge tube containing 1 ml of new nanopure water on the sip.</li>
	<li>Select a new well in CFlow program. Under &#8220;Run Limits&#8221; set time for 2 min. Under &#8220;Fluidics&#8221; select &#8220;fast&#8221; and &#8220;Run&#8221;.</li>
	<li>Once the 2 min time limit is complete, click the &#8220;Delete Sample Data&#8221; button.</li>
</ol>
6. Cell Counting using the Flow Cytometer
<ol>
	<li>Replace water with the sample to be counted. Set &#8220;Run Limits&#8221; to 30 sec, &#8220;Fluidics&#8221; to &#8220;fast&#8221; and then select &#8220;Run&#8221;.</li>
	<li>After the 30 sec time limit is complete, CFlow plus will provide the events/&#956;l under &#8220;Last Run.&#8221; Repeat count for each culture. NOTE: Before cells reach 1 x 107 cells/ml, proceed with dilution assay. Cells should be discontinued after one dilution assay is completed. Cells cultured for longer periods lose their ability to respond to environmental conditions.</li>
</ol>
7. Measuring Fluorescence using the Flow Cytometer
<ol>
	<li>To assess fluorescence, set &#8220;Run Limits&#8221; to 10,000 events, and &#8220;Fluidics&#8221; to &#8220;slow&#8221;. Select &#8220;Set threshold&#8221;. Under &#8220;Primary Threshold&#8221;, select &#8220;Permanently eliminate events on&#8221;, &#8220;FSC-H&#8221; (in drop down box) and enter less than 30,000. Click &#8220;apply&#8221;, then &#8220;close&#8221;.</li>
	<li>Select the &#8220;Histogram&#8221; button in one of the new plot windows and select &#8220;FSC-A&#8221; From the drop down list, select &#8220;FL1-A&#8221;. NOTE: FL1-A measures fluorescence in the 530 nm wavelength.</li>
	<li>Select &#8220;Run&#8221; and repeat for all cultures.</li>
	<li>Run cleaning solution through the fluidics line for 2 min and water for 2 min.</li>
</ol>
8. Dilution Assays
<ol>
	<li>Pass cells to a density of 1 x 105 cells/ml in 3 ml of SDM79 in a 25 cm2 culture flask and measure florescence immediately after passage and then at 3 hr and 24 hr.</li>
</ol>
9. Data Analysis
<ol>
	<li>Select &#8220;Analyze&#8221; tab on CFlow Plus.</li>
	<li>Click &#8220;Histogram&#8221; button under &#8220;Make a new plot.&#8221; Select &#8220;FSC-A&#8221; and a drop down list will appear. Select channel &#8220;FL1-A.&#8221;</li>
	<li>Highlight a well to analyze. Select &#8220;Gate&#8221; button, and manually draw a gate for the population of interest.</li>
	<li>Flow Plus will provide cell &#8220;Count&#8221; within this gate and &#8220;% of This Plot&#8221;.</li>
</ol>

<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. 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The authors have nothing to disclose.

Glycosomes are essential, dynamic, parasite-specific organelles. The processes that regulate the biogenesis, maintenance, proliferation and remodeling of these organelles likely include drug targets that could be exploited for therapeutic purposes. Despite the potentially high abundance of such drug targets, the field of glycosome biogenesis has lagged behind the study of similar processes in other organisms, predominately due to the lack of a tractable, high-throughput system by which to monitor rapid, dynamic, organell...

