Tau Interaction with Tubulin and Microtubules: From Purified Proteins to Cells

Microtubules (MTs) play an important role in many cellular processes and are dynamic structures regulated by an important network of microtubules-associated proteins, MAPs, such as Tau. Tau has been discovered as an essential factor for MTs formation in vitro, and its region implicated in binding to MTs has been identified. By contrast, the affinity, the stoichiometry, and the topology of Tau–MTs interaction remain controversial. Indeed, depending on the experiment conditions a wide range of values have been obtained. In this chapter, we focus on three biophysical methods, turbidimetry, cosedimentation assay, and Förster Resonance Energy Transfer to study Tau–tubulin interaction both in vitro and in cell. We highlight precautions that must be taken in order to avoid pitfalls and we detail the nature of the conclusions that can be drawn from these methods about Tau–tubulin interaction.


Introduction
Microtubules cytoskeleton plays an important role in many cellular processes such as mitosis, cellular integrity, cellular shaping, intracellular trafficking, and cell migration. Microtubules (MTs) are constantly alternating between polymerization and depolymerization phases. This process, called dynamic instability of MT [1], is highly regulated by an important network of microtubulesassociated proteins, MAPs, [2] such as Tau. Since Weingarten's discovery of Tau as an essential factor for microtubule formation in vitro [3], the interaction of Tau with tubulin has been intensively studied. However, differential experimental conditions (different buffer, Tau and tubulin peptides, Tau constructs) lead to surprising range of Tau-MT affinity values. Studying this interaction is difficult for at least two reasons: the intrinsically disordered structure of Tau and the ability of αβ-tubulin dimers to self-assemble into different oligomers depending on the temperature. MTs are hollow cylinders formed at 37 °C by a combination of linear protofilaments, which themselves are formed by αβ-tubulin dimers associated heads to tails, forming a pseudohelical lattice [4][5][6]. Tubulin can also self-assemble into other oligomers than MT. For example, in solution at 20 °C, addition of magnesium to tubulin dimers induces formation of single and doubles rings [7][8][9]. These rings were also described as depolymerization products resulting of protofilaments peeling at MTs end [10][11][12].
Human brain expresses six isoforms of Tau protein produced by alternative splicing (exons 2-3 and 10) of a single mRNA [13]. The interaction domain of Tau with microtubules, called MT binding domain (MTBR), is well documented: it is constituted of three or four (depending on the isoform) partially repeated sequences and a part of N-terminal proline-rich region flanking the MTBR [14][15][16][17]. By contrast, despite the great number of methods used to characterize this interaction, its affinity, the stoichiometry, and the topology of binding site on tubulin/MT are still under debate.
In this chapter, we focus on three biophysical methods namely turbidimetry, cosedimentation assay, and Förster Resonance Energy Transfer (FRET) in living cells used to study Tau-tubulin interaction; the use of other useful methods such as analytical ultracentrifugation or microcalorimetry has been recently described [18][19][20]. We highlight precautions that must be taken in order to avoid pitfalls and we detail the nature of the conclusions that can be drawn from these methods about Tau-tubulin interaction.

