Fig. 4: Modification of Ser102 alters the conformation of BICD2.

A Representative electron micrographs obtained for wild type (left) and S102D (right) TwinStrep-BICD2, using negatively stained samples. Several BICD2 individual molecules are highlighted within white dashed circles. Representative 2D average images are displayed at the bottom of each micrograph. The percentage of particles with a triangular or a rod-like morphology is noted in each case (30,476 BICD2 wild-type particles and 22,021 BICD2 S102D particles). Scale bars for micrographs and individual/average images represent 50 and 10 nm respectively. B Gallery of representative individual images of the wild type (left) and S102D (right) TwinStrep-BICD2 plus the corresponding average images. Scale bars, 10 nm. A and B Show results from one out of three experiments with identical results, in which 195 micrographies were acquired for BICD2 WT and 165 for BICD2 S102D. A representative image field is shown for each BICD2 form in (A). C Modification of Ser102 interferes with the interaction between BICD2 N- and C-terminal regions. HeLa cells were transfected with different forms of BICD2 N-terminus (BICD2 [1–575]) plus a fusion of GST and the C-terminal part of BICD2 (BICD2 [540–820]). GST alone or fused to an internal BICD2 region (GST-BICD2 [272–540]) were used as controls. GST pulldowns (PD) were analyzed by western blot (W) using either anti-GFP or anti-GST antibodies. Note that expression of GST-tagged forms of BICD2 fragments resulting also in the apparition of free GST in the samples, possibly as a result of degradation. GFP polypeptides in the corresponding extracts are shown in the lower panel. Mean ± SD of quantifications corresponding to three independent experiments is shown. Statistical significance analyzed using one-way ANOVA with post hoc analysis (no correction for multiple comparations, Fisher’s LSD test; WT vs S102A, P = 0.2777 (n.s); WT vs. S102D, P = 0.0038 (**); S102A vs. S102D, P = 0.0012 (**)).