Mical Regulates Actin and Microtubule Dynamics

We performed complementation analysis, genetics, molecular biology, western blotting, immunostaining and generation of transgenic animals using standard techniques4 (link). Multiple new lines of the full-length ‘short’ isoform of Mical, the Mical^Δredox mutation (Mical^G→W; ref. 4 (link)) and the other transgenic animals were generated and used for all experiments. Adult bristles were examined and quantified by crossing adults at 25 °C: adult offspring from these crosses were first sorted according to genotype and then examined under a dissecting microscope. We genotyped pupae using a Zeiss Discovery M² Bio fluorescence stereomicroscope, and all preparation, staging and dissection of pupae were done using standard approaches. We imaged, drew and quantified the adult bristles with the aid of the Discovery M² Bio stereomicroscope, a motorized focus and zoom, a Zeiss AxioCam HR camera and three-dimensional-reconstruction software (Zeiss AxioVision, version 4.6.3, and Extended Focus software). All other bright-field, dark-field, differential interference contrast and fluorescence visualization, and imaging of bristles, embryos and growth cones, was done using a Zeiss Axio Imager upright microscope with motorized focus and zoom and an ApoTome module, and images were captured and quantified using the AxioCam HR camera and AxioVision software. All electron microscopy of pupae and negative staining of purified proteins was done using a FEI Tecnai G² Spirit BioTWIN transmission electron microscope. We purified recombinant Mical proteins10 and recombinant p-hydroxybenzoate hydroxylase using our previously developed approaches. Drosophila fascin (also known as singed) complementary DNA was inserted in a bacterial expression vector, and recombinant Drosophila fascin protein was purified. All F-actin and Mical co-sedimentation assays and G-actin/F-actin ratio experiments were performed using standard approaches, as were all pyrene-labelled actin polymerization and depolymerization assays, actin bundling assays, tubulin polymerization assays and microtubule co-sedimentation assays.