Name: flim-fret measurements of protein-protein interactions in live bacteria.
Uploaded: 2020-08-25T12:30:00
Description: Watch this Scientific Journal Video about FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. at JoVE.com

We acknowledge Dr Ludovic Richert for his valuable assistance on FLIM data acquisition and for the technical maintenance and development of the FLIM setup. This work was funded by grants from Fondation pour la Recherche en Chimie (https://icfrc.fr/). VN is funded by the Fondation pour la Recherche M&#233;dicale (FRM&#8208;SPF201809006906). YM is grateful to the Institut Universitaire de France (IUF) for support and providing additional time to be dedicated to research. IJS and JG acknowledge the Institute on Drug Delivery of Strasbourg for its financial support.

1. Plasmid construction
<ol>
	<li>Amplify by two PCR (PCR1 and 3) the DNA sequences (use genomic DNA of P. aeruginosa PAO1) of the 700 base pairs upstream and downstream of the regions corresponding to the insertion site in P. aeruginosa genome with high-fidelity DNA polymerase. Add restriction sites to primers in blue and green and add an overlapping sequence with mCherry to primers in red (Figure 2).
	<ol>
		<li>For PvdA labelled at the C-terminus with eGFP, amplify the...

FLIM-FRET offers some key advantages over intensity-based FRET imaging. Fluorescence lifetime is an intrinsic parameter of the fluorophore. As a consequence, it is not dependent on local concentrations of fluorophores neither on the intensity of the light excitation. The fluorescence lifetime is additionally also poorly affected by photo-bleaching. It is particularly interesting to evidence PPIs in cells where local proteins concentrations can be highly heterogeneous throughout the subcellular compartments or regions. FL...

