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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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W2A-cadherin abrogates the spine head expansion. (A) Aggregation of L929 cells stably expressing N-cadherin (Ncad) and/or W2A-cadherin (W2Acad). The homophilic adhesiveness of N-cadherin yielded large cell aggregates. Co-expression of W2A-cadherin suppressed the adhesiveness of N-cadherin (Ncad+W2Acad). E-cadherin does not change the N-cadherin adhesiveness (Ncad+Ecad). (B) The shape of spines in the W2A-cadherin transfected neurons fixed with PFA. Green, GFP; blue, W2A-cadherin. A subpopulation of spines display extended morphology (arrowheads) as well as normal shape (arrows). (C) Examples of typical protrusion morphologies. C-1, cotyloid spine; C-2, flat apex spine; C-3, thin spine; C-4, filopodia. (D) Mean lengths of the protrusions are shown. *P < 0.01 to GFP. (E) Double immunostaining of the W2A-cadherin transfected neurons with synaptophysin. The spines are apposed with synaptophysin puncta (red, arrows). (F) Time-lapse series of a W2A-cadherin transfected neuron. Depolarization induces the rounding up and the freezing (depol) as observed in control neurons. There is no enlargement of the lateral spine size during the recovery phase (recovery 30′). (G) The SCCL (left) and spine width (right) before and 30 min after depolarization are plotted. **P < 0.0002; ***P < 0.00002. (H) Change in spine shape was detected with confocal microscopy. (I) The spine widths measured on confocal images are plotted. ****P < 0.000003. (J) The graphs show changes in spine width (left) and SCCL (right) in 30 min after depolarization. Neurons were transfected with mock (GFP), N-cadherin (N-cad), W2A-cadherin (W2A-cad), and NcadΔE. (K) Immunostaining of the neuron transfected with W2A-cadherin. GluR2/3 immunoreactivity normally localized in spines (arrowheads). The data were collected from four to five neurons (dendrites) in four to five independent experiments for each group (D, G, I, and J). The data are shown as mean ± SEM (D and J). Bar: (A) 125 μm; (B) 5.00 μm; (C) 1.56 μm; (E) 5.00 μm; (F, left) 4.50 μm; (F, right) 1.50 μm; (H) 1.50 μm; (K) 3.71 μm.
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fig7: W2A-cadherin abrogates the spine head expansion. (A) Aggregation of L929 cells stably expressing N-cadherin (Ncad) and/or W2A-cadherin (W2Acad). The homophilic adhesiveness of N-cadherin yielded large cell aggregates. Co-expression of W2A-cadherin suppressed the adhesiveness of N-cadherin (Ncad+W2Acad). E-cadherin does not change the N-cadherin adhesiveness (Ncad+Ecad). (B) The shape of spines in the W2A-cadherin transfected neurons fixed with PFA. Green, GFP; blue, W2A-cadherin. A subpopulation of spines display extended morphology (arrowheads) as well as normal shape (arrows). (C) Examples of typical protrusion morphologies. C-1, cotyloid spine; C-2, flat apex spine; C-3, thin spine; C-4, filopodia. (D) Mean lengths of the protrusions are shown. *P < 0.01 to GFP. (E) Double immunostaining of the W2A-cadherin transfected neurons with synaptophysin. The spines are apposed with synaptophysin puncta (red, arrows). (F) Time-lapse series of a W2A-cadherin transfected neuron. Depolarization induces the rounding up and the freezing (depol) as observed in control neurons. There is no enlargement of the lateral spine size during the recovery phase (recovery 30′). (G) The SCCL (left) and spine width (right) before and 30 min after depolarization are plotted. **P < 0.0002; ***P < 0.00002. (H) Change in spine shape was detected with confocal microscopy. (I) The spine widths measured on confocal images are plotted. ****P < 0.000003. (J) The graphs show changes in spine width (left) and SCCL (right) in 30 min after depolarization. Neurons were transfected with mock (GFP), N-cadherin (N-cad), W2A-cadherin (W2A-cad), and NcadΔE. (K) Immunostaining of the neuron transfected with W2A-cadherin. GluR2/3 immunoreactivity normally localized in spines (arrowheads). The data were collected from four to five neurons (dendrites) in four to five independent experiments for each group (D, G, I, and J). The data are shown as mean ± SEM (D and J). Bar: (A) 125 μm; (B) 5.00 μm; (C) 1.56 μm; (E) 5.00 μm; (F, left) 4.50 μm; (F, right) 1.50 μm; (H) 1.50 μm; (K) 3.71 μm.

Mentions: Depolarization induces the lateral expansion of spine head. Hippocampal neurons were transfected with gfp and subjected to CCD imaging on 18–22 DIV. (A) GFP uniformly labels the neuronal contour. (B) Closer magnification of a GFP-filled dendrite. Closed arrow, cotyloid spine; closed arrowhead, flat apex spine; open arrow, thin spine; open arrowhead, filopodium (Table I; Fig. 7 C). (C) The GFP-filled neurons were immunolabeled with synaptophysin (red). The presynaptic terminal labeled by synaptophysin attaches to the cotyloid face on the apex of the spine. Arrowheads indicate putative synaptic cleft. (D) GFP-labeled neurons were transiently treated with high K+ (31 mM) for 2 min, and recovered for 60 min in normal K+ solution, while images were taken serially. The spine rounded up during the depolarization (2 min), and then displayed cotyloid shape again when the stimulation was halted (recovery 5′). The lateral size of the spine became larger than in the original at 15 min after the stimulation (recovery 15′), and it lasted at least for 60 min (recovery 60′). Bar: (A) 60.00 μm; (B) 5.00 μm; (C) 1.00 μm; (D) 1.25 μm.


