Figure 6.
Figure 6. Cofilin-1 is necessary for proper spine morphology and actin turnover. (A) Mouse hippocampal neurons were transfected with GFP in the presence or absence of cofilin-1 siRNA oligonucleotides at DIV 11. On DIV 12, the cells were fixed and stained with anti–cofilin-1 antibodies (see Fig. S5). Reduction in cofilin-1 protein levels resulted in abnormal spine morphology (longer necks, branched spine heads). (B) Dendritic protrusions of wild-type and cofilin-1 siRNA-transfected cells were analyzed with NeuronIQ software (Cheng et al., 2007). Dendritic protrusion density was reduced and dendritic protrusion length was increased in cofilin-1 siRNA-transfected cells as compared with wild-type cells. Numerical data are presented in Table IV. (C) Morphology analysis revealed a decrease in the number of thin spines. Numerical data are presented in Tables I and II. Moreover, the density of branched spines was increased from 0.015 to 0.039 spines per micrometer (P < 0.001) as compared with wild-type cells. Graphs represent mean ± SEM. (D) The rate of actin turnover was analyzed from wild-type and cofilin-1 siRNA-transfected cells by FRAP. Mouse cortical neurons were transfected with GFP-actin (wt) or with GFP-actin with cofilin-1 siRNA (cof) at DIV 11, and the FRAP analysis was performed at DIV 12. The frames before (−10 s) and after bleach (from +2 to +306 s) are shown. In the spines of wild-type neurons, the fluorescence of GFP-actin recovery was nearly complete at 80 s; in cofilin-1 siRNA neurons, complete recovery was not achieved within 306 s. (E) The averaged recovery curves from nine wild-type and seven cofilin-1 siRNA cells revealed a diminished rate of actin turnover in cofilin-1 siRNA-transfected cells. Error bars represent SEMs. (F) Dynamics of spines of wild-type and cofilin-1 siRNA-transfected cells were followed by time-lapse stack confocal scan microscopy. Mouse hippocampal neurons at DIV 11 (wild-type) or DIV 10 (cofilin-1 siRNA) were transfected with GFP-actin without (wt; Video 7) or with cofilin-1 siRNA oligonucleotides (cofsiRNA; Video 9), and the time-lapse videos were acquired on the day after the transfection. Note the slow removal of the filopodia-like protrusions from the spine head in the cofilin-1–depleted cell. Bars, 1 µm.

Cofilin-1 is necessary for proper spine morphology and actin turnover. (A) Mouse hippocampal neurons were transfected with GFP in the presence or absence of cofilin-1 siRNA oligonucleotides at DIV 11. On DIV 12, the cells were fixed and stained with anti–cofilin-1 antibodies (see Fig. S5). Reduction in cofilin-1 protein levels resulted in abnormal spine morphology (longer necks, branched spine heads). (B) Dendritic protrusions of wild-type and cofilin-1 siRNA-transfected cells were analyzed with NeuronIQ software (Cheng et al., 2007). Dendritic protrusion density was reduced and dendritic protrusion length was increased in cofilin-1 siRNA-transfected cells as compared with wild-type cells. Numerical data are presented in Table IV. (C) Morphology analysis revealed a decrease in the number of thin spines. Numerical data are presented in Tables I and II. Moreover, the density of branched spines was increased from 0.015 to 0.039 spines per micrometer (P < 0.001) as compared with wild-type cells. Graphs represent mean ± SEM. (D) The rate of actin turnover was analyzed from wild-type and cofilin-1 siRNA-transfected cells by FRAP. Mouse cortical neurons were transfected with GFP-actin (wt) or with GFP-actin with cofilin-1 siRNA (cof) at DIV 11, and the FRAP analysis was performed at DIV 12. The frames before (−10 s) and after bleach (from +2 to +306 s) are shown. In the spines of wild-type neurons, the fluorescence of GFP-actin recovery was nearly complete at 80 s; in cofilin-1 siRNA neurons, complete recovery was not achieved within 306 s. (E) The averaged recovery curves from nine wild-type and seven cofilin-1 siRNA cells revealed a diminished rate of actin turnover in cofilin-1 siRNA-transfected cells. Error bars represent SEMs. (F) Dynamics of spines of wild-type and cofilin-1 siRNA-transfected cells were followed by time-lapse stack confocal scan microscopy. Mouse hippocampal neurons at DIV 11 (wild-type) or DIV 10 (cofilin-1 siRNA) were transfected with GFP-actin without (wt; Video 7) or with cofilin-1 siRNA oligonucleotides (cofsiRNA; Video 9), and the time-lapse videos were acquired on the day after the transfection. Note the slow removal of the filopodia-like protrusions from the spine head in the cofilin-1–depleted cell. Bars, 1 µm.

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