Zasp52’s differentially expressed intrinsically disordered region confers thin filament stability at the Z-disc
- Department of Biology, McGill University, Canada
Figures
Figure 1 with 2 supplements
exon 15e-containing isoforms are expressed in a specific spatiotemporal manner.
(A) A schematic of the Zasp52 locus. Exons are drawn to scale; introns are not. Coding exons are shown in orange and untranslated regions in gray. Alternative start sites are also indicated. Below, in green, are mappings of structured domains according to SMART Letunic and Bork, 2018; and also Katzemich et al., 2011, which defined LIM1 and LIM2 variants. The deletion in ex15e mutants is indicated in red. (B) Western blot using five different tissue extracts from control flies probed with a Zasp52 full-length antibody. Relative amounts of Zasp52 content, as measured in each lane, over the total protein content, as measured using the TGX Stain-free gel method, are quantified below for each tissue as mean, with error bars representing standard deviation (n=3). Exon 15e content, as measured using the protein content above the dotted blue line for each lane, over Zasp52 content, is quantified below that. Molecular weights appear higher, perhaps due to retardation caused by non-specific interactions with the gel matrix, as also seen in Watts et al., 2017. (C) RNA-seq data from Spletter et al., 2018, of indirect flight muscle (IFM) tissue across various timepoints after puparium formation; the last timepoint is of adults 1 day after eclosion. Gene expression levels in transcripts per million (TPM) are shown for Zasp52 isoforms classified according to their splicing of exon 15. The majority of Zasp52 IFM isoforms contain exon 15e.
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Figure 1—source data 1
PDF file containing the original western blot for Figure 1B, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/111101/elife-111101-fig1-data1-v1.zip
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Figure 1—source data 2
Original TIF file of western blot displayed in Figure 1B.
- https://cdn.elifesciences.org/articles/111101/elife-111101-fig1-data2-v1.zip
Figure 1—figure supplement 1
RNA-seq data of exon 15e expression.
MODENCODE RNA-seq data showing expression levels at the Zasp52 locus across various developmental stages. Exon 15e is highlighted in blue.
Figure 1—figure supplement 2
Disorder prediction of amino acids encoded by exon 15e.
(A) Various disorder prediction algorithms were used to assess the level of per-residue disorder at the Zasp52 locus. The corresponding Zasp52 locus is schematized below, with PDZ domain (blue), ZM (red), LIM domains (green), and the ABM identified in Ashour et al., 2023 (yellow). The red bar below the Zasp52 schematic indicates the extent of the deletion in ex15e. For disorder prediction algorithms (AiUPRED, Disopred, and metapredict), the higher y-values represent higher levels of disorder, while low values close to zero represent low levels of disorder (structure). AlphaFold pLDDT scores are plotted alongside in black. pLDDT scores can be used to inform about disorder, with higher scores indicating higher confidence that a region is ordered, and lower scores suggesting disorder (Ruff and Pappu, 2021; Emenecker et al., 2021). (B) Dendrogram of various insects with Zasp52 orthologs that contain a PDZ domain and four LIM domains. Canonical isoforms, which usually are also the longest ones, were used to compute the number of residues comprising the linker between the first and second LIM domains, with data represented in a heatmap. The purple bar indicates all Drosophilids, and the teal bar indicates species of the order Ephemeroptera and Odonata, which use direct flight. Though some Drosophilids contain shorter linkers, no other species contain linkers of comparable length to Drosophilids and other closely related Dipterans. NARDINI Z-score matrices of the linker region between LIM1 and LIM2 in Zasp52-PF (C) and of only the sequence encoded by exon 15e (C’). The diagonal squares represent Ω-values, which quantify the linear mixing versus segregation of all of residue type x with respect to all other residues. The off-diagonal squares represent δ-values, which quantify the linear mixing versus segregation of pairs of residue types. A high z-score thus indicates segregation/blocking of a certain residue type with respect to itself/others compared to a null model; a low score indicates even dispersion and intermixing. Polar residues µ = {S,T,N,Q,C,H}; hydrophobic residues h = {I,L,V,M}; basic residues + = {R,K}; acidic residues - = {E,D}; aromatic residues π = {F,W,Y}; alanine = A; proline = P; glycine = G.
Figure 2 with 1 supplement
A CRISPR deletion of exon 15e displays flight defects.
