Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans

Abstract

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

Figure 1. Optogenetic analysis of muscle contraction and relaxation kinetics in C. elegans.

(a) Schematic diagram for the behavioral phenotypic analysis enabled by optogenetics, microfluidics, and image processing technologies. (b) A microfluidic device having sixteen parallel microchannels for simultaneous illumination and analysis of multiple animals trapped by two pneumatically-controlled microvalves (red). Scale bar = 500 μm. (c) Microscopic and segmented images of a wild-type animal trapped in the microchannel with (top) and without (bottom) the illumination of blue light. Scale bar = 100 μm. (d) Temporal change in the body size of the wild type animals that was calculated from the segmented images and normalized by average value in the first 5 s. The light was turned on for 15 s at the 5-s time point. Data represent mean ± s.e.m. n = 88.

Figure 2. Quantitative analysis of the dynamic curves obtained from the optogenetic muscle contraction and relaxation assays in microfluidic devices.

Graphs showing the optogenetic muscle contraction and relaxation processes of (a) unc-22(e105) and (b) unc-22(e66) mutants in comparison with that of the wild-type animals. The light was turned on for 15 s at the 5-s time point. Data represent mean ± s.e.m. All the plots obtained from the experiments with sixteen mutant strains were devided into two parts – contraction (0–15 s) and relaxation (15–40 s) processes–and fitted with one-phase decay and association models, respectively, to extract four quantitative parameters to describe the dynamic curves: (c) rate constant for contraction; (d) rate constant for relaxation; (e) plateau after contraction; and (f) relative body area at steady state. n ≥ 40. ^*P < 0.01; ^**P < 0.001; ^***P < 0.0001.

Figure 3. Quantitative analysis of the minimum radius of curvature at the full contraction of body wall muscles in C. elegans sarcomere mutants on plates.

(a) Representative images showing the body postures of the wild-type, unc-96(sf18), and unc-78(gk27)) without (top) and with (bottom) the illlumination of blue light to induce the full contraction of their body wall muscles. Scale bar = 100 μm. (b) The minimum radius of curvature of the wild-type and sixteen sarcomere mutant strains at full muscle contraction and normalized by the length of the animals. n ≥ 25. ^*P < 0.01; ^**P < 0.001; ^***P < 0.0001.

Figure 4. Swimming locomotion analysis of C. elegans sarcomere mutants.

(a) Schematic diagram of swimming locomotion assays. The nematodes were put into M9 buffer solution and their swimming locomotion was recorded. Three quantitative parameters were extracted from the threshold-segmented images: (b) beating frequency, and (c) doral and (d) ventral bending amplitudes normalized by the length of the animals. n ≥ 20. ^*P < 0.01; ^**P < 0.001; ^***P < 0.0001.

Figure 5. Crawling locomotion analysis of of C. elegans sarcomere mutants.

(a) Schematic diagram of crawling locomotion assays. The nematodes were put on fresh NGM plates solution and their head was gently touched by a platinum wire to induce the backward crawling locomotion. Three quantitative parameters were extracted from the threshold-segmented images: (b) frequency, (c) wavelength, and (d) amplitude of the crawling waves normalized by the length of the animals. n ≥ 20. ^*P < 0.01; ^**P < 0.001; ^***P < 0.0001.

Figure 6. Standard hierarchical analysis based on behavioral phenotypes of C. elegans sarcomere mutants.

Clustering results of the wild-type and sixteen sarcomere mutant strains based on the Z-score of the mean values of the phenotypic profiles obtained from (a) the optogenetic muscle assays, (b) the conventional swimming and crawling locomotion assays, and (c) both assays. Only the phenotypic analysis enabled by the optogenetic muscle assays provides reliable functional relationships among the sarcomere mutants.