Figure 7. γCaMKI Translocates to the Nucleus and Is Important for CREB Phosphorylation in PV+ Cells.
(A) Image of γCaMKI staining in PV+ cells before and after 40K. Note how nucleus is more uniformly filled with γCaMKI following stimulation. White circle represents nuclear outline, traced from DAPI staining. Scale bar, 10 μm.
(B) Time course of nuc/cyt ratio of immunostaining against γCaMKI.
(C) Nuc/cyt ratio of staining against γCaMKI increased with 5 min of 40K; increase prevented by nimodipine, KN93, or STO609 but not by KN92.
(D) γCaMKI nuc/cyt ratio increased in PV+ cells following 40K; increase prevented by BAPTA but spared by EGTA.
(E) Nuc/cyt ratio of γCaMKI-GFP increased following 40K in PV+ but not excitatory neurons.
(F) Following 40K for 5 min, nuclear CaM was correlated with nuclear γCaMKI. (Pearson correlation, R2 = 0.80, p<0.001).
(G) Increased CaM nuc/cyt ratio following 40K for 5 min was prevented by shRNA knockdown of γCaMKI. 40K value for shRNA-expressing cells was not different from baseline (4K) value for control cells (p>0.3). All values normalized to baseline value for control, non-transfected cells.
(H) Following 40K for 5 min, pCREB was correlated with nuclear γCaMKI. (Pearson correlation, R2 = 0.18, p<0.05).
(I) Image of neurons transfected with γCaMKI shRNA (left, cyan) or with non-silencing control (right, cyan). Cells treated with 40K for 30 s and co-stained for PV (middle row, green) and pCREB (bottom row, red). Note that γCaMKI knockdown reduced CREB phosphorylation and dendritic branching.
(J) pCREB response to 40K for 30 s was reduced by shRNA knockdown of γCaMKI and rescued by shRNA-resistant construct (green bar). Third/fifth bar from the left, non-transfected cells on coverslips transfected with shRNA/shRNA+rescue. Data shown are representative of 4 shRNA experiments.
(K) Two-dimensional Sholl analysis of dendritic arbor complexity 7 days after transfection with γCaMKI shRNA (left) or γCaMKI-GFP overexpression construct (right). Number of intersections between branches and successive concentric circles (inset, 5 μm increments). Left, area under curve reduced by 42% in neurons expressing γCaMKI shRNA (dashed line) compared to control (solid line). Right, area under curve increased by 15% in neurons overexexpressing γCaMKI-GFP (dotted line) compared to control (solid line).
(L) Overexpression of γCaMKI-GFP (dashed line) did not affect the kinetics of pCREB.