Regulation Of Membrane Fusion Research Articles

PIP2 Is An Electrostatic Catalyst for Vesicle Fusion by Lowering the Hydration Energy: Arresting Vesicle Fusion by Masking PIP2.

Lipids are key factors in regulating membrane fusion. Lipids are not only structural components to form membranes but also active catalysts for vesicle fusion and neurotransmitter release, which are driven by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. SNARE proteins seem to be partially assembled before fusion, but the mechanisms that arrest vesicle fusion before Ca2+ influx are still not clear. Here, we show that phosphatidylinositol 4,5-bisphosphate (PIP2) electrostatically triggers vesicle fusion as an electrostatic catalyst by lowering the hydration energy and that a myristoylated alanine-rich C-kinase substrate (MARCKS), a PIP2-binding protein, arrests vesicle fusion in a vesicle docking state where the SNARE complex is partially assembled. Vesicle-mimicking liposomes fail to reproduce vesicle fusion arrest by masking PIP2, indicating that native vesicles are essential for the reconstitution of physiological vesicle fusion. PIP2 attracts cations to repel water molecules from membranes, thus lowering the hydration energy barrier.

Synaptobrevin2 monomers and dimers differentially engage to regulate the functional trans-SNARE assembly.

The precise cell-to-cell communication relies on SNARE-catalyzed membrane fusion. Among ∼70 copies of synaptobrevin2 (syb2) in synaptic vesicles, only ∼3 copies are sufficient to facilitate the fusion process at the presynaptic terminal. It is unclear what dictates the number of SNARE complexes that constitute the fusion pore assembly. The structure-function relation of these dynamic pores is also unknown. Here, we demonstrate that syb2 monomers and dimers differentially engage in regulating the trans-SNARE assembly during membrane fusion. The differential recruitment of two syb2 structures at the membrane fusion site has consequences in regulating individual nascent fusion pore properties. We have identified a few syb2 transmembrane domain residues that control monomer/dimer conversion. Overall, our study indicates that syb2 monomers and dimers are differentially recruited at the release sites for regulating membrane fusion events.

The light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibits cortical reaction in mouse oocytes.

Introduction: Cortical reaction is a secretory process that occurs after a spermatozoon fuses with the oocyte, avoiding the fusion of additional sperm. During this exocytic event, the cortical granule membrane fuses with the oocyte plasma membrane. We have identified several molecular components involved in this process and confirmed that SNARE proteins regulate membrane fusion during cortical reaction in mouse oocytes. In those studies, we microinjected different nonpermeable reagents to demonstrate the participation of a specific protein in the cortical reaction. However, the microinjection technique has several limitations. In this work, we aimed to assess the potential of cell-penetrating peptides (CPP) as biotechnological tools for delivering molecules into oocytes, and to evaluate the functionality of the permeable tetanus toxin (bound to CPP sequence) during cortical reaction. Methods: Arginine-rich cell-penetrating peptides have demonstrated the optimal internalization of small molecules in mammalian cells. Two arginine-rich CPP were used in the present study. One, labeled with 5-carboxyfluorescein, to characterize the factors that can modulate its internalization, and the other, the permeable light chain of tetanus toxin, that cleaves the SNAREs VAMP1 and VAMP3 expressed in mouse oocytes. Results: Results showed that fluorescent CPP was internalized into the oocyte cytoplasm and that internalization was dependent on the concentration, time, temperature, and maturation stage of the oocyte. Using our functional assay to study cortical reaction, the light chain of tetanus toxin bound to arginine-rich cell-penetrating peptide inhibited cortical granules exocytosis. Discussion: Results obtained from the use of permeable peptides demonstrate that this CPP is a promising biotechnological tool to study functional macromolecules in mouse oocytes.

