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Mob4-dependent STRIPAK involves the chaperonin TRiC to coordinate myofibril and microtubule network growth [1]

['Joachim Berger', 'Australian Regenerative Medicine Institute', 'Monash University', 'Clayton', 'Victoria Node', 'Embl Australia', 'Silke Berger', 'Peter D. Currie']

Date: 2022-09

Myofibrils of the skeletal muscle are comprised of sarcomeres that generate force by contraction when myosin-rich thick filaments slide past actin-based thin filaments. Surprisingly little is known about the molecular processes that guide sarcomere assembly in vivo, despite deficits within this process being a major cause of human disease. To overcome this knowledge gap, we undertook a forward genetic screen coupled with reverse genetics to identify genes required for vertebrate sarcomere assembly. In this screen, we identified a zebrafish mutant with a nonsense mutation in mob4. In Drosophila, mob4 has been reported to play a role in spindle focusing as well as neurite branching and in planarians mob4 was implemented in body size regulation. In contrast, zebrafish mob4 geh mutants are characterised by an impaired actin biogenesis resulting in sarcomere defects. Whereas loss of mob4 leads to a reduction in the amount of myofibril, transgenic expression of mob4 triggers an increase. Further genetic analysis revealed the interaction of Mob4 with the actin-folding chaperonin TRiC, suggesting that Mob4 impacts on TRiC to control actin biogenesis and thus myofibril growth. Additionally, mob4 geh features a defective microtubule network, which is in-line with tubulin being the second main folding substrate of TRiC. We also detected similar characteristics for strn3-deficient mutants, which confirmed Mob4 as a core component of STRIPAK and surprisingly implicates a role of the STRIPAK complex in sarcomerogenesis.

To gain novel insights into these processes we facilitated a zebrafish screen that resulted in the discovery of a novel molecule involved in the coordination of the contractile apparatus assembly: The highly conserved MOB family member Mob4. Whereas loss of mob4 led to impaired actin biogenesis and defects in the contractile apparatus assembly, which resembled aspects of human myopathy disorders, gain of mob4 function resulted in a higher amount of contractile apparatus. Further analyses of strn3-deficient mutants demonstrated that Mob4 functions within a protein complex called striatin-interacting phosphatases and kinases (STRIPAK). Mob4 also involves another protein complex called TRiC, which is required for actin and tubulin biogenesis. Whereas actin is the main component of the muscle’s thin filaments, tubulin constitutes the microtubule network essential for neuronal axons. Accordingly, all analysed mutants for mob4 and strn3 featured neuronal as well as muscle defects. We thus conclude that the two protein complexes STRIPAK and TRiC interact through Mob4 to coordinate growth of the myofibril and microtubule network.

Within muscle, highly organised filaments slide over each other to generate the force required for the movement of our bodies. Assembly of this contractile apparatus is not well understood and its regulation remains enigmatic.

Funding: JB and PDC were supported by the National Health and Medical Research Council of Australia (APP1144159 and APP21145821, respectively). The Australian Regenerative Medicine Institute is supported by grants from the State Government of Victoria and the Australian Government. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

To discover novel molecules involved in thin filament assembly and better understand this process, we initiated a genetic screen in zebrafish that resulted in the identification of mob4-deficient mutants with sarcomeric defects and aggregates that resembled aspects of human nemaline myopathy. We reveal that the interaction of Mob4 with TRiC is required for the coordination of sarcomere assembly. Comparable to TRiC deficiency, the microtubule network of retinal ganglion cells is compromised in mob4 geh mutants. Additionally, loss of the STRIPAK scaffold Strn3 within generated zebrafish mutants resemble the defects found in mob4- and TRiC-deficient mutants. Thus, the Mob4 component of STRIPAK might regulate TRiC function to coordinate growth of the myofibril and the microtubule network.

