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Identification and investigation of Drosophila postsynaptic density homologs.

Liebl FL, Featherstone DE - Bioinform Biol Insights (2008)

Bottom Line: These receptors reside in the postsynaptic density, a network of proteins that links the receptors to downstream signaling components and to the neuronal cytoskeleton.To determine whether the fruit fly, Drosophila melanogaster, possesses a similar array of proteins as are found at the mammalian PSD, we identified Drosophila homologs of 95.8% of mammalian PSD proteins.We investigated, for the first time, the role of one of these PSD proteins, Pod1 in GluR cluster formation at the Drosophila neuromuscular junction and found that mutations in pod1 resulted in a specific loss of A-type receptors at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, USA. fl iebl@siue.edu

ABSTRACT
AMPA receptors are responsible for fast excitatory transmission in the CNS and the trafficking of these receptors has been implicated in LTP and learning and memory. These receptors reside in the postsynaptic density, a network of proteins that links the receptors to downstream signaling components and to the neuronal cytoskeleton. To determine whether the fruit fly, Drosophila melanogaster, possesses a similar array of proteins as are found at the mammalian PSD, we identified Drosophila homologs of 95.8% of mammalian PSD proteins. We investigated, for the first time, the role of one of these PSD proteins, Pod1 in GluR cluster formation at the Drosophila neuromuscular junction and found that mutations in pod1 resulted in a specific loss of A-type receptors at the synapse.

No MeSH data available.


Related in: MedlinePlus

pod1 mutants show a loss of A-type glutamate receptors. A) Confocal images showing the NMJ on ventral longitudinal muscles 6/7 in third instar larvae (110–120 h after egg laying), visualized using antibodies against neuronal membrane (HRP, magenta) and the glutamate receptor subunit, GluRIIA (green). B) Average GluRIIA cluster size was significantly reduced in pod1 mutant third instar larvae. C) Representative recordings from control and pod1 mutant third instar larve, showing spontaneous excitatory junction currents (sEJCs) in muscle 6 of the NMJ. D) Cumulative frequency histogram of sEJC amplitudes. pod1 mutant animals (pink and purple traces) exhibited fewer large events, compared with control animals (black traces).
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f2-bbi-2008-369: pod1 mutants show a loss of A-type glutamate receptors. A) Confocal images showing the NMJ on ventral longitudinal muscles 6/7 in third instar larvae (110–120 h after egg laying), visualized using antibodies against neuronal membrane (HRP, magenta) and the glutamate receptor subunit, GluRIIA (green). B) Average GluRIIA cluster size was significantly reduced in pod1 mutant third instar larvae. C) Representative recordings from control and pod1 mutant third instar larve, showing spontaneous excitatory junction currents (sEJCs) in muscle 6 of the NMJ. D) Cumulative frequency histogram of sEJC amplitudes. pod1 mutant animals (pink and purple traces) exhibited fewer large events, compared with control animals (black traces).

Mentions: To determine whether pod1 affects GluR cluster formation, we examined GluRs in third instar pod1 mutants, which are viable until pupal stage (Rothenberg et al. 2003). Mutant synapses were examined immunocytochemically using α-horseradish peroxidase (HRP) to label the pre-synaptic motor neuron and α-GluRIIA to label postsynaptic GluRs (Fig. 2). α-HRP recognizes glycosylation of multiple neuronal proteins (Paschinger et al. 2008). Three mutant alleles were used for this analysis: pod1P{GT1}BG02604 (hereafter referred to as pod1P1), pod1Δ17, and pod1Δ96. pod1P1 contains a transposable element inserted approximately 300 bp upstream of pod1. The presence of the transposable element reduced Pod1 immunreactivity to undetectable levels (see above, data not shown). pod1Δ17 and pod1Δ96 were generated by imprecise excision of the P{GT1}BG02604 tranposable element and remove the entire coding sequence of pod1 (Rothenberg et al. 2003). Control animals exhibit distinct GluRIIA immunoreactivity visible as small clusters (green) opposite of the presynaptic motor neuron (magenta; Fig. 2A left panels). Each GluR punctum represents an individual postsynaptic density (Chen and Featherstone, 2005; Rasse et al. 2005; Schmid et al. 2008). GluR cluster area, measured immunocytochemically, is directly proportional to the number of GluRs measured electrophysiologically and independent of changes in NMJ morphology (Featherstone et al. 2002; Chen and Featherstone, 2005; Rasse et al. 2005; Schmid et al. 2008). All three pod1 mutant alleles exhibited a significant reduction in GluRIIA cluster size (Fig. 2A, B and data not shown; w1118 = 1.34 ± 0.07 μm2, n = 80 clusters from 8 animals; pod1P1 = 0.79 ± 0.05 μm2, n = 66 clusters from 7 animals, p <0.0001; pod1Δ17 = 0.53 ± 0.04 μm2, n = 80 clusters from 8 animals, p < 0.0001; pod1Δ96 = 0.62 ± 0.06 μm2, n = 70 clusters from 7 animals, p <0.0001). Measurements of total fluorescence intensity indicated there is a 34% and 36% reduction in GluRIIA immunoreactivity in pod1Δ17 and pod1Δ96 mutant animals, respectively (normalized GluRIIA fluorescence w1118 = 1.00 ± 0.16, n = 15; pod1P1 = 0.69 ± 0.08, n = 9, p = 0.12; pod1Δ17 = 0.66 ± 0.09, n = 14, p = 0.03; pod1Δ96 = 0.64 ± 0.09, n = 9, p = 0.04). These data suggest Pod1 is involved in the expression and/or localization of GluRs.


Identification and investigation of Drosophila postsynaptic density homologs.

