Name: polysome profiling in leishmania, human cells and mouse testis
Uploaded: 2018-04-08T15:00:00
Description: Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

The authors thank Ching Lee for help with audio recording.The research was supported by the Start-up funds from Texas Tech University Health Sciences Center and by the Center of Excellence for Translational Neuroscience and Therapeutics (CTNT) grant PN-CTNT 2017-05 AKHRJDHW to A.L.K.; in part by NIH grant R01AI099380 to K.Z. James C. Huffman and Kristen R. Baca were CISER (Center for the Integration of STEM Education &#38; Research) scholars and were supported by the program.

All animal treatments and handling of tissues obtained in the study were performed according to protocols approved by the Institutional Animal Care and Use Committee at the Texas Tech University Health Science Center in accordance with the National Institutes of Health animal welfare guidelines, protocol number 96005. Please sacrifice vertebrate animals and prepare tissues according to the guidelines from the Institutional Animal Care and Use Committee. If lacking such a committee, please refer to the National Institutes...

<ol>
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Polysome fractionation by sucrose gradient combined with RNA and protein analysis of fractions is a powerful method to analyze translational status of individual mRNAs or the whole translatome as well as roles of protein factors regulating translational machinery during normal physiological or disease state. Polysomal profiling is an especially suitable technique to study translational regulation in organisms such as trypanosomatids including Leishmania where transcriptional control is largely absent and gene ex...

Regulation of gene expression in cells is controlled by transcriptional, posttranscriptional and posttranslational mechanisms. Advances in deep RNA sequencing allow the study of steady-state mRNA levels on a genome-wide scale at an unprecedented level. However, recent findings revealed that steady-state mRNA level does not always correlate with protein production1,2. The fate of an individual transcript is very complex and depends on many factors like internal/external stimuli, stress, etc. Regulation of gene expression during protein synthesis provides another layer of expression control necessary for a rapid response in changing conditions. Polysome (or &#34;polyribosome&#34;) profiling, the separation and visualization of actively translating ribosomes, is a powerful method to study the regulation of protein synthesis. Although, its first experimental applications appeared in the 1960s3, polysome profiling is currently one of the most important techniques in protein translation studies4. Single mRNAs can be translated by more than one ribosome leading to the formation of a polysome. Transcripts can be stalled on ribosomes with cycloheximide5 and mRNAs containing different numbers of polysomes can be separated in the process of polysome fractionation by sucrose gradient ultracentrifugation6,7,8,9. RNA analysis of polysomal fractions then allows measurement of changes in the translational states of individual mRNAs on genome-wide scale and during different physiological conditions4,7,10. The method has been also used to reveal the roles of 5&#39;UTR and 3&#39;UTR sequences in control of mRNA translatability11, examine the role of miRNAs in translational repression12, uncover defects in ribosome biogenesis13, and understand the role of ribosome-associated proteins with human diseases14,15. During the last decade, a growing role for regulation of gene expression during translation has emerged that illustrates its importance in human diseases. The evidence for translational control in cancer, metabolic and neurodegenerative diseases is overwhelming15,16,17,18. For example, dysregulation of eIF4E-dependent translational control contributes to autism related deficits15 and FMRP is involved in stalling of ribosomes on mRNAs linked to autism14. Thus, polysomal profiling is a very important tool to study defects in translational regulation in multiple human diseases.
Protein analysis of polysomal fractions under different physiological conditions dissects the function of factors associated with ribosomes during translation. The polysome profiling technique has been used in many species including yeast, mammalian cells, plants, and protozoa10,19,20,21. Protozoan parasites like Trypanosoma and Leishmania exhibit limited transcriptional control of gene expression. Their genomes are organized into polycistronic gene clusters that lack promoter-regulated transcription22. Instead, developmental gene expression is predominantly controlled at the level of protein translation and mRNA stability in trypanosomatid species23,24. Therefore, understanding of translational control in the absence of transcriptional regulation is particularly important for these organisms. Polysomal profiling is a powerful tool to study posttranscriptional regulation of gene expression in Leishmania25,26,27,28.
The recent progress in detection of individual mRNAs levels by real time quantitative PCR (RT-qPCR) and full transcriptome by next-generation sequencing, as well as proteomics technologies, brings resolution and advantages of polysomal profiling to a new level. The use of these methods can be further extended by analysis of individual polysomal fractions by deep RNA sequencing combined with proteomic analysis to monitor the translational status of cells on a genome-wide scale. This allows the identification of new molecular players regulating translation under different physiological and pathological conditions. Here, we present a universal polysome profiling protocol used on three different models: the parasite Leishmania major, cultured human cells, and animal tissues. We present advice on the preparation of cell lysates from different organisms, optimization of gradient conditions, choice of RNase inhibitors and application of RT-qPCR, Western blot and RNA electrophoresis to analyze polysome fractions in this study.

