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 fu...

<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. 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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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<td>DMEM</td>
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<td>Fetal bovine serum</td>
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<td>6-well dishes</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

27.7K ビュー.  University of Illinois at Chicago. タンパク質の細胞内局在は、細胞シグナル伝達の時空間制御を決定する上で重要です。ここで、我々は、細胞内のタンパク質の空間的な相互作用を監視するための簡単​​な方法として、二分子蛍光相補（BiFC）を記述する。