Another macromolecular complex is the striatin-interacting phosphatases and kinases (STRIPAK) complex that interacts with a number of different signalling pathways, resulting in numerous cellular and developmental roles for STRIPAK [ 17 ]. Whereas many isoforms and paralogs of STRIPAK subunits can assemble into various STRIPAK variants, Mob4 is a core protein of STRIPAK. Also known as phocein, Mob4 belongs to the family of MOBs (monopolar spindle-one-binder proteins) that are highly conserved in eukaryotes [ 18 ]. In Drosophila, Mob4 has been reported to play a role in the focusing of microtubule-based spindles as well as in axonal microtubule organization and associated neurite branching [ 19 , 20 ]. As demonstrated in rats, the neurological functions of mob4 depend on the STRIPAK complex [ 21 ]. However, various additional roles have been attributed to Mob4, including the limitation of differentiating WNT-signalling midline muscle cells to regulate the body size of planarians [ 22 ], coordination of Hippo and insulin-like receptor signalling to reactivate neural stem cells [ 23 ], or regulation of Hippo to control proliferation of pancreatic cancer cells [ 24 ]. Interestingly, Dlg5 and Slmap within STRIPAK regulate expression of sarcomeric genes via the Hippo pathway [ 25 ], which also regulates protein synthesis in mouse muscle [ 26 ].

The functional units of the myofibril are the highly ordered sarcomeres, which are mainly comprised of interdigitating myosin-rich thick and actin-based thin filaments. A comprehensive description of sarcomere assembly has not been fully established, but different theories have been brought forward. These include the model describing independent I-Z-I complexes that recruit titin to form myofibril [ 4 , 5 ] and the premyofibril model that elucidates the maturation of Z-body-containing premyofibril into myofibril [ 6 ]. It is also established that mechanical tension is required to trigger myofibril assembly [ 7 ]. Likewise, the details of the assembly of sarcomeric thin filaments are still enigmatic. Current models suggest that this process initiates at the Z-disc of sarcomeres, where monomeric α-actin is folded by the chaperonin complex TRiC (T-complex polypeptide-1 ring complex or CCT) [ 8 ]. Folded actin is passed on to the co-chaperon Bag3 that interacts with the capping protein CapZ and the Z-disc protein α-actinin to initiate actin polymerisation along the nebulin scaffold [ 9 – 13 ]. Subsequent polymerisation dynamics and final capping of thin filaments is mediated by Leiomodins and Tropomodulin4 [ 14 , 15 ]. In addition to skeletal muscle α-actin, TRiC also folds α- and β-tubulin that polymerise to form microtubules required also for neurite formation [ 16 ]. Accordingly, TRiC loss-of-function in zebrafish is characterised by neuronal as well as sarcomeric defects provoked by a compromised microtubule and thin filament assembly, respectively [ 8 ].

Skeletal muscle has a remarkable plasticity, as it can regenerate even repeated traumas and rapidly shrinks during physical inactivity or aging (sarcopenia), making muscle weakness a major contributor to both mortality and morbidity [ 1 ]. Likewise, deficits in sarcomere assembly lead to impaired myofibril function, resulting in heterogeneous muscle diseases including myopathies [ 2 ]. However, little is known about the molecular pathways that regulate these processes and how the muscle’s sarcomeres are assembled [ 3 ].

Results

Muscle integrity is compromised in the zebrafish mutant gemütlich To study sarcomere assembly, a forward genetic screen was performed in zebrafish utilising muscle birefringence. Birefringence is a feature of the pseudo-crystalline myofibril that enables muscle fibres to appear bright under polarized light in an otherwise dark environment. Thus, myofibril defects can be readily detected under polarized light and quantified by measuring the muscle’s brightness [27]. Mutations were randomly introduced by N-ethyl-N-nitrosourea in male zebrafish and germline mutations were stabilised by out-crossing of the males over two generations [28]. 126 F2 families were established and screened for myofibril deficiencies by birefringence analysis at 3 days post fertilization (dpf). One zebrafish mutant that appeared unremarkable under brightfield conditions showed a birefringence reduction under polarised light (Fig 1A and 1B). Quantification of the birefringence at 3 dpf revealed that the birefringence of this mutant was significantly reduced compared to the siblings, indicating that the amount of organised myofibril could be diminished (Fig 1C). Accordingly, although the mutant was touch sensitive at 3 dpf (S1 Video), its motility and forward thrust was impaired compared to the siblings (S2 Video). In relation to the muscle phenotype, this mutant was named gemütlich (geh), German for laid-back. In contrast to viable siblings and starved siblings that died at 11 dpf, geh homozygotes progressively showed cardiac edema and signs of impaired movement and did not survive past 6 dpf, indicating that geh mutants potentially harbour defects in addition to a muscle-related inability to hunt for food. To further assess the muscle of geh mutants, cross sections were H&E-stained at 3 dpf. Neither signs of fibrosis nor dystrophic fibers were detected on H&E-stained sections of geh homozygotes, indicating that degradation of entire myofibres, typically seen in dystrophic muscle [29], are absent (Fig 1D). Cross-sectional areas (CSA) of geh homozygous skeletal muscle (0.028 ± 0.001 mm2) were comparable to siblings (0.0280 ± 0.0008 mm2) (n = 5, P = 0.95 calculated by Student’s t-test). PPT PowerPoint slide

