This work was supported by the Medical Research Council and the Marie-Curie International Reintegration Grant scheme (to JKV).

1. Picking cDNA Expression Clones
<ol>
 <li>The cDNA library is provided as single bacterial clones in glycerol stocks in a 96-well format. Using the Rearray command in the picker menu of the Norgen CP7200, pick bacterial clones from the source plate and inoculate into 1.5 ml LB/amp in a 96-deepwell (1 ml well) plate.</li>
 <li>Seal the deepwell plates with a gas-permeable seal and incubate overnight at 37 &deg;C in a shaker.</li>
</ol>
2. Preparation of cDNA Expression Library
<ol>
 <li>Spin the deepwell plates containing the bacteria at 2,000 rpm for 20 min using plate adapters in a table top centrifuge.</li>
 <li>Aspirate media from the wells, leaving the bacterial pellet intact.</li>
 <li>Configure the deck of the Lab automation workstation as shown in Figure 2. Solutions for plasmid preparation are distributed into troughs 1-5 and the deepwell plate is placed on the deck. A multiwell trough with elution solution is placed adjacent to the deepwell plate. Tips are loaded into the tip racks.</li>
 <li>Assemble the vacuum manifold. The plasmid binding plate is placed inside the manifold and the plasmid filter plate is placed on top of the manifold.</li>
 <li>Resuspend pellets with 250 &mu;l Solution 1 (Resuspension buffer) by pipetting up and down.</li>
 <li>Lyse the bacteria by addition of 250 &mu;l Solution 2 (Lysis buffer). The plate is sealed and inverted to allow complete mixing.</li>
 <li>Start 2 min timer.</li>
 <li>Add 350 &mu;l Solution 3 (Neutralisation buffer). Seal the plate and invert 3 times.</li>
 <li>Transfer the bacterial lysates to the plasmid filter plates.</li>
 <li>Apply vacuum for 5 min.</li>
 <li>Remove the filter plate and place the binding plate on top of the manifold. Apply vacuum for 1 min.</li>
 <li>Add 500 &mu;l of additional wash (AW) buffer.</li>
 <li>Apply vacuum for 2 min.</li>
 <li>Add 900 &mu;l of wash buffer Solution 4. Apply vacuum for 2 min.</li>
 <li>Repeat step 2.14. Apply vacuum for an additional 15 min.</li>
 <li>Place DNA collection plate inside the manifold. Add 100 &mu;l of Elution buffer to the binding plate and incubate for 2 min.</li>
 <li>Vacuum for 10 min.</li>
 <li>Remove collection plate and measure DNA concentration using the Nanodrop 8000. Typically a yield of ~100 ng/&mu;l can be expected with this method.</li>
</ol>
3. Cell Seeding
<ol>
 <li>HEK293 cells are trypsinized and counted.</li>
 <li>2 x 104 cells are dispensed into each well of a PerkinElmer Viewplate using the ThermoFisher 96-well multidrop.</li>
 <li>Cells are incubated overnight at 37 &deg;C and 5% CO2.</li>
</ol>
4. Transfection
<ol>
 <li>Prepare a mix of 100 ng GFP-Grb2 plasmid DNA with 100 ng of cDNA in 25 &mu;l serum-free medium per well in a round bottom 96-well plate.</li>
 <li>Add 25 &mu;l serum-free media containing 0.5 &mu;l Transfectin per well.</li>
 <li>Mix and start timer for 30 min.</li>
 <li>Transfer 50 &mu;l of cDNA/Transfectin mix to cells using a gentle dispensing method. Alternatively, the transfection mix can be dispensed first into plates and then cells added on top (reverse transfection).</li>
 <li>Incubate cells overnight at 37 &deg;C and 5% CO2.</li>
</ol>
5. Image Acquisition
<ol>
 <li>Start Opera software. A screen snapshot of the experiment setup is shown in Figure 3. Select the "Configuration" tab and select 20x objective and the correct plate type. Ensure "collar" is set to the correct value on the objective to allow for focusing with different plate types.</li>
 <li>Select the "Microscope" tab. Define exposure 1 as FITC (488 laser) and exposure 2 as UV (365 laser). Activate the UV filter on both exposures and assign exposure 1 to camera 1 and exposure 2 to camera 2. Set exposure times to 800 msec for exposure 1 and 40 msec for exposure 2.</li>
 <li>Select the "Microscope" tab. Define exposure 1 as FITC (488 laser) and exposure 2 as UV (365 laser). Activate the UV filter on both exposures and assign exposure 1 to camera 1 and exposure 2 to camera 2. Set exposure times to 800 msec for exposure 1 and 40 msec for exposure 2.</li>
 <li>Select exposure 1. Set focus height to 0 &mu;m. Select "Focus". Once focused, expose camera 1. Adjust the focus height to optimize the exposure plane and click on "Take height". Change the exposure times and laser power to give a maximum pixel intensity of ~3,000. Save exposure parameters. Repeat for exposure 2.</li>
 <li>Select "Experiment Definition" tab. Create layout and sublayout. Drag and drop the relevant layout, exposure, reference image, skewcrop file and sublayout. Save experiment.</li>
 <li>Select "Automatic Experiment" tab and acquire images.</li>
</ol>
6. Image Analysis (Based on ImageJ 1.46h version.)
<ol>
 <li>Convert the proprietary PerkinElmer .flex files to Tagged Image Format .tif file using the Flex2Volocity Acapella script.</li>
 <li>
 <ol>
 <li>Import images in ImageJ as an image sequence, resulting in a stack.</li>
 <li>Convert the stack into a 2 channel hyperstack by selecting the Image/Hyperstack/Stack to Hyperstack menu item. The number of slices must be set to half of the imported stack.</li>
 <li>Duplicate the GFP channel using the Image/Duplicate command: tick the Duplicate hyperstack checkbox and specify Channels: 1-1. Hereafter use the duplicated stack.</li>
 </ol>
 </li>
 <li>Select Process/Enhance Contrast with 0.01% saturated pixels, using stack histogram and convert to 8 bit with Image/Type/8-bit.</li>
 <li>Define a 15 pixel radius local adaptive segmentation using the Bernsen algorithm with contrast threshold (Parameter 1) q = 30. Tick the White objects on black background checkbox in the Image/Adjust/Auto Local Threshold menu.</li>
