Dissecting the Component Parts of the Excitation-Contraction Coupling Machinery in Drosophila melanogaster.
FASEB journal : official publication of the Federation of American Societies for Experimental Biology(2022)
Abstract
The Drosophila neuromuscular system is widely used to characterize synaptic development and function. However, little is known about how specific synaptic and muscular alterations effect neuromuscular transduction and muscle contractility that ultimately dictate behavioral output. Here a system developed to characterize excitation-contraction coupling at Drosophila larval neuromuscular junctions (NMJs) was exploited to examining how specific neuronal, synaptic, and muscle manipulations disrupt muscle contractility and locomotion. Increases in motor neuron stimulation frequency and duration increased muscle force in a predictable fashion, but show considerable plasticity between 5-40 Hz and saturating above 50 Hz. Fictive contraction recordings from larvae with CNS intact revealed motor neuron burst frequencies of 20-30 Hz, consistent with the endogenous machinery operating within the range of contractility of greatest plasticity. Experimental parameters such as saline composition (e.g. calcium and magnesium) temperature, larval size and orientation, were explored to examine their impacts on muscle force production. Subsequently, genetic and pharmacological approaches were taken to dissect several molecular pathways which contribute to muscle contraction and modulation of force. First, well-established calcium regulation pathways were manipulated via application of ryanodine, caffeine, thapsigargin, and felodipine to demonstrate the role of the ryanodine receptors, SERCA pump, and postsynaptic calcium channels in modulating the magnitude and time-course of muscle contractions. Presynaptic calcium channels and ryanodine receptor expression were genetically altered to further examine their role in muscle force production. Next using both null and knock-down (Gal4/UAS system) approaches combined with tissue-specific expression of drivers, screens for motor, synaptic, and muscle proteins contributing to excitation-contraction coupling (ECC) were conducted. Altering the expression of the motor proteins kinesin, dynein or dynactin all resulted in a significant reduction in muscle force production. Critical synaptic proteins like synaptotagmin, complexin, cacophony, gbb (homolog of BMP), significantly impacted ECC in Drosophila larvae. Several critical muscle-specific proteins alter the magnitude and time-course of muscle contractions when altering their expression in a sub-set of muscle fibers. Lastly a screen for modulators of muscle contractility led to identification and characterization of the molecular and cellular pathway by which the FMRFa peptide, TPAEDFMRFa, increases muscle performance. These findings indicate Drosophila NMJs provide a robust system to correlate synaptic dysfunction, regulation, and modulation, to alterations in excitation-contraction coupling.
MoreTranslated text
Key words
drosophila melanogaster
AI Read Science
Must-Reading Tree
Example
Generate MRT to find the research sequence of this paper
Chat Paper
Summary is being generated by the instructions you defined