This work was funded by the Ministry of Education ERC CZ grant LL1205, the Grant Agency of Czech Republic grant 18-17529S, and by ERDF/ESF project Centre for research of pathogenicity and virulence of parasites (No. CZ.02.1.01/0.0/0.0/16_019/0000759).

1. Buffers and Solutions
<ol>
	<li>Prepare the solutions listed below. Degas all buffers for liquid chromatography. Add ADP, benzamidine, and protease inhibitors just before use.
	<ol>
		<li>Prepare buffer A: 50 mM Tris buffer with hydrochloric acid (Tris-HCl) pH 8.0, 0.25 M sucrose, 5 mM benzamidine, 5 mM aminocaproic acid (ACA), and protease inhibitors (10 &#181;M amastatin, 50 &#181;M bestatin, 50 &#181;M pepstatin, 50 &#181;M leupeptin, and 50 &#181;M diprotin A).</li>
		<li>Prepare buffer B: 50 mM Tris-HCl pH 8.0, 0.25 M sucrose, 4 mM ethylenediaminetetraacetic acid (EDTA), 5 mM benzamidine, 5 mM ACA, 1 mM ADP, and protease inhibitors (10 &#181;M amastatin, 50 &#181;M bestatin, 50 &#181;M pepstatin, 50 &#181;M leupeptin, and 50 &#181;M diprotin A).</li>
		<li>Prepare Q-column buffer: 20 mM Tris-HCl pH 8.0, 4 mM EDTA, 10 mM MgSO4, 5 mM benzamidine, 5 mM ACA, and 1 mM ADP.</li>
		<li>Prepare Q-column elution buffer: Q-column buffer with 1 M NaCl.</li>
		<li>Prepare&#160;SEC&#160;buffer: 20 mM Tris-HCl pH 8.0, 10 mM MgSO4, 100 mM NaCl, 1 mM ADP.</li>
		<li>Prepare chloroform saturated with 2 M Tris-HCl pH 8.5. Mix chloroform with 2 M Tris-HCl pH 8.5 in approximately 1:1 ratio in a screw-cap bottle, shake, let the organic and aqueous phases separate, and measure pH in the upper aqueous layer with a strip of pH-indicator paper. Store at room temperature. Just before use, shake again and let the phases separate. Use the lower chloroform layer. 
		CAUTION: Chloroform is volatile and irritating to eyes and skin. Work in a fume hood. Use safety spectacles when shaking.</li>
	</ol>
	</li>
</ol>
2. Preparation of sub-mitochondrial Particles
<ol>
	<li>Resuspend mitochondrial vesicles (mitoplasts) isolated by hypotonic lysis25 from 1 x 1011 to 2 x 1011 cells of procyclic T. brucei in 5 mL of ice-cold buffer A. Keep the sample chilled until step 3.2.</li>
	<li>Determine the protein concentration in the suspension by the bicinchoninic acid (BCA) protein assay26 according to the manufacturer&#39;s instructions.
	<ol>
		<li>Use a bovine serum albumin (BSA) dilution series in ultrapure water to construct the standard curve. Dilute a small amount of sample 20 - 100 times with ultrapure water to fit into the range of BSA standards.</li>
		<li>Calculate the total protein amount in the sample and bring the protein concentration to 16 mg/mL by diluting it with additional buffer A.</li>
	</ol>
	</li>
	<li>Fragment mitoplasts into inverted vesicles and membrane pieces by sonication of the suspension 7x for 15 s with a total energy of 70 to 100 J per impulse with a microtip with a diameter of 3.9 mm. If the ultrasonic homogenizer does not display the energy output, start the optimization at 50% of the maximal power. Incubate the sample on ice for 30 s between impulses. After the sonication, the suspension becomes slightly darker.</li>
	<li>Sediment the membrane fragments by ultracentrifugation at 54,000 x g for 16 h or at 98,000 x g for 5 h at 4 &#176;C. Decant the supernatant and proceed with the chloroform extraction, or flash-freeze the sediment in liquid nitrogen and store it at -80 &#176;C.</li>
</ol>
3. Release of F1-ATPase from Membrane by Chloroform
<ol>
