Name: photoactivated localization microscopy with bimolecular fluorescence complementation (bifc-palm)
Uploaded: 2015-12-22T14:00:00
Description: Watch this Scientific Journal Video about Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM at JoVE.com

The authors thank Drs. Steven Chu and Joe W. Gray for helpful discussions, Henry Marr for his initial work on the BiFC-PALM project, and Alexis Shoemaker for her technical assistance. This work is supported by startup funds to X.N. from OHSU. Research in the Nan laboratory was also supported by NIH 5U54CA143836-05, the Damon Runyon Cancer Research Foundation, the M. J. Murdock Charitable Trust, and the FEI company.

1. Cloning
<ol>
	<li>Determine the configurations to clone and choose a linker. Tag proteins with the fragments on the N- or C-terminus as described below so they do not disrupt their proper localization. Use a flexible linker such as (GGGGS)x2.</li>
	<li>Genetically tag the proteins of interest to the PAmCherry1 fragments. As one option, use the cloning plasmids listed in the Materials List containing the fragments RN (PAmCherry1 residues 1-159) and RC (Met plus PAmCherry1 residues 160-236) with flanking MCS sequences...

<ol>
	<li>Braun, P., Gingras, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=History+of+protein-protein+interactions:+From+egg-white+to+complex+networks.">History of protein-protein interactions: From egg-white to complex networks.</a> Proteomics. 12, 1478-1498 (2012).</li><li>Lasserre, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Raft+nanodomains+contribute+to+Akt/PKB+plasma+membrane+recruitment+and+activation.">Raft nanodomains contribute to Akt/PKB plasma membrane recruitment and activation.</a> Nat. Chem. Biol. 4 (9), 538-547 (2008).</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 (4), 789-798 (2002).</li><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=Bimolecular+fluorescence+complementation+(BiFC)+analysis+as+a+probe+of+protein+interactions+in+living+cells.">Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells.</a> Annu. Rev. Biophys. 37, 465-487 (2008).</li><li>Kodama, Y., 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=Bimolecular+fluorescence+complementation+(BiFC):+a+5-year+update+and+future+perspectives.">Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives.</a> Biotechniques. 53, 285-298 (2012).</li><li>Lingwood, D., Simons, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Rafts+As+a+Membrane-Organizing+Principle.">Lipid Rafts As a Membrane-Organizing Principle.</a> Science. 327 (5961), 46-50 (2009).</li><li>Tian, T., Harding, A., Inder, K., Plowman, S., Parton, R. G., Hancock, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+membrane+nanoswitches+generate+high-fidelity+Ras+signal+transduction.">Plasma membrane nanoswitches generate high-fidelity Ras signal transduction.</a> Nat. Cell Biol. 9 (8), 905-914 (2007).</li><li>Betzig, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+intracellular+fluorescent+proteins+at+nanometer+resolution.">Imaging intracellular fluorescent proteins at nanometer resolution.</a> Science. 313 (5793), 1642-1645 (2006).</li><li>Hess, S. T., Girirajan, T. P. K., Mason, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultra-high+resolution+imaging+by+fluorescence+photoactivation+localization+microscopy.">Ultra-high resolution imaging by fluorescence photoactivation localization microscopy.</a> Biophys. J. 91 (11), 4258-4272 (2006).</li><li>Nickerson, A., Huang, T., Lin, L. J., Nan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivated+Localization+Microscopy+with+Bimolecular+Fluorescence+Complementation+(BiFC-PALM)+for+Nanoscale+Imaging+of+Protein-Protein+Interactions+in+Cells.">Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM) for Nanoscale Imaging of Protein-Protein Interactions in Cells.</a> PloS one. 9 (6), e100589 (2014).</li><li>Manley, S., Gillette, J. M., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-particle+tracking+photoactivated+localization+microscopy+for+mapping+single-molecule+dynamics.">Single-particle tracking photoactivated localization microscopy for mapping single-molecule dynamics.</a> Methods Enzymol. 475, 109-120 (2010).</li><li>Shyu, Y. J., 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=Fluorescence+complementation:+an+emerging+tool+for+biological+research.">Fluorescence complementation: an emerging tool for biological research.</a> Trends Biotechnol. 26 (11), 622-630 (2008).</li><li>Fan, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Split+mCherry+as+a+new+red+bimolecular+fluorescence+complementation+system+for+visualizing+protein-protein+interactions+in+living+cells.">Split mCherry as a new red bimolecular fluorescence complementation system for visualizing protein-protein interactions in living cells.