<table>
	<tbody>
		<tr>
			<td>Instruments:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Gradient master</td>
			<td>Biocomp Instruments Inc.</td>
			<td>108</td>
		</tr>
		<tr>
			<td>Piston Gradient Fractionator</td>
			<td>Biocomp Instruments Inc.</td>
			<td>152</td>
		</tr>
		<tr>
			<td>Fraction collector</td>
			<td>Gilson, Inc.</td>
			<td>FC203B</td>
		</tr>
		<tr>
			<td>NanoDrop One</td>
			<td>Thermo Scientific</td>
			<td>NanoDrop One</td>
		</tr>
		<tr>
			<td>Nikon inverted microscope</td>
			<td>Nikon</td>
			<td>ECLIPSE Ts2-FL/Ts2</td>
		</tr>
		<tr>
			<td>2720 Thermal Cycler</td>
			<td>Applied Biosystems by Life Technologies</td>
			<td>4359659</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Panasonic Healthcare Co.</td>
			<td>MCO-170A1CUV</td>
		</tr>
		<tr>
			<td>HERATHERM incubator</td>
			<td>Thermo Scientific</td>
			<td>51028063</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet, class II, type A2</td>
			<td>NuAire Inc.</td>
			<td>NU-543-400</td>
		</tr>
		<tr>
			<td>Revco freezer</td>
			<td>Revco Technologies</td>
			<td>ULT1386-5-D35</td>
		</tr>
		<tr>
			<td>Beckman L8-M Ultracentifuge</td>
			<td>Beckman Coulter</td>
			<td>L8M-70</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810R</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
		</tr>
		<tr>
			<td>Ultracentrifuge Rotor SW41</td>
			<td>Beckman Coulter</td>
			<td>331362</td>
		</tr>
		<tr>
			<td>Swing-bucket rotor</td>
			<td>Eppendorf</td>
			<td>A-4-62</td>
		</tr>
		<tr>
			<td>Fixed angle rotor</td>
			<td>Eppendorf</td>
			<td>F-45-30-11</td>
		</tr>
		<tr>
			<td>Quant Studio 12K Flex Real-Time PCR machine 285880228</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4470661</td>
		</tr>
		<tr>
			<td>TC20 Automated cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0102</td>
		</tr>
		<tr>
			<td>Hemacytometer</td>
			<td>Hausser Scientific</td>
			<td>02-671-51B</td>
		</tr>
		<tr>
			<td>Software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Triax software&#160;</td>
			<td>Biocomp Instruments Inc.</td>
			<td></td>
		</tr>
		<tr>
			<td>Materials:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Counting slides, dual chamber for cell counter</td>
			<td>Bio-Rad</td>
			<td>145-0011</td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tube</td>
			<td>USA Scientific</td>
			<td>1615-5500</td>
		</tr>
		<tr>
			<td>Open-top polyclear centrifuge tubes, (14 mm x 89 mm)</td>
			<td>Seton Scientific</td>
			<td>7030</td>
		</tr>
		<tr>
			<td>Syringe, 5 mL</td>
			<td>BD</td>
			<td>309646</td>
		</tr>
		<tr>
			<td>BD Syringe 3 mL23 Gauge 1 Inch Needle</td>
			<td>BD</td>
			<td>10020439</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 14 cm</td>
