Jennifer R. Morgan

Uncoating of Clathrin-Coated Vesicles in Presynaptic Terminals: Roles for Hsc70 and Auxilin

We have examined the roles of Hsc70 and auxilin in the uncoating of clathrin-coated vesicles (CCVs) during neuronal endocytosis. We identified two peptides that inhibit the ability of Hsc70 and auxilin to uncoat CCVs in vitro. When injected into nerve terminals, these peptides inhibited both synaptic transmission and CCV uncoating. Mutation of a conserved HPD motif within the J domain of auxilin prevented binding to Hsc70 in vitro and injecting this mutant protein inhibited CCV uncoating in vivo, demonstrating that the interaction of auxilin with Hsc70 is critical for CCV uncoating. These studies establish that auxilin and Hsc70 participate in synaptic vesicle recycling in neurons and that an interaction between these proteins is required for CCV uncoating.

Proteins involved in synaptic vesicle trafficking

Neurotransmitter release relies on a series of synaptic vesicle trafficking reactions. We have determined the molecular basis of these reactions by microinjecting, into ‘giant’ nerve terminals of squid, probes that interfere with presynaptic proteins. These probes affect neurotransmitter release and disrupt nerve terminal structure. From the nature of these lesions, it is possible to deduce the roles of individual proteins in specific vesicle trafficking reactions. This approach has revealed the function of more than a dozen presynaptic proteins and we hypothesize that neurotransmitter release requires the coordinated action of perhaps 50–100 proteins.

Eps15 Homology Domain-NPF Motif Interactions Regulate Clathrin Coat Assembly during Synaptic Vesicle Recycling

Although genetic and biochemical studies suggest a role for Eps15 homology domain containing proteins in clathrin-mediated endocytosis, the specific functions of these proteins have been elusive. Eps15 is found at the growing edges of clathrin-coated pits, leading to the hypothesis that it participates in the formation of coated vesicles. We have evaluated this hypothesis by examining the effect of Eps15 on clathrin assembly. We found that although Eps15 has no intrinsic ability to assemble clathrin, it potently stimulates the ability of the clathrin adaptor protein, AP180, to assemble clathrin at physiological pH. We have also defined the binding sites for Eps15 on squid AP180. These sites contain an NPF motif, and peptides derived from these binding sites inhibit the ability of Eps15 to stimulate clathrin assembly in vitro. Furthermore, when injected into squid giant presynaptic nerve terminals, these peptides inhibit the formation of clathrin-coated pits and coated vesicles during synaptic vesicle endocytosis. This is consistent with the hypothesis that Eps15 regulates clathrin coat assembly in vivo, and indicates that interactions between Eps15 homology domains and NPF motifs are involved in clathrin-coated vesicle formation during synaptic vesicle recycling.

Synaptic vesicle endocytosis: the races, places, and molecular faces.

The classical experiments on synaptic vesicle recycling in the 1970s by Heuser and Reese, Ceccarelli, and their colleagues raised opposing theories regarding the speed, mechanisms, and locations of membrane retrieval at the synapse. The Heuser and Reese experiments supported a model in which synaptic vesicle recycling is mediated by the formation of coated vesicles, is relatively slow, and occurs distally from active zones, the sites of neurotransmitter release. Because heavy levels of stimulation were needed to visualize the coated vesicles, Ceccarelli’s experiments argued that synaptic vesicle recycling does not require the formation of coated vesicles, is relatively fast, and occurs directly at the active zone in a “kiss-and-run” reversal of exocytosis under more physiological conditions. For the next thirty years, these models have provided the foundation for studies of the rates, locations, and molecular elements involved in synaptic vesicle endocytosis. Here, we describe the evidence supporting each model and argue that the coated vesicle pathway is the most predominant physiological mechanism for recycling synaptic vesicles.

Clathrin and synaptic vesicle endocytosis: studies at the squid giant synapse

The role of clathrin-mediated endocytosis in SV (synaptic vesicle) recycling has been studied by combining molecular biology, physiology and electron microscopy at the squid giant synapse. Procedures that prevent clathrin from assembling into membrane coats, such as impairment of binding of the AP180 and AP-2 adaptor proteins, completely prevent membrane budding during endocytosis. These procedures also reduce exocytosis, presumably an indirect effect of a reduction in the number of SVs following block of endocytosis. Disrupting the binding of auxilin to Hsc70 (heat-shock cognate 70) prevents clathrin-coated vesicles from uncoating and also disrupts SV recycling. Taken together, these results indicate that a clathrin-dependent pathway is the primary means of SV recycling at this synapse under physiological conditions.

Actin polymerization regulates clathrin coat maturation during early stages of synaptic vesicle recycling at lamprey synapses.

Although it is established that presynaptic actin participates in synaptic vesicle recycling at several synapses, the earliest stages at which actin polymerization is employed during this process are still unclear. To address this, we prevented actin polymerization at lamprey synapses by applying latrunculin B or swinholide A. Latrunculin and swinholide depolymerize actin by sequestering actin monomers and, in addition, swinholide can sever existing actin filaments. When injected into individual presynaptic axons of the intact spinal cord, fluorescently labeled monomeric actin rapidly incorporated in a calcium‐dependent manner into a stable, filamentous actin network concentrated at endocytic zones. This pool of actin was disrupted completely by latrunculin. At stimulated synapses, specific disruption of actin polymerization with latrunculin and swinholide induced a selective increase in unconstricted clathrin‐coated pits and, in the case of swinholide, an additional increase in the size of plasma membrane evaginations. These results indicate that actin polymerization participates initially in the maturation of clathrin‐coated pits during early stages of synaptic vesicle recycling. J. Comp. Neurol. 497:600–609, 2006.

