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Myofibril diameter is set by a finely tuned mechanism of protein oligomerization in Drosophila

Research Article
Cite this article as: eLife 2019;8:e50496 doi: 10.7554/eLife.50496
8 figures, 1 table and 2 additional files

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 with Z-discs located in the center of the actin signal. (B) Upon GFP-Zasp52-PR overexpression sarcomere structure is severely affected and big Zasp aggregates appear. (C) The overexpression of Flag-Zasp52-PP, an isoform lacking all LIM domains, has a modest sarcomere phenotype and aggregation is not observed. (D) Overexpression of a mutated form of Zasp52-PR with a deletion in the ZM domain does not form aggregates. (E) Overexpression of a mutated form of Zasp52-PR carrying a deletion in the PDZ domain causes small aggregates and sarcomere phenotypes. (F and G) The overexpression of the other Zasp paralogs, Zasp66 or Zasp67, does not cause aggregates. (H) Overexpression of GFP-Zasp52-PK, a smaller isoform of Zasp52 lacking LIM2, 3, and 4 domains affects sarcomere structure, but aggregation is infrequent. (I) Estimation of Z-disc aggregates in all overexpression conditions, n = 10 muscle fibers. (J) Confocal images of IFM in control or in mild overexpression of GFP-Zasp52-PR using UH3-Gal4. (K) Estimation of Z-disc aggregates in mild overexpression of GFP-Zasp52-PR. (L) Frequency plot of Z-disc sizes. Z-discs are bigger when GFP-Zasp52-PR is overexpressed using UH3-Gal4. Scale bars, 10 µm. p-Values in panel I and K were calculated using Welch’s two-sample t-test.

Figure 1—source data 1

Aggregate frequency estimates in Zasp overexpressions.

https://cdn.elifesciences.org/articles/50496/elife-50496-fig1-data1-v3.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 regular with Z-discs located in the center of the actin signal. (B) In α-actinin-depleted muscles sarcomere structure is severely perturbed, and Z-discs are rarely visible. (C, D) Confocal images of IFM with aggregates caused by the overexpression of Zasp52-PR. (C) Overexpression of Zasp52-PR produces aggregates. (D) Overexpression of Zasp52-PR in α-actinin-depleted muscles produces smaller aggregates. (E) Estimation of Z-disc aggregates in all overexpression conditions, n = 10 muscle fibers. Act88F-Gal4 was used in all panels. Scale bars, 10 µm. p-Values in panel E were calculated using Welch’s two-sample t-test.

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, OD: 0.01, and OD: 0.001). Zasp52-PK dimerizes with itself and with the longer isoform Zasp52-PE. Zasp66 interacts with Zasp52-PK and Zasp52-PE. Zasp67 interacts with Zasp52-PK and Zasp52-PE. Negative controls using the DNA-binding domain (bait, pGAD-T7) or the Activating domain of Gal4 (prey, pGBK-T7) are shown. (B) Y2H assays testing the interaction between all protein domains encoded by the Zasp52 gene and Zasp52-PK, Zasp66-PK, or Zasp67-PD proteins. Zasp52-PK interacts with isolated ZM and LIM domains, Zasp66-PK and Zasp67-PD interact only with some LIM domains. LIM1A, LIM1B, LIM2A, and LIM2B are different splice isoforms of LIM1 and LIM2 domains. (C) Proposed model of Zasp homo/heterodimerization. The LIM domains bind the ZM domain. Zasp52-PK dimerization occurs through two ZM/LIM pairs. The heterodimerization between Zasp52-PK and Zasp66-PK or Zasp67-PD occurs through only one ZM/LIM-binding site. Zasp66-PH is a small isoform that only contains a ZM domain. (D) Y2H assays mapping the interaction between different Zasp52 LIM domains to Zasp66-PK using truncation mutants. (E) Y2H assays testing the interaction between the ZM only isoform Zasp66-PH, and the individual protein domains encoded by Zasp52.

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 His-Zasp52-PK-Flag from bacteria was treated with the chemical crosslinker ethylene glycol bis-sulfosuccinimidyl succinate (EGS) and then analyzed by SDS-Page. In addition to monomers (M, 50 kD), dimers (D) are observed upon EGS treatment.