			<td>Thermo Scientific</td>
			<td>168381</td>
		</tr>
		<tr>
			<td>Nunclon Delta Surface plate, 9 cm</td>
			<td>Thermo Scientific</td>
			<td>172931</td>
		</tr>
		<tr>
			<td>Nalgene rapid-flow 90mm filter unit, 500 mL, 0.2 aPES</td>
			<td>Thermo Scientific</td>
			<td>569-0020</td>
		</tr>
		<tr>
			<td>BioLite 75 cm3 flasks</td>
			<td>Thermo Scientific</td>
			<td>130193</td>
		</tr>
		<tr>
			<td>Nunc 50 mL conical centrifuge tubes</td>
			<td>Thermo Scientific</td>
			<td>339653</td>
		</tr>
		<tr>
			<td>Chemicals:</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol LS</td>
			<td>Ambion by Life Technologies</td>
			<td>10296028</td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher Scientific</td>
			<td>BP310-500</td>
		</tr>
		<tr>
			<td>Trizma base</td>
			<td>Sigma</td>
			<td>T1378-5KG</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Modified Eagle&#39;s Medium-high glucose (DMEM)</td>
			<td>Sigma</td>
			<td>D6429-500ML</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Sigma</td>
			<td>F0926-50ML</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (P/S)</td>
			<td>Sigma</td>
			<td>P0781-100ML</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>11668-019</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline (DPBS)</td>
			<td>Sigma</td>
			<td>D8537-500ML</td>
		</tr>
		<tr>
			<td>Magnesium chloride hexahydrate (MgCl2x6H2O)</td>
			<td>Acros Organics</td>
			<td>AC413415000</td>
		</tr>
		<tr>
			<td>Potassium Chloride (KCl)</td>
			<td>Sigma</td>
			<td>P9541-500G</td>
		</tr>
		<tr>
			<td>Nonidet P 40 (NP-40)</td>
			<td>Fluka (Sigma-Aldrich)</td>
			<td>74385</td>
		</tr>
		<tr>
			<td>Recombinant Rnasin Ribonuclease Inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
		</tr>
		<tr>
			<td>Heparin sodium salt</td>
			<td>Sigma</td>
			<td>H3993-1MU</td>
		</tr>
		<tr>
			<td>cOmplete Mini EDTA-free protease inhibitors</td>
			<td>Roche Diagnostics</td>
			<td>11836170001</td>
		</tr>
		<tr>
			<td>Glycogen</td>
			<td>Thermo Scientific</td>
			<td>R0551</td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W4502-1L</td>
		</tr>
		<tr>
			<td>Cycloheximide</td>
			<td>Sigma</td>
			<td>C7698-1G</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Fisher Scientific</td>
			<td>194002</td>
		</tr>
		<tr>
			<td>Dithiotreitol (DTT)</td>
			<td>Fisher Scientific</td>
			<td>BP172-5</td>
		</tr>
		<tr>
			<td>Ethidium Bromide</td>
			<td>Fisher Scientific</td>
			<td>BP-1302-10</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium dehydrate (EDTA)</td>
			<td>Fisher Scientific</td>
			<td>S316-212</td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Life Technologies</td>
			<td>22600050</td>
		</tr>
		<tr>
			<td>Puromycin dihydrochloride</td>
