Abstract
Control of microtubule (MT) nucleation and dynamics is critical for neuronal function. Whether MT nucleation is regulated at presynaptic boutons and influences overall presynaptic activity remains unknown. By visualizing MT plus-end dynamics at individual excitatory en passant boutons in axons of cultured hippocampal neurons and in hippocampal slices expressing EB3-EGFP and vGlut1-mCherry, we found that dynamic MTs preferentially grow from presynaptic boutons, show biased directionality in that they are almost always oriented toward the distal tip of the axon, and can be induced by neuronal activity. Silencing of γ-tubulin expression reduced presynaptic MT nucleation, and depletion of either HAUS1 or HAUS7-augmin subunits increased the percentage of retrograde comets initiated at boutons, indicating that γ-tubulin and augmin are required for activity-dependent de novo nucleation of uniformly distally oriented dynamic MTs. We analyzed the dynamics of a wide range of axonal organelles as well as synaptic vesicles (SVs) relative to vGlut1+ stable presynaptic boutons in a time window during which MT nucleation at boutons is promoted upon induction of neuronal activity, and we found that γ-tubulin-dependent presynaptic MT nucleation controls bidirectional (SV) interbouton transport and regulates evoked SV exocytosis. Hence, en passant boutons act as hotspots for activity-dependent de novo MT nucleation, which controls neurotransmission by providing dynamic tracks for bidirectional delivery of SVs between sites of neurotransmitter release.
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