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Neurogranin regulates CaM dynamics at dendritic spines.

Petersen A, Gerges NZ - Sci Rep (2015)

Bottom Line: Interestingly, Ng did not influence the immobile fraction of CaM at recovery plateau.We have previously shown that Ng enhances synaptic strength in a CaM-dependent manner.Taken together, these data indicate that Ng-mediated enhancement of synaptic strength is due to its ability to target, rather than sequester, CaM within dendritic spines.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology and Anatomy, the Medical College of Wisconsin, Milwaukee, WI 53132.

ABSTRACT
Calmodulin (CaM) plays a key role in synaptic function and plasticity due to its ability to mediate Ca(2+) signaling. Therefore, it is essential to understand the dynamics of CaM at dendritic spines. In this study we have explored CaM dynamics using live-cell confocal microscopy and fluorescence recovery after photobleaching (FRAP) to study CaM diffusion. We find that only a small fraction of CaM in dendritic spines is immobile. Furthermore, the diffusion rate of CaM was regulated by neurogranin (Ng), a CaM-binding protein enriched at dendritic spines. Interestingly, Ng did not influence the immobile fraction of CaM at recovery plateau. We have previously shown that Ng enhances synaptic strength in a CaM-dependent manner. Taken together, these data indicate that Ng-mediated enhancement of synaptic strength is due to its ability to target, rather than sequester, CaM within dendritic spines.

No MeSH data available.


Ng Concentrates CaM in Dendritic Spines.(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.
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f4: Ng Concentrates CaM in Dendritic Spines.(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.

Mentions: We also wished to test whether increasing Ng concentration would increase CaM levels within dendritic spines. To directly test whether Ng increases CaM concentration in dendritic spines, we have co-expressed YFP-CaM with CFP-Ng or CFP alone and measured the spine-to-dendrite ratio of YFP fluorescence. Figure 4B shows that the spine-to-dendrite ratio of CaM is significantly increased (36.99 ± 0.07%, p < 0.0001) by Ng co-expression. Figure 4C shows that the cumulative distribution of YFP-CaM spine-to-dendrite ratios is shifted to the right by CFP-Ng co-expression. These data demonstrate that increasing Ng increases levels of CaM at dendritic spines.


Neurogranin regulates CaM dynamics at dendritic spines.

Petersen A, Gerges NZ - Sci Rep (2015)

Ng Concentrates CaM in Dendritic Spines.(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.
© Copyright Policy - open-access
Related In: Results  -  Collection

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f4: Ng Concentrates CaM in Dendritic Spines.(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.
Mentions: We also wished to test whether increasing Ng concentration would increase CaM levels within dendritic spines. To directly test whether Ng increases CaM concentration in dendritic spines, we have co-expressed YFP-CaM with CFP-Ng or CFP alone and measured the spine-to-dendrite ratio of YFP fluorescence. Figure 4B shows that the spine-to-dendrite ratio of CaM is significantly increased (36.99 ± 0.07%, p < 0.0001) by Ng co-expression. Figure 4C shows that the cumulative distribution of YFP-CaM spine-to-dendrite ratios is shifted to the right by CFP-Ng co-expression. These data demonstrate that increasing Ng increases levels of CaM at dendritic spines.

Bottom Line: Interestingly, Ng did not influence the immobile fraction of CaM at recovery plateau.We have previously shown that Ng enhances synaptic strength in a CaM-dependent manner.Taken together, these data indicate that Ng-mediated enhancement of synaptic strength is due to its ability to target, rather than sequester, CaM within dendritic spines.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Neurobiology and Anatomy, the Medical College of Wisconsin, Milwaukee, WI 53132.

ABSTRACT
Calmodulin (CaM) plays a key role in synaptic function and plasticity due to its ability to mediate Ca(2+) signaling. Therefore, it is essential to understand the dynamics of CaM at dendritic spines. In this study we have explored CaM dynamics using live-cell confocal microscopy and fluorescence recovery after photobleaching (FRAP) to study CaM diffusion. We find that only a small fraction of CaM in dendritic spines is immobile. Furthermore, the diffusion rate of CaM was regulated by neurogranin (Ng), a CaM-binding protein enriched at dendritic spines. Interestingly, Ng did not influence the immobile fraction of CaM at recovery plateau. We have previously shown that Ng enhances synaptic strength in a CaM-dependent manner. Taken together, these data indicate that Ng-mediated enhancement of synaptic strength is due to its ability to target, rather than sequester, CaM within dendritic spines.

No MeSH data available.