Dipping contacts - a novel type of contact site at the interface between membraneless organelles and membranes.

Liquid-liquid phase separation is a major mechanism for organizing macromolecules, particularly proteins with intrinsically disordered regions, in compartments not limited by a membrane or a scaffold. The cell can therefore be perceived as a complex emulsion containing many of these membraneless organelles, also referred to as biomolecular condensates, together with numerous membrane-bound organelles. It is currently unclear how such a complex concoction operates to allow for intracellular trafficking, signaling and metabolic processes to occur with high spatiotemporal precision. Based on experimental observations of synaptic vesicle condensates - a membraneless organelle that is in fact packed with membranes - we present here the framework of dipping contacts: a novel type of contact site between membraneless organelles and membranes. In this Hypothesis, we propose that our framework of dipping contacts can serve as a foundation to investigate the interface that couples the diffusion and material properties of condensates to biochemical processes occurring in membranes. The identity and regulation of this interface is especially critical in the case of neurodegenerative diseases, where aberrant inclusions of misfolded proteins and damaged organelles underlie cellular pathology.

The synaptic vesicle cluster as a controller of pre- and postsynaptic structure and function.

The synaptic vesicle cluster (SVC) is an essential component of chemical synapses, which provides neurotransmitter-loaded vesicles during synaptic activity, at the same time as also controlling the local concentrations of numerous exo- and endocytosis cofactors. In addition, the SVC hosts molecules that participate in other aspects of synaptic function, from cytoskeletal components to adhesion proteins, and affects the location and function of organelles such as mitochondria and the endoplasmic reticulum. We argue here that these features extend the functional involvement of the SVC in synapse formation, signalling and plasticity, as well as synapse stabilization and metabolism. We also propose that changes in the size of the SVC coalesce with changes in the postsynaptic compartment, supporting the interplay between pre- and postsynaptic dynamics. Thereby, the SVC could be seen as an 'all-in-one' regulator of synaptic structure and function, which should be investigated in more detail, to reveal molecular mechanisms that control synaptic function and heterogeneity.

Vesicle condensation induced by synapsin: condensate size, geometry, and vesicle shape deformations.

We study the formation of vesicle condensates induced by the protein synapsin, as a cell-free model system mimicking vesicle pool formation in the synapse. The system can be considered as an example of liquid-liquid phase separation (LLPS) in biomolecular fluids, where one phase is a complex fluid itself consisting of vesicles and a protein network. We address the pertinent question why the LLPS is self-limiting and stops at a certain size, i.e., why macroscopic phase separation is prevented. Using fluorescence light microscopy, we observe different morphologies of the condensates (aggregates) depending on the protein-to-lipid ratio. Cryogenic electron microscopy then allows us to resolve individual vesicle positions and shapes in a condensate and notably the size and geometry of adhesion zones between vesicles. We hypothesize that the membrane tension induced by already formed adhesion zones then in turn limits the capability of vesicles to bind additional vesicles, resulting in a finite condensate size. In a simple numerical toy model we show that this effect can be accounted for by redistribution of effective binding particles on the vesicle surface, accounting for the synapsin-induced adhesion zone.

Electric Potential at the Interface of Membraneless Organelles Gauged by Graphene.

Eukaryotic cells contain membrane-bound and membrane-less organelles that are often in contact with each other. How the interface properties of membrane-less organelles regulate their interactions with membranes remains challenging to assess. Here, we employ graphene-based sensors to investigate the electrostatic properties of synapsin 1, a major synaptic phosphoprotein, either in a single phase or after undergoing phase separation to form synapsin condensates. Using these graphene-based sensors, we discover that synapsin condensates generate strong electrical responses that are otherwise absent when synapsin is present as a single phase. By introducing atomically thin dielectric barriers, we show that the electrical response originates in an electric double layer whose formation governs the interaction between synapsin condensates and graphene. Our data indicate that the interface properties of the same protein are substantially different when the protein is in a single phase versus within a biomolecular condensate, unraveling that condensates can harbor ion potential differences at their interface.

Lipid vesicle pools studied by passive X-ray microrheology.

Vesicle pools can form by attractive interaction in a solution, mediated by proteins or divalent ions such as calcium. The pools, which are alternatively also denoted as vesicle clusters, form by liquid-liquid phase separation (LLPS) from an initially homogeneous solution. Due to the short range liquid-like order of vesicles in the pool or cluster, the vesicle-rich phase can also be regarded as a condensate, and one would like to better understand not only the structure of these systems, but also their dynamics. The diffusion of vesicles, in particular, is expected to change when vesicles are arrested in a pool. Here we investigate whether passive microrheology based on X-ray photon correlation spectroscopy (XPCS) is a suitable tool to study model systems of artificial lipid vesicles exhibiting LLPS, and more generally also other heterogeneous biomolecular fluids. We show that by adding highly scattering tracer particles to the solution, valuable information on the single vesicle as well as collective dynamics can be inferred. While the correlation functions reveal freely diffusing tracer particles in solutions at low CaCl[Formula: see text] concentrations, the relaxation rate [Formula: see text] shows a nonlinear dependence on [Formula: see text] at a higher concentration of around 8 mM CaCl[Formula: see text], characterised by two linear regimes with a broad cross-over. We explain this finding based on arrested diffusion in percolating vesicle clusters.