Protein-protein interactions (PPIs) control various key processes in cells1. The roles of PPIs differ based on protein composition, affinities functions and locations in cells2. PPIs can be investigated via different techniques3. For example, co-immunoprecipitation is a relatively simple, robust, and inexpensive technique commonly used tool to identify or confirm PPIs. However, studying PPIs can be challenging when the interacting proteins have low expression levels or when the interactions are transient or relevant only in specific environments. Studying PPIs occurring between the different enzymes of the pyoverdine pathway in P. aeruginosa requires that the repression of the general iron-co-factored repressor Fur is relieved to allow the expression of all the proteins of the pyoverdine pathway to be expressed in the cell4,5,6. This common regulation for all the proteins of the pathway results in timely expressions in the cell expected to promote their interactions. The diversity in term of size, nature, expression levels and the number of proteins of this metabolic pathway make it difficult for study in reconstituted systems6. Exploring PPIs in their cellular environment is therefore critical to further understand the biological functions of proteins in their native context.
Only few methods including fluorescence allow exploring PPIs in living cells7. Amongst the different fluorescence parameters that can be measured, the fluorescence lifetime (i.e., the average time a fluorophore remains in its excited state before emitting a photon) is likely one of the most interesting parameters to explore in living cells. The fluorescence lifetime of a fluorophore is highly sensitive to its environment and FLIM can therefore provide chemical or physical information regarding the fluorophore surroundings8. This includes the presence of F&#246;rster resonance energy transfer (FRET) that can occur in the presence of an &#8220;acceptor&#8221; of fluorescence located at a short distance of a fluorescence &#8220;donor&#8221;. Energy transfer results in significant shortening of the donor fluorescence lifetime (Figure 1A), making Fluorescence Lifetime Imaging Microscopy (FLIM) a powerful approach to explore protein-protein interactions directly in live cells. FLIM can additionally provide spatial information about where the interactions take place in cells7,8. This approach is extremely powerful for investigating PPIs in situations where the labeling with fluorophores of the two interacting partners is possible.
For FRET to occur - critical conditions on the distance between two fluorophores are required8,9. The two fluorophores should not be distant from each other by more than 10 nm. Therefore, cautions must be taken when designing FLIM-FRET experiments to ensure that the donor and the acceptor of fluorescence have a chance to be located close to each other in the interacting complex. While this may seem constraining, it is in fact a true advantage as the distance-dependence of FRET ensures that two labelled proteins undergoing FRET have to physically interact (Figure 1A). The difficulties at getting clear answers about PPI in colocalization experiments (two colocalized proteins may not necessarily interact) are therefore not an issue using FLIM-FRET.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61602/61602fig1.jpg" /> 
Figure 1: FLIM-FRET analysis principle. Each pixel of the FLIM-FRET multidimensional image contains information about the fluorescence decay recorded at this particular location (#counts = number of detected photons in the channel t). (A) The classical representation of the FLIM image is usually a false-color lifetime encoded 2D image (left). A decrease in the mean fluorescence lifetime of the donor - as seen by a change in the color scale - can be observed in the presence of FRET and is informative about the presence of PPIs in this spatial area. (B) Overlap between the donor emission spectrum and the acceptor absorption spectrum is necessary for FRET to occur. <a href="https://www.jove.com/files/ftp_upload/61602/61602fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
A second requirement for FRET is that the emission spectrum of the donor and the absorption spectra of the acceptor should overlap8 (Figure 1B). The fluorescence excitation of the donor should be at wavelengths that contribute very little to the direct fluorescence excitation of the acceptor. Not all combinations of fluorophores are possible and we additionally recommend to preferentially use donors with monoexponential fluorescence decays to facilitate FLIM-FRET interpretations10. Several couples of fluorescence proteins meet these requirements, including the popular eGFP-mCherry couple11 (for a review on the palette of available fluorescent protein FRET pairs see12,13).
FLIM-FRET allows measuring the fluorescence lifetime decay of a FRET donor at every pixel of a FLIM image (Figure 1A). There are two major techniques to determine fluorescence lifetime that differ in acquisition and analysis: frequency-domain (FD)14 and time-domain (TD). TD FLIM is more widespread and is performed using a pulsed illumination combined with different possible detection configurations including gating methods15, streak camera16 or time-correlated single photon counting (TCSPC) techniques8. For both FD and TD techniques, fluorescence lifetime is not directly measured but requires an analysis of the measured data to estimate the lifetime(s) or the presence of interactions. For TCSPC techniques, the most widely used analysis relies on fitting the decays with single or multi exponential functions using least square iterative re-convolutions that minimize the weighted sum of the residuals.
