Modifier of cell adhesion regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth - PubMed (original) (raw)
Modifier of cell adhesion regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth
Qi Chen et al. J Neurosci. 2005.
Abstract
Modifier of cell adhesion (MOCA) is a member of the dedicator of cytokinesis 180 family of proteins and is highly expressed in CNS neurons. MOCA is associated with Alzheimer's disease tangles and regulates the accumulation of amyloid precursor protein and beta-amyloid. Here, we report that MOCA modulates cell-cell adhesion and morphology. MOCA increases the accumulation of adherens junction proteins, including N-cadherin and beta-catenin, whereas reducing endogenous MOCA expression lowers cell-cell aggregation and N-cadherin expression. MOCA colocalizes with N-cadherin and actin in areas of cell-cell and cell substratum contact and is expressed in neuronal processes. MOCA accumulates during neuronal differentiation, and its expression enhances NGF-induced neurite outgrowth and morphological complexity. We conclude that MOCA regulates N-cadherin-mediated cell-cell adhesion and neurite outgrowth.
Figures
Figure 7.
Image analysis of PC12 cells. A typical neuron shape (A), its respective distance transform (B), skeleton with branches (C), and branch points (D); schematic neuron used to define arc-length (E), soma to e along neurite. F, Quantitation of arc-length distances from branch points to soma (length in micrometers). G, Quantitation of arc-length distances from extremity points to soma in micrometers. This experiment was repeated with three independent clones with similar results.
Figure 1.
MOCA-induced cell-cell aggregation is calcium dependent, trypsin sensitive, and N-cadherin dependent. A, B, Phase-contrast images of 293T cells expressing MOCA or no MOCA. C, D, Three cell lines transfected with either MOCA or empty plasmid were dissociated with EGTA or trypsin in the presence or absence of 5 m
m
calcium. The rate of aggregation was measured in the presence of 5 m
m
calcium by the disappearance of single cells as a function of time at 37°C. C, 293T cells. D, B103 cells. E, PC12 cells. ○, Wild-type (WT) cells dissociated by EGTA; ▵, MOCA-expressing cells dissociated with EGTA; X-X, wild-type cells dissociated with trypsin plus calcium; □, MOCA cells dissociated with trypsin in the presence of calcium; ♦, wild-type cells dissociated with trypsin minus calcium; ▪, MOCA cells dissociated with trypsin minus calcium. Scale bar, 25 μm. _F-_-J, 293T cells (F) or 293T cells expressing MOCA (G) were dissociated with EGTA and allowed to reaggregate in the presence of anti-E-cadherin (a-E-CAD) (H) or anti-N-cadherin (a-N-CAD) (I). The data are quantitated in J. *Significantly different from 293T control; p < 0.05.
Figure 5.
MOCA accumulates during NGF-induced differentiation. PC12 cells expressing no MOCA (A; empty plasmid) or MOCA (B) were exposed to 100 ng/ml β-NGF for 2 d, lysed in SDS sample buffer, and equal amounts of protein loaded onto 10% SDS acrylamide gels. The samples were blotted with either anti-MOCA, anti-TAG, or anti-GAPDH antibodies (C). The changes of MOCA expression by NGF were quantified by NIH Image. The data are presented as the percentage of change relative to the control without NGF treatment. PC12 cells with NGF (D, E) or HCN cells were transfected with either the control shRNA construct (F) or MOCA503i shRNA construct (D, E, insets, G), and the percentage of cells with neurites greater than one cell body diameter was quantified (H). The amount of MOCA in HCN cells was assayed after transfection (I). Scale bar, 5 μm. The data are representative of three independent experiments. *p < 0.05.
Figure 2.
Transient transfection with MOCA increases cell-cell aggregation. 293T cells were transfected with MOCA or empty plasmid and a plasmid harboring the full-length GFP gene (ratio 10:1). Thirty hours later, the cells were dissociated with EGTA and allowed to aggregate. At 0 and 2 h, the cells were plated on polylysine-coated dishes. Two hours later, they were scored for single fluorescent cells or for two or more fluorescent cells in contact as a percentage of the total fluorescent cells. A, Control, phase contrast. B, Control, fluorescent cells. C, MOCA, phase contrast. D, MOCA, fluorescent cells. G, Quantitation of above. Pairs indicate groups of two or more fluorescent cells, whereas singles indicate isolated single cells. Only fluorescent cells were scored. In some cases, MOCA-expressing 293T cells were transfected with the MOCA503i shRNA construct coexpressing GFP (E) or the control shRNA construct (F) for 3 d and aggregation determined as described above and quantified (H). Note that the magnification in E and F is much lower than in _A_-D. Scale bars: D, 10 μm; F, 25 μm. *p < 0.01, significantly different from controls.
Figure 3.