</a> Biochem. Biophys. Res. Commun. 367 (1), 47-53 (2008).</li><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=Design+and+implementation+of+bimolecular+fluorescence+complementation+(BiFC)+assays+for+the+visualization+of+protein+interactions+in+living+cells.">Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells.</a> Nat. Protoc. 1, 1278-1286 (2006).</li><li>Gomez-Martinez, M., Schmitz, D., Hergovich, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+stable+human+cell+lines+with+Tetracycline-inducible+(Tet-on)+shRNA+or+cDNA+expression.">Generation of stable human cell lines with Tetracycline-inducible (Tet-on) shRNA or cDNA expression.</a> J. Vis. Exp. March. (73), e50171 (2013).</li><li>Robida, A. M., 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=Bimolecular+fluorescence+complementation+analysis+of+inducible+protein+interactions:+effects+of+factors+affecting+protein+folding+on+fluorescent+protein+fragment+association.">Bimolecular fluorescence complementation analysis of inducible protein interactions: effects of factors affecting protein folding on fluorescent protein fragment association.</a> J. Mol. Biol. 394 (3), 391-409 (2009).</li><li>Ding, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ratiometric+biosensors+based+on+dimerization-dependent+fluorescent+protein+exchange.">Ratiometric biosensors based on dimerization-dependent fluorescent protein exchange.</a> Nat. Methods. 12 (3), (2015).</li><li>Seidman, C. E., Struhl, K., Coligan, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Introduction+of+plasmid+DNA+into+cells.">Introduction of plasmid DNA into cells.</a> Curr. Protoc. Protein Sci. Appendix 4, 4D (2001).</li><li>Morrison, S. L., Coligan, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparing+frozen+bacterial+stocks.">Preparing frozen bacterial stocks.</a> Curr. Protoc. Immunol. Appendix 3, Appendix 3M (2001).</li><li>Campeau, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+versatile+viral+system+for+expression+and+depletion+of+proteins+in+mammalian+cells.">A versatile viral system for expression and depletion of proteins in mammalian cells.</a> PloS One. 4 (8), e6529 (2009).</li><li>Liu, H., Naismith, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+one-step+site-directed+deletion,+insertion,+single+and+multiple-site+plasmid+mutagenesis+protocol.">An efficient one-step site-directed deletion, insertion, single and multiple-site plasmid mutagenesis protocol.</a> BMC Biotechnol. 8 (91), (2008).</li><li>Deschout, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precisely+and+accurately+localizing+single+emitters+in+fluorescence+microscopy.">Precisely and accurately localizing single emitters in fluorescence microscopy.</a> Nat. Methods. 11 (3), 253-266 (2014).</li><li>Nan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-molecule+superresolution+imaging+allows+quantitative+analysis+of+RAF+multimer+formation+and+signaling.">Single-molecule superresolution imaging allows quantitative analysis of RAF multimer formation and signaling.</a> Proc. Natl. Acad. Sci. USA. 110 (46), 18519-18524 (2013).</li><li>Chenouard, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+comparison+of+particle+tracking+methods.">Objective comparison of particle tracking methods.</a> Nat. Methods. 11 (3), 281-289 (2014).</li><li>Persson, F., Lind&#233;n, M., Unoson, C., Elf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracting+intracellular+diffusive+states+and+transition+rates+from+single-molecule+tracking+data.">Extracting intracellular diffusive states and transition rates from single-molecule tracking data.</a> Nat. Methods. 10 (3), 265-269 (2013).</li><li>Liu, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+and+tracking+of+protein&#8211;protein+interactions+in+sub-diffraction+cellular+space.">Super-resolution imaging and tracking of protein&#8211;protein interactions in sub-diffraction cellular space.</a> Nat. Commun. 5, 1-8 (2014).</li><li>Xia, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Super-resolution+imaging+reveals+structural+features+of+EB1+in+microtubule+plus-end+tracking.">Super-resolution imaging reveals structural features of EB1 in microtubule plus-end tracking.</a> Mol. Biol. Cell. 25 (25), 4166-4173 (2014).</li></ol>

BiFC has been a commonly used technique for detecting and visualizing PPIs in cells, while PALM is a recent single molecule superresolution microscopy technique that enables nanoscale imaging of intact biological samples. The combination of BiFC with PALM achieved selective imaging of PPIs inside a cell with nanometer spatial resolution and single molecule sensitivity. BiFC-PALM extends the utility of both techniques, and as demonstrated in this work, shows great promise in revealing the molecular details of PPIs in thei...