 <li>Segmentation quality control: generate segmented object contour overlay on the original images.</li>
 <li>Feature extraction by analysing particles: measuring area, mean and total intensity of each organelle. Save result files.</li>
 <li>Open the features result file in R and generate a histogram.</li>
</ol>
7. Representative Results
Under normal conditions Grb2 is localized throughout the entire cell. Upon stimulation of a growth factor receptor it translocates to the plasma membrane and is subsequently internalized into endosomes as shown in Figure 4. The expression of a cell surface receptor capable of binding Grb2 is sufficient to induce this translocation. In a typical screening experiment, no change in GFP-Grb2 localization is observed. However, when a protein is expressed that recruits Grb2 to a sub-cellular site, there is a change in localization that can be easily visualized by fluorescence microscopy. For instance, expression of the EGFR results in relocalization of GFP-Grb2 to endosome-like structures (Figure 4). Thus, when applying a genome-wide library, it can be expected that novel Grb2-binding cell surface proteins can be identified.
This method is not limited to the detection of cell surface proteins. For instance, Dynamin2 induces a change in sub-cellular localization of GFP-Grb2 displaying endosome-like recruitment (Figure 5). Dynamin2 binds to the C-terminal SH3 domain of Grb2 and is involved in endocytosis of this complex9,10. Thus, this approach enables the identification of general protein binding complexes and is not limited to the identification of interaction partners for the SH2 domain.
Several different types of translocation can be expected such as localization to the plasma membrane, to endosomes, other cytoplasmic vesicular structures or to the nucleus. Therefore, it is recommended that all images are also inspected by eye. However, when screening large sets of cDNAs an automated image analysis algorithm is more practical. In this case, a combination of algorithms is desirable, such as spot detection to identify endosomes, cytoplasm/nucleus detection to identify nuclear shuttling and general morphological algorithms to identify cellular shape changes. The use of these different phenotypic detection methods has been discussed in more detail elsewhere11.
<img alt="Figure 1" src="/files/ftp_upload/4382/4382fig1.jpg" /> 
Figure 1. Overall scheme of the experiment. The experiment details a complete high-content screening workflow starting with the amplification and preparation of the cDNA library, transfection of cDNA vectors plus reporter constructs, image acquisition and image analysis using the free open software IMAGEJ.
<img alt="Figure 2" src="/files/ftp_upload/4382/4382fig2.jpg" /> 
Figure 2. Configuration of the Tecan Deck. (1) On the left-hand side, five 100 ml troughs need to be filled with buffers 1 (40 ml), 2 (40 ml), 3 (50 ml), AW (60 ml) and A4 (100 ml). Trough A4 will need to be re-filled once during the procedure. (2) The vacuum manifold is located on position 14 in this example. (3) The deepwell plate with bacterial pellets is located on position 30, next to the elution buffer trough with 25 ml Elution buffer. (4) Disposable tips for the 8-channel head need to be loaded in the tip racks on the left side and tips for the multichannel head need to be filled on position 40. The Tecan FreedomEvo liquid handler that is used in this experiment is equipped with 500 &mu;l syringes. <a href="https://www.jove.com/files/ftp_upload/4382/4382fig2large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 3" src="/files/ftp_upload/4382/4382fig3.jpg" /> 
Figure 3. Automation of image acquisition on the Opera LX. A) Select the Configuration tab (1). Activate the Laser lines required for the experiment (2). Select the cameras for image acquisition (3). Select the plate type and objective lens for the experiment (4). B) Select the microscope tab (1). Define the light source and exposure time for exposure 1 (2,3). Focus on one well and take height (4). C) Select the Experiment Definition tab (1). Drag and drop exposure, skewcrop, reference, layout and sublayout files (2). Drag and drop the experiment file (3). D) Select Automatic Experiment (1). Drag and drop experiment file (2). Allocate appropriate barcode (3). Start image acquisition by clicking on the start button (4). <a href="https://www.jove.com/files/ftp_upload/4382/4382fig3large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 4" src="/files/ftp_upload/4382/4382fig4.jpg" /> 
Figure 4. GFP-Grb2 translocation by cDNA expression. GFP-Grb2 relocates from a predominant cytoplasmic localization to endosome-like structures upon overexpression of a transmembrane growth factor receptor and subsequently internalizes to endosomes. On the left, microscopy images of COS M6 cells are shown that were transiently transfected with GFP-Grb2 (top) or GFP-Grb2 plus EGFR (bottom). Co-expression of the EGFR results in relocation of GFP-Grb2 to endosome-like structures.
<img alt="Figure 5" src="/files/ftp_upload/4382/4382fig5.jpg" /> 
Figure 5. Example of cDNA-induced translocation of GFP-Grb2. The GTPase Dynamin2, which is involved in Grb2-mediated endocytosis, relocates GFP-Grb2 to endosome-like structures (circled). In this experiment, GFP-Grb2 was co-transfected with DNM2 into HEK293T cells. Cells were stained with Hoechst 33342 after 24 hr and images were acquired on the Opera LX. One field of view of the green (GFP) channel and the UV (blue; Hoechst 33342) is displayed.

