Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis

Abstract

Dynamin I is dephosphorylated at Ser-774 and Ser-778 during synaptic vesicle endocytosis (SVE) in nerve terminals. Phosphorylation was proposed to regulate the assembly of an endocytic protein complex with amphiphysin or endophilin. Instead, we found it recruits syndapin I for SVE and does not control amphiphysin or endophilin binding in rat synaptosomes. After depolarization, syndapin showed a calcineurin-mediated interaction with dynamin. A peptide mimicking the phosphorylation sites disrupted the dynamin-syndapin complex, not the dynamin-endophilin complex, arrested SVE and produced glutamate release fatigue after repetitive stimulation. Pseudophosphorylation of Ser-774 or Ser-778 inhibited syndapin binding without affecting amphiphysin recruitment. Site mutagenesis to alanine arrested SVE in cultured neurons. The effects of the sites were additive for syndapin I binding and SVE. Thus syndapin I is a central component of the endocytic protein complex for SVE via stimulus-dependent recruitment to dynamin I and has a key role in synaptic transmission.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Phosphorylation-dependent interaction of syndapin I and endophilin I with dynamin I in vitro.
Figure 2: Phosphorylation-dependent interaction with syndapin I in vivo.
Figure 3: Phosphopeptide mapping of dynamin I from synaptosomes.
Figure 4: Depolarization-regulated interaction of syndapin I with dynamin I in synaptosomes.
Figure 5: Dynamin I phospho-box peptide inhibits SVE in synaptosomes.
Figure 6: DynI-dmA and DynI-dmE inhibit SVE in cerebellar granule neurons (CGNs).
Figure 7: Overexpression of DynI-dmA and DynI-dmE arrest SVE when assayed using synaptopHluorin.

Similar content being viewed by others

References

  1. Cousin, M.A. & Robinson, P.J. The dephosphins: dephosphorylation by calcineurin triggers synaptic vesicle endocytosis. Trends Neurosci. 24, 659–665 (2001).

    Article  CAS  PubMed  Google Scholar 

  2. Tan, T.C. et al. Cdk5 is essential for synaptic vesicle endocytosis. Nat. Cell Biol. 5, 701–710 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Lee, S.Y., Wenk, M.R., Kim, Y., Nairn, A.C. & De Camilli, P. Regulation of synaptojanin 1 by cyclin-dependent kinase 5 at synapses. Proc. Natl. Acad. Sci. USA 101, 546–551 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Lee, S.Y. et al. Regulation of the interaction between PIPKIγ and talin by proline-directed protein kinases. J. Cell Biol. 168, 789–799 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Floyd, S.R. et al. Amphiphysin binds the cdk5 regulatory subunit p35 and is phosphorylated by cdk5 and cdc2. J. Biol. Chem. 276, 8104–8110 (2001).

    Article  CAS  PubMed  Google Scholar 

  6. Sweitzer, S.M. & Hinshaw, J.E. Dynamin undergoes a GTP-dependent conformational change causing vesiculation. Cell 93, 1021–1029 (1998).

    Article  CAS  PubMed  Google Scholar 

  7. Grabs, D. et al. The SH3 domain of amphiphysin binds the proline-rich domain of dynamin at a single site that defines a new SH3 binding consensus sequence. J. Biol. Chem. 272, 13419–13425 (1997).

    Article  CAS  PubMed  Google Scholar 

  8. Ringstad, N., Nemoto, Y. & De Camilli, P. The SH3p4/Sh3p8/SH3p13 protein family: binding partners for synaptojanin and dynamin via a Grb2-like Src homology 3 domain. Proc. Natl. Acad. Sci. USA 94, 8569–8574 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Qualmann, B., Roos, J., DiGregorio, P.J., Kelly, R.B. & Syndapin, I, a synaptic dynamin-binding protein that associates with the neural Wiskott-Aldrich syndrome protein. Mol. Biol. Cell 10, 501–513 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Slepnev, V.I., Ochoa, G.C., Butler, M.H., Grabs, D. & DeCamilli, P. Role of phosphorylation in regulation of the assembly of endocytic coat complexes. Science 281, 821–824 (1998).

