Using a GFP-tagged TMEM184A Construct for Confirmation of Heparin Receptor Identity

1. Design of a GFP-protein Construct

1. Purchase, or design and build, a GFP-tagged construct based on the protein in question.
   NOTE: For a purchased construct, standard vectors are available from commercial laboratories that include some or all of the following suggestions: For a membrane protein, select a C-terminal location of the GFP because it is less likely to interfere with membrane protein trafficking. Consider an extension between the gene of interest and the GFP if there is reason to believe the C-terminus of the protein in question is compactly folded into a domain of the protein. Select the construct for general eukaryotic cell expression, but allow construct production in bacterial systems. Include a cleavage site (such as for the TEV protease) by which GFP can be removed if desired for some experiments where the GFP could interfere with the protein activity. Add other inserts such as an additional tag for localization or affinity interactions (e.g., His6). This was not necessary for the assays described herein, but might facilitate other assays, or be useful directly adjacent to the gene, before the GFP, if removal of GFP is desired.

2. GFP-TMEM184A Expression in Vascular Cells

1. Culture endothelial or vascular smooth muscle cells on 0.2% gelatin coated tissue culture dishes as reported previously^1,6,7.
2. Add 5 mL cell culture trypsin solution (0.5% w/v) to a rinsed 100 mm, or larger, confluent dish of cells. Incubate at 37 °C until cells are just released from the plate, and transfer the cells to a sterile polypropylene centrifuge tube.
3. Add a trypsin inhibitor (e.g., an equal volume of regular culture medium), as used in routine culture, to the cell/trypsin solution to limit trypsin activity. Pellet the cells for 5 min at approximately 600 x g (or appropriate speed and time to just pellet the cells for standard tissue culture). Aspirate the supernatant.
4. Resuspend cells in 1 mL of HeBS (Hepes-buffered Saline) electroporation buffer, limiting the amount of time cells are in the pellet or suspension. Place the cell suspension on ice and carry out remaining steps on ice.
   NOTE: Pellet can be washed one time with HeBS prior to resuspension.
5. Add sufficient volume of GFP-tagged TMEM184A plasmid to the cells to achieve a final concentration of 20 µg/mL DNA. Use an identical protocol for a GFP construct.
6. Place approximately 0.4 mL of the cell solution in HeBS in an electroporation cuvette. Pre-chill the cuvettes prior to use.
7. Electroporate the cells using the following conditions: Exponential Decay, 500 µF, ∞ ohm, and 170 V.
   NOTE: The voltage for a given cell type should be optimized. Preliminary studies with endothelial and smooth muscle cell types were accomplished with a GFP-vinculin construct to determine the optimal voltage value noted here, e.g.¹⁰.
   1. To optimize electroporation, start with the recommendations of the equipment manufacturer (which include conditions for some standard cell types). Use the desired construct or a control fluorescent protein construct similar in size and fluorescent characteristics to the desired construct.
      NOTE: Small nucleic acids appear to enter cells more readily than larger constructs, so a construct with only a fluorescent protein may be easier to express than a larger one.
   2. Use fluorescence microscopy to determine cell viability, the percentage of cells in which the fluorescent construct is expressed after 24 and 48 h, and the intensity of expression.
      NOTE: Decrease the voltage and/or time slightly if survival is low. Aim for the shortest time possible between release of cells from the culture surface and returning cells to culture to enhance survival. Slight increases in time or a second pulse can improve construct uptake if survival is high and expression is low. Optimum conditions and expression will vary between cell types. If the percentage of cells expressing the construct is high, but intensity is low, increasing the DNA concentration and monitoring the transfected cells over time for optimal intensity, which will vary based on protein half-life as well as expression, may improve intensity. Staining intensity can be magnified for some assays, if necessary, by immunofluorescence staining of GFP with anti-GFP antibodies
8. After electroporation, seed the cells into six 30 mm tissue culture wells with coverslips for imaging or into tissue culture dishes. Culture the cells using standard cell culture procedures.
9. Optional: Replace the culture medium after 24 h to remove any HeBS electroporation buffer.

