In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

In vivo single-molecule tracking at the Drosophila presynaptic motor nerve terminal. This technique describes how single proteins may be tracked and imaged in vivo at the motor nerve terminals of third-instar Drosophila larvae. The main advantage of this technique is that it allows single proteins to be imaged, tracked, and localized at high resolutions similar to that observed in in vitro systems.

Design of transgenic Drosophila. The synaptic protein of interest is tagged with a photoconvertible fluorophore, and this is expressed in Drosophila melanogaster. Dissection of third-instar Drosophila larva.

Place a wandering third-instar larva on a cylindrical or semi-cylindrical shaped Sylgard base. Add 40 microliters of Schneider's Insect Medium onto the larva. Under a dissecting microscope, stick a minutien pin through the head and another through the tail of the larva to immobilize it.

To provide access, use a minutien pin to make a narrow incision at the midline of the dorsal side of the larva's abdominal region. From this incision point, using spring scissors, cut along the body wall in the anterior posterior direction. Remove internal organs from the larva with the aid of fine forceps and spring scissors.

Wash the dissected larva with Schneider's Insect Medium. Stick two minutien pins through each of the two body wall flaps to produce a hexagon-shaped dissected larva preparation. Use spring scissors to detach the larva brain from the ventral nerve cord.

Using curved tweezers, push the minutien pins into the Sylgard base. Single particle tracking photo-activated localization microscopy. Invert the larva's Sylgard base onto a glass-bottomed culture dish filled with two milliliters of room-temperature Schneider's Insect Medium.

Apply water onto the 63x water immersion objective of an ELYRA PS.1 microscope and then place the dish onto the stage. Switch on the transmitted light and use the eyepiece to locate the larva on the Sylgard base with the aid of the 10x air objective. Switch to the 63x water immersion objective, and identify muscle six, using bright field illumination.

Using the ZEN Black Acquisition Software, select the Total Internal Reflection Fluorescence, TIRF, configuration. Switch the 488-nanometer laser to visualize eMos2 in green. Locate the neuromuscular junctions, which look like beads on a string, and acquire a low-resolution TIRF image, simultaneously switch on the 405-nanometer laser and the 561-nanometer laser so as to photoconvert and to image eMos2 red species.

Utilize very low laser power for the 405-nanometer laser to allow for moderately constant stochastic photoconversions. Here, a 405-nanometer laser is operated at 0.09%while a 561-nanometer laser is operated at 50%In TIRF Angle options on the software, slightly slide out the TIRF Critical Angle. Set the exposure time at 30 milliseconds.

Acquire a time series of images of the neuromuscular junction. Here, a 15, 000-frame movie was acquired. Save the movie as a czi format file.

Data analysis. Analyze the acquired movies with suitable single-molecular tracking analytical software. Locate the x and y coordinates of each fluorescent protein localization by the Gaussian fit of the intensity point spread function.

To trace the trajectory of each localization, set a threshold of a minimum of eight continuous different frame appearances for each localization and a maximum of three pixel mobility between each frame. Obtain the trajectory image, the mean square displacement, as well as the diffusion coefficient of all the tracked localizations. Representative results.

The mean square displacement of individual trajectories of Syntaxin-1A-eMos2 from multiple neuromuscular junctions in three different larvae were plotted, as mean plus minus standard error of mean. The area under the mean square displacement curve was also plotted. The diffusion coefficient of Syntaxin-1A-mEos2 was similarly obtained from multiple neuromuscular junctions and plotted as a histogram of the relative frequency distribution.

The diffusion coefficient threshold revealed two populations of Syntaxin-1A, a mobile and an immobile population. The ratio of the mobile to immobile populations of Syntaxin-1A-mEos2 was calculated.Conclusion. This video should provide an understanding on how single-protein tracking may be carried out at the neuromuscular junction of Drosophila larvae.

The technique provides a way for researchers in the field of neuronal communication to visualize and observe the mobility and organization of single proteins at high resolutions in vivo.