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Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines.

Shiraishi-Yamaguchi Y, Sato Y, Sakai R, Mizutani A, Knöpfel T, Mori N, Mikoshiba K, Furuichi T - BMC Neurosci (2009)

Bottom Line: Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons.CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. f1966@cc.nagasaki-u.ac.jp

ABSTRACT

Background: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin.

Results: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.

Conclusion: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

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Cupidin influences Cdc42V12-induced changes in HeLa cell morphology. (A) Human fibroblast HeLa cells were transfected with vector alone (mock), CPD, CPDΔ191–230 (= Δ191–230), Cdc42V12 (dominant active form), Cdc42V12 + CPD, and Cdc42V12 + CPDΔ191–230. Actin cytoskeletons of transfected cells were stained by Texas Red-conjugated phalloidin. In mock-, CPD- and CPDΔ191–230-transfected cells, F-actin is concentrated in the cell periphery and in fine stress fibers that traverse in the cytoplasm. On the other hand, cells transfected with dominant-active Cdc42V12 show many filopodia-like protrusions and thin actin bundles around the cell periphery and lack cytoplasmic stress fibers. Co-expression of CPD suppresses Cdc42V12-induced filopodia formation, whereas that of CPDΔ191–230 does not. (B) Filopodia formation in (A) was statistically quantified as described in the methods. *, p < 0.01; **, p < 0.001.
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Figure 3: Cupidin influences Cdc42V12-induced changes in HeLa cell morphology. (A) Human fibroblast HeLa cells were transfected with vector alone (mock), CPD, CPDΔ191–230 (= Δ191–230), Cdc42V12 (dominant active form), Cdc42V12 + CPD, and Cdc42V12 + CPDΔ191–230. Actin cytoskeletons of transfected cells were stained by Texas Red-conjugated phalloidin. In mock-, CPD- and CPDΔ191–230-transfected cells, F-actin is concentrated in the cell periphery and in fine stress fibers that traverse in the cytoplasm. On the other hand, cells transfected with dominant-active Cdc42V12 show many filopodia-like protrusions and thin actin bundles around the cell periphery and lack cytoplasmic stress fibers. Co-expression of CPD suppresses Cdc42V12-induced filopodia formation, whereas that of CPDΔ191–230 does not. (B) Filopodia formation in (A) was statistically quantified as described in the methods. *, p < 0.01; **, p < 0.001.

Mentions: We previously showed that co-expressed Cupidin suppresses the dominant-active Cdc42-induced morphological and actin-cytoskeletal changes in HeLa cells [6]. To verify the cellular function of Cdc42 binding, we analyzed the influence of Cdc42-binding deficiency on these cellular phenotypes (Fig. 3A). In HeLa cells transfected with CPD full-length or CPDΔ191–230, stress fibers and bundles of F-actin were formed in the cytoplasm and the cell periphery, respectively, as in cells transfected with vector alone (mock). Upon expression of Cdc42V12, a dominant-active form of Cdc42, many filopodia-like protrusions with actin bundles were drastically induced around the cell periphery. Co-expression of the CPD diminished the number of Cdc42V12-induced protrusions and actin bundles in the cell periphery. By contrast, co-expression of CPDΔ191–230 did not affect Cdc42V12-induced cellular phenotypes, as statistically proven by counting spike number around the cell periphery (Fig. 3B). These results indicated that the region containing amino acids 191–230 is required for the suppression of Cdc42V12-induced morphological and actin-cytoskeletal changes in HeLa cells.


Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines.

Shiraishi-Yamaguchi Y, Sato Y, Sakai R, Mizutani A, Knöpfel T, Mori N, Mikoshiba K, Furuichi T - BMC Neurosci (2009)

Cupidin influences Cdc42V12-induced changes in HeLa cell morphology. (A) Human fibroblast HeLa cells were transfected with vector alone (mock), CPD, CPDΔ191–230 (= Δ191–230), Cdc42V12 (dominant active form), Cdc42V12 + CPD, and Cdc42V12 + CPDΔ191–230. Actin cytoskeletons of transfected cells were stained by Texas Red-conjugated phalloidin. In mock-, CPD- and CPDΔ191–230-transfected cells, F-actin is concentrated in the cell periphery and in fine stress fibers that traverse in the cytoplasm. On the other hand, cells transfected with dominant-active Cdc42V12 show many filopodia-like protrusions and thin actin bundles around the cell periphery and lack cytoplasmic stress fibers. Co-expression of CPD suppresses Cdc42V12-induced filopodia formation, whereas that of CPDΔ191–230 does not. (B) Filopodia formation in (A) was statistically quantified as described in the methods. *, p < 0.01; **, p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666743&req=5

Figure 3: Cupidin influences Cdc42V12-induced changes in HeLa cell morphology. (A) Human fibroblast HeLa cells were transfected with vector alone (mock), CPD, CPDΔ191–230 (= Δ191–230), Cdc42V12 (dominant active form), Cdc42V12 + CPD, and Cdc42V12 + CPDΔ191–230. Actin cytoskeletons of transfected cells were stained by Texas Red-conjugated phalloidin. In mock-, CPD- and CPDΔ191–230-transfected cells, F-actin is concentrated in the cell periphery and in fine stress fibers that traverse in the cytoplasm. On the other hand, cells transfected with dominant-active Cdc42V12 show many filopodia-like protrusions and thin actin bundles around the cell periphery and lack cytoplasmic stress fibers. Co-expression of CPD suppresses Cdc42V12-induced filopodia formation, whereas that of CPDΔ191–230 does not. (B) Filopodia formation in (A) was statistically quantified as described in the methods. *, p < 0.01; **, p < 0.001.
Mentions: We previously showed that co-expressed Cupidin suppresses the dominant-active Cdc42-induced morphological and actin-cytoskeletal changes in HeLa cells [6]. To verify the cellular function of Cdc42 binding, we analyzed the influence of Cdc42-binding deficiency on these cellular phenotypes (Fig. 3A). In HeLa cells transfected with CPD full-length or CPDΔ191–230, stress fibers and bundles of F-actin were formed in the cytoplasm and the cell periphery, respectively, as in cells transfected with vector alone (mock). Upon expression of Cdc42V12, a dominant-active form of Cdc42, many filopodia-like protrusions with actin bundles were drastically induced around the cell periphery. Co-expression of the CPD diminished the number of Cdc42V12-induced protrusions and actin bundles in the cell periphery. By contrast, co-expression of CPDΔ191–230 did not affect Cdc42V12-induced cellular phenotypes, as statistically proven by counting spike number around the cell periphery (Fig. 3B). These results indicated that the region containing amino acids 191–230 is required for the suppression of Cdc42V12-induced morphological and actin-cytoskeletal changes in HeLa cells.

Bottom Line: Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons.CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory for Molecular Neurogenesis, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. f1966@cc.nagasaki-u.ac.jp

ABSTRACT

Background: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin.

Results: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons.

Conclusion: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

Show MeSH
Related in: MedlinePlus