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 endophilin's 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.

The control of release probability at nerve terminals.

Exocytosis is a fundamental membrane fusion process by which the soluble or membrane-associated cargoes of a secretory vesicle are delivered to the extracellular milieu or the cell surface. While essential for all organs, the brain relies on a specialized form of exocytosis to mediate information flow throughout its vast circuitry. Neurotransmitter-laden synaptic vesicles fuse with the plasma membrane on cue with astonishing speed in a probabilistic process that is both tightly regulated and capable of a fascinating array of plasticities. Here, we examine progress in the molecular understanding of synaptic vesicle fusion and its control.

A unique C2 domain at the C terminus of Munc13 promotes synaptic vesicle priming.

Neurotransmitter release during synaptic transmission comprises a tightly orchestrated sequence of molecular events, and Munc13-1 is a cornerstone of the fusion machinery. A forward genetic screen for defects in neurotransmitter release in Caenorhabditis elegans identified a mutation in the Munc13-1 ortholog UNC-13 that eliminated its unique and deeply conserved C-terminal module (referred to as HC2M) containing a Ca2+-insensitive C2 domain flanked by membrane-binding helices. The HC2M module could be functionally replaced in vivo by protein domains that localize to synaptic vesicles but not to the plasma membrane. HC2M is broadly conserved in other Unc13 family members and is required for efficient synaptic vesicle priming. We propose that the HC2M domain evolved as a vesicle/endosome adaptor and acquired synaptic vesicle specificity in the Unc13ABC protein family.

Speed Limits for Nonvesicular Intracellular Sterol Transport.

Sterol transport between the endoplasmic reticulum (ER) and plasma membrane (PM) occurs by nonvesicular mechanisms requiring sterol transport proteins (STPs). Here we examine the idea that transport is enhanced at membrane contact sites where the ER is closely apposed to the PM. We conclude that sterol desorption from the membrane, rather than STP-mediated diffusion, is rate limiting in the cellular context, so there is no apparent kinetic benefit to having STP-mediated sterol transfer occur at contact sites. Contact sites may instead compartmentalize lipid synthesis or transport machinery, providing opportunities for regulation.

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation.

Tether proteins attach the endoplasmic reticulum (ER) to other cellular membranes, thereby creating contact sites that are proposed to form platforms for regulating lipid homeostasis and facilitating non-vesicular lipid exchange. Sterols are synthesized in the ER and transported by non-vesicular mechanisms to the plasma membrane (PM), where they represent almost half of all PM lipids and contribute critically to the barrier function of the PM. To determine whether contact sites are important for both sterol exchange between the ER and PM and intermembrane regulation of lipid metabolism, we generated Δ-super-tether (Δ-s-tether) yeast cells that lack six previously identified tethering proteins (yeast extended synatotagmin [E-Syt], vesicle-associated membrane protein [VAMP]-associated protein [VAP], and TMEM16-anoctamin homologues) as well as the presumptive tether Ice2. Despite the lack of ER-PM contacts in these cells, ER-PM sterol exchange is robust, indicating that the sterol transport machinery is either absent from or not uniquely located at contact sites. Unexpectedly, we found that the transport of exogenously supplied sterol to the ER occurs more slowly in Δ-s-tether cells than in wild-type (WT) cells. We pinpointed this defect to changes in sterol organization and transbilayer movement within the PM bilayer caused by phospholipid dysregulation, evinced by changes in the abundance and organization of PM lipids. Indeed, deletion of either OSH4, which encodes a sterol/phosphatidylinositol-4-phosphate (PI4P) exchange protein, or SAC1, which encodes a PI4P phosphatase, caused synthetic lethality in Δ-s-tether cells due to disruptions in redundant PI4P and phospholipid regulatory pathways. The growth defect of Δ-s-tether cells was rescued with an artificial "ER-PM staple," a tether assembled from unrelated non-yeast protein domains, indicating that endogenous tether proteins have nonspecific bridging functions. Finally, we discovered that sterols play a role in regulating ER-PM contact site formation. In sterol-depleted cells, levels of the yeast E-Syt tether Tcb3 were induced and ER-PM contact increased dramatically. These results support a model in which ER-PM contact sites provide a nexus for coordinating the complex interrelationship between sterols, sphingolipids, and phospholipids that maintain PM composition and integrity.

Synaptic activity regulates the abundance and binding of complexin.

Nervous system function relies on precise chemical communication between neurons at specialized junctions known as synapses. Complexin (CPX) is one of a small number of cytoplasmic proteins that are indispensable in controlling neurotransmitter release through SNARE and synaptic vesicle interactions. However, the mechanisms that recruit and stabilize CPX are poorly understood. The mobility of CPX tagged with photoactivatable green fluorescent protein (pGFP) was quantified in vivo using Caenorhabditis elegans. Although pGFP escaped the synapse within seconds, CPX-pGFP displayed both fast and slow decay components, requiring minutes for complete exchange of the synaptic pool. The longer synaptic residence time of CPX arose from both synaptic vesicle and SNARE interactions, and surprisingly, CPX mobility depended on synaptic activity. Moreover, mouse CPX-GFP reversibly dispersed out of hippocampal presynaptic terminals during stimulation, and blockade of vesicle fusion prevented CPX dispersion. Hence, synaptic CPX can rapidly redistribute and this exchange is influenced by neuronal activity, potentially contributing to use-dependent plasticity.

