This experimental system acquires high resolution 3D fluorescence images in live cells to monitor and assay the spatiotemporal dynamics of protein folding quality control. First, prepare the cells to study the rapidly moving protein complexes. Acquire a Z stack of images at high Z resolution.
Then acquire a time series composed of Zacks next, render the resulting images in a 3D movie and analyze the resulting images. Ultimately, four D imaging enables tracking the dynamic and transient phenomena in cells at different conditions. The main advantage of four D imaging over existing methods like wide field microscopy and time-lapse is that it allows us to simultaneously visualize the dynamics and spatial temporal distribution of the entire population of proteins in the cell at high resolution.
This method can help answer key questions in the field of cell biology of protein aggregation, quality control, and protein folding. These are highly dynamic and transient processes. Hence, without high temporal and spatial resolution, many of them would be impossible to observe.
This method can provide insight into aggregation quality control. It can also revolutionize the study of protein interaction and dynamics. Generally, individuals new To this method will struggle because taking 3D movies of endogenously tagged or low expressing proteins require the stability of the system.
High sensitivity and low photobleaching Transform the yeast strains with an expression plasmid for a fusion protein of Fluor linked to a cytosolic folding sensor protein, like the temperature sensitive mutant of the UBC nine. Grow the yeast in synthetic media containing 2%raffinose for 24 hours, then back dilute to 2%Galactose containing media for overnight dilute the query strain to an OD 600 of about 0.2 and culture for four to six hours until the culture reaches an OD 600 between 0.8 and 1.0 30 minutes prior to imaging. Repress the expression with synthetic media supplemented with 2%glucose.
This step allows monitoring of only the already translated and folded pool of the express protein. Now heat shock the cells for 20 minutes at 37 degrees Celsius. To induce misfolding, select an appropriate plate to maintain focus during four D.Imaging acquisition.
Cover the bottom of the plate with 0.25 milligrams per milliliter con A for 10 minutes. Con A is used to adhere cells to the slide, which enables following a single cell over time. Aspirate the con A and arrow, dry the plate in a chemical hood.
Then add 200 microliters of yeast sample. Incubate for 15 minutes to enable cells to adhere to the plate. Perform three washes with media to obtain a single layer of cells for yeast imaging.
Use a confocal microscope with a few non-standard modifications as detailed in the accompanying text. This yeast imaging system uses up to four lasers and up to four photo multiplier tubes equipped with filters. Most of our imaging is done with the green filter set for EGFP and a red filter set for m cherry and TD tomato.
Also equip the confocal with a pi nano pizo stage perfect focus system and both the galvan O and resonant scanners equilibrate the microscope system until the desired temperature is reached. Before imaging based on the refractive index of the imaging medium versus the medium of the sample, select the appropriate objective water, oil, or air. Adjust the correction collar according to the thickness of the plate.
Clean the objective using lens wipes and ethanol. Also clean the slide carrying the sample. Place the plate on the stage holder securely and verify the stability of the sample holder to ensure correct settings for every sample.
Adjust the correction collar while using fluorescent beads to visualize the point spread function with the eye port. Determine the location and orientation of the yeast. Next, switch on the required epi fluorescent light and focus on cells displaying the relevant phenotype.
Then using brightfield, assess cell health and viability according to shape and texture for cell viability evaluations. Visualize the nucleus with a TD tomato flora Fluor fused to an SV 40 and LS signal. TFP is twice the size of GFP.
Since it is above the diffusion limit of the nucleus, it works very well as a nuclear marker. Excite the TFP with a green 4 88 nanometer laser simultaneously as GFP, but collect the green and red emissions into two separate PMTs for spectral resolution. To minimize noise and saturation, adjust the laser power and pinhole diameter as detailed in the accompanying manuscript.
If different flora force emit congruent wavelength, use the spectral detector feature, which enables the choice of virtual filters proceed to acquire time-lapse images as per experimental design, the Misfolded Protein U BBC nine Ts provides a model system to follow aggregation quality control over time and space. In the cytosol at the permissive temperature, UBC nine Ts is folded and diffuse in the nucleus and cytosol upon heat induced misfolding. It initially forms rapidly diffusing small cytosolic aggregate puncta that are processed for proteasomal degradation.
When the proteasome is partially inhibited, these puncta are converted into junk and iPod inclusions over the course of about two hours. Under normal conditions, GFP UBC nine Ts is natively folded and is localized diffusely in the nucleus cytosol as seen here in green. Upon temperature shift to 37 degrees Celsius, the fusion protein is misfolded and forms cytosolic puncta aggregates upon recovery from heat shock at 23 degrees Celsius.
The thermally denatured G-F-P-U-B-C nine Ts is degraded as indicated by decreased fluorescence level. The nucleus is labeled red by N-L-S-T-F-P. When the temperature shift to 37 degrees Celsius is coupled with proteasome inhibition, the G-F-P-U-B-C nine Ts is misfolded and processed into junk and iPod inclusions.
Here, the ubiquitin protease four is over expressed to block ubiquitination inhibition of ubiquitination together with the temperature shift results in G-F-P-U-B-C nine Ts processing into the iPod Inclusion. These time-lapse images acquired at four minute intervals clearly depict the dynamics of junk and iPod formation. This technique can be used to monitor cellular proteostasis over time by using UBC nine Ts as a folding sensor.
Four D imaging can also be done in other model systems such as mammalian cells and C elegance. Four D imaging also allows insight into other subtle events in aggregation quality control, such as the formation and dynamics of soluble aggregate stress foci, and the delivery of aggregates to the junk and iPod compartments.