MOCA increases the expression levels of N-cadherin. A, Effect of MOCA on the level of protein components of the cell-cell adhesion system. Cells with or without MOCA were lysed, and the same amount of protein from the same type of cell was subjected to Western blot analysis. Actin was used as a loading control. B, MOCA-induced N-cadherin expression is not affected by cell density. Calcium was depleted by treating cells with 1 m
m
EGTA for 30 min, resulting in a single-cell suspension. N-cadherin expression in cells expressing MOCA or no MOCA was examined by Western blotting as a function of time. C, Lowering MOCA by RNA interference also lowers N-cadherin expression. siRNA duplexes targeting specific MOCA sequences were transfected into cells. Ninety-six hours after transfection, the expression of MOCA and N-cadherin was assayed by Western blot analysis. The expression of N-cadherin was altered by RNA interference of MOCA gene expression as indicated by Western blotting. Actin was used as a loading control. Lane 1, 293T; lanes 2 and 3, 293T/MOCA, treated with scrambled siRNA duplexes; lanes 4 and 5, 293T/MOCA, treated with a MOCA siRNA duplex. D, MOCA increases the formation of N-cadherin and β-catenin complexes. Cell lysates extracted from 293T cells expressing MOCA or no MOCA were immunoprecipitated with the indicated antibodies (left) and/or beads alone. The resulting precipitates were then analyzed by Western blotting with the corresponding antibodies indicated on the right. The quantitation of protein expression was determined after normalized to the actin control by measuring the optical density of respective blots using NIH Image and presented as the percentage of change ±SEM (n = 3). *p < 0.01, significantly different from controls. The data are representative of three independent experiments. Ab, Antibody; E-Cad, E-cadherin; N-Cad, N-cadherin; α-Cat, α-catenin; β-Cat, β-catenin; p120-Cat, p120-catenin; γ-Cat, γ-catenin.
Figure 4.
The level of N-cadherin mRNAs and the turnover rate, immunostaining pattern, and cellular fractionation of N-cadherin in cells expressing MOCA or no MOCA. A, Northern blot hybridization of N-cadherin mRNA in 293T cells expressing MOCA or no MOCA. The quantitation of the major N-cadherin band was determined after normalization to the actin control. The data are presented as the percentage of change ±SEM. B, RT-PCR of N-cadherin was performed using two different primer sets as described in Materials and Methods. Actin and GAPDH were used as control. C, The turnover rate of newly synthesized N-cadherin in 293T cells expressing MOCA or no MOCA. The optical density of N-cadherin expression was quantified by NIH Image, and the data are presented as the percentage of change after being normalized to the initial level of newly synthesized N-cadherin. D, Immunostaining of E-cadherin, N-cadherin, and β-catenin in 293T cells expressing MOCA or no MOCA. E-cadherin, N-cadherin, and β-catenin were stained by their corresponding antibodies and visualized by fluorescein-labeled secondary antibodies (green). Nuclei were stained by 4′,6′-diamidino-2-phenylindole (blue). Cells expressing MOCA are in the right column and expressing no MOCA in the left column. Background staining with secondary antibody alone is also shown in the top panel. Scale bar, 10 μm. E, Detergent fractions of cell extracts were immunoblotted to MOCA, N-cadherin, E-cadherin, β-catenin, γ-catenin, and p120catenin. The localization of MOCA and adherens junction proteins were assayed by sequential extraction using different detergents as described in Materials and Methods. GST is the control for cytoplasm, PDI for membrane organelles, and cytokeratin 18 for the cytoskeletal-matrix fractions (Ramsby and Makowski, 1999). F, The secretion of N-cadherin was significantly enhanced by MOCA in B103, 293T, and PC12 cells, whereas the secretion of neural cell adhesion molecule (NCAM) was not affected by MOCA. The quantitation of N-cadherin secretion was determined after normalization to the NCAM. The data are presented as the percentage of change ±SEM (n = 3). *p < 0.01, significantly different from controls. Ab, Antibody; E-Cad, E-cadherin; N-Cad, N-cadherin; β-Cat, β-catenin; p120Cat, p120-catenin; γ-Cat, γ-catenin.
Figure 6.
MOCA mediates neurite outgrowth. PC12 cells expressing or not expressing MOCA were dissociated with EGTA and treated with the various antibodies as described in Materials and Methods. Neurite outgrowth was scored either 24 h (E-G) or 48 h (A-D) later. In some cases, cells were plated on culture dishes coated for 24 h with conditioned serum-free medium from either 293T or 293T/MOCA cells in the presence or absence of antisera. A, PC12/MOCA cells, no NGF. B, PC12/MOCA cells treated with 100 ng/ml NGF. C, PC12/MOCA cells plus NGF exposed to anti-fibronectin (a-FIBRO). D, PC12/MOCA cells plus NGF exposed to anti-N-cadherin (a-NCAD). E, PC12 cells plus NGF plus 293T-coated surface (SAM). F, PC12 cells plus 293T/MOCA SAM. G, PC12 cells, plus 293T/MOCA SAM plus anti-N-cadherin (a-N-CAD). H, Quantitation of neurite lengths. +Significant difference from MOCA plus NGF, p < 0.01; #significant difference from CM 293T/MOCA, p < 0.01. CONT, Control.
Figure 8.
MOCA and F-actin colocalize at cell-cell interfaces and at the cell substrate level. MOCA-transfected PC12 cells were fixed and double labeled with anti-MOCA antibody and with rhodamine-phalloidin. In A-C, MOCA and F-actin are seen colocalized at cell-cell interfaces (arrows). In the merged image in C, areas of colocalization are yellow (arrows). In D-F, confocal images at the substratum level show colocalization of MOCA and F-actin at cell-substrate contacts (arrows) and within growth cones and filopodial tufts of cell bodies (_D_-F, arrows). In the merged image in F, areas of colocalization are yellow.
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