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Syndapin is dispensable for synaptic vesicle endocytosis at the Drosophila larval neuromuscular junction.

Kumar V, Alla SR, Krishnan KS, Ramaswami M - Mol. Cell. Neurosci. (2008)

Bottom Line: The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge.Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis.Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Smurfit Institute of Genetics and Trinity College Institute of Neuroscience, Lloyd Building, University of Dublin, Trinity College, Dublin 2, Ireland. kumarv@tcd.ie

ABSTRACT
Syndapin is a conserved dynamin-binding protein, with predicted function in synaptic-vesicle endocytosis. Here, we combine genetic mutational analysis with in vivo cell biological assays to ask whether Drosophila syndapin (Synd) is an essential component of synaptic-vesicle recycling. The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge. Contrary to expectation, we report that the Synd protein is predominantly postsynaptic, undetectable at presynaptic varicosities at Drosophila third-instar larval neuromuscular junctions. Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis. Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

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At the Drosophila larval NMJ, syndapin is a postsynaptic protein highly enriched in the subsynaptic reticulum. (A) Cartoon of the Drosophila larval NMJ showing the presynaptic axon terminal (green) embedded in a postsynaptic, tubulolamellar membrane system (pink) called the subsynaptic reticulum (SSR). (B–D) Synd does not colocalize with presynaptic dynamin. Single confocal section showing localization of Synd (B, red), dynamin (C, green) and the difference in localization of both proteins (D) within type I boutons. (E–G) Synd colocalizes with Dlg, a marker of postsynaptic SSR. Single confocal section of type I boutons of a wild-type larva, double labeled with anti-Synd (red) and anti-Dlg (green) antibodies. (G) Merged image of (E) and (F). Scale bar represents 5 μm for B–G.
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fig2: At the Drosophila larval NMJ, syndapin is a postsynaptic protein highly enriched in the subsynaptic reticulum. (A) Cartoon of the Drosophila larval NMJ showing the presynaptic axon terminal (green) embedded in a postsynaptic, tubulolamellar membrane system (pink) called the subsynaptic reticulum (SSR). (B–D) Synd does not colocalize with presynaptic dynamin. Single confocal section showing localization of Synd (B, red), dynamin (C, green) and the difference in localization of both proteins (D) within type I boutons. (E–G) Synd colocalizes with Dlg, a marker of postsynaptic SSR. Single confocal section of type I boutons of a wild-type larva, double labeled with anti-Synd (red) and anti-Dlg (green) antibodies. (G) Merged image of (E) and (F). Scale bar represents 5 μm for B–G.

Mentions: Current models for syndapin function in neurons are significantly based not only on its binding to dynamin, but also on its presumed presynaptic localization (Qualmann and Kelly, 2000). To directly determine syndapin's subcellular distribution, we analyzed Synd localization at the Drosophila larval neuromuscular junctions (NMJ). Analyses of double-labeled NMJ synapses showed no colocalization with presynaptic dynamin (Figs. 2B–D) which marks sites of synaptic-vesicle endocytosis (endocytic hot-spots) at the Drosophila NMJ (Estes et al., 1996; Roos and Kelly, 1998). The absence of any obvious colocalization between dynamin and Synd suggested that Synd is either absent, or present at very low levels in the presynaptic compartment. This suggestion is supported by analysis of Synd localization relative to HRP protein, a presynaptic membrane marker, which shows Synd to be present around, rather than within, presynaptic boutons (Supplementary Fig. S2A). Since, we could not rule out the possibility of small amount of Synd not being detected by immunofluorescence in presynaptic terminals, we overexpressed a syndapin transgene in motor neurons. However, expressing this transgene did not cause any enrichment of Synd protein at motor terminals (Supplementary Fig. S2B). This suggests that synd is not normally trafficked to boutons.


Syndapin is dispensable for synaptic vesicle endocytosis at the Drosophila larval neuromuscular junction.

