The overall goal of the following experiment is to investigate how transfected fluorescent proteins are distributed and move in the endoplasmic reticulum and to assess the effect of expression on the ultra structural architecture. To do this, cultured cells are transiently transfected with GFP tagged tail anchored ER resident proteins. Following transfection live cell confocal imaging is performed to assess fluorescence recovery after photobleaching or frap and fluorescence loss.
In photobleaching or flip, ultra structural analysis is performed using transmission electron microscopy. The resulting data demonstrate that the expression of GFP tagged B five construct dramatically alters the organization and structure of the endoplasmic reticulum inducing the formation of aggregates. The method we demonstrate here can provide insight into basic cell biology processes, such as protein and lipid mobility and trafficking.
It can also be used to study responses to pharmacological treatments to evaluate fluorescent protein, oligo modernization, or to study the role of pathogenic mutant proteins in the formation of intracellular aggregates of the origin that may be relevant to their pathogenicity, such as in atrophic lateral sclerosis. The plasmid used in this study consists of an enhanced version of GFP fused at its C terminus to the tail region of the er, isoform of rat cytochrome B five via a linker sequence. The tail region of B five contains the entire sequence that remains membrane associated after cleavage by trypsin.
This includes the 17 residue transmembrane domain, flanked by upstream and downstream polar sequences. The linker consists of the M epitope followed by GLY four S3.The entire CDNA is inserted into the Hindi three XO one sites of the mammalian expression vector PC DNA three. In preparation for transfection grow cost seven cells in supplemented DMEM at 37 degrees Celsius and 5%CO2 the day before transfection plate three times 10 to the fifth cells on round 24 millimeter glass cover slips in a six well plate the next day.
Transfect the cells with the jet PEI system as described by the manufacturer here, a jet, PEI to DNA ratio of two to one is used. This typically leads to a transfection efficiency of 70 to 80%Maintain the cells at 37 degrees Celsius and 5%CO2 for 24 hours prior to imaging. Next, use forceps to transfer a cover slip with transfected cells into the imaging chamber.
Return the tissue culture dish to the incubator to save the remaining cells. For ultra structural analysis by electron microscopy. For live cell imaging, add two milliliters of imaging medium to a steel cell culture chamber.
Xi Fluor is included in the imaging medium to prevent the samples from photo bleaching. O experiments must be performed in the presence of smide, a translation inhibitor to avoid any increase in the A fluorescence signal and consequently total fluorescence due to protein.Biosynthesis. Place the imaging chamber on the stage of a confocal microscope, equipped with a temperature controlled CO2 incubator and appropriate lasers with appropriate photo multiplier settings to perform fluorescence recovery.
After photobleaching or frack bring the cells into focus, set the acquisition parameters to avoid saturation of pixels. Also ensure that the laser power is set as low as possible to avoid photo bleaching due to image acquisition. Once the imaging parameters are set, move to a new field of view with two or more cells.
One cell in each acquisition field should remain unbleached, so it can be used to normalize the fluorescent signal of the bleached cell or cells. For bleaching. Draw a region of interest or ROI corresponding to an organized smooth endoplasmic reticulum or OSER.
Set the imaging program to bleach using an adequate number of iterations for efficient and rapid photo bleaching If needed, a combination of 488 nanometers and 405 nanometer lasers can be used. Instruct the software to capture a single frame every 10 seconds for 10 minutes over the entire course of the experiment. This includes the preble, bleach and recovery periods.
Once everything is set, initiate bleaching and capture. When the capture is complete, move to a new field of view and repeat this process. Capture four to five fields of view for each cover.
Slip next to measure fluorescent loss in photo bleaching or flip. Instruct the program to perform a sequence of bleaching and post bleaching imaging for 10 seconds every 30 seconds for 10 minutes, move to a new field of view. Draw an ROI corresponding to an OSER structure and initiate bleaching and capture as before for analysis.
Import the frap and flip images into Image J software. First, assess the fluorescence recovery of the bleached ROI over time for the frack experiments, and then determine the background fluorescence by selecting an area outside the cells and measure its mean intensity over time. Next, subtract the background signal from the fluorescent intensities of the ROIs.
Normalize the data to the total fluorescence of the bleached cell, which should be constant over time. Then fit the mono exponential equation shown here where F post is the fluorescent signal after photo bleaching. F rec is the maximum fluorescence recovery value reached after bleaching T is the final time of registration, and tau is the mid time of registration.
For analysis of the flip experiments, draw an ROI outside the bleached OSER, covering the whole cell and measure its fluorescence intensity over time. As for frack experiments, subtract background by using an area outside the cells. Normalize the data to the fluorescence levels of an ROI drawn on an unbleached cell to correct for any decrease in fluorescence caused by the imaging itself.