3. Visualization of GFP-TMEM184A Localization

1. Rinse the cells with PBS and gentle shaking after culturing for at least 24 h. Limit exposure to bright light to avoid GFP fading.
2. Fix cells with 4% paraformaldehyde (PFA) in PBS for 15 min at RT with gentle shaking. Avoid using any methanol-containing reagents which may permeabilize the cells. Fixation with methanol can be used if it is not necessary to assess ligand interactions.
   Caution: Paraformaldehyde is toxic. Wear appropriate personal protective equipment. Use in a fume hood, with care, and discard properly.
3. Rinse with PBS as above. For antibody based staining, see 3.6 below.
4. Mount coverslips to slides using Mowiol or other suitable mounting medium.
5. Image slides using a confocal or fluorescence microscope with the appropriate filter sets for GFP excitation and emission spectra. Confocal microscopy is preferred because of the ability to separate samples in the z-plane. Save images in grey scale for quantitation purposes in TIF format (in addition to full color images for illustrations).
6. For comparison with WT TMEM184A expressed by the cells, prepare other cells for immunofluorescence using primary antibodies prepared to a peptide(s) from the TMEM184A sequence, e.g.¹. Identification of GFP-TEMEM184A can also be accomplished using antibodies against GFP. Evaluate both cell samples by identical microscopy techniques.

4. Rhodamine-Heparin Binding and Colocalization with GFP-TMEM184A-transfected Cells

1. Treat GFP-TMEM184A-expressing cells with 100 µg/mL rhodamine-heparin added to culture medium, and allow cells to incubate for a specific amount of time, typically less than 10 min for A7r5 cells. Rinse and fix as in 3.1 and 3.2.
   NOTE: The optimal time and concentration of ligand depend on cell response, fluorescence intensity of the ligand and image intensity obtained. This concentration of heparin was employed because it results in more than 50% response in other assays¹¹. This concentration could be visualized using standard fluorescence microscopy techniques, so it was not necessary to increase it. Dilution of rhodamine-heparin with unlabeled heparin results in lower fluorescence in the cells, thus is more difficult to image and quantify.
2. To quantitate the rhodamine-heparin binding due to GFP-TMEM184A transfection, prepare identical cells without transfected GFP-TMEM184A.
3. Image the cells using a confocal microscope with the appropriate GFP (488 nm) and rhodamine (543 nm) excitation and emission (500 – 530 nm for GFP and greater than 560 nm for rhodamine) values to determine co-localization. Obtain images of at least 50 cells from at least three separate experiments to obtain statistics. Maintain identical settings within experiments, and employ a control sample(s) (e.g., transfected cells with no treatment) that can be used to standardize data for analysis of multiple experiments.
4. Examine the images using a computer program to determine relative rhodamine binding/uptake in each cell for quantitation of binding.
   NOTE: TIF images should be employed as they are convenient for analysis and can be converted to grey scale in many computer programs if they were not initially saved in that format. Image J (freeware) was employed in the present analysis and allows area and pixel intensity to be measured for any user-defined area in an image.
   1. Use a freehand tool to circle a cell within a fluorescent image and use the measure tool to determine the intensity within that space.
      NOTE: These measurements provide the needed information to calculate total intensity for a user-defined area (whole cell, nucleus, etc.) providing a way to compare different cells with different geometries. Mean intensity over an area of background can be reported, and that facilitates collection of background intensity for analysis.
   2. Export to a spreadsheet to calculate the area multiplied by the intensity and subtract background for that same amount of area. Average the fluorescence/cell for enough cells/experiment to obtain statistical significance.

5. Fluorescence Resonance Energy Transfer from GFP-TMEM184A to Rhodamine-Heparin

1. Prepare transfected cells and incubate with 200 µg/mL rhodamine-tagged ligand as in 3.1. Note that the incubation time with fluorescent ligand is cell-type dependent. Fix with PFA only, and mount slides for imaging.
2. First, excite at 405 nm and image for rhodamine emission (566 – 685 nm; FRET). Second, excite at 488 for GFP (image at 493 – 530), and third, excite at 561 for rhodamine (image at 566 – 685). Controls without GFP-TMEM184A or without rhodamine-heparin are critical.

6. Live-cell Imaging of Rhodamine-Heparin Uptake

1. Seed the GFP-TMEM184A transfected cells into individual dishes designed for live-cell imaging and treat as in protocol 3.
   NOTE: The number of cells required depends on cell type and density desired. To minimize the amount of time cells are in suspension, do not count cells. Seed cells from a 100 mm confluent dish into six 35 mm live imaging dishes to obtain cells near confluence in 48 h.
2. After 48 h, replace the medium with culture medium without phenol red.
3. Transfer a dish of cells to a confocal microscope with a warming stage to maintain temperature, and focus the microscope.
4. Pipet 100 µg/mL rhodamine-heparin into the dish and gently mix.
5. Immediately begin recording live images. If no cell uptake of heparin occurs within the region in view, move the dish slightly to identify cells with at least one green vesicle containing the rhodamine label.
   NOTE: Imaging excitation and emission specifics are the same as for the heparin uptake protocol (section 5). The time period between images was approximately 16 s. The conditions of the experiment and the microscope will typically determine the speed at which images can be obtained.

