Cofilin Loss in Drosophila Muscles Contributes to Muscle Weakness through Defective Sarcomerogenesis during Muscle Growth

Figures

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  Figure 1.. Muscle-Specific Knockdown of DmCFL Affects Muscle Structure and Function during Muscle Growth (A) Sarcomere organization in a 3rd-instar Drosophila larval muscle (Z disc [α-Zasp, green] and F-actin [phalloidin, magenta]) (i and ii). The blue line indicates one sarcomere in (ii). (iii) Schematic depicting a single sarcomere with its structural components: Z discs, thin filaments, and thick filaments. Drosophila proteins analyzed in this study and their location within the sarcomere are marked in corresponding colors. (B–D) Quantification of sarcomere number (B), muscle length (C), and average sarcomere length (D) in LT1 (magenta) and VL1 (green) muscles at each larval instar. One muscle hemisegment, indicating the position of both muscles is shown in (B). n = 15 muscles from ≥5 control RNAi larva/instar. (E) Z disc pattern (Zasp-GFP) at the different larval instar stages. (F) Linear scaling relationship (line) between VL muscle cell length and sarcomere number across larval stages. Pearson’s correlation coefficient (r) and R2 are indicated. (G) Crawling velocities of early 3rd-instar larvae expressing Mhc-Gal4 and the indicated RNAi. n = 15 larvae/genotype. p value by unpaired t…

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  Figure 2.. DmCFL Knockdown Results in 3 Classes of Muscle Phenotypes. Larval muscles at the 3rd-instar stage expressing either control RNAi or DmCFL RNAi (1) driven with Mhc-Gal4.. (A) Lateral view of a single larval hemisegment. Muscles are stained with phalloidin (F-actin).. (B) Classification of muscle phenotypes using VL3 muscles, labeled with phalloidin (F-actin, magenta), α-Zasp (Zasp, green), and Hoechst (nuclei, blue). n = 200 muscles from 8 larvae/genotype.. (C–G) Organization of sarcomeric proteins in control and class 3 muscle cells with severe sarcomere disorganization and protein aggregates (arrows).. (C–E) Protein aggregates in class 3 VL3 muscles contain F-actin (phalloidin) and the Z disc proteins Zasp (C, magenta), α-actinin (D, green) and kettin (E, green). The arrows indicate protein aggregates. The yellow box in (D, i) denotes the region magnified in subsequent panels showing single z slices (ii–iv).. (F and G) TEMs of larval muscles at low (F) and high (G) magnification. The control muscles show organized sarcomeres (indicated by red line, with 2 highlighted in F, 1 in G) and Z discs (red arrows). DmCFL-knockdown muscles have aberrant protein aggregates in the cell…

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  Figure 3.. DmCFL Knockdown Results in Progressive Loss of Muscle Structure and Function. (A) Sarcomere pattern (Zasp-GFP) in larval muscles, from late embryogenesis (stage 17) through the different larval instars. Yellow arrows, sarcomeric structure defects.. (B) Crawling velocities of larvae at different larval stages. p values by multiple t tests. **p < 0.02 and ****p < 0.0001. n = 15 larvae/genotype.. (C) Percentage of different muscle classes at early 3rd-instar stage. Control RNAi (100.0% ± 0% wild-type pattern) and DmCFL RNAi (1) (class 1: 65.87% ± 3.76%, class 2: 6.63% ± 0.07%, class 3: 27.49% ± 3.69%). p values by 2-way ANOVA (****p < 0.0001). n = 800 muscles/genotype, from ≥8 larvae.. (D) Crawling velocities in 3rd-instar larvae. The percentage of muscle classes is shown for 3 individual larvae. p values by unpaired t test. ****p < 0.0001, n = 15 larvae, n = 125 muscles/larva.. (E) Two individual larvae imaged over 4 consecutive days. Muscle cells/sarcomeres are visualized with Tmod-GFP (grayscale). The red arrows in control and the yellow arrows in DmCFL RNAi (1) show theprogression of a single muscle over time.. (F) Percentage of muscle classes in DmCFL-knockdown larvae at each…

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  Figure 4.. Actin Aggregates in DmCFL-Knockdown Muscles Recruit Sarcomeric Tropomodulin and Troponin. Analysis of Tmod, TnT (green), and F-actin (phalloidin, magenta) protein localization in VL3 muscles in 3rd-instar larvae expressing Mhc-Gal4 to drive control or DmCFL RNAi (1). In controls, Tmod (A, C, and D) localizes to the M-line and TnT (B, E, and F) localizes to the pointed ends of actin filaments. In class 2 muscles, Tmod and TnT localize to sarcomeres and are enriched at muscle cell poles (magenta arrows), similar to actin. The dotted red and yellow boxes in (A) and (B) are the regions magnified in (C) and (E) and quantified in (D) and (F). The dotted pink lines (B) demarcate the muscle ends. (D and F) Quantification of Tmod and TnT levels at the center of the muscle cells in pixel intensity per square micron. p values by unpaired t test (****p < 0.0001). N ≥ 10 muscles from ≥5 different larvae. Error bars: mean ± SEM (D and F). Scale bars: 25 μm (A–C and E). See also Figures S3 and S4.