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TIFF original image Download: Fig 1. The muscle integrity is compromised in gemütlich (geh) mutants. (A) At 3 dpf, geh mutants appeared unremarkable under bright-field microscopy. (B) In comparison to their siblings, (B’) 3-dpf-old geh homozygotes appeared darker in representative images taken under polarised light conditions. (C) After rescaling to siblings (100 ± 1%), the birefringence of 3-dpf-old geh homozygotes was significantly reduced to 61 ± 1%. Crosses represent averaged birefringence of clutches with a minimum of 6 larvae per genotype (n = 5 clutches). Data are presented as mean ± SEM; *** P < 0.001 calculated by Student’s t-test. (D) Fibrotic signs were not detected on H&E-stained cross sections of 3-dpf-old geh homozygotes and siblings (n = 6 per genotype). Scale bar sizes are indicated. https://doi.org/10.1371/journal.pgen.1010287.g001 Taken together, a genetic screen resulted in the isolation of the zebrafish mutant gemütlich that feature a significant reduction in birefringence.

The zebrafish mutant gemütlich harbours a nonsense mutation in mob4 To identify the phenotype-causing mutation, geh mutants were subjected to positional cloning based on SNP analysis. The offspring a single geh mapping cross was sorted using birefringence analysis and the genomic DNA of pooled homozygotes and siblings was sequenced by next generation sequencing. Sequence variants were identified via the MiModD software. Regions of homozygosity were only found on chromosome 9 with the highest peak located between 32 to 33 Mb (Fig 2A). Subsequent sequence analysis discovered a single nucleotide change (from C to T) resulting in a nonsense mutation (Q41X) in exon 2 of MOB family member 4 (mob4) located within the homozygosity region (Fig 2B and 2C). Sequence alignment of Mob4 from different species showed the protein’s high conservation and the position of the affected amino acid (S1 Fig). Other mutations, predicted to alter gene functions, were not found within the linked locus. Loss of Mob4 protein in mob4geh homozygotes was confirmed by Western blot using antibodies against human MOB4 (Fig 2D). To confirm that the muscle phenotype of mob4geh is induced by a mutated mob4 allele, knockdown of mob4 was performed with two independent morpholinos: the splice-altering morpholino mob4_3D(+93–16), which targets the splice donor of exon 3, and the translation-blocking morpholino mob4_ATG(-9+16). Administration of both morpholinos induced a significant birefringence reduction compared to control injected wildtypes, resembling the birefringence reduction of mob4geh (Fig 2E, 2F and 2G). Functionality of mob4_3D(+93–16) was confirmed by RT-PCR that demonstrated the altered splicing of mob4 within the morphants (Fig 2H). Furthermore, a second mob4 mutant allele was generated by CRISPR/Cas9 technology. Administration of a single guidance RNA targeting exon 1 led to isolation of the mob4-13 mutant line that harboured a genomic deletion of 13 base pairs (bp) annotated as coding sequence located directly downstream of the ATG translation start of mob4 (NM_001003439c.5_17del) (Figs 2I and S2). In comparison to their siblings, the birefringence of mob4-13 homozygotes as well as mob413/geh compound heterozygotes was significantly reduced, confirming that the muscle phenotype of mob4geh can be attributed to the mutations within mob4 (Fig 2J). Similar to mob4geh, mob4-13 homozygotes did not survive past 6 pdf. PPT PowerPoint slide

