The ITSN, PI3K-C2&beta;, and control vectors used in this protocol are available from the authors upon request, for non-commercial purposes only. The authors wish to acknowledge Dr. Chang-Deng Hu for kindly providing advice and the reagents used in establishing the BiFC protocol in the O'Bryan laboratory. KAW was supported by funding from the Foundation Jerome Lejeune. Work in the O'Bryan laboratory is supported by grants from the NIH (HL090651), DOD (PR080428), the St. Baldrick's Foundation, and the Foundation Jerome Lejeune.

A. BiFC Calibration
<ol>
	<li>Choose a fluorophore. There are multiple fluorophores, such as YFP and Venus, that work well as BiFC fusion partners (Table 1). Amino- and carboxy-terminal ends of Venus are able to form a complex at 37&deg;C, while the YFP BiFC fragments require a pre-incubation at 30&deg;C in order to facilitate fluorophore formation2. This incubation at a low temperature may alter some cellular processes and should be taken into account when choosing fragments. Vectors for fusing Venus to the carboxy-terminus of proteins are available from Addgene (<a href="http://www.addgene.org/pgvec1">http://www.addgene.org/pgvec1</a>; seepBiFC-VN173 and pBiFC-VC155) along with additional constructs for use as controls, e.g., <a href="http://www.addgene.org/pgvec1?f=c&amp;identifier=22012&amp;atqx=BiFC&amp;cmd=findpl">pBiFC-bJunVN173</a> and pBiFC-bFosVC1552. Additional vectors, including amino-terminal Venus vectors (pFLAG-VN173 and pHA-VC155), are available at the following site: <a href="http://people.pnhs.purdue.edu/%7Ehu1/">http://people.pnhs.purdue.edu/~hu1/</a>.</li>
	<li>Tag the protein of interest. The BiFC fragments are fused to the amino- or carboxy- terminal ends of the candidate proteins. Some proteins may not allow for tagging at either end due to disruption of protein function. For example, many members of the Ras superfamily of GTPases are lipid modified at the carboxy-terminus thus precluding attachment of the BiFC fragments at that end. Thus, it is important to have some idea of how attachment of the BiFC fragments may affect function of the proteins of interest. If it is unclear how tagging a protein will affect its function multiple combinations should be tested. In addition to the BiFC fragment, a peptide linker may be included to increase the flexibility between the fragmented fluorophore and the candidate proteins. While the multiple cloning sites (MCS) in the BiFC vectors encode short amino acid stretches that may provide sufficient flexibility, the RSIAT, KQKVMNH, and RPACKIPNDLKQKVMNH linkers have been successfully used in BiFC experiments3,9</li>
	<li>Determine transfection conditions. Before testing multiple mutants, a few control experiments should be performed. The first two BiFC combinations that should be tried are two wild type proteins that are known to interact and one wild type and mutant that do not interact. Using these two combinations, different amounts of DNA and transfection times should be tested to determine optimum conditions for detecting a BiFC signal for the candidate proteins. We suggest testing 0.25ug, 0.5ug, 1.0ug of each BiFC construct for a single well of a 6 well dish. The day after transfection monitor cells by fluorescence microscopy to determine optimum time for signal development. The <a href="http://www.addgene.org/pgvec1?f=c&amp;identifier=22012&amp;atqx=BiFC&amp;cmd=findpl">pBiFC-bJunVN173</a>and pBiFC-bFosVC1552constructs are useful positive controls for BiFC and are available from Addgene (see above). In addition, Western blot analysis should be performed to confirm equal expression of the constructs. Conditions should be chosen such that a fluorescent signal is observed between the two wild type proteins but little to no signal is observed between the wild type and mutant proteins. Finally, it is best to keep protein expression as low as possible to prevent any non-specific interactions.</li>
	<li>Determine if the addition of the BiFC fragments alters the localization of the proteins of interest. Each BiFC construct contains either an HA or FLAG epitope tag. Immunostain transefected cells for both the HA or FLAG epitope tag as well as well the endogenous protein (if possible) to determine if the BiFC tag affects localization of the proteins of interest.</li>