Kumar V, Alla SR, Krishnan KS, Ramaswami M - Mol. Cell. Neurosci. (2008)

At the Drosophila larval NMJ, syndapin is a postsynaptic protein highly enriched in the subsynaptic reticulum. (A) Cartoon of the Drosophila larval NMJ showing the presynaptic axon terminal (green) embedded in a postsynaptic, tubulolamellar membrane system (pink) called the subsynaptic reticulum (SSR). (B–D) Synd does not colocalize with presynaptic dynamin. Single confocal section showing localization of Synd (B, red), dynamin (C, green) and the difference in localization of both proteins (D) within type I boutons. (E–G) Synd colocalizes with Dlg, a marker of postsynaptic SSR. Single confocal section of type I boutons of a wild-type larva, double labeled with anti-Synd (red) and anti-Dlg (green) antibodies. (G) Merged image of (E) and (F). Scale bar represents 5 μm for B–G.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2697329&req=5

fig2: At the Drosophila larval NMJ, syndapin is a postsynaptic protein highly enriched in the subsynaptic reticulum. (A) Cartoon of the Drosophila larval NMJ showing the presynaptic axon terminal (green) embedded in a postsynaptic, tubulolamellar membrane system (pink) called the subsynaptic reticulum (SSR). (B–D) Synd does not colocalize with presynaptic dynamin. Single confocal section showing localization of Synd (B, red), dynamin (C, green) and the difference in localization of both proteins (D) within type I boutons. (E–G) Synd colocalizes with Dlg, a marker of postsynaptic SSR. Single confocal section of type I boutons of a wild-type larva, double labeled with anti-Synd (red) and anti-Dlg (green) antibodies. (G) Merged image of (E) and (F). Scale bar represents 5 μm for B–G.
Mentions: Current models for syndapin function in neurons are significantly based not only on its binding to dynamin, but also on its presumed presynaptic localization (Qualmann and Kelly, 2000). To directly determine syndapin's subcellular distribution, we analyzed Synd localization at the Drosophila larval neuromuscular junctions (NMJ). Analyses of double-labeled NMJ synapses showed no colocalization with presynaptic dynamin (Figs. 2B–D) which marks sites of synaptic-vesicle endocytosis (endocytic hot-spots) at the Drosophila NMJ (Estes et al., 1996; Roos and Kelly, 1998). The absence of any obvious colocalization between dynamin and Synd suggested that Synd is either absent, or present at very low levels in the presynaptic compartment. This suggestion is supported by analysis of Synd localization relative to HRP protein, a presynaptic membrane marker, which shows Synd to be present around, rather than within, presynaptic boutons (Supplementary Fig. S2A). Since, we could not rule out the possibility of small amount of Synd not being detected by immunofluorescence in presynaptic terminals, we overexpressed a syndapin transgene in motor neurons. However, expressing this transgene did not cause any enrichment of Synd protein at motor terminals (Supplementary Fig. S2B). This suggests that synd is not normally trafficked to boutons.

Bottom Line: The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge.Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis.Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

View Article: PubMed Central - PubMed

Affiliation: Smurfit Institute of Genetics and Trinity College Institute of Neuroscience, Lloyd Building, University of Dublin, Trinity College, Dublin 2, Ireland. kumarv@tcd.ie

ABSTRACT
Syndapin is a conserved dynamin-binding protein, with predicted function in synaptic-vesicle endocytosis. Here, we combine genetic mutational analysis with in vivo cell biological assays to ask whether Drosophila syndapin (Synd) is an essential component of synaptic-vesicle recycling. The only isoform of Drosophila syndapin (synd) is broadly expressed and at high levels in the nervous system. synd mutants are late-larval lethals, but fertile adult "escapers" frequently emerge. Contrary to expectation, we report that the Synd protein is predominantly postsynaptic, undetectable at presynaptic varicosities at Drosophila third-instar larval neuromuscular junctions. Electrophysiological and synaptopHluorin imaging in control, synd-deficient or synd-overexpressing motor neurons reveals that synd is dispensable for synaptic-vesicle endocytosis. Our work in Drosophila leads to the suggestion that syndapin may not be a general or essential component in dynamin-dependent synaptic-vesicle endocytosis.

Show MeSH
Related in: MedlinePlus