Fig. 2

APC2 clusters localize to microtubule bundles in dendrites. a Three constructs were made for these experiments based on human APC2 sequence. b Localization of RFP-APC2 was assessed throughout development of neurons at DIV3 (n = 18), DIV7 (n = 19) and DIV15 (n = 15) by measuring PI, N = 2. c Localization pattern of RFP-APC2, RFP-APC2-ΔC and RFP-APC2-C in DIV16 neurons immunostained for axon initial segment (TRIM46) and dendrites (MAP2). d RFP-APC2 localizes in a regular pattern clusters in primary dendrites. d.1 and d.2 are secondary dendrites, where RFP-APC2 often localizes to the outer dendrite. Line scans highlight the clustered pattern localization. e RFP-APC2 clusters colocalize with tyrosinated and acetylated microtubule bundles in neurons. Colocalized pixels are white, non-colocalized RFP-APC2 pixels are red and non-colocalized tubulin pixels are green (n = 21 areas, 0.42 ± 0,03 Manders’ coefficient for acetylated tubulin and 0.39 ± 0.03 Manders’ coefficient for tyrosinated tubulin, p = 0.484, Independent samples t-test). f Representative frames, color-coded maximum projections of 180 frames and kymographs of RFP-APC2 or RFP-APC2-ΔC in dendrites at DIV15–16. g Immobilized clusters were quantified as a percentage of total over 3 min movies taken every 0.5 s in 6.7 µm long stretches of dendrite. (n = 31 dendrites RFP-APC2, n = 13 dendrites RFP-APC2-ΔC, p ≤ 0.001, N = 4, Mann–Whitney U test). h Neurons transfected with RFP-APC2 were imaged on DIV16 after treatment with 10 µM nocodazole. Kymograph is of 90 min movie with frames taken every 1 min. i Fluorescence intensity of RFP-APC2 was quantified pre and post nocodazole treatment in 10 µm same areas of dendrite after bleach correction (n = 40 dendrites, p = 0.006, Wilcoxon signed-rank test). Number of RFP-APC2 clusters was quantified pre and post nocodazole treatment in 10 µm areas of dendrite (n = 40 dendrites, p ≤ 0.001, N = 4, Wilcoxon signed-rank test). j Representative images of DIV15–16 neurons transfected with RFP-APC2 and either GFP-SpvB or after 90 min 10 µM nocodazole treatment undergoing FRAP. k Quantification of normalized fluorescence recovery. Measurements were taken every 30 s over 10 min (n = 15 dendrites RFP-APC2, n = 12 dendrites RFP-APC2 + GFP-SpvB, n = 13 dendrites RFP-APC2 + Nocodazole, N = 3). Graphs represent mean ± SEM. * p ≤ 0.05. Scale bars represent 10 μm in c and e zoom out and 5 μm in rest