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TIFF original image Download: Fig 2. The function of mob4 is lost within gemütlich mutants. (A) Linkage analysis of gemütlich revealed a region of homozygosity on chromosome 9 with a peak between 32 to 33 Mb. (B) The MOB family member 4 (mob4) gene was located within the linked region. (C) Genomic sequences show that the mutant gemütlich harboured a mob4 allele with a premature stop codon in exon 2 (Q41X). (D) Western blot analysis using antibodies against human MOB4 showed epitope loss in mob4geh homozygotes. (E) Knockdown of mob4 by the morpholinos mob4_3D(+93–16) that targets the splice donor of exon 3 or (F) mob4_ATG(-9+16) that targets the translation start codon led to a reduction in birefringence. (G) Compared to control injected 3-dpf-old larvae (100 ± 1% and 100 ± 2%, respectively), administration of mob4_3D(+93–16) induced a reduction in birefringence to 71 ± 2% and mob4_ATG(-9+16) to 65 ± 6%. Crosses represent individual larvae (n = 6). (H) RT-PCR using primers targeting exons 1 and 5 of mob4 revealed altered splicing in mob4_3D(+93–16)-injected larvae. (I) The mob4-13 allele harboured a genomic deletion of 13 bp from exon 1 (g.5_17del). (J) Compared to 3-dpf-old siblings (both 100 ± 1%), the birefringence of mob4-13 homozygotes and mob4-13/geh compound heterozygotes was significantly reduced to 63 ± 1% and 61.1 ± 0.8%, respectively. Crosses represent averaged birefringence of clutches with a minimum of 6 larvae per genotype (n = 5 clutches). Data are presented as mean ± SEM; *** P < 0.001 calculated by Student’s t-test. https://doi.org/10.1371/journal.pgen.1010287.g002 In summary, the reduced birefringence of mob4geh mutants is caused by loss of mob4 function.

Mob4 locates at the sarcomere’s Z-disc within cranial and trunk myofibres, where it is involved in the regulation of myofibril growth To analyse whether Mob4 locates subcellularly at the myofibril, antibodies against human MOB4 were used on 3-dpf-old sagittal muscle sections. Within wildtype zebrafish, Mob4 protein localised to the sarcomere’s Z-discs, which were identified by co-localisation with antibodies against the Z-disc marker α-Actinin (Fig 3A). As expected from the Western blot (Fig 2D), MOB4 antibodies did not locate to a specific region in skeletal muscle of 3-dpf-old mob4geh homozygotes (S3 Fig). To further confirm that mob4 function is required for myofibril assembly, the transgenic line Tg(cry:GFP;-503unc:mob4) was generated that expressed transgenic mob4 under the control of the muscle-specific 503unc promoter [30]. Birefringence analysis at 3 dpf demonstrated that directed expression of mob4 significantly rescued the birefringence of non-transgenic mob4geh homozygotes, further verifying that the birefringence reduction of mob4geh is caused by the mutant mob4geh allele (Fig 3B). Interestingly, the birefringence of Tg(cry:GFP;-503unc:mob4) transgenic mob4geh homozygotes and siblings was significantly higher compared to non-transgenic siblings, suggesting that an increase of Mob4 levels could result in an increase in the amount of organised myofibril. This important finding suggests that mob4 function might be involved in the regulation of the amount of organised myofibril. To additionally verify the Z-disc location of Mob4 protein in live zebrafish, the transgenic line Tg(cry:GFP;-503unc:mob4-GFP) was generated to express the Mob4-GFP fusion protein in skeletal muscle. In-line with the immunohistochemical results, Mob4-GFP co-localised with t-tubules that coincide with the Z-disc and were marked with transgenic mCherry-CAAX in the transgenic background of Tg(acta1:mCherryCAAX) (Fig 3C). Importantly, functionality of GFP-tagged Mob4 was confirmed by the rescue of the birefringence reduction of mob4geh mutants as revealed by birefringence analysis at 3 dpf (S4 Fig). Similar to the transgenic Mob4, forced activation of Mob4-GFP also resulted in a birefringence that was significantly higher compared to non-transgenic mob4geh homozygotes and siblings. PPT PowerPoint slide

