The overall goal of this procedure is to monitor protein re solubilization and refolding in vivo in near real time via fluorescence microscopy and enzymatic assays. This is accomplished by first transforming cells with a plasmid containing firefly luciferase fused to green fluorescent protein or F-F-L-G-F-P. The second step is to induce expression of the F-F-L-G-F-P protein, followed by cycloheximide treatment to halt production of the protein.
Next, measure luciferase activity before and for a period of time after thermal denaturation. The final step is to visualize cells to determine solubility of the F-F-L-G-F-P protein. Ultimately fluorescence microscopy and enzymatic assays are used to quantify the efficiency of reactivation of a model unfolded enzyme in multiple strains.
The main advantage of this technique over existing methods like endpoint analysis, is that you can correlate activity of the protein with aggregation status over time. To determine the relationship between these two phenomena, this method can help answer key questions in the protein homeostasis field, such as how specific chaperones and COS chaperones contribute to disaggregation and refolding of a model protein and how these two activities in the cell are dependent on one another. The firefly luciferase green fluorescent protein fusion protein F-F-L-G-F-P is expressed from this plasmid under the control of the MET 25 methionine repressible promoter.
Using an adapted lithium acetate mediated protocol, the plasmid is transformed into two croce visier strains, a wild type strain and a strain deleted for HSP 1 0 4, A yeast disaggregates that plays a vital role in repairing aggregated misfolded proteins. Since the success of this experiment depends on it being performed in a timely manner, the following should be prepared before the induction of F-F-L-G-F-P culture tubes containing media, micro centrifuge, tubes and luciferian. The amount of Lucifer needed is based on the number of samples and desired number of replicates, plus approximately 1, 200 microliters for the tubing.
Prewarm the hot water bath to 42 degrees Celsius program. The plate reader for the flash luminescence assay set temperature to 30 degrees Celsius Set plate reader to add Luciferian shake and read each well individually for reading luminescence set to endpoint reading. Five second integration time and 180 sensitivity level for recovery assay between each reading set to shake for five minutes, delay for 20 minutes and shake for an additional five minutes.
Load delucci into the injector and set the injector to dispense 50 microliters of Lucifer to give a final concentration of approximately 23 micrograms per sample to begin the procedure for induction of F-F-L-G-F-P incubate cells in five milliliters of synthetic complete media lacking uracil SC minus YURA at 30 degrees Celsius while rotating overnight on the following day. Measure OD 600 and inoculate fresh cultures in five milliliters of SC minus yura to OD 600 of approximately 0.2. Incubate cultures with rotation at 30 degrees Celsius until they reach log phase, which is OD 600 between 0.8 and 1.0 centrifuge cells in 15 milliliter conical tubes at 4, 400 RPM for three minutes.
Decant, supernatant and resuspend the cell pellet in 500 microliters of double distilled water. Transfer resuspended cells to a micro centrifuge tube centrifuge at 6, 000 RPM for 30 seconds and discard the supernatant resus suspend cells in five milliliters of synthetic complete media lacking uracil and methionine SC minus YURA minus met incubate cells in this media while rotating at 30 degrees Celsius for one hour. The removal of methionine will induce expression of F-F-L-G-F-P After one hour centrifuge cells discouraged supernatant and and wash in DD H2O After discarding the DDH two O Resus suspend cells in five milliliters of SC minus edia containing 100 micrograms per milliliter of cycloheximide to inhibit protein expression.
Proceed immediately to the enzymatic F-F-L-G-F-P recovery assay. Begin this procedure immediately after cells are resuspended in media containing cycloheximide. Remove one milliliter from each sample from measurement of D 600 later to measure preheat shock luminescence.
First set up controls that include a water blank and cells containing an empty vector in a solid white 96 well plate then transfer 100 microliters of cells for each sample with the desired number of replicates. Start reading the plate with the programmed specifications to denature the FFL MOTY of F-F-L-G-F-P. Incubate the five milliliter culture at 42 degrees Celsius with shaking for 25 minutes.
