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Attractive axon guidance involves asymmetric membrane transport and exocytosis in the growth cone

Nature Neuroscience volume 10, pages 58–66 (2007)Cite this article

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Abstract

Asymmetric elevation of the Ca2+ concentration in the growth cone can mediate both attractive and repulsive axon guidance. Ca2+ signals that are accompanied by Ca2+-induced Ca2+ release (CICR) trigger attraction, whereas Ca2+ signals that are not accompanied by CICR trigger repulsion. The molecular machinery downstream of Ca2+ signals, however, remains largely unknown. Here we report that asymmetric membrane trafficking mediates growth cone attraction. Local photolysis of caged Ca2+, together with CICR, on one side of the growth cone of a chick dorsal root ganglion neuron facilitated the microtubule-dependent centrifugal transport of vesicles towards the leading edge and their subsequent vesicle-associated membrane-protein 2 (VAMP2)–mediated exocytosis on the side with an elevated Ca2+ concentration. In contrast, Ca2+ signals without CICR had no effect on the vesicle transport. Furthermore, pharmacological inhibition of VAMP2-mediated exocytosis prevented growth cone attraction, but not repulsion. These results strongly suggest that growth cone attraction and repulsion are driven by distinct mechanisms, rather than using the same molecular machinery with opposing polarities.

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Figure 1: Attractive, but not repulsive, Ca2+ signals facilitate the transport of endocytic vesicles to the growth cone P-domain.
Figure 2: Attractive Ca2+ signals facilitate the transport of VAMP2-containing vesicles to the growth cone P-domain.
Figure 3: FM1-43–labeled and VAMP2-containing vesicles are positioned along microtubules in the growth cone.
Figure 4: Attractive Ca2+ signals induce no detectable change in microtubule dynamics in the growth cone.
Figure 5: Attractive Ca2+ signals enhance VAMP2-mediated exocytosis in the growth cone P-domain.
Figure 6: Asymmetry of VAMP2 exocytosis induced by attractive Ca2+ signals declines more slowly than that induced by repulsive Ca2+ signals.
Figure 7: Ca2+-induced growth cone attraction, but not repulsion, requires VAMP2-mediated exocytosis.
Figure 8: Growth cone attraction, but not repulsion, induced by physiological guidance cues requires VAMP2-mediated exocytosis.

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Acknowledgements

We are grateful to M.M. Poo and A.T. Guy for their valuable comments and discussions on this manuscript. We thank R.Y. Tsien for providing the mRFP construct and V. Lemmon for the L1-Fc construct. We also thank the RIKEN Brain Science Institute's Research Resources Center for providing experimental instruments. This work was partially supported by the RIKEN Special Postdoctoral Researchers Program to T.T. and Grants-in-Aid for Young Scientists (B) (16700297, T.T.) and for Scientific Research (C) (16500210, H. Kamiguchi) from the Japan Society for the Promotion of Science.

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Author notes

  1. Yan Li

    Present address: Laboratory of Genetics, The Salk Institute, 10010 North Torrey Pines Road, La Jolla, California, 92037, USA

Authors and Affiliations

  1. Laboratory for Neuronal Growth Mechanisms, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, 351-0198, Saitama, Japan

    Takuro Tojima, Hiroki Akiyama, Rurika Itofusa & Hiroyuki Kamiguchi

  2. Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai, 200031, China

    Yan Li

  3. Laboratory for Cell Function Dynamics, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, 351-0198, Saitama, Japan

    Hiroyuki Katayama & Atsushi Miyawaki

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Contributions

T.T. performed the experiments in Figs. 1, 2, 3a, 4, 7a–e and Supplementary Figs. 1 and 2 and wrote the manuscript. H.A. performed the experiments in Figs. 5 and 6. R.I. performed the experiments in Figs. 3b, 7f, 8a–f and Supplementary Figs. 3, 4, 5. Y.L. performed the experiments in Fig. 8g,h. H. Katayama and A.M. generated Venus-VAMP2 and pHVenus-VAMP2 constructs. H. Kamiguchi designed the research project, directed the experiments and wrote the manuscript together with T.T.

Corresponding author

Correspondence to Hiroyuki Kamiguchi.

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Supplementary information

Supplementary Fig. 1

Local photolysis of DMNP-EDTA generates asymmetric Ca2+ elevation in a growth cone. (PDF 252 kb)

Supplementary Fig. 2

Attractive Ca2+ signals induce no detectable changes in filopodial dynamics and retrograde flow rates of F-actin in the growth cone. (PDF 428 kb)

Supplementary Fig. 3

TeNT blocks depolarization-induced VAMP2 exocytosis. (PDF 288 kb)

Supplementary Fig. 4

VAMP2-containing vesicles are present in the growth cone P-domain. (PDF 866 kb)

Supplementary Fig. 5

VAMP2-positive vesicles contain endocytosed NGF receptors, TrkA and p75NTR. (PDF 1767 kb)

Supplementary Video 1

Time-lapse movie of FM1-43-labeled vesicle transport in the growth cone induced by attractive Ca2+ signals on L1. (MOV 1286 kb)

Supplementary Video 2

Time-lapse movie of VAMP2-mediated exocytosis in the growth cone induced by attractive Ca2+ signals on L1. (MOV 1439 kb)

Supplementary Methods (PDF 36 kb)

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Tojima, T., Akiyama, H., Itofusa, R. et al. Attractive axon guidance involves asymmetric membrane transport and exocytosis in the growth cone. Nat Neurosci 10, 58–66 (2007). https://doi.org/10.1038/nn1814

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