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TIFF original image Download: Fig 3. Mob4 is located at the Z-disk, where it might be involved in the regulation of myofibril assembly. (A) At 3dpf, antibodies against human MOB4 colocalised with antibodies against the Z-disc protein α-Actinin (n = 6 per genotype). (B) At 3 dpf, the birefringence of mob4geh homozygotes was significantly higher in the transgenic background of Tg(cry:GFP;-503unc:mob4). Also compared to non-transgenic siblings (100 ± 2%), the birefringence of Tg(cry:GFP;-503unc:mob4) transgenic mob4geh homozygotes (108 ± 1%) and siblings (106 ± 1%) was significantly higher. Crosses represent averaged birefringence of clutches with a minimum of 4 larvae per genotype (n = 5 clutches). Data are presented as mean ± SEM; *** P < 0.001 and ** P < 0.01 by one-way ANOVA with post hoc Tukey’s test. (C) In 3-dpf-old siblings and mob4geh homozygotes, Mob4-GFP fusion protein (green) expressed via Tg(cry:GFP;-503unc:mob4-GFP) colocalised to t-tubules (red, arrowhead) marked in the Tg(acta1:mCherryCAAX) transgenic background (n = 3 per genotype). (D) Highlighting F-actin with transgenic Tg(acta1:lifeact-GFP) in green confirmed residual myofibril striation and revealed disorganised thin filaments within mob4geh homozygotes at 3 dpf. Sarcolemma and t- tubules were labelled by mCherry fluorescence (red) in the Tg(acta1:mCherryCAAX) transgenic background (n = 6 per genotype). Boxed areas are magnified. (E) Labelling of F-actin with phalloidin revealed that the robust myofibril striation of siblings was reduced in mob4geh homozygotes at 3 dpf (n = 6 per genotype). (F) At 3 dpf, antibodies against α-Actinin that mark sarcomere’s Z-disks showed the typical striation of the myofibril in siblings and mob4geh homozygotes (n = 4 per genotype). (G) Visualisation of the cephalic muscles in the transgenic Tg(−503unc:GFP) background revealed that, in contrast to siblings, a gap was formed between the two hyohyoideus (hh) muscles in mob4geh homozygotes at 3 dpf (representative Z-stacks) (n = 3 per genotype). (H) At 6 dpf, representative Z-stack projections of Alcian blue stained larvae depicted cartilage malformations in mob4geh homozygotes and a widened angle formed by the two ceratohyal cartilage structures (dotted lines) (n = 4 per genotype). Designations: ceratohyal (ch); Meckel’s cartilage (m); palatoquadrate (pq). Scale bar sizes are indicated. https://doi.org/10.1371/journal.pgen.1010287.g003 To assess the sarcomere organisation within live mob4geh mutants, mob4geh was crossed into the transgenic background of Tg(acta1:lifeact-GFP) and Tg(acta1:mCherryCAAX). The fusion protein Lifeact-GFP directed GFP fluorescence to thin filaments, and the sarcolemma as well as t-tubules were marked by the integration of mCherry-CAAX [31]. In-line with the previously documented reduction in birefringence, Lifeact-GFP detected residual myofibril striation within live mob4geh homozygotes, but also highlighted isolated and misoriented thin filaments (Fig 3D). Quantification of the combined diameter of striated myofibril within myofibres confirmed that striated myofibril is significantly reduced within mob4geh mutants (13.8 ± 0.3 μm in siblings and 8.7 ± 0.2 μm in homozygotes, n = 4, P < 0.01 calculated by Student’s t-test). To confirm the residual striation in mob4geh homozygotes, the F-actin marker phalloidin was used to expose regular striated sarcomeres within mob4geh mutants. As indicated by birefringence analysis, striation was severely reduced within mob4geh mutants and, in contrast to the siblings, abundant isolated filaments were detected (Fig 3E). Further immunohistochemistry with antibodies against the Z-disc marker α-Actinin revealed that regular sarcomeric Z-discs are formed within mob4geh mutants (Fig 3F). To assess the cranial musculature of mob4-deficient zebrafish, mob4geh homozygotes were crossed into the Tg(-503unc:GFP) transgenic background that marks myofibres with GFP fluorescence. Although the cranial musculature of mob4geh homozygotes appeared anatomically comparable to siblings at 3 dpf, a gap was formed between the two contralateral hyohyoideus muscles within mob4geh homozygotes (Fig 3G). Similarly, severe cartilage malformations were apparent in Alcian Blue stained mob4geh homozygotes at 6 dpf, which is indicative of muscle weakness as muscle force is known to affect cartilage morphology (Fig 3H). Taken together, myofibril assembly within the trunk muscle is compromised and the cranial musculature could be weakened within mob4geh mutants. The abundance of isolated thin filaments further suggests a defective processing of thin filaments, which is in-line with the Mob4 localisation at Z-discs, where thin filament assembly is initiated. In addition, the enhanced birefringence after directed activation of mob4 indicates that mob4 function might be involved in the regulation of myofibril growth.

