D.F. is supported by an NSF Bridges to the Doctorate fellowship. The microscope used in this work is supported in part by the Microscope Imaging Network core infrastructure grant from Colorado State University. Work on clathrin adaptors in the author s laboratory is supported by CSU start-up funds and American Heart Association award 09SDG2280525 to S.D.

1. Incorporation of a GFP tag and selectable marker in the SLA1 gene
Apply the Longtine method17 in order to fuse a GFP tag directly at the 3' end of the SLA1 gene open reading frame (Sla1p C-terminus) and simultaneously mark the gene with the E. coli kanr gene that allows for G418 selection.
<ol>
	<li>Generate a DNA fragment by PCR using as a template the plasmid pFA6a-GFP(S65T)-kanMX618 and the following primers: forward, 5'-CA AGG CAA GCC AAC ATA TTC AAT GCT ACT GCA TCA AAT CCG TTT GGA TTC CGG ATC CCC GGG TTA ATT AA-3', and reverse, 5'-CA TAT AGC TTG TTT TAG TTA TTA TCC TAT AAA ATC TTA AAA TAC ATT AAT GAA TTC GAG CTC GTT TAA AC-3'. The underlined sequence corresponds to the SLA1 gene specific segment immediately preceding (forward primer) and following the stop codon (reverse primer). Isolate the PCR product by agarose gel electrophoresis followed by DNA purification.</li>
	<li>Prepare a 50 ml culture S. cerevisiae SEY6210 strain (MA&#932;&alpha; ura3-52, leu2-3,112 his3-&Delta;200, trp1-&Delta;901, lys2-801, suc2-&Delta;9 GAL-MEL) 19 in YPD, growing at early logarithmic phase (OD600 =0.2-0.6). Spin at room temperature for 3 min at 2,000 x g, wash twice with sterile water.</li>
	<li>Transform the PCR fragment obtained in step 1 into the cells using the lithium acetate procedure20. Wash the cells with 1 ml sterile water, add 1 ml YPD and incubate for 4 h at 30 &deg;C in a shaker.</li>
	<li>Spread the cells on YPD-G418 plates to select for G418-resistant transformants, incubate at 30 &deg;C for 2-3 days. Pick colonies and streak them on YPD-G418 plates.</li>
	<li>Using colony-PCR, identify transformants in which the GFP-kan module was properly integrated with the SLA1 gene sequences by homologous recombination. Use a forward primer that anneals within the SLA1 open reading frame and a reverse primer that anneals inside the GFP-kan module. Identify colonies which PCR products have the expected size and verify by sequencing.</li>
	<li>Screen the colonies by fluorescence microscopy to confirm they have GFP fluorescence.</li>
</ol>
2. Mutation of the variant clathrin box (vCB) in the SLA1 gene
<ol>
	<li>Introduce an LLDLQ to AAALQ mutation in the SLA1 gene (nucleotides 2407-2415) following a two step approach15.</li>
	<li>First, amplify SLA1 nucleotides 1207-1410 and 2427-2589 by PCR and clone the fragments into NotI/BamHI and EcoRI/SalI sites of pBluescriptKS, respectively.</li>
	<li>Subclone into the BamHI/EcoRI sites a PCR fragment containing URA3.</li>
	<li>Cleave the resulting plasmid with NotI/SalI, isolate the URA3 fragment by gel purification, and introduce it by lithium acetate transformation into the Sla1-GFP strain generated in Part 1, or into TVY614 (MATa ura3-52 leu2-3112 his3-&Delta;200 trp1-&Delta;901 lys2-801 suc2-&Delta;9 pep4::LEU2 prb1::HISG prc1::HIS3)21.</li>
	<li>Spread the cells on supplemented SD plates lacking uracil. Confirm by colony-PCR and sequencing that the Ura+ colonies contain properly integrated URA3 replacing the vCB fragment.</li>
	<li>In a second step, subclone SLA1 nucleotides 1207-2589 into NotI/SalI sites of pBluescriptKS. Using the QuickChange-XL site-directed mutagenesis kit (Stratagene), mutate the vCB residues LLDLQ to AAALQ. Verify by sequencing.</li>
