The Journal of Neuroscience, September 28, 2005, ():
An Aplysia Type 4 Phosphodiesterase Homolog Localizes at the Presynaptic Terminals of Aplysia Neuron and Regulates Synaptic Facilitation
J. Neurosci. Park et al.
25: 9037
Supplemental data
Files in this Data Supplement:
- supplemental material -
Supplementary fig 1. Alignment results of the apPDE amino acid sequences and its overall structure.
A. apPDE showed the highest similarity to the long isoforms of PDE4D, such as human PDE4D8, human PDE4D7, and rat PDE4D7, as well as DrosophilacAMP-specific phosphodiesterase, dunce (dunce: 60% identity and 73% similarity; human PDE4D7, PDE4D8, and rat PDE4D7: 64% identity and 77% similarity). Isoform specific N-terminal domain (solid line), UCR1, 2 (thick dotted line), and catalytic domain (dotted line) are indicated. Except the unique N-terminal and C-terminal regions, the sequences from UCR1 to the catalytic domains of each gene were very similar to each other. At the UCR1 region, a putative protein kinase A (PKA) phosphorylation site (*) was conserved indicating that apPDE might be modulated by PKA phosphorylation.
B. Overall structure of apPDE is illustrated.
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Supplementary fig 2. apPDE dsRNA lowered the expression level of apPDE.
Double strand RNA(dsRNA)s specific for apPDE (dsApPDE) or luciferase (dsLuci) were microinjected with both apPDE-FLAG and EGFP in sensory neurons of sensory-motor coculture, and anti-FLAG antibody staining was done 48 hour after microinjection. FLAG intensity was divided with EGFP intensity to normalize the expression level of apPDE-FLAG. The graph represents the mean normalized fluorescent intensity ± SEM with arbitrary unit (AU). * p < 0.05, Student t-test.
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Supplementary fig 3. FRET efficiency test using acceptor photobleaching method.
To test whether FRET between RII-CFP and CAT-YFP probes are occurred in Aplysia neuron, we confirmed this by acceptor bleaching method and calculated FRET efficiency as described previously (Karpova et al., 2003). Three independent measurements showed that FRET efficiency (EF) at the ROI (Region of Interest) is 8.3±0.5 % but control value such as CF(FRET in control region) and NF (Noise) are 0.2 ± 1.0 and 0.1 ± 0.3, respectively. The graph represents the mean relative fluorescent intensity ± SEM of each CFP and YFP in arbitrary unit (AU).
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Supplementary fig 4. PKA activation by 5-HT in apPDE knock down synapses can be elevated by excess membrane permeable cAMP analogue.
FRET measurements was done after 5-HT (10?M) and cell permeable cAMP analog (8-CPT-cAMP, 200?M) or DMSO (0.1%) were co-treated in the synapses of cocultured neurons which was injected with apPDE dsRNA. The graph represents the mean relative CFP/YFP ratio ± SEM values from multiple varicosities in the five (5-HT+8-CPT-cAMP) and three sensory neurons (5-HT+DMSO) in three different cultures. * p < 0.01 two-way ANOVA with post tests.