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  Figure 5.. DmCFL Knockdown Affects Sarcomeric Actin Filament Addition at Growing Muscle Ends. (A) Diagram of 1 hemisegment of the larval musculature, with VL muscles 1–4 highlighted in different colors.. (B and C) Quantification of the number of Z discs (B) and sarcomere length (C), using Zasp-GFP, in VL muscles shown in (A) of the indicated genotypes. p values calculated by unpaired t test. Error bars: mean ± SEM. N ≥ 12 muscles from ≥5 larvae.. (D) VL muscles stained with phalloidin for F-actin (grayscale). In controls, adjacent VL muscles have the same length and number of sarcomeric F-actin bands (38 each). In DmCFL-knockdown muscles of the same length, a class 1 muscle contains 39 F-actin bands, while a class 2 muscle contains only 35 F-actin bands. The dotted lines demarcate the muscle ends.. (E) Quantification of sarcomeric F-actin bands in VL muscles, as indicated in (D). N ≥ 12 muscles from ≥5 larvae.. (F) Plot of muscle cell length (mm) versus the number of sarcomeric F-actin bands in VL muscles. Pearson’s correlation coefficient (r) and R2 values and best-fit lines were calculated for each muscle cell class.. (G) VL muscles stained for F-actin (magenta) and Zasp (green). (Left)…

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  Figure 6.. Modulating Proteasome Activity Affects the Progression of Structural and Functional Changes in DmCFL-Knockdown Muscles. (A) Quantification of Zasp-GFP FRAP in control and class 3 muscle cells in 3rd-instar larvae. n = 9 muscles/genotype.. (B) Mobile fraction analysis of Zasp-GFP in (A). p values by unpaired t test (p = 0.685, ns).. (C) Western blot of mono- and poly-ubiquitinated protein abundance in 3rd-instar larval carcasses. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), loading control. Quantification in (D).. (D) Quantitative measurement of (C). Ub levels normalized to GAPDH. n = 20 carcasses. p values by unpaired t test (***p < 0.0005).. (E) VL3 muscles from 3rd-instar larvae labeled for mono-and poly-ubiquitin (α-FK2, green), F-actin (phalloidin, magenta), and nuclei (Hoechst, blue). n = 8 larvae/ genotype. FK2 levels quantified in (G). Dotted boxes magnified in (F).. (F) High magnification of muscles as shown in (E). In class 3 muscles, FK2 co-localizes with sarcomeric protein aggregates (yellow arrows).. (G) Quantification of α-FK2 labeling (pixel intensity values/μm2) in whole VL muscles, as in (E). p values by unpaired t test (****p < 0.0001). N ≥ 13 muscles from…

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  Figure 7.. In Vivo and In Vitro Analyses Identify Phenotypic and Biochemical Consequences of a Specific Patient CFL2 Mutation. (A) Sequence alignment of the first 30 amino acids of the proteins encoded by specified genes. There is strong conservation between sequences (gray; entire protein sequences in Figure S7A). Val7 conserved in all 5 proteins. Val7Met (V7M) mutation in HsCFL2 seen in NM patients.. (B) Comparison of nucleotide exchange rate on 2 μM ADP-actin monomers in the presence of different concentrations of human CFL2WT and CFL2V7M.. (C) Analysis of actin filament-severing activity. Each data point is the number of cumulative severing events per micron of F-actin at that time point, averaged from 3 independent experiments (n = 20 actin filaments each).. (D) Representative time-lapse images from TIRF microscopy experiments in which tethered OG (Oregon Green)-labeled actin filaments were preassembled, and then 150 nM CFL2WT or CFL2V7M was flowed in. The severing events (yellow arrowheads) are quantified in Figure S7B. Bar, 5 μm.. (E) Crawling velocities of late 3rd-instar larvae with the indicated genotypes. p values by ordinary 1-way ANOVA (**p < 0.005).. (F) Percentage of…