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. 2020 Oct 6;11:573723. doi: 10.3389/fphys.2020.573723

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Copyright © 2020 Hsu, Linsley, Reid, Hume, Leflein and Kuwada.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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Knockdown of Dmca1D selectively in muscles reduced larval locomotion and muscle Ca²⁺ transients. (A) Anti-Dmca1D labeling of muscles of larvae with Dmca1D knocked down selectively in body-wall muscles (Mef2:GAL4 > UAS:Dmca1D^RNAi) (n = 55 stripes from 11 muscles) confirmed that Dmca1D was knocked down compared with control muscles (Mef2:GAL4 > UAS:Luciferase^RNAi) (n = 50 stripes from 10 muscles). One-tailed, unpaired t test. Fluorescence was measured in ROIs that outlined each stripe. Right images are a single focal plane of muscle 4 of a control and a Dmca1D^RNAi larvae. Arrows denote the striations of Dmca1D clusters. Scale bar, 3 μm. (B) Same as in (A) except the peak fluorescence of stripes was measured. Fluorescence of individual pixels along a longitudinal line that crossed multiple stripes of muscle fibers was measured from Mef2:GAL4 > UAS:Dmca1D^RNAi (n = 26 stripes from 8 muscles) and control muscles (Mef2:GAL4 > UAS:Luciferase^RNAi) (n = 29 stripes from 9 muscles). One tailed, unpaired t test. Right images are examples of fluorescence measurements along the longitudinal line of wt and control muscles. (C) Dmca1D^RNAi larvae in which Dmca1D was knocked down in body-wall muscles (Mef2:GAL4 > UAS:Dmca1D^RNAi) showed decreased locomotion compared with control Luciferase^RNAi larvae (Mef2:GAL4 > UAS:Luciferase^RNAi). Control n = 39, Dmca1D^RNAi n = 54. One-tailed Mann-Whitney test. (D) Example of Ca²⁺ transients from a muscle in a control larva (Mef2:GAL4 > UAS:GCaMP6f; UAS:mCD8tdTomato; UAS:Luciferase^RNAi) and a Dmca1D^RNAi larva (Mef2:GAL4 > UAS:GCaMP6f; UAS:mCD8tdTomato; UAS:Dmca1D^RNAi). (E) Expression of GCaMP6f by muscles of Dmca1D^RNAi larvae was comparable to that of control. Mann-Whitney test. (F) The peaks of Ca²⁺ transients in the muscles of Dmca1D^RNAi larvae were smaller compared with controls. One-tailed, Mann-Whitney test. (G) The number of Ca²⁺ transients over 5 min in Dmca1D^RNAi and control muscles were comparable. Unpaired t test. Data in E-G were from 13 muscles of 7 control larvae and from 11 muscles of 6 Dmca1D^RNAi larvae. **P < 0.01 and ****P < 0.0001.