Dynamin phosphorylation controls optimization of endocytosis for brief action potential bursts

Moritz Armbruster,Shawn M Ferguson,Pietro De Camilli,Mirko Messa,Timothy A Ryan

doi:10.7554/elife.00845

Moritz Armbruster, Shawn M Ferguson + Show 3 more

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https://doi.org/10.7554/elife.00845

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Abstract

Modulation of synaptic vesicle retrieval is considered to be potentially important in steady-state synaptic performance. Here we show that at physiological temperature endocytosis kinetics at hippocampal and cortical nerve terminals show a bi-phasic dependence on electrical activity. Endocytosis accelerates for the first 15-25 APs during bursts of action potential firing, after which it slows with increasing burst length creating an optimum stimulus for this kinetic parameter. We show that activity-dependent acceleration is only prominent at physiological temperature and that the mechanism of this modulation is based on the dephosphorylation of dynamin 1. Nerve terminals in which dynamin 1 and 3 have been replaced with dynamin 1 harboring dephospho- or phospho-mimetic mutations in the proline-rich domain eliminate the acceleration phase by either setting endocytosis at an accelerated state or a decelerated state, respectively. DOI:http://dx.doi.org/10.7554/eLife.00845.001.

Highlights

Synaptic transmission relies on a steady supply of release competent neurotransmitter-filled synaptic vesicles (SVs) to maintain information transmission in neural circuits
Using high-sensitivity pHluorin assays of synaptic vesicle endocytosis for individual neurons we revealed the presence of two phases of stimulus and Ca2+ dependence of synaptic vesicle endocytosis: an acceleration phase prominent for small stimuli and a slowing phase prominent for larger stimuli
While the vGlut-pHluorin reporter only tracks the internalization of vGlut, previous synaptic vesicle endocytosis studies have shown SynaptopHluorin (Vamp2), Synaptophysin-pHluorin, and Synaptotagmin-pHluorin all showing the same kinetics as vGlut-pHluorin as well as the same dependence on the clathrin adaptor AP-2 (Kim and Ryan, 2009)

Summary

Introduction

Synaptic transmission relies on a steady supply of release competent neurotransmitter-filled synaptic vesicles (SVs) to maintain information transmission in neural circuits. At hippocampal and cortical nerve terminals synaptic vesicles are retrieved largely in a dynamin (Ferguson et al, 2007; Raimondi et al, 2011), clathrin (Granseth et al, 2006) and AP-2 dependent fashion (Kim and Ryan, 2009). Numerous studies have revealed a modulatory role for Ca2+ in endocytosis at nerve terminals in different synaptic preparations (Balaji et al, 2008; Sankaranarayanan and Ryan, 2001; von Gersdorff and Matthews, 1994; Wu et al, 2009; Yamashita et al, 2010; Yao et al, 2009) the precise steps at which Ca2+ acts in this process have not been identified. The direction of the Ca2+ modulation is in dispute, some studies suggest an accelerating role of Ca2+, (Sankaranarayanan and Ryan, 2001; Wu et al, 2009) while others

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