Western blots using either the Zasp52 full-length antibody (A) or antibody against LIM2-LIM4 (A’) of either w1118 (ctrl hereinafter), ex15e, or the MiMIC line MI00979 whole flies. Relative amounts of Zasp52 content, as measured in each lane, over the total protein content, are quantified below for each genotype as mean with error bars representing standard deviation (n=3) normalized against the ctrl lane. (B) Larval crawling assay of either ctrl (n=21) or ex15e (n=21) third instar larvae showing no significant difference between the two (unpaired t-test with Welch’s correction; p=0.6006). (C) Flight assays of ctrl versus ex15e flies of three different age categories: 1-day-old (ctrl n=138 flies, ex15e n=123), 5-days-old (ctrl n=125, ex15e n=119), and 3-weeks-old (ctrl n=86, ex15e n=81). The y-axis indicates flight strength: flies were released into a tube and those that landed in the top segment (y=1) had the strongest flight strength while those that landed in the bottom dish (y=8) had the weakest. The x-axis indicates the proportion of flies that landed in that segment. Red arrows indicate the average position landed in ctrl flies; yellow arrows are for ex15e flies. Difference in flight ability was statistically significant for all age categories (Fisher’s exact test; p<0.0001).
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Figure 2—source data 1
PDF file containing the original western blots of Figure 2A and A’, with relevant bands indicated.
- https://cdn.elifesciences.org/articles/111101/elife-111101-fig2-data1-v1.zip
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Figure 2—source data 2
Original TIF files of western blots displayed in Figure 2A and A’.
- https://cdn.elifesciences.org/articles/111101/elife-111101-fig2-data2-v1.zip
Figure 2—figure supplement 1
Flight assays, gross muscle morphology, and modified terminal Z-discs in ex15e mutant.
(A) A binary flight assay across three age categories of ctrl, homozygous ex15e, and ex15e over the Zasp52 deficiency BSC427. Sample sizes were as follows: 1-day-old (ctrl n=44 flies, ex15e n=42, ex15e/BSC427 n=45), 5-days-old (ctrl n=48, ex15e n=39, ex15e/BSC427 n=28), and 3-weeks-old (ctrl n=61, ex15e n=30, ex15e/BSC427 n=23). Flight was scored manually on whether they could generate upward lift or not. There was no significant difference between the homozygous mutant or ex15e over the deficiency (Fisher’s exact test; 1 d.o. p=1.0000; 5 d.o. p=0.7843; 3 w.o. p=0.5729). Polarized light images of sagittally bisected thoraces of 3-week-old ctrl (B) and ex15e (C) revealed no obvious differences in gross morphology; females shown. (D–G’’) Modified terminal Z-discs (MTZ) were affected in ex15e. Close-up views revealed a disruption in the MTZ and adjacent myofibrils in 3-week-old ex15e mutants (E) compared to ctrl (D). The regular ‘crown’-shaped structure of the ctrl MTZ is replaced with a disordered structure of variable shape in ex15e mutants. βPS integrin and Zasp52 localization appeared unaffected in ex15e (F–G’’). Thin filaments were visualized with phalloidin in green, Z-discs with a Zasp52 full-length antibody in red, and integrin adhesion sites at MTZs with a βPS integrin antibody in blue.
Figure 3
ex15e mutant indirect flight muscle (IFM) phenotypes: bending at the Z-disc and thin filament intrusion into the H-zone.
