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Fig. 1.
Pharmacological inhibition of CN but not other ser–thr phosphatases increases neurite lengths in the presence of Ca2+. A, Neurite lengths are significantly increased by 10 nM CsA or 1 nM DM (Delt) in the presence of 10 mMCa2+. In the absence of extracellular Ca2+, inhibition of CN (PP2B) by 10 nMCsA or 1 nM DM has no effect on neurite lengths. Data are mean ± SEM (n ≥ 100). B, OA at 300 pM, a concentration that does not inhibit members of the PP1 or PP2A families of protein phosphatases, has no effect on neurite length ± Ca2+. OA at 3 nM, which inhibits members of PP2A but not PP1, inhibits neurite lengths in the absence of Ca2+, suggesting that this family promotes neurite extension in the absence of waves. OA at 30 nM, which inhibits members of both PP2A and PP1, causes no further inhibition of neurite outgrowth. C, Taut at 3 nM, a concentration that affects only PP1 and not PP2A phosphatases, has no effect on neurite extension ± Ca2+. Taut at 30 and 300 nM, which inhibit members of both PP1 and PP2A, results in Ca2+-independent inhibition of neurite lengths. Data for B and C are mean ± SEM (n ≥ 30).
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Fig. 2.
Inhibition of calcineurin suppresses the slowing of neurite outgrowth by Ca2+ transients generated by photorelease of caged Ca2+. A, UV stimulation of a growth cone elicits a Ca2+transient (right; laminin substrate) that mimics a spontaneous Ca2+ wave in a different growth cone (left; tissue culture plastic substrate).B, Induced Ca2+ transients mimic spontaneous growth cone Ca2+ waves in both duration and amplitude (mean ± SEM; n = 5).C, Growth cone Ca2+ transients evoked at 8 min intervals (arrowheads) inhibit the rate of outgrowth. D, Inhibition of calcineurin (CsA, 10 nM) negates the braking effect of Ca2+transients (arrowheads) on neurite outgrowth. Neurons were imaged to establish a baseline rate of outgrowth, followed by photorelease of Ca2+ in the growth cone at 8 min intervals to mimic growth cone waves. E, Inhibition of neurite outgrowth by Ca2+ transients is suppressed in the presence of CsA (10 nM), suggesting that activation of calcineurin slows neurite outgrowth (mean ± SEM;n ≥ 15; p > 0.05).Con, Control; Spont, spontaneous.
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Fig. 3.
Peptide inhibition of CN increases neurite lengths in the presence of Ca2+. A,Myc-tagged xCN autoinhibitory domain (A.I.) cRNA was coinjected into one cell of a two-cell stage embryo with a fluorescent lineage tracer (rhodamine-dextran) to identifyA.I.-expressing cells. Nonfluorescent neurons serve as internal controls. Arrowheads indicate growth cones.Inset shows a myc-labeled neuron, demonstrating stability of the construct at 1 d in culture, is shown. Neurons were grown ±Ca2+. Scale bars, 25 μm.B, Neurite extension in the presence of external Ca2+ is significantly increased in cells expressingA.I. or the viral CN inhibitor A238L (p < 0.01). Neurons grown in the absence of Ca2+ and expressing A.I. or A238L have neurite lengths similar to those of controls. Data are mean ± SEM (n ≥ 60).
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Fig. 4.
Expression of a constitutively active xCN construct leads to short neurites in the absence of Ca2+. A, Myc-tagged CA-xCN A cRNA was coinjected into one cell of a two-cell stage embryo with mouse CN B cRNA to enhance enzymatic activity and a fluorescent lineage tracer (FITC-dextran) to identify CA-xCN-expressing cells. Nonfluorescent neurons are internal controls. Arrowheads indicate growth cones. Inset shows a myc-labeled neuron at 1 d in culture is shown. Neurons were grown ±Ca2+. Scale bars, 25 μm. B, Neurite extension is significantly inhibited in neurons expressing CA-xCN in the absence of external Ca2+ (p < 0.01). Neurons expressing CA-xCN grown in the presence of Ca2+ have neurite lengths similar to those of controls. Data are mean ± SEM (n ≥ 100).
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Fig. 5.
