Microtubule-associated proteins from adult flies collected 16 hr after a 1 hr, 37°C heat shock to induce GAL4 expression were purified from fly extracts as described (McGrail et al.,
1995). NMJ analysis was limited to muscle 4 unless stated otherwise. Antibodies used are detailed in Supplemental Experimental Procedures. A synapse was considered to have TB anti-HRP or anti-Dhc accumulation if the fluorescence intensity within the TB was clearly much higher than in proximal boutons. Fluorescent images were acquired by using a Zeiss LSM 510 confocal Proteasome inhibitor microscope using a PLAN-APO 63×, 1.4 NA oil-immersion objective. Maximum-intensity Z projections of confocal stacks were generated and processed by using Adobe Photoshop. Intensity measurements and NMJ TB volume were obtained by thresholding with Imaris software. For scanning electron microscopy, fly heads were coated with gold:palladium by using a vacuum evaporator and imaged immediately by using a LEO/Zeiss Field-Emission SEM. SPAIM experiments were performed as described (Wong et al., 2012). For other live-imaging experiments,
we rinsed wandering third-instar larvae and pinned them in Ca2+-free HL3 on the sylgard insert of a custom-made imaging mount, placed a coverslip over the preparation, and secured it. Imaging of axonal transport was performed on a Zeiss Axio Observer with a 40× oil objective (EC Plan-Neofluar 1.3 NA) and collected on an AxioCAM charge-coupled device camera. Movies were analyzed as described high throughput screening (Louie et al., 2008). For ANF:GFP fluorescence recovery after photobleaching (FRAP) experiments, we acquired spinning-disc confocal images of dense-core vesicles at muscle 6/7 NMJs by using a Zeiss Axio Imager Z1 microscope and 63× 1.4 NA oil-immersion objective and collected them on a QuantEM 512SC camera (Photometrics). ANF:GFP in proximal boutons was bleached by using a 488 nm laser controlled
by a Mosaic Digital Illumination System (Photonic Instruments). Electrophysiological recordings from muscle 6, segment A3, were performed as described (Imlach and SB-3CT McCabe, 2009). Data are expressed as mean ± SEM. A Student’s t test was performed for pairwise comparisons between each genotype and its wild-type control by using GraphPad Prism. We are grateful to Chris Doe, Vladimir Gelfand, Tom Hays, Rod Murphey, Phil Wong, Sangyun Jeong, and Herman Aberle for reagents. We thank Ben Choi for pMad work and Manish Jaiswal and Vafa Bayat for helpful comments. We thank Erik Griffin, Geraldine Seydoux, Norm Haughey, Terry Shelley, Michele Pucak, and the NINDS Multi-photon Core Facility (MH084020) at JHMI for assistance with imaging. We also thank the BDSC, VDRC, and DGRC for fly stocks and reagents. This work was funded by the Packard Center for ALS Research (A.L.K. and T.E.L.), P2ALS (B.D.M.), NINDS K08-NS062890 to T.E.L., R01-NS35165 to A.L.K, and RO1-NS32385 to M.Y.W. and E.S.L. A.L.K.