(A–C) Analysis of bent sarcomere phenotype. Actin is visualized by phalloidin in green. Ctrl myofibrils (A) appear straight while ex15e myofibrils (B) contain a range of bending (representative 3-week-old myofibrils shown). Red arrowhead indicates a representative sarcomere with a bend of <145°, yellow arrowhead indicates 145–165°, and white arrowhead >165°. (C) Bend angles were statistically equivalent (≡) between ctrl and ex15e in 1-day-olds and 5-day-olds, but not equivalent (ne) in 3-week-olds, where ex15e displayed greater bentness. Sample sizes: 1-day-old (ctrl n=1242 sarcomeres, ex15e n=1851), 5-days-old (ctrl n=1192, ex15e n=946), and 3-weeks-old (ctrl n=1768, ex15e n=1125) (two one-sided test [TOST] with ±4.5° equivalence margin, α=0.05; 1d.o. tL = 12.95, tU = −12.07; 5d.o. tL = 6.03, tU = −8.01; 3 w.o. tL = −6.34, tU = −20.63). (D–F) Sarcomeres also exhibited actin in the H-zone phenotype (representative 3-week-old myofibrils shown). Ctrl myofibrils (D) display normal H-zones without actin, with M-lines/H-zones indicated by blue arrowheads. ex15e myofibrils (E) display actin accumulation at the H-zone; arrowheads indicate same as above. Actin is visualized by phalloidin in green and Z-discs are identified by the full-length Zasp52 antibody in red. (F) These sarcomeres with H-zone actin, which appear hypercontracted, are significantly overrepresented in ex15e compared to ctrl across all age categories. Sample sizes were as follows: 1-day-old (ctrl n=3255 sarcomeres, ex15e n=4056), 5-days-old (ctrl n=3221, ex15e n=2677), and 3-weeks-old (ctrl n=5394, ex15e n=4720) (Fisher’s exact test; p<0.0001). (G) To ascertain that sarcomeres with actin in the H-zone were indeed contracted, the Z- to Z-disc length was measured in ex15e 3-week-old flies of both sarcomeres with a bare H-zone (H-zone normal, n=89 sarcomeres) and those with actin in the H-zone (H-zone actin, n=117). H-zone actin sarcomeres were significantly smaller (unpaired t-test with Welch’s correction; p<0.0001).
Figure 4
Transmission electron microscopy (TEM) reveals defects in mutant sarcomere ultrastructure.
TEM images reveal similar phenotypes as immunofluorescent ones (3-week-old shown). Ctrl (A) sarcomeres are very structured and even. Longitudinal sections of ex15e (B, C) display broken Z-discs (red arrowhead), broken M-lines (yellow arrowhead), bending at the Z-discs (green arrowhead), splits in filaments (purple arrowhead), and fraying of peripheral filaments (blue arrowhead). Transverse sections of ex15e (D) also display some of these abnormalities (purple and blue arrowhead).
Figure 5
A putative actin-binding motif within exon 15e is not responsible for ex15e defects.
ΔABM homozygotes along with all other flies in this figure were analyzed at 3 weeks of age. ΔABM myofibrils (C) resembled those of ctrl (A) and showed none of the phenotypes seen in ex15e mutants (B). Actin is visualized by phalloidin in green. (D) Bend angles were equivalent between ctrl and ΔABM flies (n=1648) (two one-sided test [TOST] with ±4.5° equivalence margin, α=0.05; tL = 21.88, tU = −7.93). (E) The proportion of sarcomeres that were contracted was not significantly different between ctrl and ΔABM flies (n=3773) (Fisher’s exact test; p=0.1274). (F) Flight ability was only slightly decreased in ΔABM flies (n=104 flies) compared to ctrl (n=86) but significantly improved compared to ex15e (n=81). Red arrows indicate the average position landed in ctrl flies, yellow are ex15e, and green are ΔABM (Fisher’s exact test; ctrl-ΔABM p=0.0351; ctrl-ex15e and ex15e-ΔABM p<0.0001).
Figure 6
ex15e defects are restored by rescue constructs.
