Abstract
The spatial distribution and coordination of vesicular dynamics within growth cones are poorly understood. It has long been thought that membranous organelles are concentrated in the central regions of growth cones and excluded from filopodia; this view has dramatically shaped conceptual models of the cellular mechanisms of axonal growth and presynaptic terminal formation. To begin to test these models, we studied membrane dynamics within axonal growth cones of living rat cortical neurons. We demonstrate that growth cone filopodia contain vesicles that transport synaptic vesicle proteins bidirectionally along filopodia and fuse with the filopodial surface in response to focal stimulation, allowing for both local secretion of vesicular contents and rapid changes in the plasma membrane composition of individual filopodia. Our results suggest a new model in which growth cone filopodia are actively involved in both emitting and responding to local signals related to axon growth and early synapse formation.
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References
O'Connor, T.P., Duerr, J.S. & Bentley, D. Pioneer growth cone steering decisions mediated by single filopodial contacts in situ. J. Neurosci. 10, 3935–3946 (1990).
Gomez, T.M., Robles, E., Poo, M. & Spitzer, N.C. Filopodial calcium transients promote substrate-dependent growth cone turning. Science 291, 1983–1987 (2001).
Cooper, M.W. & Smith, S.J. A real-time analysis of growth cone-target cell interactions during the formation of stable contacts between hippocampal neurons in culture. J. Neurobiol. 23, 814–828 (1992).
Tennyson, V.M. The fine structure of the axon and growth cone of the dorsal root neuroblast of the rabbit embryo. J. Cell Biol. 44, 62–79 (1970).
Gordon-Weeks, P.R. Neuronal Growth Cones (Cambridge Univ. Press, Cambridge, 2000).
Bunge, M.B. Initial endocytosis of perioxidase or ferritin by growth cones of cultured nerve. J. Neurocytol. 6, 407–439 (1977).
Nuttall, R.P. & Wessells, N.K. Veils, mounds and vesicle aggregates in neurons elongating in vitro. Exp. Cell Res. 119, 163–174 (1979).
Landis, S.C. Growth cones of cultured sympathetic neurons contain adrenergic vesicles. J. Cell Biol. 78, R8–14 (1978).
Cheng, T.P. & Reese, T.S. Recycling of plasmalemma in chick tectal growth cones. J. Neurosci. 7, 752–759 (1987).
Rees, R.P. & Reese, T.S. New structural features of freeze-substituted neuritic growth cones. Neuroscience 6, 247–254 (1981).
Bridgman, P.C. & Dailey, M.E. The organization of myosin and actin in rapid frozen nerve growth cones. J. Cell Biol. 108, 95–109 (1989).
Cheng, T.P. & Reese, T.S. Polarized compartmentalization of organelles in growth cones from developing optic tectum. J. Cell Biol. 101, 1473–1480 (1985).
Forscher, P. & Smith, S.J. Actions of cytochalasins on the organization of actin filaments and microtubules in a neuronal growth cone. J. Cell Biol. 107, 1505–1516 (1988).
Forscher, P., Kaczmarek, L.K., Buchanan, J.A. & Smith, S.J. Cyclic AMP induces changes in distribution and transport of organelles within growth cones of Aplysia bag cell neurons. J. Neurosci. 7, 3600–3611 (1987).
Diefenbach, T.J., Guthrie, P.B., Stier, H., Billups, B. & Kater, S.B. Membrane recycling in the neuronal growth cone revealed by FM1-43 labeling. J. Neurosci. 19, 9436–9444 (1999).
Chang, S. & De Camilli, P. Glutamate regulates actin-based motility in axonal filopodia. Nat. Neurosci. 4, 787–793 (2001).
Kraszewski, K. et al. Synaptic vesicle dynamics in living cultured hippocampal neurons visualized with CY3-conjugated antibodies directed against the lumenal domain of synaptotagmin. J. Neurosci. 15, 4328–4342 (1995).
Hazuka, C.D. et al. The sec6/8 complex is located at neurite outgrowth and axonal synapse-assembly domains. J. Neurosci. 19, 1324–1334 (1999).
Betz, W.J. & Bewick, G.S. Optical analysis of synaptic vesicle recycling at the frog neuromuscular junction. Science 255, 200–203 (1992).
Hirokawa, N. Kinesin and dynein superfamily proteins and the mechanism of organelle transport. Science 279, 519–526 (1998).
Dent, E.W. & Kalil, K. Axon branching requires interactions between dynamic microtubules and actin filaments. J. Neurosci. 21, 9757–9769 (2001).
Tashiro, A., Dunaevsky, A., Blazeski, R., Mason, C.A. & Yuste, R. Bidirectional regulation of hippocampal mossy fiber filopodial motility by kainate receptors: a two-step model of synaptogenesis. Neuron 38, 773–784 (2003).
Takamori, S., Rhee, J.S., Rosenmund, C. & Jahn, R. Identification of a vesicular glutamate transporter that defines a glutamatergic phenotype in neurons. Nature 407, 189–194 (2000).
Washbourne, P., Bennett, J.E. & McAllister, A.K. Rapid recruitment of NMDA receptor transport packets to nascent synapses. Nat. Neurosci. 5, 751–759 (2002).
Jontes, J.D. & Smith, S.J. Filopodia, spines and the generation of synaptic diversity. Neuron 27, 11–14 (2000).