</ol>
B. Plating and Transfection of Cells
<ol>
	<li>COS cells (1.3x105) are plated each onto one glass bottom Matek plate and two wells of a 6-well plate per sample. Allow cells to settle overnight at 37&deg;C. Alternative cell types that are more relevant to the candidate proteins of interest may also be used.</li>
	<li>Prepare DNAs for transfection. We typically utilize Lipofectamine (Invitrogen) for COS transfections. However, other reagents may be more appropriate for the cell line of interest. Since the transfection mixture will be split between one glass bottom dish and two wells of a 6-well plate, use the appropriate amount of DNA to account for this division. Dilute DNA in 250uL of serum free (SF) DMEM. Add CFP at 1/5 the amount of total BiFC DNAs as a transfection control. For BiFC quantification, only cells which are positive for CFP will be analyzed for the presence of a BiFC signal. Note that the CFP spectra will overlap with some of the BiFC pairs in Table 1; therefore an alternate transfection control maybe needed. Dilute Lipofectamine in 250uL SF DMEM (10uL Lipofectamine/1ug of DNA). Mix the DNA and Lipofectamine dilutions. Incubate at room temperature for 20min. 
	Note: The Lipofectamine:DNA ratio can vary depending on cell type. Use the appropriate transfection method for the cell line of interest.</li>
	<li>Rinse cells 2x with warm SF DMEM. Add 2mL of SF DMEM to each glass bottom plate or well of a 6-well dish.</li>
	<li>Split each transfection mixture evenly between one glass bottom dish and two wells of a 6-well dish.</li>
	<li>Incubate cells at 37&deg;C for 5hrs.</li>
	<li>Remove transfection media and replace with complete media (DMEM+10%FBS).</li>
	<li>Incubate cells overnight at 37&deg;C. The length of incubation following transfection will vary depending on the expression levels of the proteins of interest. Prolonged incubation may result in non-specific interaction so this step will need to be determined empirically.</li>
</ol>
C. Preparation of Cells for Imaging
<ol>
	<li>Cells are initially examined under an epifluorescent microscope to ensure that the positive control is fluorescent. If not, it may be necessary to allow cells additional time at 37&deg;C until signal is observed.</li>
	<li>Rinse cells 3x with PBS (pH 7.4). To the cells in the glass bottom dish add 2%paraformaldehyde (pH 7.4). Fix cells for 10min on ice. Rinse cells 3x with PBS (pH 7.4). Store cells at 4&deg;C covered with 1mL PBS (pH 7.4). Cells do not have to be fixed for imaging, but once the BiFC fragments reform an intact fluorophore it is irreversible thus preventing analysis of dynamic interactions3. Also, keep in mind that unfixed cells will continue to develop signal. Lyse the cells in the 6-well dish and prepare lysates for Western blot analysis. It is important that all cells are prepared at the same time so that the lysed cells are representative of the imaged cells.</li>
</ol>
D. Imaging Cells
<ol>
	<li>The fluorescence intensity will be calculated per cell. Be sure to image individual cells.</li>
	<li>CFP was included as a tranfection control and only CFP positive cells are selected for analysis of BiFC signals. We make the assumption that if the cell is transfected with CFP, it is also transfected with the BiFC constructs. This approach ensures that imaged cells which lack a BiFC signal are negative due to a lack of an interaction between the proteins of interest and not due to absence of one or both BiFC expression constructs in that cell.</li>
	<li>We use a Zeiss LSM 510 confocal microscope for cell imaging. When using this microscope it is important to keep the zoom, pinhole, detector gain, amplifier offset, frame size, scan speed, scan average, and laser power consistent. When using any imaging system it is important to keep settings constant so that the fluorescence is comparable between samples. Also, when quantifying fluorescence it is important that pixels are not saturated.</li>
</ol>
E. Quantifying Fluorescence
<ol>
	<li>Fluorescence may be quantified using any imaging software. We utilize ImageJ which is freely available from the NIH (http://rsb.info.nih.gov/ij/). Open image files in ImageJ. Go to AnalyzeSet Measurements. Check the boxes for Area and Mean Gray Value in the Measurements box.</li>