Finally, FLIM-FRET can be performed both by using single photon or multiphoton excitations. The latest have several advantages like reducing autofluorescence and photodamage out of the focal plane. Multiphoton excitations allow also a longer excitation depth if working in thick 3D samples8. On the contrary, single photon excitation is usually more efficient as the two-photon absorption cross sections of fluorescent proteins are limited17.
Here, we propose a protocol for FLIM-FRET measurements of PPIs in live P. aeruginosa in the particular case of two interacting proteins (PvdA and PvdL) expressed with highly different numbers of copies to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. PvdA and PvdL proteins are involved in pyoverdine biosynthesis. PvdA is a L-ornithine N5-oxygenase and synthesizes the L-N5-formyl-N5-hydroxyornithine from L-ornithine by hydroxylation (PvdA) and formylation (PvdF)18. PvdL is a non-ribosomal peptide synthesis (NRPS) enzyme composed of four modules. The first module catalyzes the acylation of myristic acid. The second module catalyzes the activation of L-Glu and its condensation to the myristic-coA. Then, the third module condenses a L-Tyr amino acid that is then isomerized in D-Tyr. Finally, the fourth module binds a L-Dab (Diaminobutyric acid) amino acid to form the acylated tripeptide L-Glu/D-Tyr/L-Dab6. PvdL is thus responsible for the synthesis of the three first amino acids of the pyoverdine precursor. The interaction of PvdA protein with PvdL is surprising as PvdL, on the contrary to PvdI and PvdJ, does not carry a module specific for the L-N5-formyl-N5-hydroxyornithine. This interaction suggest that all the enzymes responsible for the pyoverdine precursor biosynthesis are arranged in large transient and dynamic multi-enzymatic complexes19,20.
In this report we explain in detail how to construct the bacterial strains expressing natively the two interacting eGFP and mCherry labelled proteins. We also describe sample preparation and conditions for efficient FLIM-FRET cell imaging. Finally, we propose a step-by-step tutorial for image analysis including a recently developed tool providing advanced visualization possibilities for straightforward interpretation of complex FLIM-FRET data. With this report, we would like to convince not only adventurous but most biologists that FRET-FLIM is an accessible and powerful technique able to address their questions about PPIs directly in the native cellular environment.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">525/50 nm band-pass filter</td>
			<td style="width:251px;">F37-516, AHF, Germany</td>
			<td style="width:139px;"></td>
			<td style="width:186px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">680 nm short pass filter</td>
			<td>F75-680, AHF, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium Sulfate (NH4)2SO4</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td>A4418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DreamTaq DNA polymerase 5U/&#956;L</td>
			<td>ThermoFisher Scientific</td>
			<td>EP0714</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">E. coli TOP10</td>
			<td>Invitrogen</td>
			<td>C404010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fiber-coupled avalanche photo-diode</td>
			<td>SPCM-AQR-14- FC, Perkin Elmer</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass coverslips (Thickness No. 1.5, 20&#215;20mm</td>
			<td style="width:251px;">Knitel glass</td>
			<td>MS0011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">High-Fidelity DNA polymerase Phusion 2U/&#956;L</td>
			<td>ThermoFisher Scientific</td>
			<td>F530S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lysogeny broth (LB)</td>
			<td>Millipore</td>
			<td>1.10285</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:327px;">Magnesium Sulfate Heptahydrate (MgSO4 . 7H2O)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td>10034-99-8</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microscope slides (25&#215;75mm)</td>
			<td style="width:251px;">Knitel glass</td>
			<td>MS0057</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NucleoSpin Gel and PCR Clean-up</td>
			<td>Macherey-Nagel</td>
			<td>740609.50</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NucleoSpin Plasmid</td>
			<td>Macherey-Nagel</td>
			<td>740588.10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Phosphate Dibasic (K2HPO4)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td>RES20765</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td>P5655</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Succinate (Disodium)</td>
			<td style="width:251px;">Sigma-Aldrich</td>
			<td>14160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SPCImage, SPCM software</td>
			<td>Becker &#38; Hickl</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile inoculating loop</td>
			<td style="width:251px;">Nunc</td>
			<td>7648-1PAK</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">T4 DNA ligase 1U/&#956;L</td>
			<td>ThermoFisher Scientific</td>
			<td>15224017</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCSPC module</td>
			<td>SPC830, Becker &#38; Hickl, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti:Sapphire laser</td>
			<td>Insight DeepSee, Spectra Physics</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tubes 50mL</td>
			<td style="width:251px;">Falcon</td>
			<td style="width:139px;">352070</td>
			<td></td>
		</tr>
	</tbody>
</table>