Trypanosoma brucei causes African sleeping sickness in humans and a wasting disease, nagana, in cattle. Drugs used in the treatment of these diseases are antiquated and extremely toxic, vaccines are not available, and the potential for the development of drug resistance necessitates the search for new drug targets1.
During its lifecycle, T. brucei, alternates between an insect vector and mammalian host; two hosts that present very different environments in which the parasite must survive. A number of metabolic and morphological changes occur as the parasite is exposed to different environmental conditions. Some of the most dramatic changes are observed in single-membrane-bounded parasite specific microbodies, termed glycosomes13.
Glucose levels are relatively high (~5 mM) in the bloodstream and bloodstream parasites (BSF) generate ATP exclusively through glycolysis while mitochondrial metabolism is repressed14. Unlike other eukaryotes in which glycolysis occurs in the cytoplasm, T. brucei compartmentalizes most of the glycolytic enzymes in glycosomes14,15. The parasites are taken up by the tsetse fly during a bloodmeal and experience a drop in glucose, which falls to undetectable levels within 15 min of being ingested by the fly. The metabolism of insect, procyclic form (PCF), parasites is more flexible and glucose, as well as amino acids such as proline, can be used in the synthesis of ATP16-18. Comparative proteomic studies reveal lifecycle dependent changes in glycosomal and mitochondrial proteins with glycolytic proteins increased in bloodstream parasites and mitochondrial proteins involved in TCA cycle and respiratory chain13,19. While many studies have focused on the differences between BSF and PCF glycosomes, little is known about the changes in PCF glycosomes that occur in response to environmental changes.
In the hindgut of the fly, glucose levels are low with transient increases during a feeding20. In most in vitro studies, PCF parasites are grown in media containing glucose. However, recent studies have demonstrated that PCF metabolism changes significantly in response to glucose availability17. In the absence of glucose, proline uptake and proline dehydrogenase activity increase18. This change in mitochondrial metabolism is likely accompanied by a change in glycosome composition and morphology, however, this has not been directly assessed.
Electron and fluorescence microscopy are common techniques used to study glycosome dynamics in T. brucei2,21-24. These protocols are time and labor intensive, expensive, and difficult to adapt to real-time studies and high throughput protocols. To overcome this limitation, a fluorescent-organelle reporter system used to study organelles in mammalian and yeast systems has been modified for use in T. brucei12.
Fluorescent-organelle reporter systems have been extensively used in higher eukaryotes such as yeast, plant, and mammalian cells25-27. In such systems, a fluorescent protein is fused to an amino acid sequence that targets the protein to specific organelles. The degradation or synthesis of the targeted proteins is measured via fluorescence and changes in organelle composition are reflected by changes in cell fluorescence.
When the open reading frame of enhanced yellow fluorescent protein (eYFP) is fused to a type II peroxisomal targeting sequence (PTS2)12, the PTS2eYFP protein is imported into mature, import-competent glycosomes and fluorescence can be monitored via flow cytometry. Variations in glycosome composition are reflected by changes in cellular fluorescence. This system can aid in resolving the mechanisms that regulate environmentally induced changes in glycosome composition.
This manuscript describes the generation of a glycosome reporter system in PCF parasites in conjunction with flow cytometry to monitor real-time glycosome dynamics in live parasites and provides an example of how it has been used to follow changes in glycosome composition in response to different environments. In summary, glycosome composition is influenced by extracellular glucose concentrations and passage of log-phase cultures into fresh media triggers changes in glycosome composition. This system can be modified to study the dynamic behavior of other organelles in trypanosomes and other parasites.

<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 is a kinetoplastid parasite that causes human African trypanosomiasis (HAT), or sleeping sickness, and a wasting disease, nagana, in cattle1. The parasite alternates between the bloodstream of the mammalian host and the tsetse fly vector. The composition of many cellular organelles changes in response to these different extracellular conditions2-5.
Glycosomes are highly specialized peroxisomes in which many of the enzymes involved in glycolysis are compartmentalized. Glycosome composition changes in a developmental and environmentally regulated manner4-11. Currently, the most common techniques used to study glycosome dynamics are electron and fluorescence microscopy; techniques that are expensive, time and labor intensive, and not easily adapted to high throughput analyses.
To overcome these limitations, a fluorescent-glycosome reporter system in which enhanced yellow fluorescent protein (eYFP) is fused to a peroxisome targeting sequence (PTS2), which directs the fusion protein to glycosomes12, has been established. Upon import of the PTS2eYFP fusion protein, glycosomes become fluorescent. Organelle degradation and recycling results in the loss of fluorescence that can be measured by flow cytometry. Large numbers of cells (5,000 cells/sec) can be analyzed in real-time without extensive sample preparation such as fixation and mounting. This method offers a rapid way of detecting changes in organelle composition in response to fluctuating environmental conditions.

