Jeremy S. Dittman

Interplay between Facilitation, Depression, and Residual Calcium at Three Presynaptic Terminals

Synapses display remarkable alterations in strength during repetitive use. Different types of synapses exhibit distinctive synaptic plasticity, but the factors giving rise to such diversity are not fully understood. To provide the experimental basis for a general model of short-term plasticity, we studied three synapses in rat brain slices at 34°C: the climbing fiber to Purkinje cell synapse, the parallel fiber to Purkinje cell synapse, and the Schaffer collateral to CA1 pyramidal cell synapse. These synapses exhibited a broad range of responses to regular and Poisson stimulus trains. Depression dominated at the climbing fiber synapse, facilitation was prominent at the parallel fiber synapse, and both depression and facilitation were apparent in the Schaffer collateral synapse. These synapses were modeled by incorporating mechanisms of short-term plasticity that are known to be driven by residual presynaptic calcium (Cares). In our model, release is the product of two factors: facilitation and refractory depression. Facilitation is caused by a calcium-dependent increase in the probability of release. Refractory depression is a consequence of release sites becoming transiently ineffective after release. These sites recover with a time course that is accelerated by elevations of Cares. Facilitation and refractory depression are coupled by their common dependence on Cares and because increased transmitter release leads to greater synaptic depression. This model captures the behavior of three different synapses for various stimulus conditions. The interplay of facilitation and depression dictates synaptic strength and variability during repetitive activation. The resulting synaptic plasticity transforms the timing of presynaptic spikes into varying postsynaptic response amplitudes.

Ubiquitin and AP180 Regulate the Abundance of GLR-1 Glutamate Receptors at Postsynaptic Elements in C. elegans

Regulated delivery and removal of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) from postsynaptic elements has been proposed as a mechanism for regulating synaptic strength. Here we test the role of ubiquitin in regulating synapses that contain a C. elegans GluR, GLR-1. GLR-1 receptors were ubiquitinated in vivo. Mutations that decreased ubiquitination of GLR-1 increased the abundance of GLR-1 at synapses and altered locomotion behavior in a manner that is consistent with increased synaptic strength. By contrast, overexpression of ubiquitin decreased the abundance of GLR-1 at synapses and decreased the density of GLR-1-containing synapses, and these effects were prevented by mutations in the unc-11 gene, which encodes a clathrin adaptin protein (AP180). These results suggest that ubiquitination of GLR-1 receptors regulates synaptic strength and the formation or stability of GLR-1-containing synapses.

Molecular Circuitry of Endocytosis at Nerve Terminals

Presynaptic terminals are specialized compartments of neurons responsible for converting electrical signals into secreted chemicals. This self-renewing process of chemical synaptic transmission is accomplished by the calcium-triggered fusion of neurotransmitter-containing vesicles with the plasma membrane and subsequent retrieval and recycling of vesicle components. Whereas the release of neurotransmitters has been studied for over 50 years, the process of synaptic vesicle endocytosis has remained much more elusive. The advent of imaging techniques suited to monitor membrane retrieval at presynaptic terminals and the discovery of the molecules that orchestrate endocytosis have revolutionized our understanding of this critical trafficking event.

Osh proteins regulate membrane sterol organization but are not required for sterol movement between the ER and PM

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by an ATP‐dependent, non‐vesicular mechanism that is presumed to require sterol transport proteins (STPs). In Saccharomyces cerevisiae, homologs of the mammalian oxysterol‐binding protein (Osh1‐7) have been proposed to function as STPs. To evaluate this proposal we took two approaches. First we used dehydroergosterol (DHE) to visualize sterol movement in living cells by fluorescence microscopy. DHE was introduced into the PM under hypoxic conditions and observed to redistribute to lipid droplets on growing the cells aerobically. Redistribution required ATP and the sterol acyltransferase Are2, but did not require PM‐derived transport vesicles. DHE redistribution occurred robustly in a conditional yeast mutant (oshΔosh4‐1ts) that lacks all functional Osh proteins at 37°C. In a second approach we used a pulse‐chase protocol to analyze the movement of metabolically radiolabeled ergosterol from the ER to the PM. Arrival of radiolabeled ergosterol at the PM was assessed in isolated PM‐enriched fractions as well as by extracting sterols from intact cells with methyl‐β‐cyclodextrin. These experiments revealed that whereas ergosterol is transported effectively from the ER to the PM in Osh‐deficient cells, the rate at which it moves within the PM to equilibrate with the methyl‐β‐cyclodextrin extractable sterol pool is slowed. We conclude (i) that the role of Osh proteins in non‐vesicular sterol transport between the PM, ER and lipid droplets is either minimal, or subsumed by other mechanisms and (ii) that Osh proteins regulate the organization of sterols at the PM.

Endophilin Functions as a Membrane-Bending Molecule and Is Delivered to Endocytic Zones by Exocytosis

Two models have been proposed for endophilin function in synaptic vesicle (SV) endocytosis. The scaffolding model proposes that endophilins SH3 domain recruits essential endocytic proteins, whereas the membrane-bending model proposes that the BAR domain induces positively curved membranes. We show that mutations disrupting the scaffolding function do not impair endocytosis, whereas those disrupting membrane bending cause significant defects. By anchoring endophilin to the plasma membrane, we show that endophilin acts prior to scission to promote endocytosis. Despite acting at the plasma membrane, the majority of endophilin is targeted to the SV pool. Photoactivation studies suggest that the soluble pool of endophilin at synapses is provided by unbinding from the adjacent SV pool and that the unbinding rate is regulated by exocytosis. Thus, endophilin participates in an association-dissociation cycle with SVs that parallels the cycle of exo- and endocytosis. This endophilin cycle may provide a mechanism for functionally coupling endocytosis and exocytosis.