Thin filament biogenesis in mob4geh results in nemaline-like bodies To study the sarcomere organisation within mob4-deficient mutants in more detail, transmission electron microscopy (TEM) was performed. Consistent with previous results, residual organised sarcomeres, comparable to the ones within siblings, were found within 3-dpf-old mob4geh homozygotes (Fig 4A and 4B). However, additional disorganised and fragmented sarcomeric structures along with isolated filaments were detected in mob4geh mutants using TEM, indicating that sarcomere assembly is disrupted (Fig 4B, 4C and 4D). Furthermore, widened Z-discs and small electron-dense structures close to Z-discs of mob4geh mutants were revealed by TEM (Fig 4B, 4C and 4D). Interestingly, the electron-dense aggregates of mob4geh mutants often featured a lattice structure, which is characteristic for nemaline bodies that define human nemaline myopathy [32]. To further characterise the detected electron-dense aggregates, Gomori trichrome staining, a clinical marker for nemaline bodies [2], was performed on cross sections of 3-dpf-old larvae. In contrast to the siblings, subsarcolemmal structures were stained in dark blue in mob4geh homozygotes (Fig 4E). Thus, although aggregates were not detected using α-Actinin antibodies and thin filament markers that typically mark nemaline bodies in humans, TEM and Gomori trichrome staining indicated the presence of nemaline-like bodies within mob4geh mutants. PPT PowerPoint slide

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TIFF original image Download: Fig 4. Sarcomere organisation is compromised in mob4geh mutants. (A) Transmission electron micrograph depicted highly organised and arrayed myofibrils in 3-dpf-old siblings (n = 3). (B) Organised sarcomeres were rarely detected within mob4geh homozygotes (n = 3). (B’) As shown in the magnification of the boxed area, sarcomeres were frequently disorganised and deposits of isolated filaments (asterisk) in addition to electron-dense structures (arrowhead), often associated with Z-disks, were found instead. (C) Electron-dense aggregates of mob4geh homozygotes often showed a lattice structure (arrowhead) and (D) fragmented sarcomeres and widened Z-disks (double-arrowhead) were detected as well. (E) At 3 dpf, Gomori trichrome staining revealed subsarcolemmal dark blue structures within mob4geh homozygotes but not siblings (n = 6 per genotype). Boxed areas are shown in higher magnification. (F) GFP fluorescence of transgenic ACTA1-GFP showed a striated pattern in 3-dpf-old siblings and a uniform pattern in mob4geh homozygotes. Expression of ACTA1D286G-GFP led to rod-shaped structures in siblings and exclusively amorphic aggregates within mob4geh homozygotes (n = 6 per genotype). Scale bar sizes are indicated. https://doi.org/10.1371/journal.pgen.1010287.g004 To further assess actin biogenesis and aggregate formation within mob4geh mutants, fusion proteins of GFP with human skeletal α-actin (ACTA1-GFP) and a mutant isoform of α-actin (ACTA1D286G-GFP) were transiently expressed under the muscle-specific 503unc promoter [30]. ACTA1-GFP has been shown to incorporate into sarcomeres of mice and zebrafish, whereas ACTA1D286G-GFP, which has been associated with nemaline myopathy in humans, has been reported to form rod-shaped nemaline bodies in addition to its sarcomere integration [33,34]. In 3-dpf-old siblings, the obtained striated GFP fluorescence pattern reported the expected ACTA1-GFP incorporation into sarcomeres (Fig 4F). In mob4geh mutants, however, ACTA1-GFP was not incorporated into residual sarcomeres as shown by the uniform GFP fluorescence, indicating that α-actin processing is affected within mob4geh mutants (Fig 4F). Expression of the mutant isoform ACTA1D286G-GFP in siblings led to the expected GFP fluorescence from GFP-positive striated sarcomeres and rod-shaped nemaline bodies. In mob4geh homozygotes, however, GFP fluorescence visualised exclusively amorphic aggregates along with a faint striated pattern, demonstrating that mob4 is also involved in the formation of rod-shaped nemaline bodies (Fig 4F). In conclusion, mob4 function is required for the incorporation of skeletal muscle α-actin into organised sarcomeres and loss of mob4 function results in aggregates, which share only some aspects of nemaline bodies present in human nemaline myopathy.