Lin, S.Y. & Constantine-Paton, M. Suppression of sprouting: an early function of NMDA receptors in the absence of AMPA/kainate receptor activity. J. Neurosci. 18, 3725–3737 (1998).
Wong, W.T. & Wong, R.O. Changing specificity of neurotransmitter regulation of rapid dendritic remodeling during synaptogenesis. Nat. Neurosci. 4, 351–352 (2001).
Hume, R.I., Role, L.W. & Fischbach, G.D. Acetylcholine release from growth cones detected with patches of acetylcholine receptor-rich membranes. Nature 305, 632–634 (1983).
Young, S.H. & Poo, M.M. Spontaneous release of transmitter from growth cones of embryonic neurones. Nature 305, 634–637 (1983).
Dailey, M.E. & Bridgman, P.C. Vacuole dynamics in growth cones: correlated EM and video observations. J. Neurosci. 13, 3375–3393 (1993).
Peretti, D., Peris, L., Rosso, S., Quiroga, S. & Caceres, A. Evidence for the involvement of KIF4 in the anterograde transport of L1-containing vesicles. J. Cell Biol. 149, 141–152 (2000).
Taylor, J., Docherty, M. & Gordon-Weeks, P.R. GABAergic growth cones: release of endogenous gamma-aminobutyric acid precedes the expression of synaptic vesicle antigens. J. Neurochem. 54, 1689–1699 (1990).
Kater, S.B. & Rehder, V. The sensory-motor role of growth cone filopodia. Curr. Opin. Neurobiol. 5, 68–74 (1995).
Banker, G. & Goslin, K. Culturing Nerve Cells (MIT Press, Cambridge, Massachusetts, 1998).
McAllister, A.K. & Stevens, C.F. Non-saturation of AMPA and NMDA receptors at hippocampal synapses. Proc. Natl. Acad. Sci. USA 97, 6173–6178 (2000).
Rochlin, M.W., Dailey, M.E. & Bridgman, P.C. Polymerizing microtubules activate site-directed F-actin assembly in nerve growth cones. Mol. Biol. Cell 10, 2309–2327 (1999).
Gallo, G. & Letourneau, P.C. Different contributions of microtubule dynamics and transport to the growth of axons and collateral sprouts. J. Neurosci. 19, 3860–3873 (1999).
Zakharenko, S. & Popov, S. Dynamics of axonal microtubules regulate the topology of new membrane insertion into the growing neurites. J. Cell Biol. 143, 1077–1086 (1998).
Acknowledgements
We would like to thank H.J. Cheng, M.P. Sceniak, P. Washbourne, A.F. Ikin and A. Huberman for helpful reading of this manuscript. We thank Jennie Bennett for technical assistance. We also thank J. Sullivan for synaptophysin-EGFP and R. Scheller for VAMP2-EGFP constructs. This work was supported by the Alfred P. Sloan Foundation (A.K.M.), the Pew Charitable Trusts (A.K.M.), the March of Dimes (A.K.M.), National Eye Institute (A.K.M.), National Institute of Neurological Disorders and Stroke (S.L.S.) and National Institute of Mental Health (S.L.S.).
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Supplementary information
Supplementary Fig. 1.
Axonal growth cones can be identified in 3-5 d.i.v. neurons by morphological criteria. A reconstruction of the major axonal and dendritic arbors of a cortical neuron (3 d.i.v.) transfected with VAMP2-CFP. The axon is easily identified since it is much longer than the dendrites, does not taper and branches less frequently. A tracing of the neuron is shown to the right; the axon is shown in red, and the dendrites in black. (JPG 103 kb)
Supplementary Fig. 2.
Clusters of VAMP2-EGFP are associated with intracellular membranes. Neurons transfected with VAMP2-EGFP were fixed 24 h after transfection then extracted with Triton X-100, a condition known to selectively extract surface VAMP239. Significant punctate immunolabeling with anti-EGFP antibodies remained after the extraction (arrows), implying VAMP2-EGFP is associated with intracellular membranes. Similar results were obtained using both rabbit (a) and mouse (b) anti-EGFP antibodies. Scale bars = 5 µm. (PDF 303 kb)
Supplementary Fig. 3.
Synaptic vesicle proteins colocalize within axons, growth cones and their filopodia. (a) Thin optical sections through an axonal growth cone transfected with both synaptophysin-EGFP (green) and VAMP2-DsRed (red) show that the two proteins colocalize well throughout the axon and growth cone. (b) The same growth cones as in panel a, but with increased gain to better show the vesicles that contain both proteins in the filopodia. Note that within the axon the signal is saturated at this increased gain, resulting in a blooming effect. In both panels, vesicles in the growth cone that contain both VAMP2-DsRed and synaptophysin-EGFP are indicated by arrows. Scale bars = 5 µm. (PDF 314 kb)
Supplementary Video.
A time-lapse recording showing that VAMP2-EGFP clusters move bi-directionally within growth cone filopodia. Images were collected every 15 s for approximately 6 min. Scale bar = 10 µm. Arrows follow moving clusters of VAMP2-EGFP. (MOV 390 kb)
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Sabo, S., McAllister, A. Mobility and cycling of synaptic protein–containing vesicles in axonal growth cone filopodia. Nat Neurosci 6, 1264–1269 (2003). https://doi.org/10.1038/nn1149
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DOI: https://doi.org/10.1038/nn1149