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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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Syndapin loss-of-function mutants show normal synaptic transmission. (A–C) NMJ synapses of third-instar larvae co-stained for axonal plasma membrane (anti-HRP, green) and syndapin (red) of: (A) wild type, (B) synd heteroallelic combination (syndΔEx22/synd1d) and (C) syndapin revertant animal (obtained from the precise molecular excision of P-element of synd1d). A strong syndapin immunoreactivity around boutons of wild type animals is absent (reduced to background levels) in the synd mutants. Note that syndapin immunoreactivity at the NMJs reverts to wild-type levels in Synd revertants. (D) Western blot analysis of lysates from third instar larval fillets of control and synd mutant confirming that the syndapin protein is absent or dramatically reduced in the mutants. (E–I) Representative traces of (E) evoked synaptic potentials (F) spontaneous mEJP from control and synd (F2-generation) mutant animals; (G) average EJP amplitude (H) average mEJP amplitude and (I) average mEJP frequency of indicated genotypes. Numbers in histogram indicate number of animals analyzed. Error bar represents standard error of the mean (s.e.m.). Scale bar represents 10 μm for A–C.
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fig3: Syndapin loss-of-function mutants show normal synaptic transmission. (A–C) NMJ synapses of third-instar larvae co-stained for axonal plasma membrane (anti-HRP, green) and syndapin (red) of: (A) wild type, (B) synd heteroallelic combination (syndΔEx22/synd1d) and (C) syndapin revertant animal (obtained from the precise molecular excision of P-element of synd1d). A strong syndapin immunoreactivity around boutons of wild type animals is absent (reduced to background levels) in the synd mutants. Note that syndapin immunoreactivity at the NMJs reverts to wild-type levels in Synd revertants. (D) Western blot analysis of lysates from third instar larval fillets of control and synd mutant confirming that the syndapin protein is absent or dramatically reduced in the mutants. (E–I) Representative traces of (E) evoked synaptic potentials (F) spontaneous mEJP from control and synd (F2-generation) mutant animals; (G) average EJP amplitude (H) average mEJP amplitude and (I) average mEJP frequency of indicated genotypes. Numbers in histogram indicate number of animals analyzed. Error bar represents standard error of the mean (s.e.m.). Scale bar represents 10 μm for A–C.

Mentions: We employed standard genetic techniques to generate loss-of-function mutations in the synd gene. Transposon-insertion lines, EP409 and EY7010 in Synd were identified and their genomic positions, respectively in the first intron and 100 bp upstream of the putative transcriptional start site, identified by sequencing the isolated flanking DNA. EP409 and EY7010 lines were viable as homozygotes, consistent with our observation that they allowed robust Synd expression (data not shown). To generate severe loss-of-function alleles in synd, we mobilized P-elements in EP409 or EY7010 and screened excision lines for the absence/reduction of Synd expression by immunostaining of larval NMJs. Two new P-alleles: synd1d and synd19R (from EP409 remobilization) and 2 deletion lines: syndΔEx22 and syndΔEx23 (from EY7010 remobilization) recovered showed absence of syndapin immunoreactivity at the NMJs and on western blots (Figs. 3A–D). The synd P-insertion were pupal lethal with few adult escapers. The escapers were behaviorally normal, often producing mutant progeny that died at the larval/pupal stage — suggesting that maternally provided Synd protein was not required for viability in early embryonic/larval stages.


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)

Syndapin loss-of-function mutants show normal synaptic transmission. (A–C) NMJ synapses of third-instar larvae co-stained for axonal plasma membrane (anti-HRP, green) and syndapin (red) of: (A) wild type, (B) synd heteroallelic combination (syndΔEx22/synd1d) and (C) syndapin revertant animal (obtained from the precise molecular excision of P-element of synd1d). A strong syndapin immunoreactivity around boutons of wild type animals is absent (reduced to background levels) in the synd mutants. Note that syndapin immunoreactivity at the NMJs reverts to wild-type levels in Synd revertants. (D) Western blot analysis of lysates from third instar larval fillets of control and synd mutant confirming that the syndapin protein is absent or dramatically reduced in the mutants. (E–I) Representative traces of (E) evoked synaptic potentials (F) spontaneous mEJP from control and synd (F2-generation) mutant animals; (G) average EJP amplitude (H) average mEJP amplitude and (I) average mEJP frequency of indicated genotypes. Numbers in histogram indicate number of animals analyzed. Error bar represents standard error of the mean (s.e.m.). Scale bar represents 10 μm for A–C.
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Related In: Results  -  Collection

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fig3: Syndapin loss-of-function mutants show normal synaptic transmission. (A–C) NMJ synapses of third-instar larvae co-stained for axonal plasma membrane (anti-HRP, green) and syndapin (red) of: (A) wild type, (B) synd heteroallelic combination (syndΔEx22/synd1d) and (C) syndapin revertant animal (obtained from the precise molecular excision of P-element of synd1d). A strong syndapin immunoreactivity around boutons of wild type animals is absent (reduced to background levels) in the synd mutants. Note that syndapin immunoreactivity at the NMJs reverts to wild-type levels in Synd revertants. (D) Western blot analysis of lysates from third instar larval fillets of control and synd mutant confirming that the syndapin protein is absent or dramatically reduced in the mutants. (E–I) Representative traces of (E) evoked synaptic potentials (F) spontaneous mEJP from control and synd (F2-generation) mutant animals; (G) average EJP amplitude (H) average mEJP amplitude and (I) average mEJP frequency of indicated genotypes. Numbers in histogram indicate number of animals analyzed. Error bar represents standard error of the mean (s.e.m.). Scale bar represents 10 μm for A–C.
Mentions: We employed standard genetic techniques to generate loss-of-function mutations in the synd gene. Transposon-insertion lines, EP409 and EY7010 in Synd were identified and their genomic positions, respectively in the first intron and 100 bp upstream of the putative transcriptional start site, identified by sequencing the isolated flanking DNA. EP409 and EY7010 lines were viable as homozygotes, consistent with our observation that they allowed robust Synd expression (data not shown). To generate severe loss-of-function alleles in synd, we mobilized P-elements in EP409 or EY7010 and screened excision lines for the absence/reduction of Synd expression by immunostaining of larval NMJs. Two new P-alleles: synd1d and synd19R (from EP409 remobilization) and 2 deletion lines: syndΔEx22 and syndΔEx23 (from EY7010 remobilization) recovered showed absence of syndapin immunoreactivity at the NMJs and on western blots (Figs. 3A–D). The synd P-insertion were pupal lethal with few adult escapers. The escapers were behaviorally normal, often producing mutant progeny that died at the larval/pupal stage — suggesting that maternally provided Synd protein was not required for viability in early embryonic/larval stages.

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