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

Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We have previously shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here, we looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc.

PK1 from Drosophila obscurin is an inactive pseudokinase with scaffolding properties.

Obscurins are large filamentous proteins with crucial roles in the assembly, stability and regulation of muscle. Characteristic of these proteins is a tandem of two C-terminal kinase domains, PK1 and PK2, that are separated by a long intrinsically disordered sequence. The significance of this conserved domain arrangement is unknown. Our study of PK1 from Drosophila obscurin shows that this is a pseudokinase with features typical of the CAM-kinase family, but which carries a minimalistic regulatory tail that no longer binds calmodulin or has mechanosensory properties typical of other sarcomeric kinases. PK1 binds ATP with high affinity, but in the absence of magnesium and lacks detectable phosphotransfer activity. It also has a highly diverged active site, strictly conserved across arthropods, that might have evolved to accommodate an unconventional binder. We find that PK1 interacts with PK2, suggesting a functional relation to the latter. These findings lead us to speculate that PK1/PK2 form a pseudokinase/kinase dual system, where PK1 might act as an allosteric regulator of PK2 and where mechanosensing properties, akin to those described for regulatory tails in titin-like kinases, might now reside on the unstructured interkinase segment. We propose that the PK1-interkinase-PK2 region constitutes an integrated functional unit in obscurin proteins.

Slik phosphorylation of Talin T152 is crucial for proper Talin recruitment and maintenance of muscle attachment in Drosophila.

Talin is the major scaffold protein linking integrin receptors with the actin cytoskeleton. In Drosophila, extended Talin generates a stable link between the sarcomeric cytoskeleton and the tendon matrix at muscle attachment sites. Here, we identify phosphorylation sites on Drosophila Talin by mass spectrometry. Talin is phosphorylated in late embryogenesis when muscles differentiate, especially on T152 in the exposed loop of the F1 domain of the Talin head. Localization of a mutated version of Talin (Talin-T150/T152A) is reduced at muscle attachment sites and can only partially rescue muscle attachment compared with wild-type Talin. We also identify Slik as the kinase phosphorylating Talin at T152. Slik localizes to muscle attachment sites, and the absence of Slik reduces the localization of Talin at muscle attachment sites causing phenotypes similar to Talin-T150/T152A. Thus, our results demonstrate that Talin phosphorylation by Slik plays an important role in fine-tuning Talin recruitment to integrin adhesion sites and maintaining muscle attachment.

Different Evolutionary Trajectories of Two Insect-Specific Paralogous Proteins Involved in Stabilizing Muscle Myofibrils.

Alp/Enigma family members have a unique PDZ domain followed by zero to four LIM domains, and are essential for myofibril assembly across all species analyzed so far. Drosophila melanogaster has three Alp/Enigma family members, Zasp52, Zasp66, and Zasp67. Ortholog search and phylogenetic tree analysis suggest that Zasp genes have a common ancestor, and that Zasp66 and Zasp67 arose by duplication in insects. While Zasp66 has a conserved domain structure across orthologs, Zasp67 domains and lengths are highly variable. In flies, Zasp67 appears to be expressed only in indirect flight muscles, where it colocalizes with Zasp52 at Z-discs. We generated a CRISPR null mutant of Zasp67, which is viable but flightless. We can rescue all phenotypes by re-expressing a Zasp67 transgene at endogenous levels. Zasp67 mutants show extended and broken Z-discs in adult flies, indicating that the protein helps stabilize the highly regular myofibrils of indirect flight muscles. In contrast, a Zasp66 CRISPR null mutant has limited viability, but only mild indirect flight muscle defects illustrating the diverging evolutionary paths these two paralogous genes have taken since they arose by duplication.

Binding partners of the kinase domains in Drosophila obscurin and their effect on the structure of the flight muscle.

Drosophila obscurin (Unc-89) is a titin-like protein in the M-line of the muscle sarcomere. Obscurin has two kinase domains near the C-terminus, both of which are predicted to be inactive. We have identified proteins binding to the kinase domains. Kinase domain 1 bound Bällchen (Ball, an active kinase), and both kinase domains 1 and 2 bound MASK (a 400-kDa protein with ankyrin repeats). Ball was present in the Z-disc and M-line of the indirect flight muscle (IFM) and was diffusely distributed in the sarcomere. MASK was present in both the M-line and the Z-disc. Reducing expression of Ball or MASK by siRNA resulted in abnormalities in the IFM, including missing M-lines and multiple Z-discs. Obscurin was still present, suggesting that the kinase domains act as a scaffold binding Ball and MASK. Unlike obscurin in vertebrate skeletal muscle, Drosophila obscurin is necessary for the correct assembly of the IFM sarcomere. We show that Ball and MASK act downstream of obscurin, and both are needed for development of a well defined M-line and Z-disc. The proteins have not previously been identified in Drosophila muscle.

Sallimus and the dynamics of sarcomere assembly in Drosophila flight muscles.

The Drosophila indirect flight muscles (IFM) can be used as a model for the study of sarcomere assembly. Here we use a transgenic line with a green fluorescent protein (GFP) exon inserted into the Z-disc-proximal portion of sallimus (Sls), also known as Drosophila titin, to observe sarcomere assembly during IFM development. Firstly, we confirm that Sls-GFP can be used in the heterozygote state without an obvious phenotype in IFM and other muscles. We then use Sls-GFP in the IFM to show that sarcomeres grow individually and uniformly throughout the fibre, growing linearly in length and in diameter. Finally, we show that limiting the amounts of Sls in the IFM using RNAi leads to sarcomeres with smaller Z-discs in their core, whilst the thick/thin filament lattice can form peripherally without a Z-disc. Thick filament preparations from those muscles show that although the Z-disc-containing core has thick filaments of a regular length, filaments from the peripheral lattice are longer and asymmetrical around the bare zone. Therefore, the Z-disc and Sls are required for thick filament length specification but not for the assembly of the thin/thick filament lattice.

