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Cadherin activity is required for activity-induced spine remodeling.

Okamura K, Tanaka H, Yagita Y, Saeki Y, Taguchi A, Hiraoka Y, Zeng LH, Colman DR, Miki N - J. Cell Biol. (2004)

Bottom Line: N-cadherin-venus fusion protein laterally dispersed along the expanding spine head.Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion.Inhibition of actin polymerization with cytochalasin D abolished the spine expansion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Osaka University Medical School, Suita, Japan.

ABSTRACT
Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.

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N-cadherin was redistributed along the expanding spine head. Neurons were transfected with N-cadherin-venus fusion cDNA and subjected to time-lapse imaging. (A) Diagram shows the construction of N-cadherin-venus. (B) Immunostaining of a N-cadherin-venus transfected neuron. N-cadherin-venus is accumulated in dendritic spines positive with PSD-95 (arrowheads). Inset, closer magnification of a spine. (C) Immunostaining for endogenous N-cadherin and PSD-95. Arrowheads indicate spines. (D) Movements of N-cadherin-venus on spine head at rest. Note that the fluorescent signal shifts laterally. (E) N-cadherin-venus expressed on the apical surfaces of the spines before (top) and 30 min after (bottom) the depolarization. N-cadherin-venus dispersed along the expanding spine head. Arrowheads indicate lateral extents of spine heads; arrows indicate an edge of a dendritic shaft. (F) The lateral N-cadherin extent of each spine before and 30 min after depolarization is plotted. The length of the curve on the top of the N-cadherin-venus positive region, which presumably corresponds to SCCL was measured. The measurements of 21 spines of nine neurons (dendrites) were collected from nine independent experiments. *P < 0.001. (G) The graph shows the area size of N-cadherin immunoreactive puncta (mean ± SEM) before and 30 min after stimulation. The data were collected from four independent experiments. **P < 0.01. Bar: (B) 10.00 μm; (B, inset) 3.00 μm; (C) 6.00 μm; (D) 1.67 μm; (E) 1.30 μm.
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fig5: N-cadherin was redistributed along the expanding spine head. Neurons were transfected with N-cadherin-venus fusion cDNA and subjected to time-lapse imaging. (A) Diagram shows the construction of N-cadherin-venus. (B) Immunostaining of a N-cadherin-venus transfected neuron. N-cadherin-venus is accumulated in dendritic spines positive with PSD-95 (arrowheads). Inset, closer magnification of a spine. (C) Immunostaining for endogenous N-cadherin and PSD-95. Arrowheads indicate spines. (D) Movements of N-cadherin-venus on spine head at rest. Note that the fluorescent signal shifts laterally. (E) N-cadherin-venus expressed on the apical surfaces of the spines before (top) and 30 min after (bottom) the depolarization. N-cadherin-venus dispersed along the expanding spine head. Arrowheads indicate lateral extents of spine heads; arrows indicate an edge of a dendritic shaft. (F) The lateral N-cadherin extent of each spine before and 30 min after depolarization is plotted. The length of the curve on the top of the N-cadherin-venus positive region, which presumably corresponds to SCCL was measured. The measurements of 21 spines of nine neurons (dendrites) were collected from nine independent experiments. *P < 0.001. (G) The graph shows the area size of N-cadherin immunoreactive puncta (mean ± SEM) before and 30 min after stimulation. The data were collected from four independent experiments. **P < 0.01. Bar: (B) 10.00 μm; (B, inset) 3.00 μm; (C) 6.00 μm; (D) 1.67 μm; (E) 1.30 μm.

Mentions: To gain insight into the involvement of N-cadherin in the activity-induced enlargement of the spine head, we pursued the redistribution of N-cadherin by expressing recombinant N-cadherin fused with the Venus fluorescent protein, a variant of GFP (Fig. 5 A; Nagai et al., 2002). Although Venus was ligated adjacent to the catenin-binding domain of N-cadherin, N-cadherin-venus normally bound to β-catenin and exhibited homophilic adhesive activity on transfected L929 cells (unpublished data). N-cadherin-venus was distributed widely throughout the spine head, whereas PSD-95, a postsynaptic density marker protein, was often restricted to the center of the spine head (Fig. 5 B). The distribution of N-cadherin-venus well fitted in with the distribution of intrinsic N-cadherin as examined by immunostaining (Fig. 5 C). N-cadherin-venus was redistributed along the laterally moving spine head in resting state (Fig. 5 D). Upon depolarization, N-cadherin-venus showed lateral dispersion along the expanding spine head (Fig. 5, E and F). The lateral extent of spinal N-cadherin changed from 3.23 ± 0.222 μm to 3.60 ± 0.274 μm in 30 min of recovery. Thus, N-cadherin seems to be redistributed in accordance with the broadening of the synaptic apposition zone.


Cadherin activity is required for activity-induced spine remodeling.

