This work was funded by Human Frontier Science Program grant RGP0024.

1. DNA Template Preparation and in vitro Transcription
<ol>
 <li>The gene of interest is cloned in any T7 and/or e.g. SP6 promoter based plasmid. To obtain RNCs of interest, C-terminus of the target polypeptide is extended by adding arrest inducing sequence of SecM FXXXXWIXXXXGIRAGP7. In order to ensure that the polypeptide fragment of interest will extrude out of the ribosomal tunnel, the C-terminal part of the target protein has to be extended by at least 30 amino acids12-14. A flexible Glycine-Serine rich linker can be introduced between the protein and the SecM arrest sequence to avoid any possible conformational constraints.</li>
 <li>For in vitro transcription, template DNA should be linearized with restriction enzyme cutting downstream of the ORF. One needs to verify complete linearization of the plasmid DNA by running the restriction digestion product on agarose gel electrophoresis.</li>
 <li>The linearized plasmid is further used for in vitro transcription reaction. Different concentration of template DNA can be tested to identify optimum DNA concentration required for in vitro transcription. Generally with Ambion's MEGAscript High yield Transcription Kit (Ambion/Life Technologies, Grand Island, NY), 1 &mu;g linearized DNA yields 40-60 &mu;g mRNA. In vitro transcription is done following manufacturer's instruction (Ambion/Life Technologies, Grand Island, NY).</li>
 <li>Following in vitro transcription, mRNA is purified by lithium chloride precipitation according to manufacturer's instruction (Ambion's MEGAscript High yield Transcription Kit, Ambion/Life Technologies, Grand Island, NY).</li>
 <li>mRNA integrity is further verified by electrophoresis using acrylamide or agarose gels.</li>
</ol>
2. In vitro Translation
For in vitro translation using the RTS 100 E. coli HY Kit (5 Prime, Gaithersburg, MD) follow the steps below:
<ol>
 <li>Prepare 100 &mu;l of in vitro translation reaction following manufacturer's instruction. Briefly, in nuclease free water add 24 &mu;l amino acid mixture minus methionine (1 mM), 10 units of Ribonuclease Inhibitor (Invitrogen/Life Technologies, Grand Island, NY), 20 &mu;Ci of radioactive [35S]-Methionine (MP Biomedicals, Solon, OH), 20 &mu;l Reaction mix, 24 &mu;l Reconstitution buffer and 24 &mu;l of E. coli Lysate.</li>
 <li>Incubate the reaction at 30 &deg;C for 5 min to pre warm the translation reaction. Add 1-2 &mu;g of mRNA to the translation reaction and incubate at 30 &deg;C for 10-15 min.</li>
 <li>Stop the reaction by placing the translation reaction on ice.</li>
</ol>
3. Isolation of Nascent Polypeptide from in vitro Translation Reaction
<ol>
 <li>To isolate RNCs, the translation reaction is layered on top of 4.5 ml of 5-30% sucrose gradient in 20 mM HEPES-KOH pH 7.5, 15 mM MgCl2, 100 mM Potassium acetate, 1 mM DTT and centrifuged using Beckman Coulter SW55-Ti rotor at 41,000 rpm, 4 &deg;C for 2 hrs.</li>
 <li>Following centrifugation, sucrose gradient is fractionated to isolate different ribosomal populations and associated nascent chains. Separation is monitored using the ISCO Programmable Density Gradient System with continuous recording at 254 nm using an ISCO UA-6 absorbance detector.</li>
 <li>The fraction(s) containing 70S ribosomes are collected and analyzed further depending on the aim of the experiment.</li>
</ol>
4. Observation of Protein Bound to Ribosome with Tris-tricine SDS PAGE
To check, whether the SecM extended protein remains attached to the 70S ribosome, gradient fractions were collected and treated as follows:
<ol>
 <li>Protein in each fraction was precipitated by adding Trichloroacetic acid (TCA) to a final concentration of 10% and incubated at 4 &deg;C overnight.</li>
 <li>After overnight incubation, samples were centrifuged at 14,000 X g for 15 min to pellet the protein. Following centrifugation supernatant was removed and the pellet washed twice with solution containing Acetone: 1 mM Tris-HCl pH 7.6 (4:1). Pellet was further air dried and dissolved in SDS-PAGE loading buffer.</li>