7. Isolation of GFP-TMEM184A and GFP from Cultured Cells

1. After removing culture medium and rinsing with PBS, add 2 mL 0.2% (w/v) CHAPS 1x PBS solution with protease inhibitors to a 150 mm plate of cells expressing GFP-TMEM184A or GFP (for control binding assays). Scrape the cells from the dish, place the cells/CHAPS solution in a 15 mL polypropylene tube on ice, and mix well by tapping. Complete all steps in bright light to ensure the GFP tag is bleached for the binding assay below.
2. To specifically bind GFP-TMEM184A (or an appropriate GFP control), add 2 µg/mL biotinylated anti-GFP antibody to the solution and incubate overnight at 4 °C with rocking.
3. Add 500 µL of streptavidin-agarose beads to at least 5 mL 0.2% CHAPS/PBS and centrifuge at approximately 600 x g for 3 to 5 min. Remove the supernatant. Repeat two more times with fresh CHAPS/PBS each time. Add beads to the antibody and cell solution and incubate overnight at 4 °C with rocking to allow the beads to bind to the biotinylated anti-GFP antibody bound to the GFP or GFP-TMEM184A.
4. Pellet the beads by centrifuging (as in 7.3) and remove the unbound material. Add at least 5 mL 0.2% CHAPS/PBS/protease inhibitors to wash. Repeat washing and centrifugation at least 3x to effectively remove any unbound protein.
5. After removing the final wash, incubate the beads with 1 mL of 0.2 M glycine/0.2% CHAPS in PBS (pH to 2.0 using HCl) on ice for 3 min to dissociate the antibody-GFP binding (resulting in free GFP-TMEM184A or free GFP). Mix gently by tapping every 30 s.
6. Centrifuge at approximately 600 x g and transfer the supernatant containing the purified protein to a new 15 mL tube followed by immediate neutralization with 5 mL of 1 M sodium bicarbonate (bring the pH to 7).
7. Concentrate the sample using a 10,000 Dalton molecular weight cut-off centrifugal concentrator (employ centrifugation speed recommended by the supplier). When the volume reaches approximately 0.5 mL, add 0.2% CHAPS/PBS. Continue concentrating and adding more 0.2% CHAPS/PBS until at least ten volumes (times the starting sample volume) of the 0.2% CHAPS/PBS have been added.
8. To determine an estimate of the amount of isolated GFP or GFP-tagged protein, obtain an absorbance reading at 280 nm and compare it to a standard curve prepared from bovine serum albumin or other control protein in the same buffer.
   NOTE: Due to extinction coefficient differences, this concentration is an estimate, but can provide an approximate protein concentration to facilitate further analysis without wasting sample. Accurate protein concentration can be determined using a micro-Lowry protein assay making certain to prepare the standard protein in the same buffer as the sample. Further analysis of the isolated protein can be accomplished by western blotting of the isolated protein (using antibody detection for GFP) and comparison to the predicted molecular weight. Alternatively, use of the TMEM184A antibodies should result in a band of the predicted construct size, but might also result in a WT TMEM184A band if dimers (or higher-order oligomers) are present in the sample. An estimate of purity can be obtained by staining a blot for total protein from the isolated sample vs total protein from an aliquot of the starting material.