	<li>Cleave the resulting construct with BsgI/AgeI, and isolate the mutant vCB containing fragment by gel purification. Cotransform the BsgI/AgeI fragment with pRS313 (HIS3)22 into the strain obtained in Part 2.3.</li>
	<li>Spread the cells on supplemented SD plates lacking histidine. Replica-plate His+ colonies onto agar medium containing 5-fluorotic acid to identify cells in which the mutant sequences replaced URA3, thus regenerating the sla1 gene containing the LLDLQ to AAALQ mutation (sla1AAA). Confirm by colony-PCR and sequencing.</li>
</ol>
3. Fluorescence microscopy
<ol>
	<li>Grow the yeast strains Sla1-GFP and sla1AAA-GFP generated in Parts 1 and 2 in 4 ml of supplemented SD media at 30&deg;C in a rotator in the dark until reaching an OD600 = 0.1-0.4. Spin 1ml of culture in microcentrifuge at 4,000 x g for 1 min at room temperature. Discard ~950 &mu;l of supernatant. Resuspend cells in the remaining liquid (~50 &mu;l).</li>
	<li>Deposit 3 &mu;l of the cell suspension onto a microscopy slide and cover with a glass coverslip. Protect the sample from light.</li>
	<li>Mount slide on a 100x oil-immersion objective of a spinning-disc confocal microscope.</li>
	<li>Locate correct focal plane of cells using brightfield illumination or 488 nm laser with appropriate filters to excite and detect fluorescence from GFP.</li>
	<li>Capture time-lapse images of Sla1-GFP and sla1AAA-GFP in cells with exposure time of 500 ms (or appropriate value) at an interval of one image per second for 150 seconds. Expected result: GFP labeled punctae will appear on the inner surface of limiting membrane of the cell, persist for several seconds, and move toward the center of the cell as the signal quickly disappears (indicating coat disassembly).</li>
	<li>Select a portion of an image in which spots are seen to appear, remain for several seconds, and disappear. Crop this section of the image for each frame of the time-lapse images.</li>
	<li>Generate a kymograph representing this section of the image in each frame of the 150 second time-lapse image by aligning the frames along an axis corresponding to time elapsed.</li>
	<li>Calculate the duration of each spot based on how many seconds (time-lapse frames) the spots is present in the kymograph. Note that each spot should "curve" toward the interior of the cell as it is disappearing, reflecting endocytosis and disassembly of the coat.</li>
	<li>Compare wild type Sla1-GFP with sla1AAA-GFP. Repeat to determine if results are statistically significant. Expected result: sla1AAA-GFP spots persist significantly longer (~80 sec) than wild type (~30 sec) indicating a defect in coat formation15.</li>
</ol>
4. Preparation of yeast cytosolic extract and total cell extract
<ol>
	<li>Inoculate 3 liters of YPD with an appropriate yeast strain, such as TVY614 21, and grow at 30 &deg;C in a shaker incubator until reaching an OD600 = 1-2.</li>
	<li>Centrifuge the yeast culture at room temperature for 20 min at 3,700 x g. Discard the supernatant, add 3-5 ml of sterile water, and pipette up and down until the pellet is completely resuspended.</li>
	<li>Pour ~150 ml of liquid nitrogen in a 250 ml plastic beaker. Flash-freeze the yeast by adding dropwise into the liquid nitrogen in a circular pattern to avoid agglutination of the frozen pellets. Do not let the pellets thaw, add more liquid nitrogen if necessary.</li>