<ol>
	<li>Sastry, L., Cao, T., King, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+Grb2-protein+complexes+in+human+cancer+cells.">Multiple Grb2-protein complexes in human cancer cells.</a> Int. J. Cancer. 70, 208-213 (1997).</li><li>Li, S., Couvillon, A. D., Brasher, B. B., Van Etten, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tyrosine+phosphorylation+of+Grb2+by+Bcr/Abl+and+epidermal+growth+factor+receptor:+a+novel+regulatory+mechanism+for+tyrosine+kinase+signaling.">Tyrosine phosphorylation of Grb2 by Bcr/Abl and epidermal growth factor receptor: a novel regulatory mechanism for tyrosine kinase signaling.</a> Embo. J. 20, 6793-6804 (2001).</li><li>Bisson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+reaction+monitoring+mass+spectrometry+reveals+the+dynamics+of+signaling+through+the+GRB2+adaptor.">Selected reaction monitoring mass spectrometry reveals the dynamics of signaling through the GRB2 adaptor.</a> Nat. Biotechnol. 29, 653-658 (2011).</li><li>Ketteler, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Feynman+trajectories:+determining+the+path+of+a+protein+using+fixed-endpoint+assays.">The Feynman trajectories: determining the path of a protein using fixed-endpoint assays.</a> J. Biomol. Screen. 15, 321-326 (2010).</li><li>Yamazaki, 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+Grb2+in+EGF-stimulated+EGFR+internalization.">Role of Grb2 in EGF-stimulated EGFR internalization.</a> J. Cell. Sci. 115, 1791-1802 (2002).</li><li>Antczak, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Domain-Based+Biosensor+Assay+to+Screen+for+Epidermal+Growth+Factor+Receptor+Modulators.">Domain-Based Biosensor Assay to Screen for Epidermal Growth Factor Receptor Modulators.</a> in Live Cells. ASSAY and Drug Development Technologies. 10, 24-36 (2012).</li><li>Su, J., Batzer, A., Sap, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+tyrosine+phosphatase+R-PTP-alpha+is+tyrosine-phosphorylated+and+associated+with+the+adaptor+protein+Grb2.">Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2.</a> J. Biol. Chem. 269, 18731-18734 (1994).</li><li>Hertog, J. d. e. n., Tracy, S., Hunter, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphorylation+of+receptor+protein-tyrosine+phosphatase+alpha+on+Tyr789,+a+binding+site+for+the+SH3-SH2-SH3+adaptor+protein+GRB-2+in+vivo.">Phosphorylation of receptor protein-tyrosine phosphatase alpha on Tyr789, a binding site for the SH3-SH2-SH3 adaptor protein GRB-2 in vivo.</a> Embo. J. 13, 3020-3032 (1994).</li><li>Miki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+Ash/Grb-2+with+dynamin+through+the+Src+homology+3+domain.">Association of Ash/Grb-2 with dynamin through the Src homology 3 domain.</a> J. Biol. Chem. 269, 5489-5492 (1994).</li><li>Seedorf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamin+binds+to+SH3+domains+of+phospholipase+C+gamma+and+GRB-2.">Dynamin binds to SH3 domains of phospholipase C gamma and GRB-2.</a> J. Biol. Chem. 269, 16009-16014 (1994).</li><li>Carpenter, A. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellProfiler:+image+analysis+software+for+identifying+and+quantifying+cell+phenotypes.">CellProfiler: image analysis software for identifying and quantifying cell phenotypes.</a> Genome Biol. 7, R100 (2006).</li><li>Seed, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developments+in+expression+cloning.">Developments in expression cloning.</a> Curr. Opin. Biotechnol. 6, 567-573 (1995).</li></ol>