	<li>Using the 'free hand selection' tool, draw an outline around the edge of the entire cell in the CFP channel.</li>
	<li>Leaving this outline in place, shift to the YFP channel. Go to AnalyzeMeasure. 
	The Mean Gray Value is the sum of the grey values of all the pixels in the selection divided by the number of pixel (i.e., the average fluorescence intensity per area of the cell).</li>
	<li>For each image draw a circle in the YFP channel in an area that does not contain a cell. Take a measurement for this area as background. Subtract the background from each image.</li>
	<li>Average the Mean Gray Value minus the background for all the cells imaged in one sample. This will be the average fluorescence intensity for a population of cells. We would suggest about 60 cells over three experiments be quantified.</li>
</ol>
F. Representative Results:
Our lab focuses on the multi-domain scaffolding protein, intersectin (ITSN) which interacts with numerous proteins to regulate multiple biochemical and signaling pathways10,11,12,13,14. ITSN contains two Eps15 homology (EH) domains, a coiled-coiled region, and five Src homology 3 (SH3) domains. The longer isoform of ITSN also contains Dbl homology (DH) and pleckstrin homology (PH) domains that act in concert as a guanine nucleotide exchange factor for Cdc4215. This modular structure promotes protein:protein interactions and makes ITSN an ideal candidate for BiFC experiments. The subcellular localization of ITSN can alter its binding partners and therefore alter the pathways regulated by ITSN (unpublished data). Recently, our lab has demonstrated that ITSN regulates neuronal survival through regulation of a novel class II PI3K, PI3K-C2&beta; 11. The amino-terminal Pro-rich domain of PI3K-C2&beta; contains two binding sites for ITSN's SH3 domains. Using co-immunoprecipitation with ITSN and PI3K-C2&beta; truncation mutants we demonstrated that ITSN's SH3A and SH3C domains interact with the amino-terminal region of PI3K-C2&beta;. Next, we used BiFC to visualize the subcellular localization of this complex. ITSN was fused to the amino-terminus of Venus (pFLAG-VN173) and PI3K-C2&beta; constructs fused to the carboxy-terminus of Venus (pHA-VC155). As another negative control, a non-specific peptide was fused to pHA-VC155. VN-ITSN and VC-PI3K-C2&beta; formed a BiFC complex with a punctuate distribution (Figure 2A, upper panels). Mutations in the Pro-rich domain of PI3K-C2&beta; that disrupt co-precipitation of ITSN and PI3K-C2&beta; decreased the BiFC signal (Figure 2A, lower panels)11. This difference in BiFC signal between ITSN and the two PI3K-C2&beta; proteins was not due to differences in protein expression (Figure 2B).
<img src="/files/ftp_upload/2643/2643fig1.jpg" alt="Figure 1" /> 
Figure 1. In BiFC, a fluorophore (in this case Venus) is split into amino(VN)- and carboxy(VC)-terminal ends. These ends are fused to two proteins of interest. When the two proteins interact, the VN and VC fragments re-associate resulting in reconstitution of the fluorophore and fluorescence at the sites of interaction. BiFC is a specific example of the protein fragment complementation assay (PCA) used to measure protein:protein interactions5.
<img src="/files/ftp_upload/2643/2643fig2.jpg" alt="Figure 2" /> 
Figure 2. ITSN and PI3K-C2&beta; form a BiFC complex. A. VN-tagged ITSN was co-tranfected with VC-tagged PI3K-C3&beta; WT or a proline-rich domain mutant (PI3K-C2&beta;-PA). ITSN and WT PI3K-C2&beta; form a complex (green). CFP (red) was used as a transfection control B. A Western blot was performed to demonstrate equal expression of the constructs. The VC-tagged constructs are HA tagged.
<img src="/files/ftp_upload/2643/2643table1.jpg" alt="Table 1" /> 
Table 1. There are multiple vectors that are compatible with BiFC. YN155: 1-155aa of YFP; YC155: 155-238aa of YFP; YN173: 1-172aa of YFP; YC173, 173-238aa of YFP; VN155: 1-154aa of Venus; VC155: 155-238 of Venus; VN173: 1-172aa of Venus; VC173: 173-238aa of Venus; CN155 1-154aa of CFP; CC155 155-238aa of CFP, GN173: 1-172aa of GFP, CitN155: 1-155aa of Citrine, CitC155: 155-238aa of Citrine, CitN173: 1-172aa of Citrine, CitC173: 173-238aa of Citrine, CerN173: 1-172aa of Cerulean 2,3,9.

