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Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation.

Bencsik N, Szíber Z, Liliom H, Tárnok K, Borbély S, Gulyás M, Rátkai A, Szűcs A, Hazai-Novák D, Ellwanger K, Rácz B, Pfizenmaier K, Hausser A, Schlett K - J. Cell Biol. (2015)

Bottom Line: In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways.Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation.We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.

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Affiliation: Department of Physiology and Neurobiology, Eötvös Loránd University, H-1117 Budapest, Hungary.

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Endogenous PKD is activated within dendritic spines during plasticity-inducing changes in vitro. (A) Schematic representation of the PKD activity reporter, containing the PKD-specific substrate sequence of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) and the EGFP sequence. The a-pS294 antibody recognizes the phosphorylated Ser294 target site. (B) Inverted fluorescent and a-pS294/EGFP ratio images of tertiary dendritic branches from control or cLTP- or KCl-treated neurons after 30 min. Arrowheads indicate mushroom spines. Bars, 1 µm. (C and D) Relative a-pS294/EGFP ratio values in mushroom spines treated with KCl (C) or cLTP (D) for the indicated time. 3 µM kbNB 142-70 (kbNB) or 1 µM PDBu was applied 1 h before other treatments or for 15 min, respectively. 10 µM MK-801, 50 µM APV, 1 µM nifedipine, and 1 µM ω-conotoxin MVIIC were applied for the indicated time periods. S/A indicates a reporter construct with a nonphosphorylatable alanine mutation. Data were obtained from three to four independent cultures and displayed as mean ± SEM. The number of analyzed spines is indicated within the graphs. Asterisks represent significance compared with control values, and $ symbols indicate significant differences between data pairs. *, P < 0.05; **, P < 0.01; *** or $$$, P < 0.001.
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fig1: Endogenous PKD is activated within dendritic spines during plasticity-inducing changes in vitro. (A) Schematic representation of the PKD activity reporter, containing the PKD-specific substrate sequence of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) and the EGFP sequence. The a-pS294 antibody recognizes the phosphorylated Ser294 target site. (B) Inverted fluorescent and a-pS294/EGFP ratio images of tertiary dendritic branches from control or cLTP- or KCl-treated neurons after 30 min. Arrowheads indicate mushroom spines. Bars, 1 µm. (C and D) Relative a-pS294/EGFP ratio values in mushroom spines treated with KCl (C) or cLTP (D) for the indicated time. 3 µM kbNB 142-70 (kbNB) or 1 µM PDBu was applied 1 h before other treatments or for 15 min, respectively. 10 µM MK-801, 50 µM APV, 1 µM nifedipine, and 1 µM ω-conotoxin MVIIC were applied for the indicated time periods. S/A indicates a reporter construct with a nonphosphorylatable alanine mutation. Data were obtained from three to four independent cultures and displayed as mean ± SEM. The number of analyzed spines is indicated within the graphs. Asterisks represent significance compared with control values, and $ symbols indicate significant differences between data pairs. *, P < 0.05; **, P < 0.01; *** or $$$, P < 0.001.

Mentions: Previously, we have described a PKD activity reporter, which is suitable to visualize endogenous PKD-mediated phosphorylation events in fixed cells (Fig. 1 A; Czöndör et al., 2009; Fuchs et al., 2009) and is present in the dendritic branches and spines of DIV12-13 hippocampal neurons (see the EGFP signal in Fig. 1 B and Fig. S1 A). To compare the extent of reporter phosphorylation in spines, ratiometric images were created by normalizing the a-pS294 to EGFP signal intensities (Fig. 1 B and Fig. S1 A, ratio images). Only mushroom spines with clearly enlarged heads were chosen for the analysis. To confirm the specificity of the pS294 antibody signal, a mutant reporter construct containing alanine instead of the target serine was also investigated (S/A mutant). In all cases, S/A mutant reporter displayed only a negligible ratio signal (Fig. 1, B–D).


Protein kinase D promotes plasticity-induced F-actin stabilization in dendritic spines and regulates memory formation.