	<li>Resuspend the pellet of mitochondrial membranes in buffer B with the aid of a small Dounce homogenizer. Calculate the volume of buffer B based on the total amount of buffer A used in steps 2.1 and 2.2 using the following formula: volume (buffer B) = volume (buffer A) x 12/21. Transfer the suspension to a 15- or 50-mL conical tube.</li>
	<li>Remove the sample from ice and, from now on, keep the sample and all solutions to be used at room temperature.</li>
	<li>Add chloroform saturated with 2 M Tris-HCl pH 8.5; the volume of chloroform to be added equals half the volume of the suspension. Close the cap tightly. Shake it vigorously for exactly 20 s. Centrifuge it immediately at 8,400 x g for 5 min at room temperature.</li>
	<li>Transfer the upper cloudy aqueous phase to 1.6-mL microtubes. Add protease inhibitors (10 &#181;M amastatin, 50 &#181;M bestatin, 50 &#181;M pepstatin, 50 &#181;M leupeptin, and 50 &#181;M diprotin A) to replace the inhibitors removed by the chloroform treatment. Centrifuge the samples at 13,000 x g for 30 min at room temperature. Transfer the supernatant to fresh microtubes and repeat the centrifugation to remove any insoluble material.</li>
</ol>
4. Anion-exchange Chromatography
<ol>
	<li>Equilibrate the 5-mL anion exchange (Q) column attached to a fast-protein liquid chromatography system with the Q-column buffer at a flow rate of 5 mL/min until the absorbance at 280 nm and the conductivity stabilize (approximately 50 mL of buffer).</li>
	<li>Load the supernatant from step 3.3 on the equilibrated column at a flow rate of 1 mL/min. Wait until the absorbance at 280 nm stabilizes at the background. Apply a 25-mL linear gradient of the Q-column elution buffer from 0% to 100% at a flow rate of 0.5 mL/min. Collect 1-mL fractions.</li>
	<li>Assay the individual fractions corresponding to the major elution peak for ATP hydrolytic activity by the Pullman ATPase assay2 at pH 8.0. Use 10 &#181;L of each fraction per 1 mL of reaction mixture.Pool the fractions that exhibit ATPase activity. Optionally, separate 10 &#181;L of each fraction on sodium dodecyl phosphate polyacrylamide gel electrophoresis (SDS-PAGE) and stain the gel by Coomassie Blue to visualize individual F1-ATPase subunits and contaminating proteins.</li>
	<li>Concentrate the pooled sample by membrane ultrafiltration using a spin column with a 100,000 MWCO PES filter to 200 - 500 &#181;L. Proceed to SEC or store the sample overnight at room temperature.</li>
</ol>
5. Size-exclusion Chromatography
<ol>
	<li>Equilibrate the SEC column attached to a liquid chromatography system with at least 48 mL (two column volumes) of the SEC buffer at a flow rate of 0.5 mL/min.</li>
	<li>Apply the sample on the column and run chromatography at a flow rate of 0.25 mL/min. Collect 0.25-mL fractions.</li>
	<li>Run 10 &#181;L of the fractions that correspond to the peaks of the UV280nm absorbance trace on SDS-PAGE and stain them by Coomassie Blue. The first major peak contains the F1-ATPase. Assay the fractions corresponding to this peak for the ATP hydrolytic activity and azide sensitivity by the Pullman ATPase assay. Determine the protein concentration by the BCA assay.</li>
	<li>Keep the purified F1-ATPase at room temperature and use it within 3 d after purification for downstream applications. Alternatively, concentrate the sample using a spin column with a 100,000 MWCO PES filter to &#62; 1.5 mg/mL, precipitate it by mixing it with saturated ammonium sulfate adjusted to pH 8.0 (1.2x the volume), and store it at 4 &#176;C.</li>
</ol>

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The authors have nothing to disclose.

