Figure 1.
Characterization of adult muscle.
The morphology, function and transcriptional activity of wild-type indirect flight muscles (IFM) was assayed at sequential time points during the first 30 days of fly life. (A) Simplified diagram showing the relaxed and contracted state of sarcomeres in a longitudinal section. The structural elements relevant to the work presented in this study have been included. Actin, or thin, filaments are shown in blue; Myosin, or thick, filaments are shown in red; Projectin is shown in green; the M-line is shown in yellow; and the Z-disc is shown in black. Note the slight shortening of the sarcomere in the contracted state, specifically in the H-zone and I-band as is characteristic of the IFM. (B) Simplified diagram of a transverse section of a sarcomere. As in (A), actin filaments are shown in blue and myosin filaments are shown in red. (C) Longitudinal Sections (LS) and (D) Transverse Sections of wild-type IFM sarcomeres show that the Z-disc, M-lines, actin and myosin filaments, and the myosin and actin lattice all exhibit consistent appearance over the first 30 days of fly life. (C: z, Z-disc. m, M-line; D: Larger circles are myosin, smaller dots are actin; Scale bars are in white and are 500 nm; see Figure 1A for schematic representations). (E) The climbing ability of flies was assayed using a negative geotaxis assay. Over the 30 day time course, the climbing ability of adult flies decreased in a linear fashion by 82%. N is 10 independent replicates each using 10 flies to assay climbing ability. (F) Key sarcomeric genes are continually transcribed in adult flies. RNA was extracted from whole flies and qPCR was used to assay for expression levels of bent, Mhc, and Act88F. GAPDH was used as the internal control for expression. Expression levels were normalized to the Day 0 timepoint. For all three genes, mRNA levels remained close, with slight variations, to the initial timepoint over the first 30 days of fly life. N≥3 for all qPCR time points, where each replicate was an independent RNA extraction using 10 flies.
Figure 2.
Sarcomeric actin undergoes turnover in adult muscles.
(A) Graphic representation of the experimental design for the pulse-chase experiments. Act88F::GFP or eGFP was expressed under the control of the TARGET system for 28 hours directly after eclosion (the pulse) and then shut off (the chase). (B, B′) Localization of eGFP (B) or Act88F::GFP (B′) when expressed throughout development and adulthood with the Mef2:GAL4 system (F-actin is counterstained with rhodamine-phalloidin). eGFP non-specifically labels the whole sarcomere while Act88F::GFP is specifically localized to the sarcomeric core. (C,D) A time series of representative images of IFMs from flies expressing eGFP (C) or Act88F::GFP (D) under the control of the TARGET system (F-actin counterstained with rhodamine-phalloidin). The locations of the Z-discs and M-lines are indicated by a ‘z’ or ‘m’ in both panels. (E) Quantification of fluorescent intensity at the Z-disc for eGFP during TARGET pulse-chase experiment. Intensity at the Z-disc increased during the 28 hr pulse phase but did not decline afterwards. Instead, intensity remained elevated. (F) Quantification of fluorescent intensity at the Z-disc for Act88F::GFP during TARGET pulse-chase experiment. Intensity at the Z-disc increased during the 28 hour pulse phase and then gradually declined. (G) The ratio of Z-disc to sarcomere body fluorescent intensity during the pulse-chase experiment shows that the increase in staining intensity is specific to the Z-disc for Act88F::GFP. Comparison of Z-disc to sarcomere body intensity ratios for eGFP (grey bars) and Act88F::GFP (black bars) expression shows that while eGFP localization is non-specific, Act88F::GFP is significantly enriched at the Z-disc. (H) Examples showing how Z-disc and sarcomere intensity was measured for both TARGET>eGFP and TARGET>Act88F::GFP myofibrils. Phalloidin counterstains were used to identify relevant areas. Red rectangles indicate the area quantified for Z-disc intensities. Irregular blue tetragons indicate the area quantified for sarcomere body intensities. For (E,F,G) N≥40 individual sarcomeres from ≥5 animals. For all panels, error bars indicate standard error; ns indicates not significant; * indicates a p-value<0.05, ** indicates a p-value<0.005, *** indicates a p-value<0.0005. TARGET>eGFP and TARGET>Act88F::GFP are abbreviated as T>eGFP and T>Act88F::GFP throughout.
Figure 3.
Results of the Mef2:GAL4 screen of the role cytoskeleton muscle function.