 <li>The treated sample was resolved and analyzed by Tris-tricine SDS-PAGE.</li>
 <li>After electrophoresis, gels were fixed, dried using vacuum gel dyer and subjected to autoradiography. The distribution of nascent polypeptides were observed using phosphoimaging.</li>
</ol>
5. Representative Results
Here we present an experiment describing the isolation of the full-length bovine Gamma-B Crystallin stably bound to the 70S ribosome. Figure 1 depicts steps involved in the isolation of bovine Gamma-B Crystallin RNCs. The C-terminus of the Gamma- B Crystallin was extended overall by 32 amino acids to ensure that full-length protein extrudes out of the ribosomal tunnel; this also includes the SecM stalling sequence placed at the very C-terminus of the fusion polypeptide. Following in vitro translation, the 70S ribosomes were isolated by sucrose gradient centrifugation (Figure 2.1). In order to ensure that the protein remains stably bound to the ribosome, the 70S containing fractions were pooled, desalted, buffer exchanged and subjected to an additional round of sucrose gradient centrifugation (Figure 2.2). The result presented in Figure 2 clearly suggests that SecM can efficiently induce translational arrest of the Gamma-B Crystallin RNCs.
<img alt="Figure 1" src="/files/ftp_upload/4027/4027fig1.jpg" /> 
Figure 1. Schematic explanation of the steps involved in isolation of RNCs. C-terminus of bovine Gamma-B Crystallin was extended by 32 amino acid sequence. This extended C-terminal regions also includes SecM arrest sequence. Gamma-B Crystallin with SecM sequence was cloned in pIVEX 2.3 (T7 based) plasmid. For in vitro transcription the template DNA was linearized by XbaI. Linearized template was further used for in vitro transcription. The T7 in the DNA template is recognized by T7 RNA polymerase that transcribes the Gamma-B Crystallin gene located downstream of the T7 promoter. mRNA was purified using lithium chloride precipitation method. The purified mRNA was then used for in vitro translation. Following incubation, the translation reaction was layered on top of 5-30% sucrose gradient and centrifuged in Beckman Coulter SW55-Ti rotor at 41,000 rpm, 4 &deg;C for 2 hrs.
<img alt="Figure 2" src="/files/ftp_upload/4027/4027fig2.jpg" /> 
Figure 2. Isolation of bovine Gamma-B Crystallin RNCs stably bound to the 70S ribosome. 1 &mu;g mRNA was mixed in 100 &mu;l reaction E. coli S30 Extract System as mentioned in the protocol section with 20 &mu;Ci [35S]-Methionine and incubated at 30 &deg;C for 15 min. Following incubation, the translation reaction was layered on top of 5-30% sucrose gradient in 20 mM HEPES-KOH pH 7.5, 15 mM MgCl2, 100 mM Potassium acetate, 1 mM DTT and centrifuged in Beckman Coulter SW55-Ti rotor at 41,000 rpm, 4 &deg;C for 2 hrs. Following velocity sedimentation, the gradient was unloaded and the ribosome pro le was obtained. The data were recorded by the PeakTrak program (ISCO gradient density gradient fractionation system). Fractions were collected, the protein was TCA precipitated and resolved on 16.5% T, 6% C Tris-Tricine PAGE gel15. The gel was dried and exposed for autoradiography. Following exposure the gel was scanned using Typhoon 9410 imaging scanner. Figure 2.1 shows that full-length Gamma-B Crystallin is present in 70S ribosome fractions. Thus, SecM stalling sequence allows the isolation of the stable bovine Gamma-B Crystallin RNCs. Data in Figure 2.2 clearly indicate that the isolated RNCs are indeed stable. In the current experiment the 70S fractions after first round of sucrose gradient centrifugation were pooled and the sucrose was removed using Amicon Ultra-4 Centrifugal Filter Unit (Millipore), followed by buffer exchange in solution containing 20 mM HEPES-KOH pH 7.5, 15 mM MgCl2, 100 mM Potassium acetate, 1 mM DTT. This sample was further subjected to an additional round of centrifugation through 5-30% sucrose gradient and fractionated. Each fraction was treated and analyzed like in Figure 2.1.

