Figure 3.
Figure 3. Role of mDia2 in spinogenesis. (A) Mouse hippocampal neurons were transfected with a plasmid expressing GFP (left) or a GFP fusion of active mDia2 construct at DIV 11, then fixed and stained with phalloidin at DIV 12. Active mDia2 localized to the filopodia tips, and its expression induced filopodia and spine head loss. (B) Mouse hippocampal neurons were transfected with GFP (left) or GFP-active mDia2 (green) and inactive Rif-myc (red) constructs at DIV 12; the cells were fixed and stained with anti-myc antibodies at DIV 13. Expression of active mDia2 overcomes the effect of inactive Rif. Arrows indicate the GFP-mDia2 tip localization. (C) Quantitative analysis of neurons expressing inactive Rif and active mDia2 (inactRif + actmDia2) showed significant reduction in dendritic protrusion density and length as compared with wild-type (wt) cells. Numerical data and p-values are presented in Table IV. (D) Dendritic protrusion morphology analysis of inactive Rif and active mDia2-expressing neurons revealed an increase in the number of filopodia and a significant decrease in the number of thin spines. Numerical data and p-values are presented in Tables I and II. (E) Mouse hippocampal neurons were transfected with GFP-actin, GFP-actin + mDia2 siRNA, or GFP-actin + inactive Rif constructs at DIV 12; the cells were fixed and stained with anti-mDia2 antibodies (Fig. S3) or anti-myc antibodies (not depicted) at DIV 13. Transfection of cells with mDia2 siRNA oligonucleotides resulted in dendritic protrusion morphology defects similar to those from expression of inactive Rif (shortened spine necks and larger spine heads). (F) Quantitative analysis of mDia2 siRNA-treated neurons showed a significant reduction in dendritic protrusion density and dendritic protrusion length as compared with wild type (wt). Numerical data and p-values are presented in Table IV. (G) Dendritic protrusion morphology analysis of mDia2 siRNA-treated neurons revealed a decrease in the number of filopodia and thin spines, and a significant increase in the number of stubby spines. Numerical data and p-values are presented in Tables I and II. Graphs represent mean ± SEM. Bars, 5 µm.

Role of mDia2 in spinogenesis. (A) Mouse hippocampal neurons were transfected with a plasmid expressing GFP (left) or a GFP fusion of active mDia2 construct at DIV 11, then fixed and stained with phalloidin at DIV 12. Active mDia2 localized to the filopodia tips, and its expression induced filopodia and spine head loss. (B) Mouse hippocampal neurons were transfected with GFP (left) or GFP-active mDia2 (green) and inactive Rif-myc (red) constructs at DIV 12; the cells were fixed and stained with anti-myc antibodies at DIV 13. Expression of active mDia2 overcomes the effect of inactive Rif. Arrows indicate the GFP-mDia2 tip localization. (C) Quantitative analysis of neurons expressing inactive Rif and active mDia2 (inactRif + actmDia2) showed significant reduction in dendritic protrusion density and length as compared with wild-type (wt) cells. Numerical data and p-values are presented in Table IV. (D) Dendritic protrusion morphology analysis of inactive Rif and active mDia2-expressing neurons revealed an increase in the number of filopodia and a significant decrease in the number of thin spines. Numerical data and p-values are presented in Tables I and II. (E) Mouse hippocampal neurons were transfected with GFP-actin, GFP-actin + mDia2 siRNA, or GFP-actin + inactive Rif constructs at DIV 12; the cells were fixed and stained with anti-mDia2 antibodies (Fig. S3) or anti-myc antibodies (not depicted) at DIV 13. Transfection of cells with mDia2 siRNA oligonucleotides resulted in dendritic protrusion morphology defects similar to those from expression of inactive Rif (shortened spine necks and larger spine heads). (F) Quantitative analysis of mDia2 siRNA-treated neurons showed a significant reduction in dendritic protrusion density and dendritic protrusion length as compared with wild type (wt). Numerical data and p-values are presented in Table IV. (G) Dendritic protrusion morphology analysis of mDia2 siRNA-treated neurons revealed a decrease in the number of filopodia and thin spines, and a significant increase in the number of stubby spines. Numerical data and p-values are presented in Tables I and II. Graphs represent mean ± SEM. Bars, 5 µm.

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