The oxoglutarate dehydrogenase complex is involved in myofibril growth and Z-disc assembly in Drosophila

Figures

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  Fig. 1.. OGDH localizes to the Z-disc. (A) Heatmap showing the ERC values from all the hits in the ERC screen ordered by automatic clustering. Note the Zasp52/Zasp66 and the Zasp52/actinin clusters. The color scale of the ERC values from −1 to +1 is at the bottom. The positions of Actn, Zasp52, Zasp66 and OGDH are indicated. (B) Confocal image of muscles carrying the OGDHMI06026-GFSTF.1 GFP trap allele. OGDH-GFP (magenta) and actin filaments (green); OGDH-GFP localizes to the Z-disc. Composite and single-channel images are shown. Asterisks show selected Z-discs in B. Scale bar: 5 µm. (C-G) Super-resolution dSTORM images of the individual subunits of the OGDH complex as intensity heat maps (see also Fig. S2C). (C) The E1 subunit, OGDH, localizes to a wide band at the Z-disc core. (D) The DLD subunit localizes to two discrete bands that overlap the periphery of the E1 subunit distribution. (E) The E2 core subunit DLST localizes to a wide band at the Z-disc core in the same way as the E1 subunit does. (F,G) Composite dSTORM images: OGDH with DLD (F) and OGDH with DLST (G). Scale bar: 500 nm. The GFP-tagged versions of the subunits were used together with a GFP antibody.

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  Fig. 2.. Depletion of OGDH results in a small Z-disc. (A,B) Confocal images of control (A) and OGDH-HM (B) muscles with Zasp52-mCherry as a Z-disc marker (magenta) and actin filaments as a reference (green). In OGDH-HM muscles, the Z-discs do not grow to their final size and appear small. Protein aggregates are occasionally present (arrow). (C-E) High-magnification confocal images of OGDH-HM muscles with different sarcomere markers. In C, the Z-disc marked by Zasp52-mCherry is smaller than the myofibril. In D, the M-line marked by Obscurin-GFP covers the entire width of the myofibril. In E, the Z-disc marked by α-actinin-GFP is smaller then the myofibril. In D and E, actin filaments are shown in green. (F,G) Transmission electron microscopy images of control and OGDH-HM sarcomeres. The Z-discs are the black electron-dense structures indicated by red arrows. The H zone is the white region at the center of the sarcomere. In the control image, the Z-disc is well defined and spans the whole diameter of the sarcomere. In OGDH-HM muscles, the Z-disc is reduced to a tiny clump at the middle of the myofibril (arrows). Asterisks indicate selected Z-discs in A and B. Scale bars: 5 µm in A and B;…

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  Fig. 3.. The LIM2 domain of Zasp52 binds OGDH through a ZM-like sequence. (A) Y2H assays show LIM2a and LIM2b are the only domains in Zasp52 that bind OGDH. (B) Cartoon of OGDH-PA and Zasp66-PA proteins with protein domains highlighted. The short sequence common to both proteins is marked by a red line. OGDH-BM has this short sequence removed. (C) Alignment of the common sequence between OGDH-PA and Zasp66-PA isoforms. (D) Y2H assays show that Zasp52-PK binds to OGDH but the interaction is lost in OGDH-BM, a mutant version that lacks the common short sequence. (E) Y2H assays show LIM2a and LIM2b bind the short sequence in OGDH. Serial dilutions are shown from left to right in A, D and E (OD 0.1, OD 0.01 and OD 0.001).

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  Fig. 4.. Zasp52 recruits OGDH to the Z-disc. (A,B) Confocal images of muscles showing BiFC fluorescence and actin staining for reference. (A) Zasp52-PK and OGDH form a complex at the Z-disc; BiFC signal is detected at the Z-disc (asterisks). (B) OGDH-BM does not bind Zasp52-PK at the Z-disc, and the BiFC signal is not detected at the Z-disc. (C) Plots of the BiFC fluorescence intensity values relative to background noise. Physical interaction is detected between Zasp52 and OGDH (blue) (P<0.0001 in all cases). (D,E) Confocal images showing the localization of OGDH-GFP in two Zasp52 mutants. (D) In Zasp52MI02988 mutants, OGDH is properly localized to the Z-disc. (E) In Zasp52MI00979 mutants, OGDH-GFP is not detected at the Z-disc (asterisks). (F) Boxplot of OGDH-GFP intensities in control and Zasp52 mutant backgrounds. The Zasp52MI00979 mutant has reduced Z-disc localization compared with the wild type and Zasp52MI02988 mutant (P=2e-29 and P=2e-27). (G) Confocal images of wild-type or binding mutant OGDH forms tagged with mCherry and box plot of the fluorescence intensity. Flies with one or two copies of OGDH-wt-mCh show Z-disc localization. The OGDH-BM-mCh form has reduced Z-disc…