No conflicts of interest declared.

Expression cloning is a powerful tool that has been used in the past to identify novel cellular components such as virus receptors and blood cell antigens12. Here, we describe a method to facilitate the identification of novel putative signal transduction receptors that bind to Grb2.
 There are a few critical steps in the protocol.
 <ol>
 <li>For automated plasmid preparation it is absolutely essential to have complete resuspension of the bacterial pellet. Sometimes, it is necessary to include a rigorous shaking step or further mixing with pipettes.</li>
 <li>Upon addition of solution 2 and 3, complete mixing is essential and we have found that manually inverting the sealed plate is more efficient than the use of an automated shaker. Pipetting should be avoided at this step as it would lead to shearing of the DNA.</li>
 <li>In all vacuum steps, one has to ensure that the liquid is completely drained from the plate. Otherwise low yields are to be expected.</li>
 <li>Upon elution, it is quite common that eluate drops form at the bottom of the plasmid binding plate. These drops need to be collected in the elution plate, so it is recommended that the plasmid binding plate is lifted carefully and any residual drops at the bottom are carefully transferred to the eluate plate.</li>
 <li>During transfection, the time of complex formation is critical. We usually adhere to 20-30 min. Longer times up to 1 hr are fine without reduction in yield, while shorter times are not recommended.</li>
 <li>For the microscopy, the choice of magnification is important. On the Opera LX system used here, resolution at 20x is sufficient to get high quality images. If higher resolution images are required, the objective can be changed, but in order to get statistically significant numbers of cells, a higher number of fields and higher acquisition times are required. In general, we aim to get at least 100 cells imaged per well.</li>
 </ol>
 The Grb2 translocation assay has been used by other groups to identify small molecule inhibitors of EGFR kinase activation6. In that case, the EGFR is specifically activated with ligand causing recruitment of Grb2 to the plasma membrane. Disruption of this interaction can then be investigated using small molecule compounds. Similarly, it can be envisaged that siRNA screens can be employed to identify endogenous genes involved in EGFR-Grb2 signaling or other Grb2-binding growth factor receptors such as c-KIT or the erythropoietin receptor. Thus, there are multiple potential applications for this technique. An analogous approach could be applied to GFP-tagged reporter systems for other adapter molecules such as Shc, Gab or IRS.
 One major advantage of using this cell-based assay in mammalian cells is that it enables the identification of physiologically relevant interactions. The assay is a readout for protein complex formation, but more importantly, the relevant interactions are monitored at the correct sub-cellular site. In this regard, this technique overcomes artifacts from other protein-protein interaction methods such as yeast-two-hybrid or in vitro assays. It should be noted though, that indirect interactions may also result in Grb2 recruitment. Similarly, the transcriptional up-regulation of binding partners may be induced by cDNA expression. To distinguish between these possibilities, it is necessary to perform the appropriate secondary assays to distinguish between direct and indirect binding effects.
 In conclusion, the GFP-adapter molecule translocation assay promises high potential for genome-wide screening and drug discovery applications.