The roles of troponin C isoforms in the mechanical function of Drosophila indirect flight muscle.

Stretch activation (SA) is a fundamental property of all muscle types that increases power output and efficiency, yet its mechanism is unknown. Recently, studies have implicated troponin isoforms as important in the SA mechanism. The highly stretch-activated Drosophila IFMs express two isoforms of the Ca(2+)-binding subunit of troponin (TnC). TnC1 (TnC-F2 in Lethocerus IFM) has two calcium binding sites, while an unusual isoform, TnC4 (TnC-F1 in Lethocerus IFM), has only one binding site. We investigated the roles of these two TnC isoforms in Drosophila IFM by targeting RNAi to each isoform. IFMs with TnC4 expression (normally ~90% of total TnC) replaced by TnC1 did not generate isometric tension, power or display SA. However, TnC4 knockdown resulted in sarcomere ultrastructure disarray, which could explain the lack of mechanical function and thus make interpretation of the influence of TnC4 on SA difficult. Elimination of TnC1 expression (normally ~10% of total TnC) by RNAi resulted in normal muscle structure. In these IFMs, fiber power generation, isometric tension, stretch-activated force and calcium sensitivity were statistically identical to wild type. When TnC1 RNAi was driven by an IFM specific driver, there was no decrease in flight ability or wing beat frequency, which supports our mechanical findings suggesting that TnC1 is not essential for the mechanical function of Drosophila IFM. This finding contrasts with previous work in Lethocerus IFM showing TnC1 is essential for maximum isometric force generation. We propose that differences in TnC1 function in Lethocerus and Drosophila contribute to the ~40-fold difference in IFM isometric tension generated between these species.

Alp/Enigma family proteins cooperate in Z-disc formation and myofibril assembly.

The Drosophila Alp/Enigma family protein Zasp52 localizes to myotendinous junctions and Z-discs. It is required for terminal muscle differentiation and muscle attachment. Its vertebrate ortholog ZASP/Cypher also localizes to Z-discs, interacts with α-actinin through its PDZ domain, and is involved in Z-disc maintenance. Human mutations in ZASP cause myopathies and cardiomyopathies. Here we show that Drosophila Zasp52 is one of the earliest markers of Z-disc assembly, and we use a Zasp52-GFP fusion to document myofibril assembly by live imaging. We demonstrate that Zasp52 is required for adult Z-disc stability and pupal myofibril assembly. In addition, we show that two closely related proteins, Zasp66 and the newly identified Zasp67, are also required for adult Z-disc stability and are participating with Zasp52 in Z-disc assembly resulting in more severe, synergistic myofibril defects in double mutants. Zasp52 and Zasp66 directly bind to α-actinin, and they can also form a ternary complex. Our results indicate that Alp/Enigma family members cooperate in Z-disc assembly and myofibril formation; and we propose, based on sequence analysis, a novel class of PDZ domain likely involved in α-actinin binding.

The function of the M-line protein obscurin in controlling the symmetry of the sarcomere in the flight muscle of Drosophila.

Obscurin (also known as Unc-89 in Drosophila) is a large modular protein in the M-line of Drosophila muscles. Drosophila obscurin is similar to the nematode protein UNC-89. Four isoforms are found in the muscles of adult flies: two in the indirect flight muscle (IFM) and two in other muscles. A fifth isoform is found in the larva. The larger IFM isoform has all the domains that were predicted from the gene sequence. Obscurin is in the M-line throughout development of the embryo, larva and pupa. Using P-element mutant flies and RNAi knockdown flies, we have investigated the effect of decreased obscurin expression on the structure of the sarcomere. Embryos, larvae and pupae developed normally. In the pupa, however, the IFM was affected. Although the Z-disc was normal, the H-zone was misaligned. Adults were unable to fly and the structure of the IFM was irregular: M-lines were missing and H-zones misplaced or absent. Isolated thick filaments were asymmetrical, with bare zones that were shifted away from the middle of the filaments. In the sarcomere, the length and polarity of thin filaments depends on the symmetry of adjacent thick filaments; shifted bare zones resulted in abnormally long or short thin filaments. We conclude that obscurin in the IFM is necessary for the development of a symmetrical sarcomere in Drosophila IFM.

Muscle type-specific expression of Zasp52 isoforms in Drosophila.

Zasp52 is a member of the PDZ-LIM domain protein family in Drosophila, which comprises Enigma, ENH, ZASP, Alp, CLP36, RIL, and Mystique in vertebrates. Drosophila Zasp52 colocalizes with integrins at myotendinous junctions and with α-actinin at Z-disks, and is required for muscle attachment as well as Z-disk assembly and maintenance. Here we document 13 Zasp52 splice variants giving rise to six different LIM domains. We demonstrate stage- and tissue-specific expression in different muscle types for Zasp52 isoforms encoding different LIM domains. In particular, LIM1b is expressed only in heart muscle and certain somatic muscles, implying muscle-specific functions in Z-disk assembly or maintenance.