Figure 8.

Characterization of morphological and trafficking parameters of mitochondria and lysosomes grown in 96-wells after treatment with the Srk-kinase inhibitor PP2. Morphological analysis with a Fiji macro of live cell images of MitoTracker stained mitochondria yielded (a) the normalized number of mitochondria (b) the aspect ratio of ellipses fitted on the mitochondria and (c) and the size of mitochondria (n ≥ 50). Characterization of mitochondria trafficking was achieved by utilization of the Fiji plugin TrackMate. Obtained tracking parameters were (d) the moving fraction, which was defined by tracks with a displacement above 1.2 µm, (e) and their respective mean speed (n ≥ 12). (f) Processivity of moving tracks was calculated by dividing their respective displacement by their total track length (n ≥ 12). The lysosomal fraction of midbrain/hindbrain neurons cultivated in 96-wells and treated with PP2 was investigated for its morphological parameters as well. (g) The normalized number of lysosomes as well as (h) their shape and (i) size, was calculated from the first image of a video stack from live cell images with LysoTracker by utilizing a macro in Fiji (n ≥ 45). The same video stack was then analyzed with TrackMate to yield (j) the fraction of motile lysosomes, which was defined by the tracks with a displacement above 1.2 µm (n ≥ 12). (k) The mean speed of moving lysosomes of neurons grown in 96-wells after treated with PP2 was extracted from the tracking parameters provided by TrackMate (n ≥ 12). (l) Processivity of the movement of lysosomal vesicles was calculated by dividing each individual tracks displacement by its track length, which were both obtained from the previously conducted analysis with TrackMate (n ≥ 12). Boxes represent 25–75 percentiles, line represents median, whiskers represent 10–90%, + represents mean. Bars represent mean ± SEM. * indicate significant differences, */**/*** represent p < 0.05/0.01/0.001