	<li>Chill a stainless steel blender container by pouring liquid nitrogen in it. Allow the liquid nitrogen to almost completely evaporate. Add the frozen yeast pellets to the cold blender container. Close the blender container with a cold rubber stopper and grind the yeast pellets for 10 seconds. Invert the container 3-4 times to mix contents and repeat the grinding step twice. The ground frozen yeast will have a powder-like appearance.</li>
	<li>Chill a funnel and a 50 ml conical tube with liquid nitrogen. Using the cold funnel, transfer the ground yeast to the tube. The frozen ground yeast can be store at -80 &deg;C or used immediately.</li>
	<li>To obtain a cytosolic extract for the GST-fusion protein affinity assay (Part 5), weight 3 g of frozen ground TVY614 yeast in a 15 ml conical tube and resuspend in 3 ml of room temperature buffer A (10 mM HEPES, pH 7.0, 150 mM NaCl, 1 mM EDTA, 1 mM DTT) containing protease inhibitor cocktail (Sigma).</li>
	<li>Cap and invert the tube several times until completely thawed and then put on ice.</li>
	<li>Ultracentrifuge the extract at 4 &deg;C for 20 min at 300,000 x g. Carefully transfer the supernatant (cytosolic extract) to a conical tube using a pipette without disturbing the pellet. Keep the cytosolic extract on ice.</li>
	<li>Add 100 &mu;l of a 50% (v/v) glutathione-Sepharose slurry in buffer A. It is convenient to cut the end of the tip in order to facilitate pipeting the beads. Rotate at 4 &deg;C for 15 min, spin-down the beads by centrifugation at 4 &deg;C for 2 min at 1,000 x g, and transfer the supernatant (cytosolic extract) to a fresh tube. Reserve 50 &mu;l of extract for input control.</li>
	<li>To obtain total cell extracts for co-immunoprecipitation experiments (Part 6), use strain TVY614 (WT SLA1), the sla1AAA strain carrying an LLDLQ to AAALQ mutation generated in Part 2 in TVY614 background, and a sla1 strain carrying a deletion of the SLA1 gene such as GPY3130 23. Weight 2 g of the corresponding frozen ground yeast in conical tubes and resuspend them in 2 ml of room temperature buffer A, containing protease inhibitor cocktail (Sigma) and 2% Triton X-100.</li>
	<li>Cap and invert the tube several times until completely thawed and then incubate on ice for 10 min, periodically mixing by inversion.</li>
	<li>Transfer the material to microcentrifuge tubes and spin at 4 &deg;C for 15 min at 16,000 x g (top speed in microcentrifuge). Carefully transfer the supernatant (total cell extract) to a conical tube on ice using a pipette without disturbing the pellet.</li>
	<li>Add 100 &mu;l of a 50% (v/v) protein A-Sepharose slurry in buffer A. Rotate at 4 &deg;C for 15 min, spin-down the beads by centrifugation at 4 &deg;C for 2 min at 1,000 x g, and transfer the supernatant (total extract) to a fresh tube. Reserve 50 &mu;l of extract for input control.</li>
</ol>
5. GST-fusion protein affinity assay
<ol>
	<li>Amplify by PCR a fragment containing the Sla1p variant clathrin-box (vCB) (residues 803-807), clone it into pGEX-5X to obtain pGEX-5X-vCB. Verify by sequencing.</li>
	<li>Using pGEX-5X-vCB as a template and the QuickChange-XL site-directed mutagenesis kit (Stratagene), mutate the vCB residues LLDLQ to AAALQ to obtain pGEX-5X-vCBmut. Verify by sequencing.</li>
	<li>Express GST and the fusion proteins GST-vCB and GST-vCBmut in E. coli (BL21 DE3), purify using glutathione-Sepharose, elute from the beads with reduced glutathione, dialyze against PBS, and determine protein concentration.</li>
	<li>Label 3 microcentrifuge tubes: GST, GST-vCB, and GST-vCBmut, and add 1 ml of PBS, 30 &mu;l of a 50% (v/v) glutathione-Sepharose slurry in buffer A, and 50 &mu;g of dialized GST, GST-vCB, or GST-vCBmut fusion proteins. </li>