Liebl FL, Featherstone DE - Bioinform Biol Insights (2008)

pod1 mutants show a loss of A-type glutamate receptors. A) Confocal images showing the NMJ on ventral longitudinal muscles 6/7 in third instar larvae (110–120 h after egg laying), visualized using antibodies against neuronal membrane (HRP, magenta) and the glutamate receptor subunit, GluRIIA (green). B) Average GluRIIA cluster size was significantly reduced in pod1 mutant third instar larvae. C) Representative recordings from control and pod1 mutant third instar larve, showing spontaneous excitatory junction currents (sEJCs) in muscle 6 of the NMJ. D) Cumulative frequency histogram of sEJC amplitudes. pod1 mutant animals (pink and purple traces) exhibited fewer large events, compared with control animals (black traces).
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2735971&req=5

f2-bbi-2008-369: pod1 mutants show a loss of A-type glutamate receptors. A) Confocal images showing the NMJ on ventral longitudinal muscles 6/7 in third instar larvae (110–120 h after egg laying), visualized using antibodies against neuronal membrane (HRP, magenta) and the glutamate receptor subunit, GluRIIA (green). B) Average GluRIIA cluster size was significantly reduced in pod1 mutant third instar larvae. C) Representative recordings from control and pod1 mutant third instar larve, showing spontaneous excitatory junction currents (sEJCs) in muscle 6 of the NMJ. D) Cumulative frequency histogram of sEJC amplitudes. pod1 mutant animals (pink and purple traces) exhibited fewer large events, compared with control animals (black traces).
Mentions: To determine whether pod1 affects GluR cluster formation, we examined GluRs in third instar pod1 mutants, which are viable until pupal stage (Rothenberg et al. 2003). Mutant synapses were examined immunocytochemically using α-horseradish peroxidase (HRP) to label the pre-synaptic motor neuron and α-GluRIIA to label postsynaptic GluRs (Fig. 2). α-HRP recognizes glycosylation of multiple neuronal proteins (Paschinger et al. 2008). Three mutant alleles were used for this analysis: pod1P{GT1}BG02604 (hereafter referred to as pod1P1), pod1Δ17, and pod1Δ96. pod1P1 contains a transposable element inserted approximately 300 bp upstream of pod1. The presence of the transposable element reduced Pod1 immunreactivity to undetectable levels (see above, data not shown). pod1Δ17 and pod1Δ96 were generated by imprecise excision of the P{GT1}BG02604 tranposable element and remove the entire coding sequence of pod1 (Rothenberg et al. 2003). Control animals exhibit distinct GluRIIA immunoreactivity visible as small clusters (green) opposite of the presynaptic motor neuron (magenta; Fig. 2A left panels). Each GluR punctum represents an individual postsynaptic density (Chen and Featherstone, 2005; Rasse et al. 2005; Schmid et al. 2008). GluR cluster area, measured immunocytochemically, is directly proportional to the number of GluRs measured electrophysiologically and independent of changes in NMJ morphology (Featherstone et al. 2002; Chen and Featherstone, 2005; Rasse et al. 2005; Schmid et al. 2008). All three pod1 mutant alleles exhibited a significant reduction in GluRIIA cluster size (Fig. 2A, B and data not shown; w1118 = 1.34 ± 0.07 μm2, n = 80 clusters from 8 animals; pod1P1 = 0.79 ± 0.05 μm2, n = 66 clusters from 7 animals, p <0.0001; pod1Δ17 = 0.53 ± 0.04 μm2, n = 80 clusters from 8 animals, p < 0.0001; pod1Δ96 = 0.62 ± 0.06 μm2, n = 70 clusters from 7 animals, p <0.0001). Measurements of total fluorescence intensity indicated there is a 34% and 36% reduction in GluRIIA immunoreactivity in pod1Δ17 and pod1Δ96 mutant animals, respectively (normalized GluRIIA fluorescence w1118 = 1.00 ± 0.16, n = 15; pod1P1 = 0.69 ± 0.08, n = 9, p = 0.12; pod1Δ17 = 0.66 ± 0.09, n = 14, p = 0.03; pod1Δ96 = 0.64 ± 0.09, n = 9, p = 0.04). These data suggest Pod1 is involved in the expression and/or localization of GluRs.

Bottom Line: These receptors reside in the postsynaptic density, a network of proteins that links the receptors to downstream signaling components and to the neuronal cytoskeleton.To determine whether the fruit fly, Drosophila melanogaster, possesses a similar array of proteins as are found at the mammalian PSD, we identified Drosophila homologs of 95.8% of mammalian PSD proteins.We investigated, for the first time, the role of one of these PSD proteins, Pod1 in GluR cluster formation at the Drosophila neuromuscular junction and found that mutations in pod1 resulted in a specific loss of A-type receptors at the synapse.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, Southern Illinois University Edwardsville, Edwardsville, IL, USA. fl iebl@siue.edu

ABSTRACT
AMPA receptors are responsible for fast excitatory transmission in the CNS and the trafficking of these receptors has been implicated in LTP and learning and memory. These receptors reside in the postsynaptic density, a network of proteins that links the receptors to downstream signaling components and to the neuronal cytoskeleton. To determine whether the fruit fly, Drosophila melanogaster, possesses a similar array of proteins as are found at the mammalian PSD, we identified Drosophila homologs of 95.8% of mammalian PSD proteins. We investigated, for the first time, the role of one of these PSD proteins, Pod1 in GluR cluster formation at the Drosophila neuromuscular junction and found that mutations in pod1 resulted in a specific loss of A-type receptors at the synapse.

No MeSH data available.


Related in: MedlinePlus