<ol>
	<li>Fedorov, A. N., Baldwin, T. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+protein+folding.">Cotranslational protein folding.</a> J. Biol. Chem. 272, 32715-32718 (1997).</li><li>Hardesty, B., Kramer, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Folding+of+a+nascent+peptide+on+the+ribosome.">Folding of a nascent peptide on the ribosome.</a> Prog. Nucleic Acid Res. Mol. Biol. 66, 41-66 (2001).</li><li>Kramer, G., Boehringer, D., Ban, N., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosome+as+a+platform+for+co-translational+processing,+folding+and+targeting+of+newly+synthesized+proteins.">The ribosome as a platform for co-translational processing, folding and targeting of newly synthesized proteins.</a> Nat. Struct. Mol. Biol. 16, 589-597 (2009).</li><li>Komar, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pause+for+thought+along+the+co-translational+folding+pathway.">A pause for thought along the co-translational folding pathway.</a> Trends Biochem. Sci. 34, 16-24 (2009).</li><li>Cabrita, L. D., Dobson, C. M., Christodoulou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+folding+on+the+ribosome.">Protein folding on the ribosome.</a> Curr. Opin. Struct. Biol. 20, 33-45 (2010).</li><li>Oliver, D., Norman, J., Sarker, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+Escherichia+coli+secA+by+cellular+protein+secretion+proficiency+requires+an+intact+gene+X+signal+sequence+and+an+active+translocon.">Regulation of Escherichia coli secA by cellular protein secretion proficiency requires an intact gene X signal sequence and an active translocon.</a> J. Bacteriol. 180, 5240-5242 (1998).</li><li>Nakatogawa, H., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ribosomal+exit+tunnel+functions+as+a+discriminating+gate.">The ribosomal exit tunnel functions as a discriminating gate.</a> Cell. 108, 629-636 (2002).</li><li>Nakatogawa, H., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secretion+monitor,+SecM,+undergoes+self-translation+arrest+in+the+cytosol.">Secretion monitor, SecM, undergoes self-translation arrest in the cytosol.</a> Mol. Cell. 7, 185-192 .</li><li>Muto, H., Nakatogawa, H., Ito, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+but+non+polypeptide+prolyl-tRNA+functions+in+the+A+site+for+SecM-mediated+ribosomal+stall.">Genetically encoded but non polypeptide prolyl-tRNA functions in the A site for SecM-mediated ribosomal stall.</a> Mol. Cell. 22, 545-552 (2006).</li><li>Cabrita, L. D., Hsu, S. T., Launay, H., Dobson, C. M., Christodoulou, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probing+ribosome-nascent+chain+complexes+produced+in+vivo+by+NMR+spectroscopy.">Probing ribosome-nascent chain complexes produced in vivo by NMR spectroscopy.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 22239-22244 (2009).</li><li>Eichmann, C., Preissler, S., Riek, R., Deuerling, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+structure+acquisition+of+nascent+polypeptides+monitored+by+NMR+spectroscopy.">Cotranslational structure acquisition of nascent polypeptides monitored by NMR spectroscopy.</a> Proc. Natl. Acad. Sci. USA. 107, 9111-9116 (2010).</li><li>Kolb, V. A., Makeyev, E. V., Spirin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+activity+of+the+ribosome-bound+nascent+polypeptide.">Enzymatic activity of the ribosome-bound nascent polypeptide.</a> FEBS Lett. 378, 166-170 (1996).</li><li>Ban, N., Nissen, P., Hansen, J., Moore, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+complete+atomic+structure+of+the+large+ribosomal+subunit+at+2.4+Å+resolution.">The complete atomic structure of the large ribosomal subunit at 2.4 Å resolution.</a> Science. 289, 905-920 (2000).</li><li>Voss, N. R., Gerstein, M., Steitz, T. A., Moore, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+geometry+of+the+ribosomal+polypeptide+exit+tunnel.">The geometry of the ribosomal polypeptide exit tunnel.</a> J. Mol. Biol. 360, 893-906 (2006).</li><li>Sch&#228;gger, H., von Jagow, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tricine-sodium+dodecyl+sulfate-polyacrylamide+gel+electrophoresis+for+the+separation+of+proteins+in+the+range+from+1+to+100+kDa.">Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa.</a> Anal. Biochem. 166, 368-379 (1987).</li><li>Komar, A. A., Kommer, A., Krasheninnikov, I. A., Spirin, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cotranslational+folding+of+globin.">Cotranslational folding of globin.</a> J. Biol. Chem. 272, 10646-10651 (1997).</li><li>Keiler, K. C., Waller, P. R., Sauer, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+a+peptide+tagging+system+in+degradation+of+proteins+synthesized+from+damaged+messenger+RNA.">Role of a peptide tagging system in degradation of proteins synthesized from damaged messenger RNA.</a> Science. 271, 990-993 (1996).</li><li>Evans, M. S., Ugrinov, K. G., Frese, M. A., Clark, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+stalled+ribosome+nascent+chain+complexes+produced+in+vivo+or+in+vitro.">Homogeneous stalled ribosome nascent chain complexes produced in vivo or in vitro.</a> Nat. Methods. 2, 757-762 (2005).</li></ol>

