The overall goal of this assay is to detect minute changes in the nucleocytoplasmic transport rate of cells in real time in the absence or presence of potential transport rate-suppressive molecules. This method can help answer key questions about specific neurogenetic diseases such as ALS, that have been proposed to negatively impact nucleocytoplasmic transport. Some advantages of this technique are that it is highly sensitive and quantitative and that it allows the real-time analysis of minute changes in the nucleocytoplasmic transport rate.
Demonstrating the procedure will be Tom Shani, a graduate student from my laboratory. Begin by thawing approximately one times 10 to the sixth mouse neuroblastoma spinal cord cells from liquid nitrogen storage in a 37 degree Celsius water bath. When the cells are fully thawed, collect them by centrifugation, and resuspend the pellet in one milliliter of fresh medium.
Then transfer the cells into 15-milliliter flask containing five milliliters of cell culture medium for an overnight incubation at 37 degrees Celsius in 5%carbon dioxide. When the cells reach 90%confluency, place eight uncoated cover slips into individual 60-millimeter culture dishes and sterilize the cover slips with 70%ethanol. Dry the cover slips under a UV light for 30 minutes.
When the glass is dry, wash the cover slips with fresh medium to remove the residual ethanol. When the cover slips are ready, dissociate the neuroblastoma cells with trypsin-EDTA for about 15 seconds in the cell culture incubator. Then rest the sells for one minute at room temperature.
Collect the cells by centrifugation. After counting, resuspend the pellet at a 3.5 times 10 to the fifth cells per three milliliters of medium concentration and seed three milliliters of cells per dish. Then place the cultures in the incubator for 24 hours to allow the cells to attach to the cover slips.
The next day, add two micrograms of each transfection plasmid to one sterile microcentrifuge tube containing 300 microliters of DMEM medium per 60-millimeter culture dish. Add four microliters of commercial transfection reagent per two micrograms of plasmid to each tube. And vortex the DNA transfection solutions.
Alternatively in cell populations where the shuttle-GFP nucleus to cytoplasmid ratio is close to a one-to-one ratio, the cells can be pre-labeled with a nuclear marker prior to their transfection to facilitate nuclear distinction. After a 25 minute incubation at room temperature, add the plasmids to each dish. And gently stir to obtain a homogenous solution.
Return the cultures to the incubator for another 48 hours. Then attach the coverslips to a coverslip holder for a gentle PBS wash to remove any detached dead cells. Next place the coverslip under an inverted microscope and identify a patch of 20 to 30 GFP-expressing cells in which the nuclei are clearly defined.
Use the imaging software to mark the nuclei within the patch. Then add 200 microliters of exportin-1 inhibitor leptomycin B buffer dropwise to the coverslip and quantify the fluorescence intensity of the marked nuclei in three-second intervals for around 400 to 500 seconds. After all the cover slips have been imaged, normalize the fluorescence intensity of each cell nuclei and generate a fluorescence intensity curve representing the changes in nuclear fluorescence as a function of time for each cell patch.
Then analyze the maximum reaction velocity of the slope of each cell patch derived from three to five independent experiments to generate one slope per experimental group. For protein expression validation 48 hours after transfection, place the culture dishes on ice and wash the cells two times with PBS. Treat the cells in each dish with 200 to 400 microliters of lysis buffer.
And use a cell scraper to remove the cells from the cover slips. Transfer the detached cells from each plate into individual microcentrifuge tubes. And further dissociate the cells with a homogenizer under ice.
After one minute, pellet the homogenized cell pieces by centrifugation and measure the protein concentration in each supernatant by Bradford assay. Then pool 30 to 70 micrograms of protein per group into new microcentrifuge tubes. Store the tubes negative 20 degrees Celsius until SDS page protein expression analysis.
Generic GFP can enter or exit the cell nucleus in the absence of a localization signal tag by diffusion only, resulting in an even distribution of the protein between the nucleus and the cytoplasm. In contrast, shuttle-GFP protein is mostly observed in the cytoplasm due to the nuclear export and nuclear localization signals directing its dispersion. Treatment with leptomycin B, a GFP protein export inhibitor, induces the accumulation of shuttle-GFP within the nucleus, allowing the shuttle-GFP import rate to be measured.
These changes in the nuclear fluorescence intensity in the absence or presence of protein export inhibition can then be quantified in real time to assess the nuclear cytoplasmic transport rate of the shuttle-GFP overtime. To confirm that the activity of nucleolar stress-inducing dipeptide repeat proteins is not affected by protein export inhibition GFP, GFP-tagged dipeptide repeat proteins were visualized by fluorescence microscopy after leptomycin B treatment revealing poly(PR)expression in the nucleus as inclusions only, excluding the possibility of the generation of a false positive nuclear import signal. Poly(GR)expression however was observed homogenously in the cytosol and as inclusions within the nucleus.
Indeed, neuroblastoma spinal cord cell transfection with poly(GR)only followed by leptmycin B treatment results in no observed shuttle movement of the expressed poly(GR)Further analysis of the nuclear cytoplasmic shuttle-GFP transport rate reveals the singular expression of either poly(PR)or poly(GR)dipeptide repeats indicating an about 50%decrease in the total nuclear import rate compared to control empty plasmid-transfected cells. We first had the idea for this method when we tried to find the quantitative assay for detecting small changes in nucleocytoplasmic dysfunction in real time. Once mastered, the microscopic analysis of the cells can be performed in approximately 10 minutes per coverslip.
The data analysis for each coverslip can be completed in a few minutes. After its development, this technique paves the way for researchers in the field of neurodegenerative diseases to explore defects in nucleocytoplasmic transport using cellular models. After watching this video, you should have a good understanding of how to detect and quantify minute changes in cellular nucleocytoplasmic transport rate in real time.
