The Journal of Neuroscience, April 14, 2004, ():
Regulation of Axonal Extension and Growth Cone Motility by Calmodulin-Dependent Protein Kinase I
J. Neurosci. Wayman et al.
24: 3786
Supplemental data
Supplemental movies and figures
Files in this Data Supplement:
- Supplemental Fig. 1
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Supplementary Figure 1. A. Specificity of monoclonal pCaMKI antibody. GST-wtCaMKI was incubated ± CaMKK for 20 min in a standard phosphorylation reaction. Increasing amounts (50pg, 150pg, 500pg, 1000pg) of CaMKI ± CaMKK were loaded onto SDS-PAGE and western blotted using pCaMKI (upper panel) or total CaMKI (lower panel). Note that the pCaMKI antibody only recognizes CaMKI after phosphorylation of Thr177 by CaMKK. B. Transfected dnCaMKI inhibits phosphorylation of Thr177 in CaMKI by CaM KK. NG108 cells were transfected with either vector control (left two lanes) or dnCaMKI (right two lanes) using lipofectamine 2000. After 24hrs the level of CaMKI phospho-Thr177 in lysates was determined by western blot using anti pCaMKI (upper panel). Total CaMKI levels (as a loading control) were determine using anti-CaMKI antibody. Expression of dnCaMKI reduced phospho-Thr177 levels of endogenous CaMKI by greater than 80%. C. Dominant-negative CaMKIV distributes throughout the cytoplasm of transfected neurons. Low-density hippocampal cultures were transfected on day 3 with plasmid encoding EGFP-dnCaMKIV (upper panels) or a construct modified by addition of a nuclear localization signal, dnCaMKIVnuc (lower panels). Low magnification confocal images of the EGFP signal are shown in the left panels. While the dnCaMKIV lacking a nuclear targeting signal filled the cytoplasm of the cell body (arrow), the whole length of the axon and dendrites, it was largely excluded from the nucleus (right panel). In contrast, EGFP-dnCaMKIVnuc was found exclusively in the nucleoplasm of the transfected neuron (arrow). Scale bar: 50 ?m. D. Nuclear restricted dnCaMKIVnuc inhibits NMDA-stimulated CRE-mediated transcription. Hippocamapal neurons (10 DIV) were transfected with a CRE-regulated luciferase reporter, a ?-actin promoter-driven ?-galactosidase reporter, and a 10-fold excess of empty vector or dnCaMKIVnuc. After 48 hours the neurons were stimulated with 5 or 10 ?M NMDA in the presence of 1 ?M glycine for 5 hours and assayed for luciferase activity. A ?-galactosidase assay was conducted using the Galacton substrate (Tropix) according to the manufacturers instructions. The data (±SEM) are expressed as the ratio of luciferase to ?-galactosidase (n = 4).
- Supplemental Movie 1
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Movie 1. Axonal growth cone motility of a 3 day-old hippocampal neuron transfected with soluble EGFP and imaged after 8 hrs of expression. Fluorescence images were acquired every 5 sec over a period of 10 min.
- Supplemental Movie 2
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Movie 2. Axonal growth cone motility of a 3 day-old hippocampal neuron transfected with EGFP-tagged wtCaMKI and imaged after 8 hrs of expression. Fluorescence images were acquired every 5 sec over a period of 10 min.
- Supplemental Movie 3
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Movie 3. Axonal growth cone motility of a 3 day-old hippocampal neuron transfected with EGFP-tagged dnCaMKI and imaged after 8 hrs of expression. Fluorescence images were acquired every 5 sec over a period of 10 min.
- Supplemental Movie 4
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Movie 4. Axonal growth cone motility of a 3 day-old hippocampal neuron transfected with EGFP-tagged dnCaMKI and imaged after 20 hrs of expression. Fluorescence images were acquired every 5 sec over a period of 10 min.
- Supplemental Movie 5
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Movie 5. Comparison of axonal growth cone motility in the same neuron at 5 min (left panel), 60 min (middle panel), and 90 min (right panel) after addition of 2.6 ?M STO-609 as assessed with phase contrast time-lapse imaging (5 sec intervals, 5 min duration).
- Supplemental Fig. 2
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Supplementary Figure 2. Hippocampal neurite growth is not suppressed by inhibition of Akt. High density cultures of rat hippocampal neurons were transfected on day 2 with plasmid encoding soluble EGFP alone (control) or in combination with dnCaKI, caCaMKI, or dnAkt. Neurons were cultured a further 2 days, then fixed and imaged. Quantification of the effects of inhibiting or activating CaMKI or inhibiting Akt are also shown. Total neurite growth was quantified as in Figure 1 (30 cells per condition). Although expression of dnCaMKI inhibited neurite outgrowth, expression of dnAkt had no effect on neurite outgrowth. * P values of < 0.01 (t-Test).
- Supplemental Fig. 3
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Supplementary Figure 3. STO-609 inhibits CaMKK but does not inhibit CaMKII or Akt in hippocampal neurons. A. STO-609 inhibits CaMKK-mediated phosphorylation of CaMKI. Hippocampal neurons (5 DIV) were incubated with vehicle or 2.6 ?M STO-609 for 90 minutes prior to incubation for 10 min ± 60 mM KCl. CaMKK activity was assessed by its phosphorylation of CaMKI on Thr177 using a phospho-Thr177 antibody (upper panel); total CaMKI was detected using anti-CaMKI antibody (lower blot). Phosphorylation of CaMKI on Thr177 by CaMKK, under basal or depolarizing condtions, was blocked almost completely by pretreatment with 2.6 ?M STO-609 B. STO-609 inhibits CaMKK-mediated phosphorylation of CaMKIV. Hippocampal neurons (20 DIV) were incubated with vehicle or the indicated concentration of STO-609 for 90 minutes prior to stimulation for 5 min with 60 mM KCl. CaMKK activity was assessed by its phosphorylation of CaM KIV on Thr196 using a phospho-Thr196 antibody (upper panel); total KIV was detected using anti-CaMKIV antibody, (Transduction Labs, lower blot). C. STO-609 does not inhibit basal Akt activity. Hippocampal neurons (4 DIV) were incubated with either vehicle, 2.6 ?M STO-609 or 10 ?M Wortmannin for 90 min. The cells were harvested, and equal amounts of total protein were loaded on 7.5% SDS-PAGE. Activity of Akt was assessed with antibodies (Cell signaling) specific to key phosphorylation sites in Akt; phospho-Thr308 Akt (upper blot), phospho-T473 Akt (middle blot) as well as total Akt (lower blot). Wortmannin, but not STO-609, efficiently blocked basal Akt activity. C. STO-609 does not inhibit CaMKII activation. Low-density hippocampal cultures (21 DIV) were treated without (top panels) or with (bottom panels) 2.6 µM STO-609 for 90 min., then treated without (left panels) or with (right panels) 90 mM KCl for 5 min. The neurons were then stained with phospho-specific antibody for Thr286 in CaMKII (pCaMKII), imaged using both phase contrast and fluorescent (pCaMKII, red) microscopy, and these images were overlaid. In the absence of KCl stimulation (left panels), anti-phospho-CaMKII staining is low in both non-treated neurons (upper left) and neurons treated with 2.6 µM STO-609 (lower left). Upon KCl stimulation, STO-609 treated neurons also show strong staining for CaMKII autophosphorylation at Thr286 throughout their cell bodies and processes (lower right), demonstrating that STO-609 does not inhibited activation of endogenous CaMKII. Scale bar: 25?m.