Mob4 interacts with TRiC to regulate myofibril growth TRiC locates at the Z-disc similar to Mob4 and both single loss-of-function mutants, mob4geh and cct3sa1761, are characterised by compromised α-actin biogenesis and nemaline-like body formation [8]. Furthermore, TRiC and Mob4 are part of a multiprotein complex, as shown by co-immunoprecipitation in human cells [35], in which they directly interact as demonstrated by genome-wide association studies in nematodes [36]. Co-localisation of Mob4 and TRiC was confirmed with antibodies against human MOB4 and human CCT5 (S5 Fig). In order to genetically evaluate the interaction of Mob4 with TRiC in vivo, mob4geh was crossed to cct3sa1761, in which cct3 deficiency results in loss of TRiC function [8]. At 3 dpf, TEM micrographs depicted highly organised sarcomeres in siblings and the expected nemaline-like aggregates in single cct3sa1761 homozygotes (Fig 5A), which were comparable in shape and location to the aggregates of mob4geh (Fig 4B). However, compound mob4geh;cct3sa1761 homozygotes were devoid of electron-dense aggregates. To further evaluate this interesting finding, birefringence analysis was performed at 3 dpf. Analysis of single cct3sa1761 homozygous siblings resulted in the expected severe birefringence reduction [8], but the birefringence of mob4geh;cct3sa1761 homozygotes was significantly higher, demonstrating a significant amelioration of the cct3sa1761 birefringence reduction (Fig 5B). The increased birefringence of compound mob4geh;cct3sa1761 homozygotes, which were devoid of aggregates, over single mutants that featured aggregates, also suggests that aggregates could contribute to the severity of myopathies. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Mob4 interacts with TRiC. (A) As depicted in transmission electron micrographs, the sarcomere organisation detected in siblings was compromised in single cct3sa1761 homozygotes as well as cct3sa1761;mob4geh compound homozygotes. Electron-dense aggregates (arrowhead) as found in cct3sa1761 homozygotes were absent in cct3sa1761;mob4geh compound homozygotes (n = 3 per genotype). Scale bar sizes are 2 μm. (B) After rescaling to siblings (100 ± 1%), the birefringence of cct3sa1761;mob4geh compound homozygotes (31.4 ± 0.6%) was significantly ameliorated compared to single cct3sa1761 homozygotes (22.7 ± 0.3%). Crosses represent averaged birefringence of clutches with a minimum of 4 larvae per genotype (n = 5 clutches). Data are presented as mean ± SEM; *** P < 0.001 by one-way ANOVA with post hoc Tukey’s test. https://doi.org/10.1371/journal.pgen.1010287.g005 In summary, analysis of compound mob4geh;cct3sa1761 mutants, in combination with the previously established molecular interaction between MOB4 and TRiC in human cells [35] and our co-localization results, is consistent with the model that the two proteins interact in vivo in zebrafish as well, and that mob4 function is required for the establishment of normal myofibril structure. Furthermore, compound mob4geh;cct3sa1761 mutants that were devoid of aggregates suggesting that aggregates could contribute to the severity of myopathies.

