Distinct modes of endocytotic presynaptic membrane and protein uptake at the calyx of Held terminal of rats and mice

Abstract

Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, compensatory retrieval of membranes and vesicular proteins is essential. We combined capacitance measurements and pH-imaging via pH-sensitive vesicular protein marker (anti-synaptotagmin2-cypHer5E), and compared the retrieval kinetics of membranes and vesicular proteins at the calyx of Held synapse. Membrane and Syt2 were retrieved with a similar time course when slow endocytosis was elicited. When fast endocytosis was elicited, Syt2 was still retrieved together with the membrane, but endocytosed organelle re-acidification was slowed down, which provides strong evidence for two distinct endocytotic pathways. Strikingly, CaM inhibitors or the inhibition of the Ca2+-calmodulin-Munc13-1 signaling pathway only impaired the uptake of Syt2 while leaving membrane retrieval intact, indicating different recycling mechanisms for membranes and vesicle proteins. Our data identify a novel mechanism of stimulus- and Ca2+-dependent regulation of coordinated endocytosis of synaptic membranes and vesicle proteins.DOI: http://dx.doi.org/10.7554/eLife.14643.001

Synaptic Vesicle Endocytosis Occurs on Multiple Timescales and Is Mediated by Formin-Dependent Actin Assembly

“…The time course and mechanism of SV endocytosis determined here closely matches observations in a variety of other systems. Capacitance measurements at the calyx of Held (Eguchi et al, 2012;Okamoto et al, 2016;Wu et al, 2014a), ribbon synapses of retinal bipolar cells (Llobet et al, 2011), and inner hair cells (Beutner et al, 2001), and of cerebellar mossy fiber synapses (Delvendahl et al, 2016) have revealed time constants for endocytosis that range from less than one second up to tens of seconds (Okamoto et al, 2016), depending on stimulation. We observe a slowing of SV endocytosis with increasing stimulus strength at physiological temperature (e.g., about 0.7 and 12 s for 2 APs, 0.7 and 26 s for 10 APs at 10 Hz, >30 s for 200 APs at 40 Hz) in agreement with prior data (Armbruster et al, 2013), although the precise t values vary depending on the experimental conditions (e.g., extracellular calcium) and the pHluorin reporter used (e.g., we observe a tendency for faster kinetics of synaptophysin compared to synaptotagmin 1 internalization in conventional pHluorin [compare Figures 2E and S2F] and in acid quench [ Figure 1E] assays).…”

Promotion of endocytosis efficiency through an ATP‐independent mechanism at rat calyx of Held terminals

“…; Okamoto et al . ) and at elevated temperature (Renden & von Gersdorff, ). Two lines of evidence suggest that ATP‐independent endocytosis retrieves pre‐existing membrane.…”

“…However, because the temporal resolution of this method is limited by the rate of vesicle acidification and sampling interval, endocytosis with τ of ∼2 s, if present, could have escaped detection (Kononenko & Haucke, ; Okamoto et al . ). Indeed, imaging with quantal dots and high‐pressure freezing electron microscopy have demonstrated that hippocampal boutons contain the rapid ‘kiss‐and‐run’ mode of endocytosis (Zhang et al .…”

“…; Okamoto et al . ), large Ca 2+ transient (Beutner et al . ) and at physiological temperature (Renden & von Gersdorff, ).…”

Integration of Synaptic Vesicle Cargo Retrieval with Endocytosis at Central Nerve Terminals

“…Biochemical enrichment of bulk endosomes immediately after their formation revealed that a series of essential SV cargo molecules were present, suggesting they were retrieved during invagination (Nicholson-Fish et al, 2015 ). Furthermore, simultaneous monitoring of endogenous SV cargo with membrane invagination in large atypical nerve terminals demonstrated that during “fast” endocytosis cargo was internalized into a slowly acidifying compartment (Okamoto et al, 2016 ). Fast endocytosis is triggered by strong stimulation and generally thought to equate to ADBE (Wu et al, 2005 ), therefore this compartment may reflect bulk endosomes.…”