Materials
Prepare all solutions using ultrapure water and analytical grade reagents.
2. 1 M ethylene glycol-bis(2-aminoethyl-ether)-N,N,NP,NPtetraacetic acid (EGTA): weigh 7.6 g EGTA in a 20 mL glass beaker and add ultrapure water to have just less than 20 mL. Transfer in a 20 mL graduated flask and adjust volume to 20 mL. Put in a plastic tube or glass bottle and store at room temperature.
3. 0.1 M guanosine triphosphate sodium salt hydrate (GTP): weigh 0.0567 g GTP in a microtube and add 970 μL of ultrapure water. Check the concentration by measuring the absorbance at 256 nm with a molecular extinction coefficient ε256 nm = 13,700 M −1 cm −1 and store at −20 °C (see Note 1). 5. 20 mM phosphate buffer (1 L): put 50 mL of 400 mM phosphate buffer in graduated flask and adjust volume to 1 L. pH must be at 6.95 at 25 °C. 6. PM buffer: 20 mM phosphate buffer, 0.5 mM MgCl 2 . In a 500 mL glass beaker, add 25 mL 400 mM phosphate buffer and 250 μL of 1 M MgCl 2 to 450 mL of ultrapure water. Adjust pH to 7.0 with 10 M NaOH. Transfer the buffer in a 500 mL graduated flask and complete volume to 500 mL. Then, transfer to a 500 mL cylinder and store at 4 °C. 7. PMS buffer: 20 mM phosphate buffer, 0.5 mM MgCl 2 , 0.24 M sucrose. In a 4 L glass beaker, add 200 mL 400 mM phosphate buffer, 2 mL 1 M MgCl 2 and 328.6 g sucrose to 3.5 L of ultrapure water. The pH must be at 6.90 without adjustment. Transfer in 4 L graduated flask and complete volume to 4 L. Put in a glass beaker to store at 4 °C.    Incubator, French press, centrifuge, FPLC apparatus, dialysis tubing: cellulose molecular weight cutoff 3500 Spectra/Por® molecular porous n°3 18 mm × 50 ft, freeze-dryer.

A Confocal Laser Scanning Microscope (CLSM) equipped
with Argon (488 nm) and Helium-Neon (543 nm) laser lines, specific dichroic filter and a spectral mode to select specific domains of the emission spectrum (see Note 9).

3.
A photobleaching module coupled to CLSM including an observation beam, a photo-destruction beam and a timeresolved recording system. 4. The public-domain ImageJ software [23] and the plug-in PixFRET [24] for image analysis.

Methods
Tubulin purification necessitates three consecutive days, one for materials and buffer preparations and two others for the purification.
1. Equilibrate the ionic exchange resin in 20 mM phosphate buffer. From stock solution 400 mM phosphate buffer, prepare 1 L of phosphate buffer 20 mM by adding 50 mL of 400 mM phosphate buffer in graduated flask and adjust volume to 1 L with ultrapure water. Then, weigh 10 g of DEAE-Sephadex A50 in a 2 L flask and add 1 L 20 mM phosphate buffer. Put this flask in boiling water and let DEAE-Sephadex A50 swell for 5 h (see Note 10).
2. G25 Preparation: pack the Sephadex G25 beads (preincubated in ultrapure water overnight) in a column of 50 cm high and 3 cm diameter (3 cm × 50 cm). Leave 2 cm at the top of the column to add the sample. Equilibrate it with 400 mL PM buffer at a 1 mL/min flow rate (see Note 11).
3. Chemical preparation: crush ammonium sulfate crystals in powder to dissolve it easier.
4. Weigh GTP samples as described in Subheading 2.1.1 and store it at −20 °C.

5.
Material and room preparation: clean and organize the cold storage room. Dispose of materials.
The brains (fifteen lamb or pig brains) should be collected just after the animal death in the slaughterhouse and put them in plastic bag and rapidly in ice box for the transport. From there, conduct all the experiments in a cold room to prevent tubulin denaturation. Nitrile gloves are used for all purification steps and workers must wear mask during brain homogenization.

Tubulin Purification (Day 2)
1. Brain homogenization: remove superficial blood vessels and meninges from brains with dissection pliers. Cut and rinse them with an equal volume of PMS (do not exceed 2.5 L) that will be eliminated through gauze. Repeat this step three times. Add PMS (total volume must not exceed 3 L to limit centrifuge run numbers) to brains and homogenize them in a blender for 30 s.