The protocol for F1-ATPase purification from T. brucei was developed based on previously published methods for the isolation of F1-ATPase complexes from other species13,14. The method does not require any genetic modification (e.g., tagging) and yields a fully active complex with all subunits present. The crucial step is the chloroform-facilitated release of the F1-ATPase from the membrane-attached part of the en...

The F-type ATP synthases are membrane-bound rotating multiprotein complexes that couple proton translocation across energy-transducing membranes of bacteria, mitochondria, and chloroplasts with the formation of ATP. Molecular details of the rotational mechanism of ATP synthesis are known mainly because of structural studies of purified bacterial and mitochondrial ATP synthases and their subcomplexes1. F-type ATP synthase is organized into membrane-intrinsic and membrane-extrinsic moieties. The membrane-extrinsic part, known as F1-ATPase, contains three catalytic sites, where the phosphorylation of adenosine diphosphate (ADP) to ATP or the reverse reaction occurs. F1-ATPase can be released experimentally from the membrane-intrinsic moiety while retaining its ability to hydrolyze, but not synthesize, ATP. The membrane-bound sector, called Fo, mediates protein translocation, which drives the rotation of the central part of the enzyme. The F1 and Fo sectors are connected by the&#160;central and peripheral stalks.
The first attempts to purify the F1-ATPase from budding yeast and bovine heart mitochondria date back to the 1960s. These protocols used extracted mitochondria, which were disrupted by sonication, fractionated by ammonium or protamine sulfate precipitation, followed by optional chromatography step(s) and heat treatment2,3,4,5,6. The purification was greatly improved and simplified by the use of chloroform, which readily releases the F1-ATPase from the mitochondrial membrane fragments7. The chloroform extraction was then used to extract F1-ATPases from various animal, plant, and bacterial sources (e.g., rat liver8, corn9, Arum maculatum10, and Escherichia coli11). Further purification of the chloroform-released F1-ATPase by affinity or size-exclusion chromatography (SEC) yielded a highly pure protein complex, which was suitable for high-resolution structure determination by X-ray crystallography, as documented by the structures of F1-ATPase from bovine heart12,13 and Saccharomyces cerevisiae14. F1-ATPase structures were also determined from organisms that are difficult to cultivate and, thus, the amount of the initial biological material was limited. In this case, the F1-ATPase subunits were artificially expressed and assembled into the complex in E. coli, and the whole heterologous enzyme was purified by affinity chromatography via a tagged subunit. Such approach led to the determination of F1-ATPase structures from two thermophilic bacterial species, Geobacillus stearothermophilus15 and Caldalkalibacillus thermarum16,17. However, this methodology is rather unsuitable for eukaryotic F1-ATPases since it relies on the prokaryotic protheosynthetic apparatus, posttranslational processing, and complex assembly.
The chloroform-based extraction was previously used to isolate F1-ATPases from unicellular digenetic parasites Trypanosoma cruzi18 and T. brucei19, important mammalian pathogens causing American and African trypanosomiases, respectively, and from monogenic insect parasite Crithidia fasciculata20. These purifications led only to a simple description of the F1-ATPases, since no downstream applications were used to fully characterize the composition, structure, and enzymatic properties of the complex. This article describes an optimized method for F1-ATPase purification from the cultured insect life cycle stage of T. brucei. The method is developed based on the established protocols for isolation of bovine and yeast F1-ATPases21,22. The procedure yields highly pure and homogeneous enzyme suitable for in vitro enzymatic and inhibitory assays, detailed proteomic characterization by mass spectrometry23, and structure determination24. The purification protocol and the knowledge of the F1-ATPase structure at the atomic level opens a possibility to design screens to identify small-molecule inhibitors, and aid in the development of new drugs against African trypanosomiases. Moreover, the protocol can be adapted to purify F1-ATPase from other organisms.

