Kymograph analysis and lysosome trafficking

To determine speed and run length, kymographs were generated with the KymoToolBox [58] and the motion of individual lysosomes traced using the Simple Neurite Tracer plug-in for ImageJ. For each kymograph paths were created as a user-defined selection, converted to a segmented line and exported as a ROI, before analysis with a Python-based script. Movement that lasted for at least 3 frames (1.5 sec), was considered a run and was followed until the next change of state. Each track was then classified as retrograde, anterograde, bidirectional or paused, and only tracks traced for at least 60 sec in the retrograde direction were considered. All analysis was done blinded to genotype.