No conflicts of interest declared.

For reproducible results, quality and concentration of the components used for in vitro transcription and translation are critical. We have used commercially available in vitro transcription and translation extracts and they give efficient and reproducible results, if handled carefully. Quality of mRNA can affect the translation, so it is of utmost importance to test the integrity of mRNA before using it for in vitro translation. Incubation time for in vitro translation varies with the...

<table border="1">
	<tbody>
		<tr>
			<td>Name of reagent/ Kit</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>MEGAscript T7 High yield Transcription Kit</td>
			<td>Ambion</td>
			<td>AM1333</td>
		</tr>
		<tr>
			<td>RTS 100 E.coli HY Kit</td>
			<td>5 Prime</td>
			<td>2401100</td>
		</tr>
		<tr>
			<td>Ribonuclease Inhibitor</td>
			<td>Invitrogen</td>
			<td>15518012</td>
		</tr>
		<tr>
			<td>Trans [35S]-Label</td>
			<td>MP Biomedicals</td>
			<td>0151006</td>
		</tr>
		<tr>
			<td>Amicon Ultra-4 Centrifugal Filter Unit</td>
			<td>Millipore</td>
			<td>UFC801008</td>
		</tr>
		<tr>
			<td>Storage phosphor autoradiography</td>
			<td>GE Healthcare</td>
			<td>Typhoon 9410 variable mode imager</td>
		</tr>
		<tr>
			<td>Density Gradient Fractionation Systems</td>
			<td>Teledyne Isco, Inc.</td>
			<td>ISCO Programmable Density Gradient System</td>
		</tr>
		<tr>
			<td>Sucrose Gradient Centrifugation</td>
			<td>Beckman Coulter</td>
			<td>Optima L-90 K Preparative Ultracentrifuge</td>
		</tr>
	</tbody>
</table>