The underlying mechanical properties of membranes tune their ability to fuse

Membrane fusion is a ubiquitous process associated with a multitude of biological events. Although it has long been appreciated that membrane mechanics plays an important role in membrane fusion, the molecular interplay between mechanics and fusion has remained elusive. For example, although different lipids modulate membrane mechanics differently, depending on their composition, molar ratio, and complex interactions, differing lipid compositions may lead to similar mechanical properties. This raises the question of whether (i) the specific lipid composition or (ii) the average mesoscale mechanics of membranes acts as the determining factor for cellular function. Furthermore, little is known about the potential consequences of fusion on membrane disruption. Here, we use a combination of confocal microscopy, time-resolved imaging, and electroporation to shed light onto the underlying mechanical properties of membranes that regulate membrane fusion. Fusion efficiency follows a nearly universal behavior that depends on membrane fluidity parameters, such as membrane viscosity and bending rigidity, rather than on specific lipid composition. This helps explaining why the charged and fluid membranes of the inner leaflet of the plasma membrane are more fusogenic than their outer counterparts. Importantly, we show that physiological levels of cholesterol, a key component of biological membranes, has a mild effect on fusion but significantly enhances membrane mechanical stability against pore formation, suggesting that its high cellular levels buffer the membrane against disruption. The ability of membranes to efficiently fuse while preserving their integrity may have given evolutionary advantages to cells by enabling their function while preserving membrane stability.

Membrane Fusion and SNAREs: Interaction with Ras Proteins.

The superfamily of Ras proteins comprises different molecules belonging to the GTPase family. They normally cycle between an active state bound to GTP which activates effectors while the protein is membrane-associated, and an inactive GDP-bound state. They regulate the intracellular trafficking and other cellular processes. The family of Rab proteins includes several members and they have been found, among other Ras proteins, to be fundamental for important biological processes, such as endocytosis and exocytosis. SNARE proteins control the fusion of vesicles by forming quaternary complexes which are divided into two small groups on the two different compartments. Generally, the association of three SNARE proteins on the donor compartment with the one on the target compartment determines the formation of the SNARE complex, the opening of the fusion pore and the formation of one single bigger vesicle. Interestingly, novel interactions between other molecules involved in intracellular trafficking, endosomal fusion and maturation have recently been found, such as the interaction between invariant chain and the Qb SNARE vti1b, and more functional connections between Rab proteins and SNAREs are supposed to be fundamental for the regulation of membrane fusion.

All-optical monitoring of excitation-secretion coupling demonstrates that SV2A functions downstream of evoked Ca2+ entry.

SV2A, an essential transporter-like synaptic vesicle protein, is a major target for antiepileptic drugs and a receptor for clostridial neurotoxins including Botox. While SV2A is required for normal levels of evoked neurotransmitter release, the mechanism underlying this role remains unclear. Here, we introduce a new chemogenetic approach for all-optical monitoring of excitation-secretion coupling, and we demonstrate its use in characterizing the SV2A knockout (KO) phenotype in cultured hippocampal neurons. This method employs the HaloTag system to target a robust small-molecule Ca2+ indicator, JF646 -BAPTA, to the presynaptic compartment. The far-red fluorescence of this indicator enables multiplexing with the fluorescent glutamate sensor iGluSnFR for detection of presynaptic Ca2+ influx and glutamate release at the same axonal boutons. Evoked glutamate release probability was reduced in SV2A KO neurons without a change in presynaptic Ca2+ entry, suggesting that SV2A supports vesicle fusion by increasing the functional availability, or efficiency, of the Ca2+ -regulated membrane fusion machinery. KEY POINTS: One of the most prescribed antiepileptic medications, levetiracetam, acts by binding a protein of uncertain molecular function. This transporter-like protein, SV2A, is trafficked to synaptic vesicles and acts to support neurotransmitter release, but the mechanism underlying this function has not been determined In this study, we sought to establish whether SV2A changes Ca2+ signalling at nerve terminals, which is a key regulatory system for synaptic vesicle exocytosis. To do so, we adapted new chemogenetic tools to perform all-optical measurements of presynaptic Ca2+ and glutamate release in neurons lacking SV2A. Our measurements showed that loss of SV2A reduces glutamate release without reducing Ca2+ influx at hippocampal nerve terminals, demonstrating that SV2A increases the likelihood that Ca2+ will trigger synaptic vesicle fusion.