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 (oshΔ osh4-1(ts)) 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-ß-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-ß-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.

Complexin Membrane Interactions: Implications for Synapse Evolution and Function.

The molecules and mechanisms behind chemical synaptic transmission have been explored for decades. For several of the core proteins involved in synaptic vesicle fusion, we now have a reasonably detailed grasp of their biochemical, structural, and functional properties. Complexin is one of the key synaptic proteins for which a simple mechanistic understanding is still lacking. Living up to its name, this small protein has been associated with a variety of roles differing between synapses and between species, but little consensus has been reached on its fundamental modes of action. Much attention has been paid to its deeply conserved SNARE-binding properties, while membrane-binding features of complexin and their functional significance have yet to be explored to the same degree. In this review, we summarize the known membrane interactions of the complexin C-terminal domain and their potential relevance to its function, synaptic localization, and evolutionary history.

Phospholipids are imported into mitochondria by VDAC, a dimeric beta barrel scramblase.

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identify the Voltage Dependent Anion Channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act to import lipids into mitochondria.

Two successive oligomeric Munc13 assemblies scaffold vesicle docking and SNARE assembly to support neurotransmitter release.

The critical presynaptic protein Munc13 serves numerous roles in the process of docking and priming synaptic vesicles. Here we investigate the functional significance of two distinct oligomers of the Munc13 core domain (Munc13C) comprising C1-C2B-MUN-C2C. Oligomer interface point mutations that specifically destabilized either the trimer or lateral hexamer assemblies of Munc13C disrupted vesicle docking, trans-SNARE formation, and Ca 2+ -triggered vesicle fusion in vitro and impaired neurotransmitter secretion and motor nervous system function in vivo. We suggest that a progression of oligomeric Munc13 complexes couples vesicle docking and assembly of a precise number of SNARE molecules to support rapid and high-fidelity vesicle priming.

Perforin-2 clockwise hand-over-hand pre-pore to pore transition mechanism.

Perforin-2 (PFN2, MPEG1) is a pore-forming protein that acts as a first line of defense in the mammalian immune system, rapidly killing engulfed microbes within the phagolysosome in macrophages. PFN2 self-assembles into hexadecameric pre-pore rings that transition upon acidification into pores damaging target cell membranes. Here, using high-speed atomic force microscopy (HS-AFM) imaging and line-scanning and molecular dynamics simulation, we elucidate PFN2 pre-pore to pore transition pathways and dynamics. Upon acidification, the pre-pore rings (pre-pore-I) display frequent, 1.8 s-1, ring-opening dynamics that eventually, 0.2 s-1, initiate transition into an intermediate, short-lived, ~75 ms, pre-pore-II state, inducing a clockwise pre-pore-I to pre-pore-II propagation. Concomitantly, the first pre-pore-II subunit, undergoes a major conformational change to the pore state that propagates also clockwise at a rate ~15 s-1. Thus, the pre-pore to pore transition is a clockwise hand-over-hand mechanism that is accomplished within ~1.3 s. Our findings suggest a clockwise mechanism of membrane insertion that with variations may be general for the MACPF/CDC superfamily.

Membrane-mediated protein interactions drive membrane protein organization.

The plasma membrane's main constituents, i.e., phospholipids and membrane proteins, are known to be organized in lipid-protein functional domains and supercomplexes. No active membrane-intrinsic process is known to establish membrane organization. Thus, the interplay of thermal fluctuations and the biophysical determinants of membrane-mediated protein interactions must be considered to understand membrane protein organization. Here, we used high-speed atomic force microscopy and kinetic and membrane elastic theory to investigate the behavior of a model membrane protein in oligomerization and assembly in controlled lipid environments. We find that membrane hydrophobic mismatch modulates oligomerization and assembly energetics, and 2D organization. Our experimental and theoretical frameworks reveal how membrane organization can emerge from Brownian diffusion and a minimal set of physical properties of the membrane constituents.

Control of Gαq signaling dynamics and GPCR cross-talk by GRKs.

Numerous processes contribute to the regulation of G protein-coupled receptors (GPCRs), but relatively little is known about rapid mechanisms that control signaling on the seconds time scale or regulate cross-talk between receptors. Here, we reveal that the ability of some GPCR kinases (GRKs) to bind Gαq both drives acute signaling desensitization and regulates functional interactions between GPCRs. GRK2/3-mediated acute desensitization occurs within seconds, is rapidly reversible, and can occur upon local, subcellular activation. This rapid desensitization is kinase independent, insensitive to pharmacological inhibition, and generalizable across receptor families and effectors. We also find that the ability of GRK2 to bind G proteins also enables it to regulate the extent and timing of Gαq-dependent signaling cross-talk between GPCRs. Last, we find that G protein/GRK2 interactions enable a novel form of GPCR trafficking cross-talk. Together, this work reveals potent forms of Gαq-dependent GPCR regulation with wide-ranging pharmacological and physiological implications.