	<li>Rotate at room temperature for 30 min to allow for binding. </li>
	<li>Wash the beads 2 times with PBS and 1 time with buffer A. Add 1 ml of yeast cytosolic extract - prepared as described in Part 4 - to each tube. </li>
	<li>Rotate the tubes 1 h at 4 &deg;C, spin 10 sec at 5,000 x g to pellet the beads, discard supernatant, and quickly wash the beads 3 times with buffer A containing 0.1% Triton X-100, and 1 time with buffer A. </li>
	<li>Spin down the beads one more time and using a gel loading tip remove as much liquid as possible. At this point the samples can be stored at -20 &deg;C to continue at a later time.</li>
	<li>Add 15 &mu;l of 2x Laemmli sample buffer to each tube, incubate at 96 &deg;C for 5 min, and spin 10 sec at 5,000 x g. </li>
	<li>Analyze the samples by immunoblotting using an antibody to the clathrin heavy chain. Expected results: clathrin binds to GST-vCB but not to GST or GST-vCBmut. Also analyze the samples by SDS-PAGE to confirm similar loading of the GST fusion proteins.</li>
</ol>
6. Co-immunoprecipitation assay
<ol>
	<li>Label 3 microcentrifuge tubes: WT (wild type SLA1), sla1AAA, and sla1&Delta;, and add 1 ml of PBS, 30 &mu;l of a 50% (v/v) protein A-Sepharose slurry in buffer A, and 2 &mu;g of a rabbit anti-Sla1p antibody. </li>
	<li>Rotate at room temperature for 30 min. </li>
	<li>Wash the beads twice with PBS and 1 time with buffer A containing 1% Triton X-100.</li>
	<li>Add 1 ml of the corresponding total yeast extracts prepared in Part 4: SLA1, sla1AAA, and sla1&Delta;. Rotate 1 h at 4 &deg;C. </li>
	<li>Spin 10 sec at 5,000 x g to pellet the beads, discard supernatant, and quickly wash the beads 3 times with buffer A containing 0.1% Triton X-100, and 1 time with buffer A. </li>
	<li>Spin down the beads one more time and using a gel loading tip remove as much liquid as possible. At this point the samples can be stored at -20 &deg;C to continue at a later time.</li>
	<li>Add 15 &mu;l of 2x Laemmli sample buffer to each tube, incubate at 96 &deg;C for 5 min, and spin 10 sec at 5,000 x g. </li>
	<li>Analyze the samples by immunoblotting using an antibody to the clathrin heavy chain. Expected results: clathrin co-immunoprecipitates with wild type Sla1p but not to with sla1AAA, or in the sla1&Delta; sample.</li>
</ol>
7. Representative Results
<img src="/files/ftp_upload/2352/2352fig1.jpg" alt="Figure 1" /> Figure 1. Analysis of Sla1p clathrin adaptor at endocytic sites by live cell fluorescence microscopy. One frame (left) from a video and corresponding kymographs (right) of yeast cells expressing Sla1-GFP or sla1AAA-GFP carrying an LLDLQ to AAALQ mutation. Both wild type and mutant Sla1-GFP were expressed from the endogenous SLA1 locus. Endocytic sites are observed as bright spots distributed in the cell periphery (left images). The area between white lines corresponds to the region from which kymographs were generated. Movies were taken at a frame rate of 1 frame/sec. Notice the longer lifetime of Sla1-GFP in the LLDLQ to AAALQ mutant (sla1AAA) compared to wild type (SLA1).
<img src="/files/ftp_upload/2352/2352fig2.jpg" alt="Figure 2" /> Figure 2. Physical interaction between clathrin and the Sla1p-variant clathrin box. GST fused to Sla1p fragment aa798-813 containing the sequence LLDLQ (GST-vCB), the corresponding AAALQ mutant (GST-vCBmut), or GST alone (GST) were bound to glutathione-Sepharose beads and incubated with a cytosolic extract from wild-type yeast cells. The associated proteins were eluted and analyzed by immunoblotting (IB) for clathrin heavy chain.