Start recovery assay luminescence readings immediately after cells are removed from the incubator, which is time 0.0. Measure luminescence for the first time point and every 30 minutes for 90 minutes. To prepare cells for fluorescence microscopy centrifuge one milliliter of cells at 6, 000 RPM for 30 seconds and remove the supernatant resus.
Suspend the cell pellet in two microliters of DDH two o. Mix two microliters of cells plus two microliters of 2%low melt agros on a slide and add a cover slip. In order to obtain a single plane of cells lightly press a finger down in the center of the cover slip and rotate until the cover slip is difficult to move.
Cells are visualized on a fluorescent microscope using a 100 x oil objective. Use DIC to visualize cells and the Fitz filter to visualize GFP fluorescence. Take multiple pictures for each strain at each time point.
For quantitation fields for pictures should contain approximately 15 to 30 cells for quantitative post experimental analysis. To begin this procedure, induce F-F-L-G-F-P expression and heat shock cells as demonstrated previously, and then centrifuge cells in a 15 milliliter conical tube at 4, 400 RPM for two minutes. Discard supernatant and wash cells with 500 microliters of water.
After discarding the water resus suspend cells in 10 to 15 microliters of SC minus yura. For each strain, cut out a five by five millimeter section of an SC minus yura agar plate. Place the agar section on an approximately 55 millimeter number zero glass bottom dish with the agar surface that was at the bottom of the plate.
Face up pipette four microliters of cells and spread it around the agar surface with the pipette tip. Let stand for about one minute. Next, use tweezers to place the agar section face down on the same glass bottom dish.
Press down lightly visualize cells on a fluorescent microscope using a 100 x oil objective. Use DIC to visualize cells and the fit e filter to visualize GFP fluorescence set coordinates for two to four focal planes for each strain, depending on the density of the culture. Set the camera to take a Z stack of pictures with the appropriate range every five minutes for a 90 minute period.
To investigate the role of the yeast HSP 1 0 4 protein in refolding heat denatured proteins luminescence flash assay was used to monitor the activity of F-F-L-G-F-P in wild type and HSP 1 0 4 delta cells prior to heat shock and immediately after heat shock for each time point indicated, the results revealed a stepwise increase in activity over 90 minutes that ultimately led to a greater than 80%recovery in wild type cells. The HSP 1 0 4 delta strain recovered 19%of the original activity over the same timeframe. Moreover, the extent of initial inactivation was much higher in the HSP 1 0 4 delta strain as indicated by the 26%activity in wild type versus 11%in HSP 1 0 4 delta.
This suggests that HS P 1 0 4 may be serving a protective role prestress or rapidly associates with denaturing F-F-L-G-F-P during the thermal inactivation step to reduce loss of enzymatic activity. Population microscopy corroborated. The enzymatic activity assay cells were collected at 0, 30, 60, and 90 minutes after heat shock and visualized by fluorescence microscopy representative images of each time point are shown here.
The bright green puncta within the cells represent the F-F-L-G-F-P aggregates. Quantitation was done by counting about 100 cells in four to five fields and calculating the percent of cells containing aggregates. It was observed that nearly all cells in the HSP 1 0 4 delta mutant strain maintained at least one F-F-L-G-F-P aggregate compared to the number of aggregates in the wild type strain, which decreased by 62%within 90 minutes after heat shock single cell.
Automated microscopy revealed that in wild type versus HSP 1 0 4 delta cells, F-F-L-G-F-P was re solubilized at a higher rate. This figure shows representative still images projected from a ZS stack acquired during a 90 minute period. In addition, the dynamics of the protein aggregates were very different in wild type and HS P 1 0 4, delta cells in the wild type cells aggregates tended to fuse before being solubilized as illustrated in this movie.
In HSP 1 0 4 delta cells, the aggregates do not fuse before being solubilized and the rate of solubilization is lower than in wild type cells Once mastered. This technique can be done in approximately three hours, starting from mid logarithmic phase cells.
