Erbin controls dendritic morphogenesis by regulating localization of delta-catenin

Abstract

The LAP [leucine-rich and postsynaptic density-95/Discs large/zona occludens-1 (PDZ)] protein erbin and delta-catenin, a component of the cadherin-catenin cell adhesion complex, are highly expressed in neurons and associate through PDZ-mediated interaction, but have incompletely characterized neuronal functions. We show that short hairpin RNA-mediated knockdown of erbin and knockdown or genetic ablation of delta-catenin severely impaired dendritic morphogenesis in hippocampal neurons. Simultaneous loss of erbin and delta-catenin does not enhance severity of this phenotype. The dendritic phenotype observed after erbin depletion is rescued by overexpression of delta-catenin and requires a domain in delta-catenin that has been shown to regulate dendritic branching. Knockdown of delta-catenin cannot be rescued by overexpression of erbin, indicating that erbin is upstream of delta-catenin. delta-Catenin-null neurons have no alterations in global levels of active Rac1/RhoA. Knockdown of erbin results in alterations in localization of delta-catenin. These results suggest a critical role for erbin in regulating dendritic morphogenesis by maintaining appropriate localization of delta-catenin.

Figure 1.

Knockdown of erbin impairs dendritic morphogenesis in hippocampal neurons. A, B, Rat hippocampal neurons were transfected at DIV 11 with vector or shRNA 1 to erbin (A) or vector or shRNA 2 to erbin and examined at DIV 17 (B). Representative images show dendritic morphology. C, Anti-MAP2 staining at DIV 17 of cultured rat hippocampal neurons expressing erbin shRNA 1. D–F, Quantification of total average dendritic length (vector 1, 2019.99 ± 99.87 μm; shRNA 1, 760.56 ± 70.13 μm; p < 0.0001; vector, 1663.59 ± 91.10 μm; shRNA 2, 741.23 ± 30.89 μm; p < 0.0001; D), dendritic end points (vector, 30.78 ± 1.6; shRNA 1, 11.13 ± 0.668; p < 0.0001, vector 2, 25.8 ± 1.16; shRNA 2, 13.79 ± 0.72; p < 0.0001; E), and primary dendrites (vector, 5.3 ± 0.22; shRNA 1, 5 ± 0.29; p = 0.41; vector, 5.28 ± 0.31; shRNA 2, 4.97 ± 0.24; p = 0.43; F) from rat hippocampal neurons transfected at DIV 11 with vector, shRNA 1, vector or shRNA 2, and examined at DIV 17 (n > 21 neurons for each; ***p < 0.0001). NS, Not significant. Scale bars, 20 μm. Error bars indicate SEM.

Figure 2.

Overexpression of the C terminus or the PDZ domain of erbin reduces dendritic length in rat hippocampal neurons. A, Rat hippocampal neurons were transfected with vector that also encodes GFP or vector and the C terminus of erbin. Representative images show dendritic morphology of neurons transfected at DIV 11 and examined at DIV 17. B, Rat hippocampal neurons were transfected with vector or vector and the PDZ domain of erbin. Representative images show dendritic morphology of neurons transfected at DIV 11 and examined at DIV 17. Scale bar, 20 μm.

Figure 3.

Hippocampal neurons from δ-catenin-null mice exhibit reduced dendritic complexity. Mouse hippocampal neurons from δ-catenin+/+ and δ-catenin−/− neurons were cultured, transfected with a plasmid encoding GFP, and examined at DIV 17. Representative images show dendritic morphology. Scale bar, 20 μm.

Figure 4.

Acute knockdown of δ-catenin impairs dendritic morphogenesis. A, Rat hippocampal neurons were transfected at DIV 11 with vector or δ-catenin shRNA and examined at DIV 17. Representative images show dendritic morphology. B, Anti-MAP2 staining at DIV 17 of cultured rat hippocampal neurons expressing δ-catenin shRNA. Scale bars, 20 μm.

Figure 5.

Nonadditive effects of erbin and δ-catenin depletion on dendritic length. A, Hippocampal neurons from wild-type (δ-catenin+/+) or δ-catenin-null (δ-catenin−/−) mice were transfected with a vector or erbin shRNA at DIV 11 and examined at DIV 17. Representative images of neuronal morphology are shown. B, Quantification of total average dendritic length from δ-catenin+/+ and δ-catenin−/− hippocampal neurons expressing vector or erbin shRNA (δ-catenin+/+ + vector, 2186.32 μm ± 193.05 μm; δ-catenin+/+ + shRNA, 987.88 μm ± 158.25 μm; p < 0.0005; δ-catenin−/− + vector, 982.29 μm ± 91.56; δ-catenin−/− + shRNA, 804.40 μm ± 98.31; p = 0.2; n > 10 neurons). ***p < 0.0005. C, Rat hippocampal neurons were transfected at DIV 11 with the indicated constructs and fixed at DIV 17. Representative images of neuronal morphology are shown. D, Quantification of total average dendritic length from neurons expressing the indicated constructs (vector, 1669.14 ± 104.56 μm; erbin shRNA, 549.83 ± 60.71 μm; δ-catenin shRNA, 707.67 ± 58.17 μm; p < 0.00001 for vector vs erbin shRNA and vector vs δ-catenin shRNA; n > 22 neurons). ***p < 0.0001. NS, Not significant. Scale bars, 20 μm. Error bars indicate SEM.