using secm arrest sequence as a tool to isolate ribosome bound polypeptides

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway that polypeptide chain follows during co-translational folding to achieve its functional form is still an enigma. In order to understand this process and to determine the exact conformation of the co-translational folding intermediates, it is essential to develop techniques that allow the isolation of RNCs carrying nascent chains of predetermined sizes to allow their further structural analysis.
SecM (secretion monitor) is a 170 amino acid E. coli protein that regulates expression of the downstream SecA (secretion driving) ATPase in the secM-secA operon6. Nakatogawa and Ito originally found that a 17 amino acid long sequence (150-FSTPVWISQAQGIRAGP-166) in the C-terminal region of the SecM protein is sufficient and necessary to cause stalling of SecM elongation at Gly165, thereby producing peptidyl-glycyl-tRNA stably bound to the ribosomal P-site7-9. More importantly, it was found that this 17 amino acid long sequence can be fused to the C-terminus of virtually any full-length and/or truncated protein thus allowing the production of RNCs carrying nascent chains of predetermined sizes7. Thus, when fused or inserted into the target protein, SecM stalling sequence produces arrest of the polypeptide chain elongation and generates stable RNCs both in vivo in E. coli cells and in vitro in a cell-free system. Sucrose gradient centrifugation is further utilized to isolate RNCs.
The isolated RNCs can be used to analyze structural and functional features of the co-translational folding intermediates. Recently, this technique has been successfully used to gain insights into the structure of several ribosome bound nascent chains10,11. Here we describe the isolation of bovine Gamma-B Crystallin RNCs fused to SecM and generated in an in vitro translation system.

Watch this Scientific Journal Video about Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides at JoVE.com

Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

We describe here a technique that is now routinely used to isolate stably bound ribosome nascent chain complexes (RNCs). This technique takes advantage of the discovery that a 17 amino acid long SecM "arrest sequence" can halt translation elongation in a prokaryotic (E. coli) system, when inserted into (or fused to the C-terminus) of virtually any protein.

Extensive research has provided ample evidences suggesting that protein folding in the cell is a co-translational process1-5. However, the exact pathway ...

using-secm-arrest-sequence-as-tool-to-isolate-ribosome-bound

Research

JoVE Journal

Biology

12.3K 次观看.  Cleveland State University. 我们在这里描述，现在经常使用的孤立稳定结合的核糖体新生肽链复合物（RNC）中的技术。这种技术发现了17个氨基酸长SECM“逮捕序列”可以停止在原核翻译延伸的优势（ E。大肠杆菌）系统，几乎所有的蛋白质（或C-末端融合）插入时。

视频: Using SecM Arrest Sequence as a Tool to Isolate Ribosome Bound Polypeptides

Microfabricated Post-Array-Detectors (mPADs): an Approach to Isolate Mechanical Forces

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated through myosin-actin interactions and play an important role in our physiology.  During development, they enable cells to move from one location to the next in order to form the early structures of tissue.  Traction forces help in the healing processes.  They are necessary for the proper closure of wounds or the migration and crawling of leukocytes through our body.  These same forces can be detrimental to our health in the case of cancer metastasis or vascular growth towards a tumor.  The most common method by which to study cells in vitro has been to use a glass or polystyrene dish.  However, the rigidity of the substrates makes it impossible to physically measure cell traction forces, and there are relatively few methods to study traction forces.  Our lab has developed a technique to overcome these limitations.  The method is based on a vertical array of flexible cantilevers, the stiffness and size scale of which are such that individual cells spread across many cantilevers and deflect them in the process.  The pillars we use are 3 &mu;m in diameter, 10 &mu;m tall, and are configured in a regular array with 9 &mu;m center-to-center spacing.  But these physical dimensions can be readily varied to accommodate a variety of studies.  We start with a silicon master, but the final posts are made out of silicone rubber called poly (dimethyl siloxane), or PDMS.  We can measure the deflections under a microscope and calculate the magnitude and direction of traction forces required to produce the observed deflections.  We call these substrates microfabricated post-array-detectors, or mPADs.  Here, we will show you how we fabricate and use the mPADs to assess modulations of cellular contractility.