8. In Vitro Heparin Binding Assay

1. Prepare a black avidin-coated 96-well plate by washing with 200 µL 0.2% CHAPS/PBS three times for 5 min with shaking. Prepare an identical black noncoated 96-well plate employing the same washing procedure. Prepare a layout for the experimental samples (in triplicate) to follow during the assay. Include wells for standard heparin concentrations, buffer controls, and wells with GFP samples without heparin to confirm bleaching of GFP.
   NOTE: If optimized isolation or ligand interaction buffers are different for the protein in question, do all of the plate preparations and washing with the optimal buffer system.
2. Add 100 µL of 60 pmol/well biotinylated anti-GFP to all wells in the avidin-coated plate where GFP binding is desired. The amount of antibody is sufficient to just saturate the high-affinity avidin in the wells.
   1. Add buffer only to all wells used for control purposes (no GFP or GFP-TMEM184A binding desired). Seal the wells with a plate cover to prevent evaporation. Incubate for 2 h at RT, with shaking.
      NOTE: Volumes added to wells and incubation times are based on recommendations from the provider.
3. Wash all wells three times for 5 min with 200 µL 0.2% CHAPS/PBS.
4. Add 100 µL of 5 nmol/well GFP-TMEM184A or GFP to appropriate wells in the avidin-coated plate and incubate for 1 h at RT with shaking. Wash again as in 8.3.
   NOTE: This concentration of protein was chosen to ensure that all sites were saturated with excess GFP or GFP-TMEM184A. This is important to ensure that the same amount of protein is bound to each well. Lower amounts of protein might also saturate the sites, a possibility that could be examined by evaluating unbound protein remaining after incubating several concentrations of protein with the plate and comparing unbound protein and ligand binding assays.
5. Prepare various concentrations of fluorescein-labeled heparin, e.g., 10, 25, 50, 75, 100, 200 µg/mL, in 0.2% CHAPS/PBS. Do this and all remaining work in the dark.
   NOTE: Prior to preparing concentrations, specific concentrations for other ligands should be determined for the protein target in question. The lowest concentration should be detectable in the plate reader. Therefore it is important to first determine the range that can be detected accurately in the buffer system employed. Then determine the range needed to obtain measurable binding. Concentrations of fluorescein-heparin below 10 µg/mL did not consistently register in the plate reader under the buffer conditions employed. Similarly, while rhodamine-heparin such as used in the other assays, could be visualized in the plate reader, in the buffer conditions and at the concentrations required for binding, the fluorescence readings for standards were not reproducibly different with that fluorophore.
6. Add 100 µL of fluorescein-heparin to appropriate test wells in the avidin-coated plate and concentration standard wells in both the avidin-coated and the non-coated plate. Incubate for 10 min at RT with shaking. Keep plates in the dark (or foil covered).
7. Using a plate reader, record the initial fluorescence emission from the heparin in the control wells of both plates and the initial fluorescence (total) emission from wells with immobilized GFP-TMEM184A or GFP in the avidin-coated plate. If necessary, adjust the instrument gain to ensure detection of fluorescent heparin between the lowest and highest concentrations.
   NOTE: Make certain the plate reader reads wells from above and reads at the optimum location in the wells if that is adjustable. For the study in question, the optimum location was about 75% down the wells. Information available with the plate reader should provide suggestions that are unique to the plate reader in question for reading assays of bound sample in black plates.
8. Remove unbound fluorescent heparin from wells with immobilized GFP-TMEM184A or GFP and place into corresponding wells in the non-coated black plate. Immediately read the fluorescence emission.
9. Add 100 µL fresh 0.2% CHAPS/PBS back into wells from which the fluorescein-heparin was removed, and read fluorescence emission.
10. Repeat steps 8.8 – 8.9 3x.
    NOTE: These readings from the coated plate indicate fluorescein heparin remaining in the GFP or GFP-TMEM184A wells. If significant fluorescence is found in the third unbound sample, an additional wash should occur. If fluorescence continues to be removed in each wash, binding of ligand might be low affinity, and the conditions should be re-evaluated. The final reading constitutes the bound heparin reading.
11. Record fluorescence emission from the removed, unbound, samples in the noncoated plate, obtaining numbers for each wash. These are the "unbound" readings.
12. Use total heparin emission values obtained in 8.7 to confirm the correct levels of heparin added to each well. Subtract average background readings from the values.
    NOTE: Wells without any fluorescein-heparin serve as background due to antibody, GFP and buffer. Average the background repeats to obtain the background value. Subtract average background readings from the bound and unbound readings to get actual values.
13. Employ a plot of emission vs. total fluorescein-heparin added to determine the scale of emission vs. amount of heparin.
    NOTE: Antibody alone, or antibody and antigen resulted in some quenching of fluorescence. Therefore, total heparin was determined based on the total added heparin as noted in 8.7.
14. Plot the corrected bound heparin vs. the added heparin for the triplicates in both the GFP-TMEM184A case and the GFP case.
15. Determine free ligand by adding the unbound readings from all washes for a specific concentration.