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Dual regulation of neuronal morphogenesis by a delta-catenin-cortactin complex and Rho.

Martinez MC, Ochiishi T, Majewski M, Kosik KS - J. Cell Biol. (2003)

Bottom Line: Under conditions when tyrosine phosphorylation is reduced, delta-catenin binds to cortactin and cells extend unbranched primary processes.When RhoA is inhibited, delta-catenin enhances the effects of Rho inhibition on branching.We conclude that delta-catenin contributes to setting a balance between neurite elongation and branching in the elaboration of a complex dendritic tree.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Neurology, Brigham and Women's Hospital and Harvard Medical School, Harvard Institute of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

ABSTRACT
Delta-catenin is a neuronal protein that contains 10 Armadillo motifs and binds to the juxtamembrane segment of classical cadherins. We report that delta-catenin interacts with cortactin in a tyrosine phosphorylation-dependent manner. This interaction occurs within a region of the delta-catenin sequence that is also essential for the neurite elongation effects. Src family kinases can phosphorylate delta-catenin and bind to delta-catenin through its polyproline tract. Under conditions when tyrosine phosphorylation is reduced, delta-catenin binds to cortactin and cells extend unbranched primary processes. Conversely, increasing tyrosine phosphorylation disrupts the delta-catenin-cortactin complex. When RhoA is inhibited, delta-catenin enhances the effects of Rho inhibition on branching. We conclude that delta-catenin contributes to setting a balance between neurite elongation and branching in the elaboration of a complex dendritic tree.

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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.
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fig4: 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.

Mentions: Immunostaining with δ-catenin antibodies demonstrated that the endogenous protein is coextensive with filamentous actin (Fig. 4Figure 4.


Dual regulation of neuronal morphogenesis by a delta-catenin-cortactin complex and Rho.

Martinez MC, Ochiishi T, Majewski M, Kosik KS - J. Cell Biol. (2003)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172717&req=5

fig4: 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.
Mentions: Immunostaining with δ-catenin antibodies demonstrated that the endogenous protein is coextensive with filamentous actin (Fig. 4Figure 4.

Bottom Line: Under conditions when tyrosine phosphorylation is reduced, delta-catenin binds to cortactin and cells extend unbranched primary processes.When RhoA is inhibited, delta-catenin enhances the effects of Rho inhibition on branching.We conclude that delta-catenin contributes to setting a balance between neurite elongation and branching in the elaboration of a complex dendritic tree.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Neurology, Brigham and Women's Hospital and Harvard Medical School, Harvard Institute of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.

ABSTRACT
Delta-catenin is a neuronal protein that contains 10 Armadillo motifs and binds to the juxtamembrane segment of classical cadherins. We report that delta-catenin interacts with cortactin in a tyrosine phosphorylation-dependent manner. This interaction occurs within a region of the delta-catenin sequence that is also essential for the neurite elongation effects. Src family kinases can phosphorylate delta-catenin and bind to delta-catenin through its polyproline tract. Under conditions when tyrosine phosphorylation is reduced, delta-catenin binds to cortactin and cells extend unbranched primary processes. Conversely, increasing tyrosine phosphorylation disrupts the delta-catenin-cortactin complex. When RhoA is inhibited, delta-catenin enhances the effects of Rho inhibition on branching. We conclude that delta-catenin contributes to setting a balance between neurite elongation and branching in the elaboration of a complex dendritic tree.

Show MeSH