Drosophila Nesprin-1 Isoforms Differentially Contribute to Muscle Function

Figures

• 

  Figure 1. Msp300 isoforms. (A) Schematic representation of Msp300 isoforms. Isoform diversity results from the use of different start and stop exons (respectively exon 1, 3, 18 and 21 for the start and exon 26, 29, 40 and 41 for the stop) but also from alternative splicing events. For simplicity, isoforms H, I, J and K, all starting at exon1 and containing the C-terminal KASH domain have been represented as a single transcript despite the use of few alternative exons within the coding sequence (top transcript). Similarly, isoforms D and L differ by one alternative splice event and have been represented by a single transcript (second top transcript). Isoform M is represented here although RNAseq tissue-specific analysis suggests that it is not expressed in larval muscle [27]. Exon structure is represented at scale (coding region in orange, non-coding in grey). RNAi amplicons are shown in red under the targeted exon. ex32[RNAi] was used in a previous work and positioned here for discussions sake. Protein features are positioned above the isoforms schemes: Calponin Homology domains (CH) are shown in blue, KASH domain in red, the region containing the Spectrin Repeats (SR) in dark grey and the…

• 

  Figure 2. Msp300 isoforms differentially contribute to larval locomotion. (A–D) Box plot representation of muscular Msp300[RNAi] efficiency and impact on non-targeted isoform groups by RT-qPCR analysis. The expression level of each isoform group was measured using primers in the exon targeted by the specific Msp300[RNAi] and one neighbouring exon (primer localisation shown in Figure S1). For each primer couple and RNAi condition, mRNA levels were normalised to control larvae to allow comparison between the different conditions. For a given RNAi, expression levels of all isoform groups were tested. Box plots of the RT-qPCR assessing the RNAi effect on its targeted exon are shown in red, box plots corresponding to the non-targeted isoform subsets are in black. (A) ex2[RNAi], (B) ex17[RNAi], (C) ex23[RNAi], (D) ex28[RNAi]. (E) Box plot representation of the global displacement of 48 h AEL old larvae following 2 min of wandering relative to larval size. For comparison purposes between the different RNAi lines, muscle expressing Msp300[RNAi] larvae locomotion is normalised to respective control larvae locomotion. For each condition, one representative experiment is shown (WT, n = 7, ex2[RNAi],…

• 

  Figure 3. Msp300 isoform depletion differentially alters nuclei positioning in the muscle. (A–C) Nuclei clustering quantification in fibres 4 (A), 6 (B) and 7 (C), comparing the effects of muscular expression of either lacZ[RNAi] or Msp300[RNAi] and showing the percentage of total nuclei found isolated or in groups of two to more than seven nuclei. Percentage of nuclei involved in clusters and size of the clusters vary with the RNAi and the muscle type used. Number of scored larvae and total number of counted nuclei are: lacZ[RNAi]: 9 larvae, 402/483/281nuclei (F4/F6/F7); ex2[RNAi]: 7 larvae, 244/326/188 nuclei; ex17[RNAi]: 6 larvae, 186/263/143 nuclei; ex23[RNAi]: 11 larvae, 357/416/259 nuclei; ex28[RNAi]: 9 larvae, 352/402/246 nuclei. (D–H) Immunostaining of muscles 6 and 7 expressing lacZ[RNAi] (D), ex2[RNAi] (E), ex17[RNAi] (F), ex23[RNAi] (G), ex28[RNAi] (H) showing representative nuclei clustering phenotypes. Nuclei labelled with LaminA antibody (green) and contractile apparatus with phalloidin (red). Fibres are outlined with white dotted lines for ease of identification. In each panel, Fibre 6 on top and 7 below, anterior left. Scale bar on ex28[RNAi] panel: 100 μm.

• 

  Figure 4. Nuclei fail to reach the contractile apparatus surface upon Msp300 isoform depletion. (A–E) Immunostaining of muscle 4 expressing lacZ[RNAi] (A), ex2[RNAi] (B), ex17[RNAi] (C), ex23[RNAi] (D) and ex28[RNAi] (E) showing nuclei position with respect to the contractile apparatus (respectively labelled with anti-LaminA in grey and phalloidin in magenta). For each panel, orthogonal views of the muscle are shown on the side and below. XZ views are marked by a dashed orange line and shown below the panel while YZ views are marked by a dashed cyan line and shown on the right of the XY view. While nuclei are positioned at the periphery of the muscle (above the sarcomeres) in lacZ and ex28[RNAi] conditions, they are found intermingled with myofibrils in the other conditions. Orthogonal views show myofibrils above nuclei as well as the distorted shape of nuclei. For each orthogonal view, the muscle surface is either on the top (XZ view at bottom of each panel) or on the right (YZ view on left of each panel). Scale bar on lacZ[RNAi] panel: 10 μm. (F–I) Schematic representations of muscle orthogonal views showing the nuclei position with respect to myofibrils and the plasma membrane in…

• 

  Figure 5. Depletion in CH containing Msp300 isoforms results in disorganisation of the myofibrils. Muscle 4 expressing Msp300-Venus protein-trap together with lacZ[RNAi] (A–C), ex2[RNAi] (D–F), ex17[RNAi] (G–I), or ex28[RNAi] (J–L) and showing the contractile apparatus organisation. (A,D,G,J): Msp300-Venus localisation. (B,E,H,K): phalloidin labelling in magenta and Msp300-Venus fluorescence in green. (C,F,I,L): Kettin immunostaining in magenta and Msp300-Venus fluorescence in green. In control lacZ[RNAi] (A–C), sarcomeres present a regular organisation with alternate (A)–band, lacking phalloidin staining, and Z-discs, showing bright phalloidin staining (B). Myofibrils are registered as seen with the large domains of sarcomeres’ alignment. Msp300-Venus ((A) in grey, (B,C) in green) presents a typical “beads-on-string” pattern (arrow heads) and localises at Z-discs as shown with the co-localisation with Kettin antibody ((C), in magenta). This organisation is conserved following ex28[RNAi] (J–L) muscular expression. Msp300-Venus “beads-on-string” labelling at Z-discs is largely lost following ex2[RNAi] although occasional short Msp300-Venus strings can still be observed ((D,E), arrow heads).…

• 

  Figure 6. Depletion of CH containing Msp300 isoforms with ex17[RNAi] or ex23[RNAi] results in Z-discs weakening. (A–L) Muscle 4 from larvae expressing Msp300-Venus protein-trap together with lacZ (A–C), ex17[RNAi] (D–F) or ex23[RNAi] (G–L) and showing the contractile apparatus organisation. phalloidin labelling in grey (A,D,G,J) or magenta (C,F,I,L) and Kettin immunostaining in grey (B,E,H,K) or green (C,F,I,L). In addition to important disorganisation of the myofibrils, ex23[RNAi], and to a lesser extent ex17[RNAi], lead to splittings of the Z-discs (arrows in (D–I)) as well as ruptures of myofibrils (brackets in (D–F) and (J–L)). These ruptures occur at Z-discs, suggesting that CH+ Msp300 isoform depletion weakens Z-discs’ cohesion. (M) Quantification of Z-disc defects following Msp300[RNAi]. Scale bar: 10 μm.