<ol>
	<li>Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+molecular+interactions+by+fluorescence+complementation.">Visualization of molecular interactions by fluorescence complementation.</a> Nat Rev Mol Cell Biol. 7, 449-456 (2006).</li><li>Shyu, Y. J., Liu, H., Deng, X., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+new+fluorescent+protein+fragments+for+bimolecular+fluorescence+complementation+analysis+under+physiological+conditions.">Identification of new fluorescent protein fragments for bimolecular fluorescence complementation analysis under physiological conditions.</a> Biotechniques. 40, 61-66 (2006).</li><li>Hu, C. D., Chinenov, Y., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+interactions+among+bZIP+and+Rel+family+proteins+in+living+cells+using+bimolecular+fluorescence+complementation.">Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation.</a> Mol Cell. 9, 789-798 (2002).</li><li>Tsien, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+green+fluorescent+protein.">The green fluorescent protein.</a> Annu Rev Biochem. 67, 509-544 (1998).</li><li>Michnick, S. W., Remy, I., Campbell-Valois, F. X., Vallee-Belisle, A., Pelletier, J. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+protein-protein+interactions+by+protein+fragment+complementation+strategies.">Detection of protein-protein interactions by protein fragment complementation strategies.</a> Methods Enzymol. S328, 208-230 (2000).</li><li>Michnick, S. W., MacDonald, M. L., Westwick, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+genetic+strategies+to+delineate+MAP+kinase+signaling+pathways+using+protein-fragment+complementation+assays+(PCA).">Chemical genetic strategies to delineate MAP kinase signaling pathways using protein-fragment complementation assays (PCA).</a> Methods. 40, 287-293 (2006).</li><li>Piston, D. W., Kremers, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent+protein+FRET:+the+good,+the+bad+and+the+ugly.">Fluorescent protein FRET: the good, the bad and the ugly.</a> Trends Biochem Sci. 32, 407-414 (2007).</li><li>Ciruela, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+methods+in+the+study+of+protein-protein+interactions+in+living+cells.">Fluorescence-based methods in the study of protein-protein interactions in living cells.</a> Curr Opin Biotechnol. 19, 338-343 (2008).</li><li>Hu, C. D., Kerppola, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+visualization+of+multiple+protein+interactions+in+living+cells+using+multicolor+fluorescence+complementation+analysis.">Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis.</a> Nat Biotechnol. 21, 539-545 (2003).</li><li>Martin, N. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersectin+regulates+epidermal+growth+factor+receptor+endocytosis,+ubiquitylation,+and+signaling.">Intersectin regulates epidermal growth factor receptor endocytosis, ubiquitylation, and signaling.</a> Mol Pharmacol. 70, 1643-1653 (2006).</li><li>Das, M. <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+neuron+survival+through+an+intersectin-phosphoinositide+3'-kinase+C2beta-AKT+pathway.">Regulation of neuron survival through an intersectin-phosphoinositide 3'-kinase C2beta-AKT pathway.</a> Mol Cell Biol. 27, 7906-7917 (2007).</li><li>Mohney, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersectin+activates+Ras+but+stimulates+transcription+through+an+independent+pathway+involving.">Intersectin activates Ras but stimulates transcription through an independent pathway involving.</a> JNK. J Biol Chem. 278, 47038-47045 (2003).</li><li>Tong, X. K., Hussain, N. K., Adams, A. G., O'Bryan, J. P., McPherson, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersectin+can+regulate+the+Ras/MAP+kinase+pathway+independent+of+its+role+in+endocytosis.">Intersectin can regulate the Ras/MAP kinase pathway independent of its role in endocytosis.</a> J Biol Chem. 275, 29894-29899 (2000).</li><li>Adams, A., Thorn, J. M., Yamabhai, M., Kay, B. K., O'Bryan, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intersectin+an+adaptor+protein+involved+in+clathrin-mediated+endocytosis,+activates+mitogenic+signaling+pathways.">Intersectin an adaptor protein involved in clathrin-mediated endocytosis, activates mitogenic signaling pathways.</a> J Biol Chem. 275, 27414-27420 (2000).</li><li>O'Bryan, J. P., Mohney, R. P., Oldham, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogenesis+and+endocytosis:+What's+at+the+INTERSECTIoN?.">Mitogenesis and endocytosis: What's at the INTERSECTIoN?.</a> Oncogene. 20, 6300-6308 (2001).</li><li>Shyu, Y. J., Suarez, C. D., Hu, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+AP-1+NF-kappaB+ternary+complexes+in+living+cells+by+using+a+BiFC-based+FRET.">Visualization of AP-1 NF-kappaB ternary complexes in living cells by using a BiFC-based FRET.</a> Proc Natl Acad Sci U S A. 105, 151-156 (2008).</li></ol>