2.
To eliminate membranes and cellular fragments, homogenate is centrifuged in 12 tubes (250 mL) at 13,000 × g for 30 min at 4 °C (see Note 12). During this centrifugation, add GTP to PMG buffer, PMG 0.4 M KCl and PMG 0.8 M KCl. Equilibrate G25 with 400 mL PMG at a flow rate of 1 mL/min. Begin DEAE-Sephadex A50 equilibration: get rid of the buffer by filtration with sintered-glass filter and vacuum flask. Add 250 mL PMG buffer to Sephadex beads and increase pH to 6.7 with few drops of 10 M NaOH. Get rid of the buffer as described above and add 250 mL PMG buffer.
3. Ammonium sulfate fractionation 32 %: discard pellets (see Note 13) and measure supernatant volume with cylinder. Put it in a 2 L glass beaker and add slowly 177 g of ammonium sulfate per liter of supernatant. When ammonium sulfate is completely dissolved, wait for 10 min.
4. Centrifuge the precipitate at 13,000 × g for 30 min at 4 °C in six tubes of 250 mL (see Note 12). During this second centrifugation, continue to equilibrate DEAE-Sephadex A50: increase pH to 6.8 with 10 M NaOH, throw out buffer and add 250 mL PMG buffer. Increase pH to 6.9 and throw out buffer. Add 250 mL PMG buffer. DEAE-Sephadex A50 is ready to use.
5. Ammonium sulfate fractionation 43 %: discard pellets (see Note 13) and measure the supernatant volume with cylinder. Put it in a 2 L glass beaker and add slowly 71 g of ammonium sulfate per liter of supernatant. When ammonium sulfate is completely dissolved, wait for 10 min. 11. Centrifuge at 9,000 × g for 10 min at 4 °C (see Note 12). During this centrifugation, add 500 μL 0.1 M GTP to the 500 mL of PMG sucrose buffer.
12. Collect pellets and dissolve them in a minimum volume of PMG sucrose buffer (do not exceed 5 mL) with gentle agitation (see Note 14).
13. The solution is placed in a dialysis tubing (cellulose molecular weight cutoff 3500, Spectra/Por® molecular porous n°3 18 mm × 50 ft) and dialyzed for 16 h against PMG sucrose buffer supplemented with 0.1 mM GTP.
1. Collect tubulin from the dialysis tubing and centrifuge it at 12,000 × g for 30 min at 4 °C (see Note 12). Discard pellets and keep the supernatant. Make 300 μL aliquot in cryotubes and stock them in liquid nitrogen. Store 2 mL of dialysis buffer.
2. Tubulin concentration is determined by spectrophotometry. Put 5 μL of dialysis buffer in 500 μL of 6 M Guanidine-HCl and make the blank. Put in triplicate 5 μL of tubulin sample in 500 μL and measure the absorbance at 275 nm. Determine the concentration using the Lambert-Beer law with a molecular extinction coefficient of ε 275 nm = 1.09 mL mg −1 cm −1 taking into account the dilution factor (here 101).

Tubulin Purification and Storage (Day 3)
3. Test the tubulin capacity to form microtubules using a turbidimetry assay (see Subheading 3.3.2).
At the end of the purification, you should have 200-300 mg of pure tubulin at 40-80 mg/mL and an absorbance variation of about 0.5 for 30 μM (3.0 mg/mL) of tubulin corresponding to microtubule formation (Fig. 1).
1. Switch on the "Hoffman heater" 30 min before the beginning of the bacterial growth (see Note 16).
2. Near the "Hoffman heater" in a 500 mL Erlenmeyer flask, mix 100 mL sterilized medium Luria Broth Miller with 100 μL Ampicillin and 100 μL stock bacteria. Place it in an incubator at 37 °C overnight with agitation.
1. Switch on the "Hoffman heater" 30 min before the beginning of bacterial growth (see Note 16).
2. Near the "Hoffman heater" first, remove 5 mL medium without bacteria to make blank to read absorbance and second, in each two 2 L Erlenmeyer flask, 500 mL sterilized medium Luria Broth Buffered are mixed with 1.85 g glucose, 100 μL ampicillin, and 10 mL pregrowth bacteria. Place them in an incubator at 37 °C with agitation for few hours. Read absorbance at 2. Start to collect 5 mL fractions and inject sample with equilibration buffer at a flow rate of 0.5 mL/min. Absorbance increases during sample deposit (resin-protein interaction). Rinse column with equilibration buffer as far as absorbance comes back to baseline level and then begin elution.

