This method can help answer key questions in the field of fluorescent proteins such as how fluorescent proteins can affect their fusion partners. The main advantage of this technique is that it provides a rapid and easily scalable assessment of the effects of the fluorescent proteins and their fusion partners. Though this method can provide insight into fluorescent protein behavior, it can also be applied to other genetically encoded tags.
Also demonstrating this procedure will be Sonja Di Gregorio, who is a graduate student at Western University. Each fluorescent protein that is tested by this method is first cloned into a yeast expression vector that encodes a galactose inducible version of FLAG-tagged HTT exon one harboring either the non-toxic 25Q repeat or the Huntington Disease associated toxic 72Q repeat. Clones are selected and verified by sequencing and subsequently transformed into yeast.
To prepare cell cultures for the various assays, streak the yeast clones carrying 25Q or 72Q tag with a fluorescence protein of interest on an agar plate containing yeast selection medium with glucose as the carbon source. At the same time, streak yeast carrying 25Q or 72Q yeast optimized monomeric superfolder GFP to serve as a positive control. Incubate the plates at 30 degrees Celsius for two to three days.
Select up to the three single colonies from each plate and inoculate five milliliters of synthetic complete medium supplemented with 2%glucose as the carbon source. Incubate the cultures at 30 degrees Celsius overnight. On the following day, transfer 200 microliters of each overnight culture to a microcentrifuge tube and centrifuge to pellet the cells.
Wash at least three times with sterile distilled water. It's important to wash the cells well to eliminate all traces of glucose that could contribute to repressing the induction of the GAL1 promoter. Prepare the cells as demonstrated previously and re-suspend them in synthetic complete medium containing 2%galactose as the carbon source to induce the expression of polyQ fusions.
As a control, re-suspend the cells in glucose-containing media. Incubate the cells at 30 degrees Celsius in a tube rotator overnight. On the following morning, measure the optical density at 600 nanometers of each culture using a spectrophotometer.
Equalize the cell densities to an optical density 600 of 2 in 100 microliters of synthetic complete medium in a sterile 96-well plate. Prepare four five-fold dilutions of each sample by pipetting 20 microliters of the sample from the previous well into 80 microliters of media in the next well. Use a yeast pinning tool to spot the cells onto selective plates and incubate at 30 degrees Celsius for two days.
Image the plates with an image documentation device. Prepare the cell cultures for this assay and then measure the OD600 of each culture using a spectrophotometer. Dilute the cells to an OD600 of 1 in 300 microliters of media in the 96-well plate.
Prepare each sample in triplicate. Incubate the plate in a plate reader incubator with shaking capabilities. Set the number of samples, the temperature at 30 degrees Celsius, the absorbance at 600 nanometers, the length of the experiments to 24 hours, and the measurement intervals to 15 minutes.
Select the continuous shaking mode. When the experiment is done, create the growth curve and quantify the area under the curve using scientific graphing software. Paste the data into an XY table with three replicate values.
The growth curve will be shown under the graphs folder at the left side. To quantify the area under the curve, select analyze at the top left and click area under curve in XY analyses. Start this procedure by diluting the prepared cells 10-fold in growth medium.
Transfer 200 microliters of each sample to an eight-well imaging chamber. Image the cells using a standard wide-field fluorescent microscope. Adjust the imaging settings for image acquisition.
Since the 72Q aggregates are much brighter than the diffused 25Q signal, it is often required to use a different acquisition setting between the different plasmids in order to avoid saturation of the fluorescent signal. Process the images using a suitable image-processing software. In this protocol, dot blot is used to examine protein expression levels.
Prepare the buffer for generating protein lysates by adding four micromolar of phenylmethylsulfonyl fluoride and a protease inhibitor cocktail to the lysis buffer. Pellet five milliliters of each overnight culture by centrifugation. Re-suspend the cells in 200 microliters of glass beads and 200 microliters of lysis buffer.
Vortex for 30 seconds for 12 rounds, icing in between rounds. Centrifuge at 12, 000 times G at four degrees Celsius for 10 minutes and collect the supernatant. Use a microfiltration apparatus to spot equal amounts of total protein on a nitrocellulose membrane.
Pre-wet the membrane with PBS and assemble the apparatus. Connect it to a vacuum source and make sure the screws are tightened. Load the samples, turn on the vacuum, and let the sample filter through the membrane by gravity.
Blot the membrane in PBS 05%Tween 5%fat-free milk for 30 minutes. Incubate the membrane with primary anti-flag antibody at four degrees Celsius overnight. On the following day, wash the membrane three times for 10 minutes each with PBS 05%Tween.
Incubate the membrane with a fluorescently labeled secondary antibody in PBS 05%Tween 5%fat free milk at room temperature for one hour. Wash the membrane three times for 10 minutes each with PBS 05%Tween. Subsequently, image the membrane using an amino blot documentation system.
Yeast expressing either 25Q or 72Q HTT exon one fused to yeast optimized monomeric superfolder GFP or yeast optimized monomeric tag BFP2 was cultured in glucose or galactose medium overnight and either spotted on agar plates or incubated further in liquid media. While 72Q yeast optimized monomeric superfolder GFP induces a significant growth defect. 72Q yeast optimized monomeric tag BFP2 displays a growth phenotype similar to the non-toxic 25Q counterparts indicating that the nature of the fluorescent tag can impede polyQ expansion behavior in cells.
Assessment of aggregation of the fluorescent polyQ fusions by fluorescent microscopy shows that 72Q yeast optimized monomeric superfolder GFP displays significant aggregation, while 72Q yeast optimized monomeric tag BFP2 displays a diffused cytoplasmic signal similar to the non-toxic 25Q counterparts. Since expression levels of the various polyQ fusions could affect toxicity, dot blots were performed to assess protein levels. Results from five-fold dilutions of the cell lysates are shown.
Don't forget that this technique allows for rapid comparison of uncharacterized and fluorescent proteins with the GFP variants, but it cannot directly assess oligomerization. Following this procedure, other measures, for example, the ulcer assay in mammalian cells can be performed to answer additional questions such as the monomeric status of the fluorescent proteins.
