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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 rate of endocytosis (A, B) Synaptic depression was not observed in Synd mutants during high-frequency nerve stimulation. Continuous EJP recordings from (A) control and (B) synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. (C) Representative traces of EJPs at indicated time points during 10 Hz stimulation in 1.5 mM Ca2+ containing HL3 saline. (D) Normalized EJP amplitudes in control (black lines) and synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. No significant difference in EJP amplitude was observed at any time points. (E) Representative wild-type SpH responses to 50 Hz stimulation before, during and after stimulation. (F) Indistinguishable SpH responses in control (blue, n = 25 boutons, 5 animals; Elav3E, UAS-SpH, synd1d/+) and mutant synapses (black, n = 25 boutons, 6 animals; Elav3E, UAS-SpH, synd1d/syndΔEx22) to a 50 Hz, 10 s stimulus train in HL3 containing 2.0 mM Ca2+. The horizontal bar represents the duration of stimulus. SpH fluorescence intensity was normalized to the image prior to the onset of stimulus. (G) Same data as in F, however, values normalized to the peak ΔF/F intensity, for visual comparison of endocytosis rate after the stimulus train. Error bars are standard error of the mean (s.e.m.).
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fig4: Syndapin loss-of-function mutants show normal rate of endocytosis (A, B) Synaptic depression was not observed in Synd mutants during high-frequency nerve stimulation. Continuous EJP recordings from (A) control and (B) synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. (C) Representative traces of EJPs at indicated time points during 10 Hz stimulation in 1.5 mM Ca2+ containing HL3 saline. (D) Normalized EJP amplitudes in control (black lines) and synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. No significant difference in EJP amplitude was observed at any time points. (E) Representative wild-type SpH responses to 50 Hz stimulation before, during and after stimulation. (F) Indistinguishable SpH responses in control (blue, n = 25 boutons, 5 animals; Elav3E, UAS-SpH, synd1d/+) and mutant synapses (black, n = 25 boutons, 6 animals; Elav3E, UAS-SpH, synd1d/syndΔEx22) to a 50 Hz, 10 s stimulus train in HL3 containing 2.0 mM Ca2+. The horizontal bar represents the duration of stimulus. SpH fluorescence intensity was normalized to the image prior to the onset of stimulus. (G) Same data as in F, however, values normalized to the peak ΔF/F intensity, for visual comparison of endocytosis rate after the stimulus train. Error bars are standard error of the mean (s.e.m.).

Mentions: Electrophysiological analyses showed “F2-generation” syndapin mutants (synd1d/syndΔEx22) to be indistinguishable from controls in basic aspects of synaptic transmission (Figs. 3E–I). Thus, synd1d/syndΔEx22 have normal: a) mEJP amplitude (0.92 ± 0.05 mV compared to 0.80 ± 0.07 mV in controls; P > 0.19); b) EJP amplitude (49.8 ± 2.3 mV compared to 46.55 ± 2.4 mV in controls; P > 0.34) and mEJP frequency (3.6 ± 0.22 compared to 3.3 ± 0.3 in controls; P > 0.38). In addition, the observation that mutant synapses can sustain 5 min of high frequency (10 Hz) stimulation in 1.5 mM Ca2+ would not be expected if synd mutants have substantially reduced rates of synaptic-vesicle recycling (Figs. 4A–D) (Stimson et al., 2001; Verstreken et al., 2002; Marie et al., 2004). Synd overexpression in the motor neuron also has no significant effect on synaptic transmission (Supplementary Figs. S2C–F): thus, mEJP amplitude (0.77 ± 0.04 mV in controls compared to 0.79 ± 0.043 mV in Elav-Gal4; UAS-Synd, P > 0.75) and EJP amplitude (42.8 ± 2.0 mV in controls compared to 43.1 ± 1.54 mV in Elav-Gal4; UAS-Synd, P > 0.9) were indistinguishable between control and Synd overexpressing motor neurons. These observations indicate that syndapin is largely dispensable for synaptic transmission, at least under the conditioned tested in our experiments.