No conflicts of interest declared.

BiFC is an excellent method for visualizing protein:protein interactions in whole cells and determining the subcellular localization of these complexes. The advantages of BiFC are that only interacting proteins are fluorescent, transient interactions are stabilized, and post-processing of the imaging data is minimal. Two disadvantages of this method are the maturation time for the fluorophore and the irreversibility of fluorophore complex. Under some applications this irreversibility can be used as an advantage. For exam...

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		<th>Material Name</th>
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		<th>Catalogue Number</th>
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<td>DMEM</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-013</td>
<td>&nbsp;</td>
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<td>Fetal bovine serum</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>35-011-CV</td>
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<td>Glass Bottom Microwell dishes</td>
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<td>Matek</td>
<td>P35G-1.5-14C</td>
<td>&nbsp;</td>
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<td>6-well dishes</td>
<td>&nbsp;</td>
<td>Falcon</td>
<td>35-3846</td>
<td>&nbsp;</td>
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<td>Lipofectamine</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>18324020</td>
<td>&nbsp;</td>
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<td>PBS</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>21-031-CV</td>
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<td>Paraformaldehyde</td>
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<td>Sigma</td>
<td>P6148</td>
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<td>Confocal Microscope</td>
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<td>Zeiss</td>
<td>LSM510 META</td>
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bimolecular fluorescence complementation

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as immunoprecipitation do not provide information on the spatial localization of complexes. In contrast, BiFC monitors the interaction and subcellular compartmentalization of protein complexes. In this method, a fluororescent protein is split into amino- and carboxy-terminal non-fluorescent fragments which are then fused to two proteins of interest. Interaction of the proteins results in reconstitution of the fluorophore (Figure 1)1,2. A limitation of BiFC is that once the fragmented fluorophore is reconstituted the complex is irreversible3. This limitation is advantageous in detecting transient or weak interactions, but precludes a kinetic analysis of complex dynamics. An additional caveat is that the reconstituted flourophore requires 30min to mature and fluoresce, again precluding the observation of real time interactions4. BiFC is a specific example of the protein fragment complementation assay (PCA) which employs reporter proteins such as green fluorescent protein variants (BiFC), dihydrofolate reductase, b-lactamase, and luciferase to measure protein:protein interactions5,6. Alternative methods to study protein:protein interactions in cells include fluorescence co-localization and F&ouml;rster resonance energy transfer (FRET)7. For co-localization, two proteins are individually tagged either directly with a fluorophore or by indirect immunofluorescence. However, this approach leads to high background of non-interacting proteins making it difficult to interpret co-localization data. In addition, due to the limits of resolution of confocal microscopy, two proteins may appear co-localized without necessarily interacting. With BiFC, fluorescence is only observed when the two proteins of interest interact. FRET is another excellent method for studying protein:protein interactions, but can be technically challenging. FRET experiments require the donor and acceptor to be of similar brightness and stoichiometry in the cell. In addition, one must account for bleed through of the donor into the acceptor channel and vice versa. Unlike FRET, BiFC has little background fluorescence, little post processing of image data, does not require high overexpression, and can detect weak or transient interactions. Bioluminescence resonance energy transfer (BRET) is a method similar to FRET except the donor is an enzyme (e.g. luciferase) that catalyzes a substrate to become bioluminescent thereby exciting an acceptor. BRET lacks the technical problems of bleed through and high background fluorescence but lacks the ability to provide spatial information due to the lack of substrate localization to specific compartments8. Overall, BiFC is an excellent method for visualizing subcellular localization of protein complexes to gain insight into compartmentalized signaling.