<table border="1">
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 <td>Comments (optional)</td>
 </tr>
 <tr>
 <td>cDNA library</td>
 <td>Origene</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>LB+amp</td>
 <td></td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Gas-permeable seals</td>
 <td>ThermoScientific</td>
 <td>AB-0718</td>
 <td></td>
 </tr>
 <tr>
 <td>Nucleospin 96 Plasmid Kit</td>
 <td>Macherey-Nagel</td>
 <td>740625.4</td>
 <td></td>
 </tr>
 <tr>
 <td>Transfectin</td>
 <td>BioRad</td>
 <td>170-3351</td>
 <td></td>
 </tr>
 <tr>
 <td>Hoechst33342</td>
 <td>Molecular Probes</td>
 <td>H3570</td>
 <td></td>
 </tr>
 <tr>
 <td>Viewplate 96 F TC</td>
 <td>PerkinElmer</td>
 <td>6005182</td>
 <td></td>
 </tr>
 <tr>
 <td>RapidPick</td>
 <td>Hudson</td>
 <td></td>
 <td>Norgren CP7200</td>
 </tr>
 <tr>
 <td>Freedom Evo</td>
 <td>Tecan</td>
 <td></td>
 <td>With vacuum manifold</td>
 </tr>
 <tr>
 <td>Multidrop 384</td>
 <td>Thermo</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Opera LX</td>
 <td>PerkinElmer</td>
 <td></td>
 <td></td>
 </tr>
 </table>

a high-content imaging workflow to study grb2 signaling complexes by expression cloning

Signal transduction by growth factor receptors is essential for cells to maintain proliferation and differentiation and requires tight control. Signal transduction is initiated by binding of an external ligand to a transmembrane receptor and activation of downstream signaling cascades. A key regulator of mitogenic signaling is Grb2, a modular protein composed of an internal SH2 (Src Homology 2) domain flanked by two SH3 domains that lacks enzymatic activity. Grb2 is constitutively associated with the GTPase Son-Of-Sevenless (SOS) via its N-terminal SH3 domain. The SH2 domain of Grb2 binds to growth factor receptors at phosphorylated tyrosine residues thus coupling receptor activation to the SOS-Ras-MAP kinase signaling cascade. In addition, other roles for Grb2 as a positive or negative regulator of signaling and receptor endocytosis have been described. The modular composition of Grb2 suggests that it can dock to a variety of receptors and transduce signals along a multitude of different pathways1-3.
	Described here is a simple microscopy assay that monitors recruitment of Grb2 to the plasma membrane. It is adapted from an assay that measures changes in sub-cellular localization of green-fluorescent protein (GFP)-tagged Grb2 in response to a stimulus4-6. Plasma membrane receptors that bind Grb2 such as activated Epidermal Growth Factor Receptor (EGFR) recruit GFP-Grb2 to the plasma membrane upon cDNA expression and subsequently relocate to endosomal compartments in the cell. In order to identify in vivo protein complexes of Grb2, this technique can be used to perform a genome-wide high-content screen based on changes in Grb2 sub-cellular localization. The preparation of cDNA expression clones, transfection and image acquisition are described in detail below. Compared to other genomic methods used to identify protein interaction partners, such as yeast-two-hybrid, this technique allows the visualization of protein complexes in mammalian cells at the sub-cellular site of interaction by a simple microscopy-based assay. Hence both qualitative features, such as patterns of localization can be assessed, as well as the quantitative strength of the interaction.