Structural and functional analysis of p47 cofactor binding on the p97 disease mutant.

Human p97/VCP, an AAA+ ATPase, plays a pivotal role in the regulation of multiple cellular activities by interacting with various cofactor proteins. p97 is involved in the ubiquitin-dependent protein quality control and regulation of membrane-fusion in the Golgi apparatus in the presence of cofactor p47. Heterozygous missense mutations of p97 have been implicated in neurodegenerative diseases, such as IBMPFD (Inclusion body myopathy with early-onset Paget's disease and frontotemporal dementia) and ALS. The single amino-acid mutation of R155H on the N-domain is the highest mutated sites, leading to a rare degenerative disease MSP1, and resulting in abnormal ATPase activity and cofactor dysregulation. Our study aims to characterize the unknown protein interactions between p97R155H and p47 cofactor and identify the key complex structure to answer disease relevance of the p97R155H . Biochemical characterization along with single-particle cryo-electron microscopy (cryo-EM). Performed ATPase activity assays and fluorescent labeling to detect the temperature-related intensity change signals for quantitative determination of binding affinities of p97R155H-p47. First cryo-EM structures of the full-length p97R155H-p47 complex in different nucleotide-binding states. Without nucleotides, p97R155H is stable either as a hexamer or stacked as p97R155H-dodecamer. The p97R155H-dodecamer revealed a highly ordered C-terminal tail density and does not bind to p47 or nucleotides. It bears close resemblance to the inhibitor bound CB-5083:p97 structure, implying that the dodecameric form is inactive. In the full-length p97R155H-p47 complex structure without nucleotides, four N-domain densities and one p47UBX domain were identified interacting in an asymmetric manner implying a dynamic equilibrium. For the nucleotide-bound states, the flexibility of the p47UBX bound N-domains were less for the p97R155H|ATP𝛾S-p47, whereas fragmented in p97R155H|ADP-p47. Superimposition of the D1 and D2 domains of the three nucleotide states revealed that the D1-domains exhibit negligible structural change, whereas the D2-HTH motifs of the complexes with nucleotides are tilted downward. The C-terminal tails of the p97R155H|ATP𝛾S-p47 structure points in an opposite direction than that of p97R155H-p47/p97R155H|ADP-p47. The D1 and D2 arginine fingers play a critical role for the elevated p97R155H ATPase activity. These recent findings give us an insight into the disease mechanism of the R155H p97 mutation regarding its role in its pathological functions.

Cooperation of Conical and Polyunsaturated Lipids to Regulate Initiation and Processing of Membrane Fusion.

The shape of lipids has long been suspected to be a critical determinant for the control of membrane fusion. To experimentally test this assertion, we used conical and malleable lipids and measured their influence on the fusion kinetics. We found that, as previously suspected, both types of lipids accelerate fusion. However, the implicated molecular mechanisms are strikingly different. Malleable lipids, with their ability to change shape with low energy cost, favor fusion by decreasing the overall activation energy. On the other hand, conical lipids, with their small polar head relative to the area occupied by the hydrophobic chains, tend to make fusion less energetically advantageous because they tend to migrate towards the most favorable lipid leaflet, hindering fusion pore opening. They could however facilitate fusion by generating hydrophobic defects on the membranes; this is suggested by the similar trend observed between the experimental rate of fusion nucleation and the surface occupied by hydrophobic defects obtained by molecular simulations. The synergy of dual-process, activation energy and nucleation kinetics, could facilitate membrane fusion regulation in vivo.

