The Journal of Neuroscience, October 12, 2005, ():
Zn2+ Inhibits Mitochondrial Movement in Neurons by Phosphatidylinositol 3-Kinase Activation
J. Neurosci. Malaiyandi et al.
25: 9507
Supplemental data
Files in this Data Supplement:
- supplemental material
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Supplemental Video 1. Movie of zinc inhibition of mitochondrial movement in mt-eYFP-transfected cortical neurons. 3 µM ZnCl2 in the presence of 20 µM sodium pyrithione perfusion begins at 7:00 minutes.
- supplemental material
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Supplemental Figure 1. Contribution of reactive oxygen species to Zn2+-induced movement inhibition. Primary neurons expressing mt-eYFP were perfused for 10 minutes with various concentrations of 2,2’-DTDP (a) or H2O2 (b) followed by 50 µM TPEN. Mitochondrial movement was analyzed after the stimulus (clear bars) and after TPEN (solid bars). Neurons perfused simultaneously with 30 µM 2,2’-DTDP and TPEN (c) or 100 µM H2O2 and TPEN (d) for 10 minutes followed by additional perfusion with TPEN alone. Mitochondrial movement is measured after the combined stimulus (clear bars) and after TPEN (solid bars). (e) Mitochondrial movement was measured in neurons perfused 5 minutes with Zn2+, 2,2’-DTDP or H2O2 followed by 1 mM dithiothreitol (DTT). Mitochondrial movement was analyzed after stimulus (clear bars) and after DTT (solid bars). Results are representative of 4-6 different cultures.
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Supplemental Figure 2. Investigating the involvement of Zn2+-initiated signaling pathways in movement inhibition. To identify the protein kinase involved in the Zn2+-mediated movement inhibition, various protein kinase inhibitors were used to prevent the Zn2+ effect: PKC inhibitors (GF1 and RO), MEK inhibitor U0126, PI 3-kinase inhibitors (wortmannin and LY294002), tyrosine kinase receptor inhibitor genistein, and p38 inhibitor SB203580. Cultures were pretreated for 10 minutes with kinase inhibitors prior to 3 µM Zn2+. Mitochondrial movement was measured after inhibitor (clear bars) and Zn2+ exposure (solid bars). ? p < 0.005 by one-way ANOVA with Bonferroni’s Multiple Comparison Test. Results are representative of 4-6 different cultures.
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Supplemental Figure 3. Identifying targets of PI 3-kinase involved in Zn2+-induced movement inhibition. To identify the downstream player that is phosphorylated by PI 3-kinase in the Zn2+-mediated movement event, we examined the role of candidate protein kinases by preventing their activation and analyzing mitochondrial movement in the presence of Zn2+. Cultures were transiently transfected with the plasmid (provided by Dr. Daniel Altschuler, University of Pittsburgh) containing dominant negative Akt (K179M) to inhibit Akt activity. To modulate ARF6 activity, the following plasmids (provided by Dr. Jes Klarlund, University of Pittsburgh) were also transiently transfected: wild type ARF6 which is active when phosphorylated by PI 3-kinase, constitutively active ARF6 (Q67L) and dominant negative ARF6 (T27N). Three days post transfection, cells were acutely perfused for 10 minutes with 3 µM Zn2+. To inhibit TOR activity, cultures were acutely pretreated for 10 minutes with rapamycin (100 nM) prior to Zn2+ exposure. The extent of Zn2+-mediated movement inhibition is normalized to movement under control (buffer alone) conditions. These values were compared with Zn2+ treatment alone (negative control) and Zn2+ in the presence of wortmannin (positive control). p < 0.005 by Student’s t test. Results are representative of 4-6 different cultures.
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