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 Zasp66-GFP intensities in control and Zasp52 mutant backgrounds. (E, F) Examples of a negative BiFC control (F) and a positive BiFC signal (E) suggesting Zasp52-PK dimerizes at the Z-disc. (G, H) Plots of the BiFC fluorescence intensity values relative to background noise. Positive BiFC fluorescence is detected between Zasp52-PK and Zasp52-PK (G) and between Zasp52-PK and Zasp66-PH (H). Act88F-Gal4 was used to drive expression of NYFP- or CYFP-tagged proteins. Scale bar, 5 µm. p-Values in panels D, G, and H were calculated using Welch’s two-sample t-test.

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 consists of a splice acceptor followed by stop codons for all three reading frames. (B) Confocal images of Actn-GFP IFM in control and Zasp52 mutant backgrounds. Actn-GFP levels are comparable between the control and Zasp52 mutants. (C) Boxplot of Actn-GFP intensities in control and Zasp52 mutant backgrounds. p-Values were calculated using one-sided Welch’s two-sample t-test. (D) Representation of the BiFC principle. (E) The ZM-only Zasp66-PH isoform tagged with a Venus fluorescent protein localizes to the Z-disc, marked by Zasp52-mCherry.

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 phylogenetic tree of selected eukaryotic lineages with annotated presence of the ZM domain from the PFAM domain database (PFAM: PF15936). The ZM domain is restricted to bilateral animals.

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 growing isoforms use their free LIM domains to recruit more Zasp molecules through the ZM domain, thus growing the Z-disc. At later stages, growth is downregulated by the incorporation of blocking isoforms of Zasp, which are recruited to the Z-disc but cannot recruit further proteins because they lack LIM domains. (B) RNAseq data plots showing the expression of selected Zasp isoforms at different developmental timepoints. The y-axis corresponds to the log of the number of transcripts per million (TPM) and the x-axis to hours after pupa formation. Isoforms were classified as blocking isoforms (0 or 1 LIM domain) or growing isoforms (two or more LIM domains). (C) Confocal microscopy image of IFM cross sections expressing Zasp52-GFP (green) and stained for actin (magenta). (D) Differential distribution of Zasp growing and blocking isoforms throughout the Z-disc. Images of false-colored denoised Z-discs. Zasp52-growing isoforms are more concentrated at the center of the disc, Zasp66 and Zasp67 are mostly concentrated in the periphery. Color scale is shown at the right. (E) Profile plots of relative intensity values at the Z-disc diameters from at least 10 individual Z-discs. Growing isoforms peak at the center of the Z-discs, blocking isoforms have two peaks. Scale bars in D, 1 µm.

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 from gray look-up table mode to physics look-up table mode. Higher concentration of Zasp52-GFP at the center of the disc is observed at this stage (A; false colored). Higher concentration of Zasp67-GFP at the periphery of the disc is observed at this stage (B; false colored). Then, we rotated the Z-disc image by 2 degrees 180 times, and we calculated the average from all the resulting images (average image). Finally, we calculated the relative fluorescence intensities. (C) An artificially generated disc image from random noise treated in the same way as panels A and B does not show any obvious pattern.

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) Frequency plot of Z-disc sizes in control, Zasp52MI02988, and Zasp52MI00979 mutants. Small Z-discs are only observed in the Zasp52MI00979 mutant. (D) Overexpression of the Zasp52-PP blocking isoform also results in smaller Z-discs. The small Z-disc phenotype is not observed in Zasp52-Stop143, lacking the ZM domain. (E) Small Z-disc phenotypes are observed upon overexpression of Zasp66 and Zasp67. (F and G) Boxplots of the Zasp52 fluorescence intensities upon overexpression of different blocking isoforms. (F) Zasp52-mCherry levels decrease upon overexpression of Zasp52-PP but are restored if the ZM domain of Zasp52-PP is deleted (Zasp52-*143). (G) Zasp52-mCherry levels also decrease upon overexpression of Zasp66 and Zasp67. Act88F-Gal4 was used for overexpression experiments in panels D-G. Scale bars, 5 µm. p-Values in panels C-E were calculated using Fisher's exact test for count data. p-Values in panels F and G were calculated using Welch’s two-sample t-test.

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 overexpression backgrounds. Relative fluorescence levels are similar in all conditions. (C) Boxplot of the Actn-GFP fluorescence intensities upon overexpression of different blocking isoforms. Act88F-Gal4 was used for overexpression experiments. Scale bars, 5 µm. p-Values were calculated using Welch’s two-sample t-test with Bonferroni correction for multiple comparisons.