			<td>Sigma</td>
			<td>P8833-100MG</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Fisher Scientific</td>
			<td>S5-3KG</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Sigma</td>
			<td>T4049-100ML</td>
		</tr>
		<tr>
			<td>Hgh Capacity cDNA Reverse Transcriptase Kit</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4368814</td>
		</tr>
		<tr>
			<td>Power SYBR Green PCR Master Mix</td>
			<td>Applied Biosystems by life technologies</td>
			<td>4367659</td>
		</tr>
		<tr>
			<td>HCl</td>
			<td>Fisher Scientific</td>
			<td>A144SI-212</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Fisher Scientific</td>
			<td>BP26324</td>
		</tr>
		<tr>
			<td>Potassium Hydroxide (KOH)</td>
			<td>Sigma</td>
			<td>221473-500G</td>
		</tr>
		<tr>
			<td>Anti-RPL11 antibody</td>
			<td>Abcam</td>
			<td>ab79352</td>
		</tr>
		<tr>
			<td>Ribosomal protein S6 (C-8) antibody</td>
			<td>Santa Cruz Biotechnology Inc.</td>
			<td>sc-74459</td>
		</tr>
		<tr>
			<td>1xM199</td>
			<td>Sigma</td>
			<td>M0393-10X1L</td>
		</tr>
		<tr>
			<td>Lithium cloride</td>
			<td>Sigma</td>
			<td>L-9650</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Fisher Scientific</td>
			<td>D128-500</td>
		</tr>
		<tr>
			<td>Gel Loading Buffer II</td>
			<td>Thermo Scientific</td>
			<td>AM8546G</td>
		</tr>
		<tr>
			<td>UltraPure Agarose</td>
			<td>Thermo Scientific</td>
			<td>16500-100</td>
		</tr>
		<tr>
			<td>Trichloracetic acid (TCA)</td>
			<td>Fisher Scientific</td>
			<td>A322-100</td>
		</tr>
		<tr>
			<td>SuperSignal West Pico PLUS chemiluminescent substrate</td>
			<td>Thermo Scientific</td>
			<td>34580</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>BP531-500</td>
		</tr>
		<tr>
			<td>Sodium Dodecyl Sulfate (SDS)</td>
			<td>Sigma</td>
			<td>L5750-1KG</td>
		</tr>
		<tr>
			<td>Phenylmethylsulfonyl fluoride (PMSF)</td>
			<td>Sigma</td>
			<td>P7626-5G</td>
		</tr>
		<tr>
			<td>RNeasy Mini kit</td>
			<td>Qiagen</td>
			<td>74104</td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate (ATP)</td>
			<td>Sigma</td>
			<td>A1852-1VL</td>
		</tr>
		<tr>
			<td>Cytosine 5&#39;-triphosphate disodium salt hydrate (CTP)</td>
			<td>Sigma</td>
			<td>C1506-250MG</td>
		</tr>
		<tr>
			<td>Uridine 5&#39;-triphosphate trisodium salt hydrate (UTP)</td>
			<td>Sigma</td>
			<td>U6625-100MG</td>
		</tr>
		<tr>
			<td>Guanosine 5&#39;-triphosphate sodium salt hydrate (GTP)</td>
			<td>Sigma</td>
			<td>G8877-250MG</td>
		</tr>
		<tr>
			<td>SP6 RNA Polymerase</td>
			<td>NEB</td>
			<td>M0207S</td>
		</tr>
		<tr>
			<td>Pyrophoshatase</td>
			<td>Sigma</td>
			<td>I1643-500UN</td>
		</tr>
		<tr>
			<td>Spermidine</td>
			<td>Sigma</td>
			<td>S0266-1G</td>
		</tr>
	</tbody>
</table>