Protein-protein interactions (PPIs) are fundamental to biology1 and are tightly regulated via spatiotemporal mechanisms across many time and length scales. Studies on cell signaling on the cell membrane, for example, have revealed dynamic, nanoscale spatial compartments that facilitate specific PPIs and cellular processes2. Hence, the ability to probe PPIs in biological systems with sufficient spatial and temporal resolutions, high specificity, and high sensitivity is key to achieving a mechanistic understanding of biology.
Bimolecular fluorescence complementation (BiFC) is one of the few existing tools for visualizing PPIs in a cell with subcellular resolution and live-cell compatibility3&#8211;5. The technique is relatively straightforward and involves splitting of a fluorescence protein into two non-fluorescent fragments; when genetically tagged to two interacting proteins and brought into proximity, the fragments can reconstitute to form a complete fluorescent protein, yielding fluorescent signal. When properly designed, BiFC probes should not spontaneously reconstitute in the absence of PPIs. As such, the fluorescence signal in a BiFC assay will only arise in the presence of PPIs, which enables direct visualization of PPIs with high specificity. Additional benefits of using fluorescence as the readout are high sensitivity, subcellular resolution, and compatibility with high throughput and high content screening assays, among others. For these benefits, a number of BiFC probes based on different parent fluorescent proteins have been developed. As in all other detection techniques based on conventional light microscopy, however, the spatial resolution of BiFC is limited to ~250 nm by the diffraction of light. This makes it a challenge to study the regulation of PPIs at the nanoscale, which, as alluded earlier and exemplified by lipid rafts6&#160;and Ras nanoclusters7, is a critical length scale to understanding many cellular processes such as signaling.
BiFC has been combined with photoactivated localization microscopy (PALM)8,9 to overcome this limit in spatial resolution for imaging PPIs10. PALM is a recent superresolution microscopy technique that circumvents the diffraction limit in fluorescence imaging through stochastic activation and subdiffractive localization of single fluorescent molecules. In each activation cycle, a fluorescent molecule emits a few hundred to a few thousand photons and gives rise to a single molecule image on the detector. While the image is diffraction-limited (~250 nm in width), its centroid can be determined with much higher precision, typically on the order of 10-50 nm depending on the number of photons detected. By activating and localizing each fluorescent molecule in the sample, a high resolution image can be reconstructed. Performed on living cells, single molecule tracking (smt-) PALM further permits acquisition of thousands of protein diffusion trajectories from a single cell11. Importantly, PALM uses specialized fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs) to achieve stochastic activation. Since both BiFC and PALM use fluorescent proteins, they were combined by splitting PAmCherry1, a commonly used PA-FP for PALM, into two fragments between amino acids 159 and 160.
The BiFC system based on split PAmCherry1 shows low background signal from spontaneous reconstitution of the two fragments. When genetically tagged to a pair of interacting proteins, the two fragments (RN = residues 1-159; RC = Met + residues 160-236) reconstituted efficiently to form complete PAmCherry1 proteins even at 37 &#176;C and without incubating at lower temperatures, which is not the case for other BiFC pairs12 such as the parent mCherry13. Furthermore, the reconstituted PAmCherry1 protein retained the photophysical properties of the parent PAmCherry1, such as high contrast ratio, medium photon yield, and fast photoactivation, among others, which are critical for accurate single molecule localization and high-resolution PALM imaging.
In this protocol, the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells by using split PAmCherry1 (Figure 1A) is described. The first step is to design the constructs for expressing fusion proteins between PAmCherry1 fragments (i.e., RN and RC) and the proteins of interest. In theory, for each pair of candidate proteins (A and B), there are eight pairs of fusion proteins to be tested: RN-A/RC-B; RN-A/B-RC; RC-A/RN-B; RC-A/B-RN; A-RN/RC-B; A-RN/B-RC; A-RC/RN-B; and A-RC/B-RN. This process can often be simplified by taking into account the structural or biochemical properties of the candidate proteins. In the case of Ras, the protein is post-translationally modified at the C-terminal CAAX box (C=Cys; A=aliphatic; X=any), after which the AAX motif is cleaved off. Hence, RN or RC can only be fused to the N-terminus of Ras; this reduces the number of fusion protein pairs to four (Figure 1B). For Raf, the Ras-binding domain (RBD; residues 51-131) is used and can be tagged on either end. These four fusion configurations were generated: RN-KRas/RC-Raf RBD; RN-KRas/Raf RBD-RC; RC-KRas/RN-Raf RBD; and RC-KRas/Raf RBD-RN.