Finally, plot the results using GraphPad prism or similar software. Many of the reagents used in preparing the samples for electro microscopy are toxic. Therefore, the following steps should be carried out wearing a lab coat and gloves while working under a fume hood.
After removing the cover slip from the Petri dish for optical microscopy, fix the remaining cells grown on the bottom of the dish as a monolayer for 10 minutes at room temperature. Next, using a Teflon scraper, detach the cells and transfer them into 1.5 milliliter micro centrifuge tubes pellet the cells by centrifugation at 9, 000 times G for 10 minutes following the spin. Remove the supernatant, add fresh fixative and leave overnight at four degrees Celsius the next day.
Wash the pellets by gently aspirating the fixative with a past pipette and adding cate buffer. To avoid disturbing the pellet, allow the buffer to run down the sidewall of the tube as it is added. Aspirate the wash solution and post fix by adding cells in a solution of 1%Osmium tetroxide in Cate Buffer for one hour at room temperature.
Next, rinse with Milli Q water and add 1%urinal acetate in distilled water directly to the micro centrifuge tube. Unblock stain for 20 minutes to an hour, dehydrate the samples in an increasing ethanol series of 70%80%90%100%and 100%again for 10 minutes each. Then wash the pellets twice in propylene oxide for 15 minutes each time.
Infiltrate the samples by substituting the propylene oxide with the one-to-one mixture of propylene oxide and epon incubate for at least two hours and as long as overnight. Next, to embed the samples, place the cell pellet fragments in embedding molds that are prefilled with epon epoxy resin cure at 60 degrees Celsius for at least 24 hours. The next day, mount the resin block on an ultra microtome equipped with a 45 degree diamond knife.
Using a trimming knife, manually trim the resin blocks to remove the portions of the resin block in which the embedded sample is not present. Then using the ultra microtome, obtain 60 to 70 nanometer sections. Collect the sections on 300 mesh copper grids to stain the sections.
Place the grids on 30 microliter drops of a saturated solution of urinal, acetate, and incubate for 20 minutes. Then rinse three times in milli Q water and incubate the grids again on small drops of lead citrate for seven minutes. After the incubation, thoroughly wash the grids by immersing them in milli Q water and allow them to dry at room temperature.
Observe the stained grids using a transmission electron microscope and capture images using a bottom mounted CCD camera at final magnification, ranging from 6, 000 to 39, 000 x to investigate lateral diffusion of D-E-G-F-P ER in organized, smooth endoplasmic reticulum. Transiently transfected caused seven cells from three cover slips were analyzed by frap as described in this video as shown in this time-lapse sequence, two OSER structures were bleached and fluorescence Recovery was recorded over time. Clear fluorescence recovery was detected one minute after bleaching with a further increase in signal four minutes later to determine the recovery halftime and quantify the mobile fraction of D-E-G-F-P-E-R.
The captured images were analyzed as described in this video as can be seen here. The fluorescent intensity of the bleached area increased over time, demonstrating that the fluorescent protein is highly mobile within the aggregates. To study the continuity between different subdomains of the endoplasmic reticulum flip experiments were performed as shown in this time lapse sequence.
The continuous bleaching of an OSER indicated here by the yellow arrow causes a progressive decrease in fluorescence in the rest of the ER and in other OSER structures within the same cell. The red arrow indicates a portion of an unbleached cell in which the fluorescent signal is constant over time. Quantitative analysis of these data revealed a progressive and rapid emptying of the reticular ER, demonstrating that the aggregates are physically connected with the rest of the ER To examine the fine architecture of these aggregates cells expressing high levels of D-E-G-F-P ER were observed using a transmission electron microscope.
This micrograph shows a low magnification view of cell cytoplasm containing an aggregate of membranes. The aggregate consists of stack cyi and undulating sinusoidal membranes. As can be seen here.
Mitochondria can be seen clustered around the OSER structures, while ribosomes indicated by the arrowheads decorate only the membranes of the outermost cyi. Note that the 11 nanometer thick electron dense space between the membranes is continuous with the cytoplasm. An OS ER may be formed by lamellar er, which are stacks of flattened er cyi that can be continuous or fragmented in their appearance in thin sections, vesicles, budding from the outermost cistern of the stack can occasionally be observed as shown here.
Taken together these data demonstrate that the fluorescent aggregates observed in cultured cells transfected with D-E-G-F-P ER represent patches of smooth and flattened er steri that spatially organize themselves into well-defined 3D geometries. While obtaining this procedure, it's important to remember to acquire all images without saturated pixels, which may alter the fluorescent recovery and consequently the protein mobility analysis. Always be sure to normalize the fluorescent signal in the bleach, the region of interest to the total fluorescence of the same cell in order to consider fluorescence intensity variations due to bleaching during image acquisition or small changes in the focus plane.