flim-fret measurements of protein-protein interactions in live bacteria.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster resonance energy transfer (FRET) provide accurate information about PPIs in live cells. FLIM-FRET relies on measuring the fluorescence lifetime decay of a FRET donor at each pixel of the FLIM image, providing quantitative and accurate information about PPIs and their spatial cellular organizations. We propose here a detailed protocol for FLIM-FRET measurements that we applied to monitor PPIs in live Pseudomonas aeruginosa in the particular case of two interacting proteins expressed with highly different copy numbers to demonstrate the quality and robustness of the technique at revealing critical features of PPIs. This protocol describes in detail all the necessary steps for PPI characterization - starting from bacterial mutant constructions up to the final analysis using recently developed tools providing advanced visualization possibilities for a straightforward interpretation of complex FLIM-FRET data.

Empirical cumulative distribution functions (ecdf) of the fluorescence lifetimes measured for the different bacterial strains are shown in Figure 8. If FRET occurs, the ecdfs are shifted towards the shorter-lived lifetimes (Figure 8A,8B). Note that when the interaction of the two proteins results in a long distance between the two fluorophores, no FRET can occur (Figure 8C). This situation cannot be distinguished fr...

Watch this Scientific Journal Video about FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria. at JoVE.com

FLIM-FRET Measurements of Protein-Protein Interactions in Live Bacteria.

We describe here a protocol to characterize protein-protein interactions between two highly-differently expressed proteins in live Pseudomonas aeruginosa using FLIM-FRET measurements. The protocol includes bacteria strain constructions, bacteria immobilization, imaging and post-imaging data analysis routines.

Protein-protein interactions (PPIs) control various key processes in cells. Fluorescence lifetime imaging microscopy (FLIM) combined with F&#246;rster ...

FILM-FRET is a powerful technique that makes it possible to confirm suspected or predicted protein-protein interactions. In this protocol, we present a way to analyze data in particular cases of unbalanced donor and acceptor quantities. FILM-FRET is able to provide information about the protein-protein interactions directly in live cells.It is important to investigate personal-protein interactions in native cellular environment, in particular when fluorescent protein are expressed in the endogenous level. Begin by growing P.aeruginosa, E.coli TOP10, and E.coli helper bacteria each in five milliliters of LB without antibiotic at 30 degrees Celsius while shaking overnight. On the next day, measure the optical density of the cultures and mix an equal quantity of P.aeruginosa, E.coli TOP10 and E.coli helper in a 1.5 milliliter microtube.Centrifuge the tube for five minutes at 9, 300 times G to pellet the bacteria, then discard the supernatant and resuspend the pellet in 50 microliters of LB.Plate a spot of the mixture on preheated LB agar and incubate it for five hours at 37 degrees Celsius. After the incubation, scrape the spot with a sterile inoculation loop and resuspend it in one milliliter of LB.Plate 100 microliters of this bacterial suspension on LB agar containing 10 micrograms per milliliter chloramphenicol and 30 micrograms per milliliter gentamycin and incubate two days at 37 degrees Celsius to eliminate E.coli TOP10 and E.coli helper bacteria. Resuspend one colony in one milliliter of LB and incubate it at 37 degrees Celsius with orbital shaking for four hours, then centrifuge the tube for three minutes at 9, 300 times G and discard 950 microliters of supernatant.Resuspend the pellet in 50 microliters of LB and isolate the mixture on LB agar containing sucrose and no sodium chloride. Incubate the plate overnight at 30 degrees Celsius. Spot isolated colonies on LB agar and LB agar containing 15 milligrams per milliliter gentamycin in order to check for gentamycin sensitivity.Place a microscope glass slide on a flat horizontal surface and arrange two glass slides topped with two layers of adhesive tape around it. Pipette a 70 microliter droplet of 1%melted agarose onto the glass slide and place a fourth slide on top to flatten the agarose droplet. Press down gently for about a minute.Take off the upper slide and drop about three microliters of bacteria in three to four spots at different locations on the agarose pad. Cover the agarose pad with a microscopy glass coverslip and fix it with melted paraffin to seal it onto the glass slide starting with the four corners. Place the microscopy slide on the stage with the coverslip facing the objective.Turn the filter cube turret to select the eGFP cube and open the fluorescence lamp shutter, then send the fluorescence light towards the eyepiece of the microscope. Focus the objective on the bacteria using the microscope knob. Select a region of interest in the sample using the joystick that controls the motorized stage.Send the fluorescence emission path back towards the detector, then turn back the filter cube turret to select the dichroic cube for the 930 nanometer laser and set the laser power to 20 milliwatts. Set the size of the region of interest to 30 micrometers which adjusts the voltage operating the galvo mirrors and defines the range of their movements. Turn on the detector and start scanning the sample.Choose the field of view for imaging by finely moving the stage from the computer interface. This can be done on the setup by moving the cross on the image in the microscope control software which will define the new center of the image and pressing move stage. A good field of view for acquisition should contain 10 to 30 immobile bacteria all in focus.If interested in extracting single cells FILM-FRET data, ensure that the bacteria are well-individualized. Open the imaging software and check that the photon count rate is not too high to avoid a pileup effect that can influence lifetime measurements. If necessary, lower the laser intensity to keep the photon count rate low.Adjust acquisition parameters including the acquisition collection time. Press the start button and wait for the acquisition to complete. To analyze the data, open RStudio and create a new project.Create a new folder in the main project folder and name it data, then move all analysis ask files into this folder. Open a new script file or the supplementary script flim_analysis.r. Install the dedicated FlimDiagRam package for film data analysis and call the package in the workspace.Empirical cumulative distribution functions of the fluorescence lifetimes measured for the different bacterial strains are shown here. If FRET occurs, the functions are shifted towards the shorter lived lifetimes. It is important to note that FRET can't occur when the interaction of the two proteins results in a long distance between the two flurophores, which can't be distinguished from the absence of interaction.Therefore, it may be necessary to label the proteins at different positions to maximize the chances of probing the interaction. Due to the large difference in protein expressions between PvdA and PvdL, the same complex does not result in similar FILM-FRET data. Unbalanced stoichiometries lead to differences in the contribution of the free as compared to the bound donor-labeled proteins in the recorded fluorescence lifetime distribution.The diagram plot can be used to provide critical information about the stoichiometry. In the PvdA-eGFP mCherry-PvdL mutant, the quantity of donor-labeled PvdA is much higher than the quantity of mCherry-PvdL. Amongst all donors present in the sample, only a few of them are interacting with PvdL.The single tau one value centered at approximately 2.3 nanoseconds suggests that each PvdA-eGFP donor can only transfer with one mCherry-PvdL acceptor. When the labeling is reversed, most of the eGFP-PvdL proteins are expected to interact with PvdA-mCherry which is confirmed by the higher alpha one values. The tau one values became more distributed suggesting that multiple PvdA proteins may bind to a single PvdL protein.The FLIM setup is becoming available in an increasing number of imaging facilities. Imaging samples in a FILM-FRET is no more complicated than imaging in video microscopy.