Abstract 105: Novel Functions For Synaptosomal-associated Protein 29 (snap29) In Cardiac Arrhythmias

Human genetic studies and mouse models have classically linked mutations/deficiencies in components of the desmosomal cell-cell junction to arrhythmogenic right ventricular cardiomyopathy (ARVC). However, growing evidence points to the importance of the desmosome in cardiac diseases beyond ARVC, which include electrical diseases that have no impact on cardiac structure/morphology. The mechanisms of how defects in cardiac desmosomal protein homeostasis drive these distinct forms of cardiac disease remain elusive to the field. To uncover mechanisms that underlie the distinct pathophysiology (structural versus non-structural) encompassed by desmosomal mutations/loss, we performed an unbiased yeast-two-hybrid screen using an adult human heart cDNA library and the desmosomal protein, desmoplakin (DSP) to uncover new regulators of cardiac desmosomal protein homeostasis. We identified synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein in the adult human heart. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome and in the heart is undefined. We show that SNAP29 is a subcomponent of the cardiac desmosome, as it co-localizes with DSP in the adult heart and DSP-deficient hearts harbor loss of SNAP29. Cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. We show that a loss of a subset of desmosomal proteins and connexin43 as well as upregulation of selective autophagy-mediated degradation underlie SNAP29 deficient cardiomyocyte arrhythmias. In line with this, acute blockade of autophagy was sufficient to rescue desmosomal and connexin43 protein levels as well as arrhythmias in SNAP29 deficient cardiomyocytes. In conclusion, SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias. Thus, loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical nature.

BioID reveals an ATG9A interaction with ATG13-ATG101 in the degradation of p62/SQSTM1-ubiquitin clusters.

ATG9A, the only multi-pass transmembrane protein among core ATG proteins, is an essential regulator of autophagy, yet its regulatory mechanisms and network of interactions are poorly understood. Through quantitative BioID proteomics, we identify a network of ATG9A interactions that includes members of the ULK1 complex and regulators of membrane fusion and vesicle trafficking, including the TRAPP, EARP, GARP, exocyst, AP-1, and AP-4 complexes. These interactions mark pathways of ATG9A trafficking through ER, Golgi, and endosomal systems. In exploring these data, we find that ATG9A interacts with components of the ULK1 complex, particularly ATG13 and ATG101. Using knockout/reconstitution and split-mVenus approaches to capture the ATG13-ATG101 dimer, we find that ATG9A interacts with ATG13-ATG101 independently of ULK1. Deletion of ATG13 or ATG101 causes a shift in ATG9A distribution, resulting in an aberrant accumulation of ATG9A at stalled clusters of p62/SQSTM1 and ubiquitin, which can be rescued by an ULK1 binding-deficient mutant of ATG13. Together, these data reveal ATG9A interactions in vesicle-trafficking and autophagy pathways, including a role for an ULK1-independent ATG13 complex in regulating ATG9A.

Loss of dysferlin or myoferlin results in differential defects in excitation\u2013contraction coupling in mouse skeletal muscle

Muscular dystrophies are disorders characterized by progressive muscle loss and weakness that are both genotypically and phenotypically heterogenous. Progression of muscle disease arises from impaired regeneration, plasma membrane instability, defective membrane repair, and calcium mishandling. The ferlin protein family, including dysferlin and myoferlin, are calcium-binding, membrane-associated proteins that regulate membrane fusion, trafficking, and tubule formation. Mice lacking dysferlin (Dysf), myoferlin (Myof), and both dysferlin and myoferlin (Fer) on an isogenic inbred 129 background were previously demonstrated that loss of both dysferlin and myoferlin resulted in more severe muscle disease than loss of either gene alone. Furthermore, Fer mice had disordered triad organization with visibly malformed transverse tubules and sarcoplasmic reticulum, suggesting distinct roles of dysferlin and myoferlin. To assess the physiological role of disorganized triads, we now assessed excitation contraction (EC) coupling in these models. We identified differential abnormalities in EC coupling and ryanodine receptor disruption in flexor digitorum brevis myofibers isolated from ferlin mutant mice. We found that loss of dysferlin alone preserved sensitivity for EC coupling and was associated with larger ryanodine receptor clusters compared to wildtype myofibers. Loss of myoferlin alone or together with a loss of dysferlin reduced sensitivity for EC coupling, and produced disorganized and smaller ryanodine receptor cluster size compared to wildtype myofibers. These data reveal impaired EC coupling in Myof and Fer myofibers and slightly potentiated EC coupling in Dysf myofibers. Despite high homology, dysferlin and myoferlin have differential roles in regulating sarcotubular formation and maintenance resulting in unique impairments in calcium handling properties.

