Fig. 6

Mechanism of APC2 C-terminus microtubule binding. a Cos7 cells were transfected with GFP-EB3 and either RFP-APC2-C, RFP-APC2-C(SxNN), mCherry-PRC1 or mCherry-TRIM46. One microtubule bundle per cell was photo-ablated and imaged for 90 s stream for microtubule polymerization. Kymographs are color-coded for opposite directions of assembling microtubule plus ends. b Lag time from photo-ablation to first spotted microtubule assembly via GFP-EB3 was measured from live movies (n = 52 bundles RFP-APC2-C, n = 19 bundles RFP-APC2-C(SxNN), n = 28 bundles mCherry-PRC1, n = 23 bundles mCherry-TRIM46, N = 2, Kruskal–Wallis Test, Dunn’s post-hoc). c Cos7 cells expressing either RFP-APC2-C or RFP-APC2-C1 were fixed and immunostained for tyrosinated or acetylated tubulin. Colocalized pixels are white, while non-colocalized RFP pixels are red and non-colocalized tubulin pixels are green. d Pearson’s coefficient was quantified from colocalization of RFP and GFP channel from maximum of three different regions per cell (n = 24 areas RFP-APC2-C tyr. tubulin, n = 28 areas RFP-APC2-C acetyl. tubulin, n = 30 all other conditions; tyr. tubulin p < 0.001, N = 3, Independent samples t-test, acetyl. tubulin p < 0.001, Independent samples t-test). e Cos7 cells were transfected with FKBP-eGFP-CAAX and either FRB-RFP-APC2-C, FRB-RFP-APC2-C1 or FRB-RFP-APC2-C2 and imaged 24 h later. Kymographs are of 40 min movies with frames taken every 30 s post rapalog addition. RFP and GFP channels are presented in fire scale (schematic drawn by O.I.K.). f Percentage of fluorescence arising from bundles localized to FKBP-eGFP-CAAX at the cell membrane 40 min post rapalog addition was measured by subtracting mean fluorescence from non-bundled regions of FKBP-eGFP-CAAX from total mean fluorescence intensity of the cell (n = 18 cells FRB-RFP-APC2-C and C2, n = 16 cells FRB-RFP-APC2-C1, N = 2, One-Way ANOVA, Tukey post-hoc). g Representative still frames and maximum projections of 3 min movies taken every 0.5 sec from the same cell expressing FKBP-eGFP-CAAX and FRB-RFP-APC2-C2 before and after rapalog addition. Graphs represent mean ± SEM. *p ≤ 0.05. Scale bar is 10 µm in a and 5 µm in c, e and g