Figure 3.
Disruption of Rab2, Syd, and dynein result in similar DCV axonal transport phenotypes. (A) Representative kymographs showing transport of ILP2-GFP–positive DCVs in motor axons in the A7 nerve of third instar larvae with the indicated genotypes. KD, motor neuron-specific knockdown (driven by OK6-Gal4). Time-lapse confocal imaging was performed immediately after bleaching the areas indicated with red bars. Each kymograph depicts a single recording, except for Arl8/Df, where 20 superimposed recordings from nine larvae are shown (see Materials and methods). Scale bar: 10 µm. (B–D) Directional distributions showing the relative frequency of DCV transport velocities, expressed as the angle between the DCV trajectories and the vertical axis in the kymographs in A (see inset in B). Actual DCV velocities converted from angles have been added to the x axis in B. For each genotype except Arl8/Df, the directional distribution was averaged from n larvae, where n is equal to the number of data points in E–H (specified below). The directional distribution of Arl8/Df was produced from the superimposed Arl8/Df recordings in A. (E) Logarithmic ratio of the retrograde to anterograde peak amplitude in the directional distributions in B–D (the retrograde and anterograde peak amplitude are the maximal relative frequency of angles lower than −46° and higher than 46°, respectively). (F) The static peak amplitude relative to the sum of the static, retrograde, and anterograde peak amplitudes (the static peak amplitude is the maximal relative frequency of angles between −13° and 13°, located centrally on the x axis). (G) Counts of DCVs entering from the sides into the field of view in the anterograde or retrograde directions and of static vesicles in the central unbleached area. Counts were done over 30 s and multiplied by two, converting the dynamic vesicle counts to DCV flux in vesicles per minute. (H) Percentage of static vesicle counts relative to total vesicle counts for each genotype in G. Results involving Arl8/Df represent reanalysis of data published earlier (Lund et al., 2021). ANOVA followed by Dunnett’s test (E, G, and H), Steel with control test (F). Number of larvae analyzed (n) in B–D and E–H: control 29, Rab2 10, sydz4/Df 12, Dhc-KD 12, Khc-KD 8, Arl8/Df 9, and unc-104P350/O3.1 12.

Disruption of Rab2, Syd, and dynein result in similar DCV axonal transport phenotypes. (A) Representative kymographs showing transport of ILP2-GFP–positive DCVs in motor axons in the A7 nerve of third instar larvae with the indicated genotypes. KD, motor neuron-specific knockdown (driven by OK6-Gal4). Time-lapse confocal imaging was performed immediately after bleaching the areas indicated with red bars. Each kymograph depicts a single recording, except for Arl8/Df, where 20 superimposed recordings from nine larvae are shown (see Materials and methods). Scale bar: 10 µm. (B–D) Directional distributions showing the relative frequency of DCV transport velocities, expressed as the angle between the DCV trajectories and the vertical axis in the kymographs in A (see inset in B). Actual DCV velocities converted from angles have been added to the x axis in B. For each genotype except Arl8/Df, the directional distribution was averaged from n larvae, where n is equal to the number of data points in E–H (specified below). The directional distribution of Arl8/Df was produced from the superimposed Arl8/Df recordings in A. (E) Logarithmic ratio of the retrograde to anterograde peak amplitude in the directional distributions in B–D (the retrograde and anterograde peak amplitude are the maximal relative frequency of angles lower than −46° and higher than 46°, respectively). (F) The static peak amplitude relative to the sum of the static, retrograde, and anterograde peak amplitudes (the static peak amplitude is the maximal relative frequency of angles between −13° and 13°, located centrally on the x axis). (G) Counts of DCVs entering from the sides into the field of view in the anterograde or retrograde directions and of static vesicles in the central unbleached area. Counts were done over 30 s and multiplied by two, converting the dynamic vesicle counts to DCV flux in vesicles per minute. (H) Percentage of static vesicle counts relative to total vesicle counts for each genotype in G. Results involving Arl8/Df represent reanalysis of data published earlier (Lund et al., 2021). ANOVA followed by Dunnett’s test (E, G, and H), Steel with control test (F). Number of larvae analyzed (n) in B–D and E–H: control 29, Rab2 10, sydz4/Df 12, Dhc-KD 12, Khc-KD 8, Arl8/Df 9, and unc-104P350/O3.1 12.

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