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. 2020 Feb 5;9:e52009. doi: 10.7554/eLife.52009

Figure 1. The kinase Trc regulates neurite outgrowth and microtubule sliding.

(A) Example images of timelapse imaging to measure microtubule sliding in Drosophila S2 cells. Microtubules are shown at time zero in green. Magenta indicates photoconverted region. Photoconverted region is highlighted by dotted yellow line at other timepoints. Red box indicates region shown in inset in A’ Scale bar 10 µm. (B) Quantification of microtubule sliding rate shows an increase upon Pavarotti or Trc depletion. n = 16–20 cells. Ctrl = 1.0 ± 0.1 upper 95% CI = 1.2, lower 95% CI = 0.8, Pav RNAi = 3.4 ± 1.0, 5.5, 1.4 Trc RNAi = 2.1 ± 0.4, 3.0, 1.2. Ctrl vs Trc RNAi p=0.03. (C) Representative images of 3rd instar larvae cultured neurons under control or elav > Trc RNAi conditions. (D) Quantification of total neurite length per cell over time in culture. The total neurite length is increased from control upon Trc depletion. 24 hr; ctrl = 137.8 ± 12.0 µm, Trc RNAi 254 ± 19.1 µm. p=0.0001. 48 hr; ctrl = 163.7 ± 15.3 µm, Trc RNAi = 251.2 ± 19.9 µm, p=0.001, 72 hr; ctrl = 156.3 ± 13.8 µm, TrcRNAi = 314.4 ± 36.5 µm, p=0.009. N = 11–23 cells from three independent experiments. (E) Example images from timelapse imaging of photoconverted microtubules in neurons under control conditions or upon Trc depletion. Tubulin was labeled with tdMaple3 alpha tubulin84b. After photoconversion, cells were imaged every minute for 10 min. Scale bar = 5 µm. (F) Quantification of microtubule sliding rates. Trc depletion leads to an increase in microtubule sliding rates in neurons. Ctrl = 1.0 ± 0.14, TrcRNAi = 1.69 ± 0.30. N = 20 cells from three independent experiments. p=0.04 Student’s T-test.

Figure 1—source data 1. Sliding rates for control, Pav RNAi and Trc RNAi-treated S2 cells.
Related to Figure 1A and B.
Figure 1—source data 2. Neurite length for neurons cultured from control larvae or Trc RNAi larvae.
Related to Figure 1C and D.
Figure 1—source data 3. Sliding rates for control, and Trc RNAi neurons.
Related to Figure 1E and F.

Figure 1.

Figure 1—figure supplement 1. Domain structure of Pavarotti and demonstration of Trc knockdown.

Figure 1—figure supplement 1.

(A) Schematic showing domain structure and location of proposed Trc phosphorylation site inDrosophila Pavarotti and human MKLP1. (B) Lysates from S2 cells demonstrate efficient knockdown of Trc with dsRNA treatment. Pavarotti protein levels are not affected. (C) Lysates from brains dissected from 3rd instarDrosophilalarvae confirm neuron-specific knockdown of Trc.