Watch this Scientific Journal Video about A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning at JoVE.com

A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Signal  transduction by growth factor receptors is essential for cells to maintain  proliferation and differentiation and requires tight control. Signal  ...

a-high-content-imaging-workflow-to-study-grb2-signaling-complexes

Microsoft Bing

Research

JoVE Journal

Biology

10.5K Views.  University College London. A high-content screening method for the identification of novel signaling competent transmembrane receptors is described. This method is amenable to large-scale automation and allows predictions about in vivo protein binding and the sub-cellular localization of protein complexes in mammalian cells.

Video: A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Using the Gene Pulser MXcell Electroporation System to Transfect Primary Cells with High Efficiency

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser MXcell electroporation system and Gene Pulser electroporation buffer (Bio-Rad) were specifically developed to easily transfect nucleic acids into mammalian cells and difficult-to-transfect cells, such as primary and stem cells. We will demonstrate how to perform a simple experiment to quickly identify the best electroporation conditions. We will demonstrate how to run several samples through a range of electroporation conditions so that an experiment can be conducted at the same time as optimization is performed. We will also show how optimal conditions identified using 96-well electroporation plates can be used with standard electroporation cuvettes, facilitating the switch from electroporation plates to electroporation cuvettes while maintaining the same electroporation efficiency. In the video, we will also discuss some of the key factors that can lead to the success or failure of electroporation experiments.

This procedure shows how to use the Gene Pulser MXcell electroporation system to rapidly and easily identify the best electroporation conditions for mouse embryonic fibroblasts (MEFs) or other primary cells. Considerations for troubleshooting are also discussed in the associated video.

It is becoming increasingly apparent that electroporation is the most effective way to introduce plasmid DNA or siRNA into primary cells. The Gene Pulser ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Using an Automated Cell Counter to Simplify Gene Expression Studies: siRNA Knockdown of IL-4 Dependent Gene Expression in Namalwa Cells

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only to study the effects of downregulating single genes, but also to interrogate signaling pathways and other complex interaction networks.  These pathway analyses require both the use of relevant cellular models and methods that cause less perturbation to the cellular physiology.  Electroporation is increasingly being used as an effective way to introduce siRNA and other nucleic acids into difficult to transfect cell lines and primary cells without altering the signaling pathway under investigation. There are multiple critical steps to a successful siRNA experiment, and there are ways to simplify the work while improving the data quality at several experimental stages.  To help you get started with your siRNA mediated gene knockdown project, we will demonstrate how to perform a pathway study complete from collecting and counting the cells prior to electroporation through post transfection real-time PCR gene expression analysis.  The following study investigates the role of the transcriptional activator STAT6 in IL-4 dependent gene expression of CCL17 in a Burkitt lymphoma cell line (Namalwa).  The techniques demonstrated are useful for a wide range of siRNA-based experiments on both adherent and suspension cells.  We will also show how to streamline cell counting with the TC10 automated cell counter, how to electroporate multiple samples simultaneously using the MXcell electroporation system, and how to simultaneously assess RNA quality and quantity with the Experion automated electrophoresis system.

This procedure describes a quick and easy workflow to introduce siRNA into difficult to transfect cell lines and follow gene expression by real-time PCR. Use of an automated cell counter, multi-well electroporation plate, and automated electrophoresis station provide quick and reliable results without the need for expensive robotic handling.

The use of siRNA mediated gene knockdown is continuing to be an important tool in studies of gene expression.  siRNA studies are being conducted not only ...

