Our lab uses Drosophila melanogaster to identify and characterize genes and pathways that regulate mitochondrial function during development and physiological homeostasis. The protocol presented here uses live imaging with addition of hydrogen peroxide to visualize the effects of oxidative damage on the localization and dynamics of subcellular structures in the adult Drosophila ovary. We frequently use imaging protocols to study how mutations alter normal mitochondrial localization and movement in female germ cells.
In the course of our studies we found that stress introduced by dissection or other cell stressors had a profound impact on mitochondrial localization. As a control to identify whether or not general stress, as opposed to experimental treatment, is the cause of any changes to the localization or behavior of subcellular structures. We developed this protocol to introduce control stress using hydrogen peroxide.
In this video, we will demonstrate how hydrogen peroxide induced oxidative damage changes the distribution of mitochondria and the ribonucleoprotein Clu. The media best suited for this live imaging technique contained Schneider's Drosophila media containing 15%heat inactivated fetal bovine serum, 0.6 X Pen-Strep, and 200 micrograms per microliter bovine insulin, hereafter referred to as complete Schneider's media. Performing the media preparation under sterile conditions will ensure that it does not become contaminated.
Mix the contents well and store overnight at four degrees. Insulin does not dissolve completely in the complete Schneider's media and you will notice a precipitate settle in the bottom of the tube. Make aliquots, being sure to leave this precipitate as it will interfere with imaging.
This solution may be used for one month if stored in aliquots at four degrees. For optimal female germ cell imaging, first prepare a vial containing a dab of wet yeast paste that is the consistency of peanut butter. This ensures the females are well fed and will produce all follicle developmental stages for imaging.
For optimally healthy flies, collect zero to one day old females and transfer with males into a fly food vial containing wet yeast paste. Make sure the sleeping flies do not contact the yeast paste as they can stick to it. Feed the flies three to seven days, changing the yeast paste daily.
Make sure the yeast paste contacts the fly food, so it does not dry out. To induce oxidative damage, we will use a stock solution of 30%hydrogen peroxide and to visualize mitochondria, we will use a stock solution of 100 millimolar TMRE. It is important to prepare the media solutions fresh because hydrogen peroxide is susceptible to oxidation and TMRE degrades over time.
Right before dissection, in complete Schneider's media, prepare a fresh aliquot of two micromolar hydrogen peroxide, a fresh aliquot of 46 nanomolar TMRE, and a fresh aliquot of 46 nanomolar TMRE plus two micromolar hydrogen peroxide. For ovary dissection you'll need two pairs of fine forceps and a pair of electrolytically sharpened tungsten needles. To dissect the ovaries fill a watch glass with complete Schneider's media that has been warmed to room temperature, then place a single fly in the media using forceps.
Gently grasp the fly by the thorax using one pair of fine forceps. With the other forceps, grasp the posterior and gently pull to remove the ovaries. Remove any extraneous cuticle or tissue, then transfer the ovary to a new well containing fresh media.
The ovaries should still be moving from the surrounding muscle sheath. Using the sharpened tungsten needles, gently tease apart the ovarioles, taking care to remove the surrounding muscle sheath. If the muscle sheath is not removed, as demonstrated here, the ovaria will twitch, causing problems with image acquisition.
Once the ovarioles have been cleanly dissected, transfer them in a 100 microliter drop of media to a MatTek dish if visualizing endogenously labeled structures. Transfer the ovarioles in a 100 microliter drop of TMRE media to a MatTek dish if visualizing mitochondria labeled with TMRE. The ovarioles will sink to the bottom of the droplet.
It is important to practice dissections in order to start imaging the tissue as quickly as possible. Once the ovarioles are mounted, place the dish on the inverted microscope and acquire still images or brief videos as a record of pretreatment conditions. Once this is done, pause the live imaging, remove the lid, and carefully add 100 microliters of the two micromolar hydrogen peroxide prepared solution.
Avoid breaking the surface of the existing media droplet or adding the solution too quickly, so as not to displace the ovarioles resting on the bottom. Replace the dish cover, relocate and refocus the desired ovariole, and resume imaging. Take care to resume imaging as quickly as possible, as the experimental treatment is time-sensitive.
Shown here are still images from a movie, taken post-hydrogen peroxide treatment. Mitochondria and female germ cells are dispersed throughout the cytoplasm when the flies are well fed and healthy. After hydrogen peroxide addition, the oxidative damage causes mitochondria to clump, which can be seen as quickly as 10 minutes post-treatment.
After 20 minutes, the damage causes the mitochondrial membrane potential to dissipate. Thus, the majority of the mitochondria fade. In this live movie of the process, mitochondria can be seen quickly clumping and the majority of mitochondria ultimately fade.
The pulsing blinking in the cytoplasm, particularly in the surrounding follicle cells, appears characteristic of TMRE addition. As mentioned before, imaging must take place quickly after hydrogen peroxide addition. As shown here, if too much time elapses, the mitochondrial membrane potential is already lost and the TMRE labeling dissipates, making the data uninterpretable.
In these video still images, GFP endogenously labeled Clu particles are robust and abundant in the germplasm of well fed females. After hydrogen peroxide addition, Clu particles disperse within 10 minutes. This is demonstrated in the accompanying video.
After hydrogen peroxide addition, the Clu bliss particles quickly disperse. We have found this protocol to be a useful tool to ensure accurate imaging of wild-type localization of mitochondria and Clu bliss particles in Drosophila female germ cells. This protocol should be applicable for a wide range of live imaging studies and could be used to treat tissues before fixation as well.
The most important steps are to isolate ovarioles free of the muscle sheath, to gently add the hydrogen peroxide, so as not to dislodge the ovarioles, and to image quickly after hydrogen peroxide addition. We hope you will find this a useful control for live imaging a variety of subcellular structures. Thank you for watching and good luck with your experiments.