polysome profiling in leishmania, human cells and mouse testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

In this study, we describe the application of the polysomal profiling technique to three different sources: parasitic Leishmania major, cultured human cells, and mouse testis. Leishmania cells freely grow in the liquid media in suspension, cultured human cells grow in the adherent monolayer on plates, and the mouse testis represents a tissue sample. The method can be easily adjusted to other types of freely grown cells in suspension, different types of tissues, or from a...

Watch this Scientific Journal Video about Polysome Profiling in Leishmania, Human Cells and Mouse Testis at JoVE.com

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

The overall goal of this technique is to study engagement of indivdual mRNA's with polysomes during translation. Polysome profiling is a powerful technology to study mRNA engagement with ribosomes and polysomes. The technique is important for studies of protein synthesis regulation, translation activation, and repression.Here our team presents the techniques for polysomal fractionation, and analysis of fractions on the example of three organisms. Parasite Leishmania major, cultured human cells, and an animal tissue. This approach consists of four major steps, lysate preparation, sucrose gradient preparation and ultracentrifugation, polysome fractionation, and sample collection.Analysis of the fractions. Cells from different sources are collected, washed and lysed in lysis buffer by passage through a needle or Dounce homogenizer. Centrifugation is used to remove cell debris clarifying the lysate.A continuous sucrose gradient is formed by the mixing of 10%and 50%sucrose solutions in a gradient maker. Lysate is loaded on the top of the gradient. Ultracentrifugation separates mrNA's associated with different number of ribosomes.Which is monitored by a UV Detector during fractionation, forming distinct absorbant spectrums. Lysate preparation. There are some differences in the preparation of the lysate depending on the source.After lysate preparation, procedures are identical regardless of the sample origin. Leishmania major Cytoplasmic Lysate preparation. Place Leishmania major cells in 30 milliliter of medium at the density of 100, 000 cells per milliliter.This step is conducted in a biosafety cabinet. Place the flask with Leishmania culture in the incubator and grow cells at 27 degrees Celsius, until the logarithmic phase. It usually takes about two days to grow.Leishmania major cells are easily visible under the microscope. Add Cycloheximide to the grown Leishmania culture to a final concentration of 100 micrograms per milliliter, to arrest ribosomes on translated mRNAs. Place cells back in the incubator for ten minutes at 27 degrees Celsius.After cycloheximide treatment is completed transfer cells to a 50 milliliter conical tube and spin it 1, 800 g for 8 minutes. Discard supernatant. Wash cells with 30 milliliter of pbs and centrifuge.Discard the supernatant and re-suspend the cells in one milliliter of pbs. Take an aliquot of cells and mix it with the formaldehyde solution. Then count cells by hemocytometer.Transfer the desired number of cells into microfuge tube. Spin the cells at 1, 800 g for 8 minutes at four degrees Celsius. Then discard the supernatant.Re-suspend the cell pellet in one milliliter of lysis buffer containing protease inhibitors and RNAsin. Pass the cells through a 23 gauge needle three times. After passing through the needle, the lysate becomes transparent.Centrifuge at 11, 200 g for ten minutes at four degrees Celsius to clarify lysate. Transfer the clarified lysate to a fresh microfuge tube and keep it on ice until sucrose gradient ultracentrifugation. Cytoplasmic lysate preparation from cultured human cells.Grow HeLa cells in a large plate in the incubator at 37 degrees Celsius, with 5%CO2. Add cycloheximide to grown HeLa cells to the final concentration of 100 micrograms per milliliter to arrest the ribosomes on translated mRNA's. Incubate cells for ten minutes at 37 degrees Celsius with 5%CO2.Aspirate medium and wash plate on ice twice with cold pbs. Add 500 microliters of lysis buffer to the plate and scrape the cells. Transfer the lysed cells to the microfuge tube.Pass the cells through a 23 gauge needle three times. Spin at 11, 200 g for eight minutes to clarify lysate. After centrifugation move the tubes on ice, and transfer supernatant to the new tube.Take an aliquot, dilute it, and measure absorbance at 260 nanometer using a nano drop. If there are multiple samples, adjust the lysates to the same optical density and keep the samples on ice until sucrose gradient ultracentrifugation. Cytoplasmic lysate preparation from mouse testis.Dissect the mouse testis. Make a small incision in the tunica albugenia and collect the seminiforous tubules of the testis and transfer them in the conical tube containing five milliliters of pbs. Mix the tissue vigorously.And allow the tissue to settle on ice for five minutes. Remove the buffer, and wash it with pbs two more times. Transfer the seminiferous tubules in a two milliliter tube and spin them down at 500 g for one minute.Remove the supernatant. Add 500 microliter lysis buffer to the tubules and use a pipet to disrupt the tissue. Transfer the suspension to a small volume dounce homogenizer.Disrupt the tissue with seven to eight strokes of the glass pestle. Transfer the lysate to a micro centrifuge tube. Centrifuge the sample at 12, 000 g at four degrees Celsius for eight minutes to clear the lysate.Transfer the supernatant into a new tube, and keep it on ice. Then, proceed with sucrose gradient preparation. Sucrose gradient preparation.Place an ultracentrifuge tube into