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Mobility and cycling of synaptic protein–containing vesicles in axonal growth cone filopodia

Nature Neuroscience volume 6, pages 1264–1269 (2003)Cite this article

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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Figure 1: Vesicles containing synaptic-vesicle proteins are found in axonal growth cone filopodia.
Figure 2: Vesicles move rapidly and bidirectionally along axonal growth cone filopodia.
Figure 3: Movement of vesicles within axonal growth cone filopodia depends on microtubules.
Figure 4: Filopodial vesicles fuse with the axonal growth cone plasma membrane.

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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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  1. Center for Neuroscience, University of California, Davis, 1544 Newton Court, Davis, 95616, California, USA

    Shasta L Sabo & A Kimberley McAllister

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  1. Shasta L Sabo
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Correspondence to A Kimberley McAllister.

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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 Methods (PDF 9 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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