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)

W2A-cadherin abrogates the spine head expansion. (A) Aggregation of L929 cells stably expressing N-cadherin (Ncad) and/or W2A-cadherin (W2Acad). The homophilic adhesiveness of N-cadherin yielded large cell aggregates. Co-expression of W2A-cadherin suppressed the adhesiveness of N-cadherin (Ncad+W2Acad). E-cadherin does not change the N-cadherin adhesiveness (Ncad+Ecad). (B) The shape of spines in the W2A-cadherin transfected neurons fixed with PFA. Green, GFP; blue, W2A-cadherin. A subpopulation of spines display extended morphology (arrowheads) as well as normal shape (arrows). (C) Examples of typical protrusion morphologies. C-1, cotyloid spine; C-2, flat apex spine; C-3, thin spine; C-4, filopodia. (D) Mean lengths of the protrusions are shown. *P < 0.01 to GFP. (E) Double immunostaining of the W2A-cadherin transfected neurons with synaptophysin. The spines are apposed with synaptophysin puncta (red, arrows). (F) Time-lapse series of a W2A-cadherin transfected neuron. Depolarization induces the rounding up and the freezing (depol) as observed in control neurons. There is no enlargement of the lateral spine size during the recovery phase (recovery 30′). (G) The SCCL (left) and spine width (right) before and 30 min after depolarization are plotted. **P < 0.0002; ***P < 0.00002. (H) Change in spine shape was detected with confocal microscopy. (I) The spine widths measured on confocal images are plotted. ****P < 0.000003. (J) The graphs show changes in spine width (left) and SCCL (right) in 30 min after depolarization. Neurons were transfected with mock (GFP), N-cadherin (N-cad), W2A-cadherin (W2A-cad), and NcadΔE. (K) Immunostaining of the neuron transfected with W2A-cadherin. GluR2/3 immunoreactivity normally localized in spines (arrowheads). The data were collected from four to five neurons (dendrites) in four to five independent experiments for each group (D, G, I, and J). The data are shown as mean ± SEM (D and J). Bar: (A) 125 μm; (B) 5.00 μm; (C) 1.56 μm; (E) 5.00 μm; (F, left) 4.50 μm; (F, right) 1.50 μm; (H) 1.50 μm; (K) 3.71 μm.
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Related In: Results  -  Collection

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fig7: W2A-cadherin abrogates the spine head expansion. (A) Aggregation of L929 cells stably expressing N-cadherin (Ncad) and/or W2A-cadherin (W2Acad). The homophilic adhesiveness of N-cadherin yielded large cell aggregates. Co-expression of W2A-cadherin suppressed the adhesiveness of N-cadherin (Ncad+W2Acad). E-cadherin does not change the N-cadherin adhesiveness (Ncad+Ecad). (B) The shape of spines in the W2A-cadherin transfected neurons fixed with PFA. Green, GFP; blue, W2A-cadherin. A subpopulation of spines display extended morphology (arrowheads) as well as normal shape (arrows). (C) Examples of typical protrusion morphologies. C-1, cotyloid spine; C-2, flat apex spine; C-3, thin spine; C-4, filopodia. (D) Mean lengths of the protrusions are shown. *P < 0.01 to GFP. (E) Double immunostaining of the W2A-cadherin transfected neurons with synaptophysin. The spines are apposed with synaptophysin puncta (red, arrows). (F) Time-lapse series of a W2A-cadherin transfected neuron. Depolarization induces the rounding up and the freezing (depol) as observed in control neurons. There is no enlargement of the lateral spine size during the recovery phase (recovery 30′). (G) The SCCL (left) and spine width (right) before and 30 min after depolarization are plotted. **P < 0.0002; ***P < 0.00002. (H) Change in spine shape was detected with confocal microscopy. (I) The spine widths measured on confocal images are plotted. ****P < 0.000003. (J) The graphs show changes in spine width (left) and SCCL (right) in 30 min after depolarization. Neurons were transfected with mock (GFP), N-cadherin (N-cad), W2A-cadherin (W2A-cad), and NcadΔE. (K) Immunostaining of the neuron transfected with W2A-cadherin. GluR2/3 immunoreactivity normally localized in spines (arrowheads). The data were collected from four to five neurons (dendrites) in four to five independent experiments for each group (D, G, I, and J). The data are shown as mean ± SEM (D and J). Bar: (A) 125 μm; (B) 5.00 μm; (C) 1.56 μm; (E) 5.00 μm; (F, left) 4.50 μm; (F, right) 1.50 μm; (H) 1.50 μm; (K) 3.71 μm.
Mentions: Depolarization induces the lateral expansion of spine head. Hippocampal neurons were transfected with gfp and subjected to CCD imaging on 18–22 DIV. (A) GFP uniformly labels the neuronal contour. (B) Closer magnification of a GFP-filled dendrite. Closed arrow, cotyloid spine; closed arrowhead, flat apex spine; open arrow, thin spine; open arrowhead, filopodium (Table I; Fig. 7 C). (C) The GFP-filled neurons were immunolabeled with synaptophysin (red). The presynaptic terminal labeled by synaptophysin attaches to the cotyloid face on the apex of the spine. Arrowheads indicate putative synaptic cleft. (D) GFP-labeled neurons were transiently treated with high K+ (31 mM) for 2 min, and recovered for 60 min in normal K+ solution, while images were taken serially. The spine rounded up during the depolarization (2 min), and then displayed cotyloid shape again when the stimulation was halted (recovery 5′). The lateral size of the spine became larger than in the original at 15 min after the stimulation (recovery 15′), and it lasted at least for 60 min (recovery 60′). Bar: (A) 60.00 μm; (B) 5.00 μm; (C) 1.00 μm; (D) 1.25 μm.

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