Microfabricated Post-Array-Detectors mPADs: an Approach to Isolate Mechanical Forces

In this video, we demonstrate how to fabricate and utilize microfabricated post array detectors (mPADs) to assess modulations of cellular contractility.

In this video, we will present our approach to measure cellular traction forces using a microfabricated array of posts.  Traction forces are generated ...

Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity necessary for proper cellular function. Understanding how the enzymes, proteins, and cytoskeletal components govern and maintain this biochemical segregation is therefore of paramount importance. The use of fluorescently tagged molecules to localize to and/or perturb subcellular compartments has yielded a wealth of knowledge and advanced our understanding of cellular regulation. Imaging techniques such as fluorescent and confocal microscopy make ascertaining the position of a fluorescently tagged small molecule relatively straightforward, however the resolution of very small structures is limited 1.
On the other hand, electron microscopy has revealed details of subcellular morphology at very high resolution, but its static nature makes it difficult to measure highly dynamic processes with precision 2,3. Thus, the combination of light microscopy with electron microscopy of the same sample, termed Correlative Light and Electron Microscopy (CLEM) 4,5, affords the dual advantages of ultrafast fluorescent imaging with the high-resolution of electron microscopy 6. This powerful technique has been implemented to study many aspects of cell biology 5,7. Since its inception, this procedure has increased our ability to distinguish subcellular architectures and morphologies at high resolution. 
Here, we present a streamlined method for performing rapid microinjection followed by CLEM (Fig. 1). The microinjection CLEM procedure can be used to introduce specific quantities of small molecules and/or proteins directly into the eukaryotic cell cytoplasm and study the effects from millimeter to multi-nanometer resolution (Fig. 2). The technique is based on microinjecting cells grown on laser etched glass gridded coverslips affixed to the bottom of live cell dishes and imaging with both confocal fluorescent and electron microscopy. Localization of the cell(s) of interest is facilitated by the grid pattern, which is easily transferred, along with the cells of interest, to the Epon resin used for immobilization of samples and sectioning prior to electron microscopy analysis (Fig. 3). Overlay of fluorescent and EM images allows the user to determine the subcellular localization as well as any morphological and/or ultrastructural changes induced by the microinjected molecule of interest (Fig. 4). This technique is amenable to time points ranging from &le;5 s up to several hours, depending on the nature of the microinjected sample.

Correlative Light and Electron Microscopy CLEM as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets

The CLEM technique has been adapted to analyze ultrastructural morphology of membranes, organelles, and subcellular structures affected by microinjected molecules. This method combines the powerful techniques of micromanipulation/microinjection, confocal fluorescent microscopy, and electron microscopy to allow millimeter to multi-nanometer resolution. This technique is amenable to a wide variety of applications.


The eukaryotic cell relies on complex, highly regulated, and functionally distinct membrane bound compartments that preserve a biochemical polarity ...

