(A) Examples of dendritic spines from neurons expressing either EGFP-CaM (top) or EGFP-PSD95 (bottom) and undergoing FRAP. Representative confocal images are shown before photobleaching (“pre-bleach”), right after photobleaching (“0.6 s”) and at different times during fluorescence recovery, as indicated. Bleached regions are indicated with squares in the “pre-bleach” panels. (B) Recovery curves of EGFP-CaM and EGFP-PSD95 were fit by one-phase exponential equations (n = 11 EGFP-CaM, n = 21 EGFP-PSD95). (C) The bar graph represents the immobile fraction of EGFP-CaM and PSD95 at the plateau. The immobile fraction of EGFP-CaM is significantly smaller than that of EGFP-PSD95 (p < 0.0001). D) The bar graph shows the recovery constant (K) of EGFP-PSD95 (0.03 ± 0.01/s) and EGFP-CaM (0.30 ± 0.04/s, p < 0.0001).

(A) Examples of dendritic spines from neurons co-expressing YFP-CaM with either CFP alone (top) or CFP-Ng (bottom) and undergoing FRAP. Representative confocal images are shown before photobleaching (“pre-bleach”), right after photobleaching (“0.6 s”) and at different times during fluorescence recovery, as indicated. Bleached regions are indicated with squares in the “pre-bleach” panels. (B) Recovery curves of YFP-CaM in the presence of CFP alone or CFP-Ng were fit with a one-phase exponential decay equation (n = 10 CFP, n = 7 CFP-Ng). Inset shows the initial 10 seconds of fluorescence recovery. (C) The bar graph represents the recovery constant (K) of YFP-CaM in presence of CFP alone or CFP-Ng (0.51 ± 0.08/s and 0.20 ± 0.04, respectively, p < 0.05). (D) The bar graph shows percent recovery of YFP-CaM fluorescence at 1.8–2.8 s after photobleaching in the presence of CFP alone (55.74 ± 3.56%) or CFP-Ng (38.86 ± 3.12%, p < 0.05). (E) The bar graph shows the immobile fraction of YFP-CaM fluorescence at the recovery plateau in the presence of CFP alone (20.37 ± 1.78%) or CFP-Ng (19.74 ± 2.94%, p = 0.8). To note, the immobile fraction of YFP-CaM is not affected by the presence of CFP-Ng.

The bar graphs represent average dendritic spine dimensions measured from neurons expressing EGFP (n = 9), EGFP-Ng (n = 13), EGFP-CaM (n = 17) or EGPF-Ng with pCI-CaM (n = 16). (A) The bar graph shows the width of dendritic spine heads in neurons expressing EGFP (784 ± 41 nm), EGFP-Ng (731 ± 28 nm), EGFP-CaM (875 ± 87 nm) and EGFP-Ng with pCI-CaM (737 ± 33 nm) (p = 0.6). (B) The bar graph shows the width of dendritic spine shafts in neurons expressing EGFP (429 ± 15 nm), EGFP-Ng (425 ± 13 nm), EGFP-CaM (418 ± 14 nm) and EGFP-Ng with pCI-CaM (378 ± 13 nm) (p = 0.3). (C) The bar graph shows the length of dendritic spine heads in neurons expressing EGFP (777 ± 38 nm), EGFP-Ng (763 ± 29 nm), EGFP-CaM (921 ± 48 nm) and EGFP-Ng with pCI-CaM (785 ± 32 nm) (p = 0.1). (D) The bar graph shows the length of dendritic spine shafts in neurons expressing EGFP (840 ± 72 nm), EGFP-Ng (952 ± 150 nm), EGFP-CaM (892 ± 77 nm) and EGFP-Ng with pCI-CaM (996 ± 106 m) (p = 0.7). (E) The bar graph shows the total length of dendritic spines in neurons expressing EGFP (1389 ± 89 nm), EGFP-Ng (1379 ± 116 nm), EGFP-CaM (1472 ± 89 nm) and EGFP-Ng with pCI-CaM (1475 ± 97 nm) (p = 0.5).

(A) Representative confocal image of a spine and the adjacent dendritic shaft from a neuron transfected with YFP-CaM and CFP (left) or with YFP-CaM and CFP-Ng (right), as indicated. Scale bar: 1 μm. Lower panels: representative line plot profiles of YFP fluorescence intensities across dendrite–spine pairs. (B) Spine-to-dendrite ratios are calculated from the corresponding peaks of fluorescence intensity at spines and adjacent dendritic shafts. Fluorescence intensity of YFP-CaM in spines was measured and normalized to fluorescence in adjacent dendrite area in the presence of either CFP or CFP-Ng (1.00 ± 0.05 and 1.37 ± 0.07, respectively, p < 0.0001). (C) Cumulative distribution of YFP-CaM fluorescence spine-to-dendrite ratio. A right shift of the distribution indicates an overall increase of YFP-CaM spine-to-dendrite ratio in the presence of CFP-Ng.

(A) Examples of dendritic spines from neurons co-expressing YFP-CaM with either CFP-Ng-SD (top) or CFP-Ng-SA (bottom) and undergoing FRAP. Representative confocal images are shown before photobleaching (“pre-bleach”), right after photobleaching (“0.6 s”) and at different times during fluorescence recovery, as indicated. Bleached regions are indicated with squares in the “pre-bleach” panels. (B) Recovery curves of YFP-CaM in the presence of CFP-Ng-SD or CFP-Ng-SA were fit with a one-phase exponential decay equation (n = 14 CFP-Ng-SD, n = 20 CFP-Ng-SA). Inset shows the initial 10 seconds of fluorescence recovery. (C) The bar graph represents the recovery constant (K) of YFP-CaM in presence of CFP-Ng-SD or CFP-Ng-SA (0.44 ± 0.05/s and 0.22 ± 0.02/s, respectively, p < 0.0001. (D) The bar graph shows the percent recovery of YFP-CaM fluorescence at 1.8–2.8 s after photobleaching in the presence of CFP-Ng-SD (57.25 ± 2.54%) or CFP-Ng-SA (41.10 ± 2.40%, p < 0.0001). (E) The bar graph shows immobile fraction of YFP-CaM fluorescence at recovery plateau in the presence of CFP-Ng-SD (16.5 ± 1.33%) or CFP-Ng-SA (18.57 ± 1.47%, p = 0.3203).