Article Figures & Data
Figures
- Figure 1.
The dynamics of dendritic protrusions are increased in sdy neurons. Hippocampal neurons from wild-type or sdy mice were transfected at 14 DIV with the Venus construct and imaged every minute for 30 min at 17 DIV. A, C, E, Representative images superimposed from those taken at 4 adjacent time points during the 30 min live-imaging period. In A, red dots represent protrusion retraction, yellow dots indicate protrusion addition; in C, red dots represent the transformation from mushroom/thin spines to filopodia, yellow dots indicate the transformation in the opposite direction; in E, red dots represent the conversion from mushroom/thin to stubby spines, yellow dots indicate the conversion in the opposite direction. B, D, F, Quantification of A, C, E. G, Cumulative frequency plot of total dynamic events occurring during the 30 min imaging period. Scale bar, 5 μm. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 2.
The change to dendritic protrusion density in sdy and dysbindin knockdown neurons. Cultured hippocampal neurons were prepared from sdy mice or their wild-type littermates (A, B) or rats (F, G), and transfected with the Venus construct alone (A, B) or along with lentivirus expressing dysbindin siRNA and siRNA-resistant dysbindin (mDys) at 14 DIV. A, F, Representative images of neurons and dendrites. Scale bars: neurons, 20 μm; dendrites, 5 μm. B, G, Quantification for A and F; n = 15 cells in each group; data are presented as mean ± SEM; *p < 0.05, **p < 0.01. C, E, Cultured wild-type cortical neurons were infected with lentivirus expressing EGFP alone or along with siRNAs targeting dysbindin mRNAs at 5 DIV and harvested for RT-PCR and immunoblotting analysis for endogenous dysbindin mRNAs and proteins at 2 weeks after infection. D, HEK 293 cells were transfected with plasmids expressing HA-tagged dysbindin, tropomodulin 2- (Tmod2, containing no dysbindin siRNA-2 binding sites) or siRNA-resistant dysbindin along with the dysbindin siRNA construct, and harvested at 2 d after transfection for immunoblotting.
- Figure 3.
CaMKIIα is required for the stabilization of dendritic protrusions. A, B, Hippocampi of 2- to 3-week-old sdy mice and their wild-type littermates were removed for analyses of CaMKIIα and CaMKIIα phosphorylated at Thr286 in whole-cell lysates (WCL) and the P2 fraction. C–I, Hippocampal neurons were transfected at 14 DIV with the Venus construct along with the CA-CaMKIIα or the CaMKIIα siRNA oligonucleotides, and imaged every minute for 30 min at 17 DIV. A, Representative blots. B, Quantification for A; n = 4 experiments. C, E, G, Representative images superimposed from those taken at 4 contiguous time points during the 30 min live-imaging period. Red dots represent protrusion retraction (C), the transformation from mushroom/thin spines to filopodia (E), and the conversion from mushroom/thin to stubby spines (G); yellow dots indicate the protrusion addition (C), the transformation from filopodia to mushroom/thin spines (E), and the conversion from stubby to mushroom/thin spines (G). D, F, H, Quantification of C, E, G. I, Cumulative frequency plot for the total number of all types of dynamic events taking place during the 30 min period. Scale bar, 5 μm. Data are presented as mean ± SEM;*p < 0.05, **p < 0.01.
- Figure 4.
CaMKIIα controls the number of mushroom/thin spines. Cultured hippocampal neurons from rats were transfected at 14 DIV with indicated plasmids. A, Representative images of neurons (top) and dendrites (bottom). B, Quantification for A, n = 15 neurons for each group. Data are presented as mean ± SEM; *p < 0.05. Scale bars: neurons, 20 μm; dendrites, 5 μm.
- Figure 5.
CaMKIIα(T286D) corrects the hyperactivity of dendritic protrusions in sdy neurons. Hippocampal neurons from wild-type or sdy mice were transfected at 14 DIV with the Venus construct alone or along with the CaMKIIα(T286D) construct, and imaged every minute for 30 min at 17 DIV. A, C, E, Representative images superimposed from those taken at four contiguous time points during the 30 min live-imaging period. Red dots represent protrusion retraction (A), the transformation from mushroom/thin spines to filopodia (C), the conversion from mushroom/thin to stubby spines (E); yellow dots indicate protrusion addition (A), the transformation from filopodia to mushroom/thin spines (C), the conversion from stubby to mushroom/thin spines (E). B, D, F, Quantification of A, C, E. G, Cumulative frequency plot for the total number of all types of dynamic events taking place during the 30 min period. Scale bar, 5 μm. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01.
- Figure 6.
CaMKIIα restores spine density in sdy neurons. Cultured hippocampal neurons from sdy or wild-type mice were transfected with the Venus construct alone or along with the CaMKIIα(T286D) construct at 14 DIV and imaged at 17 DIV. A, Representative images of neurons (top) and dendrites (bottom). B, Quantification for A; n = 15 neurons for each group. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01.
- Figure 7.
Abi1 forms more complexes with CaMKIIα in sdy mice. The hippocampal tissue removed from WT and sdy mice (3-week-old) was used for coimmunoprecipitation with an antibody against Abi1. A, Representative immunoblots. B–D, Quantification of Abi1 and CaMKα in whole-cell lysates (B), the P2 fraction (C) and the immunoprecipitate (D). Data represent the average of three independent experiments using three pairs of wild-type and sdy mice, and are presented as mean ± SEM; *p < 0.05, **p < 0.01.
- Figure 8.
Abi1 eliminates protrusion hyperdynamics of sdy neurons. Hippocampal neurons from wild-type and sdy mice were transfected at 14 DIV with the Venus construct alone or along with the Abi1 construct, and imaged every minute for 30 min at 17 DIV. A, C, E, Representative images superimposed from those taken at four contiguous time points during the 30 min live-imaging period. Red dots represent protrusion retraction (A), the transformation from mushroom/thin spines to filopodia (C), the conversion from mushroom/thin to stubby spines (E); yellow dots indicate protrusion addition (A), the transformation from filopodia to mushroom/thin spines (C), the conversion from stubby to mushroom/thin spines (E). B, D, F, Quantification of A, C, E. G, Cumulative frequency plot for the total number of all types of dynamic events taking place during the 30 min period. Scale bar, 5 μm. Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.
- Figure 9.
Abi1 restores the densities of mushroom/thin spines and filopodia in sdy neurons. Cultured hippocampal neurons from sdy or wild-type mice were transfected with the Venus construct alone or along with the Abi1 construct at 14 DIV and imaged at 17 DIV. A, Representative images of neurons (top) and dendrites (bottom). B, Quantification for A; n = 15 neurons for each group. Data are presented as mean ± SEM; ***p < 0.001.