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No conflicts of interest declared.

Clathrin-coated vesicles (CCV) participate in endocytosis and transport from the trans Golgi network and endosomes, conserved pathways that are fundamental to eukaryotic cell biology. The approaches described in this methods paper are useful to study the molecular mechanisms responsible for CCV formation, in particular interactions between adaptor proteins and clathrin. GST-fusion protein affinity and co-immunoprecipitation assays allow testing for physical association between an adaptor (candidate) and clathrin...

<table border="1">
 <th>Material</th>
 <th>Company</th>
 <tbody>
 <tr>
 <td>QuickChange-XL site-directed mutagenesis kit</td>
 <td>Stratagene</td>
 </tr>
 <tr>
 <td>protease inhibitor cocktail</td>
 <td>Sigma</td>
 </tr>
 </tbody>
</table>

in vivo and in vitro studies of adaptor-clathrin interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs are distinguished by a polyhedral lattice of clathrin that coats the vesicle membrane and serves as a mechanical scaffold. Clathrin coats are assembled during vesicle formation from individual clathrin triskelia , the soluble form of clathrin composed of three heavy and three light chain subunits7,8. Because the triskelion does not have the ability to bind to the membrane directly, clathrin-binding adaptors are critical to link the forming clathrin lattice to the membrane through association with lipids and/or membrane proteins9. Adaptors also package transmembrane protein cargo, such as receptors, and can interact with each other and with other components of the CCV formation machinery9.
Over twenty clathrin adaptors have been described, several are involved in clathrin mediated endocytosis and others localize to the trans Golgi network or endosomes9. With the exception of HIP1R (yeast Sla2p), all known clathrin adaptors bind to the N-terminal -propeller domain of the clathrin heavy chain9. Clathrin adaptors are modular proteins consisting of folded domains connected by unstructured flexible linkers. Within these linker regions, short binding motifs mediate interactions with the clathrin N-terminal domain or other components of the vesicle formation machinery9. Two distinct clathrin-binding motifs have been defined: the clathrin-box and the W-box9. The consensus clathrin-box sequence was originally defined as L[L/I][D/E/N][L/F][D/E]10 but variants have been subsequently discovered11. The W-box conforms to the sequence PWxxW (where x is any residue).
Sla1p (Synthetic Lethal with Actin binding protein-1) was originally identified as an actin associated protein and is necessary for normal actin cytoskeleton structure and dynamics at endocytic sites in yeast cells12. Sla1p also binds the NPFxD endocytic sorting signal and is critical for endocytosis of cargo bearing the NPFxD signal13,14. More recently, Sla1p was demonstrated to bind clathrin through a motif similar to the clathrin box, LLDLQ, termed a variant clathrin-box (vCB), and to function as an endocytic clathrin adaptor15. In addition, Sla1p has become a widely used marker for the endocytic coat in live cell fluorescence microscopy studies16. Here we use Sla1p as a model to describe approaches for adaptor-clathrin interaction studies. We focus on live cell fluorescence microscopy, GST-pull down, and co-immunoprecipitation methods.

Watch this Scientific Journal Video about In vivo and in vitro Studies of Adaptor-clathrin Interaction at JoVE.com

In vivo and in vitro Studies of Adaptor-clathrin Interaction

Clathrin-mediated endocytosis depends on adaptor proteins that coordinate cargo selection and clathrin coat assembly. Here we describe procedures to study adaptor-clathrin physical interaction and live cell imaging approaches using as a model the yeast endocytic adaptor protein Sla1p.

In vivo and in vitro Studies of Adaptor-clathrin Interaction

A major endocytic pathway initiates with the formation of clathrin-coated vesicles (CCVs) that transport cargo from the cell surface to endosomes1-6. CCVs ...

in-vivo-and-in-vitro-studies-of-adaptor-clathrin-interaction

Research

JoVE Journal

Biology

14.4K Views.  Colorado State University. Клатрин эндоцитоза зависит от адаптера белков, которые координируют отбор грузов и клатрин сборки пальто. Здесь мы описываем процедуры для изучения адаптера клатрин физического взаимодействия и живые изображения ячейки подходов, используя в качестве модел?...

Video: In vivo and in vitro Studies of Adaptor-clathrin Interaction

In vitro and in vivo Bioluminescence Reporter Gene Imaging of Human Embryonic Stem Cells

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative medicine. However, direct injection of hESCs, and cells differentiated from hESCs, into living organisms has thus far been hampered by significant cell death, teratoma formation, and host immune rejection. Understanding the in vivo hESC behavior after transplantation requires novel imaging techniques to longitudinally monitor hESC localization, proliferation, and viability. Molecular imaging has given investigators a high-throughput, inexpensive, and sensitive means for tracking in vivo cell proliferation over days, weeks, and even months. This advancement has significantly increased the understanding of the spatio-temporal kinetics of hESC engraftment, proliferation, and teratoma-formation in living subjects.

A major advance in molecular imaging has been the extension of noninvasive reporter gene assays from molecular and cellular biology into in vivo multi-modality imaging platforms. These reporter genes, under control of engineered promoters and enhancers that take advantage of the host cell s transcriptional machinery, are introduced into cells using a variety of vector and non-vector methods. Once in the cell, reporter genes can be transcribed either constitutively or only under specific biological or cellular conditions, depending on the type of promoter used. Transcription and translation of reporter genes into bioactive proteins is then detected with sensitive, noninvasive instrumentation (e.g., CCD cameras) using signal-generating probes such as D-luciferin.