SNAP29 Restricts Cardiac Arrhythmias by Insulating A Subset of Desmosomal Proteins and Connexin43 from Autophagic Degradation

Desmosomes are strong cell-cell adhesion junctions that connect the cytoskeleton of one cell to another (via intermediate filaments) to maintain the mechanical integrity of tissues under constant stress, such as the heart. Classically, human genetic studies and mouse models have linked components of the desmosome mutations/deficiencies to arrhythmogenic right ventricular cardiomyopathy (ARVC), an inherited cardiac disease of the desmosome that impacts cardiac structure/morphology. However, growing evidence points to a role for loss of desmosomal integrity in cardiac electrical diseases (eg., Brugada syndrome, long QT, sinus node arrhythmias) that have no impact on cardiac structure/morphology; however, how desmosomal protein loss results in these distinct forms of cardiac disease remains unclear. We exploited an unbiased yeast-two-hybrid screen using the desmosomal protein, desmoplakin (DSP) as a bait, to uncover novel desmosomal protein-protein interactions that may help further explain the pathobiology (structural versus non-structural) encompassed by desmosomal mutations/loss. Through this screen, we identified the SNARE protein, synaptosomal-associated protein 29 (SNAP29), as a novel DSP-interacting protein. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy; however, its role at the desmosome in the heart is undefined. We hypothesized that SNAP29 is a new subcomponent of the cardiac desmosome that regulates desmosomal protein degradation and thus, may have important roles in desmosomal based diseases. We show that SNAP29 co-localizes with DSP in adult mouse heart and that cardiac-specific DSP deficient mice hearts harbor loss of SNAP29, validating SNAP29 as a subcomponent of the cardiac desmosome. By generating novel cardiomyocyte-specific SNAP29 knockout (SNAP29-cKO) mice (using Xenopus myosin light chain 2-Cre), we show that SNAP29-cKO mouse hearts displayed baseline and pacing-induced ventricular arrhythmias in an age-dependent manner in the absence of cardiac structural and functional deficits. Molecular analyses further revealed that adult SNAP29-cKO hearts displayed a loss of a subset of desmosomal (DSP, plakophilin2) as well as gap junction (connexin43) proteins when compared to controls. These cell-cell junction defects were accompanied by an accumulation of autophagic markers (p62 and LC3BII), as well as machinery (autophagic vesicles), which functionally resulted in cardiac arrhythmias. We show that acute blockade of autophagy was sufficient to rescue levels of desmosomal and gap junction proteins as well as arrhythmias in SNAP29 deficient cardiomyocytes. In summary, our data suggest that SNAP29 insulates a subset of desmosomal and gap junction proteins from selective autophagy-mediated degradation to restrict cardiac arrhythmias and that loss of SNAP29-desmosome-gap junction interactome may predispose the heart to desmosomal based diseases of an electrical (non-structural) nature. Collectively, our results identify defects in cardiac autophagy in desmosomal disease pathogenesis, while providing a novel role for SNARE proteins in cardiac arrhythmias.

Localization of the Interaction Site of Herpes Simplex Virus Glycoprotein D (gD) on the Membrane Fusion Regulator, gH/gL.