Okamura K, Tanaka H, Yagita Y, Saeki Y, Taguchi A, Hiraoka Y, Zeng LH, Colman DR, Miki N - J. Cell Biol. (2004)

N-cadherin was redistributed along the expanding spine head. Neurons were transfected with N-cadherin-venus fusion cDNA and subjected to time-lapse imaging. (A) Diagram shows the construction of N-cadherin-venus. (B) Immunostaining of a N-cadherin-venus transfected neuron. N-cadherin-venus is accumulated in dendritic spines positive with PSD-95 (arrowheads). Inset, closer magnification of a spine. (C) Immunostaining for endogenous N-cadherin and PSD-95. Arrowheads indicate spines. (D) Movements of N-cadherin-venus on spine head at rest. Note that the fluorescent signal shifts laterally. (E) N-cadherin-venus expressed on the apical surfaces of the spines before (top) and 30 min after (bottom) the depolarization. N-cadherin-venus dispersed along the expanding spine head. Arrowheads indicate lateral extents of spine heads; arrows indicate an edge of a dendritic shaft. (F) The lateral N-cadherin extent of each spine before and 30 min after depolarization is plotted. The length of the curve on the top of the N-cadherin-venus positive region, which presumably corresponds to SCCL was measured. The measurements of 21 spines of nine neurons (dendrites) were collected from nine independent experiments. *P < 0.001. (G) The graph shows the area size of N-cadherin immunoreactive puncta (mean ± SEM) before and 30 min after stimulation. The data were collected from four independent experiments. **P < 0.01. Bar: (B) 10.00 μm; (B, inset) 3.00 μm; (C) 6.00 μm; (D) 1.67 μm; (E) 1.30 μm.
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fig5: N-cadherin was redistributed along the expanding spine head. Neurons were transfected with N-cadherin-venus fusion cDNA and subjected to time-lapse imaging. (A) Diagram shows the construction of N-cadherin-venus. (B) Immunostaining of a N-cadherin-venus transfected neuron. N-cadherin-venus is accumulated in dendritic spines positive with PSD-95 (arrowheads). Inset, closer magnification of a spine. (C) Immunostaining for endogenous N-cadherin and PSD-95. Arrowheads indicate spines. (D) Movements of N-cadherin-venus on spine head at rest. Note that the fluorescent signal shifts laterally. (E) N-cadherin-venus expressed on the apical surfaces of the spines before (top) and 30 min after (bottom) the depolarization. N-cadherin-venus dispersed along the expanding spine head. Arrowheads indicate lateral extents of spine heads; arrows indicate an edge of a dendritic shaft. (F) The lateral N-cadherin extent of each spine before and 30 min after depolarization is plotted. The length of the curve on the top of the N-cadherin-venus positive region, which presumably corresponds to SCCL was measured. The measurements of 21 spines of nine neurons (dendrites) were collected from nine independent experiments. *P < 0.001. (G) The graph shows the area size of N-cadherin immunoreactive puncta (mean ± SEM) before and 30 min after stimulation. The data were collected from four independent experiments. **P < 0.01. Bar: (B) 10.00 μm; (B, inset) 3.00 μm; (C) 6.00 μm; (D) 1.67 μm; (E) 1.30 μm.
Mentions: To gain insight into the involvement of N-cadherin in the activity-induced enlargement of the spine head, we pursued the redistribution of N-cadherin by expressing recombinant N-cadherin fused with the Venus fluorescent protein, a variant of GFP (Fig. 5 A; Nagai et al., 2002). Although Venus was ligated adjacent to the catenin-binding domain of N-cadherin, N-cadherin-venus normally bound to β-catenin and exhibited homophilic adhesive activity on transfected L929 cells (unpublished data). N-cadherin-venus was distributed widely throughout the spine head, whereas PSD-95, a postsynaptic density marker protein, was often restricted to the center of the spine head (Fig. 5 B). The distribution of N-cadherin-venus well fitted in with the distribution of intrinsic N-cadherin as examined by immunostaining (Fig. 5 C). N-cadherin-venus was redistributed along the laterally moving spine head in resting state (Fig. 5 D). Upon depolarization, N-cadherin-venus showed lateral dispersion along the expanding spine head (Fig. 5, E and F). The lateral extent of spinal N-cadherin changed from 3.23 ± 0.222 μm to 3.60 ± 0.274 μm in 30 min of recovery. Thus, N-cadherin seems to be redistributed in accordance with the broadening of the synaptic apposition zone.

Bottom Line: N-cadherin-venus fusion protein laterally dispersed along the expanding spine head.Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion.Inhibition of actin polymerization with cytochalasin D abolished the spine expansion.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Osaka University Medical School, Suita, Japan.

ABSTRACT
Neural activity induces the remodeling of pre- and postsynaptic membranes, which maintain their apposition through cell adhesion molecules. Among them, N-cadherin is redistributed, undergoes activity-dependent conformational changes, and is required for synaptic plasticity. Here, we show that depolarization induces the enlargement of the width of spine head, and that cadherin activity is essential for this synaptic rearrangement. Dendritic spines visualized with green fluorescent protein in hippocampal neurons showed an expansion by the activation of AMPA receptor, so that the synaptic apposition zone may be expanded. N-cadherin-venus fusion protein laterally dispersed along the expanding spine head. Overexpression of dominant-negative forms of N-cadherin resulted in the abrogation of the spine expansion. Inhibition of actin polymerization with cytochalasin D abolished the spine expansion. Together, our data suggest that cadherin-based adhesion machinery coupled with the actin-cytoskeleton is critical for the remodeling of synaptic apposition zone.

Show MeSH
Related in: MedlinePlus