Figure 6.

Erbin is upstream of δ-catenin in regulating dendritic morphogenesis. A, Rat hippocampal neurons were transfected at DIV 11 with vector, erbin shRNA 1, or erbin shRNA 1 plus EGFP δ-catenin and fixed at DIV 17. Representative images show dendritic morphology. Scale bar, 20 μm. B, Quantification of dendritic length from neurons transfected at DIV 11 with the indicated constructs and fixed at DIV 17 (vector, 1871.7 ± 119.56 μm; shRNA 1, 590.26 ± 39.67 μm; shRNA 1 + δ-catenin, 1252.68 ± 97.59 μm; p < 0.0001 for vector vs shRNA 1; p < 0.0001 for shRNA 1 vs shRNA 1 + δ-catenin; vector, 1780.54 ± 127.96 μm; shRNA 2, 806.37 ± 69.07 μm; shRNA 2 + δ-catenin, 1397.63 ± 142.13 μm; p < 0.0001 for vector vs shRNA 2; p < 0.001 for shRNA 2 vs shRNA 2 + δ-catenin). C, Quantification of total average dendritic length from neurons transfected with vector 1, erbin shRNA 1, or erbin shRNA 1 and p120ctn (vector, 1666.96 ± 87.86 μm; shRNA 1, 586.72 ± 69.82 μm; shRNA 1 + p120ctn, 600.29 ± 44.47 μm; p < 0.0001 for vector vs shRNA 1; p = 0.87 for shRNA 1 vs shRNA 1 + p120ctn; n > 18 neurons each). D, Quantification of total dendritic length in neurons expressing vector, δ-catenin (δ-cat) shRNA, or δ-catenin shRNA plus full-length erbin (n > 20 neurons each). **p < 0.001; ***p < 0.0001. NS, Not significant. Error bars indicate SEM.

Figure 7.

The C-terminal region of δ-catenin is necessary for δ-catenin to rescue the loss of function of the erbin phenotype. A, Schematic of constructs of δ-catenin. B, Immunoblots from tsa201 cells expressing the constructs in A with anti-δ-catenin. C, Rat hippocampal neurons in culture were transfected with the indicated constructs at DIV 11 and fixed at DIV 17. Representative images show dendritic morphology. D, Quantification of total average dendritic length from neurons expressing the indicated constructs (vector, 1644.93 ± 116.91 μm; shRNA, 510.51 ± 50.56 μm; shRNA + N-term, 560 ± 37.86; shRNA + N-Arm, 563.35 ± 45.33 μm; shRNA + Δ-PDZ, 932.52 ± 76.30 μm; p values vs shRNA: vector, <0.0001; shRNA + N-term, 0.43; shRNA + N-Arm, 0.44; shRNA + Δ-PDZ, <0.0001; n > 19 neurons). ***p < 0.0001. Scale bar, 20 μm. Error bars indicate SEM.

Figure 8.

Erbin knockdown or overexpression of the C terminal or PDZ domain of erbin leads to altered localization of δ-catenin. A–F, Altered localization (arrowheads) of EGFP-δ-catenin in hippocampal neurons that express EGFP-δ-catenin and vector (A), EGFP-δ-catenin and erbin shRNA (B), EGFP-δ-catenin (C, E), EGFP-δ-catenin and the C terminus of erbin (D), and EGFP δ-catenin and the PDZ domain of erbin (F). Images are pseudocolored for fluorescence intensity (EGFP-δ-catenin + vector: diffuse distribution, n = 12; punctate distribution, n = 4; EGFP-δ-catenin + erbin shRNA 1: diffuse distribution, n = 1; punctate distribution, n = 17; EGFP-δ-catenin: diffuse distribution, n = 19; punctate distribution, n = 3; EGFP-δ-catenin + C terminus of erbin: diffuse distribution, n = 0; punctate distribution, n = 28; EGFP-δ-catenin: diffuse distribution, n = 18; punctate distribution, n = 2; EGFP-δ-catenin + PDZ domain (dom) of erbin: diffuse distribution, n = 0; punctate distribution, n = 8). Scale bar, 10 μm.