A cascade of protein-protein interactions between four herpes simplex virus (HSV) glycoproteins (gD, gH/gL, and gB) drive fusion between the HSV envelope and host membrane, thereby allowing for virus entry and infection. Specifically, binding of gD to one of its receptors induces a conformational change that allows gD to bind to the regulatory complex gH/gL, which then activates the fusogen gB, resulting in membrane fusion. Using surface plasmon resonance and a panel of anti-gD monoclonal antibodies (MAbs) that sterically blocked the interaction, we previously showed that gH/gL binds directly to gD at sites distinct from the gD receptor binding site. Here, using an analogous strategy, we first evaluated the ability of a panel of uncharacterized anti-gH/gL MAbs to block binding to gD and/or inhibit fusion. We found that the epitopes of four gD-gH/gL-blocking MAbs were located within flexible regions of the gH N terminus and the gL C terminus, while the fifth was placed around gL residue 77. Taken together, our data localized the gD binding region on gH/gL to a group of gH and gL residues at the membrane distal region of the heterodimer. Surprisingly, a second set of MAbs did not block gD-gH/gL binding but instead stabilized the complex by altering the kinetic binding. However, despite this prolonged gD-gH/gL interaction, "stabilizing" MAbs also inhibited cell-cell fusion, suggesting a unique mechanism by which the fusion process is halted. Our findings support targeting the gD-gH/gL interaction to prevent fusion in both therapeutic and vaccine strategies against HSV.IMPORTANCE Key to developing a human HSV vaccine is an understanding of the virion glycoproteins involved in entry. HSV employs multiple glycoproteins for attachment, receptor interaction, and membrane fusion. Determining how these proteins function was resolved, in part, by structural biology coupled with immunological and biologic evidence. After binding, virion gD interacts with a receptor to activate the regulator gH/gL complex, triggering gB to drive fusion. Multiple questions remain, one being the physical location of each glycoprotein interaction site. Using protective antibodies with known epitopes, we documented the long-sought interaction between gD and gH/gL, detailing the region on gD important to create the gD-gH/gL triplex. Now, we have identified the corresponding gD contact sites on gH/gL. Concurrently we discovered a novel mechanism whereby gH/gL antibodies stabilize the complex and inhibit fusion progression. Our model for the gD-gH/gL triplex provides a new framework for studying fusion, which identifies targets for vaccine development.

A kinase cascade on the yeast lysosomal vacuole regulates its membrane dynamics: conserved kinase Env7 is phosphorylated by casein kinase Yck3

Membrane fusion/fission is a highly dynamic and conserved process that responds to intra- and extracellular signals. Whereas the molecular machineries involved in membrane fusion/fission have been dissected, regulation of membrane dynamics remains poorly understood. The lysosomal vacuole of budding yeast (Saccharomyces cerevisiae) has served as a seminal model in studies of membrane dynamics. We have previously established that yeast ENV7 encodes an ortholog of STK16-related kinases that localizes to the vacuolar membrane and downregulates vacuolar membrane fusion. Additionally, we have previously reported that Env7 phosphorylation in vivo depends on YCK3, a gene that encodes a vacuolar membrane casein kinase I (CKI) homolog that nonredundantly functions in fusion regulation. Here, we report that Env7 physically interacts with and is directly phosphorylated by Yck3. We also establish that Env7 vacuole fusion/fission regulation and vacuolar localization are mediated through its Yck3-dependent phosphorylation. Through extensive site-directed mutagenesis, we map phosphorylation to the Env7 C terminus and confirm that Ser-331 is a primary and preferred phosphorylation site. Phospho-deficient Env7 mutants were defective in negative regulation of membrane fusion, increasing the number of prominent vacuoles, whereas a phosphomimetic substitution at Ser-331 increased the number of fragmented vacuoles. Bioinformatics approaches confirmed that Env7 Ser-331 is within a motif that is highly conserved in STK16-related kinases and that it also anchors an SXXS CKI phosphorylation motif (328SRFS331). This study represents the first report on the regulatory mechanism of an STK16-related kinase. It also points to regulation of vacuolar membrane dynamics via a novel Yck3-Env7 kinase cascade.

