Transient docking of synaptic vesicles: Implications and mechanisms

Abstract

As synaptic vesicles fuse, they must continually be replaced with new docked, fusion-competent vesicles to sustain neurotransmission. It has long been appreciated that vesicles are recruited to docking sites in an activity-dependent manner. However, once entering the sites, vesicles were thought to be stably docked, awaiting calcium signals. Based on recent data from electrophysiology, electron microscopy, biochemistry, and computer simulations, a picture emerges in which vesicles can rapidly and reversibly transit between docking and undocking during activity. This "transient docking" can account for many aspects of synaptic physiology. In this review, we cover recent evidence for transient docking, physiological processes at the synapse that it may support, and progress on the underlying mechanisms. We also discuss an open question: what determines for how long and whether vesicles stay docked, or eventually undock?

Figure 1.

The strict definition of synaptic vesicle docking. What is meant by ‘docking’ in synaptic ultrastructure varies from study to study. This term is often used for all vesicles within 30–40 nm of the plasma membrane at the active zone (measuring the nearest distance between the edge of the vesicle membrane and plasma membrane). Here, we refer to docking by a strict definition: structurally, docking is the closest synaptic vesicles can get to the plasma membrane at the active zone before fusion as observed by electron microscopy. (a) In high-pressure frozen and freeze-substituted samples, docked vesicles make a ‘point contact’ with the plasma membrane, visible in both (a) 2D thin sectioning EM and (b) 3D electron tomography (solid arrowheads indicate vesicles with visible plasma membrane contact in the tomograph slice shown, hollow arrowheads indicate vesicles that are docked and make contact with the plasma membrane, but the contact is not visible in this slice; green vesicles in the 3D rendering are docked), with no apparent space between vesicle membrane and plasma membrane down to the effective resolution of this technique (0–2 nm) [28,43]. (c) In cryo-electron tomography, which visualizes the native state of tissue under vitreous ice without any staining, dehydration, or fixation, the closest vesicles get to the plasma membrane in synapses at rest is ~5 nm [–86], and by our definition these constitute docked vesicles. This means the apparent 0–2 nm distance in freeze substituted samples is likely an artifact. However, the two characteristic distances in these techniques are likely both meaningful and correspond to the same vesicles. ~75% of all vesicles within 20 nm of the plasma membrane accumulate at this closest distance in cultured hippocampal synapses, regardless of which technique is used [28,84]. [27,82]. Accumulation at this specific distance is unique to docking, as undocked vesicles within 100 nm are roughly evenly distributed in distance from the active zone. Only this closest stage of approach requires SNARE complex assembly [27,43], only docked vesicles are depleted by stimulation [26,28], and in cryo-electron tomography only these vesicles are connected to the membrane by a stereotyped protein density that may correspond to the docking/fusion machinery [84]. All these lines of evidence together strongly argue that docked vesicles, and only docked vesicles, are at the final stage of priming and readiness for fusion. Vesicles that are close to the plasma membrane, but not docked, we refer to simply as undocked or as ‘replacement vesicles’ (these vesicles are sometimes referred to as ‘tethered’). In terms of distance from the plasma membrane by EM, our definition of docking corresponds to the term ‘tightly docked’ often used in the field [7]. Note that any studies using traditional chemical fixation for electron microscopy, rather than fast freezing, cannot resolve the distinctions discussed here. Aldehyde fixation of living tissue causes severe deformations in cellular structures [87] and directly triggers synaptic vesicle exocytosis [88], making evaluation of fine structure near the active zone inaccurate. For example, under chemical fixation, preventing SNARE complex assembly has no apparent effect on docking [89]. (a) and (c) are reproduced, with permission, from [43] and [84], respectively. (b) is reproduced from [26].

Figure 2.

Proposed scheme for docking and undocking of synaptic vesicles at rest and during activity. Middle row: At steady state, vesicles reversible dock and undock as the trans-SNARE complex tightens and loosens, shuttling between a ‘docking site’ and ‘replacement site. Upon calcium binding to Syt1, docked vesicles fuse. Top row: in ‘two-step’ release, calcium binding to a calcium sensor(s) triggers docking then immediate fusion, perhaps giving rise to asynchronous release. While Syt1 is shown here, other higher-affinity calcium sensors may mediate docking or fusion during two-step release. Bottom row: docking is enhanced during activity as high-affinity calcium sensors such as Syt3 and Syt7 (and/or other signaling molecules) push vesicles into the dock state, or lock them there. Biasing the reaction coordinate towards docking makes more docked vesicles available for the next round of fusion, giving rise to synaptic potentiation and resistance to synaptic depression. For simplicity, only the SNARE complex, and not other essential parts of the docking machinery like Munc13, is shown. Adapted from an unpublished figure by Erik M. Jorgensen, with inspiration from [7]. Note that molecular structures are hypothetical.