Developmental expression of Xenopuscalcineurin transcripts is visualized by in situhybridization of whole-mount albino Xenopus embryos with an xCN-specific antisense probe. A, xCN mRNA is not detected at the neural plate stage (stage 15). B,Message is first observed at the neural tube stage in the presumptive brain (stage 22). C, Transcripts are upregulated in the brain and spinal cord by the early tailbud stage (stage 26).D, Expression increases posteriorly as development progresses to the late tailbud stage (stage 32). E, A control tailbud stage embryo hybridized with xCN-specific sense probe does not reveal staining, indicating specificity of the antisense. For all embryos anterior is to the left, and dorsal isup. Scale bar, 500 μm.
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Fig. 6.
Ca2+ wave frequency and Ca2+-dependent expression of GABA are not affected by inhibition or activation of CN. A, Spontaneous Ca2+ waves are shown. Intracellular Ca2+ was monitored using the Ca2+indicator dye Fluo 3, and images were acquired at 10 sec intervals in the presence of 1 nM DM. Arrows indicate events scored as waves, the asterisk denotes a Ca2+ spike, and the dashed lineindicates the event threshold. B, Inhibition of CN does not significantly affect the frequency of Ca2+ waves: control, 7 ± 3/hr; CsA (10 nM), 6 ± 3/hr; DM (Delt, 1 nM), 7 ± 3/hr. Neurons expressing CA-xCN, indicated by the fluorescence of fura-2-dextran, generate spontaneous Ca2+ waves at normal frequencies in growth cones: control, 7 ± 3/hr; CA-xCN, 7 ± 2/hr. Neurons were imaged for 30 min intervals. Data are mean ± SEM (n≥ 15). C, Neurons treated with either 10 nMCsA or 1 nM DM were assayed for expression of GABA with a rabbit anti-GABA polyclonal antibody. CN-inhibited neurons express the same extent of GABA immunoreactivity as controls. Reciprocal experiments with neurons expressing CA-xCN, confirmed by myc immunoreactivity, demonstrate no effect on GABA expression. Data are mean ± SEM (n ≥ 100).
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Fig. 7.
Ca2+ waves act via calcineurin to dephosphorylate GAP-43. A Western blot of extracts of cultured neurons grown in the presence of Ca2+ and Ca2+ + DM (Delt, 1 nM) and in the absence of Ca2+ demonstrates that GAP-43 phosphorylation is lower in the presence of Ca2+ and that inhibiting calcineurin returns this phosphorylation to levels similar to those observed in the absence of Ca2+(n = 7).
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Fig. 8.
Disruption or stabilization of actin mimics the effects of activation or inhibition of CN. A, Shaded horizontal barsindicate neurite lengths in the presence of Ca2+ and CN activity and in their absence (see Fig. 1). Disruption of actin with Cyto D (50 nM) results in shorter neurites in the absence of CN activity, whereas stabilization of actin with Jasp (25 nM) generates longer neurites in the presence of CN activity. See text for further details. B,C, Dose–response analyses reveal the concentrations of Cyto D and Jasp (arrowheads) that mimic the effect of activating or inhibiting CN on neurite outgrowth. Disruption of actin filaments with Cyto D (50 nM; B) inhibits outgrowth in the absence of Ca2+ but not in its presence, whereas Jasp (25 nM; C) increases neurite lengths in its presence of Ca2+ but not in its absence. D, E, Dose–response analyses demonstrate that destabilization or stabilization of microtubules with colcemide (D) or taxol (E) results in a Ca2+-independent inhibition of neurite outgrowth. Data are mean ± SEM (n ≥ 30).CSA, CsA.
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Fig. 9.
Model of the signal transduction cascade by which Ca2+ waves act via CN to regulate neurite extension. Ca2+ waves activate CN, shifting cytoskeletal-regulating proteins to a dephosphorylated state, destabilizing actin filaments, and inhibiting neurite elongation. CN is not activated in the absence of Ca2+ waves, shifting cytoskeletal-regulating proteins to a phosphorylated state, stabilizing actin filaments, and promoting neurite outgrowth. Activating or inhibiting this cascade at different points increases or decreases neurite extension. Delt, DM.
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ppp3ca ( protein phosphatase 3, catalytic subunit, alpha isozyme ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 22, lateral view, anterior left, dorsal up.
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ppp3ca ( protein phosphatase 3, catalytic subunit, alpha isozyme ) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 26, lateral view, anterior left, dorsal up.
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