EMC1 Is Required for the Sarcoplasmic Reticulum and Mitochondrial Functions in the Drosophila Muscle

Abstract

EMC1 is part of the endoplasmic reticulum (ER) membrane protein complex, whose functions include the insertion of transmembrane proteins into the ER membrane, ER-mitochondria contact, and lipid exchange. Here, we show that the Drosophila melanogaster EMC1 gene is expressed in the somatic musculature and the protein localizes to the sarcoplasmic reticulum (SR) network. Muscle-specific EMC1 RNAi led to severe motility defects and partial late pupae/early adulthood lethality, phenotypes that are rescued by co-expression with an EMC1 transgene. Motility impairment in EMC1-depleted flies was associated with aberrations in muscle morphology in embryos, larvae, and adults, including tortuous and misaligned fibers with reduced size and weakness. They were also associated with an altered SR network, cytosolic calcium overload, and mitochondrial dysfunction and dysmorphology that impaired membrane potential and oxidative phosphorylation capacity. Genes coding for ER stress sensors, mitochondrial biogenesis/dynamics, and other EMC components showed altered expression and were mostly rescued by the EMC1 transgene expression. In conclusion, EMC1 is required for the SR network's mitochondrial integrity and influences underlying programs involved in the regulation of muscle mass and shape. We believe our data can contribute to the biology of human diseases caused by EMC1 mutations.

Keywords: endoplasmic reticulum membrane protein complex; mitochondria; musculature.

Figure 1

RNAi-mediated silencing of EMC1 in the Drosophila muscle leads to pupal lethality and severe motor disabilities in adults and larvae. (A) EMC1 transcript levels were calculated as described in the Section 2 for individuals of the indicated genotype, using either the VDRC RNAi line V8477 or the BDSC RNAi line 34581. Transcripts were normalized by the levels of EMC1 in the Mef2>+ control at the indicated developmental time. (B) Viability was calculated as the percentage of individuals of the indicated genotype that reached the next developmental stage. (C) The adult eclosion process is represented from frames of movies recorded for Mef2>+ control and Mef2>EMC1 RNAi V8477 individuals with various degrees of phenotype severity (#1, #2, and #3). The initial time represents the first adult movements still inside the puparium, until the animals leave the pupal case (WT and phenotype #1) or notably fail to do so (#2 and 3). See details in Videos S1A–C. (D) Locomotion of adult individuals of the indicated genotypes and ages was evaluated as the average (±S.D.) climbing abilities. *, **, and **** indicate, respectively, p < 0.05, p < 0.01, and p < 0.0001, according to Student’s t-tests applied between Mef2>+ controls and Mef2>EMC1 RNAi animals. Adult life span (E) and larval locomotion (F) analyses were performed as described in the Section 2 using Mef2>+ controls and Mef2>EMC1 RNAi V8477 individuals. Individual larval crawling paths are represented in different colors in (F) (left panel).

Figure 2

EMC1 knockdown causes muscle deformations and reduced size. Immunofluorescent confocal microscopy images of representative musculatures of stage 16 embryos (A–D), third-instar larvae (E–H), and 2-day-old adults (I–K) of the indicated genotypes. Samples were treated with rhodamine-phalloidin and DAPI to stain F-actin fibers and nuclei, as described in the Section 2. Images in (B,D) are enlargements of (A,C), respectively. Arrows indicate the zones of fiber insertion with a strong F-actin staining in Mef2>EMC1 RNAi animals. (L–O) Quantification of data in (A–K) as averages ± S.D. *** and **** represent, respectively, p < 0.001 and p < 0.0001, according to Student´s t-tests.

Figure 3

EMC1 is required for maintenance of the sarcoplasmic reticulum network and cytosolic calcium homeostasis. Immunofluorescent confocal microscopy images (Z-stacks) of representative indirect flight muscles of Mef2>+ and Mef2>EMC1−RNAi adults stained with anti−EMC1 and anti-calreticulin; right panel, average (±S.D.) level of calreticulin relative fluorescence intensity. (A), and of Mef2>mitoGFP and Mef2>mitoGFP;EMC1−RNAi adults stained with anti-EMC1, anti-calreticulin, rhodamine-phalloidin, and DAPI (B). (C–E) Transcript levels of the indicated genes were calculated as described in the Section 2 for 2-day-old adult individuals of the indicated genotypes, normalized by the levels in the Mef2>+ control. (F) Left panel, representative real-time Fluo−4 AM measurements of calcium mobilization in the thoracic muscle of 2-day-old adults of the indicated genotypes; right panel, average (±S.D.) level of intracellular calcium at the timepoint of signal saturation (indicated by a vertical dashed line on the left panel). ΔRFU, change in relative fluorescence units. * and ** represent, respectively, p < 0.05 and p < 0.01, according to Student´s t-tests applied between the Mef2>+ controls and Mef2>EMC1−RNAi, Mef2>EMC1−OE or Mef2>EMC1−OE;EMC1-RNAi animals. For RT−qPCR experiments, five thoraces were used for each biological replicate, with a total of three replicates utilized per phenotype and one hundred thoraces from 2-day-old flies were divided into five groups for the calcium experiments.

Figure 4

EMC1 silencing results in severely altered mitochondrial morphology, lower membrane potential, and lower oxidative phosphorylation in adult indirect flight muscles. Immunofluorescent confocal microscopy images (A) and transmission electron microscopy of longitudinal sections (B) of representative thoracic indirect flight muscles of adult flies of the indicated genotype and age. Samples were stained with rhodamine-phalloidin in (A). (C) Quantification of intermyofibrillar mitochondria data shown in (A,B). (D) TMRE signal from isolated thoraces of 2-day-old adults was obtained through fluorescence reading using the FlexStation 3 Benchtop Multi-Mode Microplate Reader, indicating the genotype and showing relative mitochondrial membrane potential. RFU, relative fluorescence units. (E) Representative traces (left panels) and quantification (right panels, average ±S.D.) of oxygen consumption rates of 1- to 3-day-old adult thoraces. Dissected tissues were incubated in the Mir05 respiration buffer in the presence of pyruvate, malate, and glutamate (Leak (n) respiratory state), followed by addition of ADP (OXPHOS respiratory state), oligomycin (Leak (Omy) respiratory state), and finally antimycin A (Ant, non-mitochondrial respiration), as described in the Section 2. * and ** represent, respectively, p < 0.05 and p < 0.01, according to Student´s t-tests applied between the Mef2>+ controls and Mef2>EMC1-RNAi animals.