Figure 5: LMTK3 phosphorylation of RCP is necessary for HGF-driven EphA2 trafficking and cell scattering. | Nature Communications

Figure 5: LMTK3 phosphorylation of RCP is necessary for HGF-driven EphA2 trafficking and cell scattering.

From: Phosphorylation of Rab-coupling protein by LMTK3 controls Rab14-dependent EphA2 trafficking to promote cell:cell repulsion

Figure 5

(a) H1299 cells were transfected with siRNAs targeting LMTK3 (si-LMTK3) or a non-targeting control (si-Nt) (left panel). Alternatively cells were transfected with GFP-RCP or GFP-RCP435A (right panel). Internalization of EphA2 in the presence and absence of HGF was then determined as for Fig. 3b. Values are mean±s.e.m. from three independent experiments. ***P<0.001 (si-LMTK3+HGF versus si-Nt+HGF) **P<0.01 (RCP435A+HGF versus RCPWT+HGF); two-way ANOVA, Bonferroni post-test. (b) H1299 cells were transfected with EphA2-GFP in combination with mCherry-RCP or mCherry-RCP435A, in the presence or absence of siRNAs targeting LMTK3 (si-LMTK3) or non-targeting control (si-Nt) as indicated. Trafficking of EphA2-GFP and mCherry-RCPs in the presence and absence of HGF (added 30 min before collecting the movies) was visualized by fluorescence confocal time-lapse microscopy and co-localization was quantified as for Fig. 3e. Stills were extracted from movies at the indicated time points following the collection of the first frame of the movie, and these display the region of interest indicated by the white box. Bar, 10 μm. Values are mean±s.e.m. from three separate experiments incorporating >60 cells per condition. ***P<0.001; one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparison Test). (c) H1299 cells were transfected with siRNAs targeting LMTK3 (si-LMTK3), LMTK1 (si-LMTK1) or a non-targeting control (si-Nt). Alternatively cells were transfected with GFP-RCP or GFP-RCP435A. Cell scattering in the presence and absence of HGF was then experimentally determined and quantified as for Fig. 2d,e. Scattering is expressed as the accumulated distance travelled by cells from their starting point over 6 h. In the right panel, GFP-RCP and GFP-RCP435A–expressing cells were plated subconfluently and the migration speed of non-contacting cells was determined in the presence and absence of HGF. Data are represented as box and whiskers plots (whiskers: 10–90 percentile,+represents the mean). ***P<0.001; one-way ANOVA (Kruskal-Wallis test, Dunn’s Multiple Comparison Test). Data are from three independent experiments with >50 cells being tracked per condition.

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