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 rate of endocytosis (A, B) Synaptic depression was not observed in Synd mutants during high-frequency nerve stimulation. Continuous EJP recordings from (A) control and (B) synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. (C) Representative traces of EJPs at indicated time points during 10 Hz stimulation in 1.5 mM Ca2+ containing HL3 saline. (D) Normalized EJP amplitudes in control (black lines) and synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. No significant difference in EJP amplitude was observed at any time points. (E) Representative wild-type SpH responses to 50 Hz stimulation before, during and after stimulation. (F) Indistinguishable SpH responses in control (blue, n = 25 boutons, 5 animals; Elav3E, UAS-SpH, synd1d/+) and mutant synapses (black, n = 25 boutons, 6 animals; Elav3E, UAS-SpH, synd1d/syndΔEx22) to a 50 Hz, 10 s stimulus train in HL3 containing 2.0 mM Ca2+. The horizontal bar represents the duration of stimulus. SpH fluorescence intensity was normalized to the image prior to the onset of stimulus. (G) Same data as in F, however, values normalized to the peak ΔF/F intensity, for visual comparison of endocytosis rate after the stimulus train. Error bars are standard error of the mean (s.e.m.).
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Related In: Results  -  Collection

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fig4: Syndapin loss-of-function mutants show normal rate of endocytosis (A, B) Synaptic depression was not observed in Synd mutants during high-frequency nerve stimulation. Continuous EJP recordings from (A) control and (B) synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. (C) Representative traces of EJPs at indicated time points during 10 Hz stimulation in 1.5 mM Ca2+ containing HL3 saline. (D) Normalized EJP amplitudes in control (black lines) and synd heteroallelic larvae (syndΔEx22/synd1d) stimulated at 10 Hz for 5 min. No significant difference in EJP amplitude was observed at any time points. (E) Representative wild-type SpH responses to 50 Hz stimulation before, during and after stimulation. (F) Indistinguishable SpH responses in control (blue, n = 25 boutons, 5 animals; Elav3E, UAS-SpH, synd1d/+) and mutant synapses (black, n = 25 boutons, 6 animals; Elav3E, UAS-SpH, synd1d/syndΔEx22) to a 50 Hz, 10 s stimulus train in HL3 containing 2.0 mM Ca2+. The horizontal bar represents the duration of stimulus. SpH fluorescence intensity was normalized to the image prior to the onset of stimulus. (G) Same data as in F, however, values normalized to the peak ΔF/F intensity, for visual comparison of endocytosis rate after the stimulus train. Error bars are standard error of the mean (s.e.m.).
Mentions: Electrophysiological analyses showed “F2-generation” syndapin mutants (synd1d/syndΔEx22) to be indistinguishable from controls in basic aspects of synaptic transmission (Figs. 3E–I). Thus, synd1d/syndΔEx22 have normal: a) mEJP amplitude (0.92 ± 0.05 mV compared to 0.80 ± 0.07 mV in controls; P > 0.19); b) EJP amplitude (49.8 ± 2.3 mV compared to 46.55 ± 2.4 mV in controls; P > 0.34) and mEJP frequency (3.6 ± 0.22 compared to 3.3 ± 0.3 in controls; P > 0.38). In addition, the observation that mutant synapses can sustain 5 min of high frequency (10 Hz) stimulation in 1.5 mM Ca2+ would not be expected if synd mutants have substantially reduced rates of synaptic-vesicle recycling (Figs. 4A–D) (Stimson et al., 2001; Verstreken et al., 2002; Marie et al., 2004). Synd overexpression in the motor neuron also has no significant effect on synaptic transmission (Supplementary Figs. S2C–F): thus, mEJP amplitude (0.77 ± 0.04 mV in controls compared to 0.79 ± 0.043 mV in Elav-Gal4; UAS-Synd, P > 0.75) and EJP amplitude (42.8 ± 2.0 mV in controls compared to 43.1 ± 1.54 mV in Elav-Gal4; UAS-Synd, P > 0.9) were indistinguishable between control and Synd overexpressing motor neurons. These observations indicate that syndapin is largely dispensable for synaptic transmission, at least under the conditioned tested in our experiments.

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