Neuronal connectivity is compromised in mob4geh Drosophila dMob4 has been reported to play a role in microtubule organization, which is essential for neurite branching and axonal transport [19]. Besides α-actin, TRiC also folds α- and β-tubulin and accordingly neuronal neurite formation is severely reduced in TRiC-deficient zebrafish [8,16]. To analyse neurons within mob4geh, cranial sections were stained with toluidine blue at 3 dpf. Although the size of the retina of mob4geh homozygotes (23’800 ± 200 μm2) was significantly smaller compared to their siblings (25’000 ± 100 μm2) (S6 Fig), the morphology appeared largely comparable to their siblings (Fig 6A). However, in contrast to their siblings, pyknotic nuclei were detected dispersed throughout the retina and the tectum of mob4geh homozygotes (Fig 6A). To study the cell death in more detail, Terminal deoxynucleotidyl transferase dUTP nick-end labelling (TUNEL) staining was performed on cryopreserved sections to detect apoptosis. Whereas apoptotic signals were barely detected in 3-dpf-old siblings, abundant apoptotic cells were present within the retina and tectum of mob4geh homozygotes (Fig 6B). Neuron viability depends on neurites to innervate their target tissues and the axons of the retinal ganglion cells project through the optic chiasm to the contralateral tectum. To highlight retinal ganglion cells with GFP fluorescence in live zebrafish, mob4geh was crossed into the transgenic background of Tg(atoh7:GFP) [37]. Consistent with the tectonal and retinal apoptosis, retinal ganglion cells of 3-dpf-old siblings were found to project via the optic chiasm to the optic tectum, however within mob4geh homozygotes only somas of retinal ganglion cells were detected (Fig 6C and 6D). To assess the microtubule network required for axon formation, immunostaining with antibodies against acetylated α-tubulin was performed. In contrast to the siblings, a severe reduction in tectonal neurites was detected within mob4geh homozygotes and the intertectal commissure was completely absent, indicating that microtubules are compromised within mob4geh (Fig 6E). The intertectal commissure was also lost in mob4-13 homozygotes (S7 Fig), suggesting that in addition to the muscle phenotype also the neuronal phenotype of mob4-13 homozygotes phenocopied mob4geh mutants. PPT PowerPoint slide

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TIFF original image Download: Fig 6. Loss of mob4 function compromises neuronal connectivity. (A) At 3 dpf, toluidine blue-stained sections displayed pyknotic nuclei (arrowhead) dispersed throughout the retina and tectum of mob4geh homozygotes, not siblings (n = 4 per genotype). (B) Abundant apoptosis within the retina and tectum of 3-dpf-old mob4geh homozygotes was detected by TUNEL assay (n = 8 per genotype). (C) In representative ventral views (Z-stack), the optic chiasm (asterisk) was highlighted by Tg(ath7:GFP) within 3-dpf-old siblings but not mob4geh homozygotes (n = 3 per genotype). (D) In representative Z-stacks, axons of retinal ganglion cell marked by Tg(ath7:GFP) project contralaterally via the optic chiasm (asterisk) from the retina (arrowhead) onto the tectum (arrow) of 3-dpf-old siblings (n = 4). In contrast, axons were not formed by retinal ganglion cells (arrowhead) of mob4geh homozygotes (n = 4). (E) In Z-stacks of 3-dpf-old larvae, antibodies against acetylated α-tubulin revealed defective neurite formation within the tectum of mob4geh homozygotes. Boxed areas are shown in higher magnification (n = 3 per genotype). Scale bar sizes are indicated. https://doi.org/10.1371/journal.pgen.1010287.g006 In conclusion, it could be speculated that the compromised microtubule network within mob4-deficient zebrafish could lead to the severely reduced axon formation of retinal ganglion cells, which could consequently result in the detected retinal and tectonal apoptosis and the diminished retina size.

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[1] Url: https://journals.plos.org/plosgenetics/article?id=10.1371/journal.pgen.1010287

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