Myofibril diameter is set by a finely tuned mechanism of protein oligomerization in Drosophila
- MGill University, Canada
Figures
Figure 1 with 2 supplements
Aggregate formation upon Zasp overexpression.
(A–H) Confocal images of IFM in control or Zasp overexpression conditions using Act88F-Gal4; actin filaments are marked in magenta and Z-discs in green. (A) In control flies, sarcomeres are regular …
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Figure 1—source data 1
Aggregate frequency estimates in Zasp overexpressions.
- https://cdn.elifesciences.org/articles/50496/elife-50496-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Differences in Zasp isoforms.
Cartoon of selected Zasp protein isoforms with annotated protein domains. Isoforms are named according to FlyBase. Numbers at each end represent the length in amino acids. ZM, Zasp-like motif.
Figure 1—figure supplement 2
Zasp-mediated aggregation depends only partially on α-actinin.
(A, B) Confocal images of IFM in control or α-actinin depleted conditions; actin filaments are marked in magenta and Z-discs marked by Zasp66-GFP are shown in green. (A) Control sarcomeres are …
Figure 2 with 1 supplement
Y2H assays reveal domains involved in homo/heterodimerization.
(A) Yeast two-hybrid assays between the three Zasp paralogs. Images of double-transformed yeast grown in selective -Ade/-His/-Leu/-Trp plates. Serial dilutions are shown from left to right (OD: 0.1, …
Figure 2—figure supplement 1
Zasp self-interaction by co-IP and chemical crosslinking.
(A) Endogenous Zasp52 tagged with GFP was purified from thorax extracts via GFP beads. GFP-Zasp52 co-immunoprecipitates Flag-Zasp52-PK. GFP alone was used as negative control. (B) Purified …
Figure 3 with 1 supplement
Zasp interaction in vivo at the Z-disc.
(A–C) Confocal images of Zasp66-GFP IFM in control and Zasp52 mutant backgrounds. Zasp66-GFP levels are lower in Zasp52MI00979 mutant than in the control or in Zasp52MI02988 mutants. (D) Boxplot of …
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Figure 3—source data 1
Zasp66 levels and BiFC values.
- https://cdn.elifesciences.org/articles/50496/elife-50496-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Zasp52 gene map and BiFC.
(A) Cartoon representing the Zasp52 genomic locus with selected transcripts, two alternative transcription start sites, protein domains, and three MiMIC lines introducing an artificial exon which …
Figure 4
The evolution of the ZM domain.
(A) Protein sequence alignment between the three Zasp paralogs in flies and the ZM consensus sequence SMART:SM00735. Similar amino acids to the consensus are highlighted in gray. (B) Radial …
Figure 5 with 1 supplement
Balance of ZM to LIM domains sets the diameter of the Z-disc.
(A) Proposed model of Z-disc growth and aggregate formation. In nascent Z-bodies, Zasp binds α-actinin and forms a homodimer between its ZM and LIM domains. During the early growth phase, Zasp …
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Figure 5—source data 1
Line intensity profile plots.
- https://cdn.elifesciences.org/articles/50496/elife-50496-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Rotation smoothing of cross section Z-disc images.
(A–C) Steps for obtaining relative Zasp concentrations throughout single Z-discs. First, a single properly oriented Z-disc is selected from a confocal image (raw image). Then, we changed the display …
Figure 6 with 1 supplement
Balance of isoforms dictates Z-disc size.
(A and B) Confocal microscopy images of control and Zasp52MI00979 mutant. Actin filaments are marked in magenta and Z-discs in green. The Zasp52MI00979 mutant has smaller and frayed Z-discs. (C) …
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Figure 6—source data 1
Z-disc diameter estimates and Zasp52 levels.
- https://cdn.elifesciences.org/articles/50496/elife-50496-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Actinin levels in different Zasp overexpression backgrounds.
(A) Confocal images of Zasp52-mCherry in control and different Zasp overexpression backgrounds. Quantification shown in Figure 6F,G. (B) Confocal images of Actn-GFP in control and different Zasp …
Figure 7
Isoform imbalance leads to Z-disc aggregation.
(A–C) Confocal microscopy images of Zasp66 and Zasp67 single null mutants and the Zasp66 Zasp67 double mutant. Aggregates are outlined by a stippled red line. (D) Plot of aggregate frequencies of …
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Figure 7—source data 1
Z-disc diameter estimates and aggregate estimates.
