Investigating the Spreading and Toxicity of Prion-like Proteins Using the Metazoan Model Organism C. elegans

The overall goal of the following experiments is to examine the spreading and complex toxicity of PreOn like proteins using the Metazoan model organism C elegance. This is achieved by monitoring the intra and intracellular transport of vesicles containing the PreOn like protein, using time-lapse fluorescence microscopy. Next tissue specific folding sensors and ubiquitously expressed stress.

Reporters are used to evaluate cell autonomous and non-cell autonomous effects on cellular fitness. Then a genome-wide RNAi screen is performed in order to identify new modifiers of PreOn induced toxicity. The screen is performed in liquid culture using thrashing as a readout for toxicity.

Finally, worm tracker, A plugin for image J is used to quantify movement of C elegance in combination. These methods allow teasing apart of genetic pathways involved in the intercellular movement of PreOn like proteins and their non-cell autonomous toxicity. After preparing synchronized sea elegance control and PreOn domain transgenic lines and 10%agros pads, according to the accompanying text protocol, pipette three microliters of 100 nanograms of polystyrene beads and three microliters of four millimolar limassol into the agros pad.

Using a platinum wire pick transfer worms at the desired age from a plate into the solution and use a cover slip to cover to image the worms at the subcellular level. Place a slide into the microscope slide holder of a confocal microscope and use a 63 x or 100 x 1.4 NA oil objective. Next, open METAMORPH and select multidimensional acquisition.

And under the main tab, check the boxes for time lapse and run journals. Under the saving tab, select or create the directory folder for saving files and assign a name to the file Under the wavelength tab, use Yoko quad red or an equivalent illumination of MRFP imaging and adjust the exposure to between 100 and 300 milliseconds and the camera gain to between 100 and 300. Next, under the time lapse tab, set time interval to one second, duration to five minutes, and the number of time points to 301.

Under the journals tab. For journal select A FC set, Z hold, and A FC return to Z hold for type select special twice and for initial point, select start of time point and end of time point. When the setup is done, press acquire Following this procedure, other methods like particle tracking applications can be used in order to measure the dynamic properties of dis cus.

After synchronizing prion domain transgenic and while type nematodes and copying the inger RNAI library plates according to the text protocol, incubate duplicated RNAi plates at 37 degrees Celsius and 300 RPM to use HT one 15 E coli bacteria harboring the empty vector L 4 4 4 0 as a control. Grow it separately in a culture tube and then use a multi-channel pipette to transfer 65 microliters of the culture into one quarter of the wells of 2 96. Well plates with an automated workstation containing a multi-channel head dispensed.

10 microliters of five, millimolar of diluted IPTG into each well of the RNAi bacteria and control plates. Reseal them before incubating them for three hours while the bacteria are incubating. Mix synchronized wild type control and transgenic L one larvae and M nine buffer well and pipette five to 10 microliters onto a glass microscope.

Slide count the worms and calculate the number of worms per microliter. After the three hour bacterial incubation, remove the plates from the incubator and allow them to come to room temperature to avoid heat stressing the worms. Next, supplement the M nine buffer with IPTG cholesterol and antibiotics according to the text protocol.

And add the appropriate volume of PreOn domain transgenic worm solution to a final concentration of 15 worms per microliter mix to evenly distribute. Then dispense 50 microliters into each well of the RNAi plates and the wells of one of the empty vector control plates. Dispense wild type worm solution into the second control plate.

Then leave all the plates unsealed to allow aeration and keep the liquid culture from evaporating by stacking four to five plates together, wrapping them with a damp paper towel followed by aluminum foil. Incubate the plates at 20 degrees Celsius and 200 RPM for four days. To the plates, use a Falcon four M 60 camera connected to a computer with a monitor to visually screen for increased thrashing as compared with control treated PreOn domain transgenic animals.