Watch this Scientific Journal Video about Bimolecular Fluorescence Complementation at JoVE.com

Bimolecular Fluorescence Complementation

The subcellular localization of proteins is important in determining the spatio-temporal regulation of cell signaling. Here, we describe bimolecular fluorescence complementation (BiFC) as a straightforward method for monitoring the spatial interactions of proteins in the cell.

Defining the subcellular distribution of signaling complexes is imperative to understanding the output from that complex. Conventional methods such as ...

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Biology

27.8K vistas.  University of Illinois at Chicago. La localización subcelular de las proteínas es importante para determinar la regulación espacio-temporal de la señalización celular. Aquí se describe la complementación bimolecular de fluorescencia (BiFC) como un método sencillo para el control de la interacción espacial de las proteínas en la célula.

Video: Bimolecular Fluorescence Complementation

Fluorescence Activated Cell Sorting of Plant Protoplasts

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental stimuli in any multicellular organism. In situ hybridization and reporter gene visualization can to a limited extent be used to this end but for high resolution quantitative RT-PCR or high-throughput transcriptome-wide analysis the isolation of RNA from particular cell types is requisite. Cellular dissociation of tissue expressing a fluorescent protein marker in a specific cell type and subsequent Fluorescence Activated Cell Sorting (FACS) makes it possible to collect sufficient amounts of material for RNA extraction, cDNA synthesis/amplification and microarray analysis.
An extensive set of cell type-specific fluorescent reporter lines is available to the plant research community. In this case, two marker lines of the Arabidopsis thaliana root are used: PSCR::GFP (endodermis and quiescent center) and PWOX5::GFP (quiescent center). Large numbers (thousands) of seedlings are grown hydroponically or on agar plates and harvested to obtain enough root material for further analysis. Cellular dissociation of plant material is achieved by enzymatic digestion of the cell wall. This procedure makes use of high osmolarity-induced plasmolysis and commercially available cellulases, pectinases and hemicellulases to release protoplasts into solution.
FACS of GFP-positive cells makes use of the visualization of the green versus the red emission spectra of protoplasts excited by a 488 nm laser. GFP-positive protoplasts can be distinguished by their increased ratio of green to red emission. Protoplasts are typically sorted directly into RNA extraction buffer and stored for further processing at a later time.
This technique is revealed to be straightforward and practicable. Furthermore, it is shown that it can be used without difficulty to isolate sufficient numbers of cells for transcriptome analysis, even for very scarce cell types (e.g. quiescent center cells). Lastly, a growth setup for Arabidopsis seedlings is demonstrated that enables uncomplicated treatment of the plants prior to cell sorting (e.g. for the cell type-specific analysis of biotic or abiotic stress responses). Potential supplementary uses for FACS of plant protoplasts are discussed.

A method for isolating specific cell types from plant material is demonstrated. This technique employs transgenic marker lines expressing fluorescent proteins in particular cell types, cellular dissociation and Fluorescence Activated Cell Sorting. Additionally, a growth setup is established here that facilitates treatment of Arabidopsis thaliana seedlings prior to cell sorting.

