The overall goal of this experiment is to identify protein transport routes in primary cilia. This method can help answer key question in the cilia field, such as protein transport route differences in normal and dysfunctional cilia. The main advantage of this technique is that it has high enough resolution to see subdiffraction protein transport routes in live cells.
To begin, thaw frozen stock of specially engineered of NIH-3T3 cells at 37 degrees Celsius, one and a half weeks in advanced of the experiment. Transfer the thawed cells to a 25 centimeter squared cell cultured flask containing three milliliters of pre-warmed media. Maintain the cells at 37 degrees Celsius in a 5%carbon-dioxide incubator.
Culture the cells until they reach 80%confluency and then split the cells. In order to split the cells, trypsinize them with 0.25%Trypsin for two minutes at 37 degrees Celsius. Following the brief incubation, aspirate the Trypsin and replace it with two milliliters of pre-warmed culture media.
Pipette the media to break up any cell clusters, then remove 75%of the cells from the flask and bring total volume of media back up to three milliliters. Split the cells at least three times before the experiment to ensure homogeneity of their cell cycle. Two days before the experiment, split the cells and plate them 60-70%confluency on a 35 millimeter glass bottom plate with 1.5 milliliters of the same culture medium.
Then, one day before the experiment, chemically transfect the cells with the desired plasmid. Mix 500-1000 nanograms of the plasmid in a one to 2.5 ratio with transfection reagent and 0.25 milliliters of reduced serum media without antibiotics for 30 minutes. Once the transfection complex has formed, aspirate the media from the 35 millimeter glass bottom dish and replace it with the 0.25 millimeters transfection mixture and an extra 1.25 millimeters of reduced serum media without antibiotics and return the plate to the incubator.
For cells with externally labeled SSTR3 construct, remove the media from the glass bottom dish one hour before the experiment. Wash the cells five times with one milliliter of PBS and add one milliliter of reduced serum media supplemented with one micromolar of Alexa 647 conjugated streptavidin. No more than 15 minutes before the experiment, remove media from the glass bottom dish and wash the cells five times with one milliliter of PBS.
Then add one milliliter of the imaging buffer into the glass bottom dish. Affix the glass bottom plate to the stage in the microscope. Find a GFP express cell using the GFP filter set and locate a cell that is properly expressing SSTR3-GFP in a primary cilia.
Once a suitable cell has been found, align the NPHP4-mCherry spot at the base of the primary cilia with the location on the imaging plane that corresponds to the laser's single point illumination. Using the snap function, in the camera tab of the focus controls window, capture an epifluorescence image of NPHP4-mCherry and SSTR3 proteins tagged with green fluorescence protein. Once the reference images are obtained, locally reduce the concentration of labeled single molecules by photobleaching the transition zone with a one milliwatt laser illumination for 20 seconds.
To prepare for single molecule tracking, reduce the laser illumination power to 0.5 milliwatts for molecules labeled with Alexa 647. Set the gain and intensification to maximum and the frame rate to two milliseconds. Then click the stream button in the camera tab of the focus controls window and engage the appropriate illumination laser.
Record the non-photobleached labeled single molecules as they are transported through the photobleached region of the transition zone. And remember to record no more than two minutes of video to avoid the effects of ciliary drift. After capturing the single molecules, process the videos using a 2D Gaussian fitting algorithm such as Glimpse by the Gelles lab, which precisely localizes the centroid of each single molecule's excitation point, spread function, and an encompassing area of interest.
Finally, select all single molecule locations with a precision better than 10 nanometers and correct the center of the cilia based on the distribution of single molecule locations fitted with a 2D Gaussian function. As described in this video, single point illumination can be used to track an AF647 tagged SSTR3 molecule through the transition zone marked by NPHP4-mCherry. Before data analysis, superimposing the single molecule video on the wide field illumination is a useful validation step to make sure the laser is properly aligned, and that there is a adequate signal-to-noise ratio.
Imaged here is a cell with a primary cilium identified by the staining. IFT20 is component of the intraflagellar transport complex which carries cargo along microtubules inside primary cilia, marked by Arl13b-mCherry. Once identified, speed microscopy is used to track individual IFT20-GFP proteins.
Next, and 2D to 3D transformation algorithm is used to produce the spatial probability density distribution of the protein inside primary cilia along a single dimension. This high density region likely colocalizes with the axonemal microtubules in agreement with the known location of the intraflagellar transport route. While attempting this procedure, it's important to precisely align the sample with the laser to minimize systematic error.
After watching this video, you should have a good understanding of how to do single molecule tracking with speed microscopy and you will know how to obtain single molecule transport route in primary cilia.