The main advantage of using an automated liquid handling workstation to dispense the worms into the R RNAi plates is that it greatly reduces the variability and the number of worms per well. To perform a motility assay on solid plates and validate candidate RNAi clones grow RNAi bacteria overnight according to the text protocol. Then use one millimolar IPTG to induce the expression of DS RNA for three hours.

Use 150 microliters of each RNAi bacterial clone to seed previously prepared NGM plates spreading into a thin layer. Allow the bacteria to dry for two days at room temperature in the dark under a stereo microscope. Determine the amount of synchronized worms and suspension as described earlier and add 25 to 30 L one larvae into each experimental plate.

Incubate the nematodes at 20 degrees Celsius for four days until the worms reach. Day two of adulthood to quantitate worm motility, turn on the camera and the simple PCI imaging software. Click on the camera icon to enable adjustment of imaging conditions.

Next, set up the imaging condition as follows. Set the gain to zero light mode to high speed index to one and binning to two. Then click live and auto expose and adjust the lighting conditions by moving the microscope mirrors and the brightness and contrast dials.

Click on time, scan and type in a folder and a file name set field delay to 20 milliseconds and stop at time to 30 seconds. Press live review in order to choose the area of the plate to record. Then tap the plate three or four times on the stage.

Quickly confirm its position in the field of view and press start after the movie finishes recording right click on the image and choose export montage sequence to export the movie from a CXD format to an A VI file. To analyze the motility videos, open image J choose the plugins tab, then choose worm tracker and select Worm Tracker batch, and select the directory containing all the files to be analyzed in the main input window of Worm Tracker batch load the input values as detailed here. An explanation for each of the parameters can be found in the instructions that accompany the plugin.

Click okay and allow the movement analysis to run. Finally, to curate the results, confirm that all detected tracks are from actual sea elegance and eliminate artifacts. Open the TXT files created for each movie and copy the information to a data analysis software file.

Open the labels zip files created and run the resulting labels TIFF to manually check and eliminate false worm tracks. Yesper Peterson, a former postdoc in the lab, first had the idea for the worm tracker as a substitute for our manual assessment of CL Elegance motil, which is a readout for toxicity. The worm tracker allows us to do these experiments in an automated way with no user bias while assessing more conditions in a shorter amount of time Using time-lapse imaging.

The RFP tagged R two E two, which accumulates in tubular vesicles was seen transported within and between cells by coex expressing a folding sensor or stress reporter. Together with the PreOn like protein cell, autonomous and non-cell autonomous effects can be assessed. The expression of R two E two in muscle cells leads to an earlier onset of Q 44 aggregation in the intestine revealing that R two E two has non-cell autonomous effects on proteostasis co-expression of R two E two With the SOD three PGFP Transcriptional oxidative stress reporter leads to upregulation of the reporter in different tissues, demonstrating that the PreOn domain induces an oxidative stress response non-cell autonomously following the whole genome screen.

This movie demonstrates that high resolution imaging and visualization on the computer screen enable the screening of one quarter of a plate simultaneously Once mastered, those screening protocol can be done in 12 rounds of 25 plates per session. If two sessions are performed per week, the entire RNAi library can be screened within six weeks. The worm tracker was used to quantify C elegance motility as seen here.

Worm Tracker converts an original video into a binary format and each worm is assigned a track. Processed videos can be viewed with tracks superimposed to manually eliminate any artifacts. The body lengths per second parameter normalizes for potential size differences.Nematodes.

This method identified 35 suppressors of PreOn induced toxicity that increased the motility phenotype to at least 133%and up to 256%compared to control treated worms. While leasing the worm tracker, it's important to remember to manually verify the obtain worm tracks. The software does a great job in identifying the worms, but sometimes the background can still be mistakenly identified as an animal.

Therefore, remember to check your videos after running worm tracker on them. These methods can help answer key questions in the field of neurodegenerative diseases such as whether a certain disease link protein is able to spread from cell to cell, similar prence, or whether there is a link between the intercellular transmission and the complex toxicity of these proteins.Kids.