Bacterial Culture
3. Apply a 30 % NaCl gradient: increase the salt concentration to 30 % in two column volumes (10 mL) with a flow rate of 1 mL/min. Apply it as long as absorbance is constant.
4. Increase the salt gradient to 70 % in one column volume (5 mL) with a flow rate of 1 mL/min. Tau is released at this step. When Tau protein has been released from the column, a 100 % gradient is applied to wash column for few minutes at a flow rate of 1 mL/min.

5.
To verify that Tau is present in fractions or to know if there is no Tau in flow through, you may take 20 μL of each fraction, add 5 μL of loading buffer 5× and depose them on SDS PAGE 12 % acrylamide (see Note 17).
6. Fractions containing Tau are pooled in a dialysis tubing (cellulose molecular weight cutoff 3500, Spectra/Por® molecular porous n°3 18 mm × 50 ft) and dialyzed for 2 h, three times against ultrapure water. Then, Tau is collected and frozen at −80 °C to be lyophilized. Once lyophilized, Tau can be stored at −20 °C for several months.

Tau Purification
Tau Interaction with Tubulin and Microtubules: From Purified Proteins to Cells 7. Before use, Tau is dissolved in the desired buffer, centrifuged at 3,000 × g for 10 min and its concentration is determined by spectrophotometry using the extinction coefficient ε 280nm = 7,700 M −1 cm −1 after light scattering correction (Fig. 2).
Microtubule formation is a cooperative system that needs in vitro a tubulin minimum concentration called "tubulin critical concentration" (Cr). Cr is defined as the inverse of the apparent MT-elongation constant K app [25]. To determine Cr, we measured microtubule amount by turbidimetry or sedimentation assay (tubulin concentration in the pellet) as a function of total tubulin concentration and fit the results to a linear regression. The Cr corresponds in this case to the value of x (tubulin concentration) found for y = 0 (no microtubule formation). More accurately, measuring the concentration found in the supernatant of sedimentation assay of microtubule solution leads directly the Cr value. This value is dependent of the experimental conditions. It is well known that magnesium ions, glycerol, dimethyl sulfoxide, sucrose are positive factors that reduce the Cr. For example, Cr of tubulin is 7 μM  [26] and 20 μM in 80 mM K-Pipes, 1 mM MgCl 2 , 1 mM EGTA, GTP 1 mM pH 6.8 [27]. In these conditions, called in this chapter as "assembly conditions buffer," presence of Tau leads to a microtubule formation facilitated by ligand binding. In 2004, we found experimental conditions in which the presence of Tau induces the microtubule formation in the absence of any other known polymerization factors (20 mM phosphate buffer pH 6.5, 0.1 mM GTP, 1 mM TCEP) (see Note 6).