Additionally, the linker between the fragments and the proteins of interest will need to be considered. A flexible linker of about ten amino acids is often used as it provides sufficient freedom for complementation to occur. One such linker is (GGGGS)x2, although there are many others that have been successfully applied, including random sequences generated from a multiple cloning site (MCS)14. The length of the linkers may need to be optimized depending on the size of the proteins of interest and their orientations when interacting.
The PAmCherry1 fragments are contained in a small cloning backbone with flanking MCSs (see Materials List). Genes of interest can be inserted via the restriction sites or with a ligation-independent method. After cloning and sequence verification, the expression cassette is transferred to an expression vector using a recombinase reaction, a cloning process with high fidelity and high efficiency.
Next, the resulting expression constructs are transfected into the target cell line or, if a stable cell line is desired, packaged into lentivirus for infecting the target cell line. Transient transfection allows for quick validation of the BiFC configurations, but potential problems must be noted. Transfection using chemicals often stresses the cells, resulting in high autofluorescence; while the use of total internal reflection fluorescence (TIRF) microscopy can mitigate this background signal by limiting the illumination volume, TIRF ideal when the PPIs take place on the cell membrane. Additionally, transient transfections often lead to high levels of protein expression, far exceeding those of the endogenous proteins, which may cause artifacts in detecting the PPIs. Hence, it is recommended that stable cell lines be established after an appropriate BiFC configuration has been determined through initial testing. Stable cell lines also have the potential for tunable expression via doxycycline induction15.
After infection, cells are selected with antibiotics, typically puromycin and neomycin, one for each construct. The subsequent steps for sample preparation, image acquisition, and data analysis will be described in detail in the protocols.
With this approach, the formation of nanoscale clusters in BiFC-PALM images of Ras-Raf RBD are routinely observed (Figure 2A). Consistently, smt-PALM trajectories of Ras-Raf RBD show a heterogeneous distribution in the diffusion states (Figure 2B-D). These results suggest that Ras-Raf complexes exist in multiple states on the cell membrane, presumably as monomers and clusters, with potential biological implications. This work demonstrates the power of BiFC-PALM in selective imaging of PPIs in cells with nanometer spatial resolution and single molecule sensitivity, which would be difficult to obtain with conventional BiFC, fluorescence co-localization, or fluorescence resonance energy transfer (FRET).
When designing BiFC experiments and interpreting the results, it is important to keep in mind that the BiFC process is irreversible in most cases, including split PAmCherry1. Once the two fragments combine and form a complete PAmCherry1 protein, the linkage between the two fragments, and thus the PPI becomes permanent. This limits the use of BiFC and BiFC-PALM for monitoring the dynamics of the PPIs (i.e., binding kinetics and not the diffusion dynamics of the protein complex once it is formed) and at times could even lead to mislocalization of the PPI complexes.
An additional factor to consider when designing BiFC-PALM experiments is the delay in chromophore maturation that is inherent in fluorescent proteins. Once two proteins interact and the FP fragments come together, they typically refold on the order of seconds (half-time of 60 sec for EYFP3). However, subsequent chromophore maturation and fluorescent signal develop on the order of minutes. While fluorescence may be detectable within 10 min&#160;after reconstitution of split-Venus, a fast-folding YFP variant, the half-time for maturation in general is often around 60 min16. Split-PAmCherry1 was observed to have similar rates. Hence, BiFC and BiFC-PALM are currently poor choices for monitoring real-time PPI kinetics; other methods, such as FRET and a recently developed dimerization dependent fluorescent protein17, may be more suited for this purpose.

