Presented here is a protocol to monitor degradation of the protein huntingtin fused to the photoconvertible fluorophore Dendra2.
This protocol allows for tracking and quantification of the degradation of a protein of interest in live and aging C.elegans. This in vivo and noninvasive technique requires only little microscopy set up to reliably convert Dendra2 into a bright and stable fluorophore. The method is adaptable to mammalian systems, as well as zebrafish.
To study, put in turnover within the proteostatis network as well as transport and trafficking. Take care not to provoke unwanted conversion of Dendra2 while scanning with the blue light laser, and to test and optimize the acquisition settings and conversion settings for each system and fusion protein. Begin by growing age-matched nematodes at 20 degrees Celsius to the desired age for the experiment.
On the day of the imaging experiment, melt general grade agarose at a 3%concentration in double distilled water. While the agarose is cooling, cut the tip of a one milliliter pipette tip to facilitate aspiration of approximately 400 microliters of the melted agarose. Carefully add a few drops of agarose onto a clean glass slide and immediately place another slide on top of the drops to create a thin pad of agarose between the two glass pieces.
When the agarose has dried, carefully remove the top slide. When enough slides have been prepared, open the confocal microscope software and set the light path for excitation and emission. For green Dendra2 add 486 to 553 nanometers, and for red Dendra2 add 580 to 740 nanometers.
Adjust the power and gain of both of the channels according to the intensity of the fluorophore and set the pinhole to fully open. Select a sequential channel mode and set the track to switch every frame. Set the scan mode as frame and the frame size as 1024 by 1024 with a line step of one.
Set the averaging to two and to average by the mean method and mode of unidirectional line. Set the bit depth to eight bits. Define the multidimensional acquisition setting for the conversion of Dendra2.
For conversion and bleaching, use the 405 nanometer diode laser set to 60%energy power. Select a time series of two cycles with a 0.0 millisecond interval between the cycles and a normal start and stop. Set the bleaching to start after scan one of two, to repeat for 30 iterations and to stop when the intensity drops to 50%Then, define the speed of the acquisition pixel dwell to fast for conversion and to medium for capturing a snap image.
To mount the nematodes onto the slides, use a permanent marker to draw and number a window with four squares on the opposite of the agarose pad on each slide. Add 15 microliters of levamisole to the middle of the agarose pad and use a stereo microscope and a wire pick to transfer for age-matched nematodes into the droplet. Use an eyelash pick to gently move each nematode into one window of the square.
When all of the nematodes have been repositioned, wait until the worms have stopped moving before placing a cover slip over the liquid to immobilize the nematodes during imaging. Then, place the slide onto the confocal microscope stage. For green Dendra2 conversion, use the eyepiece of the 20 X objective under green fluorescence to locate the first nematode and focus on the head or tail.
Switch to confocal mode and increase or decrease the gain and laser power to obtain a saturated, but not overexposed image. Draw a region of interest around the selected neuron and draw a second larger region of interest around the tail that includes the first region of interest. Set the first region to be acquired, bleached, and analyzed.
Set the second region of interest to be acquired and analyzed, but not bleached. Set the speed of scanning to maximum and start the scan to convert the selected Dendra2 neurons. Immediately after conversion, begin live scanning with the green 561 nanometer laser to visualize Dendra2 in the red channel and find the focus and respective maximum projection of the converted neuron.
Quickly set the scan rate to a lower pixel dwell speed and acquire a snapshot image of both channels at a higher resolution. This image is considered at time zero after conversion. Then, save the scan with an identifiable name and or number, followed by the time zero label.
To track Dendra2 degradation over time, open the times zero image of the respective nematode neuron. Using the same image setting, begin scanning live in the red channel and bring the converted red neuron into focus. Then, without changing any acquisition parameters, obtain a snapshot at the same speed as the first image.
Be sure to define your conversion setting carefully to convert Dendra2 efficiently and sufficiently and to maintain the same acquisition parameters throughout different time points. To analyze the converted Dendra2 images, open the time zero and second time point images in Fiji and open Analyze and Set Measurements. Select the Area and Integrated Density functions and select the image obtained with the red channel.
Using the Polygon Selection Tool, draw a region of interest around the time zero converted neuron. To properly identify the contours of the neuron, select Image, Adjust, and Threshold to highlight the intensity thresholds. Once the selection has been defined, click Analyze and Measure.
A pop up results window will appear, including the area, integrated density, and raw integrated density values for the selected region of interest. Perform the same process of selection and measurement for the image from the second time point. Then, copy the values into the spreadsheet.
In this representative analysis before conversion, no red signal was visible when the sample was excited in the red channel. Upon irradiation, the green signal diminished as HTT-D2 was converted and a red signal subsequently appeared. HTT-D2 expression degraded over time, resulting in a reduction in the levels of red HTT-D2 signal and a possible increase of the green HTT-D2 signal as more fusion protein was newly synthesized.
Notably, the neurons of the tail region were determined to be significantly more active compared with those of the head. Further, there was significantly more degradation of control HTTQ25-D2 24 hours after conversion, then after two hours in both the head and tail neurons. This difference was not observed in pathogenic HTTQ97-D2, however, suggesting that the proteostasis network was unable to remove HTT-D2 containing longer glutamine stretches throughout the nervous system.
HTTQ97-D2 was not degraded as efficiently as HTTQ25-D2 in very old nematodes, highlighting the inability of the proteostasis network to remove aggregated and possibly toxic species of Huntingtin in older animals. Importantly, tail neurons were able to cope with toxic HTTQ97-D2 in young nematodes, suggesting that various concomitant proteostasis network mechanisms might be at work to remove HTT-D2. For each sample acquire and non-overexposed and non-saturated image immediately after conversion and reuse the same setting for that sample throughout time.
With this protocol, it is possible to test changes in a proteostatis network, and also, with the help of super-resolution microscopy, to track the spreading of disease associated proteins.