High-resolution, cell type-specific analysis of gene expression greatly enhances understanding of developmental regulation and responses to environmental ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation (BiFC) System

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein (YFP) into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest. These constructs can then be co-transformed into Nicotiana benthamiana via Agrobacterium mediated transformation, allowing the transit expression of fusion proteins. The reconstitution of YFP signal only occurs when the inquest proteins interact 1-7. To test and validate the protein-protein interactions, BiFC can be used together with yeast two hybrid (Y2H) assay. This may detect indirect interactions which can be overlooked in the Y2H. Gateway technology is a universal platform that enables researchers to shuttle the gene of interest (GOI) into as many expression and functional analysis systems as possible8,9. Both the orientation and reading frame can be maintained without using restriction enzymes or ligation to make expression-ready clones. As a result, one can eliminate all the re-sequencing steps to ensure consistent results throughout the experiments. We have created a series of Gateway compatible BiFC and Y2H vectors which provide researchers with easy-to-use tools to perform both BiFC and Y2H assays10. Here, we demonstrate the ease of using our BiFC system to test protein-protein interactions in N. benthamiana plants.

Detection of Protein Interactions in Plant using a Gateway Compatible Bimolecular Fluorescence Complementation BiFC System

We have developed a technique to test protein-protein interactions in plant. A yellow fluorescent protein (YFP) is split into two non-overlapping fragments. Each fragment is cloned in-frame to a gene of interest via Gateway system, enabling expression of fusion proteins. Reconstitution of YFP signal only occurs when the inquest proteins interact.

We have developed a BiFC technique to test the interaction between two proteins in vivo. This is accomplished by splitting a yellow fluorescent protein ...

Cryosectioning Yeast Communities for Examining Fluorescence Patterns

Microbes typically live in communities. The spatial organization of cells within a community is believed to impact the survival and function of the community1. Optical sectioning techniques, including confocal and two-photon microscopy, have proven useful for observing spatial organization of bacterial and archaeal communities2,3. A combination of confocal imaging and physical sectioning of yeast colonies has revealed internal organization of cells4. However, direct optical sectioning using confocal or two-photon microscopy has been only able to reach a few cell layers deep into yeast colonies. This limitation is likely because of strong scattering of light from yeast cells4.
Here, we present a method based on fixing and cryosectioning to obtain spatial distribution of fluorescent cells within Saccharomyces cerevisiae communities. We use methanol as the fixative agent to preserve the spatial distribution of cells. Fixed communities are infiltrated with OCT compound, frozen, and cryosectioned in a cryostat. Fluorescence imaging of the sections reveals the internal organization of fluorescent cells within the community.
Examples of yeast communities consisting of strains expressing red and green fluorescent proteins demonstrate the potentials of the cryosectioning method to reveal the spatial distribution of fluorescent cells as well as that of gene expression within yeast colonies2,3. Even though our focus has been on Saccharomyces cerevisiae communities, the same method can potentially be applied to examine other microbial communities.

We present a protocol for freezing and cryosectioning yeast communities to observe internal patterns of fluorescent cells. The method relies on methanol-fixing and OCT-embedding to preserve the spatial distribution of cells without inactivating fluorescent proteins within a community.

Microbes typically live  in communities. The spatial organization  of cells within a community is believed to impact the survival and function of the  ...

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC is based on the production of fluorescence using two non-fluorescent fragments of a fluorescent protein (Venus, a Yellow Fluorescent Protein variant, is used here). Non-fluorescent Venus fragments (VN and VC) are fused to two interacting proteins (in this case, AKAP-Lbc and PDE4D3), yielding fluorescence due to VN-AKAP-Lbc-VC-PDE4D3 interaction and the formation of a functional fluorescent protein inside cells.
BiFC provides information on the subcellular localization of protein complexes and the strength of protein interactions based on fluorescence intensity. However, BiFC analysis using microscopy to quantify the strength of protein-protein interaction is time-consuming and somewhat subjective due to heterogeneity in protein expression and interaction. By coupling flow cytometric analysis with BiFC methodology, the fluorescent BiFC protein-protein interaction signal can be accurately measured for a large quantity of cells in a short time. Here, we demonstrate an application of this methodology to map regions in PDE4D3 that are required for the interaction with AKAP-Lbc. This high throughput methodology can be applied to screening factors that regulate protein-protein interaction.