Bencsik N, Szíber Z, Liliom H, Tárnok K, Borbély S, Gulyás M, Rátkai A, Szűcs A, Hazai-Novák D, Ellwanger K, Rácz B, Pfizenmaier K, Hausser A, Schlett K - J. Cell Biol. (2015)

Endogenous PKD is activated within dendritic spines during plasticity-inducing changes in vitro. (A) Schematic representation of the PKD activity reporter, containing the PKD-specific substrate sequence of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) and the EGFP sequence. The a-pS294 antibody recognizes the phosphorylated Ser294 target site. (B) Inverted fluorescent and a-pS294/EGFP ratio images of tertiary dendritic branches from control or cLTP- or KCl-treated neurons after 30 min. Arrowheads indicate mushroom spines. Bars, 1 µm. (C and D) Relative a-pS294/EGFP ratio values in mushroom spines treated with KCl (C) or cLTP (D) for the indicated time. 3 µM kbNB 142-70 (kbNB) or 1 µM PDBu was applied 1 h before other treatments or for 15 min, respectively. 10 µM MK-801, 50 µM APV, 1 µM nifedipine, and 1 µM ω-conotoxin MVIIC were applied for the indicated time periods. S/A indicates a reporter construct with a nonphosphorylatable alanine mutation. Data were obtained from three to four independent cultures and displayed as mean ± SEM. The number of analyzed spines is indicated within the graphs. Asterisks represent significance compared with control values, and $ symbols indicate significant differences between data pairs. *, P < 0.05; **, P < 0.01; *** or $$$, P < 0.001.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig1: Endogenous PKD is activated within dendritic spines during plasticity-inducing changes in vitro. (A) Schematic representation of the PKD activity reporter, containing the PKD-specific substrate sequence of phosphatidylinositol 4-kinase IIIβ (PI4KIIIβ) and the EGFP sequence. The a-pS294 antibody recognizes the phosphorylated Ser294 target site. (B) Inverted fluorescent and a-pS294/EGFP ratio images of tertiary dendritic branches from control or cLTP- or KCl-treated neurons after 30 min. Arrowheads indicate mushroom spines. Bars, 1 µm. (C and D) Relative a-pS294/EGFP ratio values in mushroom spines treated with KCl (C) or cLTP (D) for the indicated time. 3 µM kbNB 142-70 (kbNB) or 1 µM PDBu was applied 1 h before other treatments or for 15 min, respectively. 10 µM MK-801, 50 µM APV, 1 µM nifedipine, and 1 µM ω-conotoxin MVIIC were applied for the indicated time periods. S/A indicates a reporter construct with a nonphosphorylatable alanine mutation. Data were obtained from three to four independent cultures and displayed as mean ± SEM. The number of analyzed spines is indicated within the graphs. Asterisks represent significance compared with control values, and $ symbols indicate significant differences between data pairs. *, P < 0.05; **, P < 0.01; *** or $$$, P < 0.001.
Mentions: Previously, we have described a PKD activity reporter, which is suitable to visualize endogenous PKD-mediated phosphorylation events in fixed cells (Fig. 1 A; Czöndör et al., 2009; Fuchs et al., 2009) and is present in the dendritic branches and spines of DIV12-13 hippocampal neurons (see the EGFP signal in Fig. 1 B and Fig. S1 A). To compare the extent of reporter phosphorylation in spines, ratiometric images were created by normalizing the a-pS294 to EGFP signal intensities (Fig. 1 B and Fig. S1 A, ratio images). Only mushroom spines with clearly enlarged heads were chosen for the analysis. To confirm the specificity of the pS294 antibody signal, a mutant reporter construct containing alanine instead of the target serine was also investigated (S/A mutant). In all cases, S/A mutant reporter displayed only a negligible ratio signal (Fig. 1, B–D).

Bottom Line: In nonneuronal cells, protein kinase D (PKD) has an important role in stabilizing F-actin via multiple molecular pathways.Consequently, impaired PKD functions attenuate activity-dependent changes in hippocampal dendritic spines, including LTP formation, cause morphological alterations in vivo, and have deleterious consequences on spatial memory formation.We thus provide compelling evidence that PKD controls synaptic plasticity and learning by regulating actin stability in dendritic spines.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Neurobiology, Eötvös Loránd University, H-1117 Budapest, Hungary.

Show MeSH
Related in: MedlinePlus