<table><tbody><tr><td>&lt;strong&gt;Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adenosin Diphosphate Disodium Salt (ADP)</td><td>Applichem</td><td>A0948</td><td></td></tr><tr><td>Amastatin Hydrochloride</td><td>Glantham Life Sciences</td><td>GA1330</td><td></td></tr><tr><td>Aminocaproic Acid</td><td>Applichem</td><td>A2266</td><td></td></tr><tr><td>BCA Protein Assay Kit</td><td>ThermoFischer Scientific/Pierce</td><td>23225</td><td></td></tr><tr><td>Benzamidine Hydrochloride</td><td>Calbiochem</td><td>199001</td><td></td></tr><tr><td>Bestatin Hydrochloride</td><td>Sigma Aldrich/Merck</td><td>B8385</td><td></td></tr><tr><td>Chloroform</td><td>Any supplier</td><td></td><td></td></tr><tr><td>cOmplete Tablets, Mini EDTA-free</td><td>Roche</td><td>4693159001</td><td>Protease inhibitor cocktail tablets</td></tr><tr><td>Ethylenediaminetetraacetic Acid (EDTA)</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Any supplier</td><td></td><td>For pH adjustment</td></tr><tr><td>Ile-Pro-Ile</td><td>Sigma Aldrich/Merck</td><td>I9759</td><td>Alias Diprotin A</td></tr><tr><td>Leupeptin</td><td>Sigma Aldrich/Merck</td><td>L2884</td><td></td></tr><tr><td>Magnesium Sulfate Heptahydrate</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Pepstatin A</td><td>Sigma Aldrich/Merck</td><td>P5318</td><td></td></tr><tr><td>Protein Electrophoresis System</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Sodium Chloride</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Sucrose</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Tris</td><td>Any supplier</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Centrifuge Tubes for SW60Ti, Polyallomer</td><td>Beckman Coulture</td><td>328874</td><td></td></tr><tr><td>DounceTissues Homogenizer 2 mL</td><td>Any supplier</td><td></td><td></td></tr><tr><td>Glass Vacuum Filtration Device</td><td>Sartorius</td><td>516-7017</td><td>Degasing solutions for liquid chromatography</td></tr><tr><td>HiTrap Q HP, 5 mL</td><td>GE Healthcare Life Sciences</td><td>17115401</td><td>Anion exchange chromatography column</td></tr><tr><td>Regenaretad Cellulose Membrane Filters, pore size 0.45 &amp;mu;m, diameter 47 mm</td><td>Sartorius</td><td>18406--47------N</td><td>Degasing solutions for liquid chromatography</td></tr><tr><td>Superdex 200 Increase 10/300 GL</td><td>GE Healthcare Life Sciences</td><td>29091596</td><td>Size-exclusion chromatography column</td></tr><tr><td>Vivaspin 6 MWCO 100 kDa PES</td><td>Sartorius</td><td>VS0641</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AKTA Pure 25</td><td>GE Healthcare Life Sciences</td><td>29018224</td><td>Or similar FPLC system</td></tr><tr><td>Spectrophotometer Shimadzu UV-1601</td><td>Shimadzu</td><td></td><td>Or similar spectrophotometer with kinetic assay mode</td></tr><tr><td>Ultracentrifuge Beckman Optima with SW60Ti Rotor</td><td>Beckman Coulture</td><td></td><td>Or similar ultracentrifuge and rotor</td></tr><tr><td>Ultrasonic Homogenizer with Thin Probe, Model 3000</td><td>BioLogics</td><td>0-127-0001</td><td>Or similar ultrasonic homogenizer</td></tr></tbody></table>