flim-fret-measurements-protein-protein-interactions-live

Research

JoVE Journal

Biology

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has been proposed that the microenvironment that the pathogen encounters in the commensal layer is important in determining the extent of colonization. Current culture methods for investigating pathogen colonization are not well suited for investigating this hypothesis as they do not enable co-culture of bacteria and epithelial cells in a manner that mimics the GI tract microenvironment. Here we describe a microfluidic co-culture model that enables independent culture of eukaryotic cells and bacteria, and testing the effect of the commensal microenvironment on pathogen colonization. The co-culture model is demonstrated by developing a commensal Escherichia coli biofilm among HeLa cells, followed by introduction of enterohemorrhagic E. coli (EHEC) into the commensal island, in a sequence that mimics the sequence of events in GI tract infection.

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

The human gastrointestinal (GI) tract is a unique environment in which intestinal epithelial cells and non-pathogenic (commensal) bacteria coexist. It has ...

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of methodological options for addressing molecular interactions in cells have been developed, but most of these methods suffer from being rather indirect and therefore hardly quantitative. On the contrary, a few high-end quantitative approaches were introduced, which however are difficult to extend to high throughput. To combine high throughput capabilities with the possibility to extract quantitative information, we recently developed a new concept for identifying protein-protein interactions (Schwarzenbacher et al., 2008). Here, we describe a detailed protocol for the design and the construction of this system which allows for analyzing interactions between a fluorophore-labeled protein ("prey") and a membrane protein ("bait") in-vivo. Cells are plated on micropatterned surfaces functionalized with antibodies against the bait exoplasmic domain. Bait-prey interactions are assayed via the redistribution of the fluorescent prey. The method is characterized by high sensitivity down to the level of single molecules, the capability to detect weak interactions, and high throughput capability, making it applicable as screening tool.

In-vivo Detection of Protein-protein Interactions on Micro-patterned Surfaces

This video shows experiments with subsequent analysis of protein-protein interactions by the use of micro-patterned surfaces. The approach offers the possibility to detect protein interactions in living cells and combines high throughput capabilities with the possibility to extract quantitative information.

Unraveling the interaction network of molecules in-vivo is key to understanding the mechanisms that regulate cell function and metabolism. A multitude of ...

