Phylogenetic origin of dRyR, its body wall muscle associated expression and role in locomotion.

(A) Evolutionary analysis by the maximum likelihood method of ryanodine receptor genes (RYR) from different taxa. The tree with the highest log likelihood (−95718.65) is shown. The percentage of trees in which the associated taxa clustered is shown next to the branches. (B) A wide view (upper panels) and a zoomed view (lower panels) of ventral VL3 larval muscle stained for dRyR (green) and Dlg (red) that labels T-tubules. (C) A high-magnification view showing dRyR dots at the interface of T-tubules (arrows). (D) Scheme presenting subcellular location of dRyR receptor at the sarcoplasmic reticulum (SR) membrane in a close vicinity of T-tubules. The dotted line refers to the optical level of confocal view in (C). (E-G) Larval muscle targeted dRyR knockdown (C57>dRyRRNAi) leads to a marked decline in muscle performance compared to control (C57>mCherryRNAi). Three muscle performance tests were applied: (E) righting test, (F) motility test, and (G) locomotor test. Overexpression of dRyR in larval muscle (C57>dRyR) impacts muscle performance measured by the locomotor and motility tests (F, G). Scale bar: 20 μm. All statistical analyses were performed using Prism. The one-way ANOVA test was used for comparisons of datasets. Bar plot represent the mean and the standard deviation. On the figures, statistical comparisons of sample vs control are indicated as ****: P ≤ 0.0001; ***: P ≤ 0.001; **: P ≤ 0.01; *: P ≤ 0.05; ns > 0.05.

dRyR transcript expression in larval muscles.

(A) Exon/intron organization of dRyR isoforms. FISH-HCR probes and targeted alternative exons are indicated by boxes with a color code. (B-E) Confocal views of dRyR transcript patterns in VL3 larval muscles revealed with FISH-HCR probes (white). Identities of the FISH-HCR probes and targeted dRyR isoforms are indicated above panels. Note highly similar subcellular distribution of different dRyR transcripts. (D’) Exon11 FISH-HCR (white) with nuclear DAPI staining (blue). (E’) Exon23 FISH-HCR (white) with phalloidin staining (red) to reveal sarcomeric pattern. Scale bar: 20 μm.

dRyR is expressed in the heart tube and is required for correct heartbeat.

(A-A”‘) Adult heart tube labeled for dRyR (green) and actin (red). (A’, A’’’) Zoomed views revealing dRyR expression in circular fibers. (B,D) M-modes of control Hand/+ (B) and Hand>dRyR RNAi (D) contexts showing a slow heart rate induced by dRyR attenuation. Compared with control (C), circular fibers in Hand>dRyR RNAi (E) context showed a fuzzy pattern suggesting an affected sarcomeric organisation. (F-M) Heartbeat variables in cardiac dRyR knockdown (Hand>dRyR RNAi) and cardiac dRyR overexpression contexts (Hand>dRyR). Scale bar: 50 μm in A, A’’; 10 μm in A’, A’’’, C, E. All statistical analyses were performed using Prism. The one-way ANOVA test was used for comparisons of datasets. Bar plot represent the mean and the standard deviation. On the figures, statistical comparisons of sample vs control are indicated as ****: P ≤ 0.0001 *: P ≤ 0.05; ns > 0.05.

Muscle targeted dRyR loss and gain of function impacts body size and structural muscle properties.

(A-D) General views of 3rd instar larva in control (A, C), muscle-targeted dRyR attenuation (B) or overexpression (D). (E-H) Representative VL3 and VL4 ventral longitudinal muscle views from age-matched 3rd instar larvae in control (E, G), C57>dRyR RNAi (F) and C57>dRyR (H) contexts. Muscle fibers and nuclei were revealed with phalloidin (red) and DAPI (blue), respectively. (I-L) Zoomed views of VL3 muscles of control (I, K), C57>dRyR RNAi (J) and C57>dRyR (L) larvae triple stained for phalloidin (red), DAPI (blue) and Kettin/D-Titin (green). (M-P) Z band profiles (Kettin signal intensity plot) from zoomed views of VL3 muscles presented in (I-L). (Q)Statistical representation of 3rd instar larva length. (R-T) Statistical representation of VL3 muscle characteristics: (R)VL3 muscle length; (S) number of nuclei and (T) sarcomere size. Scale bar: 1 mm in A-C; 50 μm in D-F; 20 μm in G-I. Bar plots represent the mean and the standard deviation. All statistical analyses were performed using Prism. The one-way ANOVA test was used for comparisons of datasets. Bar plot represent the mean and the standard deviation. On the figures, statistical comparisons of sample vs control are indicated as ****: P ≤ 0.0001; ***: P ≤ 0.001; **: P ≤ 0.01; *: P ≤ 0.05; ns > 0.05.

dRyR is required for correct mitochondria pattern.

(A-D) Zoomed views of a 3rd instar VL3 muscle in control (A, C), muscle-targeted dRyR attenuation (B) or overexpression (D) stained for ATP5 to reveal mitochondria pattern. (E) Statistical representation of ATP5 staining area in control, C57>dRyR RNAi and C57>dRyR contexts. “% of total” means the percentage of the measured muscle area that is positive for ATP5a staining. Scale bar: 20 μm. All statistical analyses were performed using Prism. The one-way ANOVA test was used for comparisons of datasets. Bar plot represent the mean and the standard deviation. On the figures, statistical comparisons of sample vs control are indicated as; ***: P ≤ 0.001.

Developmental dRyR protein pattern in embryos.