isolation of f1-atpase from the parasitic protist trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to produce adenosine triphosphate (ATP). The isolation of the intact F1-ATPase from its native source is an essential prerequisite to characterize the enzyme&#39;s protein composition, kinetic parameters, and sensitivity to inhibitors. A highly pure and homogeneous F1-ATPase can be used for structural studies, which provide insight into molecular mechanisms of ATP synthesis and hydrolysis. This article describes a procedure for the purification of the F1-ATPase from Trypanosoma brucei, the causative agent of African trypanosomiases. The F1-ATPase is isolated from mitochondrial vesicles, which are obtained by hypotonic lysis from in vitro cultured trypanosomes. The vesicles are mechanically fragmented by sonication and the F1-ATPase is released from the inner mitochondrial membrane by the chloroform extraction. The enzymatic complex is further purified by consecutive anion exchange and size-exclusion chromatography. Sensitive mass spectrometry techniques showed that the purified complex is devoid of virtually any protein contaminants and, therefore, represents suitable material for structure determination by X-ray crystallography or cryo-electron microscopy. The isolated F1-ATPase exhibits ATP hydrolytic activity, which can be inhibited fully by sodium azide, a potent inhibitor of F-type ATP synthases. The purified complex remains stable and active for at least three days at room temperature. Precipitation by ammonium sulfate is used for long-term storage. Similar procedures have been used for the purification of F1-ATPases from mammalian and plant tissues, yeasts, or bacteria. Thus, the presented protocol can serve as a guideline for the F1-ATPase isolation from other organisms.

A typical purification (Figure 1) starts with mitochondrial vesicles (mitoplasts) isolated on the Percoll gradient from hypotonically lysed 1 x 1011 to 2 x 1011 procyclic T. brucei cells25 cultured in standard glucose-rich SDM-79 medium27. The mitoplasts are fragmented by sonication, spun, and the matrix-containing supernatant is discarded. Mitochondrial membranes are treated with ...

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Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

This protocol describes the purification of F1-ATPase from the cultured insect stage of Trypanosoma brucei. The procedure yields a highly pure, homogeneous, and active complex suitable for structural and enzymatic studies.

Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

F1-ATPase is a membrane-extrinsic catalytic subcomplex of F-type ATP synthase, an enzyme that uses the proton motive force across biological membranes to ...

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7.2K Views.  Czech Academy of Science, Institute of Parasitology. De zuivering van F1-ATPase is gebaseerd op een klassieke biochemische methode die cruciaal bleek te zijn voor ons begrip van het mechanisme van ATP-productie door andere ATP-synthases. Het belangrijkste voordeel van deze techniek is dat het een zeer zuiver enzym produceert dat geschikt is voor structuurbepaling door röntgenkristallografie of cryo-elektronenmicroscopie. Hoewel dit protocol is geoptimaliseerd voor de isolatie van F1-ATPase van trypanosomen, kan het worden aangepast aan andere ...

Video: Isolation of F1-ATPase from the Parasitic Protist Trypanosoma brucei

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate for telomerase for de novo telomere DNA synthesis and is therefore important for telomere maintenance. T. brucei is a protozoan parasite that causes sleeping sickness in humans and nagana in cattle. Once infected mammalian host, T. brucei cell regularly switches its surface antigen to evade the host's immune attack. We have recently demonstrated that the T. brucei telomere structure plays an essential role in regulation of surface antigen gene expression, which is critical for T. brucei pathogenesis. However, T. brucei telomere structure has not been extensively studied due to the limitation of methods for analysis of this specialized structure. We have now successfully adopted the native in-gel hybridization and ligation-mediated primer extension methods for examination of the telomere G-overhang structure and an adaptor ligation method for determination of the telomere terminal nucleotide in T. brucei cells. Here, we will describe the protocols in detail and compare their different advantages and limitations.

Examination of the Telomere G-overhang Structure in Trypanosoma brucei

Telomeres are essential for chromosome stability and the telomere G-overhang structure is essential for telomerase-mediated telomere maintenance. We have recently adopted two methods for detecting the telomere G-overhang structure in Trypanosoma brucei, which are native in-gel hybridization and ligation-mediated primer extension, which will be described.

The telomere G-overhang structure has been identified in many eukaryotes including yeast, vertebrates, and Trypanosoma brucei. It serves as the substrate ...

