The rate of protein synthesis is perturbed during disease and aging. Fluorescence recovery after photobleaching, or FRAP, allows the measurement of rate of protein synthesis in vivo. We use transparent C.elegans worms, which express GFP, as a model to monitor the novel protein synthesis after photobleaching.
We provide practical guidelines to measure fluorescence recovery by using transgenic worms, which express GFP under different promoters, either widely expressed or in specific cells or tissues. Also, this method offers protein synthesis speed monitoring in real time. Use a dissecting stereomicroscope to assess the developmental stages and growth of wild-type and mutant transgenic nematodes.
Pick 10 L4 larvae of wild-type and mutant transgenic animals carrying the desired fluorescent reporter onto nematode growth media plates seeded with E.coli. Incubate and grow the nematodes at the standard temperature of 20 degrees Celsius. Four days later, the plate contains a mixed population of transgenic worms.
Select and transfer 15 L4 larvae of each strain on freshly seeded OP50 NGM plates. Perform FRAP assay, and monitor protein synthesis rate on day one of adulthood. The next day, prepare and use cycloheximide-containing NGM plates as a positive control.
Kill bacteria by exposing the seeded NGM plates for 15 minutes with UV light. Add cycloheximide on the top of bacterial-seeded plates too 500 microgram per mL final concentration in the agar volume. Allow plates to dry.
Transfer transgenic nematodes expressing GFP on vehicle and cycloheximide-containing plates. Incubate animals for two hours at the standard temperature of 20 degrees Celsius. Pick and transfer one-day adult transgenic animals to individual NGM plates seeded with a 20 microliters OP50 drop in the center.
Remove the lid and place each plate under a 20H objective lens of an epifluorescent microscope. Focus and capture a reference image before photobleaching. Photobleach each sample for 10 minutes.
Capture an image after photobleaching. Keep animals in individual NGM plates, and let them to recover. Image and record the recovery of each fluorescent reporter every one hour for at least six hours at an epifluorescent stereomicroscope.
Prepare 2%agarose pads, and add 10 microliters drop of M9 buffer at the center of the agarose pad. Transfer five transgenic nematodes expressing pan-neuronal cytoplasmic GFP into a drop of M9 buffer. Use an eyelash to spread the liquid.
Animals display reduced movements within two minutes because of M9 absorbance into the agar. Change nematode's position by using an eyelash pick. Place the sample at the 40H objective lens of an epifluorescent microscope without using a cover slip.
Focus and capture a reference image. Photobleach a targeted area of interest for 90 seconds. Capture an image after photobleaching.
Add a 10 microliters drop of M9 buffer on photobleached nematodes. Let the animals to recover for five minutes. Use the eyelash pick or a pipette to transfer the nematodes to individual NGM plates seeded with 20 microliters OP50 drop in the center.
Capture an image of each sample every one hour under an epifluorescent stereomicroscope. Wild-type and fe-2 mutant worms expressing cytoplasmic GFP throughout their somatic tissues by using the fe-2 promoter were compared before, immediately after photobleaching, and five hours post-recovery. Wild-type animals fully recovered while fe-2 mutants had the minis recovery capacity.
Thus wild type worms initiate the normal protein synthesis after photobleaching, while worms lacking mRNA translation initiation factor fe-2 are unable to do so, indicating that the rate of fluorescent recovery illustrates the rate of protein synthesis in vivo. Cycloheximide, a specific inhibitor of mRNA translation can be used as a positive control for protein translation inhibition. Indeed, cycloheximide-treated transgenic animals expressing cytoplasmic GFP under the promoter do not recover their fluorescence upon photobleaching.
mRNA processing bodies affect the rate of protein translation. Wild-type and edc-3 mutant nematodes expressing GFP pan-neuronally were examined for their recovery capacity upon targeted photobleaching at the head region. Fluorescent recovery is much slower in edc-3-deficient animals compared to wild-type worms.
Protein synthesis modulation is essential for organismal homeostasis. During aging, global as well as specific protein synthesis is perturbed. Protein translation balance directly controls senescence and aging.
Specifically, core components of the translation machinery as well as P-body interacting components accelerate the aging process. Perturbation of translation initiation decelerates aging. Hence, measuring global protein synthesis rates by FRAP is a direct readout of the aging process.