Tau-Tubulin/ Microtubule Interaction in Solution
In this condition, called in this chapter "non-assembly condition," presence of Tau leads to a microtubule formation induced by ligand binding [28].
Before using tubulin it is necessary to eliminate sucrose. Tubulin is equilibrated (see Note 19) in the appropriate buffer (assembly or non-assembly condition) on gel filtration Sephadex G25 resin on a gravity column (1 cm*25 cm).
1. Equilibrate the column with cold 40 mL of appropriate buffer at a flow rate of 1 mL/min and defreeze rapidly tubulin sample.
2. When the buffer is removed (without completely drying the resin), put tubulin solution (see Note 20) on the top. When all the protein entered in the resin, put the buffer and start elution at a flow rate of 1 mL/min. Collect 500 μL fractions and put them on ice. The tubulin concentrations into the tubes with meniscus (see step 10 in Subheading 3.1.2) are determined by spectrophotometry as described in Subheading 3.1.3 with elution as control instead of dialysis buffer.
Tubulin prepared as described above is diluted to 7.5 μM in the appropriate buffer to be at a tubulin final concentration of 5 μM (this concentration is under the 7 μM Cr for the assembly condition). Sample volume and number of samples depend on the spectrophotometer (for us, it is 300 μL and six samples).
1. Before starting the time-driven microtubule formation, make blank on the spectrophotometer with buffer alone.
2. Put 200 μL of tubulin at 7.5 μM in the six different cuvettes and put them on ice.
3. Put rapidly the cuvettes in the spectrophotometer thermostated at 37 °C and measure the absorbance time course at 350 nm for 5 min. You may have to wipe condensation off.
4. Add rapidly in each cuvettes 100 μL of buffer for control or different Tau concentrations (from 0.5 to 5 μM). Do not use Tau concentration above the tubulin concentration because it induces microtubule bundling [28]. The signal should increase rapidly indicating microtubule formation. The turbidity is

Turbidimetry Assay
Tau Interaction with Tubulin and Microtubules: From Purified Proteins to Cells observed for 30 min (Fig. 2). The samples then can be used for cosedimentation assay and/or electron microscopy. To quantify free and bound tubulin and free and bound Tau, loading of each sample must be readjusted to obtain comparable and non-saturated spots. Protein quantification is determined with band intensity quantification by ImageJ.

Cosedimentation Assays: Microtubules Bound Tau, Free Tau, and Free Tubulin Separation
The experiments must be done three times to have a representative average. Because Tau concentration in the pellet represents MT-bound Tau concentration and that in the supernatant the free one, Scatchard plot can be used to determine affinity constant and the stoichiometry of the reaction. For analysis used the following equation: (2) Equation 1 is characterized by an apparent elongation constant K app = K 2 (1 + K 1 [Tau] free ) (for more details, see the appendix of ref. [29]) and Eq. 2 is characterized by an apparent elongation constant K app = K 1 K 2 [Tau] free /(1 + K 1 [Tau] free ). As already mentioned in Subheading 3.3, the K app is related to 1/Cr and the Cr corresponds to the free tubulin concentration in the supernatant of the cosedimentation assay. So plot K app in function of free Tau concentration and fit the data with the equation of K app described above. Use Eq. 1 in assembly condition and Eq. 2 in non-assembly condition.  Collect three images of cells on the field. An alternative method to quantify the absolute efficiency of FRET between the donor EGFP-tau and the acceptor mCherry-α-tubulin is to measure the EGFP emission before and after selective photobleaching of the mCherry [30]. 4. Adjust gain and offset of mCherry-α-tubulin channel with cells expressing mCherry-α-tubulin alone. The pixel intensities must be linear with the total dynamic scale of fluorescence intensities (for example 256-grey scale for an 8-bit image depth).