Electroantennographic Bioassay as a Screening Tool for Host Plant Volatiles

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host plant.1,2 When a host plant is an important agronomic commodity feeding damage by insect pests can inflict serious economic losses to growers. Accordingly, kairomones can be used as attractants to lure or confuse these insects and, thus, offer an environmentally friendly alternative to pesticides for insect control.3 Unfortunately, plants can emit a vast number volatiles with varying compositions and ratios of emissions dependent upon the phenology of the commodity or the time of day. This makes identification of biologically active components or blends of volatile components an arduous process. To help identify the bioactive components of host plant volatile emissions we employ the laboratory-based screening bioassay electroantennography (EAG). EAG is an effective tool to evaluate and record electrophysiologically the olfactory responses of an insect via their antennal receptors. The EAG screening process can help reduce the number of volatiles tested to identify promising bioactive components. However, EAG bioassays only provide information about activation of receptors. It does not provide information about the type of insect behavior the compound elicits; which could be as an attractant, repellent or other type of behavioral response. Volatiles eliciting a significant response by EAG, relative to an appropriate positive control, are typically taken on to further testing of behavioral responses of the insect pest. The experimental design presented will detail the methodology employed to screen almond-based host plant volatiles4,5 by measurement of the electrophysiological antennal responses of an adult insect pest navel orangeworm (Amyelois transitella) to single components and simple blends of components via EAG bioassay. The method utilizes two excised antennae placed across a "fork" electrode holder. The protocol demonstrated here presents a rapid, high-throughput standardized method for screening volatiles. Each volatile is at a set, constant amount as to standardize the stimulus level and thus allow antennal responses to be indicative of the relative chemoreceptivity. The negative control helps eliminate the electrophysiological response to both residual solvent and mechanical force of the puff. The positive control (in this instance acetophenone) is a single compound that has elicited a consistent response from male and female navel orangeworm (NOW) moth. An additional semiochemical standard that provides consistent response and is used for bioassay studies with the male NOW moth is (Z,Z)-11,13-hexdecadienal, an aldehyde component from the female-produced sex pheromone.6-8

A method to rapidly screen host plant volatiles by measurement of the electrophysiological response of adult navel orangeworm (Amyelois transitella) antennae to single components and blends via electroantennographic analysis is demonstrated.

Plant volatiles play an important role in plant-insect interactions. Herbivorous insects use plant volatiles, known as kairomones, to locate their host ...

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the messagefor review see 1. However, it's now widely accepted that synonymous codon usage is the primary cause of non-uniform translational elongation rates1. Synonymous codons are not used with identical frequency. A bias exists in the use of synonymous codons with some codons used more frequently than others2. Codon bias is organism as well as tissue specific2,3. Moreover, frequency of codon usage is directly proportional to the concentrations of cognate tRNAs4. Thus, a frequently used codon will have higher multitude of corresponding tRNAs, which further implies that a frequent codon will be translated faster than an infrequent one. Thus, regions on mRNA enriched in rare codons (potential pause sites) will as a rule slow down ribosome movement on the message and cause accumulation of nascent peptides of the respective sizes5-8. These pause sites can have functional impact on the protein expression, mRNA stability and protein foldingfor review see 9. Indeed, it was shown that alleviation of such pause sites can alter ribosome movement on mRNA and subsequently may affect the efficiency of co-translational (in vivo) protein folding1,7,10,11. To understand the process of protein folding in vivo, in the cell, that is ultimately coupled to the process of protein synthesis it is essential to gain comprehensive insights into the impact of codon usage/tRNA content on the movement of ribosomes along mRNA during translational elongation.
Here we describe a simple technique that can be used to locate major translation pause sites for a given mRNA translated in various cell-free systems6-8. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA. The rationale is that at low-frequency codons, the increase in the residence time of the ribosomes results in increased amounts of nascent peptides of the corresponding sizes. In vitro transcribed mRNA is used for in vitro translational reactions in the presence of radioactively labeled amino acids to allow the detection of the nascent chains. In order to isolate ribosome bound nascent polypeptide complexes the translation reaction is layered on top of 30% glycerol solution followed by centrifugation. Nascent polypeptides in polysomal pellet are further treated with ribonuclease A and resolved by SDS PAGE. This technique can be potentially used for any protein and allows analysis of ribosome movement along mRNA and the detection of the major pause sites. Additionally, this protocol can be adapted to study factors and conditions that can alter ribosome movement and thus potentially can also alter the function/conformation of the protein.

Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA

A technique to identify translational pause sites on mRNA is described. This procedure is based on isolation of nascent polypeptides accumulating on ribosomes during in vitro translation of a target mRNA, followed by the size analysis of the nascent chains using a denaturing gel electrophoresis.