the microblock and draw the line along the upper level of the block. Then transfer the tube into a stable rack. Take a ten milliliter syringe, with the layering device attached and fill the syringe with 10%sucrose solution.Gently release it at the bottom of the ultracentrifuge tube until sucrose solution reaches the mark. Fill another syringe with 50%sucrose solution and carefully insert its layering device through the 10%sucrose layer to the bottom of the tube. Gently release sucrose solution, starting from the bottom until it reaches the mark on the tube.Then seal the tube with provided cap. To prepare sucrose gradient, turn the gradient maker device on. Then level the plate using the up or down buttons and press done.After leveling the plate press grad. Go to the list of gradient menu and select the SW41 rotor. Then choose desired sucrose gradient.Press use. Place magnet base tube holder on the gradient maker. Then transfer the tube into the holder.Up to six gradients can be prepared at the same time. Press run. The gradient maker rotates the tubes at the program speed and angles, forming a linear gradient.It will take only a few minutes to prepare the gradient. When the process is completed, place the tube in a rack. Remove the volume, equal of sample volume from the top of the ultracentrifuge tubes with sucrose gradients.Carefully load 500 microliters of the lysate on top. Place the tubes in rotor buckets and balance them. Centrifuge at 260, 000 g for two hours at four degrees Celsius.Polysome fractionation and sample collection. After ultracentrifugation, place the tubes in the rotor buckets on ice. Turn the fraction collector and the fractionator on.Click scan on the fractionator menu. Put a rack with collection tubes into fraction collector. Fill up a rinse reservoir, on the side of the fractionator with deionized water.Press rinse key for ten seconds to rinse the pump. Attach a rinse adapter with the syringe filled with water to the piston for the calibration. Open the fractionator software on the computer.Press calibrate. Use the default setting and press Ok.Be ready to inject water from the syringe. Press Ok to do calibration.Immediately start injecting water for the next five seconds. The sign Zero calibration completed will appear. Remove a rinse adapter with the syringe and attach a tip to the piston of the fractionator.Open the brass air valve and press air key for ten seconds to dry air tubing and flow cell. Close the air valve. Take the gradient tube from the rotor bucket and place it in the rack.Apply the tube holder cap to the top of the tube, and carefully move the tube into the tube holder and lock it. Place the holder under the piston. Often, polysomal bands can be seen by eye.Introduce the desired settings for fraction numbers and volume. Name the file and press Ok, then go to graph button. In the next window press start scan.Settings will appear, and press Ok.The collector will move from the gutter to the first fraction, and the piston will move into the tube. When the piston will reach the top of the gradient it will slow to your selected speed, and fractions will be collected. Usually, we collect 24 fractions at 500 microliters each.When completed, the piston will move out of the gradient tube. Open the brass air valve. Press air key on the fractionator to retrieve the last fraction.At the end of the run you will see the absorbance profile of the gradient. Analysis of the fractions. Fractions obtained can be used for RNA and Protein analysis.RNA can be analyzed by electrophoresis followed by Northern Blot or used for cDNA production, followed by RT-qPCR reaction to analyze association of individual mRNA's with polysomes. Next generation sequencing can be used to analyze the translational status of mRNA's on a transcriptome scale. For protein analysis of polysomal fractions, proteins are precipitated with tricluricedic acid to concentrate them.Then proteins are analyzed by Western Blotting, or by Mass Spec at the proteome level.Results. Here we present the results of polysome fractionation and analysis on the examples of three organisms. Leishmania, cultured human cells, and mouse tissue.Polysome fractionation was used to study Sherp and Tubulin mRNA's association with ribosomes during translation and Leishmania major. The absorbance graph for the fractionation has a distinct shape with typical peaks for ribosome sub-units 40S and 60S, monosomes 80S, and polysomes. Aliquots of the fractions were combined into three groups.Pre-polysomes, light polysomes, and heavy polysomes. The mRNA relative levels were studied by a real time quantitative PCR. The data suggests that the Tubulin mRNA is actively translated.As it was mostly associated with heavy and light polysomes. While the translation of Sherp mRNA is less active. It was mostly found in association with pre-polysomes and light polysomes.Thus, this experiment clearly demonstrates the ability of the technique to study an individual mRNA engagement in translation. This figure presents a typical absorbance graph of the HeLa cells polysome fractionation. The proteins were concentrated by precipitation with 10%trichloroacetic acid.The small sub unit ribosomal protein RPS6, and large sub unit ribosomal protein RPL11 were detected by Western Blot. Their distribution correlated well with the distinct peaks, on the absorbance spectrum. On this figure, we present an example of the detection of ribosomal RNAs by electrophoresis in fractionated polysomes form the mouse testis.Conclusions.Polysomal profiling is a versatile technique, that can be used to analyze translational state of individual mRNAs, examine ribosome-associated proteins, and study translational regulation in different model organisms under different conditions.

