Filamin actin-binding and titin-binding fulfill distinct functions in Z-disc cohesion

Fig 1. Cher localizes to the Z-disc in the IFM and is necessary for sarcomere structural stability. (A) cher genomic region showing several transcripts and several protein isoforms that can be grouped based on their molecular weight into 4 groups (CherA-D). Green triangles represent transgenic insertion sites of protein-trap lines, each bearing a splice acceptor site followed by a Venus and a Flag tag. The protein domains encoded by different exons are indicated at the bottom: CH (ABD), calponin homology domain 1 and 2 comprises the actin-binding domain; Ig, immunoglobulin-like domains; DD, dimerization domain. Asterisks denote location of the premature stop point mutants in cherQ1042x and cherQ1415sd. The cherΔ5 deleted region is denoted by a black line (B) Immunoblot from cherCPTI1399 thoraces incubated with anti-Flag antibody reveals 4 Cher isoforms, specifically depleted in Mef2-Gal4>UAS-cher-JF RNAi. (C, D) IFM confocal images from heterozygous Flag-tagged Cher traps stained with anti-Flag antibody to visualize the tagged isoforms. In all images, asterisks mark Z-discs of a selected myofibril immediately above the asterisks. (C) All Cher isoforms, revealed by the cherCPTI1403…

Fig 2. Distinct sarcomere phenotypes revealed by electron microscopy. (A) TEM image of a wild type sarcomere. Z stands for the Z-disc; H stands for the H-zone and is indicated by orange brackets; (B, C) Mef2-Gal4, UAS-cher-JF sarcomeres show widened Z-discs (green box, shown enlarged on the right), smaller than normal Z-discs (red box, shown enlarged on the right) and actin accumulation at the H-zone (orange brackets). Light green bracket denotes the width of the Z-disc. Numbers correspond to (1) a widened Z-disc, (2) a smaller or fractured Z-disc, and (3) actin incorporation into the H-zone. Scale bars: 500 nm.

Fig 3. Disruption of Cher-actin binding accounts for the widened Z-disc phenotype. (A, B) Electron micrographs of cherΔ5 mutant sarcomeres show Z-discs ripped apart (enlarged in A’, blue arrowheads indicate the left and right borders of a ripped-apart Z-disc) and actin accumulation at the H-zone (blue arrows), without affecting the M-line (light blue arrowheads). (C-E) Confocal images of IFM stained with phalloidin to visualize actin thin filaments in green, anti-Kettin antibody to visualize Z-discs in red, and anti-Flag antibody to visualize Cher isoforms in blue. (C, D) Both cherΔ5 and cher1 mutants show widened Z-discs with reduced actin staining in many sarcomeres. (E) The cher1403 actin-binding mutant also phenocopies the widened Z-disc phenotype. Scale bars: 5 μm.

Fig 4. Genetic interaction between Act88F and cher. (A, B) Confocal images of IFM stained with phalloidin to visualize actin thin filaments in green and anti-Kettin antibody to visualize Z-discs in red. (A) Act88F heterozygotes have a moderate sarcomere phenotype. (B) Transheterozygous Act88F/cherΔ5 sarcomeres display Kettin aggregates and stronger phenotypes, including widened Z-discs. White brackets denote area of completely frayed and thus uncountable sarcomeres, purple brackets show recognizable sarcomeres. Scale bars: 5 μm. (C) Quantification of recognizable sarcomeres in Act88F heterozygotes or in transheterozygous Act88F cher mutants. Only mutants affecting the actin-binding CherD isoform genetically interact with Act88F. Statistical significance assessed by one-way ANOVA with post hoc Tukey: **** = P ≤ 0.0001.

Fig 5. C-terminal Ig domains maintain Z-disc cohesion perpendicular to the myofibril. (A, B) TEM of two cher C-terminal mutants. (A) cherQ1415sd mutant sarcomere shows actin accumulation at the H-zone and a smaller Z-disc (purple box, enlarged in A’). (A’) Z-disc material (red arrowhead) and actin filaments (blue arrowhead) are observed detaching from the myofibril. (B) cherQ1042x mutant sarcomere displays mild defects, with Z-disc material pulled into the H-zone (enlarged in B’). (C) Quantification of the number of widened Z-discs in different cher mutants. Only mutants affecting the actin-binding CherD isoform display a widened Z-disc in the IFM. Statistical significance assessed by one-way ANOVA with post hoc Tukey: n.s. = not significant, **** = P ≤ 0.0001. (D) Schematic representation of the laser tachometer used to measure wing beat frequency. (E) All cher homozygous mutants have very short flying times, compared to the wild type control OregonR. The average of 10 flies with standard deviation is shown.