(A-A’) lateral view of a stage 12 embryo. dRyR (green) could be detected in the somatic and visceral muscle precursors (arrows in A) also revealed by Actin (red) (A’). (B-C’) dorso-lateral views of stage 14 (B,B’) and stage 16 (C,C’) embryos. dRyR accumulates in body wall muscle precursors (arrows in B and C) and in visceral muscle of the midgut (arrowhead in B) and in the dorsally aligned cardioblasts (double-head arrow in C). (D,D’) Subcellular dRyR pattern in ventral muscle precursors at embryonic stage 16. Note granular cytoplasmic distribution of dRyR. Scale bar: 50 μm.

Embryonic expression of dRyR transcript isoforms.

(A) Exon/intron organization of dRyR isoforms. Blue, green, brown and red boxes indicate alternative exons 10, 11, 22 and 23 (FISH-HCR probes). (B-E) Representative views of stage 13 and stage 16 embryos stained for four FISH-HCR probes (white/green) and actin (red) to reveal somatic muscles. Note that Ex10 and Ex22 probes consistently labeled dRyR transcript isoforms listed at the top of panels that accumulate in the developing muscle precursors. No or only faint dRyR expression was detected with Ex11 and Ex23 probes. Scale bar: 50 μm.

dRyR is required for correct embryonic muscle development.

(A, B) ventro-lateral views of stage 16 embryos stained for actin to reveal embryonic muscle pattern in wild-type (A) and in homozygous dRyR16 mutant embryo (B). Note a wide range of developmental muscle defects that could be observed in dRyR loss-of-function context. Asterisks in B pinpoint muscle fiber loss, arrowheads indicate the myofibers that failed to extend and remained as myospheres and a series of arrows point to supernumerary lateral transverse myofibers (6 instead of 4). (C-E) Effects of lateral transverse (LT) muscle-targeted attenuation (D) and overexpression (E) of dRyR. LT muscles were revealed by targeted expression of LifeActinGFP (LA) transgene using LT-specific Lms-GAL4 driver. (C) Four LT muscles (arrows) are seen in a control Lms>LA;LacZ context. (D) dRyRRNAi attenuation led to misshaped thin LTs (arrowheads) – major phenotype and to an occasional LT muscle split phenotype (6 LTs indicated by arrows). (E) LT targeted overexpression of dRyR resulted mainly in LT muscle splitting (arrows). (F) Cam attenuation induced mostly LT muscle splitting (arrows) while (G) SERCA RNAi knockdown lead to affected myofiber growth with thin LT muscle phenotype (arrowheads). (H) Statistical representation of LT muscle phenotypes in dRyR mutants and LT targeted dRyR knockdown, gain-of-function and Cam and SERCA attenuation contexts. The statistical analyses were performed using Prism - contingency test; 50-60 segments/genotype. Scale bar: 10 μm.

Live imaging of developing LT muscles and GCAMP-revealed calcium levels in control and dRyR loss-of-function contexts.

Time-lapse images of developing LT muscles revealed by LifeActin-GFP (green) and myonuclei revealed by DsRed NLS (red) in (A) control Lms>LA; DsRed NLS; LacZ and (B) Lms>LA; DsRed NLS; dRyR RNAi embryos. (B) Arrows point to a growth-defective LT3 muscle and asterisks show the area where it is missing within the segment. Note a reduced myonuclei signal in dRyR RNAi context compared to control (red staining) indicating fewer myonuclei in LTs after dRyR attenuation. (C-D) Representative lateral views of LT muscles from late-stage 15 embryos stained with anti-GFP antibody to reveal cytoplasmic calcium detectable on GCaMP reporter binding. Calcium-dependent GCaMP signal was at a high level in control LTs (C) and comparatively weaker in dRyR16 mutant LTs (D). Scale bar 10 μm.

Schematic comparison of amino acid sequence of human RYR and Drosophila dRyR proteins.

(A) Schematic representation of alignment of human RYR2 and Drosophila dRyR proteins with focus on Ins, MIR, RIH, SPRY, RYR, EF-hand and ITD protein domains. Percentage of identity for each domain is indicated on the dRyR protein scheme. (B) Schematic representation of pathogenic mutations in human RYR1 and RYR2. Black vertical lines denote positions of mutations clustered into three hot spots. For the color code, see identity heat map. Position of variant mutation of unknown significance (VUS) (p.M4881I) is indicated with respect to distribution of pathogenic RYR mutations and dRyR conservation heat map. Below an extraction from the dRyR and RYR1 sequence alignment encompassing the VUS mutation. Mutated amino acid is in blue.

Modeling human RYR1 variant mutation in Drosophila.

(A,B) Age-matched 3rd instar wild-type (A) and RyR1 p.Met4881Ile mutant (B) larvae. Note a reduced size of larvae carrying RYR1 variant mutation. (C,D) Representative views of ventral longitudinal (VL) muscles in wild type (C) and RYR1 variant mutant larvae (D). Note slightly reduced VL3 muscle length and reduced number of myonuclei in mutant condition. (E-H) Z band profile revealing reduction of sarcomere length in RYR1 variant context (F,H)) compared to control (E,G). Kettin is in green, Phalloidin in red and DAPI in blue. (I-L) Statistical representation of larva length (I) and structural muscle variables (J-L) in wild-type and p.Met4881Ile RYR1 variant mutation contexts. (M-O) Statistical assessment of functional larval muscle performance using righting test (M), motility test (N) and locomotor test (O) in wild-type and RYR1 mutant conditions. Scale bar: 1 mm in A-B; 50 mm in C-D; 20 mm in E-F. All statistical analyses were performed using Prism. The t test was used to compare control to variant context. Bar plot represent the mean and the standard deviation. On the figures, statistical comparisons of sample vs control are indicated as ****: P ≤ 0.0001; *: P ≤ 0.05.