Defining the Homo- and Heterodimerization Propensities of Metabotropic Glutamate Receptors.

The eight metabotropic glutamate receptors (mGluRs) serve critical modulatory roles throughout the nervous system. The molecular diversity of mGluRs is thought to be further expanded by the formation of heterodimers, but the co-expression of mGluR subtypes at the cellular level and the relative propensities of heterodimer formation are not well known. Here, we analyze single-cell RNA sequencing data and find that cortical pyramidal cells express multiple mGluR subtypes with distinct profiles for different receptor combinations. We then develop quantitative, fluorescence-based assays to define the relative homo- and heterodimer propensities across group-I, -II, and -III mGluRs. We find a strong preference for heterodimerization in a number of cases, including mGluR2 with mGluR3, which we confirm in frontal cortex using in situ RNA hybridization and co-immunoprecipitation. Together, our findings support the biological relevance of mGluR heterodimerization and highlight the complex landscape of mGluR populations in the brain.

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 GABA(B) 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.

Unc13: a multifunctional synaptic marvel.

Nervous systems are built on synaptic connections, and our understanding of these complex compartments has deepened over the past quarter century as the diverse fields of genetics, molecular biology, physiology, and biochemistry each made significant in-roads into synaptic function. On the presynaptic side, an evolutionarily conserved core fusion apparatus constructed from a handful of proteins has emerged, with Unc13 serving as a hub that coordinates nearly every aspect of synaptic transmission. This review briefly highlights recent studies on diverse aspects of Unc13 function including roles in SNARE assembly and quality control, release site building, calcium channel proximity, and short-term synaptic plasticity.

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.

Mechanism-based rescue of Munc18-1 dysfunction in varied encephalopathies by chemical chaperones.

Heterozygous de novo mutations in the neuronal protein Munc18-1 are linked to epilepsies, intellectual disability, movement disorders, and neurodegeneration. These devastating diseases have a poor prognosis and no known cure, due to lack of understanding of the underlying disease mechanism. To determine how mutations in Munc18-1 cause disease, we use newly generated S. cerevisiae strains, C. elegans models, and conditional Munc18-1 knockout mouse neurons expressing wild-type or mutant Munc18-1, as well as in vitro studies. We find that at least five disease-linked missense mutations of Munc18-1 result in destabilization and aggregation of the mutant protein. Aggregates of mutant Munc18-1 incorporate wild-type Munc18-1, depleting functional Munc18-1 levels beyond hemizygous levels. We demonstrate that the three chemical chaperones 4-phenylbutyrate, sorbitol, and trehalose reverse the deficits caused by mutations in Munc18-1 in vitro and in vivo in multiple models, offering a novel strategy for the treatment of varied encephalopathies.

A C1-C2 Module in Munc13 Inhibits Calcium-Dependent Neurotransmitter Release.

Almost all known forms of fast chemical synaptic transmission require the synaptic hub protein Munc13. This essential protein has also been implicated in mediating several forms of use-dependent plasticity, but the mechanisms by which it controls vesicle fusion and plasticity are not well understood. Using the C. elegans Munc13 ortholog UNC-13, we show that deletion of the C2B domain, the most highly conserved domain of Munc13, enhances calcium-dependent exocytosis downstream of vesicle priming, revealing a novel autoinhibitory role for the C2B. Furthermore, C2B inhibition is relieved by calcium binding to C2B, while the neighboring C1 domain acts together with C2B to stabilize the autoinhibited state. Selective disruption of Munc13 autoinhibition profoundly impacts nervous system function in vivo. Thus, C1-C2B exerts a basal inhibition on Munc13 in the primed state, permitting calcium- and lipid-dependent control of C1-C2B to modulate synaptic strength.

Unique Structural Features of Membrane-Bound C-Terminal Domain Motifs Modulate Complexin Inhibitory Function.

Complexin is a small soluble presynaptic protein that interacts with neuronal SNARE proteins in order to regulate synaptic vesicle exocytosis. While the SNARE-binding central helix of complexin is required for both the inhibition of spontaneous fusion and the facilitation of synchronous fusion, the disordered C-terminal domain (CTD) of complexin is specifically required for its inhibitory function. The CTD of worm complexin binds to membranes via two distinct motifs, one of which undergoes a membrane curvature dependent structural transition that is required for efficient inhibition of neurotransmitter release, but the conformations of the membrane-bound motifs remain poorly characterized. Visualizing these conformations is required to clarify the mechanisms by which complexin membrane interactions regulate its function. Here, we employ optical and magnetic resonance spectroscopy to precisely define the boundaries of the two CTD membrane-binding motifs and to characterize their conformations. We show that the curvature dependent amphipathic helical motif features an irregular element of helical structure, likely a pi-bulge, and that this feature is important for complexin inhibitory function in vivo.