ABSTRACT
Drosophila larval musculature is a genetically and optically accessible system to study muscle development. Each larval muscle is a single fiber with conserved cytoarchitecture, including its sarcomere structure and composition. Here, we present a workflow for systematically analyzing muscle structure and function at discrete larval stages, as well as throughout the larval instars, using both newly developed and adapted methods. For complete details on the use and execution of this protocol, please refer to Balakrishnan et al. (2020).
Subject(s)
Muscle Development , Sarcomeres/metabolism , Animals , Drosophila melanogaster , Larva , Sarcomeres/geneticsABSTRACT
The sarcomere is the basic contractile unit of muscle, composed of repeated sets of actin thin filaments and myosin thick filaments. During muscle development, sarcomeres grow in size to accommodate the growth and function of muscle fibers. Failure in regulating sarcomere size results in muscle dysfunction; yet, it is unclear how the size and uniformity of sarcomeres are controlled. Here we show that the formin Diaphanous is critical for the growth and maintenance of sarcomere size: Dia sets sarcomere length and width through regulation of the number and length of the actin thin filaments in the Drosophila flight muscle. To regulate thin filament length and sarcomere size, Dia interacts with the Gelsolin superfamily member Flightless I (FliI). We suggest that these actin regulators, by controlling actin dynamics and turnover, generate uniformly sized sarcomeres tuned for the muscle contractions required for flight.
Subject(s)
Drosophila Proteins/physiology , Formins/physiology , Gelsolin/physiology , Sarcomeres/ultrastructure , Animals , Drosophila/genetics , Drosophila/physiology , Drosophila/ultrastructure , Drosophila Proteins/genetics , Flight, Animal , Formins/genetics , Gene Knockdown Techniques , Muscles/ultrastructureABSTRACT
Sarcomeres, the fundamental contractile units of muscles, are conserved structures composed of actin thin filaments and myosin thick filaments. How sarcomeres are formed and maintained is not well understood. Here, we show that knockdown of Drosophila cofilin (DmCFL), an actin depolymerizing factor, disrupts both sarcomere structure and muscle function. The loss of DmCFL also results in the formation of sarcomeric protein aggregates and impairs sarcomere addition during growth. The activation of the proteasome delays muscle deterioration in our model. Furthermore, we investigate how a point mutation in CFL2 that causes nemaline myopathy (NM) in humans affects CFL function and leads to the muscle phenotypes observed in vivo. Our data provide significant insights to the role of CFLs during sarcomere formation, as well as mechanistic implications for disease progression in NM patients.
Subject(s)
Actin Depolymerizing Factors/metabolism , Drosophila melanogaster/metabolism , Muscle Development , Muscle Weakness/metabolism , Muscles/metabolism , Muscles/pathology , Organogenesis , Sarcomeres/metabolism , Actin Cytoskeleton/metabolism , Actins/metabolism , Amino Acid Sequence , Animals , Cofilin 2/chemistry , Cofilin 2/genetics , Gene Knockdown Techniques , Humans , Myopathies, Nemaline/genetics , Phenotype , Point Mutation , Proteasome Endopeptidase Complex/metabolism , Protein Aggregates , Tropomodulin/metabolism , Troponin/metabolismABSTRACT
In this issue of Developmental Cell, Tan et al. (2018) show how a novel player in myonuclear positioning, the ubiquitin ligase Ari-1, regulates levels of Koi, a member of the LINC mechanosensing complex, and affects nuclear morphology and positioning in both Drosophila muscles and human vascular smooth muscle cells.
Subject(s)
Cell Nucleus , Nuclear Envelope , Aneurysm , Animals , Drosophila , Humans , MusclesABSTRACT
In the skeletal muscle, nuclei are positioned at the periphery of each myofiber and are evenly distributed along its length. Improper positioning of myonuclei has been correlated with muscle disease and decreased muscle function. Several mechanisms required for regulating nuclear position have been identified using the fruit fly, Drosophila melanogaster. The conservation of the myofiber between the fly and vertebrates, the availability of advanced genetic tools, and the ability to visualize dynamic processes using fluorescent proteins in vivo makes the fly an excellent system to study myonuclear positioning. This chapter describes time-lapse and fixed imaging methodologies using both the Drosophila embryo and the larva to investigate mechanisms of myonuclear positioning.