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 single and double mutants. (E) Frequency plot of Z-disc size categories in single and double mutants (for categories see Figure 6). (F–H) Confocal microscopy images of muscles overexpressing the growing GFP-Zasp52-PR isoform and selected Zasp blocking isoforms or a LacZ control. (F) In the control, GFP-Zasp52-PR overexpression produces aggregates. (G) The aggregation phenotype is suppressed by co-overexpression of the blocking isoform Zasp52-PP. (H) The aggregation phenotype is not rescued upon co-overexpression of Zasp52-Stop143, which lacks the ZM domain. (I) Aggregation frequency in different co-overexpression backgrounds. Act88F-Gal4 was used for overexpression experiments in panels F-I. Scale bars in A-C and F-J, 10 µm. p-Values in panel E were calculated using Fisher's exact test for count data. p-Values in panels D and I were calculated using Welch’s two-sample t-test.

Figure 7—source data 1

Z-disc diameter estimates and aggregate estimates.

https://cdn.elifesciences.org/articles/50496/elife-50496-fig7-data1-v3.xlsx
Author response image 1

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Drosophila melanogaster)Zasp52FBgn0265991
Gene (Drosophila melanogaster)Zasp66FBgn0035917
Gene (Drosophila melanogaster)Zasp67FBgn0036044
Gene (Drosophila melanogaster)ActnFBgn0000667
Genetic reagent (Drosophila melanogaster)Act88F-Gal4RM Cripps PMID: 22008792FBal0268407
Genetic reagent (Drosophila melanogaster)UH3-Gal4
(P[GawB]HkUH3)
Anja Katzemich PMID: 23505387FBti0148868
Genetic reagent (Drosophila melanogaster)UAS-LacZBDSCRRID:BDSC_3356
Genetic reagent (Drosophila melanogaster)UAS-GFP-Zasp52-PRCurrent studyN/AExpresses 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-PKCurrent studyN/AExpresses 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-PRKuo An Liao PMID: 27783625FBal0323349Expresses 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-ΔPDZKuo An Liao PMID: 27783625FBal0323350Expresses 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-ΔZMKuo An Liao PMID: 27783625FBal0323351Expresses 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-PPCurrent studyN/AExpresses 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-Stop143Current studyN/AExpresses 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-HACurrent studyN/ATransgenic made from the UFO11045 plasmid from DGRC. The landing site is ZH-attP-58A.
Genetic reagent (Drosophila melanogaster)UAS-Zasp67-PE-Flag-HACurrent studyN/ATransgenic 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-mCherryNicanor Gonzalez-Morales
PMID: 29423427
PMID: 29423427Replacement of the MIMIC02988 cassette in Zasp52 with an in-frame mCherry tag.
Genetic reagent (Drosophila melanogaster)Zasp52-GFP Zasp52[ZCL423]BDSCRRID:BDSC_58790
Genetic reagent (Drosophila melanogaster)Zasp66-GFP Zasp66[ZCL0663]BDSCRRID:BDSC_6824
Genetic reagent (Drosophila melanogaster)Zasp67-GFP fTRGVDRCv318355
Genetic reagent (Drosophila melanogaster)UAS-Actn-KK (RNAi)VDRCv110719; FBst0482284
Genetic reagent (Drosophila melanogaster)Zasp52[MI02988]BDSCRRID:BDSC_41034
Genetic reagent (Drosophila melanogaster)Zasp52[MI07547]BDSCRRID:BDSC_43724
Genetic reagent (Drosophila melanogaster)Zasp52[MI00979]BDSCRRID:BDSC_33099
Genetic reagent (Drosophila melanogaster)Zasp66[KO]PMID: 31123042PMID: 31123042CRISPR null mutant of Zasp66