polysome-profiling-in-leishmania-human-cells-and-mouse-testis

Research

JoVE Journal

Biochemistry

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug used to treat depression and anxiety by binding to the serotonin transporter with high-affinity, blocking serotonin reuptake. Here we report an efficient procedure and a set of tools to stabilize, express, purify, and crystallize serotonin transporter-antibody complexes bound to S-citalopram and other antidepressants. Mutations which stabilize the serotonin transporter were identified using an S-citalopram binding assay. Serotonin transporter expressed in baculovirus-transduced HEK293S GnTI- cells, was reconstituted into proteoliposomes and used to raise high-affinity antibodies. We have developed a strategy to discover antibodies that are useful for structural studies. A straightforward approach for the expression of antibody fragments in Sf9 cells has also been established. Transporter-antibody complexes purified using this procedure are well-behaved and readily crystallize, producing complexes with S-citalopram that diffract X-rays to 3-4 &#197; resolution. The strategies developed here can be utilized to determine the structure of other challenging membrane proteins.

Thermostabilization, Expression, Purification, and Crystallization of the Human Serotonin Transporter Bound to S-citalopram

This manuscript describes how to screen for thermostabilizing mutations, purify the human serotonin transporter, generate high affinity antibodies, and crystallize the serotonin transporter-antibody complex bound to the antidepressant drug S-citalopram. This protocol can be adapted to the study of other challenging membrane transporters, receptors, and channels.

The serotonin transporter is a sodium and chloride-coupled transporter that &#34;pumps&#34; extracellular serotonin into cells. S-citalopram is a drug ...

Genome-wide Quantification of Translation in Budding Yeast by Ribosome Profiling

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription reflect changes in protein synthesis, but many exceptions have been observed. Recently, a technique called ribosome profiling (or Ribo-Seq) has emerged as a powerful method that allows identification, with high accuracy, which regions of mRNA are translated into proteins and quantification of translation at the genome-wide level. Here, we present a generalized protocol for genome-wide quantification of translation using Ribo-Seq in budding yeast. In addition, combining Ribo-Seq data with mRNA abundance measurements allows us to simultaneously quantify translation efficiency of thousands of mRNA transcripts in the same sample and compare changes in these parameters in response to experimental manipulations or in different physiological states. We describe a detailed protocol for generation of ribosome footprints using nuclease digestion, isolation of intact ribosome-footprint complexes via sucrose gradient fractionation, and preparation of DNA libraries for deep sequencing along with appropriate quality controls necessary to ensure accurate analysis of in vivo translation.

Translational regulation plays an important role in the control of protein abundance. Here, we describe a high-throughput method for quantitative analysis of translation in the budding yeast Saccharomyces cerevisiae.

Translation of mRNA into proteins is a complex process involving several layers of regulation. It is often assumed that changes in mRNA transcription ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

High-Resolution Complexome Profiling by Cryoslicing BN-MS Analysis

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed complexes. The latter can be elegantly resolved according to molecular size using native polyacrylamide gel electrophoresis (BN-PAGE). Coupling of such separations to sensitive mass spectrometry (BN-MS) has been well-established and theoretically allows for exhaustive assessment of the extractable complexome in biological samples. However, this approach is rather laborious and provides limited complex size resolution and sensitivity. Also, its application has remained restricted to abundant mitochondrial and plastid proteins. Thus, for a majority of proteins, information regarding integration into stable protein complexes is still lacking. Presented here is an optimized approach for complexome profiling comprising preparative-scale BN-PAGE separation, sub-millimeter sampling of broad gel lanes by cryomicrotome slicing, and mass spectrometric analysis with label-free protein quantification. The procedures and tools for critical steps are described in detail. As an application, the report describes complexome analysis of a solubilized endosome-enriched membrane fraction from mouse kidneys, with 2,545 proteins profiled in total. The results demonstrate identification of uniform, low-abundance membrane proteins such as intracellular ion channels as well as high resolution, complex protein assembly patterns, including glycosylation isoforms. The results are in agreement with independent biochemical analyses. In summary, this methodology allows for comprehensive and unbiased identification of protein (super)complexes and their subunit composition, providing a basis for investigating stoichiometry, assembly, and interaction dynamics of protein complexes in any biological system.

A versatile cryoslicing BN-MS protocol using a microtome is presented for high-resolution complexome profiling.

Proteins generally exert biological functions through interactions with other proteins, either in dynamic protein assemblies or as a part of stably formed ...

