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. 2018 Jul 21;75(23):4341–4356. doi: 10.1007/s00018-018-2875-z

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© The Author(s) 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

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Fig. 7 — Evaluation of muscle performance and NMJ morphology in Mef2 > Hsp67BcWT-Venus, Mef2 > Hsp67BcR126E-Venus and Mef2 > Hsp67BcR126N-Venus third instar larvae. a Muscle crawling test shows reduced motility of Mef2 > Hsp67BcR126E-Venus larvae (red bars) in comparison with control Mef2 > Venus larvae (black bars), Mef2 > Hsp67BcWT-Venus larvae (gray bars), and Mef2 > Hsp67BcR126N-Venus larvae (green bars). Error bars indicate SEM (standard error of mean). Tests were repeated 3 times for each individual, n = 15 flies per genotype, *P < 0.05 (Student’s t test/ANOVA). b Muscle contractility test shows affected contractility of Mef2 > Hsp67BcR126E-Venus larvae (red bars) in comparison with control Mef2 > Venus larvae (black bars), Mef2 > Hsp67BcWT-Venus larvae (gray bars), and Mef2 > Hsp67BcR126N-Venus larvae (green bars). The fiber contractility index (CI) was calculated from the following formula: CI = (size of relaxed fibers − size of contracted fibers)/size of relaxed fibers. Error bars indicate SEM, n = 15 flies per genotype; *P < 0.05 (Student’s t test/ANOVA). c Muscle staining with MitoTracker Red CMXRos (Thermo Fisher), whose accumulation is dependent upon membrane potential. We observed a reduction of the mitochondrial signal, indicating an abrupt respiratory function in the case of Mef2 > Hsp67BcR126E-Venus larvae compared to Mef2 > Hsp67BcWT-Venus or Mef2 > Hsp67BcR126N-Venus larvae. An asterisk indicates a region containing reduction of the mitochondrial signal. Magnified longitudinal optical sections of ventral longitudinal VL4. d NMJ (neuromuscular junction) formation is affected in Mef2 > Hsp67BcR126E-Venus and Mef2 > Hsp67BcR126N-Venus larvae, which is reflected in the abnormal number of boutons. The muscle-target expression of Hsp67BcR126E-Venus significantly reduces the number of boutons formed during development (indicated by four asterisks), whereas the presence of Hsp67BcR126N-Venus affects it to a lesser extent, causing an increase of the number of boutons (indicated by one asterisk). Error bars indicate SEM, n = 30 NMJs per genotype; *P < 0.05, ****P < 0.00001 (Student’s t test/ANOVA). e The muscle-targeted expression of Hsp67BcR126E-Venus and Hsp67BcR126N-Venus does not affect bouton diameter. Error bars indicate SEM, n = 30 NMJs per genotype; *P < 0.05 (Student’s t test/ANOVA). f The muscle-targeted expression of Hsp67BcR126E-Venus and Hsp67BcR126N-Venus does not affect the number of branches per NMJ. Error bars indicate SEM, n = 30 NMJs per genotype; *P < 0.05 (Student’s t test/ANOVA). g Magnified longitudinal optical sections of lateral longitudinal LL1 surface area from Mef2 > Hsp67BcWT-Venus, Mef2 > Hsp67BcR126E-Venus or Mef2 > Hsp67BcR126N-Venus larvae. 3D reconstruction of confocal scans through synaptic boutons within the NMJ (Olympus FV1000 software). Anti-DLG (DSHB)