This study investigates the effects of human RNA-binding protein variants on the motor function of Caenorhabditis elegans. By analyzing the movement of worms expressing different protein levels, the research highlights the correlation between protein aggregation and motor impairment.
Take Petri dishes containing a uniform distribution of worm food on nematode growth media.
Flip each plate and mark the center.
Using a microscope, introduce Caenorhabditis elegans worms to the centrally-marked spot.
Then, introduce age-matched worms onto the remaining plates.
These worms express either the wild-type or mutant forms of a human RNA-binding protein, which forms toxic aggregates in the neuronal cytoplasm, and leads to neuronal death.
This reduces the worms' ability to control muscle movements, resulting in impaired motor function.
Allow the worms to crawl for a set period.
Using a microscope, mark each worm's final location.
Measure the radial distance traveled by the worms from the central spot to their final positions.
Compared to controls, worms expressing lower levels of the wild-type protein show mild motor impairment, those with higher levels of the wild-type protein show moderate impairment, and those with the mutant protein display severe impairment.
Flip the NGM assay plates upside down, and label the bottom with an identifier for the C. elegans strains to be assayed. Make a small dot with the marker in the center of the upside down plate. While working with a dissecting microscope, transfer worms to the center of the assay plate, and set a timer for 30 minutes. Put the lid back on the plate, and set it aside. Continue transferring worms until all strains are on the designated assay plates.
After 30 minutes, begin scoring the first plate by removing the lid and placing the plate face down under the dissecting microscope. Adjust the microscope focus until all worms are visible through the agar. Using a different colored felt tip pen from the center point, put a small dot at the location of each worm.
Check the edge of the plate, as some worms may end up there. Also, count and record how many worms did not move from the center point. Measure the distance from the center point to the final location markings for each worm, and record the distance using a ruler. The first worm point is marked with a dash. Record length data for each dot consecutively by rotating the plate in a clockwise fashion.
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