To avoid the need for excitatory light to track stem cells in vivo as is required for fluorescence imaging, bioluminescence reporter gene imaging systems require only an exogenously administered probe to induce light emission. Firefly luciferase, derived from the firefly Photinus pyralis, encodes an enzyme that catalyzes D-luciferin to the optically active metabolite, oxyluciferin. Optical activity can then be monitored with an external CCD camera. Stably transduced cells that carry the reporter construct within their chromosomal DNA will pass the reporter construct DNA to daughter cells, allowing for longitudinal monitoring of hESC survival and proliferation in vivo. Furthermore, because expression of the reporter gene product is required for signal generation, only viable parent and daughter cells will create bioluminescence signal; apoptotic or dead cells will not. 

In this video, the specific materials and methods needed for tracking stem cell proliferation and teratoma formation with bioluminescence imaging will be described.

With the growing interest in stem cell therapies, molecular imaging techniques are ideal for monitoring stem cell behavior after transplantation. Luciferase reporter genes have enabled non-invasive, repetitive assessment of cell survival, location, and proliferation in vivo. This video will demonstrate how to track hESC proliferation in a living mouse.

The discovery of human embryonic stem cells (hESCs) has dramatically increased the tools available to medical scientists interested in regenerative ...

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many degenerative human diseases. The recent discovery of MG53 as a key component of the membrane resealing machinery allows for a better molecular understanding of the basic biology of tissue repair, as well as for potential translational applications in regenerative medicine. Here we detail the experimental protocols for exploring the in vivo function of MG53 in repair of muscle injury using treadmill exercise protocols on mouse models, for testing the ex vivo membrane repair capacity by measuring dye entry into isolated muscle fibers, and for monitoring the dynamic process of MG53-mediated vesicle trafficking and cell membrane repair in cultured cells using live cell confocal microscopy.

Visualization of MG53-mediated Cell Membrane Repair Using in vivo and in vitro Systems

Described here are protocols used to visualize the dynamic process of MG53-mediated cell membrane repair in whole animals and at the cellular level. These methods can be applied to investigate the cell biology of plasma membrane resealing and regenerative medicine.

Repair of acute injury to the cell membrane is an elemental process of normal cellular physiology, and defective membrane repair has been linked to many ...

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 ...

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA (dsDNA) which has the requisite characteristics to stimulate the cytoplasmic DNA sensing pathways can be generated cheaply and with ease. By the concatemerization of short, synthetic oligonucleotides (which lack CpG motifs), dsDNA can be generated to be of sufficient length to activate the cytosolic DNA sensing pathway. This protocol involves blunt end ligation of the oligonucleotides in the presence of polyethylene glycol (PEG), which provides an environment for efficient ligation to occur. The dsDNA concatemers can be used, following purification by phenol/chloroform extraction, to simulate the innate immune response in vitro by standard transfection protocols. This DNA can also be used to stimulate innate immunity in vivo by intradermal injection into the ear pinna of a mouse, for example. By standardizing the concatemerization process and the subsequent stimulation protocols, a reliable and reproducible activation of the innate immune system can be produced.

Stimulation of Cytoplasmic DNA Sensing Pathways In Vitro and In Vivo

The aim of the protocol is to use optimal methods for stimulating the cytoplasmic DNA sensing pathways in cells and in vivo. This is achieved by improving the generation of long, double-stranded DNA during blunt-end ligation. Cells or mice are then transfected using a lipid transfection reagent.

In order to efficiently stimulate an innate immune response, DNA must be of sufficient length and purity. We present a method where double stranded DNA ...