<table>
	<tbody>
		<tr>
			<td>TIRF Microscope</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>60X oil immersion TIRF objective with 1.49 NA</td>
			<td>Nikon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD camera</td>
			<td>Andor</td>
			<td>iXon Ultra 897</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>405 nm laser</td>
			<td>Coherent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm dichroic mirror</td>
			<td>Semrock</td>
			<td>&#160;Di01-R405/488/561/635-25x36</td>
			<td></td>
		</tr>
		<tr>
			<td>561 nm filter</td>
			<td>Semrock</td>
			<td>FF01-525/45-25</td>
			<td></td>
		</tr>
		<tr>
			<td>405/561 nm notch filter</td>
			<td>Semrock</td>
			<td>NF01-405/488/568-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature and CO2 controlled stage</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR-D-TOPO-PAmCherry1_1-159-MCS</td>
			<td>Addgene</td>
			<td>60545</td>
			<td></td>
		</tr>
		<tr>
			<td>pENTR-D-TOPO-PAmCherry160-236-MCS</td>
			<td>Addgene</td>
			<td>60546</td>
			<td></td>
		</tr>
		<tr>
			<td>pcDNA3.2-DEST</td>
			<td>Life Technologies</td>
			<td>12489-019</td>
			<td></td>
		</tr>
		<tr>
			<td>pLenti-DEST</td>
			<td>Addgene</td>
			<td></td>
			<td>http://www.addgene.org/Eric_Campeau/</td>
		</tr>
		<tr>
			<td>Phusion High-Fidelity DNA Polymerase</td>
			<td>Thermo Scientific</td>
			<td>F-531</td>
			<td></td>
		</tr>
		<tr>
			<td>In-Fusion HD Cloning</td>
			<td>Clontech</td>
			<td>639649</td>
			<td></td>
		</tr>
		<tr>
			<td>LR Clonase</td>
			<td>Life Technologies</td>
			<td>11791</td>
			<td></td>
		</tr>
		<tr>
			<td>Vira Power Lentivirus Packaging</td>
			<td>Life Technologies</td>
			<td>K497500</td>
			<td></td>
		</tr>
		<tr>
			<td>X-tremeGENE Transfection Reagent</td>
			<td>Roche</td>
			<td>13873800</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab-Tek II Chambered Coverglass</td>
			<td>Thermo Scientific</td>
			<td>155409</td>
			<td>#1.5 glass bottom dishes</td>
		</tr>
		<tr>
			<td>U2OS cells</td>
			<td>ATCC</td>
			<td>HTB-96</td>
			<td></td>
		</tr>
		<tr>
			<td>293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM with phenol red</td>
			<td>Life Technologies</td>
			<td>11995</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM no phenol red</td>
			<td>Life Technologies</td>
			<td>21063</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Life Technologies</td>
			<td>10082</td>
			<td></td>
		</tr>
		<tr>
			<td>Leibovit&#39;s L-15, no phenol red</td>
			<td>Life Technologies</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr>
			<td>Reduced serum medium</td>
			<td>Life Technologies</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Life Technologies</td>
			<td>14040</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>BD Biosciences</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter</td>
			<td>Millipore</td>
			<td>SLHV033RB</td>
			<td></td>
		</tr>
		<tr>
			<td>Lentiviral concentrator</td>
			<td>Clontech</td>
			<td>631231</td>
			<td></td>
		</tr>
		<tr>
			<td>Retroviral concentrator</td>
			<td>Clontech</td>
			<td>631455</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm culture dish</td>
			<td>BD Biosciences</td>
			<td>353003</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well culture plate</td>
			<td>BD Biosciences</td>
			<td>353046</td>
			<td></td>
		</tr>
		<tr>
			<td>Polybrene</td>
			<td>Sigma</td>
			<td>107689</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin</td>
			<td>Life Technologies</td>
			<td>A11138</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418</td>
			<td>Calbiochem</td>
			<td>345812</td>
			<td>Neomycin</td>
		</tr>
		<tr>
			<td>Doxycyline</td>
			<td>Fisher</td>
			<td>BP2653</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td>Fisher</td>
			<td>BP152</td>
			<td></td>
		</tr>
		<tr>
			<td>EDTA</td>
			<td>Sigma</td>
			<td>EDS</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Hydroxide</td>
			<td>Sigma</td>
			<td>S5881</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>G6257</td>
			<td></td>
		</tr>
		<tr>
			<td>PIPES</td>
			<td>Sigma</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034</td>
			<td></td>
		</tr>
		<tr>
			<td>EGTA</td>
			<td>Sigma</td>
			<td>3777</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma</td>
			<td>M2643</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Hydroxide</td>
			<td>Sigma</td>
			<td>221473</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium chloride</td>
			<td>Fisher</td>
			<td>BP358</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Fisher</td>
			<td>M33</td>
			<td></td>
		</tr>
		<tr>
			<td>100 nm gold particles</td>
			<td>BBI Solutions</td>
			<td>EM.GC100</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular grade water</td>
			<td>Life Technologies</td>
			<td>10977</td>
			<td></td>
		</tr>
		<tr>
			<td>Dpn1</td>
			<td>New England Biolabs</td>
			<td>R0176</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR purification kit</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td></td>
		</tr>
		<tr>
			<td>Miniprep kit</td>
			<td>Qiagen</td>
			<td>27104</td>
			<td></td>
		</tr>
		<tr>
			<td>Midiprep kit</td>
			<td>Macherey-Nagel</td>
			<td>740410</td>
			<td></td>
		</tr>
		<tr>
			<td>0.6 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-120</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL microcentrifuge tubes</td>
			<td>Fisher</td>
			<td>05-408-137</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tubes</td>
			<td>Fisher</td>
			<td>05-539-12</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL&#160; polypropylene round-bottom tubes</td>
			<td>BD Biosciences</td>
			<td>352063</td>
			<td></td>
		</tr>
		<tr>
			<td>14 mL polypropylene round-bottom tubes</td>
			<td>BD Biosciences</td>
			<td>352059</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL tube</td>
			<td>BD Biosciences</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tube</td>
			<td>GeneMate</td>
			<td>C-3328-1</td>
			<td></td>
		</tr>
		<tr>
			<td>SOC medium</td>
			<td>Life Technologies</td>
			<td>15544</td>
			<td></td>
		</tr>
		<tr>
			<td>LB broth</td>
			<td>BD Biosciences</td>
			<td>244610</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin sulfate</td>
			<td>Fisher</td>
			<td>BP906</td>
			<td></td>
		</tr>
		<tr>
			<td>Competent cells</td>
			<td>Life Technologies</td>
			<td>C4040</td>
			<td></td>
		</tr>
		<tr>
			<td>Matlab</td>
			<td>Mathworks</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

