δ-Catenin induces branching in PC12 cells. (A) GFP (a) and GFP-δ-catenin constructs (b) transfected in PC12 cells. Cells were fixed 36 h after transfection, and NGF was added at the time of the transfection. Bar, 5 μm. (B) PC12 cells (a) and δ-PC12 cells (b) were treated with NGF for 36 h. Cells were fixed and stained for actin. Quantification of the total process length and the number of primary and secondary processes is shown for PC12 and δ-PC12 cells after 36 h of NGF treatment. Bar, 5 μm.

The COOH terminus of δ-catenin is necessary for neurite formation. (A) Schematic representation of δ-catenin and cortactin deletion constructs. (a) δ-Catenin deletion constructs fused to GFP (as described in Materials and methods). Constructs are named by the number of amino acids deleted from the COOH terminus. (b) Schematic representation of the cortactin GST fusion constructs. (B) Hippocampal neurons in culture for 8 d (a) and NGF-treated PC12 cells (b) were transfected with the GFP, FLδ, ΔC205, and ΔC99 constructs. Quantification of the number and length of protrusions for the hippocampal neurons and the number of primary branches and total summed length of all processes for PC12 cells are shown.

Cortactin binds to the COOH terminal region of δ-catenin. (A) COS1 cells were transfected with GFP–δ-catenin (FLδ), ΔC99 (top), ΔC205 (middle), and ΔC332 (bottom) constructs. Transfected cells were lysed and subjected to pull down with GST and GST–cortactin (GSTCort) constructs. (B) COS1 cells were transfected with either full-length δ-catenin (Flδ) or a full-length construct from which the polyproline tract was deleted (FLδΔP6), pulled down with GST or GST–cortactin, and blotted with mAb δ-catenin (top). COS1 cells were transfected with FLδ or ΔC99 and pulled down with GST or GST-cortactin, or GST–CTcortactin (GSTCtCort, aa 287–509 of cortactin), which retains the SH3 domain (bottom).

Tyrosine phosphorylation of δ-catenin. (A) δ-PC12 cells (pretreated with NGF for 36 h) and hippocampal neurons were treated with 25 μm PP2 for 1 h; additionally, hippocampal neurons were treated with 200 μM H₂O₂ for 5 min, δ-catenin was immuno- precipitated with a mAb δ-catenin, and its phosphorylation state was tested with antiphosphotyrosine. (B) δ-PC12 cells were transfected with constructs corresponding to Fyn or a dominant-negative Fyn (FynK299M). Cells were lysed, and lysates were subjected to immunoprecipitation with a mAb δ-catenin. The immunocomplexes were sequentially immunoblotted with a mAb δ-catenin and a phospho- tyrosine antibody. (C) In the top panel, COS 1 cells were transfected with FLδ or FLδΔP6. Cell lysates were incubated with GST–SH3Lck and GST–SH3Fyn fusion proteins bound to glutathione-sepharose beads. Lysates incubated with glutathione beads alone served as a control. In the bottom panel, GST–SH3Lck and GST–SH3Fyn fusion proteins bound to glutathione sepharose beads, or beads alone were incubated with lysates from 3-wk-old hippocampal neurons. In all lanes, eluted proteins were blotted with a mAb δ-catenin.

Inhibition of Rho in the presence of δ-catenin enhances branch formation. (A) PC12 cells treated with NGF for 36 h were transfected with GFP (a), GFP and RhoV14 (b), GFP and C3 exotoxin (c). A second set of NGF-treated PC12 cells were transfected with GFP–δ-catenin (d), GFP–δ-catenin plus RhoV14 (e), or GFP–δ-catenin plus C3 exotoxin (f). Note the small branches in GFP–δ-catenin–C3-transfected cells (f, arrows). Bar, 5 μM. (B, a) PC12 and δ-PC12 cells transfected with GFP alone or cotransfected with GFP and C3 exotoxin. Neurite length and number of primary and secondary processes of C3-treated cells were quantified and expressed relative to those of untreated cells. (b) GFP-transfected PC12 and δ-PC12 cells were treated with the Rho kinase inhibitor Y27632 (10 μM) for 1 h before fixation. Total process length and number of primary and secondary processes were quantified relative to those of untreated cells. (C) Hippocampal neurons plated for 8 d were transfected with GFP–δ-catenin (d), GFP–δ-catenin plus RhoV14 (e), or GFP–δ-catenin plus C3 (f). As control, GFP (a), GFP plus RhoV14 (b), and GFP plus C3 (c) were transfected. Cells were fixed 24 h after transfection. Bar, 15 μM. Quantification of the length and number of protrusions with asterisks indicating significant differences relative to transfections with δ-catenin alone indicated by GFP-δ.

