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First published online 22 November 2005
doi: 10.1242/jcs.02679

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Neurite outgrowth involves adenomatous polyposis coli protein and ß-catenin

Violet Votin^*, W. James Nelson and Angela I. M. Barth

Department of Biological Sciences, and Department of Molecular and Cellular Physiology, Stanford University, Beckman Center B121, Stanford, CA 94305, USA

View larger version (40K): [in a new window]   Fig. 1. APC in neurite tips is along microtubules and changes during neuronal polarization. (A) An axonal growth cone of a hippocampal neuron immunostained for tubulin and APC. Projection of deconvolved planes is shown. (Note that deconvolution resolves APC enrichment into puncta.) (B) Immunostaining for the axon marker dephospho-tau (Tau) and APC show APC enrichment at several neurite tips in an unpolarized neuron and only at the tip of the axon (arrowhead), which is marked by dephospho-tau staining (arrow), in a polarized neuron. Magnified images below also show phase-contrast images of neurite tips N3 and N4 from the unpolarized neuron and the axon tip from the polarized neuron. A, axon; D, dendrite; N, neurite. (C) Line scans quantifying fluorescence intensity of APC (red) and dephospho-tau (Tau, green) along corresponding neurites from B. (D) From line scans of polarized neurons (including that in C), we calculated the ratio of maximum APC intensity at the neurite tip to that of equivalent length of the neurite shaft. Shown is the mean±s.e.m. of the axon and the longest dendrite of 13 neurons. ***P<0.001 compared to APC enrichment in axons by Student's t-test. Bar, 2 µm (A); 20 µm (whole-cell images in B); 2.5 µm (magnified images in B).

View larger version (18K): [in a new window]   Fig. 2. APC is enriched at the axon tip prior to significant length increase. (A) Hippocampal neurons immunostained for APC and dephospho-tau were classified by two neuronal polarization criteria: length + indicates that the length of the longest neurite is greater than or equal to twice the length of the next-longest neurite; tau + indicates increased dephospho-tau staining (represented by green) in one of the neurites. Polarizing neurons were defined as dephosphotau +, length – neurons (see Results). (B) Unpolarized, polarizing and polarized neurons (left, middle and right bars, respectively) were further classified by which neurite tips had enriched APC. Black, longest extension only or most intensely; gray, two or more neurites evenly; white, no neurites or only one neurite other than the longest. Shown is the mean±s.e.m. of three independent experiments; n, total number of cells in each category.

View larger version (62K): [in a new window]   Fig. 3. Localization of endogenous ß-catenin with APC at neurite tips. APC and ß-catenin immunostaining in (A) hippocampal neurons (2 days in vitro) and (B) PC12 cells (treated for 4 days with NGF) as deconvolved planes. Arrow, no ß-catenin enrichment at neurite tip; solid arrowhead, complete overlap of ß-catenin enrichment with APC (see also magnified images i in A and i, ii and iii in B); open arrowhead, partial or no overlap (see also magnified images ii in A and i in B). Bar, 10 µm.

View larger version (77K): [in a new window]   Fig. 4. Stabilized ß-catenin accumulates with APC clusters independently of the transcriptional activation domain. Transfected PC12 cells were treated with NGF for 3-4 days to induce extensions. (A,B) Deconvolved images of immunostaining for APC (first column) and KT3 epitope tag of *ß-catenin or *ß-catC-term (middle column). (C) Immunostaining for tubulin (first column) and myc tag of ß-cat-eng (middle column). *ß-catenin, full-length stabilized ß-catenin; *ß-catC-term, stabilized ß-catenin lacking the C-terminal transcriptional transactivation domain; ß-cat-eng, ß-catenin lacking the transactivation domain and fused to a transcriptional repression domain from Engrailed. Arrows indicate neurite tips magnified in inset and arrowheads indicate all other neurite tips. Compared to endogenous ß-catenin (Fig. 3B), stabilized ß-catenin is enriched in APC clusters at neurite tips (A,B). Bar, 10 µm.

View larger version (29K): [in a new window]   Fig. 5. Transcriptionally active and inactive stabilized ß-catenin mutants inhibit neurite outgrowth. PC12 cells were transfected with GFP alone or one of three stabilized ß-catenin constructs shown on the left. Transcriptional activation (A) and NGF-induced neurite formation (B) and neurite growth (C) were measured. (A) Co-transfection of pTOPFLASH Tcf/Lef-driven luciferase reporter (gray bars, TOPFLASH) indicates that only full-length stabilized ß-catenin (*ß-catenin) increased Tcf/Lef-mediated transcription. pFOPFLASH (black bars, FOPFLASH) is a negative control for the reporter assay, with mutated Tcf/Lef-binding sites. Luciferase activity was normalized for transfection efficiency (see Materials and Methods). Shown is the mean±s.e.m. of three independent experiments. (B,C) One day after transfection, PC12 cells were passaged and treated with NGF for 3.5 days. Cells were stained for tubulin and >250 cells were counted for the presence of neurites (see Table 1). Expression of each stabilized ß-catenin mutant reduced the percentage of cells with neurites of any length (B) and cells with neurites longer than twice cell body width (C). For the GFP control, *ß-cat and ß-cat-eng bars show the mean±s.e.m. of three independent experiments. For *ß-catC-term, the bar represents the average of two independent experiments. Each mutant was significantly different from the GFP control by binomial test (*P<0.05). (D) Representative images of PC12 cells expressing CFP-tagged *ß-catC-term and induced with NGF for 24 hours. High-expressing cells (arrowheads) usually had short or no neurites in contrast to untransfected cells. (E) Quantification of neurite length in cells, including those in D, which express varying levels of *ß-catC-term and *ß-catenin. For each cell, higher mean fluorescence intensity correlated with decreased length of the longest neurite. Bar, 10 µm.

View larger version (19K): [in a new window]   Fig. 6. A model for APC/ß-catenin interaction during neurite outgrowth. (A) In uninduced cells, active GSK3ß may phosphorylate APC and ß-catenin. Phosphorylated APC (APCP) has a higher affinity for ß-catenin (Rubinfeld et al., 1996) and a lower affinity for microtubules (striped bar) (Zumbrunn et al., 2001). Phosphorylated ß-catenin (ßP) is degraded. (B) In NGF-induced cells, NGF locally inactivates GSK3ß (Zhou et al., 2004), so GSK3ß may be inactive at the cell periphery and eventually the neurite tips, leading to local ß-catenin stabilization. Unphosphorylated APC has lower affinity for ß-catenin, so ß-catenin in the APC complex turns over rapidly. Unphosphorylated APC also binds and bundles microtubules, and this function at neurite tips may be regulated by its binding to ß-catenin. The ratio of free versus ß-catenin-bound APC could thereby determine microtubule bundling and neurite growth rate. The equilibrium would further shift toward neurite growth if ß-catenin levels are decreased, for example by being sequestered at adhesion sites, or if APC levels are increased, for example in response to NGF (Dobashi et al., 1996).

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