    Article  CAS  PubMed  Google Scholar 

  11. Gad, H. et al. Fission and uncoating of synaptic clathrin-coated vesicles are perturbed by disruption of interactions with the SH3 domain of endophilin. Neuron 27, 301–312 (2000).

    Article  CAS  PubMed  Google Scholar 

  12. Verstreken, P. et al. Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating. Neuron 40, 733–748 (2003).

    Article  CAS  PubMed  Google Scholar 

  13. Jockusch, W.J., Praefcke, G.J., McMahon, H.T. & Lagnado, L. Clathrin-dependent and clathrin-independent retrieval of synaptic vesicles in retinal bipolar cells. Neuron 46, 869–878 (2005).

    Article  CAS  PubMed  Google Scholar 

  14. Peter, B.J. et al. BAR domains as sensors of membrane curvature: the amphiphysin BAR structure. Science 303, 495–499 (2004).

    Article  CAS  PubMed  Google Scholar 

  15. Itoh, T. et al. Dynamin and the actin cytoskeleton cooperatively regulate plasma membrane invagination by BAR and F-BAR proteins. Dev. Cell 9, 791–804 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Robinson, P.J. et al. Dynamin GTPase regulated by protein kinase C phosphorylation in nerve terminals. Nature 365, 163–166 (1993).

    Article  CAS  PubMed  Google Scholar 

  17. Larsen, M.R., Graham, M.E., Robinson, P.J. & Roepstorff, P. Improved detection of hydrophilic phosphopeptides using graphite powder micro-columns and mass spectrometry: evidence for in vivo doubly phosphorylated dynamin I and dynamin III. Mol. Cell. Proteomics 3, 456–465 (2004).

    Article  CAS  PubMed  Google Scholar 

  18. Solomaha, E., Szeto, F.L., Yousef, M.A. & Palfrey, H.C. Kinetics of SH3 domain association with the proline rich domain of dynamins: specificity, occlusion and the effects of phosphorylation. J. Biol. Chem. 280, 23147–23156 (2005).

    Article  CAS  PubMed  Google Scholar 

  19. Tomizawa, K. et al. Cophosphorylation of amphiphysin I and dynamin I by Cdk5 regulates clathrin-mediated endocytosis of synaptic vesicles. J. Cell Biol. 163, 813–824 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Liu, J.P., Sim, A.T.R. & Robinson, P.J. Calcineurin inhibition of dynamin I GTPase activity coupled to nerve terminal depolarization. Science 265, 970–973 (1994).

    Article  CAS  PubMed  Google Scholar 

  21. Cousin, M.A. et al. Synapsin I-associated phosphatidylinositol 3-kinase mediates synaptic vesicle delivery to the readily releasable pool. J. Biol. Chem. 278, 29065–29071 (2003).

    Article  CAS  PubMed  Google Scholar 

  22. Cousin, M.A. & Robinson, P.J. Ca2+ inhibition of dynamin arrests synaptic vesicle recycling at the active zone. J. Neurosci. 20, 949–957 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Tibbs, G.R., Dolly, J.O. & Nicholls, D.G. Evidence for the induction of repetitive action potentials in synaptosomes by K +-channel inhibitors: an analysis of plasma membrane ion fluxes. J. Neurochem. 67, 389–397 (1996).

    Article  CAS  PubMed  Google Scholar 

  24. Cousin, M.A. & Robinson, P.J. Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals. J. Neurochem. 75, 1645–1653 (2000).

    Article  CAS  PubMed  Google Scholar 

  25. Miesenbock, G., De Angelis, D.A. & Rothman, J.E. Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 (1998).

    Article  CAS  PubMed  Google Scholar 

  26. Kessels, M.M. & Qualmann, B. The syndapin protein family: linking membrane trafficking with the cytoskeleton. J. Cell Sci. 117, 3077–3086 (2004).