Tau-Tubulin Interactions by FRET in Living Cells
5. Collect two images of cells with the 488 and 543 nm excitation lines with donor and acceptor settings as previously described in steps 3 and 4 (see Note 28).
7. Recollect two images of cells with the 488 and 543 nm excitation lines after the mCherry-α-tubulin photobleaching in the ROI.
The ImageJ plug-in PixFRET (Center for Integrative Genomics, Faculty of biology and medicine, University of Lausanne, Switzerland) [24] can generate images of sensitized-emission FRET, and hence show where FRET occurs within a cell or a group of cells, by computing pixel by pixel the images of a sample acquired in a three channel setting. 3. Calculate the intensity ratio of the uFRET on donor images; the mean value of all intensity ratios corresponds to the donor SBT parameter A (see Note 31).
4. Repeat steps 1-3 to calculate the acceptor SBT parameter B with images of cells expressing mCherry-α-tubulin alone.
5. Before starting the PixFRET plug-in, create a stack of three images of the cell or the group of cells expressing both the donor EGFP-tau and the acceptor mCherry-α-tubulin: the first one in the uFRET setting, the second one in the donor setting and the third one in the acceptor setting.
6. Launch PixFRET from the plug-ins menu in ImageJ.
7. Enter the value for the donor and acceptor SBTs in the Donor and Acceptor Models bookmarks, respectively (see Note 32), and click on the FRET bookmark to select a ROI on the stack in order to determine the background outside cells; press Get button.
8. In the parameter box below, select "no Gaussian Blur" and a value of 1.0 for threshold correction factor (see Note 33), as well as the type of normalization of the FRET values among 4 possibilities: by dividing by the donor or the acceptor intensity [31], by the product of donor and acceptor intensities [32] or by the square root of the product of donor and acceptor intensities [33]; users can also generate an image displaying the FRET efficiencies in the sample. For example, the methodology reported in ref. [30] was to normalize corrected FRET (Fc) to the donor intensity.
9. Click on "Compute FRET" button to generate the normalized FRET image, in addition to the Fc image calculated from the formula described by ref. [34]: Where I FRET , I donor , and I acceptor are pixel intensities (subtracted from their background intensity) in cells under the distinct uFRET, donor, and acceptor settings, respectively. The parameters A and B were calculated in steps 3 and 4.
10. To reduce background noise, prefer to use a nonlinear filtering of the computed FRET image with for example a 1-pixel-range median filter than a Gaussian filter [35,36].
11. In the Image menu of ImageJ, select an adequate LookUp Table (LUT) and adjust brightness and contrast to take over all the dynamic scale of fluorescence intensities (see Note 34).
12. Save the computed FRET images as a TIFF file.
1. With ImageJ software [23], create a stack of four images of cells expressing EGFP-tau and mCherry-α-tubulin before and after the acceptor photobleaching.
2. On the post-bleaching image of mCherry-α-tubulin, select manually a ROI overlaying the bleached zone.

Notes
1. GTP is dissolved in water. Make Aliquots of 100 μL and store them at -20 °C. GTP should be added freshly to buffer to avoid hydrolysis of GTP into GDP which is inactive for MTs formation.
2. Glycerol is viscous. Precautions should be taking to be sure that it was accurately mixed with buffer or water.

FRET Calculation for FRET by the Acceptor Photobleaching
3. Add a pinch of azide to prevent bacteria or fungus growth. After use, unpacked resin, wash ten times with ultrapure water. It can be used ten times.
4. In the case of guanidine-HCl, the volume increases when it is dissolved and the reaction is endothermic. Do not put a volume of water larger than the half of the total volume.

