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

Figure 3. Trc regulates microtubule sliding via phosphorylation of Pavarotti.

(A) Sliding experiments in S2 cells show a decrease in microtubule sliding with Trc overexpression. Trc overexpression in conjunction with depletion of Pavarotti increases sliding beyond control levels Scale bar 5 µm. (B) Quantification of sliding experiments in A. n = 27–41 cells per condition. Ctrl = 1.0 ± 0.11, Upper 95% CI = 1.2, Lower 95% CI = 0.8, Pav RNAi = 1.6 ± 0.25, 2.1, 1.0, Trc OE = 0.61 ± 0.0.8, 0.8, 0.44, Pav RNAi + Trc OE = 1.4 ± 0.22, 1.9, 0.9. Ctrl vs Trc OE p=0.04, Trc OE vs Pav RNAi + Trc OE p=0.003. One-way ANOVA with Sidak’s post-hoc test. (C) Sliding experiments in S2 cells show an increase in microtubule sliding with Pavarotti depletion. The effect can be rescued with WT Pavarotti but not with Phospho null mutant S745A. (D) Quantification of sliding experiments shown in C. n = 39–48 cells from 4 independent experiments. Ctrl = 1 ± 0.10 Upper 95% CI = 1.2, Lower 95% CI = 0.79, Pav RNAi = 3.16 ± 0.26, 2.65, 3.70, Pav RNAi + WT = 1.96 ± 0.28, 1.40, 2.52 Pav RNAi + Pav S745A = 3.49 ± 0.48, 2.53, 4.44. Ctrl vs Pav RNAi p=0.0001, Pav RNAi vs pav RNAi + WT p=0.04, Pav RNAi vs Pav RNAi + S745A p=0.94, Pav RNAi + WT vs Pav RNAi + S745A p=0.0025. One-way ANOVA with Sidak’s post hoc correction. (E) Example images of extracted S2 cells expressing mCherry Tubulin and WT Pavarotti GFP under control or Trc RNAi conditions. (F) Quantification of microtubule area colocalized with Pavarotti. n = 18–26 cells from three independent experiments. Ctrl = 23.5 ± 2.4%, Upper 95% CI = 28.6, Lower 95% CI = 18.3 Trc RNAi = 12.78 ± 1.9%, 16.62, 8.94. p=0.001 Student’s T-test Scale bar = 10 µm. (G) Example images of extracted S2 cells expressing mCherry Tubulin and WT Pavarotti GFP or Pavarotti S745A GFP. Endogenous Pavarotti was depleted with dsRNA targeting non-coding regions. (H) Quantification of microtubule area colocalized with Pavarotti. n = 12–17 cells from three independent experiments. Scale bar = 10 µm. WT = 29.4 ± 5.0%, Upper 95% CI = 40.4, Lower 95% CI = 18.4, S745A = 7.79 ± 1.8%, 11.5, 4.08. p=0.0001 Student’s T-test (I) Western blot of S745 phospho-Pavarotti from HEK cell lysates shows an increase in this species with Trc overexpression. (J) Western blot of immunoprecipitated Pavarotti prepared from dissected 3rd instar larvae brains shows a decrease in phospho Pavarotti at S745 upon depletion of Trc.

Figure 3—source data 1. Microtubule sliding rates for control, Pav knockdown and Trc overexpressing cells.
Related to Figure 3A and B.
Figure 3—source data 2. Microtubule sliding rates for Pavarotti knockdown and rescue experiments.
Related to Figure 3C and D.
Figure 3—source data 3. Pavarotti localization in control and Trc RNAi-treated cells.
Related to Figure 3E and F.
Figure 3—source data 4. Pavarotti WT and S745A localization.
Related to Figure 3G and H.

Figure 3.

Figure 3—figure supplement 1. Pavarotti knockdown and rescue.

Figure 3—figure supplement 1.

(A) Western blot of S2 cell lysate confirming Pavarotti knockdown with dsRNA targeting a non-coding region. (B) Immunofluorescence showing expression of WT or S745A GFP Pavarotti. (C) Quantification of Pavarotti GFP signal normalized to mCherry tubulin expression shows equal expression of WT and S745A Pavarotti.
Figure 3—figure supplement 1—source data 1. Pavarotti WT and S745A expression levels.
Related to B and C.
Figure 3—figure supplement 2. Method for determining Pav-positive microtubule area.

Figure 3—figure supplement 2.

Related to Figures 3 and 4. (A) Schematic detailing approach for quantifying Microtubule area positive for Pavarotti GFP. (B) Each channel of the example images presented for Pavarotti localization experiments including the generated Pavarotti-positive microtubule area images.