In this system, a glucose-dependent change in glycosome composition was observed. When cells are grown in glucose containing media, two populations are observed; one bright and one dim (Figure 2A). Dim cells harbor immature glycosomes, which have not imported the PTS2eYFP while bright cells harbor a mixture of mature and immature glycosomes12. When glucose is present in the media, mislocalization of glycosome proteins is lethal15,28 and glycosome protein expression is likely coupled...

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.

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

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.

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

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.

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.

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

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

The obligate intracellular bacterium Chlamydia elicits a great burden on global public health. C.&#160;trachomatis is the leading bacterial cause of sexually transmitted infection and also the primary cause of preventable blindness in the world. An essential determinant for successful infection of host cells by Chlamydia is the bacterium's ability to manipulate host cell signaling from within a novel, vacuolar compartment called the inclusion. From within the inclusion, Chlamydia acquire nutrients required for their 2-3 day developmental growth, and they additionally secrete a panel of effector proteins onto the cytosolic face of the vacuole membrane and into the host cytosol. Gaps in our understanding of Chlamydia biology, however, present significant challenges for visualizing and analyzing this intracellular compartment. Recently, a reverse-imaging strategy for visualizing the inclusion using GFP expressing host cells was described. This approach rationally exploits the intrinsic impermeability of the inclusion membrane to large molecules such as GFP. In this work, we describe how GFP- or mCherry-expressing host cells are generated for subsequent visualization of chlamydial inclusions. Furthermore, this method is shown to effectively substitute for costly antibody-based enumeration methods, can be used in tandem with other fluorescent labels, such as GFP-expressing Chlamydia, and can be exploited to derive key quantitative data about inclusion membrane growth from a range of Chlamydia species and strains.

Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells

A live cell fluorescent protein based method for illuminating cellular vacuoles (inclusions) containing Chlamydia is described. This strategy enables rapid, automated determination of Chlamydia infectivity in samples and can be used to quantitatively investigate inclusion growth dynamics.

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

The Mesenteric Lymph Duct Cannulated Rat Model: Application to the Assessment of Intestinal Lymphatic Drug Transport

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine via a series of lymphatic vessels and nodes that converge at the superior mesenteric lymph duct. Cannulation of the mesenteric lymph duct thus enables the collection of mesenteric lymph flowing from the intestine. Mesenteric lymph consists of a cellular fraction of immune cells (99% lymphocytes), aqueous fraction (fluid, peptides and proteins such as cytokines and gut hormones) and lipoprotein fraction (lipids, lipophilic molecules and apo-proteins). The mesenteric lymph duct cannulation model can therefore be used to measure the concentration and rate of transport of a range of factors from the intestine via the lymphatic system. Changes to these factors in response to different challenges (e.g., diets, antigens, drugs) and in disease (e.g., inflammatory bowel disease, HIV, diabetes) can also be determined. An area of expanding interest is the role of lymphatic transport in the absorption of orally administered lipophilic drugs and prodrugs that associate with intestinal lipid absorption pathways. Here we describe, in detail, a mesenteric lymph duct cannulated rat model which enables evaluation of the rate and extent of lipid and drug transport via the lymphatic system for several hours following intestinal delivery. The method is easily adaptable to the measurement of other parameters in lymph. We provide detailed descriptions of the difficulties that may be encountered when establishing this complex surgical method, as well as representative data from failed and successful experiments to provide instruction on how to confirm experimental success and interpret the data obtained.

Here we describe a technique to cannulate the mesenteric lymph duct in rats which enables quantification of lipid and drug transport via the lymphatic system following intestinal delivery. The technique can be adapted to assess mesenteric lymph concentrations and/or transport of fluid, immune cells, peptides, proteins and lipophilic molecules.

The intestinal lymphatic system plays key roles in fluid transport, lipid absorption and immune function. Lymph flows directly from the small intestine ...

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.

