Computational protocol: JNK-Interacting Protein 3 Mediates the Retrograde Transport of Activated c-Jun N-Terminal Kinase and Lysosomes

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[…] RNA in situ hybridization was performed as described . Digoxygenin-labeled antisense RNA probes were generated for jip3 and jnk3 using the full-length cDNA cloned. Whole mount immunohistochemistry was performed following established protocols . The following antibodies were used: anti-GFP (1∶1000; Invitrogen #A11122), anti-pJNK (1∶100; Cell Signaling #9251S), anti-tJNK (1∶100; Cell Signaling #9252), anti-p150glued (1∶100; Signal Transduction Labs #610473), anti-dynein heavy chain (1∶100; gift of R. Vallee; ), anti-Rab7 (1∶100; Sigma #R8779), anti-Lamp1 (1∶100; Developmental Studies Hybridoma Bank), anti-LC3 (1∶100; Novus #NB100-2331), anti-TrkB (1∶100; Santa Cruz Biotechnology #sc-12) and Alexa-488/568/647 (1∶750; Invitrogen). Antibodies not used previously in zebrafish were validated by Western blot analysis (see below: –; Rab7–24 kD, LC3–14.5 kD, TrkB-69 kD and 18 kD, Lamp1–27 kD). For TUNEL labeling, embryos were processed as previously described with minor modifications according to the manufacturer's instructions (In situ cell death kit, Roche). For Lysotracker red vital dye staining, 4–5 dpf larvae were incubated in Lysotracker red (1∶10,000; Invitrogen) for 15 minutes in embryo media, washed briefly, embedded in 1.2% low-melt agarose, and imaged. All fluorescently labeled embryos were imaged using a FV1000 laser scanning confocal system (Olympus). Brightfield or Nomarski microscopy images were collected using a Zeiss Imager Z1 system. Images were processed using ImageJ software . Brightness and contrast were adjusted in Adobe Photoshop and figures were compiled in Adobe Illustrator. [...] Zygotes were injected with plasmid DNA encoding fluorescently tagged cargos of interest with expression driven by the 5kbneurod promoter . At 30 hpf, 2 dpf, or 5 dpf, embryos or larvae were sorted under epifluorescence to identify individuals with tagged cargo expression in a few cells of the pLL ganglion. For imaging, embryos were mounted in 1.2% low melting point agarose on a glass coverslip, submerged in embryo media containing 0.02% tricaine and imaged using a 60X/NA = 1.2 water objective on an upright Fluoview1000 confocal microscope (Olympus). For each embryo, a region of interest (30–200 µm) was selected in the pLL nerve in which a long stretch of axon was observable in a single plane. Scans were taken at the fastest possible speed (3–5 frames per second) for 600 to 2500 frames. Embryos were subsequently released from agarose and processed for genotyping. For co-transport, embryos expressing both constructs in a single cell were selected and imaged as described above using sequential imaging of the 488 and 568 nm excitation channels. 600 frames were collected at 2–3 frames per second.Transport parameters were analyzed using kymograph analysis in the MetaMorph software package (Molecular Devices, Inc.). Kymographs were generated from each imaging session and used to determine distance moved in individual bouts of movement (uninterrupted straight lines) and velocity of movement (slope of uninterrupted straight lines). Typically, 10–50 traces were analyzed in each kymograph and these were averaged within individual embryos for statistical analysis. The number of particles moving in each direction was estimated based on traces on the kymographs and then normalized to length of axonal segment and total imaging time. […]

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