Live-cell neuron imaging and analysis

One hour before imaging, HaloTag ligands and/or most SNAP-tag ligands were applied for 15 min at a final concentration of 100 nM, followed by a 30–45-min washout. SNAP-tag ligand Blue 430 was applied for 30 min at a final concentration of 2 µM, followed by a 30-min washout. In applicable experiments, neurons were incubated with Lysotracker (25 nM) for 15–30 min, which was then removed for imaging. In applicable experiments, BafilomycinA1 (100 nM) or DMSO was added 2 h before imaging, and then neurons were imaged in the third hour of continued treatment. Neurons were imaged in Imaging Media (HibernateE [Brain Bits] supplemented with 2% B27 and 33 mM D-glucose). Autophagosome behavior was monitored in the proximal (<100 µm from the soma), distal (<100 µm from the distal tip), or mid-axon of 6–8 DIV neurons imaged at a rate of 1 time point/s for 2–3 min. Neurons were imaged in an environmental chamber at 37°C on a PerkinElmer UltraView Vox spinning-disk confocal on a Nikon Eclipse Ti Microscope with an Apochromat 100 × 1.49 NA oil-immersion objective and a Hamamatsu EMCCD C9100-50 camera driven by Volocity (PerkinElmer). Only cells expressing moderate levels of fluorescent proteins were imaged to avoid overexpression artifacts or aggregation. It should be noted that the quality of the primary neuron dissections affected autophagosomal motility, but compared conditions were always collected from the same dissections and imaging sessions.

Kymographs were generated in ImageJ (https://imagej.net/ImageJ2) using the MultiKymograph plugin (line width, 5) and analyzed either in ImageJ or using the MatLab program KymoSuite (J. Nirschl, University of Pennsylvania, Philadelphia, PA). Puncta were classified as either anterograde (moving ≥10 µm toward the axon tip), retrograde (moving ≥10 µm toward the soma), or stationary/bidirectional (net movement <10 µm during the video). Because fluorescent LC3 is cytosolic (as well as punctate) and neurites occasionally crossed in culture, raw videos were referenced throughout kymograph analysis to ensure only real puncta (≥1.5 SD from the axon mean) were included in analyses. All comigration analyses were performed using kymographs. Line scans were generated for presentation purposes from raw video stills and normalized either within that line (for positive channels) or to the local region (for negative channels; surrounding ∼10-µm area).