Fig. 5. Adenomatous polyposis coli (APC) accumulates at the tip of the presumptive axon. (A) Endogenous APC was visualised in stage-2 neurons transfected with a GFP expression construct. Phase-contrast, APC-immuno-reactivity, GFP fluorescence and the resulting ratio image are shown. (B) Localisation of endogenous APC in stage-2+ neurons in the largest and longest tip. (C) Preferential axonal localisation of endogenous APC in stage-3 neurons is shown in a representative neuron. (D) In the same neuron as shown in C, the intensity of APC fluorescence along the axon (labelled 1) and two minor neurites (labelled 2 and 3) was measured to demonstrate that APC accumulates strongly in the axonal tip but not in the tips of minor neurites. (E) Quantification of the preferential APC accumulation in one neurite tip. The fluorescent APC signal was divided by the signal obtained in the same cell from a soluble cytoplasmic marker GFP or from DTAF, and the ratio of intensities at neurite tips was measured, normalised and sorted in descending order according to intensity. As a control for the specificity of APC accumulation, the fluorescence intensity of a soluble protein that does not accumulate in individual tips (p38) was used (mean ± s.e.m.,^**P<0.001, ^*P<0.01). (F) A neuron 48 hours after GSK-3 inhibition is shown. Here, endogenous APC is distributed symmetrically in the tips of all long neurites. (G) In the same neurons as shown in F, the signal of APC fluorescence intensity was measured along the three longest neurites. Here, APC accumulates in the tip of three neurites. (H) Quantification of the results shown in F and G, comparing the accumulation of APC in neurites of control stage-3 neurons versus neurons in which GSK-3 was inhibited for 48 hours. The APC signal was normalised against GFP or DTAF, and the ratio intensity at neurite tips calculated, normalised and sorted in descending according to intensity (mean ± s.e.m., ^**P<0.001). (I) Localisation of endogenous APC in neurons isolated from GSK-3/ß^{21A/21A/9A/9A} mice is preferentially restricted to one neurite, similar to wild type neurons. (J) Localisation of APC and EB1 in an individual neuron. Bars, 10 µm.