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

Figure 4. Phospho-Pavarotti brakes microtubule sliding via interaction with 14-3-3 proteins.

(A) Western blot from HEK cell lysate showing co immunoprecipitation of Pavarotti and 14-3-3. The interaction is lost upon mutation of S745 to Alanine and increased upon co-expression of the kinase Trc. (B) Sliding experiments in S2 cells show the ability of Pavarotti to brake microtubule sliding is dependent upon 14-3-3 proteins. Scale bar = 10µm. (C) Quantification of sliding experiments in B. n=21-26 cells from three independent experiments. Ctrl = 1 ± 0.14 Upper 95% CI = 1.28, Lower 95% CI = 0.72, PavOE = 0.35 ± 0.08, 0.52, 0.18 14-3-3 RNAi = 1.05 ± 0.17, 1.41, 0.70, 14-3-3RNAi + PavOE = 1.20 ± 0.20, 1.6, 0.8. ctrl vs pav OE p = 0.01, ctrl vs 1433RNAi p = 0.99, ctrl vs 1433 RNAi + PavOE p = 0.78, Pav OE vs 1433 RNAi p = 0.007, pav OE vs 1433 RNAi + Pav OE p = 0.0004, 1433 RNAi vs 1433 RNAi + Pav OE p = 0.91 One-way ANOVA with Tukey’s post-hoc correction. (D) Example images of extracted S2 cells expressing mCherry Tubulin and WT Pavarotti GFP. Depletion of 14-3-3s decreases microtubule area decorated with Pavarotti. Scale bar = 10µm (E). Quantification of microtubule area colocalized with Pavarotti. n = 22-26 cells from three independent experiments. Ctrl = 15.4 ± 2.1, Upper 95% CI = 19.72, Lower 95% CI = 11.07, 14-3-3 RNAi = 5.54 ± 2.0, 9.78, 1.30. p = 0.0017 Student’s T-test. (F) Western blot from S2 cell lysate demonstrating knockdown of 14-3-3.

Figure 4—source data 1. Sliding rates for 14-3-3 RNAi and Pavarotti overexpression experiments.
Related to Figure 4B and C.
Figure 4—source data 2. Pavarotti localization with 14-3-3 RNAi.
Related to Figure 4D and E.

Figure 4.

Figure 4—figure supplement 1. Confirming GFP Pavarotti expression.

Figure 4—figure supplement 1.

Expression of GFP Pavarotti can be detected after photoconversion of EOS tubulin. GFP Pavarotti does not convert upon exposure to UV light so is still visible in the 488 channel. See within photoconversion zone in dotted yellow circle. White in the merged image shows GFP Pavarotti colocalized with microtubules.