Structural Changes in Apis mellifera Indirect Flight Muscle Thick Filaments During Maturation

This is the authors' abstract. We don't add key points for this paper.

Honeybee (Apis mellifera) workers undergo a well-defined series of developmental stages, transitioning from nursing duties for recently emerged bees to guarding and ultimately foraging, the final stage before death. A previous study using high resolution cryoEM on the fruit fly, Drosophila melanogaster, of the insect order Diptera, showed age related changes in its indirect flight muscle thick filament structure [1]. This study explores whether similar age-related structural changes can be observed in flight muscle thick filaments of a species from another insect order, Hymenoptera, that has also evolved indirect flight muscle. We identify a correlation between bee maturation and the organization of myosin heads within thick filaments that may influence muscle physiology [1]. Thoraces from three developmental stages of Apis mellifera were collected and dissected on the same day. After dissection, the thoraces were stored in a relaxing buffer containing 80% glycerol at -80°C [2, 3]. Thick filaments were isolated through tissue homogenization, centrifugation, and Triton washes, followed by calpain digestion to remove the Z-disk components [2, 3]. The filaments were sheared using a syringe, pelleted, and treated with gelsolin to eliminate actin contamination. cryo-EM grids were plunge frozen on a Vitrobot Mark IV. Data collection occurred at NCCAT using a Titan Krios G4 with a Falcon 4i camera. Newly emerged bees were imaged at a pixel size of 0.73 Å (38,365 movies), foragers, which are generally older than 28 days, at 0.927 Å (16,982 movies), and pre-emerged adults at 0.927 Å (34,000 movies). The final reconstructions used 319,900 filament segments (particles) for newly emerged bees <45 min old, 470,880 segments for forager bees, and 127,445 segments for pre-emerged teneral adult bees. The order of myosin heads on the thick filament varied across the three developmental stages. Based on 2-D classifications and micrographs (Figs. 1A-F, 2A-F), thick filaments in all 3 stages have a solid backbone most likely due to the presence of a complete annulus of paramyosin. Notably, the thick filament protein stretchin-klp was not seen on the backbone surface of filaments in any of the three developmental stages. The non-myosin protein flightin, which is positioned among the coiled coil myosin tails, appeared in the extended conformation of all three age groups (Fig. 3D). Across all three age groups we found similarities in the structure of backbone including the myosin coiled coils and the non-myosin proteins. The chief difference occurs in the ordering of the myosin heads which increased as the bees aged (Fig. 3A-C), being greatest in the oldest sample, the forager bees, and least ordered in the pre- and emerged bees, which were the youngest. Changes with age in Drosophila melanogaster flight muscle thick filaments include a transition from tubular to solid filaments, loss of stretchin-klp, change in flightin conformation and ordering of the myosin heads on the filament backbone [1]. Although the transition toward more ordered heads in Apis thick filaments is not as pronounced as found in Drosophila, the transition to the more ordered head arrangement, may be understandable in terms of ATP consumption, which is greater when the heads are disordered even when the muscle is not active [7]. Electron micrographs of myosin thick filaments from (A) forager adult bees, (B) newly emerged adult bees, and (C) pre-emerged teneral adult bees, showing myosin heavy chains organized around the solid backbone. 2D classification results depicting the structural organization of the myosin thick in (A) Forager adult bees, (B) Newly emerged adult bees, and (C) pre-emerged teneral adult bees. Density of the myosin heads is highest in the foragers, but not absent in the other age groups. Detailed longitudinal views of segmented maps, showing myosin and non-myosin protein distribution in thick filaments with extended flightin (A) forager adult bees, (B) newly emerged adult bees, and (C) pre-emerged teneral adult bees. There are no dramatic changes in the 3D structure of the myosin thick filament backbone throughout developmental aging. However, as the bees age, there is a gradual transition to a more ordered relaxed filament structure. (D) The extended shape of flightin is observed in all three structures.