Synaptic vesicle endocytosis: fast and slow modes of membrane retrieval

Abstract

Several modes of synaptic vesicle release, retrieval and recycling have been identified. In a well-established mode of exocytosis, termed 'full-collapse fusion', vesicles empty their neurotransmitter content fully into the synaptic cleft by flattening out and becoming part of the presynaptic membrane. The fused vesicle membrane is then reinternalized via a slow and clathrin-dependent mode of compensatory endocytosis that takes several seconds. A more fleeting mode of vesicle fusion, termed 'kiss-and-run' exocytosis or 'flicker-fusion', indicates that during synaptic transmission some vesicles are only briefly connected to the presynaptic membrane by a transient fusion pore. Finally, a mode that retrieves a large amount of membrane, equivalent to that of several fused vesicles, termed 'bulk endocytosis', has been found after prolonged exocytosis. We are of the opinion that both fast and slow modes of endocytosis co-exist at central nervous system nerve terminals and that one mode can predominate depending on stimulus strength, temperature and synaptic maturation.

Figure 1

SNARE complexes and exo-endocytosis. Classical (clathrin-mediated) and alternative routes for vesicular membrane retrieval. (a) The diagram shows that vesicle docking and fusion is mediated by the formation of SNARE (soluble N-ethylmaleimide sensitive factor (NSF) attachment protein receptor) complexes. A t-SNARE (target-SNARE or plasma membrane SNARE; dark blue) binds loosely or tightly to a v-SNARE molecule (vesicular-SNARE; purple) to form a SNARE complex [31,72]. Once a synaptic vesicle is docked and primed for exocytosis it proceeds with fusion via tight SNARE complex formation (coiled SNAREs are tightly bound and in a stable low energy state; right hand pathway [81]) or via loose SNARE complex formation which can also lead to fusion [31,32] (left hand pathway). The chaperone protein NSF (plus ATP) can uncoil and reverse the tight SNARE complex after fusion. It is, thus, required to disassemble used tight SNARE complexes and, thus, recycle SNARE proteins. NSF might also act as an ATPase before the fusion event [73]. An influx of Ca²⁺ ions triggers vesicle fusion via the formation and expansion of a fusion pore. Neurotransmitters are then released in <100 μs by the rapid expansion of the fusion pore in full-collapse fusion. In kiss-and-run exocytosis the fusion pore expands rapidly to a size sufficient for rapid transmitter release (e.g. a pore conductance of 300 pS) and then closes within approximately a second (or a few 100 μs). This mode of endocytosis is, thus, thought to be ‘fast.’ After full-collapse fusion the slow retrieval mode of endocytosis (time constant: τ = 10–50 s) is hypothesized to be mediated by clathrin-coated-pit formation and dynamin binding to the fission pore as a collar that constricts the pore neck via GTP hydrolysis. The fast mode of endocytosis (τ = 1–10 s) is thought to be clathrin-independent and is not well understood at a molecular level. Question marks indicate hypothesized pathways. (b) Multivesicular exo-endocytosis. Strong and prolonged stimulation of synapses might lead to copious exocytosis. It might also lead to compound exocytosis in which vesicles fuse with each other before they fuse to the plasma membrane. The large amount of vesicle membrane might be subsequently endocytosed as one large membrane invagination (bulk endocytosis). Parts of the plasma membrane might also be reinternalized via this mode of endocytosis, which could lead to a fast step-like rate of endocytosis because large amounts of membrane are fissioned at once from the plasma membrane. There is good evidence for this mode of endocytosis after strong stimulation in synaptosomes, ribbon-type synapses and at the NMJ [64,65]. Clathrin-mediated budding of vesicles from the endosome formed after bulk endocytosis can then produce synaptic vesicles that enter the reserve and/or recycling pool of vesicles.

Figure 2

The kinetics of endocytosis at a ribbon-type synapse. Capacitance (C_m) measurements from an isolated goldfish bipolar cell synaptic terminal embedded in a retinal slice. The C_m jump is triggered by a depolarization from −60 mV to 0 mV for 200 ms (arrow), which elicits the Ca current (I_Ca) shown in the inset. The C_m decay has both fast and slow components with normal 15-mM Cl⁻ in the patch pipette (red data points; double exponential fit with τ_f = 0.5 s [fast component; 34% contribution to overall fit] and τ_s = 5.5 s [slow component]). However, with 125-mM internal Cl⁻, endocytosis has no fast component (blue data points; C_m decay fit by a single exponential with τ = 18.2 s). High internal Cl⁻, thus, blocks fast endocytosis selectively, indicating that it is due to a molecular mechanism distinct from slow endocytosis. The internal Ca buffer was 5-mM ethylene glycol tetraacetic acid (EGTA); so high EGTA does not slow the rate of endocytosis per se, as long as the Ca currents are of large amplitude [18]. Reproduced, with permission, from Ref. [18].

