Szilárd Szikora

Myofibrils are structurally highly conserved elements of the cardiac and striated muscles. The striking commonalities in their basic organization principles emerge during myofibrillogenesis when the initially formed premyofibrils grow in length and width to attain their final dimensions, characteristic of each muscle type. Although the molecular composition of the myofibrils is well known, the mechanisms governing their growth remain poorly understood, particularly those driving peripheral thickening. Here, we show that two cardiac disease associated proteins, Flightless I and LRRFIP, are required for lateral integration of the myofilaments that is essential for circumferential myofibril growth in the Drosophila flight muscle. Genetic and biochemical analysis reveal that these proteins form multimeric complexes enriched at the barbed end of the actin filaments. Furthermore, we found that the Flightless I/LRRFIP complex acts in a formin dependent manner. Together, our findings demonstrate that Flightless I and LRRFIP cooperate to promote the spatially controlled integration of peripheral actin filaments at the Z-disc, uncovering a key step in myofibril maturation.

Position of the nucleus is dynamically controlled to ensure a variety of cellular functions in a broad range of organisms form yeast to human. Nuclear positioning in Drosophila nurse cells is crucial during dumping when cells transfer their entire cytoplasmic content into the oocyte. An important prerequisite of effective dumping is the formation of an array of actin cables which holds the nucleus in a central position, thereby allowing transmission of the cytoplasmic cargo. Here we report the identification of FRL, a formin type of actin assembly factor, as a novel determinant of cytoplasmic actin bundle formation. We found that FRL and the formerly described Ena protein display a differential requirement. Comparison of the frl and ena loss of function situations revealed that FRL is mainly required for creation of the cytoplasmic actin subpopulation at stage 10B, while Ena mostly promotes formation of a ring canal attached actin array, already present at stage 7 and persists till dumping. Upon the concurrent absence of FRL and Ena the nuclear positioning actin cables are completely missing, strongly suggesting that nuclear positioning in the nurse cells requires the coordinated action of two spatially distinct actin networks.

The indirect flight muscle is a widely used model for studying sarcomere structure and muscle development due to its extremely regular architecture. Nevertheless, precise measurement of the basic sarcomeric parameters remains a challenge even in this greatly ordered tissue. In this study, we identified several factors affecting measurement reliability and developed a software tool for precise, high-throughput measurement of sarcomere length and myofibril width. The accuracy of this new tool was validated against simulated images and blinded manual measurements. To extend the scope of this morphometric analysis to the sub-sarcomeric scale, we used electron and super-resolution microscopy to quantify myofilament number and filament length during myofibrillogenesis. Our findings revealed the dynamics of thin and thick filament elongation, as well as the addition of myofilaments at the sarcomere periphery during myofibrillogenesis. We precisely measured the dimensions of the Z-disc, I-band and H-zone during development, enabling us to construct refined models of sarcomere growth at the level of individual myofilaments, providing a spatial framework for interpreting nanoscopic localization data. These models deepen our understanding of sarcomere growth and lay the groundwork for future studies on the molecular mechanisms driving myofilament elongation and assembly.

The indirect flight muscle is a widely used model for studying sarcomere structure and muscle development due to its extremely regular architecture. Nevertheless, precise measurement of the basic sarcomeric parameters remains a challenge even in this greatly ordered tissue. In this study, we identified several factors affecting measurement reliability and developed a software tool for precise, high-throughput measurement of sarcomere length and myofibril width. The accuracy of this new tool was validated against simulated images and blinded manual measurements. To extend the scope of this morphometric analysis to the sub-sarcomeric scale, we used electron and super-resolution microscopy to quantify myofilament number and filament length during myofibrillogenesis. These results provided novel insights into the dynamics of sarcomere growth, and enabled us to construct a refined model of sarcomere growth reaching to the level of individual myofilaments and providing a spatial framework for interpreting molecular localization data. These findings enhance our understanding of sarcomere assembly and offer a foundation for future studies of muscle development and function. SUMMARY STATEMENT: This study identifies factors influencing sarcomere size measurements, introduces a validated tool for precise analysis, and presents a comprehensive model of Drosophila flight muscle sarcomere growth during muscle development.

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.

The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial components of the contractile apparatus in muscles. The thin filaments are organized into densely packed lattices interdigitated with myosin-based thick filaments. The crossbridge interactions between these myofilaments drive muscle contraction, and the degree of myofilament overlap is a key factor of contractile force determination. As such, the optimal length of the thin filaments is critical for efficient activity, therefore, this parameter is precisely controlled according to the workload of a given muscle. Thin filament length is thought to be regulated by two major, but only partially understood mechanisms: it is set by (i) factors that mediate the assembly of filaments from monomers and catalyze their elongation, and (ii) by factors that specify their length and uniformity. Mutations affecting these factors can alter the length of thin filaments, and in human cases, many of them are linked to debilitating diseases such as nemaline myopathy and dilated cardiomyopathy.

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 myofibril cables. Muscle contraction is achieved by the simultaneous shortening of sarcomeres and for a highly coordinated contraction to occur all sarcomeres should have the same size. Muscles have evolved a variety of ways to ensure sarcomere homogeneity, one example being the controlled oligomerization of Zasp proteins that sets the diameter of the myofibril. To understand how Zasp proteins effect myofibril growth, we looked for Zasp-binding proteins at the Z-disc. We found that the E1 subunit of the oxoglutarate dehydrogenase complex is recruited to the Z-disc by Zasp52 and is required to sustain myofibril growth. By making specific mutants, we show that its enzymatic activity is important for myofibril growth, and that the other two subunits of the complex are also required for myofibril formation. Using super resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Then, using metabolomic analysis, we uncovered an amino acid balance defect affecting protein synthesis, that we also confirmed by genetic tools. In summary, we show that Zasp controls the local amino acid pool responsible for myofibril growth by recruiting the OGDH complex to the Z-disc.

Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data. Bio-protocol is an online peer-reviewed protocol journal. Its mission is to make life science research more efficient and reproducible by curating and hosting high quality, free access protocols.