This method can help answer key questions about the direct role of tau in the nuclear compartment while excluding any potential cytoplasmic tau contamination. The main advantage of this technique is that it is broadly applicable to the study on the nuclear function of tau in other cell types and under different cellular conditions. Demonstrating the procedure will be Giacomo Siano, a post-doc from my laboratory.
Begin by plating four times 10 to the five SH-SY5Y human neuroblastoma cell line cells to be transfected with the empty control vector, four times 10 to the five cells to be transfected with untagged tau, four times 10 to the five cells to be transfected with tau-NLS, and four times 10 to the five cells to be transfected with tau-NES into individual wells of a six-well plate. The day after plating, separately incubate 400 nanograms of DNA and the appropriate volume of cationic lipids into one tube of 250 microliters of reduced serum per well. After five minutes at room temperature, combine each vector with one volume of cationic lipid and incubate the DNA lipid complexes for 20 minutes at room temperature.
At the end of the incubation, replace the supernatants in each well with two milliliters of fresh complete culture medium and transfect each well with the appropriate DNA lipid complex solution for an overnight incubation at 37 degrees Celsius. The next day, replace the supernatants with fresh differentiation medium. For Western Blot analysis, wash the transfected and differentiated cells in each well with PBS and incubate the cells with 500 microliters of 0.1%Trypsin per well for four minutes at 37 degrees Celsius.
When the cells have detached, stop the reaction with an equal volume of complete medium and transfer the cell suspension from each well into individual tubes for centrifugation. After carefully removing the supernatants, resuspend the pellets in one milliliter of PBS per tube for a second centrifugation and remove the supernatants again. For total protein extracts, incubate the pellets for 30 minutes on ice in 50 to 100 microliters of lysis buffer supplemented with protease and phosphatase inhibitors.
At the end of the incubation, centrifuge the extract at 16, 000 times g for 15 minutes and quantify the protein concentration by any standard quantification assay. Then mix 20 micrograms of each protein sample with five microliters of 4X Laemmli buffer to a total volume of 20 microliters and boil the samples at 100 degrees Celsius for five minutes. For subcellular fractionation, resuspend the cells in complete medium for counting and spin down one times 10 to the six cells per sample.
To isolate the cytosolic fraction, incubate the pellets in 100 microliters of ice cold cytoplasmic extraction buffer supplemented with protease inhibitors at four degrees Celsius with gentle mixing for 10 minutes. At the end of the incubation, collect the samples by centrifugation and transfer the supernatants into pre-chilled tubes. Add 100 microliters of ice cold membrane extraction buffer supplemented with protease inhibitors to the pellet for a 10 minute incubation at four degrees Celsius with gentle mixing.
Then centrifuge the pellets for five minutes at four degrees Celsius and 3, 000 times g and collect the supernatants into new tubes. To collect the soluble nuclear fraction, add 50 microliters of nuclear extraction buffer supplemented with protease inhibitors to the pellets with vortexing before incubating the samples at four degrees Celsius for 30 minutes. At the end of the incubation, centrifuge the samples and collect the supernatants.
To collect the insoluble nuclear fraction, add 50 microliters of nuclear extraction buffer supplemented with protease inhibitors, calcium chloride and micrococcal nuclease to the pellets with vortexing. Incubate the samples for five minutes at 37 degrees Celsius before vortexing and centrifuging. Then collect the supernatants.
To collect the cytoskeletal fraction, add 50 microliters of cytoskeletal extraction buffer supplemented with protease inhibitors to the pellets with vortexing. After a 10 minute incubation at room temperature, centrifuge the samples and collect the supernatants. For SDS-PAGE analysis, add seven microliters of 4X Laemmli buffer to 20 microliters of each of the collected subcellular fraction samples and boil the samples at 100 degrees Celsius for five minutes.
At the end of the incubation, load the samples onto an acrylamide gel and perform the electrophoresis at a constant voltage of 120 volts. Then transfer the proteins to a nitrocellulose membrane at 250 milliamps for 90 minutes. Incubate the membrane for five minutes in Ponceau staining solution to check for proper protein gel electrophoresis and a successful blotting.
At the end of the incubation, rinse the membrane in distilled water until the background is clean and remove the stain with continuous washing with Tris-buffered saline supplemented with Tween 20 for 10 minutes on a shaker. Next, incubate the membrane with blocking solution for one hour at room temperature with shaking before washing three times with Tris-buffered saline plus Tween 20 for five minutes per wash. After the last wash, hybridize the membrane with the primary antibody of interest in blocking solution overnight at four degrees Celsius followed by three washes with Tris-buffered saline plus Tween 20 for five minutes.
After the last wash, hybridize the membrane with an appropriate horseradish peroxidase conjugated secondary antibody in blocking solution for one hour at room temperature before washing the membrane with Tris-buffered saline plus Tween 20 three times for five minutes per wash. After the last wash, detect the protein band using chemiluminescence and quantify the intensity of Western Blot bands by ImageJ. In the absence of retinoic acid and brain-derived neurotrophic factor, undifferentiated SH-SY5Y cells assume a rounder morphology and form cell clumps.
As expected, after five days of treatment with retinoic acid, the clumps unwind and the cells spread out. After three additional days of treatment with brain-derived neurotrophic factor, the cells appear uniformly distributed and interconnected via a network of branched neurites. After tau-NLS or tau-NES transfection, tau subcellular localization can be detected by immunofluorescence with anti-tau antibodies.
Depending on the efficiency of the transfection, the cells display a strong nuclear staining merging with the DAPI signal after tau-NLS transfection or a cytoplasmic staining with the empty nuclei after tau-NES. Western Blot analysis reveals the enrichment of actin within the cytoskeletal fraction, tubulin within the cytoplasmic and cytoskeletal fractions, and H2B within the nuclear fractions. Western Blot analysis also confirms the expression of tagged and untagged tau within the nuclear compartment and vesicular glutamate transporter one expression in the total extract.
Indeed, the ratio of tau in the soluble nuclear and cytoplasmic fractions highlights the fact that tau-NLS is highly enriched in the soluble nuclear fraction while tau-NES is decreased. Moreover, tau-NLS is enriched in the chromatin-bound fraction with respect to the cytoplasmic fraction while tau-NES is decreased. Be sure to carefully check the number of cells used for the fractionation of each sample and to verify the enrichment of the housekeeping genes in the proper fractions.
After watching this video, you should be able to isolate cellular compartments and to evaluate the functional role of tau in the nucleus.