Immunology and Infection

Identification of Inositol Phosphate or Phosphoinositide Interacting Proteins by Affinity Chromatography Coupled to Western Blot or Mass Spectrometry

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal transduction, metabolism, and development. These metabolites perform this regulatory activity by binding to proteins, thereby changing protein conformation, catalytic activity, and/or interactions. The method described here uses affinity chromatography coupled to mass spectrometry or Western blotting to identify proteins that interact with inositol phosphates or phosphoinositides. Inositol phosphates or phosphoinositides are chemically tagged with biotin, which is then captured via streptavidin conjugated to agarose or magnetic beads. Proteins are isolated by their affinity of binding to the metabolite, then eluted and identified by mass spectrometry or Western blotting. The method has a simple workflow that is sensitive, non-radioactive, liposome-free, and customizable, supporting the analysis of protein and metabolite interaction with precision. This approach can be used in label-free or in amino acid-labelled quantitative mass spectrometry methods to identify protein-metabolite interactions in complex biological samples or using purified proteins. This protocol is optimized for the analysis of proteins from Trypanosoma brucei, but it can be adapted to related protozoan parasites, yeast or mammalian cells.

This protocol focuses on the identification of proteins that bind to inositol phosphates or phosphoinositides. It uses affinity chromatography with biotinylated inositol phosphates or phosphoinositides that are immobilized via streptavidin to agarose or magnetic beads. Inositol phosphate or phosphoinositide binding proteins are identified by Western blotting or mass spectrometry.

Inositol phosphates and phosphoinositides regulate several cellular processes in eukaryotes, including gene expression, vesicle trafficking, signal ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

Neuroscience

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.

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 ...

High-throughput Gene Tagging in Trypanosoma brucei

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

High-throughput Gene Tagging in Trypanosoma brucei

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

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

Mitochondrial Fractionation in Small Tissue Samples for Supercomplex Analysis

Isolation of Mitochondria for Mitochondrial Supercomplex Analysis from Small Tissue and Cell Culture Samples

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial electron transport chain organization, giving rise to the proposal of the "plasticity model." This model postulates the coexistence of different proportions of SCs and complexes depending on the tissue or the cellular metabolic status. The dynamic nature of the assembly in SCs would allow cells to optimize the use of available fuels and the efficiency of electron transfer, minimizing reactive oxygen species generation and favoring the ability of cells to adapt to environmental changes. 
More recently, abnormalities in SC assembly have been reported in different diseases such as neurodegenerative disorders (Alzheimer's and Parkinson's disease), Barth Syndrome, Leigh syndrome, or cancer. The role of SC assembly alterations in disease progression still needs to be confirmed. Nevertheless, the availability of enough amounts of samples to determine the SC assembly status is often a challenge. This happens with biopsy or tissue samples that are small or have to be divided for multiple analyses, with cell cultures that have slow growth or come from microfluidic devices, with some primary cultures or rare cells, or when the effect of particular costly treatments has to be analyzed (with nanoparticles, very expensive compounds, etc.). In these cases, an efficient and easy-to-apply method is required. This paper presents a method adapted to obtain enriched mitochondrial fractions from small amounts of cells or tissues to analyze the structure and function of mitochondrial SCs by native electrophoresis followed by in-gel activity assays or western blot.

Author Spotlight: Unveiling Oxidative Phosphorylation System Dynamics and Mitochondrial Roles in Health and Disease

This protocol describes a technique for the analysis of respiratory supercomplexes when only small amounts of samples are available.

Over the last decades, the evidence accumulated about the existence of respiratory supercomplexes (SCs) has changed our understanding of the mitochondrial ...