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)

Phagocytosis is a mechanism used by specialized cells to internalize and eliminate microorganisms or cellular debris. It relies on profound rearrangements of the actin cytoskeleton that is the driving force for plasma membrane extension around the particle. In addition, efficient engulfment of large material relies on focal exocytosis of intracellular compartments. This process is highly dynamic and numerous molecular players have been described to have a role during phagocytic cup formation. The precise regulation in time and space of all of these molecules, however, remains elusive. In addition, the last step of phagosome closure has been very difficult to observe because inhibition by RNA interference or dominant negative mutants often results in stalled phagocytic cup formation. 
We have set up a dedicated experimental approach using total internal reflection fluorescence microscopy (TIRFM) combined with epifluorescence to monitor step by step the extension of pseudopods and their tips in a phagosome growing around a particle loosely bound to a coverslip. This method allows us to observe, with high resolution the very tips of the pseudopods and their fusion during closure of the phagosome in living cells for two different fluorescently tagged proteins at the same time.

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

We describe an experimental setup to visualize with unprecedented high resolution phagosome formation and closure in three dimensions in living macrophages, using total internal reflection fluorescence microscopy. It allows monitoring of the base of the phagocytic cup, the extending pseudopods, as well as the precise site of phagosome scission.

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

Currently, it is possible to construct recombinant forms of various viruses, such as human immunodeficiency virus 1 (HIV-1) and hepatitis C virus (HCV), that carry foreign genes such as a reporter or marker protein in their genomes. These recombinant viruses usually faithfully mimic the life cycle of the original virus in infected cells and exhibit the same host range dependence. The development of a recombinant virus enables the efficient screening of inhibitors and the identification of specific host factors. However, to date the construction of recombinant hepatitis B virus (HBV) has been difficult because of various experimental limitations. The main limitation is the compact genome size of HBV, and a fairly strict genome size that does not exceed 1.3 genome sizes, that must be packaged into virions. Thus, the size of a foreign gene to be inserted should be smaller than 0.4 kb if no deletion of the genome DNA is to be performed. Therefore, to overcome this size limitation, the deletion of some HBV DNA is required. Here, we report the construction of recombinant HBV encoding a reporter gene to monitor the early stage of the HBV replication cycle by replacing part of the HBV core-coding region with the reporter gene by deleting part of the HBV pol coding region. Detection of recombinant HBV infection, monitored by the reporter activity, was highly sensitive and less expensive than detection using the currently available conventional methods to evaluate HBV infection. This system will be useful for a number of applications including high-throughput screening for the identification of anti-HBV inhibitors, host factors and virus-susceptible cells.

Here we describe a newly developed hepatitis B virus (HBV) reporter system to monitor the early stages of the HBV life cycle. This simplified in vitro system will aid in the screening of anti-HBV agents using a high-throughput strategy.

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

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple condensation reactions. DHDDS (dehydrodolichyl diphosphate synthase) is a eukaryotic long-chain cis-PT (forming cis double bonds from the condensation reaction) that catalyzes chain elongation of farnesyl diphosphate (FPP, an allylic diphosphate) via multiple condensations with isopentenyl diphosphate (IPP). DHDDS is of biomedical importance, as a non-conservative mutation (K42E) in the enzyme results in retinitis pigmentosa, ultimately leading to blindness. Therefore, the present protocol was developed in order to acquire large quantities of purified DHDDS, suitable for mechanistic studies. Here, the usage of protein fusion, optimized culture conditions and codon-optimization were used to allow the overexpression and purification of functionally active human DHDDS in E. coli. The described protocol is simple, cost-effective and time sparing. The homology of cis-PT among different species suggests that this protocol may be applied for other eukaryotic cis-PT as well, such as those involved in natural rubber synthesis.

Overexpression and Purification of Human Cis-prenyltransferase in Escherichia coli

A simple protocol for overexpression and purification of codon-optimized, human cis-prenyltransferase, under non-denaturing conditions, from Escherichia coli, is described, along with an enzymatic activity assay. This protocol can be generalized for production of other cis- prenyltransferase proteins in quantity and quality suitable for mechanistic studies.

Prenyltransferases (PT) are a group of enzymes that catalyze chain elongation of allylic diphosphate using isopentenyl diphosphate (IPP) via multiple ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...