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.
Highlights
The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins
Quantitative study of muscle contraction and relaxation kinetics in C. elegans was enabled by optogenetically-induced activation of motor neurons that induce muscle contraction
Many of our mutants were chosen that have minimal known defects on nematode locomotion (unc-22(e105), mak-1, atn-1, scpl-1, dim-1, uig-1, lim-9). This criteria was used so that we might determine if optogenetic assays might reveal more subtle defects in muscle function
Summary
The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Krajacic et al quantified various biomechanical properties such as bending frequency and amplitude of swimming in synaptic mutants[17] These methods allow more nuanced phenotyping, low user bias, and potentially can extract more information; it is possible that assays that measure other attributes of muscle contraction and relaxation may expand the repertoire of descriptors of sarcomere functions. We employed a worm strain carrying unc-17p::ChR2(H134R)::YFP, which expresses the light-induced channelrhodopsin-2 (ChR2) in its cholinergic motor neurons, to control the contractile activity of the body wall muscle cells and the locomotion of the whole nematode, as first described by Liewald et al.[20]. Quantitative parameters, including rate constants for contraction and relaxation, could be extracted from these optogenetic experiments This was performed on wild type nematodes and loss of function mutants for 15 genes that encode proteins of the sarcomere. Our approach should provide a complement to existing behavioral assays for the analysis of C. elegans muscle mutants
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