Purification of the Sarco-Endoplasmic Reticulum Ca2+-ATPase from Rabbit Muscle

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific physicochemical conditions during purification to obtain them with high purity and structural quality and in a catalytically active form. The disaccharide trehalose is a compatible solute that is synthesized and accumulated in high concentrations in the yeast cytoplasm to stabilize the membranes and proteins. The use of trehalose as an additive in the protocol for the purification of plasma membrane H+-ATPase results in a high-quality preparation, the hexameric structure of which is shown by biochemical analytical methods. Trehalose can, therefore, be used as a stabilizing additive for the purification of membrane proteins (P-ATPases). This protocol describes the modification of the classical protocol for SERCA purification by subjecting SERCA to centrifugation on a trehalose concentration gradient. The inclusion of this carbohydrate led to the purification of SERCA in a catalytically active form with high purity and, importantly, in a stable form. Partial biochemical characterization of the purified SERCA (SDS-PAGE, enzyme kinetics, FITC labeling, circular dichroism spectroscopy) showed that the enzyme is suitable for functional and structural studies. The use of trehalose in the purification protocol of P-type ATPases and other labile membrane (and cytosolic) proteins is suggested.

This protocol describes an improved SERCA purification method, which includes the disaccharide trehalose in the final centrifugation step. This carbohydrate stabilizes proteins under harsh conditions. The purified SERCA was catalytically active and displayed high purity, making it suitable for structural and functional studies.

Some P-type ATPases, such as sarco/endoplasmic reticulum Ca2+-ATPase (SERCA), are inherently labile membrane proteins that require specific ...

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers (Diethylaminoethyl-cellulose Columns)

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is the best method for diagnosis of first stage HAT and furthermore this parasite purification method permits serological and research investigations.
HAT is caused by Tsetse fly transmitted Trypanosoma brucei gambiense and T. b. rhodesiense. Related trypanosomes are the causative agents of animal trypanosomiasis. Trypanosome detection is essential for HAT diagnosis, treatment and follow-up. The technique described here is the most sensitive parasite detection technique, adapted to field conditions for the diagnosis of T. b. gambiense HAT and can be completed within one hour. Blood is layered onto an anion-exchanger column (DEAE cellulose) previously adjusted to pH 8, and elution buffer is added. Highly negatively charged blood cells are adsorbed onto the column whereas the less negatively charged trypanosomes pass through. Collected trypanosomes are pelleted by centrifugation and observed by microscopy. Moreover, parasites are prepared without cellular damage whilst maintaining their infectivity.
Purified trypanosomes are required for immunological testing; they are used in the trypanolysis assay, the gold standard in HAT serology. Stained parasites are utilized in the card agglutination test (CATT) for field serology. Antigens from purified trypanosomes, such as variant surface glycoprotein, exoantigens, are also used in various immunoassays. The procedure described here is designed for African trypanosomes; consequently, chromatography conditions have to be adapted to each trypanosome strain, and more generally, to the blood of each species of host mammal.
These fascinating pathogens are easily purified and available to use in biochemical, molecular and cell biology studies including co-culture with host cells to investigate host-parasite relationships at the level of membrane receptors, signaling, and gene expression; drug testing in vitro; investigation of gene deletion, mutation, or overexpression on metabolic processes, cytoskeletal biogenesis and parasite survival.

Purification of Extracellular Trypanosomes, Including African, from Blood by Anion-Exchangers Diethylaminoethyl-cellulose Columns

This method of trypanosome separation from blood depends on their surface charge being less negative than mammalian blood cells. Infected blood is placed and treated on an anion-exchanger column. This method, the most fitting diagnostic for African trypanosomiasis, provides purified parasites for immunological, biological, biochemical, pharmaceutical and molecular biology investigations.

This method allows the separation of trypanosomes, parasites responsible for animal and human African trypanosomiasis (HAT), from infected blood. This is ...

Development of pHluorin2-Based Sensor for Glycosomal pH in Trypanosoma brucei

Measuring Dynamic Glycosomal pH Changes in Living Trypanosoma brucei

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

Author Spotlight: Advancements in Glycosomal pH Monitoring in Trypanosoma brucei Using pHluorin2 Biosensor

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

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

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Isolation of F₁-ATPase from the Parasitic Protist Trypanosoma brucei

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