Behavioral Impact of Neurotransmitter-Activated G-Protein-Coupled Receptors: Muscarinic and GABAB Receptors Regulate Caenorhabditis elegans Locomotion

Neurotransmitter released from presynaptic terminals activates both ligand-gated ion channels (ionotropic receptors) and a variety of G-protein-coupled receptors (GPCRs). These neurotransmitter receptors are expressed on both presynaptic and postsynaptic cells. Thus, each neurotransmitter acts on multiple receptor classes, generating a large repertoire of physiological responses. The impact of many ionotropic receptors on neuronal activity and behavior has been clearly elucidated; however, much less is known about how neurotransmitter-gated GPCRs regulate neurons and circuits. In Caenorhabditis elegans, both acetylcholine (ACh) and GABA are released in the nerve cord and mediate fast neuromuscular excitation and inhibition during locomotion. Here we identify a muscarinic receptor (GAR-2) and the GABAB receptor dimer (GBB-1/2) that detect synaptically released ACh and GABA, respectively. Both GAR-2 and GBB-1/2 inhibited cholinergic motor neurons when ACh and GABA levels were enhanced. Loss of either GPCR resulted in movement defects, suggesting that these receptors are activated during locomotion. When the negative feedback provided by GAR-2 was replaced with positive feedback, animals became highly sensitive to ACh levels and locomotion was severely impaired. Thus, conserved GPCRs act in the nematode motor circuit to provide negative feedback and to regulate locomotory behaviors that underlie navigation.

Synaptic Vesicles Position Complexin to Block Spontaneous Fusion

Synapses continually replenish their synaptic vesicle (SV) pools while suppressing spontaneous fusion events, thus maintaining a high dynamic range in response to physiological stimuli. The presynaptic protein complexin can both promote and inhibit fusion through interactions between its α-helical domain and the SNARE complex. In addition, complexins C-terminal half is required for the inhibition of spontaneous fusion in worm, fly, and mouse, although the molecular mechanism remains unexplained. We show here that complexins C-terminal domain binds lipids through a novel protein motif, permitting complexin to inhibit spontaneous exocytosis in vivo by targeting complexin to SVs. We propose that the SV pool serves as a platform to sequester and position complexin where it can intercept the rapidly assembling SNAREs and control the rate of spontaneous fusion.

Constitutive phospholipid scramblase activity of a G protein-coupled receptor

Opsin, the rhodopsin apoprotein, was recently shown to be an ATP-independent flippase (or scramblase) that equilibrates phospholipids across photoreceptor disc membranes in mammalian retina, a process required for disc homeostasis. Here we show that scrambling is a constitutive activity of rhodopsin, distinct from its light-sensing function. Upon reconstitution into vesicles, discrete conformational states of the protein (rhodopsin, a metarhodopsin II-mimic, and two forms of opsin) facilitated rapid (>10,000 phospholipids per protein per second) scrambling of phospholipid probes. Our results indicate that the large conformational changes involved in converting rhodopsin to metarhodopsin II are not required for scrambling, and that the lipid translocation pathway either lies near the protein surface or involves membrane packing defects in the vicinity of the protein. Additionally, we demonstrate that β2-adrenergic and adenosine A2A receptors scramble lipids, suggesting that rhodopsin-like G protein-coupled receptors may play an unexpected moonlighting role in re-modeling cell membranes.

Membrane curvature sensing by the C-terminal domain of complexin

Complexin functions at presynaptic nerve terminals to inhibit spontaneous SNARE-mediated synaptic vesicle exocytosis, while enhancing stimulated neurotransmitter release. The C-terminal domain (CTD) of complexin is essential for its inhibitory function and has been implicated in localizing complexin to synaptic vesicles via direct membrane interactions. Here we show that complexins CTD is highly sensitive to membrane curvature, which it senses via tandem motifs, a C-terminal motif containing a mix of bulky hydrophobic and positively charged residues, and an adjacent amphipathic region that can bind membranes in either a disordered or a helical conformation. Helix formation requires membrane packing defects found on highly curved membrane surfaces. Mutations that disrupt helix formation without disrupting membrane binding compromise complexins inhibitory function in vivo. Thus, this membrane curvature-dependent conformational transition, combined with curvature sensitive binding by the adjacent C-terminal motif, constitute a novel mechanism for activating complexins inhibitory function on the surface of synaptic vesicles.

The accessory helix of complexin functions by stabilizing central helix secondary structure

The presynaptic protein complexin (CPX) is a critical regulator of synaptic vesicle fusion, but the mechanisms underlying its regulatory effects are not well understood. Its highly conserved central helix (CH) directly binds the ternary SNARE complex and is required for all known CPX functions. The adjacent accessory helix (AH) is not conserved despite also playing an important role in CPX function, and numerous models for its mechanism have been proposed. We examined the impact of AH mutations and chimeras on CPX function in vivo and in vitro using C. elegans. The mouse AH fully restored function when substituted into worm CPX suggesting its mechanism is evolutionarily conserved. CPX inhibitory function was impaired when helix propagation into the CH was disrupted whereas replacing the AH with a non-native helical sequence restored CPX function. We propose that the AH operates by stabilizing CH secondary structure rather than through protein or lipid interactions. DOI: http://dx.doi.org/10.7554/eLife.04553.001