C. elegans Muscle Area Measurements: A Standardized Method to Quantify Striated Muscle Morphology

- C. elegans have two types of muscles. The single sarcomere or non-striated muscles include those in the pharynx and vulva. The multiple sarcomere or obliquely striated muscles are rhomboid-shaped body wall muscles named for the regular striated pattern of their myosin fibers.

They are arranged in staggered pairs as four bundles oriented longitudinally in the four quadrants of the worm body viewed in cross section. Each cell has many equally spaced points of attachment to the hypodermis and cuticle. This arrangement helps to translate waves of muscle contractions into smooth locomotion.

To visualize these muscles, image immobilized worms with transgenically GFP- labeled myosin by fluorescent confocal microscopy. Use analysis software to measure the area of a striated muscle cell and any gaps in the fiber pattern of that cell. Gaps in muscle fiber organization could indicate disruption by fiber degradation or the accumulation of cellular debris. Calculate the ratio of the gap area to the total area to define the amount of disruption. In the example protocol, we will quantitatively assess body wall muscle morphology using Fiji.

- To measure muscle cell area, open the image in Fiji software and use Polygon Selection to carefully trace around a single oblique muscle cell. Adjust the line of the polygon at the end by dragging the anchor dot to improve tracing. Navigate to the Analyze tab at the top of the software and click Measure to calculate the selected area.

For muscle cells with a degenerated or missing region, trace the missing area with the polygon selection tool and click Measure again. If there are multiple gaps, trace each one separately. Calculate the ratio of the gap area to the area of the entire cell. A high ratio indicates a higher extent of muscle degeneration, and if there are no missing regions, the ratio is calculated as zero.