(A) Phenotypic breakdown of results from the Mef2:GAL4 screen. The Mef2:GAL4 screen expressed the RNAi constructs throughout both development and adulthood. By assaying or developmental lethality or climbing ability, we observed that 106 genes had no role in muscle development or maintenance (None, brown), 20 were Embryo Lethal, 68 were Pupal Lethal, and 44 caused a Climbing Defect. (B) Enrichment analysis of Gene Ontology (GO) terms by phenotype. Enrichment ratios were calculated by comparing frequency of a term in a specific phenotypic class to the frequency of the same term in the entire screened set. GO terms are listed on the left. An enrichment ratio of 0 indicates that a given term did not appear in the phenotypic group. An enrichment ratio of <1 indicates that the frequency for the term was reduced in the phenotypic class compared to the whole screened set. An enrichment ratio of 1 indicates that the frequency for the term was the same in the phenotypic class compared to the whole screened set. An enrichment ratio of >1 indicates that the frequency for the term was the enriched in the phenotypic class compared to the whole screened set.
Figure 4.
Results of the TARGET Screen for adult muscle maintenance.
(A) Phenotypic breakdown of results from the TARGET screen. In the TARGET screen, RNAi constructs were only expressed after flies had eclosed. We identified 46 genes that caused climbing defects in adults as assayed by a negative geotaxis assay. The remainder, 86 genes, had no identifiable climbing defect. (B) As in Figure 3, we performed a GO term enrichment analysis by phenotype. GO terms are listed on the left. Note the enrichment in sarcomere-associated terms as well as “integrin complex”. Enrichment ratios were calculated by comparing frequency of a term in a specific phenotypic class to the frequency of the same term in the entire screened set. An enrichment ratio of 0 indicates that a given term did not appear in the phenotypic group. An enrichment ratio of <1 indicates that the frequency for the term was reduced in the phenotypic class compared to the whole screened set. An enrichment ratio of 1 indicates that the frequency for the term was the same in the phenotypic class compared to the whole screened set. An enrichment ratio of >1 indicates that the frequency for the term was enriched in the phenotypic class compared to the whole screened set.
Figure 5.
Sample assay graphs from TARGET screen.
Severity of the climbing phenotypes identified in the TARGET screen were classified based on when flies lost the ability to climb. Loss of climbing ability between Day 0 and Day 9 were classified as ‘Severe’; between Day 12 and Day 21 were classified as ‘Intermediate’; between Day 24 and Day 30 were classified as ‘Weak’; and ‘None’ if climbing ability was not altered. Representative samples for each of the four classes were selected. (A-C) Examples of ‘Severe’ phenotypes. up RNAi (A), Mhc RNAi (B), and Act88F RNAi (C). In total, 5 genes had caused ‘Severe’ climbing defects. (D-F) Examples of ‘Intermediate’ phenotypes. bent RNAi (D), act79B RNAi (E) and rab5 RNAi (F). In total, 13 genes caused ‘Intermediate’ phenotypes. (G-I) Examples of ‘Weak’ phenotypes. EB1 RNAi (G), myo28B RNAi (H) and actr13E RNAi (I). In total, 28 genes caused Weak' phenotypes. J-L. Examples of ‘None’ phenotypes. shot RNAi (J), msps RNAi (K) and βTub60D RNAi (L). In total, 86 genes had no effect on adult climbing ability. For all graphs, blue lines are the control and red lines are the RNAi-mediated gene knockdown line. Error bars are standard error. For all lines, climbing ability was normalized to the first timepoint, Day 0, before RNAi construct expression was started.
Figure 6.
Genetic interaction network of TARGET screen hits.
Genetic and biochemical interactions of the 46 genes identified in the TARGET screen were mined from the Drosophila Interactions Database (www.droidb.org). Lines linking each gene indicate direct genetic or protein-based interactions. Reflexive loops indicate a direct genetic or protein-based interaction with itself. Genes were then organized into functional groups based on GO term analysis. Each gene in the network was colour-coded based on the severity of the climbing phenotype caused by RNAi-mediated gene knockdown (see Figure 5). Red indicates a “Severe” phenotype; orange indicates a “Intermediate” phenotype; and blue indicates a “Weak” phenotype. The gene rhea is coloured gray as it was not part of this screen but was previously identified, using the same approach, to be required for muscle maintenance. Note cluster of genes that caused “Severe” or “Intermediate” phenotypes in the ‘Sarcomeric’ group.
Figure 7.
Loss of bent, Mhc or Act88F does not disrupt sarcomeric architecture.
