In mammalian cells, the endoplasmic reticulum or ER plays an important role in protein biogenesis and calcium signaling. The heterotrimeric sex 61 complex in the ER membrane provides an aqueous path for newly synthesized polypeptides into the lumen of the er. Recent data suggests that sex 61 may also form transient calcium leak channels to directly address the role of the Sex 61 complex as a potential calcium leak channel.
And to characterize its putative regulatory mechanisms, cells are first treated with siRNAs directed against the sex 61 A one gene. In complementation analysis, the cells are cot transfected with an iris GFP vector that allows for the irna resistant expression of the wild type sex 61 A one gene. Then the cells are loaded with the ratio metric calcium indicator RO two and changes in the cytosolic calcium concentration are monitored via fluorescence microscopy.
As the calcium levels are measured, solution changes are performed to assess the impact of various agents such as calmodulin antagonists and fabs argon on calcium leakage. Quantitative analysis of the resulting images reveals changes in calcium F flux induced by the silencing of sex 61 A one. In this video, a model for the role of sex 61 A one in calcium F flux will be presented following demonstration of the procedure and presentation of the results.
The main advantage of this technique of existing methods like planet lipid bio experiments, or single channel measurements is that candidate membrane proteins can be analyzed in the native environment and without tedious purification and typically inefficient functional reconstitution. This method can help to answer key questions in the calcium signaling field, such as, for instance, what is the contribution of a specific ER membrane proteins to the calcium ffl from the ER in different cell types and how are they controlled? Demonstrating the procedure will be sway long and NICO two talented graduate students from our laboratories Begin this procedure by preparing the solutions and reagents to be used First, prepare calcium free buffer according to the instructions in the accompanying written protocol.
Call next, dilute a stock solution of the calcium indicator. Die fira 2:00 AM into one milliliter of DMEM containing 10%FBS and 1%penicillin streptomycin to a final concentration of four micromolar dilute a one millimolar Apso Garin stock solution in calcium free buffer to a final concentration of three micromolar also have on hand 200 micromolar olein A and 20 micromolar to reduce the risk of studying off-target effects of a particular irna. Two different irna are used to silence each gene to assess possible side effects of the transfection procedure.
Negative control siRNAs, which are composed of a random sequence of nucleotides are transfected in parallel with the experimental siRNAs. It is important that the effect of irna mediated silencing can be rescued by expression of wild type CD NA.Therefore, the siRNAs used for the experiment are specifically selected so that the rescue plasmid is not affected by the siRNAs. In this experiment, siRNAs are directed against both the coating and non-coating region or untranslated region, abbreviated UTR of the gene of interest.
When siRNAs are directed against the UTR rescue, plasmids without the endogenous UTR can be used to perform transfect in hela cells. Prepare the cells for the transfection by placing a 25 millimeter polyol lysine coated cover slip in one six centimeter cell culture dish for each transfection after one hour, add 5.2 times 10 to the fifth hela cells and supplemented DMEM to each dish. To transfect the cells label one micro centrifuge tube.
For each transfection reaction to each tube, add four microliters of 20 micromolar S irna dissolved in 80 microliters of optimum here, SEC 61 A one, sex 61 A one UTR and negative control irna are prepared to each tube. Add 20 microliters of hyper perfect transfection reagent gently vortex, then incubate the reactions at room temperature for 10 minutes. To transfect the cells, add the irna mix 104 microliters in total dropwise to the seated heli cells and gently swirl the dish to ensure uniform distribution of the transfection complexes.
Incubated 37 degrees Celsius in a humidified environment with 5%carbon dioxide. After 24 hours, replace the medium with 3.9 milliliters of fresh medium. Then to increase transfection efficiency, perform the transfection a second time, using the same irna mix to evaluate silencing, perform western blot analysis.
Silencing should be above 80%as shown here for sex 61 A one. The maximum silencing effect is typically seen 96 hours after the first transfection, 48 hours after the first transfection prepared to rescue the phenotype resulting from sex. 61 silencing begin by labeling a new micro centrifuge tube.
For each reaction, the cells will now be transfected with either empty vector or wild type sex. 61 A one expression plasmid. The wild type expression plasmid used here was generated by inserting sex 61 A one cDNA into the multiple cloning site of a pcd.
A three internal ribosomal entry GFP vector containing the cytomegalovirus promoter, the iris plus the green fluorescent protein coating sequence. Prepare the transfection reagent by adding four micrograms of each plasmid dissolved in 80 microliters of optimum to each tube. Then to this add 16 microliters of FU gene HD transfection reagent gently vortex this solution and incubated at room temperature for 10 minutes.
Then after exchanging the medium, add the plasmid mix dropwise to the seated 5.2 times 10 to the fifth heli cells and gently swirl the dish to ensure uniform distribution of the transfection complexes. Place the cells at 37 degrees Celsius and 5%carbon dioxide and incubate for a further 48 hours. To determine transfection efficiency, evaluate GFP expression by fluorescence microscopy.