    Article  CAS  PubMed  Google Scholar 

  27. Witke, W. et al. In mouse brain profilin I and profilin II associate with regulators of the endocytic pathway and actin assembly. EMBO J. 17, 967–976 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. McNiven, M.A. et al. Regulated interactions between dynamin and the actin-binding protein cortactin modulate cell shape. J. Cell Biol. 151, 187–198 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Kessels, M.M., Engqvist-Goldstein, A.E.Y., Drubin, D.G. & Qualmann, B. Mammalian Abp1, a signal-responsive F-actin-binding protein, links the actin cytoskeleton to endocytosis via the GTPase dynamin. J. Cell Biol. 153, 351–366 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Orth, J.D., Krueger, E.W., Cao, H. & McNiven, M.A. The large GTPase dynamin regulates actin comet formation and movement in living cells. Proc. Natl. Acad. Sci. USA 99, 167–172 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Lee, E. & De Camilli, P. Dynamin at actin tails. Proc. Natl. Acad. Sci. USA 99, 161–166 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kessels, M.M. & Qualmann, B. Syndapins integrate N-WASP in receptor-mediated endocytosis. EMBO J. 21, 6083–6094 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Shupliakov, O. et al. Impaired recycling of synaptic vesicles after acute perturbation of the presynaptic actin cytoskeleton. Proc. Natl. Acad. Sci. USA 99, 14476–14481 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Sankaranarayanan, S., Atluri, P.P. & Ryan, T.A. Actin has a molecular scaffolding, not propulsive, role in presynaptic function. Nat. Neurosci. 6, 127–135 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Farsad, K. et al. Generation of high curvature membranes mediated by direct endophilin bilayer interactions. J. Cell Biol. 155, 193–200 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Cao, H., Garcia, F. & McNiven, M.A. Differential distribution of dynamin isoforms in mammalian cells. Mol. Biol. Cell 9, 2595–2609 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Brymora, A., Valova, V.A., Larsen, M.R., Roufogalis, B.D. & Robinson, P.J. The brain exocyst complex interacts with RalA in a GTP-dependent manner: identification of a novel mammalian Sec3 gene and a second Sec15 gene. J. Biol. Chem. 276, 29792–29797 (2001).

    Article  CAS  PubMed  Google Scholar 

  38. Xue, J. et al. Phosphorylation of G-Septin on Ser-91 by cyclic GMP-dependent protein kinase-I in nerve terminals. Biochem. J. 381, 753–760 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Dunkley, P.R., Jarvie, P.E., Heath, J.W., Kidd, G.J. & Rostas, J.A. A rapid method for isolation of synaptosomes on Percoll gradients. Brain Res. 372, 115–129 (1986).

    Article  CAS  PubMed  Google Scholar 

  40. Di Paolo, G. et al. Decreased synaptic vesicle recycling efficiency and cognitive deficits in amphiphysin 1 knockout mice. Neuron 33, 789–804 (2002).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank the large number of our colleagues who have generously provided materials for this study. We particularly wish to thank J. Xue, A. Quan and G. Evans for technical advice and assistance, and E. van Dam, R. Duncan, M. Shipston and P. Rowe for critical reading of the manuscript. This work was supported by grants from the National Health and Medical Research Council of Australia, the Wellcome Trust (Ref: GR070569), an Australian Bicentennial Scholarship (V.A.), a University of Sydney Postgraduate Award (V.A.) and a University of Edinburgh Medical Faculty Scholarship (K.J.S.).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Phillip J Robinson.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

DynIdmA and DynIdmE cause SVE arrest in cerebellar granule neurons. (PDF 109 kb)

Supplementary Methods (PDF 113 kb)

Supplementary Discussion (PDF 109 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Anggono, V., Smillie, K., Graham, M. et al. Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis. Nat Neurosci 9, 752–760 (2006). https://doi.org/10.1038/nn1695

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn1695

This article is cited by

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing