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Neuronal communication crucially relies on exocytosis of neurotransmitters from synaptic vesicles (SVs) which are clustered at synapses. To ensure reliable neurotransmitter release, synapses need to maintain an adequate pool of SVs at all times. Decades of research have established that SVs are clustered by synapsin 1, an abundant SV-associated phosphoprotein. The classical view postulates that SVs are crosslinked in a scaffold of protein-protein interactions between synapsins and their binding partners. Recent studies have shown that synapsins cluster SVs via liquid-liquid phase separation (LLPS), thus providing a new framework for the organization of the synapse. We discuss the evidence for phase separation of SVs, emphasizing emerging questions related to its regulation, specificity, and reversibility.

Contains fulltext : 292891.pdf (Publisher’s version ) (Open Access) Endosomal Sorting Complex Required for Transport (ESCRT) proteins can be transiently recruited to the plasma membrane for membrane repair and formation of extracellular vesicles. Here, we discovered micrometer-sized worm-shaped ESCRT structures that stably persist for multiple hours at the plasma membrane of macrophages, dendritic cells, and fibroblasts. These structures surround clusters of integrins and known cargoes of extracellular vesicles. The ESCRT structures are tightly connected to the cellular support and are left behind by the cells together with surrounding patches of membrane. The phospholipid composition is altered at the position of the ESCRT structures, and the actin cytoskeleton is locally degraded, which are hallmarks of membrane damage and extracellular vesicle formation. Disruption of actin polymerization increased the formation of the ESCRT structures and cell adhesion. The ESCRT structures were also present at plasma membrane contact sites with membrane-disrupting silica crystals. We propose that the ESCRT proteins are recruited to adhesion-induced membrane tears to induce extracellular shedding of the damaged membrane.

Alpha-synuclein (aSyn) is an intrinsically disordered protein associated with Parkinson’s disease and other synucleinopathies, where it accumulates in pathognomonic protein inclusions in the brain. However, both the physiological function and the pathological role of aSyn are still elusive. Unlike other proteins also prone to aggregation, expression of aSyn in cell models does not readily result in inclusion formation, and additional insults are required to induce aggregation. In this study, we report for the first time a model that recapitulates the aggregation cascade of aSyn. Our study sheds light on the role of synphilin-1 (Sph1) in aSyn assembly and localization. We show that Sph1 acts as a protein-adaptor for aSyn, and that the coiled-coil domain of Sph1 is essential for inducing inclusion formation. We also demonstrate that the interaction between Sph1 and aSyn can be modulated (genetically, protein levels and heat shock proteins), and that altering the expression levels of each protein affects inclusion size and number per cell. Our study highlights the potential importance of membrane binding for inclusion formation and the presence of lysosomes and AP-1 vesicles in the inclusions. Finally, our study demonstrates that further investigation is necessary to decipher the role of Sph1 and other aSyn-interacting proteins on its biology and pathobiology.

Maternal autoantibodies can be transmitted diaplacentally, with potentially deleterious effects on neurodevelopment. Synapsin 1 (SYN1) is a neuronal protein that is important for synaptic communication and neuronal plasticity. While monoallelic loss of function (LoF) variants in theSYN1gene result in X-linked intellectual disability (ID), learning disabilities, epilepsy, behavioral problems, and macrocephaly, the effect of SYN1 autoantibodies on neurodevelopment remains unclear. We recruited a clinical cohort of 208 mothers and their children with neurologic abnormalities and analyzed the role of maternal SYN1 autoantibodies. We identified seropositivity in 9.6% of mothers, and seropositivity was associated with an increased risk for ID and behavioral problems. Furthermore, children more frequently had epilepsy, macrocephaly, and developmental delay, in line with the SYN1 LoF phenotype. Whether SYN1 autoantibodies have a direct pathogenic effect on neurodevelopment or serve as biomarkers requires functional experiments.

Anti-neuronal autoantibodies can be transplacentally transferred during pregnancy and may cause detrimental effects on fetal development. It is unclear whether autoantibodies against synapsin-I, one of the most abundant synaptic proteins, are associated with developmental abnormalities in humans. We prospectively recruited a cohort of 263 pregnant women and detected serum synapsin-I IgG autoantibodies in 13.3%. Seropositivity was strongly associated with abnormalities of fetal development including intrauterine growth retardation. This finding indicates that these autoantibodies may be clinically useful developmental biomarkers and/or even directly participate in the disease process, thus being amenable to antibody-targeting interventional strategies in the future.

Deposition of amyloid beta (Aβ) along with glia cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer’s disease (AD). In recent years, impairment of autophagy has been found to be another important feature, contributing to AD progression and aging. Therefore, we assessed the effect of the autophagy activator Spermidine, a small body-endogenous polyamine often used as dietary supplement and known to promote longevity, on glia cell-mediated neuroinflammation. Spermidine reduced TLR3- and TLR4- mediated inflammatory processes in microglia and astrocytes by decreasing cytotoxicity, inflammasome activity and NF-κB signaling. In line with these anti-inflammatory effects, oral treatment of the amyloid prone AD-like APPPS1 mice with Spermidine reduced neuroinflammation and neurotoxic soluble Aβ. Mechanistically, single nuclei sequencing revealed microglia as one of the main targets of Spermidine treatment, with increased expression of genes implicated in cell motility and phagocytosis. Thus, Spermidine provides a promising therapeutic potential to target glia cells in AD progression.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that leads to the death of upper and lower motor neurons. While most cases of ALS are sporadic, some of the familial forms of the disease are caused by mutations in the gene encoding for the RNA-binding protein FUS. Under physiological conditions, FUS readily phase separates into liquid-like droplets in vivo and in vitro. ALS-associated mutations interfere with this process and often result in solid-like aggregates rather than fluid condensates. Yet, whether cells recognize and triage aberrant condensates remains poorly understood, posing a major barrier to the development of novel ALS treatments. Using a combination of ALS-associated FUS mutations, optogenetic manipulation of FUS condensation, chemically induced stress, and pH-sensitive reporters of organelle acidity, we systematically characterized the cause-effect relationship between the material state of FUS condensates and the sequestering of lysosomes. From our data, we can derive three conclusions. First, regardless of whether we use wild-type or mutant FUS, expression levels (i.e., high concentrations) play a dominant role in determining the fraction of cells having soluble or aggregated FUS. Second, chemically induced FUS aggregates recruit LAMP1-positive structures. Third, mature, acidic lysosomes accumulate only at FUS aggregates but not at liquid-condensates. Together, our data suggest that lysosome-degradation machinery actively distinguishes between fluid and solid condensates. Unraveling these aberrant interactions and testing strategies to manipulate the autophagosome-lysosome axis provides valuable clues for disease intervention.

The occurrence of linear cholesterol-recognition motifs in alpha-helical transmembrane domains has long been debated. Here, we demonstrate the ability of a genetic algorithm guided by coarse-grained molecular dynamics simulations—a method coined evolutionary molecular dynamics (Evo-MD)—to directly resolve the sequence which maximally attracts cholesterol for single-pass alpha-helical transmembrane domains (TMDs). We illustrate that the evolutionary landscape of cholesterol attraction in membrane proteins is characterized by a sharp, well-defined global optimum. Surprisingly, this optimal solution features an unusual short hydrophobic block, consisting of typically only eight small hydrophobic amino acids, surrounded by three successive lysines. Owing to the membrane thickening effect of cholesterol, cholesterol-enriched ordered phases favor TMDs characterized by a long rather than a too short hydrophobic length (a negative hydrophobic mismatch). However, this short hydrophobic pattern evidently offers a pronounced net advantage for the attraction of free cholesterol in both coarse-grained and atomistic simulations. We illustrate that optimal cholesterol attraction is in fact based on the superposition of two distinct thermodynamic attraction mechanisms. In addition, we explore the evolutionary occurrence and feasibility of these two mechanisms by analyzing existing databases of membrane proteins and through the direct expression of analogous short hydrophobic sequences in live cell assays. The puzzling sequence variability of proposed linear cholesterol-recognition motifs is indicative of a sub-optimal membrane-mediated attraction of cholesterol which markedly differs from ligand binding based on shape compatibility.Significance StatementOur work demonstrates how a synergy between evolutionary algorithms and high-throughput coarse-grained molecular dynamics can yield fundamentally new insights into the evolutionary fingerprints of protein-mediated lipid sorting. We illustrate that the evolutionary landscape of cholesterol attraction in isolated transmembrane domains is characterized by a well-defined global optimum. In contrast, sub-optimal attraction of cholesterol is associated with a diverse solution space and features a high sequence variability despite acting on the same unique molecule. The contrasting physicochemical nature of the resolved attraction optimum suggests that cholesterol attraction via linear motifs does not pose a dominant pressure on the evolution of transmembrane proteins.

The material properties of biomolecular condensates play pivotal roles in many biological and pathological processes. Despite the rapid increase in the number of biomolecules identified that undergo liquid-liquid phase separation (LLPS), quantitative studies of the resulting condensates have been severely lagging behind. Here, we develop a micropipette-based technique, which uniquely allows quantifications of both the surface tension and viscosity of biomolecular condensates, independent of labeling and surface wetting effects. We demonstrate the accuracy and versatility of this technique by measuring condensates of LAF-1 RGG domains and a polymer-based aqueous two-phase system (ATPS). We anticipate this technique will be widely applicable to biomolecular condensates and will resolve several limitations regarding current approaches.

Phase separation in the cytoplasm is emerging as a major principle in intracellular organization. In this process, sets of macromolecules assemble themselves into liquid compartments that are distinct from the surrounding medium but are not delimited by membrane boundaries. Here, we discuss how phase separation, in which a component of one of the two phases is vesicles rather than macromolecules, could underlie the formation of synaptic vesicle (SV) clusters in proximity to presynaptic sites. The organization of SVs into a liquid phase could explain how SVs remain tightly clustered without being stably bound to a scaffold so that they can be efficiently recruited to release site by active zone components.

Neurotransmission relies on the tight spatial and temporal regulation of the synaptic vesicle (SV) cycle. Nerve terminals contain hundreds of SVs that form tight clusters. These clusters represent a distinct liquid phase in which one component of the phase are SVs and the other synapsin 1, a highly abundant synaptic protein. Another major family of disordered proteins at the presynapse includes synucleins, most notably α-synuclein. The precise physiological role of α-synuclein in synaptic physiology remains elusive, albeit its role has been implicated in nearly all steps of the SV cycle. To determine the effect of α-synuclein on the synapsin phase, we employ the reconstitution approach using natively purified SVs from rat brains and the heterologous cell system to generate synapsin condensates. We demonstrate that synapsin condensates recruit α-synuclein, and while enriched into these synapsin condensates, α-synuclein still maintains its high mobility. The presence of SVs enhances the rate of synapsin/α-synuclein condensation, suggesting that SVs act as catalyzers for the formation of synapsin condensates. Notably, at physiological salt and protein concentrations, α-synuclein alone is not able to cluster isolated SVs. Excess of α-synuclein disrupts the kinetics of synapsin/SV condensate formation, indicating that the molar ratio between synapsin and α-synuclein is important in assembling the functional condensates of SVs. Understanding the molecular mechanism of α-synuclein interactions at the nerve terminals is crucial for clarifying the pathogenesis of synucleinopathies, where α-synuclein, synaptic proteins and lipid organelles all accumulate as insoluble intracellular inclusions.

Contains fulltext : 172064.pdf (Publisher’s version ) (Open Access) Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P-2) is a minor component of total plasma membrane lipids, but it has a substantial role in the regulation of many cellular functions, including exo- and endocytosis. Recently, it was shown that PI(4,5)P-2 and syntaxin 1, a SNARE protein that catalyzes regulated exocytosis, form domains in the plasma membrane that constitute recognition sites for vesicle docking. Also, calcium was shown to promote syntaxin 1 clustering in the plasma membrane, but the molecular mechanism was unknown. Here, using a combination of superresolution stimulated emission depletion microscopy, FRET, and atomic force microscopy, we show that Ca2+ acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P-2 complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca2+ concentrations is likely to be important for Ca2+-regulated secretion.

Long-range correlated motions in proteins are candidate mechanisms for processes that require information transfer across protein structures, such as allostery and signal transduction. However, the observation of backbone correlations between distant residues has remained elusive, and only local correlations have been revealed using residual dipolar couplings measured by NMR spectroscopy. In this work, we experimentally identified and characterized collective motions spanning four β-strands separated by up to 15 A in ubiquitin. The observed correlations link molecular recognition sites and result from concerted conformational changes that are in part mediated by the hydrogen-bonding network.

SNARE-mediated membrane fusion is a key step in signal transmission between neurons. In this biological process strongly curved synaptic vesicles fuse with a planar presynaptic membrane to release neurotransmitter molecules into the synaptic cleft. Although there is a considerable number of different in vitro assays to analyse neuronal SNARE-mediated fusion, these assays do not imitate the in vivo situation accurately enough because they are mostly based on vesicle bulk fusion where two curved membranes fuse with each other. Considering the geometries of the membranes of interest in the synapse we developed a model system based on SNARE-mediated fusion of large unilamellar vesicles (LUVs) with a planar pore-spanning membrane (PSM). PSMs containing syntaxin 1A and SNAP25 were obtained by spreading giant unilamellar vesicles on gold covered 6-mercapto-1-hexanol functionalized porous silicon nitride substrates with pore diameters of 1.2 µm. Both the lipids and the SNAREs were fully mobile in the PSMs, which is prerequisite for the formation of active fusion complexes. Synaptobrevin 2 doped LUVs with a mean diameter of 325 ± 20 nm were prepared by extrusion and the highly SNARE specific single vesicle fusion events with pore-spanning membranes were analyzed by two color confocal laser scanning microscopy in a time resolved manner. From the recorded time dependent intensities of the two fluorophors, it was possible to unambiguously distinguish between vesicle docking, hemifusion and full fusion.

Artificial SNARE analogues derived from SNARE proteins, which mediate synaptic membrane fusion, are of interest. They mimic the tetrameric α-helix bundle of the SNARE motif with various bio-oligomer recognition units. Interaction between complementary oligomers linked to the respective membrane by lipid or peptide anchors leads to proximity of vesicles and to fusion of lipid bilayers. β-Peptide nucleic acids were introduced as hybrid oligomers with the native SNARE protein transmembrane/linker sequence, in order to evaluate a fusion system that allows distance tuning of approaching membranes. Formation of a four-base pair β-PNA double strand with 20 A length is sufficient for vesicle membrane fusion. Elongation of the recognition β-PNA duplex in the linker region yielded a 40 A β-peptide duplex and provided a vesicle-vesicle distance that only supported hemifusion of vesicle membranes.

Even though a number of different in vitro fusion assays have been developed to analyze protein mediated fusion, they still only partially capture the essential features of the in vivo situation. Here we established an in vitro fusion assay that mimics the fluidity and planar geometry of the cellular plasma membrane to be able to monitor fusion of single protein-containing vesicles. As a proof of concept, planar pore-spanning membranes harboring SNARE-proteins were generated on highly ordered functionalized 1.2 μm-sized pore arrays in Si3N4. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Fusion was analyzed by two color confocal laser scanning fluorescence microscopy in a time resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. The importance of the membrane geometry on the fusion process was highlighted by comparing SNARE-mediated fusion with that of a minimal SNARE fusion mimetic.

The clustering of proteins and lipids in distinct microdomains is emerging as an important principle for the spatial patterning of biological membranes. Such domain formation can be the result of hydrophobic and ionic interactions with membrane lipids as well as of specific protein–protein interactions. Here using plasma membrane-resident SNARE proteins as model, we show that hydrophobic mismatch between the length of transmembrane domains (TMDs) and the thickness of the lipid membrane suffices to induce clustering of proteins. Even when the TMDs differ in length by only a single residue, hydrophobic mismatch can segregate structurally closely homologous membrane proteins in distinct membrane domains. Domain formation is further fine-tuned by interactions with polyanionic phosphoinositides and homo and heterotypic protein interactions. Our findings demonstrate that hydrophobic mismatch contributes to the structural organization of membranes., Clustering of proteins in the plasma membrane plays an important role in the regulation of both cellular signalling and membrane remodelling. Milovanovic et al. demonstrate that mismatch between transmembrane domain length and the lipid bilayer thickness is sufficient to drive clustering of SNARE proteins.

Contains fulltext : 117424.pdf (Publisher’s version ) (Closed access) Synaptic-vesicle exocytosis is mediated by the vesicular Ca(2+) sensor synaptotagmin-1. Synaptotagmin-1 interacts with the SNARE protein syntaxin-1A and acidic phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2). However, it is unclear how these interactions contribute to triggering membrane fusion. Using PC12 cells from Rattus norvegicus and artificial supported bilayers, we show that synaptotagmin-1 interacts with the polybasic linker region of syntaxin-1A independent of Ca(2+) through PIP2. This interaction allows both Ca(2+)-binding sites of synaptotagmin-1 to bind to phosphatidylserine in the vesicle membrane upon Ca(2+) triggering. We determined the crystal structure of the C2B domain of synaptotagmin-1 bound to phosphoserine, allowing development of a high-resolution model of synaptotagmin bridging two different membranes. Our results suggest that PIP2 clusters organized by syntaxin-1 act as molecular beacons for vesicle docking, with the subsequent Ca(2+) influx bringing the vesicle membrane close enough for membrane fusion.

The genetic structure of Drosophila subobscura from the Balkan Peninsula was studied with respect to restriction site polymorphism of mitochondrial DNA in populations from the Derventa River Gorge and Sicevo Gorge (Serbia). To investigate the role of cytonuclear interactions in shaping mitochondrial DNA variability in natural populations of this species, the study was complemented with the analysis of linkage disequilibria between mitochondrial haplotypes and chromosomal inversion arrangements. Similar to other populations of D. subobscura, two main haplotypes (I and II) were found, as well as a series of less common ones. The frequencies of haplotypes I and II accounted for 25.8% and 71.0%, respectively, in the population from the Derventa River Gorge, and for 32.4% and 58.1%, respectively, in the population from Sicevo Gorge. One of the haplotypes harbored a large insertion (2.7 kb) in the A+T rich region. The frequency distribution of both haplotypes did not depart from neutrality. Contrary to prior studies, we did not detect any significant linkage disequilibrium between the two most frequent mtDNA haplotypes and any of the chromosomal arrangements in either of the populations. We conclude that linkage disequilibrium is not a general occurrence in natural populations of D. subobscura, and we discuss how transient coadaptations, ecologically specific selective pressures, and demographics could contribute to population-specific patterns of linkage disequilibrium.

In most physiological fusion processes, a strongly to moderately curved membrane (e.g. synaptic vesicle, virion) fuses with a planar membrane (e.g. (neuronal) plasma membrane). Despite these geometric considerations of the fusing membranes in nature, most in vitro results on fusion have been obtained from bulk vesicle assays, where two curved membranes fuse with each other resulting in an increase in vesicle size, i.e. the membrane's curvature changes during the fusion process.To establish a fusion assay that resembles the in vivo situation more closely, we aimed at a planar membrane architecture that is long-term stable and provides laterally mobile membrane components. Moreover, this membrane system should allow for the detection of single vesicle fusion events, should be accessible from both sides and should provide enough space for the incoming lipid material during the fusion process. One membrane system that suffice these requirements are pore-spanning membranes.We demonstrate that planar pore-spanning membranes harboring neuronal SNARE-proteins can be successfully generated on highly ordered and functionalized micrometer-sized pore arrays in silicon nitride by fusion of giant unilamellar vesicles. Full mobility of the membrane components was demonstrated by fluorescence correlation spectroscopy. Individual fusion events of unilamellar vesicles were analyzed by confocal laser scanning fluorescence microscopy in a time-resolved manner allowing to readily distinguish between vesicle docking, intermediate states such as hemifusion and full fusion. With this assay in hand, the impact of individual components, such as the PIP2 concentration in the planar membrane, on the fusion process can be analyzed in depth.