Using phalloidin and antibody labelling, IFM integrity was examined before and after bent, Mhc or Act88F RNAi expression. (A-C) qPCR analysis confirmed that expression of RNAi constructs targeting bent, Mhc or Act88F leads to substantial reduction in transcript levels for all three genes compared to control flies of equivalent age. For (A-C), 3 independent RNA extractions using 10 flies for each time-point were performed. GAPDH was used as an internal expression control. (D-G) Phalloidin stainings of sarcomeric F-actin before and after RNAi expression showed no defects in thin filament organization after 6 days of RNAi construct expression for all three genes. Z-discs are bright lines and M-lines are dark lines as indicated by the labels ‘z’ and ‘m’ in the TARGET>OR Day 0 image. (H-K) To examine the integrity of key sarcomeric structures, we used antibodies to label the Z-disc and the M-line. Z-discs were labelled with an α-actinin antibody (green) and M-lines were labelled with an Obscurin antibody (red). No disruption to the integrity of either structure or to sarcomeric actin was observed for any of the three genes. For all panels, scale bars are given below all images in black and are 5 µm; error bars indicate standard error; n.s. indicates not significant; * indicates a p-value<0.05, ** indicates a p-value<0.005, *** indicates a p-value<0.0005.
Figure 8.
Loss of bent, Mhc or Act88F does not disrupt sarcomeric ultrastructure.
We more closely examined the Z-disc and filament lattice integrity in bent, Mhc, and Act88F RNAi flies using transmission electron microscopy. The IFMs from bent, Mhc and Act88F knockdown flies were imaged before RNAi expression (Day 0) and again after 6 days. (A-D) Electron micrographs of Longitudinal Sections and Transverse Sections of IFM sarcomeres before and after RNAi expression showing no defects to Z-disc integrity or myosin filament lattice architecture. For LS: ‘z’ indicates Z-discs; for TS: larger circles are myosin, smaller dots are actin. For all panels, scale bars are overlayed in black and are 500 nm. See Figure 1A for schematic representations of longitudinal and transverse sections of IFMs.
Figure 9.
bent, Mhc and Act88F are required to maintain correct sarcomere length.
Sarcomere length was measured from M-line to M-line using the Obscurin antibody as this provided a more precise definition of sarcomere length compared to the Z-disc antibody. For all three genes, sarcomere length was measured before RNAi construct induction and after six days of RNAi construct expression. (A) Schematic showing how sarcomere length was measured. Length was measured from the centre of each M-line to the centre of the adjacent M-line in straight myofibrils. White lines marked by asterisks indicate the measured distance of two adjacent sarcomeres. Scale bar is in black, 5 µm. (B) Sarcomere length for control and bent RNAi flies. Loss of bent caused sarcomeres to shorten significantly compared to those of control flies which, by contrast, showed no difference over the same time course. (C) Sarcomere length for control and Mhc RNAi flies. Loss of Mhc causes sarcomeres to lengthen significantly after six days of RNAi construct expression. The length of sarcomeres in the control flies does not change. (D) Sarcomere length for control and Act88F RNAi flies. Loss of Act88F causes sarcomeres to lengthen significantly after six days of RNAi construct expression. The length of sarcomeres in the control flies does not change. Representative images of Obscurin stainings can be found in Figure 7H-K. For B-D error bars indicate standard deviation; ns indicates a p-value>0.05, *** indicates a p-value = <0.0005. For B-D, N≥30 sarcomeres from 5 animals at each time-point.
Figure 10.
Sarcomere length defects are due to disruption of thin filament length.
Distribution of actin across the sarcomere was used to determine sarcomere length, thin filament length and H-zone width. (A) Sample image of a single sarcomere labeled with phalloidin to show actin filaments. Black overlay lines indicate quantified parameters. (B) Sample profile of a sarcomere showing the normalized fluorescent intensities of pixels found along the overlay line labeled ‘Sarcomere’ in A. Measured parameters, “Sarcomere Length”, “Thin Filament Length” and “H-zone Width”, are indicated. (C-E) Quantification of Sarcomere Length, Thin Filament Length, and H-zone width respectively in control and bent RNAi construct-expressing flies. Sarcomere and thin filament length does not significantly change in the control flies, but significantly shortens in the bent RNAi construct-expressing flies over six days. (F-H) Quantification of Sarcomere Length, Thin Filament Length, and H-zone width respectively in control and Mhc RNAi construct-expressing flies. Sarcomere and thin filament does not significantly change in the control flies, but significantly increases in the Mhc RNAi construct-expressing flies over six days. (I-K) Quantification of Sarcomere Length, Thin Filament Length, and H-zone width respectively in control and Mhc RNAi construct-expressing flies. Sarcomere and thin filament length does not significantly change in the control flies, but significantly increases in the Mhc RNAi construct-expressing flies over six days. Representative images of phalloidin stainings can be found in Figure 7D-G For all panels, error bars indicate standard error; n.s. indicates a p-value>0.05, *** indicates a p-value<0.0005. For (C-K) N≥30 sarcomeres from 5 animals at each time-point.