Imaging Protein-protein Interactions in vivo

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, preventing their identification and analysis through traditional biochemical methods such as co-immunoprecipitation. In this regard, the genetically encodable fluorescent proteins (GFP, RFP, etc.) and their associated overlapping fluorescence spectrum have revolutionized our ability to monitor weak interactions in vivo using F&ouml;rster resonance energy transfer (FRET)1-3. Here, we detail our use of a FRET-based proximity assay for monitoring receptor-receptor interactions on the endothelial cell surface.

Imaging Protein-protein Interactions in vivo

This protocol describes how to image protein-protein interactions using a FRET-based proximity assay.

Protein-protein interactions are a hallmark of all essential cellular processes. However, many of these interactions are transient, or energetically weak, ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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 ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy (f3D-SIM)

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the diffraction of light allowing super-resolution of live samples. There are currently three main types of super-resolution techniques &#8211; stimulated emission depletion (STED), single-molecule localization microscopy (including techniques such as PALM, STORM, and GDSIM), and structured illumination microscopy (SIM). While STED and single-molecule localization techniques show the largest increases in resolution, they have been slower to offer increased speeds of image acquisition. Three-dimensional SIM (3D-SIM) is a wide-field fluorescence microscopy technique that offers a number of advantages over both single-molecule localization and STED. Resolution is improved, with typical lateral and axial resolutions of 110 and 280 nm, respectively and depth of sampling of up to 30 &#181;m from the coverslip, allowing for imaging of whole cells. Recent advancements (fast 3D-SIM) in the technology increasing the capture rate of raw images allows for fast capture of biological processes occurring in seconds, while significantly reducing photo-toxicity and photobleaching. Here we describe the use of one such method to image bacterial cells harboring the fluorescently-labelled cytokinetic FtsZ protein to show how cells are analyzed and the type of unique information that this technique can provide.

Super-resolution Imaging of the Cytokinetic Z Ring in Live Bacteria Using Fast 3D-Structured Illumination Microscopy f3D-SIM

Spatiotemporal information about dynamic proteins inside live cells is crucial for understanding biology. A type of super-resolution microscopy called fast 3D-structured illumination microscopy (f3D-SIM) reveals unique information about the cytokinetic Z ring in bacteria: both its bead-like appearance and the rapid dynamics of FtsZ within the ring.

Imaging of biological samples using fluorescence microscopy has advanced substantially with new technologies to overcome the resolution barrier of the ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance (SPR)

Protein-protein interactions are pivotal to most, if not all, physiological processes, and understanding the nature of such interactions is a central step in biological research. Surface Plasmon Resonance (SPR) is a sensitive detection technique for label-free study of bio-molecular interactions in real time. In a typical SPR experiment, one component (usually a protein, termed 'ligand') is immobilized onto a sensor chip surface, while the other (the 'analyte') is free in solution and is injected over the surface. Association and dissociation of the analyte from the ligand are measured and plotted in real time on a graph called a sensogram, from which pre-equilibrium and equilibrium data is derived. Being label-free, consuming low amounts of material, and providing pre-equilibrium kinetic data, often makes SPR the method of choice when studying dynamics of protein interactions. However, one has to keep in mind that due to the method's high sensitivity, the data obtained needs to be carefully analyzed, and supported by other biochemical methods. 
SPR is particularly suitable for studying membrane proteins since it consumes small amounts of purified material, and is compatible with lipids and detergents. This protocol describes an SPR experiment characterizing the kinetic properties of the interaction between a membrane protein (an ABC transporter) and a soluble protein (the transporter's cognate substrate binding protein).

Real Time Measurements of Membrane Protein:Receptor Interactions Using Surface Plasmon Resonance SPR

Surface Plasmon Resonance (SPR) is a label-free method for detecting bio-molecular interactions in real time. Herein, a protocol for a membrane protein:receptor interaction experiment is described, while discussing the pros and cons of the technique.

Protein-protein interactions are pivotal to most, if not all, physiological processes, and  understanding the nature of such interactions is a central ...