Classical studies commonly use fluorescent intact proteins for exon transport visualization, but endogenous cargo behavior differs. Endogenous protein levels allow making visualization challenging. We offer ideas to optimize the acquisition conditions for better visualization of endogenous GFP Rab-3, which labels synaptic vesicle precursors.
The abundance of cytosolic proteins in the axonal transport field makes it difficult to visualize their dynamics using conventional fluorescence microscopy. This protocol suggests using ablation step along with temporally controlling labeling approaches to overcome these challenges. We have recently been able to visualize the axonal transport of the endogenous spectrate, by optimizing the acquisition parameters described in this protocol, paired our temporal control labeling approach.
This allowed us to identify the first components of the machinery that mediate the transport. To begin, take out the nematode growth medium plates with Caenorhabditis elegans grown on a lawn of bacteria. Using a platinum wire pick, transfer worms to a droplet of 0.5 millimolar levamisole and incubate them for 20 minutes.
Pipette 12 microliter M9 medium to a 10%agarose patch on a glass slide. Using a hair pick, mount 10 to 20 nematodes from the levamisole droplet into the M9 droplet on the agarose patch. Place a cover slip on top of the droplet and rotate it by 45 degrees to image transport in the DA-9 axon.
Afterward, seal the space between the cover slip and the glass slide with a viscous gel-like petrolatum. To begin, take the Caenorhabditis elegans slide prepared for imaging. Observe the slide under four-X to 20-X magnification lens to locate nematodes.
Remember their positions, and then switch to a 63-X magnification lens for detailed imaging. Capture an initial image to evaluate acquisition parameters. Monitor signal intensities using the intensity histogram, and increase excitation laser intensity if necessary.
Next, using a 488 nanometer laser, bleach the region of the axon to be analyzed. Compare the signal intensity before and after the bleaching step to determine the bleaching efficiency. Use two-by-two binning to enhance the signal intensity of individual transport events.
Enhance the exposure time and use a temporal resolution of 300 to 700 milliseconds between consecutive time points to follow RAB-3 transport events. Record a time lapse of one to three minutes based on the signal intensity and frequency of transport events. To begin, record a time-lapse of endogenous GFP RAB-3 in the asynaptic zone of the DA-9 axon in a live Caenorhabditis elegans.
Import the time-lapse data file into Fiji. Then, check for movement or slight drift of the animal or axon segment during image acquisition. Use the StackReg plugin with a rigid body transformation option to correct for any movement or drifting.
Employ the segmented line tool and adjust the line width to match the axon diameter. Double-click with the left mouse button on the segmented line icon to draw a line along the axon segment for analysis. Now using the Fiji plugin KymoReslice Wide, generate the kymograph, setting the maximum intensity value across the width of the line in its parameter settings.
Using the straight line tool, trace transport events in the kymograph. Trace individual transport events and save each line to the ROI manager. In Fiji, select measurement parameters such as area, bounding rectangle, mean gray value, and Feret's diameter.
Then, paste the results table into a spreadsheet for calculation. Multiply the width by the resolution of the camera to get the run length in micrometers. To determine the duration of a movement event, multiply the height by the acquisition time between consecutive time points.
Calculate pausing time as the run duration between two consecutive movements where velocity is zero. Normalize the total events to the total length of the kymograph for event number per minute and axon length segment. Using the Feret angle tool, categorize events into anterograde and retrograde transport.
For kymographs generated by drawing the segmented line from proximal to distal, use the Feret angle to determine the directionality of each event.