photoactivated localization microscopy with bimolecular fluorescence complementation (bifc-palm)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and sensitivity in cells because the resolution of conventional light microscopy is diffraction-limited to ~250 nm. By combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM), PPIs can be visualized in cells with single molecule sensitivity and nanometer spatial resolution. BiFC is a commonly used technique for visualizing PPIs with fluorescence contrast, which involves splitting of a fluorescent protein into two non-fluorescent fragments. PALM is a recent superresolution microscopy technique for imaging biological samples at the nanometer and single molecule scales, which uses phototransformable fluorescent probes such as photoactivatable fluorescent proteins (PA-FPs). BiFC-PALM was demonstrated by splitting PAmCherry1, a PA-FP compatible with PALM, for its monomeric nature, good single molecule brightness, high contrast ratio, and utility for stoichiometry measurements. When split between amino acids 159 and 160, PAmCherry1 can be made into a BiFC probe that reconstitutes efficiently at 37 &#176;C with high specificity to PPIs and low non-specific reconstitution. Ras-Raf interaction is used as an example to show how BiFC-PALM helps to probe interactions at the nanometer scale and with single molecule resolution. Their diffusion can also be tracked in live cells using single molecule tracking (smt-) PALM. In this protocol, factors to consider when designing the fusion proteins for BiFC-PALM are discussed, sample preparation, image acquisition, and data analysis steps are explained, and a few exemplary results are showcased. Providing high spatial resolution, specificity, and sensitivity, BiFC-PALM is a useful tool for studying PPIs in intact biological samples.

The BiFC-PALM example shown is KRas G12D mutant interacting with the Ras binding domain (RBD) of CRaf (Figure 1A). As discussed, the RN or RC fragments were not tagged to the C-terminus of Ras because it would disrupt the membrane localization and hence the biological activity of Ras. This reduced the possible combinations from eight to four (Figure 1B). Each of these four combinations was introduced into U2OS cells using lentiviral infection. The cells were fixed as detailed in the prot...

Watch this Scientific Journal Video about Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM at JoVE.com

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation BiFC-PALM

Protein-protein interactions are visualized in cells with nanometer spatial resolution by combining bimolecular fluorescence complementation (BiFC) with photoactivated localization microscopy (PALM). Described here is the use of BiFC-PALM for imaging Ras-Raf interactions in U2OS cells for visualizing the nanoscale clustering and diffusion of individual Ras-Raf complexes.

Photoactivated Localization Microscopy with Bimolecular Fluorescence Complementation (BiFC-PALM)

Protein-protein interactions (PPIs) are key molecular events to biology. However, it remains a challenge to visualize PPIs with sufficient resolution and ...

Research

JoVE Journal

Bioengineering

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