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of muscle specification, formation and function in model systems has provided valuable insight into muscle physiology. Therefore, identifying and characterizing molecular mechanisms that underlie muscle damage is critical. The structure of adult Drosophila multi-fiber muscles resemble vertebrate striated muscles 1 and the genetic tractability of Drosophila has made it a great system to analyze dystrophic muscle morphology and characterize the processes affecting muscular function in ageing adult flies 2. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. These preparations allow for the tissue to be stained with classical histological stains and labeled with protein detecting dyes, and specifically cryosections are ideal for immunohistochemical detection of proteins in intact muscles. This allows for analysis of muscle tissue structure, identification of morphological defects, and detection of the expression pattern for muscle/neuron-specific proteins in Drosophila adult muscles. These techniques can also be slightly modified for sectioning of other body parts.

Paraffin-Embedded and Frozen Sections of Drosophila Adult Muscles

Identification of mechanisms underlying muscle damage is crucial. Here we present the histological technique for preparing paraffin-embedded and frozen sections of Drosophila thoracic muscles. This allows analysis of muscle morphology and localization of protein and other muscle cell components.

The molecular characterization of muscular dystrophies and myopathies in humans has revealed the complexity of muscle disease and genetic analysis of ...

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a crucial role in transepithelial fluid homeostasis1-3. CFTR has been implicated in two major diseases: cystic fibrosis (CF)4 and secretory diarrhea5. In CF, the synthesis or functional activity of the CFTR Cl- channel is reduced. This disorder affects approximately 1 in 2,500 Caucasians in the United States6. Excessive CFTR activity has also been implicated in cases of toxin-induced secretory diarrhea (e.g., by cholera toxin and heat stable E. coli enterotoxin) that stimulates cAMP or cGMP production in the gut7.
Accumulating evidence suggest the existence of physical and functional interactions between CFTR and a growing number of other proteins, including transporters, ion channels, receptors, kinases, phosphatases, signaling molecules, and cytoskeletal elements, and these interactions between CFTR and its binding proteins have been shown to be critically involved in regulating CFTR-mediated transepithelial ion transport in vitro and also in vivo8-19. In this protocol, we focus only on the methods that aid in the study of the interactions between CFTR carboxyl terminal tail, which possesses a protein-binding motif [referred to as PSD95/Dlg1/ZO-1 (PDZ) motif], and a group of scaffold proteins, which contain a specific binding module referred to as PDZ domains. So far, several different PDZ scaffold proteins have been reported to bind to the carboxyl terminal tail of CFTR with various affinities, such as NHERF1, NHERF2, PDZK1, PDZK2, CAL (CFTR-associated ligand), Shank2, and GRASP20-27. The PDZ motif within CFTR that is recognized by PDZ scaffold proteins is the last four amino acids at the C terminus (i.e., 1477-DTRL-1480 in human CFTR)20. Interestingly, CFTR can bind more than one PDZ domain of both NHERFs and PDZK1, albeit with varying affinities22. This multivalency with respect to CFTR binding has been shown to be of functional significance, suggesting that PDZ scaffold proteins may facilitate formation of CFTR macromolecular signaling complexes for specific/selective and efficient signaling in cells16-18.
Multiple biochemical assays have been developed to study CFTR-involving protein interactions, such as co-immunoprecipitation, pull-down assay, pair-wise binding assay, colorimetric pair-wise binding assay, and macromolecular complex assembly assay16-19,28,29. Here we focus on the detailed procedures of assembling a PDZ motif-dependent CFTR-containing macromolecular complex in vitro, which is used extensively by our laboratory to study protein-protein or domain-domain interactions involving CFTR16-19,28,29.

In Vitro Analysis of PDZ-dependent CFTR Macromolecular Signaling Complexes

Cystic fibrosis transmembrane conductance regulator (CFTR), an epithelial chloride channel, has been reported to interact with various proteins and regulate important cellular processes; among them the CFTR PDZ motif-mediated interactions have been well documented. This protocol describes methods we developed to assemble a PDZ-dependent CFTR macromolecular signaling complex in vitro.

Cystic fibrosis transmembrane conductance regulator (CFTR), a chloride channel located primarily at the apical membranes of epithelial cells, plays a ...

