Eps8 interacts with IRTKS and is involved in the generation of dorsal morphology. Eps8 interacts with IRTKS and is involved in the generation of dorsal.

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Eps8 interacts with IRTKS and is involved in the generation of dorsal morphology. Eps8 interacts with IRTKS and is involved in the generation of dorsal morphology. (A) Identification of IRTKS partners using mitochondrial localization. Mitochondria-targeted IRTKS–mRuby was coexpressed with GFP-tagged WAVE1, WAVE2, Mena, Eps8, VASP, Espin, mDia1, mDia2, N-WASP, Ezrin, Dyn1 or Dyn2 in HeLa cells. Dual-channel fluorescence live-cell imaging was then carried out. Regions of interest (ROIs) were drawn and Pearson's colocalization coefficients (PC) were calculated using Metamorph software. Pearson's colocalization values above 0.7 were taken as indicative of colocalization (images shown in Fig. S2B). (B) IRTKS interacts with Eps8. SBP–IRTKS was coexpressed with Flag-tagged Eps8, Eps8L1, Eps8L2, Eps8L3, GFP or TccP in 293T cells and pairwise interactions analyzed by SBP pulldown of IRTKS and immunoblotting (WB) with anti-Flag antibody. Note that Eps8L2 gave variable expression in 293T cells and in some experiments could not be seen. Hence we were not able to ascertain its ability to co-immunoprecipitate with IRTKS. L1, L2 and L3 are related independent genes of Eps8. IRTKS was detected using anti-SBP antibody. The strong ECL signal obtained when detecting SBP–IRTKS using HRP-conjugated anti-SBP antibody results in signal bleaching on X-ray films that gives the appearance of multiple bands (bottom right panel). (C) IRTKS interacts with Eps8 by FLIM-FRET. Representative FLIM images of HeLa cells expressing GFP–Eps8, GFP–mRFP, coexpression of GFP–Eps8 and mRFP, and GFP–Eps8 and mRFP–IRTKS. Numbers on images represent the respective lifetimes in ns. Scale bars: 10 µm. Data is expressed as mean±s.d. (n=5–7 cells). (D) Effect of Eps8 KO on Rif–IRTKS induction of dorsal morphology as shown by SEM. A GFP, mRuby and mCherry control is shown in the top left and top middle panels, a GFP–Eps8 rescue is shown in the top right panel, mCherry–RifL77 and IRTKS–mRuby are shown in the middle left and bottom left panels, and mCherry–RifL77 and IRTKS–mRuby with Eps8 rescue are shown in the middle left and bottom left panels. Scale bar: 1 µm. (E) Statistical analysis of data shown in D, cells were scored for dorsal membrane ruffles, dorsal filopodia and long dorsal filopodia as described in the Materials and Methods. (F) The mean count, mean length and total length of dorsal membrane ruffling per cell was determined, and expressed as mean±s.d. (n=3–4 cells). Images shown in D were used to assess the size of dorsal membrane ruffles. To score for dorsal membrane ruffling, manual tracing was performed. *P<0.001 (two-tailed t-test). Thankiah Sudhaharan et al. J Cell Sci 2016;129:2829-2840 © 2016. Published by The Company of Biologists Ltd