The conserved protein Kank directly interacts with EB1 <i>in vitro</i> and requires EB1 to localise to microtubule ends in cultured S2 cells.

<p>(A) <i>Drosophila</i> Kank contains the conserved structural elements of Kank family proteins. <i>Drosophila</i> Kank also contains putative, conserved motifs including nuclear localisation signals (NLS in green), nuclear export signals (NES in grey) and SxIP/EB1 binding motifs (in purple). (B) Kank produced by <i>in vitro</i> transcription/translation in the presence of ³⁵S-methionine was successfully pulled down by bacterially expressed MBP-EB1 but not by MBP alone. Autoradiograph and protein staining are shown at the top and bottom panels respectively. The input is equivalent to 30% of the pull downs. (C) Cultured S2 cells were transfected with GFP-Kank and immunostained for GFP and α-tubulin. The GFP signal localises to the cytoplasm, largely to microtubule ends (yellow arrows). Minor localisation was also observed at the cell periphery. (D) S2 cells transfected with GFP-Kank were immunostained for GFP and EB1. The GFP signal largely co-localised with EB1 comets (yellow arrows). (E) Immunoblotting confirmed the reduction of EB1 by RNAi. (F) Kank requires EB1 to localise to microtubule ends. RNAi of EB1 in S2 cells led to delocalisation of GFP-Kank from microtubule ends, compared to control β-lactamase RNAi cells. The percentage of cells with GFP-Kank signal localised to the majority of visible EB1 comets (>50%, estimate) was counted. The GFP-Kank signal at EB1 comets was significantly reduced in EB1 RNAi cells compared to control RNAi cells and control cells without RNAi. Error bars show the standard error of the mean. For all microscopy images yellow boxes are areas magnified in images shown below and co-localisation is indicated by yellow arrows. Scale bars = 5 µm.