Profiling Ubiquitin and Ubiquitin-like Dependent Post-translational Modifications and Identification of Significant Alterations

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the cell by controlling protein stability, activity, interactions, and intracellular localization. They enable the cell to respond to signals and to adapt to changes in its environment. Alterations within these mechanisms can lead to severe pathological situations such as neurodegenerative diseases and cancers. The aim of the technique described here is to establish ub/ubls dependent PTMs profiles, rapidly and accurately, from cultured cell lines. The comparison of different profiles obtained from different conditions allows the identification of specific alterations, such as those induced by a treatment for example. Lentiviral mediated cell transduction is performed to create stable cell lines expressing a two-tags (6His and Flag) version of the modifier (ubiquitin or a ubl such as SUMO1 or Nedd8). These tags permit the purification of ubiquitin and therefore of ubiquitinated proteins from the cells. This is done through a two-step purification process: The first one is performed in denaturing conditions using the 6His tag, and the second one in native conditions using the Flag tag. This leads to a highly specific and pure isolation of modified proteins which are subsequently identified and semi-quantified by liquid chromatography followed by tandem mass spectrometry (LC-MS/MS) technology. Easy informatics analysis of MS data using Excel software enables the establishment of PTM profiles by eliminating background signals. These profiles are compared between each condition in order to identify specific alterations which will then be studied more specifically, starting with their validation by standard biochemistry techniques.

This protocol aims at establishing ubiquitin (Ub) and ubiquitin-likes (Ubls) specific proteomes in order to identify alterations of these kind of post-translational modifications (PTMs), associated with a specific condition such as a treatment or a phenotype.

Ubiquitin (ub) and ubiquitin-like (ubl) dependent post-translational modifications of proteins play fundamental biological regulatory roles within the ...

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (&#62; 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (&#60; 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.

Phosphoproteomic Strategy for Profiling Osmotic Stress Signaling in Arabidopsis

Presented here is a phosphoproteomic approach, namely stop and go extraction tip based phosphoproteomic, which provides high-throughput and deep coverage of Arabidopsis phosphoproteome. This approach delineates the overview of osmotic stress signaling in Arabidopsis.

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based ...

Ganglioside Extraction, Purification and Profiling

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially abundant in the brain. Expressed primarily on the outer leaflet of the plasma membranes of cells, they modulate the activities of cell surface proteins via lateral association, act as receptors in cell-cell interactions and are targets for pathogens and toxins. Genetic dysregulation of ganglioside biosynthesis in humans results in severe congenital nervous system disorders. Because of their amphipathic nature, extraction, purification, and analysis of gangliosides require techniques that have been optimized by many investigators in the 80 years since their discovery. Here, we describe bench-level methods for the extraction, purification, and preliminary qualitative and quantitative analyses of major gangliosides from tissues and cells that can be completed in a few hours. We also describe methods for larger scale isolation and purification of major ganglioside species from brain. Together, these methods provide analytical and preparative scale access to this class of bioactive molecules.

Gangliosides are sialic acid-bearing glycosphingolipids that are particularly abundant in the brain. Their amphipathic nature requires organic/aqueous extraction and purification techniques to ensure optimal recovery and accurate analyses. This article provides overviews of analytic and preparative scale ganglioside extraction, purification, and thin layer chromatography analysis.

Gangliosides are glycosphingolipids that contain one or more sialic acid residues. They are found on all vertebrate cells and tissues but are especially ...

Polysome Profiling without Gradient Makers or Fractionation Systems

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs being translated by ribosomes, and analyze polysome associated factors. While automated gradient makers and gradient fractionation systems are commonly used with this technique, these systems are generally expensive and can be cost-prohibitive for laboratories that have limited resources or cannot justify the expense due to their infrequent or occasional need to perform this method for their research. Here, a protocol is presented to reproducibly generate polysome profiles using standard equipment available in most molecular biology laboratories without specialized fractionation instruments. Moreover, a comparison of polysome profiles generated with and without a gradient fractionation system is provided. Strategies to optimize and produce reproducible polysome profiles are discussed. Saccharomyces cerevisiae is utilized as a model organism in this protocol. However, this protocol can be easily modified and adapted to generate ribosome profiles for many different organisms and cell types.

This protocol describes how to generate a polysome profile without using automated gradient makers or gradient fractionation systems.

Polysome fractionation by sucrose density gradient centrifugation is a powerful tool that can be used to create ribosome profiles, identify specific mRNAs ...