The Parkinson’s Disease Protein LRRK2 Interacts with the GARP Complex to Promote Retrograde Transport to the trans-Golgi Network

SUMMARYMutations in Leucine-rich repeat kinase 2 (LRRK2) cause Parkinson’s disease (PD). However, the precise function of LRRK2 remains unclear. We report an interaction between LRRK2 and VPS52, a subunit of the Golgi-associated retrograde protein (GARP) complex that identifies a function of LRRK2 in regulating membrane fusion at the trans-Golgi network (TGN). At the TGN, LRRK2 further interacts with the Golgi SNAREs VAMP4 and Syntaxin-6 and acts as a scaffolding platform that stabilizes the GARP-SNAREs complex formation. Therefore, LRRK2 influences both retrograde and post-Golgi trafficking pathways in a manner dependent on its GTP binding and kinase activity. This action is exaggerated by mutations associated with Parkinson’s disease and can be blocked by kinase inhibitors. Disruption of GARP sensitizes dopamine neurons to mutant LRRK2 toxicity in C. elegans, showing that these pathways are interlinked in vivo and suggesting a link in PD.

Diverse roles of Synaptotagmin-7 in regulating vesicle fusion.

Synaptotagmin 7 (Syt7) is a multifunctional calcium sensor expressed throughout the body. Its high calcium affinity makes it well suited to act in processes triggered by modest calcium increases within cells. In synaptic transmission, Syt7 has been shown to mediate asynchronous neurotransmitter release, facilitation, and vesicle replenishment. In this review we provide an update on recent developments, and the newly emerging roles of Syt7 in frequency invariant synaptic transmission and in suppressing spontaneous release. Additionally, we discuss Syt7's regulation of membrane fusion in non-neuronal cells, and its involvement in disease. How such diversity of functions is regulated remains an open question. We discuss several potential factors including temperature, presynaptic calcium signals, the localization of Syt7, and its interaction with other Syt isoforms.

Cardiomyocyte SNAP29 Insulates Desmosomes from Targeted Protein Degradation by Selective Autophagy to Restrict Arrhythmias

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a fatal and untreatable inherited cardiomyopathy associated with ventricular dysfunction and cardiac arrhythmias leading to sudden cardiac death in the young. Early detection is hindered as people present with complex disease patterns and harbors a “concealed phase”, where lethal arrhythmias are observed in the absence of overt structural changes to the heart. Mutations and loss of components of the desmosome, a cardiac mechanical cell‐cell junction, have been linked to the development of ARVC through human and mouse genetic studies. However, limited information exists on how desmosomal protein dysregulation/loss can trigger the complex features of ARVC, especially the asymptomatic “concealed phase”. Using a yeast two‐hybrid screen we identified Synaptoso‐malassociated protein 29 (SNAP29) as a novel desmosomal (desmoplakin, DSP)‐interacting protein. Traditional functions of SNAP29 are to regulate membrane fusion and play a role in autophagy in non‐cardiomyocytes; however, its role at the desmosome and/or heart is undefined. We hypothesize that SNAP29 may play a critical role in maintaining desmosomal protein homeostasis and have direct relevance to ARVC disease features, as it is a disease associated with desmosomal protein dissolution. We show that SNAP29 co‐localizes with DSP in both the adult mouse and human heart. Cardiac‐specific SNAP29‐deficient mice (SNAP29‐cKO) displayed baseline and pacing‐ induced ventricular arrhythmias in an age‐dependent manner in the absence of cardiac structural and functional deficits. Furthermore, molecular analyses revealed that the arrhythmias found in SNAP29‐cKO hearts and cardiomyocytes were associated with a molecular loss of a subset of desmosomal proteins (DSP and plakophilin‐2) and the gap junction protein, connexin43. Furthermore, an accumulation of autophagic markers (p62 and LC3BII), as well as machinery, was observed at the cardiac cell‐cell junction in SNAP29 deficient cardiomyocytes. Acute blockade of autophagy using specific inhibitors (chloroquine and bafilomycin A1) was sufficient to rescue levels of desmosomal and gap junction proteins as well as arrhythmias in SNAP29 deficient cardiomyocytes. Our data suggest that SNAP29 insulates a subset of desmosomal proteins from selective autophagy‐mediated degradation to restrict cardiac arrhythmias. Thus, SNAP29 loss may directly contribute to the “concealed phase” of ARVC. Our results further identify autophagy defects as an underlying mechanism in desmosomal disease pathogenesis and highlight new potential therapeutic targets for ARVC.Support or Funding InformationNIH/NHLBI (HL09780)