δ-Catenin induces protrusions along the distal dendritic shafts of hippocampal neurons. (A) Rat hippocampal neurons in culture for 8 d were transfected with GFP (a) and GFP–δ-catenin constructs (b) and fixed 24 h later. Small branches and filopodial-like protrusions on dendrites are indicated by arrows in b. Bar, 15 μm. (B) Hippocampal neurons in culture for 8 d were transfected with GFP–δ-catenin (d–f) and stained for MAP2 (a), actin (b), and tubulin (c). Panels g–i show colocalization. Bar, 5 μm. (C) Quantification of the number and length of protrusions for GFP and GFP–δ-catenin–transfected neurons. Detail of the protrusions is shown for GFP- (a) and GFP–δ-catenin– (b) transfected neurons. Bar, 5 μm.

δ-Catenin associates with actin and cortactin in hippocampal neurons. (A) Hippocampal neurons in culture for 5–7 d (a–h) or 3 wk (i–l) were double labeled with a mAb δ-catenin (green) and either phalloidin (red, a–d) or cortactin (red, e–l). Boxed areas in a, e, and i are shown at high power in images below. Panels b, f, and j show the red labels, and panels c, g, and k show the green labels. Colocalization in yellow is shown in panels a, e, and i for the low magnification images and in panels d, h, and l for the high magnification images. Bar: (a, e, and i) 50 μm; (the remaining images) 5 μm. (B) δ-Catenin coimmunoprecipitates with cortactin in hippocampal neurons. Hippocampal neurons plated for 3 d, 7 d, and 4 wk were lysed and immunoprecipitated with δ-catenin pAb 62 or with an unrelated antibody mAb (HA). The immunocomplexes were sequentially immunoblotted with a cortactin antibody and mAb δ-catenin. Hippocampal neurons plated for 9 d were reverse immunoprecipitated with a cortactin antibody or with mouse IgG as negative control. Complexes were immunoblotted with cortactin and mAb δ-catenin antibodies. (C) Coimmunoprecipitation of δ-catenin and cortactin cotransfected into COS1 cells. Cells were lysed, and immunoprecipitation was performed with pAb anti–δ-catenin 62 or anti-mAb HA. Transfection of δ-catenin alone into COS1 cells shows that the δ-catenin bands comigrate. For a reverse coimmunoprecipitation, the cotransfected COS1 cells were immunoprecipitated with an anti-mAb Flag antibody or anti-mAb HA as negative control. Transfection of Flag–cortactin alone into COS1 cells shows that the Flag–cortactin bands comigrate. Blots were visualized with cortactin and mAb δ-catenin antibodies. (D) GST–cortactin pulls down δ-catenin from hippocampal neurons. Pull down with GST and GST–full-length cortactin (GSTCort) was performed in lysates from hippocampal neurons plated for 6 d or 4 wk. Cell lysates were incubated with glutathione sepharose beads bound to GST or GST–cortactin fusion constructs. Beads were eluted in SDS buffer and loaded for electrophoresis. Blots were visualized with mAb δ-catenin or actin antibody.

δ-Catenin–cortactin interaction is regulated by tyrosine phosphorylation. (A) PC12 and δ-PC12 cells were lysed and immunoprecipitated with mAb δ-catenin. Blots were immunolabeled with a mAb δ-catenin or a phosphotyrosine antibody (p-tyr). (B) Hippocampal neurons in culture for 10 d were treated for 5 min with 200 μM H₂O₂ or 200 μM H₂O₂ plus 1 mM sodium orthovanadate. Cells were lysed and immunoprecipitated by a mAb δ-catenin. Blots were immunolabeled for mAb δ-catenin, phosphotyrosine, cortactin, and N-cadherin. (C) Hippocampal neurons in culture for 3 wk were treated with BDNF (50 ng/ml) for 1 h, and δ-catenin was immunoprecipitated with a mAb δ-catenin. Blots of the immunoprecipitated products were labeled with mAb δ-catenin, phosphotyrosine, and cortactin antibodies. (D) δ-PC12 cells (pretreated with 100 ng/ml NGF for 36 h) were treated with PP2 (25 μM for 1 h). Immunoprecipitated products were blotted with mAb δ-catenin, phosphotyrosine, and cortactin antibodies.

Inhibition of Src family kinases enhances δ-catenin– induced primary process elongation. (A) δ-PC12 cells were treated with 100 ng/ml NGF for 36 h and then left untreated (a) or treated with PP2 for 1 h (b). Cells were labeled with a mAb δ-catenin. Bar, 5 μm. (B) Quantification of neurite length and number of primary and secondary processes before and after PP2 treatment. No secondary processes were observed in the absence or presence of PP2.

δ-Catenin and primary process extension versus branching. Two different pathways regulate the effects of δ-catenin on process elaboration. Extracellular signals such as neurotrophins acting through regulation of Src family kinases can lead to δ-catenin–cortactin complex formation. The complex becomes competent to participate in primary process elongation by recruiting the Arp2/3 complex. Rho inhibition can be amplified by δ-catenin, which leads to branching.