After 48 hours, at least 80%of the cells should be transfected. Prior to imaging. Use forceps to transfer the cover slips with the transfected hela cells to a new 3.5 centimeter cell culture dish and add one milliliter of DMEM with four micromolar.
If you're a 2:00 AM incubate for 45 minutes at 25 degrees Celsius in the dark. To perform imaging, transfer the cover slip with the fewer two loaded cells to a metal recording chamber and fix it into place by screwing the two parts of the chamber together. Using a pipette add and remove 300 microliters of calcium free buffer to the cells to wash them.
Repeat then mount the recording chamber on an I MIC microscope with polychrome five cremator. Using a filter set for URA two, find a field containing at least 20 cells in order to estimate the calcium independent fluorescence of FU A two. The excitation wavelength used in this step is 360 nanometers.
Once a suitable field is selected, set up the imaging software to excite at 340 nanometers and 380 nanometers and to measure the emitted fluorescence at 510 nanometers every three seconds, set the exposure time to produce fluorescence intensities around 1000 arbitrary units for the setup shown here. A five millisecond exposure time is set. Additionally, mark the regions of interest or ROIs for a cell-free background region and up to 20 cell contours.
Initiate life cell imaging while the experiment is running, the system will present the ratio metric images for fewer two signals as well as the real-time F three 40 F three 80 ratios for each marked ROI as a line chart where F three 40 and F three 80 correspond to the fluorescence intensity within the ROIs at 340 and 380 nanometers respectively. After one minute, add test solutions to the imaging chamber to test different experimental parameters. In these experiments, the Cal Modlin antagonist, tri fluorine, and bollin A are tested after ratio metric measurements are carried out for an additional 10 minutes.
For CAL modular antagonists, add DSA gargan and continue the measurements. Record 400 paired images per experiment. Once images have been collected, review the images and mark the ROIs for at least 20 cells for each field of view.
Subsequently use the kinetic analysis option in the software to create a sequence of background subtracted ratio images and a plot showing the background subtracted calcium ratio kinetics. For each ROI finally export the data to excel and origin using an established calibration method. The cytosol calcium concentration can be calculated from ratio measurements to examine whether sex 61 A one gene affects calcium leakage from the er.
The sex 61 A one gene was silenced by two different RNAs and helo cells for 96 hours. Silencing was evaluated by Western blood analysis of similar cell numbers in triplicate as shown here, there is a strong 6 61 alpha signal in lanes one through three and a significantly weaker signal in lanes four through six and seven through nine that correspond to the 6 61 A one silence cells. To quantify the results, the sex 61 alpha signals were normalized to beta actin signals greater than 80%silencing was achieved to test the role of calmodulin in ER calcium F flux.
URA two imaging of sex 61 A one sir A treated helo cells was performed in the presence or absence of the Cal Modin antagonist. Bollin A.This movie shows control cells treated with bollin a 10 minutes after bollin. A treatment calcium release was initiated by applying FSO Gargan, a circa inhibitor that unmasked calcium release from the ER in the absence of external calcium as can be seen here.
The addition of FSA argon led to a transient but significant increase in fluorescence indicating calcium F flux from the er. In contrast, calcium F flux following the addition of FSO garin in PHE bone, A treated sex 61 A one silent cells is much less pronounced as can be seen here. This difference can be seen clearly in this diagram, which shows estimated cytosol calcium over time in a series of movies, the quantitative results shown here confirm increased calcium flx in control cells in response to thic argon in the presence offi bolin A as can be seen in this bar diagram.
The same phenomenon was seen for a second cam antagonist triazine. However, the effects of the cam antagonists were not seen when sex 61 complex was depleted from the cells by either one of the two sex. 61 A one specific siRNAs partial rescue of this phenotype can be seen when sex 61 A one silent cells were transfected with sex.
61 a one expression plasmid as can be seen here. Calcium ffl in response to Cal Modin agonists is partially restored with transfection of the irna insensitive wild type plasmid. These results suggest that Cal Modlin is involved in limiting ER calcium flx at the level of a sex 61 complex in cells.
If so, transfection of sex, 61 A one gene with a double mutation in the Cal Modin binding site should not rescue the irna induced phenotype. Indeed, when cells were transfected using a sex 61 A one expression plasmid with a double mutation in the IQ motif, there was no rescue of the phenotype taken together. These data indicate that the release of nascent chains from the sex 61 complex indeed leads to calcium release from the ER and the formation of a calcium nano domain around the cyto select mouth of the sex 61 complex.
This calcium is bound by Cal Moulin and calcium Cal Moulin closes the sex 61 complex. After watching this video, you should have a good understanding of how to combine as IRNA mediated gene silencing in human cells with lifestyle calcium imaging Following this procedure. Other methods, like the expression of CDNAs with disease link mutations can be performed in order to answer additional questions like the potential impact of disease link mutations in irrelevant components.
And don't forget that working with drugs such as tap, again, can be extremely hazardous and precursion, such as the use of gloves should always be taken while performing this procedure.