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial scientific task. Many disease causing genes in humans have a fly homologue, making Drosophila a good model to study signaling pathways involved in the development of different disorders. Additionally, the tractability of Drosophila simplifies genetic screens to aid in identifying novel therapeutic targets that may regulate metabolism. In order to perform such a screen a simple and fast method to identify changes in the metabolic state of flies is necessary. In general, carbon dioxide production is a good indicator of substrate oxidation and energy expenditure providing information about metabolic state. In this protocol we introduce a simple method to measure CO2 output from flies. This technique can potentially aid in the identification of genetic perturbations affecting metabolic rate.

Measurement of Metabolic Rate in Drosophila using Respirometry

Metabolic disorders are among one of the most common diseases in humans. The genetically tractable model organism D. melanogaster can be used to identify novel genes that regulate metabolism. This paper describes a relatively simple method which allows studying the metabolic rate in flies by measuring their CO2 production.

Metabolic disorders are a frequent problem affecting human health. Therefore, understanding the mechanisms that regulate metabolism is a crucial ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Workflow for High-content, Individual Cell Quantification of Fluorescent Markers from Universal Microscope Data, Supported by Open Source Software

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly dependent on modes of single-cell analysis. Methods which deliver composite data reflecting the mean values of biomarkers from cell populations risk losing subpopulation dynamics that reflect the heterogeneity of the studied biological system. In keeping with this, traditional approaches are being replaced by, or supported with, more sophisticated forms of cellular assay developed to allow assessment by high-content microscopy. These assays potentially generate large numbers of images of fluorescent biomarkers, which enabled by accompanying proprietary software packages, allows for multi-parametric measurements per cell. However, the relatively high capital costs and overspecialization of many of these devices have prevented their accessibility to many investigators.
Described here is a universally applicable workflow for the quantification of multiple fluorescent marker intensities from specific subcellular regions of individual cells suitable for use with images from most fluorescent microscopes. Key to this workflow is the implementation of the freely available Cell Profiler software1&#160;to distinguish individual cells in these images, segment them into defined subcellular regions and deliver fluorescence marker intensity values specific to these regions. The extraction of individual cell intensity values from image data is the central purpose of this workflow and will be illustrated with the analysis of control data from a siRNA screen for G1 checkpoint regulators in adherent human cells. However, the workflow presented here can be applied to analysis of data from other means of cell perturbation (e.g., compound screens) and other forms of fluorescence based cellular markers and thus should be useful for a wide range of laboratories.

Presented is a flexible informatics workflow enabling multiplexed image-based analysis of fluorescently labeled cells. The workflow quantifies nuclear and cytoplasmic markers and computes marker translocation between these compartments. Procedures are provided for perturbation of cells using siRNA and reliable methodology for marker detection by indirect immunofluorescence in 96-well formats.

Advances in understanding the control mechanisms governing the behavior of cells in adherent mammalian tissue culture models are becoming increasingly ...

Imaging Spatial Reorganization of a MAPK Signaling Pathway Using the Tobacco Transient Expression System

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular fluorescent complementation (BiFC) assay in tobacco epidermal cells, from testing protein-protein interaction to monitoring spatial distribution of signal transduction in plant cells. In this protocol, we used the BiFC assay to show that the interaction and the signaling between the Arabidopsis MAPKKK YODA and MAPK6 occur at the plasma membrane. When the scaffold protein BASL was co-expressed, the YODA-MAPK interaction redistributed and spatially co-polarized with CFP-BASL. This modified tobacco expression system allows for quick examination of signaling localization and dynamic changes (less than 4 days) and can accommodate multiple of fluorescent protein colors (at least three). We also presented detailed methods to quantify protein distribution (asymmetric spatial localization, or &#34;polarization&#34;) in tobacco cells. This advanced tobacco expression system has a potential to be used widely for quick testing of dynamic signaling events in live plant cells.

At the subcellular level, signaling events are dynamically modulated by developmental and environmental cues. Here we describe a protocol that employs the tobacco transient expression system to monitor dynamic protein-protein interaction and to disclose spatial organization of signal transduction in plant cells.

Visualization of dynamic signaling events in live cells has been a challenge. We expanded an established transient expression system, the biomolecular ...