- https://cdn.elifesciences.org/articles/50496/elife-50496-fig7-data1-v2.xlsx
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | Zasp52 | FBgn0265991 | ||
| Gene (Drosophila melanogaster) | Zasp66 | FBgn0035917 | ||
| Gene (Drosophila melanogaster) | Zasp67 | FBgn0036044 | ||
| Gene (Drosophila melanogaster) | Actn | FBgn0000667 | ||
| Genetic reagent (Drosophila melanogaster) | Act88F-Gal4 | RM Cripps PMID: 22008792 | FBal0268407 | |
| Genetic reagent (Drosophila melanogaster) | UH3-Gal4 (P[GawB]HkUH3) | Anja Katzemich PMID: 23505387 | FBti0148868 | |
| Genetic reagent (Drosophila melanogaster) | UAS-LacZ | BDSC | RRID:BDSC_3356 | |
| Genetic reagent (Drosophila melanogaster) | UAS-GFP-Zasp52-PR | Current study | N/A | Expresses full length Zasp52-PR isoform with a N terminal GFP tag under UAS. The landing site is ZH-attP-86Fb. |
| Genetic reagent (Drosophila melanogaster) | UAS-GFP-Zasp52-PK | Current study | N/A | Expresses Zasp52-PK isoform with a N terminal GFP tag under UAS. The landing site is ZH-attP-86Fb. |
| Genetic reagent (Drosophila melanogaster) | UAS-Flag-Zasp52-PR | Kuo An Liao PMID: 27783625 | FBal0323349 | Expresses full length Zasp52-PR isoform with a N terminal Flag tag under UAS. The landing site is ZH-attP-86Fb. |
| Genetic reagent (Drosophila melanogaster) | UAS-Flag-Zasp52-PR-ΔPDZ | Kuo An Liao PMID: 27783625 | FBal0323350 | Expresses Full length Zasp52 without the PDZ domain and a N terminal Flag tag under UAS. The landing site is ZH-attP-86Fb. |
| Genetic reagent (Drosophila melanogaster) | UAS-Flag-Zasp52-PR-ΔZM | Kuo An Liao PMID: 27783625 | FBal0323351 | Expresses full length Zasp52-PR isoform without the ZM domain and a N terminal Flag tag under UAS. The landing site is ZH-attP-86Fb. |
| Genetic reagent (Drosophila melanogaster) | UAS-Flag-Zasp52-PP | Current study | N/A | Expresses smallest Zasp52 isoform with a N terminal Flag tag under UAS. The landing site is ZH-attP-86Fb. |
| Genetic reagent (Drosophila melanogaster) | UAS-Flag-Zasp52-Stop143 | Current study | N/A | Expresses Zasp52-PK isoform with a N terminal Flag tag and a stop codon at position 143 under UAS. The landing site is ZH-attP-86Fb. |
| Genetic reagent (Drosophila melanogaster) | UAS-Zasp66-PK-Flag-HA | Current study | N/A | Transgenic made from the UFO11045 plasmid from DGRC. The landing site is ZH-attP-58A. |
| Genetic reagent (Drosophila melanogaster) | UAS-Zasp67-PE-Flag-HA | Current study | N/A | Transgenic from Zasp67 sequence synthesized by Genscript and cloned into a pUASattb vector. The landing site is ZH-attP-58A. |
| Genetic reagent (Drosophila melanogaster) | Zasp52-MI02988-mCherry | Nicanor Gonzalez-Morales PMID: 29423427 | PMID: 29423427 | Replacement of the MIMIC02988 cassette in Zasp52 with an in-frame mCherry tag. |
| Genetic reagent (Drosophila melanogaster) | Zasp52-GFP Zasp52[ZCL423] | BDSC | RRID:BDSC_58790 | |
| Genetic reagent (Drosophila melanogaster) | Zasp66-GFP Zasp66[ZCL0663] | BDSC | RRID:BDSC_6824 | |
| Genetic reagent (Drosophila melanogaster) | Zasp67-GFP fTRG | VDRC | v318355 | |
| Genetic reagent (Drosophila melanogaster) | UAS-Actn-KK (RNAi) | VDRC | v110719; FBst0482284 | |
| Genetic reagent (Drosophila melanogaster) | Zasp52[MI02988] | BDSC | RRID:BDSC_41034 | |
| Genetic reagent (Drosophila melanogaster) | Zasp52[MI07547] | BDSC | RRID:BDSC_43724 | |
| Genetic reagent (Drosophila melanogaster) | Zasp52[MI00979] | BDSC | RRID:BDSC_33099 | |
| Genetic reagent (Drosophila melanogaster) | Zasp66[KO] | PMID: 31123042 | PMID: 31123042 | CRISPR null mutant of Zasp66 |
| Genetic reagent (Drosophila melanogaster) | Zasp67[KO] | PMID: 31123042 | PMID: 31123042 | CRISPR null mutant of Zasp67 |
| Genetic reagent (Drosophila melanogaster) | UAS-Zasp66-PH-Venus | Current study | N/A | The Zasp66-PH isoform that contains only a ZM domain cloned into pBID-UAS-GV vector. The landing site is ZH-attP-58A. |
| Genetic reagent (Drosophila melanogaster) | UAS-Zasp66-PH-NYFP (CYFP) | Current study | N/A | The Zasp66-PH isoform fused to either NYFP or CYFP. |
