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Assembly of presynaptic active zones from cytoplasmic transport packets

Abstract

Little is known about presynaptic assembly during central nervous system synaptogenesis. Here we used time-lapse fluorescence imaging, immunocytochemistry and electron microscopy to study hippocampal neuronal cultures transfected with a fusion construct of the presynaptic vesicle protein VAMP and green fluorescent protein. Our results suggest that major cytoplasmic and membrane-associated protein precursors of the presynaptic active zone are transported along developing axons together as discrete packets. Retrospective electron microscopy demonstrated varied vesicular and tubulovesicular membrane structures. Packets containing these heterogeneous structures were stabilized specifically at new sites of dendrite- and axon-initiated cell–cell contact; within less than one hour, evoked vesicle recycling was observed at these putative nascent synapses. These observations suggest that substantial membrane remodeling may be necessary to produce the uniform vesicles typical of the mature active zone, and that many presynaptic proteins may be united early in their biogenesis and sorting pathways.

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Figure 1: VAMP–GFP distribution in transfected hippocampal neurons.
Figure 2: Transport packet dynamics and presynaptic function in cells transfected with VAMP–GFP.
Figure 3: Dendrite-initiated contact formation in d.i.v. 10 neuron.
Figure 4: Dendrite-initiated contact formation in d.i.v. 8 neuron.
Figure 5: Axon-initiated contact formation in d.i.v. 8 neuron.
Figure 6: Vesicle ultrastructure in transfected and control neurons.
Figure 7: Colocalization of synaptic proteins with mobile VAMP–GFP transport packets.

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Acknowledgements

We thank R. Scheller for the gift of the VAMP–GFP fusion construct, D. Foletti for help with transfection procedures and R. Holz, K. Micheva, W.J. Nelson, R. Scheller and R.W. Tsien for discussions. This work was supported by grants from the National Institutes of Mental Health (NS28587), the National Institute of Mental Health (Silvio Conte Center for Neuroscience Research, MH48108) and the Mathers Foundation. S. Ahmari is supported by the Medical Scientist Training Program, which is funded by a training grant from the National Institute of General Medical Sciences (GM07365).

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Correspondence to Stephen J Smith.

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Ahmari, S., Buchanan, J. & Smith, S. Assembly of presynaptic active zones from cytoplasmic transport packets . Nat Neurosci 3, 445–451 (2000). https://doi.org/10.1038/74814

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