Site-specific phosphorylation of villin remodels the actin cytoskeleton to regulate Sendai viral glycoprotein-mediated membrane fusion.

Connivance of cellular factors during virus-host cell membrane fusion is poorly understood. We have recently shown that cellular villin plays an important role during membrane fusion of reconstituted Sendai virosomes with hepatocytes. Here, we employed villin-null Chinese Hamster Ovary (CHO) cells, where villin expression led to an increased fusion with virosomes, which was further enhanced due to tyrosine phosphorylation in the presence of c-src. However, the villin RRI mutant, lacking actin-severing function, failed to augment membrane fusion. Furthermore, quantitative mass spectrometry and detailed analysis revealed Tyr499 to be the key phosphorylation site of villin responsible for the enhancement of virosome-CHO cell fusion. Overall, our results demonstrate a critical role for villin and its cell-type dependent phosphorylation in regulating membrane fusion.

Vaccinia viral A26 protein is a fusion suppressor of mature virus and triggers membrane fusion through conformational change at low pH.

Vaccinia mature virus requires A26 envelope protein to mediate acid-dependent endocytosis into HeLa cells in which we hypothesized that A26 protein functions as an acid-sensitive membrane fusion suppressor. Here, we provide evidence showing that N-terminal domain (aa1-75) of A26 protein is an acid-sensitive region that regulates membrane fusion. Crystal structure of A26 protein revealed that His48 and His53 are in close contact with Lys47, Arg57, His314 and Arg312, suggesting that at low pH these His-cation pairs could initiate conformational changes through protonation of His48 and His53 and subsequent electrostatic repulsion. All the A26 mutant mature viruses that interrupted His-cation pair interactions of His48 and His 53 indeed have lost virion infectivity. Isolation of revertant viruses revealed that second site mutations caused frame shifts and premature termination of A26 protein such that reverent viruses regained cell entry through plasma membrane fusion. Together, we conclude that viral A26 protein functions as an acid-sensitive fusion suppressor during vaccinia mature virus endocytosis.

Mutations in the DI-DII linker of the NDV fusion protein conferred hemagglutinin-neuraminidase-independent cell fusion promotion.

Newcastle disease (ND), which is caused by Newcastle disease virus (NDV), is a highly contagious disease in chickens and is a great threat to the poultry industry. Fusion of the viral and target cell membranes is a prerequisite for NDV's entry into host cells. This process is directly mediated by the fusion (F) protein. Although several domains of F are known to regulate membrane fusion activity, the roles of the DI-DII linker (residues 376-381) of the NDV F protein in membrane fusion still remain unclear. To investigate the roles of this linker in NDV F-induced cell-cell fusion, mutations were engineered into this linker by site-directed mutagenesis. These mutants were analysed with respect to cell surface expression and membrane fusion activity. Each of the mutated F proteins in this linker was expressed at the cell surface at a similar level to wild-type (WT) F. However, most of them resulted in significant alterations in fusion activity. In particular, the mutants G377S, A378D, L379A and T380P were able to independently mediate cell fusion in the absence of HN protein in BHK-21 cells. Taken together, the results indicated that the DI-DII linker region has an important effect on the fusion activity of NDV F and mutants in this region could alter the requirement for HN for the promotion of membrane fusion.