The rate of translational elongation is non-uniform. mRNA secondary structure, codon usage and mRNA associated proteins may alter ribosome movement on the ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Examination of Proteins Bound to Nascent DNA in Mammalian Cells Using  BrdU-ChIP-Slot-Western Technique

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic knockdown system, we showed novel functions for HDAC1,2 in replication fork progression and nascent chromatin maintenance in mammalian cells. Additionally, we used a BrdU-ChIP-Slot-Western technique that combines chromatin immunoprecipitation (ChIP) of bromo-deoxyuridine (BrdU)-labeled DNA with slot blot and Western analyses to quantitatively measure proteins or histone modification associated with nascent DNA.
Actively dividing cells were treated with HDAC1,2 selective inhibitor or transfected with siRNAs against Hdac1 and Hdac2 and then newly synthesized DNA was labeled with the thymidine analog bromodeoxyuridine (BrdU). The BrdU labeling was done at a time point when there was no significant cell cycle arrest or apoptosis due to the loss of HDAC1,2 functions. Following labeling of cells with BrdU, chromatin immunoprecipitation (ChIP) of histone acetylation marks or the chromatin-remodeler was performed with specific antibodies. BrdU-labeled input DNA and the immunoprecipitated (or ChIPed) DNA was then spotted onto a membrane using the slot blot technique and immobilized using UV. The amount of nascent DNA in each slot was then quantitatively assessed using Western analysis with an anti-BrdU antibody. The effect of loss of HDAC1,2 functions on the levels of newly synthesized DNA-associated histone acetylation marks and chromatin remodeler was then determined by normalizing the BrdU-ChIP signal obtained from the treated samples to the control samples.

In this protocol, we describe a novel BrdU-ChIP-Slot-Western technique to examine proteins and histone modifications associated with newly synthesized or nascent DNA.

Histone deacetylases 1 and 2 (HDAC1,2) localize to the sites of DNA replication. In the previous study, using a selective inhibitor and a genetic ...

Laser-assisted Microdissection (LAM) as a Tool for Transcriptional Profiling of Individual Cell Types

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the level of individual cell types. While the methods described here are generally applicable to a wide range of species and cell types, our research focuses on plant reproduction. Plant cultivation and seed production is of crucial importance for human and animal nutrition. A detailed understanding of the regulatory networks that govern the formation of the reproductive lineage (germline) and ultimately of seeds is a precondition for the targeted manipulation of plant reproduction. In particular, the engineering of apomixis (asexual reproduction through seeds) into crop plants promises great improvements, as it leads to the formation of clonal seeds that are genetically identical to the mother plant. Consequently, the cell types of the female germline are of major importance for the understanding and engineering of apomixis. However, as the corresponding cells are deeply embedded within the floral tissues, they are very difficult to access for experimental analyses, including cell-type specific transcriptomics. To overcome this limitation, sections of individual cells can be isolated by laser-assisted microdissection (LAM). While LAM in combination with transcriptional profiling allows the identification of genes and pathways active in any cell type with high specificity, establishing a suitable protocol can be challenging. Specifically, the quality of RNA obtained after LAM can be compromised, especially when small, single cells are targeted. To circumvent this problem, we have established a workflow for LAM that reproducibly results in high RNA quality that is well suitable for transcriptomics, as exemplified here by the isolation of cells of the female germline in apomictic Boechera. In this protocol, procedures are described for tissue preparation and LAM, also with regard to RNA extraction and quality control.

Laser-assisted Microdissection LAM as a Tool for Transcriptional Profiling of Individual Cell Types

Here we present a protocol for laser-assisted microdissection of specific plant cell types for transcriptional profiling. While the protocol is suitable for different species and cell types, the focus is on highly inaccessible cells of the female germline important for sexual and apomictic reproduction in the crucifer genus Boechera.

The understanding of developmental processes at the molecular level requires insights into transcriptional regulation, and thus the transcriptome, at the ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...