Sniffing calcium from the outside: an extracellular calcium sensor for synaptic vesicle recycling

In order for neurons to maintain communication with one another, following exocytosis, synaptic vesicles within the nerve terminal must be internalized from the plasma membrane and be refilled with neurotransmitter molecules for subsequent bouts of transmitter release. Calcium influx into the nerve terminal, which triggers exocytosis, may be an important temporal regulator of vesicle recycling. Several studies designed to address the calcium dependence of synaptic vesicle endocytosis have led to conflicting conclusions that intracellular calcium speeds up (Klingauf et al. 1998; Sankaranarayanan & Ryan, 2001), slows down (von Gersdorff & Matthews, 1994) or has little effect (Wu & Betz, 1996) on the rate of vesicle recycling. However, it is difficult to study the calcium dependence of endocytosis in isolation from exocytosis because these processes proceed concurrently and because exocytosis itself requires calcium influx. Thus, the role of calcium during synaptic vesicle recycling is still unclear. In this issue of The Journal of Physiology, a study of snake motor boutons measures uptake of the membrane dye sulphorhodamine 101 (SR101) to show that calcium regulates vesicle recycling via an extracellular sensor (Teng & Wilkinson, 2003). The calcium dependence of vesicle recycling was revisited by temporally separating this process from the immediate effects of calcium influx and exocytosis. To do so, the preparation was cooled to 7 °C following stimulation in order to delay membrane retrieval well beyond the time when calcium had returned to resting levels and exocytosis was complete. The ‘endocytotic debt’ incurred by delaying endocytosis lasted for several hours and was recovered at any time by warming the preparation to room temperature. When the magnitude and time course of dye uptake were measured in the presence of increasing extracellular calcium concentrations, delayed endocytosis proceeded more rapidly. Large changes in extracellular calcium had no observable effect on resting intraterminal calcium levels, as measured by a calcium indicator that reports nanomolar calcium concentrations. Therefore, calcium is a positive regulator of the rate of membrane retrieval, and these experiments implicate an extracellular calcium sensor in synaptic vesicle recycling. These same authors reported previously that delayed endocytosis is mediated by a temperature blockade of clathrin-coated vesicle formation and uncoating (Teng & Wilkinson, 2000). Thus, the current results suggest that extracellular calcium might regulate the rate of coated vesicle formation. This hypothesis is supported by the results of an electron microscope study of the lamprey giant synapse, in which synaptic vesicle recycling from the plasma membrane was temporarily blocked by the removal of extracellular calcium following stimulation (Gad et al. 1998). Subsequent recovery of synaptic vesicles was triggered via a rapid burst in coated vesicle formation by simply adding back 11 μM extracellular calcium. Although the interpretation was that minimal intracellular calcium is needed for synaptic vesicle recycling, these experiments may have inadvertently measured the affinity of the putative extracellular calcium sensor. The mechanism by which the endocytotic calcium sensor translocates a signal across the plasma membrane to trigger coated vesicle formation remains unclear. In the endocrine system, a G-protein-coupled extracellular calcium receptor regulates parathyroid hormone release by activating a cascade of pathways including protein kinase C and mitogen-activated protein kinase (Tfelt-Hansen et al. 2003), and the mechanisms for detecting extracellular calcium at synapses might be similar. During synaptic activity, a nerve terminal is exposed to changes in both extracellular and intracellular calcium, and thus the extracellular calcium sensor may coordinate with intracellular calcium effectors to regulate the early and late stages of coated vesicle formation. Calcium influx into the presynaptic terminal is likely to decrease calcium transiently in the synaptic cleft. As a result, vesicle recycling would be slowed until exocytosis is complete and extracellular calcium is returned to basal levels. Meanwhile, the entering calcium could trigger a cascade of events leading to the dephosphorylation of clathrin-associated proteins and their consequent assembly into multimolecular endocytotic complexes (Slepnev et al. 1998). Restoration of basal extracellular calcium levels could then trigger the extracellular sensor and speed up the early stages of endocytosis at a time when the intracellular endocytotic complexes are prepared to promote a burst of coated vesicle formation. In this way, changes in both intracellular and extracellular calcium could regulate synaptic vesicle recycling via coated vesicle formation. The current study by Teng & Wilkinson (2003) also highlights several other features of synaptic vesicle endocytosis in snake motor boutons. First, only half of the membrane retrieval was mediated by delayed endocytosis, indicating that a second mode of endocytosis exists, as is seen in other nerve terminals (e.g. Aravanis et al. 2003; Gandhi & Stevens, 2003). In snake nerve terminals, the second mode of membrane retrieval is likely to be mediated by macropinocytosis, which resembles bulk endocytosis and from which coated vesicles emanate (Teng & Wilkinson, 2000). Whether calcium affects the rate of macropinocytosis is unknown. Second, the ‘endocytotic debt’ incurred by delaying endocytosis was completely restored within 1 min, indicating that coated vesicle formation can proceed rapidly. Finally, because removal of extracellular calcium never completely blocked delayed endocytosis, calcium must be a regulator rather than an essential component of membrane retrieval. Identification of the putative extracellular calcium sensor and the mechanism by which it regulates the speed of synaptic vesicle recycling awaits further investigation.