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell proliferation, migration, and alignment to form tubular structures followed by recruitment of pericytes to provide mural support and facilitate vessel maturation. Current in vitro cell culture approaches cannot fully reproduce the complex biological environment where endothelial cells and pericytes interact to produce functional vessels. We present a novel application of the in vivo matrix gel plug assay to study endothelial-pericyte interactions and formation of functional blood vessels using severe combined immune deficiency mutation (SCID) mice. Briefly, matrix gel is mixed with a solution containing endothelial cells with or without pericytes followed by injection into the back of anesthetized SCID mice. After 14 days, the matrix gel plugs are removed, fixed and sectioned for histological analysis. The length, number, size and extent of pericyte coverage of mature vessels (defined by the presence of red blood cells in the lumen) can be quantified and compared between experimental groups using commercial statistical platforms. Beyond its use as an angiogenesis assay, this matrix gel plug assay can be used to conduct genetic studies and as a platform for drug discovery. In conclusion, this protocol will allow researchers to complement available in vitro assays for the study of endothelial-pericyte interactions and their relevance to either systemic or pulmonary angiogenesis.

In Vivo Study of Human Endothelial-Pericyte Interaction Using the Matrix Gel Plug Assay in Mouse

We present a protocol to study human endothelial-pericyte interactions in mouse using a variation of the matrix gel plug angiogenesis assay.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. New vessel growth requires coordinated endothelial cell ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

In Vitro Analysis of E3 Ubiquitin Ligase Function

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.

In Vitro Analysis of E3 Ubiquitin Ligase Function

The present study provides detailed in vitro ubiquitylation assay protocols for the analysis of E3 ubiquitin ligase catalytic activity. Recombinant proteins were expressed using prokaryotic systems such as Escherichia coli culture.

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...

Double-Staining Method to Detect Pectin in Plant-Fungus Interaction

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an efficient inducible mechanism to isolate the fungal haustoria from the plant cell protoplast. Conversely, pectin, one of the polymeric components of the cell wall, is a target of several pectolytic enzymes in necrotrophic interactions. Here, a protocol to detect pectin and fungal hyphae through optical microscopy is presented. The pectin-rich encapsulation in the cells of coffee leaves infected by the rust fungus Hemileia vastatrix and the mesophyll cell wall modification induced by Cercospora coffeicola are investigated. Lesioned leaf samples were fixed with the Karnovsky solution, dehydrated, and embedded in glycol methacrylate for 2-4 days. All steps were followed by vacuum-pumping to remove air in the intercellular spaces and improve the embedding process. The embedded blocks were sectioned into 5-7 &#181;m thick sections, which were deposited on a glass slide covered with water and subsequently heated at 40 &#176;C for 30 min. Next, the slides were double-stained with 5% cotton blue in lactophenol to detect the fungus and 0.05% ruthenium red in water to detect pectin (acidic groups of polyuronic acids of pectin). Fungal haustoria of Hemileia vastatrix were found to be encapsulated by pectin. In coffee cercosporiosis, mesophyll cells exhibited dissolution of cell walls, and intercellular hyphae and conidiophores were observed. The method presented here is effective to detect a pectin-associated response in the plant-fungi interaction.

This protocol describes a microscopical method to detect pectin in coffee-fungus interaction.

Plant cells use different structural mechanisms, either constitutive or inducible, to defend themselves from fungal infection. Encapsulation is an ...

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; however, there are currently no treatment or therapeutic options for vascular calcification. Calcification begins within specialized extracellular vesicles (EVs), which serve as nucleating foci by aggregating calcium and phosphate ions. This protocol describes methods for obtaining and assessing calcification in murine aortas and analyzing the associated extracted EVs. First, gross dissection of the mouse is performed to collect any relevant organs, such as the kidneys, liver, and lungs. Then, the murine aorta is isolated and excised from the aortic root to the femoral artery. Two to three aortas are then pooled and incubated in a digestive solution before undergoing ultracentrifugation to isolate the EVs of interest. Next, the mineralization potential of the EVs is determined through incubation in a high-phosphate solution and measuring the light absorbance at a wavelength of 340 nm. Finally, collagen hydrogels are used to observe the calcified mineral formation and maturation produced by the EVs in vitro.

Analysis of Extracellular Vesicle-Mediated Vascular Calcification Using In Vitro and In Vivo Models

This paper presents the methodology for obtaining and assessing vascular calcification by isolating murine aortas followed by extracting calcified extracellular vesicles to observe the mineralization potential.

Cardiovascular disease is the leading cause of death in the world, and vascular calcification is the most significant predictor of cardiovascular events; ...