Three-Dimensional Reconstruction and Segmentation of Intact Drosophila by Ultramicroscopy

Genetic mutants are invaluable for understanding the development, physiology and behaviour of Drosophila. Modern molecular genetic techniques enable the rapid generation of large numbers of different mutants. To phenotype these mutants sophisticated microscopy techniques are required, ideally allowing the 3D-reconstruction of the anatomy of an adult fly from a single scan. Ultramicroscopy enables up to cm fields of view, whilst providing micron resolution. In this paper, we present ultramicroscopy reconstructions of the flight musculature, the nervous system, and the digestive tract of entire, chemically cleared, drosophila in autofluorescent light. The 3D-reconstructions thus obtained verify that the anatomy of a whole fly, including the filigree spatial organization of the direct flight muscles, can be analysed from a single ultramicroscopy reconstruction. The recording procedure, including 3D-reconstruction using standard software, takes no longer than 30 min. Additionally, image segmentation, which would allow for further quantitative analysis, was performed

The Drosophila blood brain barrier is maintained by GPCR-dependent dynamic actin structures

Formation of actin-rich structures along the lateral borders of subperineurial glial cells are induced and maintained by the G protein–coupled receptor Moody

AIDing-targeted protein degradation in Drosophila

International audienceConditional protein depletion is highly desirable for investigating protein functions in complex organisms. In this issue, Bence and colleagues combined auxin-inducible degradation with CRISPR, establishing an elegant tool to control protein levels. They achieve precise spatio-temporal control of protein degradation during Drosophila oogenesis and early embryogenesis by combining suitable GAL4 drivers (spatial control) with auxin feeding protocols (temporal control)

Mechanobiology: Forging a strong matrix at tendons

International audienceNature faces the challenge of stably attaching soft muscles to a stiff skeleton. A new study combines live imaging and fly genetics to reveal that mechanical tension and a putative intracellular chaperone assist in assembling the gigantic extracellular matrix protein Dumpy at fly tendon–skeleton interfaces

Mechanobiology of muscle and myofibril morphogenesis

International audienceMuscles generate forces for animal locomotion. The contractile apparatus of muscles is the sarcomere, a highly regular array of large actin and myosin filaments linked by gigantic titin springs. During muscle development many sarcomeres assemble in series into long periodic myofibrils that mechanically connect the attached skeleton elements. Thus, ATP-driven myosin forces can power movement of the skeleton. Here we review muscle and myofibril morphogenesis, with a particular focus on their mechanobiology. We describe recent progress on the molecular structure of sarcomeres and their mechanical connections to the skeleton. We discuss current models predicting how tension coordinates the assembly of key sarcomeric components to periodic myofibrils that then further mature during development. This requires transcriptional feedback mechanisms that may help to coordinate myofibril assembly and maturation states with the transcriptional program. To fuel the varying energy demands of muscles we also discuss the close mechanical interactions of myofibrils with mitochondria and nuclei to optimally support powerful or enduring muscle fibers

Gene Tagging Strategies To Assess Protein Expression, Localization, and Function in Drosophila

International audienceAnalysis of gene function in complex organisms relies extensively on tools to detect the cellular and subcellular localization of gene products, especially proteins. Typically, immunostaining with antibodies provides these data. However, due to cost, time, and labor limitations, generating specific antibodies against all proteins of a complex organism is not feasible. Furthermore, antibodies do not enable live imaging studies of protein dynamics. Hence, tagging genes with standardized immunoepitopes or fluorescent tags that permit live imaging has become popular. Importantly, tagging genes present in large genomic clones or at their endogenous locus often reports proper expression, subcellular localization, and dynamics of the encoded protein. Moreover, these tagging approaches allow the generation of elegant protein removal strategies, standardization of visualization protocols, and permit protein interaction studies using mass spectrometry. Here, we summarize available genomic resources and techniques to tag genes and discuss relevant applications that are rarely, if at all, possible with antibodies

The transmembrane protein Kon-tiki couples to Dgrip to mediate myotube targeting in Drosophila

Directed cell migration and target recognition are critical for the development of both the nervous and muscular systems. Molecular mechanisms that control these processes in the nervous system have been intensively studied, whereas those that act during muscle development are still largely uncharacterized. Here we identify a transmembrane protein, Kon-tiki (Kon), that mediates myotube target recognition in the Drosophila embryo. Kon is expressed in a specific subset of myotubes and is required autonomously for these myotubes to recognize their tendon cell targets and to establish a stable connection. Kon is enriched at myotube tips during targeting and signals through the intracellular adaptor Dgrip in a conserved molecular pathway. Forced overexpression of Kon stimulates muscle motility. We propose that Kon promotes directed myotube migration and transduces a target-derived signal that initiates the formation of a stable connection