Fig. 3
Energy transfer between EGFP-tau and mCherry-a-tubulin revealed by the acceptor photobleaching in living cells. The quenched and unquenched EGFP-tau emission is compared after specific photobleaching of the mCherry-α-tubulin fluorophore (respectively, upper and lower panels in false colors under Pre-and Postbleaching titles). Prior to the acceptor photobleaching, microtubules and uniformly labeled with EGFP-tau; after photobleaching, the fluorescence emission of EGFP-tau increased in spots along microtubules (black arrows in upper panel) confirming FRET in the initial state; no significant fluorescence intensity fluctuation is observed inside the control region. Scale bar: 10 mm; side bars: color pixel scoring for fluorescence intensities from 0 (dark purple) to 255 (red); EGFP-tau and mCherry-α-tubulin are excited at 488 and 543 nm respectively, using the multi-tracking mode of CLSM system 5. All FPLC buffers can be stored at room temperature for few months.
6. TCEP powder is very difficult to dissolve at concentration of 1 M. TCEP is preferred to DTT to reduce disulfide bonds because it is more stable. It is used to maintain Tau monomeric and have no effect on MTs formation.
7. This solution must be filtered just before use to remove crystals or possible pollutants which perturb coloration. To use uranyl acetate use gloves.
8. Add 2 mM l-glutamine and nonessential amino acids if absent in media both to stimulate growth and to prolong the viability of cells in culture.
9. We use a Leica DMI6000CS inverted microscope coupled to a CLSM Leica TCS SP5, with the TD488/543/633 dichroic filter equipping the system. 10. During DEAE swelling, you must check there is always enough water and that it is boiling.
11. The resin must not be dried. Keep an eye on column when already 500 mL went through.
12. Think to thermostat centrifuge and rotor and tubes at the good temperature before run.
13. Collect all wastes in a special bag to eliminate them in rules respect.
14. Wash all tubes meticulously with spatula and collect all volumes, as little as they are, to optimize the output. Start using spatula to crash pellet and then use a magnetic stirring bar which has almost the same length as glass beaker diameter and finish by gently by gently pipetting in and out the sample with a 1 mL pipetman without making bubbles. It is very important to suspend every tiny particle. 15. When you add buffer on the top of mixing solution a biphasic layer formed. It is the sign that tubulin is here and concentrated. 23. Use samples expressing the donor or the acceptor fluorophores alone to maximize uFRET signals, to minimize SBT, to avoid widespread saturation, to avoid unintended photobleaching, and to minimize background noise [34]. 24. Avoid images showing pixels with saturated intensity indicating an excessive excitation laser power. 25. The same PMT settings in donor and acceptor channels and low laser power are recommended, in order to generate a robust uFRET signal at donor excitation in the acceptor channel, without at the same time saturating the donor emission signal in the donor channel.
26. Do not modify gain and offset of the uFRET channel, otherwise to calculate wrong donor and acceptor SBT (parameters A and B in the FRET calculation in the Subheading 3.4.4). 27. We recommend adjusting gain and offset of the donor channel in order to spread fluorescence intensities on two-thirds of the dynamic intensity scale.
28. Users can focus on two cells in the same image, a first cell on which users irradiate a ROI to photobleach the acceptor and another cell as control.
29. On our CLSM with a 63 × 1.4 NA objective, these conditions destroy mCherry with very little or no direct bleaching of the EGFP donor. The optimal exposure conditions that bleach acceptor maximally with minimal direct effect on donor should be determined for each microscope system and filter set, using for example droplets of donor and acceptor under oil, cells transfected with donor and acceptor fusion proteins alone, or cells co-transfected with non-interacting donor and acceptor fusion proteins. When the acceptor alone is expressed and excited at both 488 and 543 nm.
32. Keep the value 0 for FRET and Donor/Acceptor boxes in the Background sections; press Accept button; in the Model Donor/Acceptor boxes, select "no Gaussian Blur" to compute FRET images from native uFRET, donor and acceptor images; press on Constant button just below and fill the box with the calculated parameters A (in the Donor Model bookmark) and B (in the Acceptor Model bookmark); press Accept button. 33. The Fc and normalized FRET images are calculated only if pixel values in each image are above a given threshold, set by default to average background values; the threshold correction factor is a multiplication factor applied to background values in order to modify this threshold; the pixels below the threshold are displayed in blue (or black in the new user's LUT) in the computed Fc and normalized FRET images.
34. The computed Fc and normalized FRET images are encoded on 32 bits but visualized on 256 color levels; the minimum value is displayed in black and the maximum value in white. Hence, an artificially amplified FRET signal may be observed in a cell where no FRET occurs (negative control). Users should therefore always: -Check the amplitude of the FRET signal by performing a profile plot through the cell of interest and compare this amplitude to that of a negative control (such as with cells expressing the donor-acceptor couple free EGFP and mCherry-α-tubulin for example) and to that a positive control (such as with cells expressing the donor-acceptor couple EGFP-β-tubulin and mCherry-α-tubulin). -Stack the image of interest with an image of a negative and a positive control for FRET in order to visualize both images with the same LUT scale.
35. This procedure uses each cell or sub-region as its own internal standard; in addition, users can calculate %E in a neighboring cell that should be similar to the one of the internal standard.
36. The significance of %E is discussed in ref. [38].