Zasp52-PF or Zasp52-PR was heterozygously overexpressed with a UH3-GAL4 driver in a homozygous ex15e background in a rescue attempt. In this figure, all myofibrils had actin visualized with phalloidin in green. All experiments were done at 3 weeks of age, and ctrl and ex15e data are the same as previous. (A) Schematic showing Zasp52-PF, which is the canonical full-length isoform, and Zasp52-PR, which contains all structured domains but lacks exon 15e. PDZ domain (blue), ZM (red), LIM domains (green). (B–E) Myofibrils display bending, Z-disc disruption, and overall myofibrillar disorganization in Zasp52-PR overexpressions (D), similar to ex15e (C), while Zasp52-PF overexpression (E) appears similar to ctrl (B). (F) Bend angles were equivalent (≡) between ex15e and ex15e; UH3>Zasp52-PR, and between ctrl and ex15e; UH3>Zasp52-PF. Sample sizes: ex15e; UH3>Zasp52-PR n=2049 sarcomeres, ex15e; UH3>Zasp52-PF n=2221 (two one-sided test [TOST] with ±4.5° equivalence margin, α=0.05; ex15e-ex15e; UH3>Zasp52-PR tL = 1.92, tU = −10.16; ctrl-ex15e; UH3>Zasp52-PF tL = 17.65, tU = −8.51). (G) The proportion of sarcomeres that were contracted (i.e. actin in H-zone) was not significantly different between ex15e and ex15e; UH3>Zasp52-PR, whereas no such sarcomeres were found in ex15e; UH3>Zasp52-PF. Sample sizes: ex15e; UH3>Zasp52-PR n=2843 sarcomeres, ex15e; UH3>Zasp52-PF n=2480 (Fisher’s exact test; p=0.0521). (H) Flight ability was significantly improved in ex15e; UH3>Zasp52-PF (n=85) compared to ex15e; UH3>Zasp52-PR (n=66), with ex15e; UH3>Zasp52-PR showing no significant difference from ex15e but ex15e; UH3>Zasp52-PF showing a significant difference from ctrl. Red arrows indicate the average position landed in ctrl flies, yellow are ex15e, blue are ex15e; UH3>Zasp52-PR, and green are ex15e; UH3>Zasp52-PF (Fisher’s exact test; ex15e; UH3>Zasp52-PR-ex15e; UH3>Zasp52-PF p<0.0001; ex15e-ex15e; UH3>Zasp52-PR p=0.2668; ctrl-ex15e; UH3>Zasp52-PF p=0.0129).
Figure 7
Large H-zones demonstrate a genetic interaction between exon 15e and Act88F.
(A–D) Myofibrils of 1-day-old flies of various genotypes. Actin was visualized with phalloidin in green. Ctrl (A) and heterozygous ex15e myofibrils (B) appear indifferentiable; heterozygous Act88FKM88 (C) are slightly disrupted with fraying and slight narrowing. Representative ex15e/+; Act88FKM88/+ myofibrils (D) with large H-zones are shown. (E) We quantified the ratio of twice the thin filament length denoted distance ‘A’ to the corresponding sarcomere’s length denoted as ‘B’ for all four genotypes. A schematic illustrates the measurement of these distances. Sample sizes were as follows: ctrl n=119 sarcomeres, ex15e/+ n = 122, Act88FKM88/+ n = 119, ex15e/+; Act88FKM88/+ n = 119. The ratio A/B represents the proportion of the sarcomere length occupied by thin filaments and is inversely correlated with H-zone length; ex15e/+; Act88FKM88/+ sarcomeres have significantly larger H-zones than all others (unpaired t-test with Welch’s correction; ctrl-ex15e/+ p=0.0502; ctrl-Act88FKM88/+ p=0.2770; ctrl-ex15e/+; Act88FKM88/+ p<0.0001; ex15e/+-Act88FKM88 p=0.4938; ex15e/+-ex15e/+; Act88FKM88 p<0.0001; Act88FKM88/+- ex15e/+; Act88FKM88 p<0.0001). All sarcomeres with an Act88FKM88 allele were radially narrower than ctrl, but a synthetic enhancement could not be concluded from this observation.
Figure 8
Fluorescence recovery after photobleaching (FRAP) reveals defects in ex15e protein dynamics.
(A) GFP-tagged exon 15e localized to the indirect flight muscle (IFM) Z-discs in 1-day-old females. (B) FRAP experiments visualizing recovery of either GFP-tagged exon 15e (UH3>GFPexon15e, n=8) or GFPZasp52-PR (UH3>GFPZasp52-PR, n=12) were performed on 1-day-old female IFM Z-discs. (C) Recovery of GFPZasp52-PR was also assessed in 1-day-old female IFM Z-discs either in our mutant background (ex15e;UH3>GFPZasp52-PR, n=9 flies) or wild-type background (same data as in B). Ctrl UH3>GFPZasp52-PR Z-discs fixed with 4% paraformaldehyde for 10 min (labeled ‘Fixed’, n=7) displayed no recovery as expected. (D) Plateaus of fitted curves were significantly different, with a greater mobile fraction in the mutant background (unpaired t-test with Welch’s correction; p<0.0001). (E) Representative snapshots of bleached Z-disc recovery illustrate this difference between genotypes.
Figure 9
ex15e defects are rescued by immobilization.