Figure 3

Endocytosis at a calyx nerve terminal: effects of temperature and synaptic maturation. Membrane capacitance at the calyx of Held nerve terminal was monitored before and after a short depolarization to 0 mV (arrows). This depolarization activated a Ca²⁺ current (inset in A) and synaptic vesicle exocytosis, causing a capacitance (C_m) jump. Compensatory endocytosis is marked by a decrease in membrane capacitance as vesicle membrane fissions from the cell surface. (a) Increasing temperature accelerates endocytosis. Recording from a calyceal terminal (postnatal day [P] 10) at room temperature (RT) and then at physiological temperature (PT) reveals the addition of a kinetically distinct form of endocytosis at PT. A short depolarization at RT (2 ms, black circles) increased membrane capacitance by 110 fF, followed by a monoexponential return to baseline. At PT, this same calyceal terminal responded to the same depolarization with a larger C_m jump (270 fF, red crosses), and endocytosis was fit by a double exponential composed of a fast and slow component. Shown are the averages of 5 sweeps at RT and 4 sweeps at PT. Ca²⁺ currents from the step depolarizations are shown in the inset. Ca²⁺ influx increases at PT owing to the faster activation kinetics for the voltage sensitive Ca²⁺ channels. (b) The capacity for fast endocytosis increases with synaptic maturation. Short depolarizations (1 ms and 5 ms to 0 mV, arrows) in calyx terminals from young (P7–10, black circles) and more mature (P14–18, blue diamonds) animals at RT result in very different exo-endocytosis profiles. Young terminals show small capacitance jumps (23 fF for 1-ms depolarizing pulses, which is equivalent to the fusion of ~300 synaptic vesicles or that elicited by 1 or 2 action potentials; 120 fF for 5 ms) and slow endocytosis (τ = 15 s). Exocytosis in more mature terminals is greatly increased (77 fF at 1 ms; 211 fF at 5 ms) whereas the endocytotic rate is augmented slightly for large depolarizations, resulting in a similar time course for membrane recovery. Because more fused vesicle membrane is retrieved at similar or faster rates, the capacity for endocytosis is apparently increased at mature nerve terminals, perhaps because they have a larger, or more functional, pool of endocytotic proteins available to promote vesicle membrane fission. Reproduced, with permission, from Ref. [21].

Figure 4

Methodological summary of recent investigations supports the existence of both classical and kiss-and-run endocytosis. (a) Cell-attached capacitance measurements follow fusion pore opening and closing in real-time. This technique supports kiss-and-run exo-endocytosis. Capacitance flickers in a cell-attached recording from posterior pituitary nerve terminals correspond to kiss-and-run exocytosis of microvesicles (50-nm in diameter [26]). (b) The majority of the paired flicker durations for capacitance up and down steps in cell-attached recording from the release face of the calyx of Held are <1 s. The inset shows two examples (46 nm diameter synaptic vesicles [27]). (c,d) Fluorescently tagged proteins enable observation of single synaptic vesicle exo-endocytosis events. (c) Labeling of the vesicular transporter showed single vesicle dwell time varied for different fusion events as shown in these four example traces indicating different modes of endocytosis. The 0-s dwell-time (t_dwell ~ 0 s) event is consistent with kiss-and-run exocytosis and fast endocytosis. (d) Average synaptophysin–GFP signal from ~3000 synapses (middle trace) stimulated by a single field stimulus (at the arrow) decayed with τ = 19 s. This is consistent with a single mode of endocytosis. The synapses with highest and lowest fluorescence decayed with similar rates (upper and lower traces, respectively). (e) The total releasable pool (TRP) as retained FM1-43 dye after exocytosis is quenched by bromophenol blue (red trace), consistent with kiss-and-run. (f) 15-nm quantum dots (Qdot; red trace) escape from vesicles more slowly than FM4-64 signal (blue trace), using either field stimulation (FS) (i) or four rounds of high K⁺ (90 mM K⁺), starting at (ii) and ending at (iii), consistent with Qdots reporting only full-collapse fusion and FM reporting both full-collapse and kiss-and-run exo-endocytosis. Part (c) modified from [50], (d) modified from [49], (e) modified from [51] and (f) modified from [52], with permission.