Genetic reagent (Drosophila melanogaster)Zasp67[KO]PMID: 31123042PMID: 31123042CRISPR null mutant of Zasp67
Genetic reagent (Drosophila melanogaster)UAS-Zasp66-PH-VenusCurrent studyN/AThe 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 studyN/AThe Zasp66-PH isoform fused to either NYFP or CYFP.
Genetic reagent (Drosophila melanogaster)UAS-Zasp52-PK-NYFP (CYFP)Current studyN/AThe Zasp52-PK isoform fused to either NYFP or CYFP.
Recombinant DNA reagentPlasmid: pGADT7Clontech630442
Recombinant DNA reagentPlasmid: pBD-Gal4-Zasp67DGRCCT33772-BD
Recombinant DNA reagentPlasmid: pOAD-Zasp67DGRCCT33772-AD
Recombinant DNA reagentPlasmid: pENTRY-Zasp52-PKDGRCDGRC: GEO02280
Recombinant DNA reagentPlasmid: pENTRY-Zasp52-PE GEO12859DGRCDGRC: GEO12859
Recombinant DNA reagentPlasmid: pENTRY-Zasp66-PH GEO14752DGRCDGRC: GEO14752
Recombinant DNA reagentPlasmid: pGBKT7-GWAddgene61703
Recombinant DNA reagentPlasmid: pGADT7-GWAddgene61702
Recombinant DNA reagentPlasmid: pGADT7-Zasp66-PKCurrent studyN/AZasp66-PK isoform cloned into pGADT7.
Recombinant DNA reagentPlasmid: pGADT7-Zasp66-PK with stop codonsCurrent studyN/AStop codons were introduced in the Zasp66-PK-pGADT7 plasmid by site-directed mutagenesis (Genscript).
Recombinant DNA reagentPlasmid: pGBKT7- Zasp52 individual domains: PDZ, ZM, LIM1a, LIM1b, LIM2a, LIM2b, LIM3 and LIM4Current studyN/AAll individual domains of Zasp52 cloned into pGBKT7.
Recombinant DNA reagentPlasmid: pGBKT7GW-Zasp52-PKCurrent studyN/AZasp52-PK isoform cloned into pGBKT7GW using Gateway cloning.
Recombinant DNA reagentPlasmid: pGADT7GW-Zasp52-PKCurrent studyN/AZasp52-PK isoform cloned into pGADT7GW using Gateway cloning.
Recombinant DNA reagentPlasmid: pGBKT7GW-Zasp52-PECurrent studyN/AZasp52-PE isoform cloned into pGBKT7GW using Gateway cloning
Recombinant DNA reagentPlasmid: pGADT7GW-Zasp52-PECurrent studyN/AZasp52-PE isoform cloned into pGADT7GW using Gateway cloning.
Recombinant DNA reagentPlasmid: pGBKT7GW-Zasp66-PHCurrent studyN/AZasp66-PH isoform cloned into pGBKT7GW using Gateway cloning
Recombinant DNA reagentPlasmid: pGADT7GW-Zasp66-PHCurrent studyN/AZasp66-PH isoform cloned into pGADT7GW using Gateway cloning.
Recombinant DNA reagentPlasmid: pDEST- pUAS-RfB-HA-CYFP-attBSven Bogdan PMID: 20937809FBrf0212496
Recombinant DNA reagentPlasmid: pDEST- pUAS-RfB-myc-NYFP-attBSven Bogdan PMID: 20937809FBrf0212496
Strain, strain background (Saccharomyces cerevisiae)Matchmaker Y2HGoldClontech630498
Strain, strain background (E. coli)BL21NEBC2530H
AntibodyAnti-FlagSIGMAF31651:200
Software, algorithmR Project for Statistical Computing: base and ape packageshttps://cran.r-project.org/RRID:SCR_001905
Software, algorithmSalmonhttps://combine-lab.github.io/salmon/RRID:SCR_017036
Software, algorithmImageJ/Fiji distributionhttps://fiji.sc/RRID: SCR_002285
Software, algorithmGalaxyhttps://usegalaxy.org/RRID:SCR_006281
Chemical compound, drugethylene glycol bis-sulfosuccinimidyl succinate (EGS)Fisher Scientific21565
Chemical compound, drugYPDA mediumClontech630464
Chemical compound, drugMinimal SD BaseClontech630411
Chemical compound, drug-Leu /- Trp DO SupplementClontech630417
Chemical compound, drug-Ade /- His /- Leu/-Trp DO SupplementClontech630428
Chemical compound, drugActi-stain 488 phalloidinCYTOSKELETON, INCPHDG1-A
Chemical compound, drugAlexa633-PhalloidinFisher ScientificA22284
Chemical compound, drugRhodamine-phalloidinFisher Scientific10063052

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