Abstract
Synaptic vesicle (SV) docking is a dynamic multi-stage process that is required for efficient neurotransmitter release in response to nerve impulses. Although the steady-state SV docking likely involves the cooperation of Synaptotagmin-1 (Syt1) and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), where and how the docking process initiates remains unknown. Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) can interact with Syt1 and SNAREs to contribute to vesicle exocytosis. In the present study, using the CRISPRi-mediated multiplex gene knockdown and 3D electron tomography approaches, we show that in mouse hippocampal synapses, SV docking initiates at ∼12nm to the active zone (AZ) by Syt1. Furthermore, we demonstrate that PI(4,5)P2 is the membrane partner of Syt1 to initiate SV docking, and disrupting their interaction could abolish the docking initiation. In contrast, the SNARE complex contributes only to the tight SV docking within 0-2nm. Therefore, Syt1 interacts with PI(4,5)P2 to loosely dock SVs within 2-12nm to the AZ in hippocampal neurons.
Highlights
During neuronal communication, action potential (AP)-triggered Ca2+ influx into the presynaptic terminals induce the fusion of synaptic vesicles (SVs) into the plasma membrane of the active zone (AZ)
The sensitive factor attachment protein receptors (SNAREs) complex has been suggested to be essential for the steady-state Synaptic vesicle (SV) docking (Imig et al, 2014), it has remained unclear where and how SV docking is initiated
To understand the orchestration that the molecular engine performed to initiate SV docking, we employed multiplex CRISPRi tools to achieve simultaneous inactivation of proteins likely involved in SV docking, including Syt1 and SNAREs, in 14–17 days in vitro (DIV) cultured mouse hippocampal neurons (Chang et al, 2018; Imig et al, 2014; Zheng et al, 2018)
Summary
Synaptic vesicle (SV) docking is a dynamic multi-stage process that is required for efficient neurotransmitter release in response to nerve impulses. The steady-state SV docking likely involves the cooperation of Synaptotagmin-1 (Syt1) and soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), where and how the docking process initiates remains unknown. Phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) can interact with Syt and SNAREs to contribute to vesicle exocytosis. In the present study, using the CRISPRi-mediated multiplex gene knockdown and 3D electron tomography approaches, we show that in mouse hippocampal synapses, SV docking initiates at $12 nm to the active zone (AZ) by Syt. We demonstrate that PI(4,5)P2 is the membrane partner of Syt to initiate SV docking, and disrupting their interaction could abolish the docking initiation. The SNARE complex contributes only to the tight SV docking within 0–2 nm. Syt interacts with PI(4,5)P2 to loosely dock SVs within 2–12 nm to the AZ in hippocampal neurons
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