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  Fig. 5.. Myofibril defects observed after targeting the individual OGDH complex subunits through tissue-specific CRISPR-Cas9. (A-D) Confocal images of muscles with CRISPR-induced mutations of the OGDH complex subunits. Flies are 1-2 days old. Negative control without a gRNA (A), the E1 subunit OGDH (B), the E2 subunit DLST (C) and the E3 subunit DLD (D). Mutations in any of the OGDH complex subunits result in very thin myofibrils that tend to stack on each other. Zasp52 marks the Z-disc in magenta and actin filaments are shown in green. Scale bar: 5 µm.

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  Fig. 6.. Myofibril defects observed after depletion of individual TCA cycle enzymes in the IFM. (A) Cartoon of the TCA cycle in Drosophila; the enzyme-encoding genes are orange and the metabolites are black. The TCA cycle is a loop of chemical reactions and constitutes a metabolic buffering system. The cycle starts when citrate synthase combines acetyl-CoA with oxaloacetate. Aconitase then converts citrate into isocitrate, followed by two oxidative decarboxylation steps. The homodimeric isocitrate dehydrogenase converts isocitrate into oxoglutarate. In the second decarboxylation step, the OGDH complex, which is composed of three subunits (OGDH/E1, DLST/E2 and DLD/E3), converts oxoglutarate into succinyl-CoA, which is converted to succinate by succinyl-CoA synthetase. Succinate dehydrogenase, which is also part of the electron transport chain, then oxidizes succinate to fumarate. Fumarase converts fumarate to malate and, finally, malate dehydrogenase reconstitutes the oxaloacetate. (B) Stacked bar plot of the ratio of myofibrils that appear normal (blue), have mild defects (orange) and have strong defects (red) in different RNAi conditions. (C-H) Confocal images showing the myofibril…

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  Fig. 7.. Gas chromatography-mass spectrometry metabolomic analysis of OGDH-HM reveals a defect in amino acid balance. (A) Volcano plot of all the metabolites analyzed in control (Act88F-Gal4, Zasp52-GFP) and OGDH-HM (Act88F-Gal4, UAS-OGDH-HM, Zasp52-GFP) samples. Significance −log10(P-value) and fold-change log2 are on the y- and x-axis, respectively. Each dot is a unique metabolite; the green dots are metabolites with P<0.05 and absolute log2FoldChange values>1. The names of selected metabolites are shown. There is an accumulation of oxoglutarate and an imbalance in many amino acids. (B) Box plots of selected metabolites in control and OGDH-HM (OGDHi) samples. The green panel shows oxoglutarate is approximately three times more concentrated in OGDH-HM than in controls, validating the approach. The blue panel shows the reduction of β-alanine, homoserine, aspartic acid and histidine in OGDH-HM compared with the control samples. The red panel shows the accumulation of several amino acids. Sarcosine is an amino acid intermediate of glycine metabolism. The box represents the 25–75th percentiles, and the median is indicated. The whiskers show the minimum and maximum values.

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  Fig. 8.. A general block in protein synthesis results in smaller but otherwise normal myofibrils. (A) IFM in which general translation is inhibited during metamorphosis for a maximum time of 48 h. The time at which the translation block was initiated is indicated at the top of each image. Blocking ribosome function using temperature-sensitive ricin at different time points results in the differential blocking of myofibril diameter growth. Blocking ribosome function at ∼28 h after pupa formation (APF) completely abolishes striation. Blocking ribosome function at ∼32 h APF results in myofibril striation, but diameter growth is impaired. Blocking ribosome function at ∼56 h APF results in smaller diameters but otherwise normal myofibrils. Blocking ribosome function at ∼80 h APF does not affect myofibril appearance much. (B-F) Confocal images of control and OGDH-HM muscles expressing a GFP-tagged version of RpS5a (Ribosome-GFP); actin filaments are also shown. Flies are 1-2 days old. (B) In control samples (Act88F-Gal4, UAS-RpS5a-GFP), ribosomes are spread in the cytoplasm but accumulate at the Z-disc. (C) In OGDH-HM samples (Act88F-Gal4, UAS-RpS5a-GFP, UAS-OGDH-HM), ribosomes are mostly…