| Genetic reagent (Drosophila melanogaster) | UAS-Zasp52-PK-NYFP (CYFP) | Current study | N/A | The Zasp52-PK isoform fused to either NYFP or CYFP. |
| Recombinant DNA reagent | Plasmid: pGADT7 | Clontech | 630442 | |
| Recombinant DNA reagent | Plasmid: pBD-Gal4-Zasp67 | DGRC | CT33772-BD | |
| Recombinant DNA reagent | Plasmid: pOAD-Zasp67 | DGRC | CT33772-AD | |
| Recombinant DNA reagent | Plasmid: pENTRY-Zasp52-PK | DGRC | DGRC: GEO02280 | |
| Recombinant DNA reagent | Plasmid: pENTRY-Zasp52-PE GEO12859 | DGRC | DGRC: GEO12859 | |
| Recombinant DNA reagent | Plasmid: pENTRY-Zasp66-PH GEO14752 | DGRC | DGRC: GEO14752 | |
| Recombinant DNA reagent | Plasmid: pGBKT7-GW | Addgene | 61703 | |
| Recombinant DNA reagent | Plasmid: pGADT7-GW | Addgene | 61702 | |
| Recombinant DNA reagent | Plasmid: pGADT7-Zasp66-PK | Current study | N/A | Zasp66-PK isoform cloned into pGADT7. |
| Recombinant DNA reagent | Plasmid: pGADT7-Zasp66-PK with stop codons | Current study | N/A | Stop codons were introduced in the Zasp66-PK-pGADT7 plasmid by site-directed mutagenesis (Genscript). |
| Recombinant DNA reagent | Plasmid: pGBKT7- Zasp52 individual domains: PDZ, ZM, LIM1a, LIM1b, LIM2a, LIM2b, LIM3 and LIM4 | Current study | N/A | All individual domains of Zasp52 cloned into pGBKT7. |
| Recombinant DNA reagent | Plasmid: pGBKT7GW-Zasp52-PK | Current study | N/A | Zasp52-PK isoform cloned into pGBKT7GW using Gateway cloning. |
| Recombinant DNA reagent | Plasmid: pGADT7GW-Zasp52-PK | Current study | N/A | Zasp52-PK isoform cloned into pGADT7GW using Gateway cloning. |
| Recombinant DNA reagent | Plasmid: pGBKT7GW-Zasp52-PE | Current study | N/A | Zasp52-PE isoform cloned into pGBKT7GW using Gateway cloning |
| Recombinant DNA reagent | Plasmid: pGADT7GW-Zasp52-PE | Current study | N/A | Zasp52-PE isoform cloned into pGADT7GW using Gateway cloning. |
| Recombinant DNA reagent | Plasmid: pGBKT7GW-Zasp66-PH | Current study | N/A | Zasp66-PH isoform cloned into pGBKT7GW using Gateway cloning |
| Recombinant DNA reagent | Plasmid: pGADT7GW-Zasp66-PH | Current study | N/A | Zasp66-PH isoform cloned into pGADT7GW using Gateway cloning. |
| Recombinant DNA reagent | Plasmid: pDEST- pUAS-RfB-HA-CYFP-attB | Sven Bogdan PMID: 20937809 | FBrf0212496 | |
| Recombinant DNA reagent | Plasmid: pDEST- pUAS-RfB-myc-NYFP-attB | Sven Bogdan PMID: 20937809 | FBrf0212496 | |
| Strain, strain background (Saccharomyces cerevisiae) | Matchmaker Y2HGold | Clontech | 630498 | |
| Strain, strain background (E. coli) | BL21 | NEB | C2530H | |
| Antibody | Anti-Flag | SIGMA | F3165 | 1:200 |
| Software, algorithm | R Project for Statistical Computing: base and ape packages | https://cran.r-project.org/ | RRID:SCR_001905 | |
| Software, algorithm | Salmon | https://combine-lab.github.io/salmon/ | RRID:SCR_017036 | |
| Software, algorithm | ImageJ/Fiji distribution | https://fiji.sc/ | RRID: SCR_002285 | |
| Software, algorithm | Galaxy | https://usegalaxy.org/ | RRID:SCR_006281 | |
| Chemical compound, drug | ethylene glycol bis-sulfosuccinimidyl succinate (EGS) | Fisher Scientific | 21565 | |
| Chemical compound, drug | YPDA medium | Clontech | 630464 | |
| Chemical compound, drug | Minimal SD Base | Clontech | 630411 | |
| Chemical compound, drug | -Leu /- Trp DO Supplement | Clontech | 630417 | |
| Chemical compound, drug | -Ade /- His /- Leu/-Trp DO Supplement | Clontech | 630428 | |
| Chemical compound, drug | Acti-stain 488 phalloidin | CYTOSKELETON, INC | PHDG1-A | |
| Chemical compound, drug | Alexa633-Phalloidin | Fisher Scientific | A22284 | |
| Chemical compound, drug | Rhodamine-phalloidin | Fisher Scientific | 10063052 |
Additional files
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Supplementary file 1
Detailed genotypes for all figures.
- https://cdn.elifesciences.org/articles/50496/elife-50496-supp1-v2.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/50496/elife-50496-transrepform-v2.docx