Flow cytometric analysis of Bimolecular Fluorescence Complementation provides a high throughput quantitative method to study protein-protein interaction. This methodology can be applied to mapping protein binding sites and for screening factors that regulate protein-protein interaction.

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

Rapid Analysis and Exploration of Fluorescence Microscopy Images

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized image analysis tools are available, most traditional image-analysis-based workflows have steep learning curves (for fine tuning of analysis parameters) and result in long turnaround times between imaging and analysis. In particular, cell segmentation, the process of identifying individual cells in an image, is a major bottleneck in this regard.
Here we present an alternate, cell-segmentation-free workflow based on PhenoRipper, an open-source software platform designed for the rapid analysis and exploration of microscopy images. The pipeline presented here is optimized for immunofluorescence microscopy images of cell cultures and requires minimal user intervention. Within half an hour, PhenoRipper can analyze data from a typical 96-well experiment and generate image profiles. Users can then visually explore their data, perform quality control on their experiment, ensure response to perturbations and check reproducibility of replicates. This facilitates a rapid feedback cycle between analysis and experiment, which is crucial during assay optimization. This protocol is useful not just as a first pass analysis for quality control, but also may be used as an end-to-end solution, especially for screening. The workflow described here scales to large data sets such as those generated by high-throughput screens, and has been shown to group experimental conditions by phenotype accurately over a wide range of biological systems. The PhenoBrowser interface provides an intuitive framework to explore the phenotypic space and relate image properties to biological annotations. Taken together, the protocol described here will lower the barriers to adopting quantitative analysis of image based screens.

Here we describe a workflow for rapidly analyzing and exploring collections of fluorescence microscopy images using PhenoRipper, a recently developed image-analysis platform.

Despite rapid advances in high-throughput microscopy, quantitative image-based assays still pose significant challenges. While a variety of specialized ...

Protein-protein Interactions Visualized by Bimolecular Fluorescence Complementation in Tobacco Protoplasts and Leaves

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular fluorescence complementation (BiFC) is an in vivo method to monitor such interactions in plant cells. In the presented protocol the investigated candidate proteins are fused to complementary halves of fluorescent proteins and the respective constructs are introduced into plant cells via agrobacterium-mediated transformation. Subsequently, the proteins are transiently expressed in tobacco leaves and the restored fluorescent signals can be detected with a confocal laser scanning microscope in the intact cells. This allows not only visualization of the interaction itself, but also the subcellular localization of the protein complexes can be determined. For this purpose, marker genes containing a fluorescent tag can be coexpressed along with the BiFC constructs, thus visualizing cellular structures such as the endoplasmic reticulum, mitochondria, the Golgi apparatus or the plasma membrane. The fluorescent signal can be monitored either directly in epidermal leaf cells or in single protoplasts, which can be easily isolated from the transformed tobacco leaves. BiFC is ideally suited to study protein-protein interactions in their natural surroundings within the living cell. However, it has to be considered that the expression has to be driven by strong promoters and that the interaction partners are modified due to fusion of the relatively large fluorescence tags, which might interfere with the interaction mechanism. Nevertheless, BiFC is an excellent complementary approach to other commonly applied methods investigating protein-protein interactions, such as coimmunoprecipitation, in vitro pull-down assays or yeast-two-hybrid experiments.

Formation of protein complexes in vivo can be visualized by bimolecular fluorescence complementation. Interaction partners are fused to complementary parts of fluorescent tags and transiently expressed in tobacco leaves, resulting in a reconstituted fluorescent signal upon close proximity of the two proteins.

Many proteins interact transiently with other proteins or are integrated into multi-protein complexes to perform their biological function. Bimolecular ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay PCA in Living Cells

Proteins interact with each other and these interactions determine in a large part their functions. Protein interaction partners can be identified at high-throughput in vivo using a yeast fitness assay based on the dihydrofolate reductase protein-fragment complementation assay (DHFR-PCA).

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein&#8217;s function, regulation and localization often ...