Rescues were attempted by immobilizing flies, thereby preventing indirect flight muscle (IFM) use. ex15e myofibrils in flies that were immobilized (C) resembled those of ctrl (A), both showing no phenotypes compared to ex15e (B). (D) Bend angles were equivalent between ctrl and ex15e immobilized flies (n=2119) (two one-sided test [TOST] with ±4.5° equivalence margin, α=0.05; tL = 15.36, tU = −12.00). (E) The proportion of sarcomeres that were contracted was not significantly different between ctrl and immobilized flies (n=2246) (Fisher’s exact test; p=0.9037). (F) Flight ability was rescued with immobilized flies (n=67 flies) having no significant flight defect compared to ctrl (n=86) but being significantly improved from their non-immobilized counterparts (n=81) (Fisher’s exact test; ctrl-immobilized p=0.0640; ctrl-ex15e and ex15e-immobilized p<0.0001).
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | Zasp52 | FlyBase | FBgn0265991 | |
| Gene (Drosophila melanogaster) | Act88F | FlyBase | FBgn0000047 | |
| Strain, strain background (D. melanogaster) | ex15e | This paper | See Materials and methods – Fly strains and husbandry | |
| Strain, strain background (D. melanogaster) | Zasp52MI00979 | BDSC | RRID:BDSC_33099 | |
| Strain, strain background (D. melanogaster) | Df(2R)BSC427 | BDSC | RRID:BDSC_24931 | |
| Strain, strain background (D. melanogaster) | ΔABM | This paper | See Materials and methods – Fly strains and husbandry | |
| Strain, strain background (D. melanogaster) | UAS-Zasp52-PF | This paper | See Materials and methods – Fly strains and husbandry | |
| Strain, strain background (D. melanogaster) | UAS-Zasp52-PR | This paper | See Materials and methods – Fly strains and husbandry | |
| Strain, strain background (D. melanogaster) | UH3-GAL4 | Singh et al., 2014 | FBti0148868 | |
| Strain, strain background (D. melanogaster) | Act88FKM88 | FlyBase | FBal0000272 | |
| Strain, strain background (D. melanogaster) | UAS-GFPexon15e | This paper | See Materials and methods – Fly strains and husbandry | |
| Strain, strain background (D. melanogaster) | UAS-GFPZasp52-PR | González-Morales et al., 2019 | FBtp0140762 | |
| Strain, strain background (D. melanogaster) | w1118 | FlyBase | FBal0018186 | |
| Antibody | Anti-Zasp52 full-length (Rabbit polyclonal) | Jani and Schöck, 2007 | 1:200 | |
| Antibody | Anti-Zasp52LIM234 (Rabbit polyclonal) | This paper | See Materials and methods – Western blotting | |
| Antibody | Anti-βPS integrin (Mouse monoclonal) | DSHB | CF.6G11 | 1:200 |
| Antibody | Goat anti-rabbit IgG (H+L) HRP | Thermo Fisher Scientific | Cat 31460 | 1:10000 |
| Antibody | Alexa Fluor 647 goat anti-rat | Thermo Fisher Scientific | Cat A-21247 | 1:400 |
| Antibody | Alexa Fluor 488 goat anti-rabbit | Thermo Fisher Scientific | Cat A-11008 | 1:400 |
| Antibody | Acti-stain 555 fluorescent phalloidin | Cytoskeleton Inc | Cat PHDH1 | 1:400 |
| Antibody | FluoTag-X4 anti-GFP | NanoTag Biotechnologies | Cat N0304 | 1:200 |
| Software, algorithm | IBS 2.0 | Xie et al., 2022 | ||
| Software, algorithm | SMART | Letunic and Bork, 2018 | ||
| Software, algorithm | Salmon 1.10.0 | Patro et al., 2017 | ||
| Software, algorithm | AIUPred v0.1 | Erdős and Dosztányi, 2024 | ||
| Software, algorithm | DISOPRED3 | Jones and Cozzetto, 2015 | ||
| Software, algorithm | metapredict online v3.0 | Emenecker et al., 2021 | ||
| Software, algorithm | AlphaFold v2.0 | Jumper et al., 2021 | ||
| Software, algorithm | iTOL v.6 | Letunic and Bork, 2024 | ||
| Software, algorithm | NARDINI | Cohan et al., 2022 |
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Zasp52’s differentially expressed intrinsically disordered region confers thin filament stability at the Z-disc
eLife 15:RP111101.
https://doi.org/10.7554/eLife.111101.3