Fig 6. Actin accumulation in the H-zone in different cher mutants. (A-D) Confocal images of IFM stained with phalloidin to visualize actin thin filaments and GFP fluorescence marking Sls at the Z-disc. (A) Transheterozygous sls-GFP Df(3R)Exel6176 mildly accumulate actin at the H-zone. (B-D) Sarcomeres from sls-GFP Df(3R)Exel6176 in combination with different cher mutants result in actin aggregates at the H-zone, while Sls-GFP remains at the Z-disc. (E, F) Confocal images of IFM stained with phalloidin to visualize actin thin filaments, anti-Zasp52 to visualize Z-disks, and GFP fluorescence marking Sls at the Z-disc. (E) sls-GFP heterozygotes show GFP at the Z-disc together with Zasp52. (F) In sls-GFP Df(3R)Exel6176/cherQ1042x sarcomeres Sls-GFP and Zasp52 still colocalize at the Z-disc. (G) Quantification of H-zone actin accumulation in different cher mutant backgrounds shown as box plots. Statistical significance assessed by one-way ANOVA with post hoc Tukey: **** = P ≤ 0.0001. Scale bars: 5 μm.

Fig 7. Cher interaction with Sls is essential for maintaining sarcomere structure. (A-D) Confocal images of IFM stained with phalloidin to visualize actin thin filaments and anti-Kettin antibody to visualize Z-discs. (A, B) sls1 and slsj1D7 heterozygotes show predominantly normal sarcomere structure. (C, D) When sls mutant alleles are combined with Mef2>cher-JF a complete loss of sarcomere structure is observed. Scale bars: 5 μm. (E) Quantification of recognizable sarcomeres in different sls heterozygotes alone or in combination with Mef2>cher-JF. (F) Mef2>cherJF-associated lethality is also aggravated by addition of sls mutant alleles. Statistical significance assessed by one-way ANOVA with post hoc Tukey: * = P ≤ 0.05, ** = P ≤ 0.01, *** = P ≤ 0.001, **** = P ≤ 0.0001. (G-M) Wing beat frequency recordings in different cher and sls heterozygotes. The average of 10 flies with standard deviation is shown. (G, H, I) Heterozygous cher mutants can fly. (J) slsj1D7 heterozygotes are also able to sustain flight. (K, L) However, cherQ1415sd slsj1D7 and cherΔ5 slsj1D7 transheterozygotes are not capable of sustained flight. (M) cherQ1042x slsj1D7 transheterozygotes can fly.

Fig 8. The Sls isoform Kettin binds Cher and recruits Cher to the Z-disc. (A) Pull-down of His-tagged Sls co-immunoprecipitates Cher-Flag, in contrast to pull-down with wild type thorax extract. (B) Pull-down of Cher-Flag co-immunoprecipitates the Sls isoform Kettin, whereas wild type thorax extract does not. (C, D) Confocal images of IFM stained with phalloidin to visualize actin thin filaments and anti-Flag antibody to visualize Cher. GFP fluorescence marks Sls at the Z-disc. (C) In slsZCL2144 heterozygotes Sls-GFP and Cher-Flag colocalize at the Z-disc. (D) In slsZCL2144 homozygotes Cher no longer localizes at the Z-disc and is instead recruited to Sls-GFP aggregates (magenta dotted line). Scale bar: 5 μm.

Fig 9. C-terminal Ig domains 19–22 of Cher bind to the Sls isoform Kettin. (A) Cartoon showing 4 epitopes in Sls whose precise localization in the I-band has been determined in IFM (short I-band sarcomeres) and leg muscles (long I-band sarcomeres). (B) Coevolutionary rate variation analysis of Cher and Sls coding regions. Coevolution scores (-1 to 1) are color-coded, values close to 1 indicate positive coevolution and appear as dark blue. Filamin alignment blocks are shown on the y axis and are labeled, Sls alignment blocks are shown on the x axis and are unlabeled. A large area of dark blue values is observed in the Sls area corresponding to Kettin and Cher C-terminal Ig domains 8–14, and even more pronounced with Ig 15–22. To visualize Sls domains, the position of known epitopes is indicated in colored circles together with their location within A-band, I-band, and Z-disc above the graph. The extent of known Sls isoforms is indicated below the graph. Zormin localizes to Z-disc and M-line. (C) Pull-down assay using fly-purified Sls and bacterially expressed Ig domains of Cher. Cher Ig 19–22, but not Cher Ig 9–11 interacts with Sls.

Fig 10. Model of Cher/Filamin function in stabilizing the Z-disc. (A) Filamin dimers (blue) localize to the Z-disc or Z-disc edge, where they serve to stabilize the Z-disc in two directions. A filamin domain close to or identical to the dimerization domain binds titin in one sarcomere, while the two CH domains bind actin thin filaments from the adjacent sarcomere. Thin filaments are in different shades of red in the right half sarcomere to indicate their location in different planes. This configuration allows Cher to stabilize the Z-disc in two directions: parallel and perpendicular to the myofibril. (B-D) Models showing one filamin dimer binding two thin filaments and titin. (B) Wild type. (C) In cher mutants that disrupt actin binding, perpendicular stability is partially maintained but anchorage to the Z-disc is lost. Thus, an actin-free and widened Z-disc is often observed. (D) In mutants which compromise Sls binding and dimerization, the Z-disc ruptures perpendicular to the myofibril, causing Z-disc material and thin filaments to detach. In all mutants except cherQ1042x, individual thin filaments can invade the H-zone owing to myosin power strokes and improper anchorage of thin…