Spermidine reduces neuroinflammation and soluble amyloid beta in an Alzheimer's disease mouse model.

BACKGROUND: Deposition of amyloid beta (Aß) and hyperphosphorylated tau along with glial cell-mediated neuroinflammation are prominent pathogenic hallmarks of Alzheimer's disease (AD). In recent years, impairment of autophagy has been identified as another important feature contributing to AD progression. Therefore, the potential of the autophagy activator spermidine, a small body-endogenous polyamine often used as dietary supplement, was assessed on Aß pathology and glial cell-mediated neuroinflammation. RESULTS: Oral treatment of the amyloid prone AD-like APPPS1 mice with spermidine reduced neurotoxic soluble Aß and decreased AD-associated neuroinflammation. Mechanistically, single nuclei sequencing revealed AD-associated microglia to be the main target of spermidine. This microglia population was characterized by increased AXL levels and expression of genes implicated in cell migration and phagocytosis. A subsequent proteome analysis of isolated microglia confirmed the anti-inflammatory and cytoskeletal effects of spermidine in APPPS1 mice. In primary microglia and astrocytes, spermidine-induced autophagy subsequently affected TLR3- and TLR4-mediated inflammatory processes, phagocytosis of Aß and motility. Interestingly, spermidine regulated the neuroinflammatory response of microglia beyond transcriptional control by interfering with the assembly of the inflammasome. CONCLUSIONS: Our data highlight that the autophagy activator spermidine holds the potential to enhance Aß degradation and to counteract glia-mediated neuroinflammation in AD pathology.

Calcium Promotes the Formation of Syntaxin 1 Mesoscale Domains through Phosphatidylinositol 4,5-Bisphosphate.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) 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)P2and 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 Ca(2+)acts as a charge bridge that specifically and reversibly connects multiple syntaxin 1/PI(4,5)P2complexes into larger mesoscale domains. This transient reorganization of the plasma membrane by physiological Ca(2+)concentrations is likely to be important for Ca(2+)-regulated secretion.

Distance Regulated Vesicle Fusion and Docking Mediated by ß-Peptide Nucleic Acid SNARE Protein Analogues.

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. ß-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 ß-PNA double strand with 20 Šlength is sufficient for vesicle membrane fusion. Elongation of the recognition ß-PNA duplex in the linker region yielded a 40 Šß-peptide duplex and provided a vesicle-vesicle distance that only supported hemifusion of vesicle membranes.

Aberrant tau condensates as catalytic microcompartments propel tau fibrillation.

In this issue of Structure, Soeda et al.1 employed optogenetic tools and demonstrate that an N-terminal truncation of tau and microtubule-binding deficiency lead to the formation of tau condensates, accelerating its fibrillation.

Synaptotagmin-1 in phase: Condensate biology reveals new insights into the synaptic calcium sensor.

Two recent papers by Mehta et al. and Zhu et al. in this issue (https://doi.org/10.1083/jcb.202311191) discover that synaptotagmin-1, the primary calcium sensor at the synapse, forms biomolecular condensates, identifying a new layer of regulation in calcium-triggered synaptic vesicle exocytosis.

Defining a Lewy Body: Running Up the Hill of Shifting Definitions and Evolving Concepts.

Lewy bodies (LBs) are pathological hallmarks of Parkinson's disease and dementia with Lewy bodies, characterized by the accumulation of α-synuclein (αSyn) protein in the brain. While LBs were first described a century ago, their formation and morphogenesis mechanisms remain incompletely understood. Here, we present a historical overview of LB definitions and highlight the importance of semantic clarity and precise definitions when describing brain inclusions. Recent breakthroughs in imaging revealed shared features within LB subsets and the enrichment of membrane-bound organelles in these structures, challenging the conventional LB formation model. We discuss the involvement of emerging concepts of liquid-liquid phase separation, where biomolecules demix from a solution to form dense condensates, as a potential LB formation mechanism. Finally, we emphasize the need for the operational definitions of LBs based on morphological characteristics and detection protocols, particularly in studies investigating LB formation mechanisms. A better understanding of LB organization and ultrastructure can contribute to the development of targeted therapeutic strategies for synucleinopathies.

Tear down this wall: phosphorylation regulates the internal interfaces of postsynaptic condensates.

Can the fusion/fission of biomolecular condensates be regulated in cells? In a recent study, Wu et al. show that phosphorylation of a key scaffold protein that drives condensates in postsynaptic densities modulates the apparent miscibility of underlying components, thus enabling intracondensate demixing-to-mixing transitions.

The Lack of Synapsin Alters Presynaptic Plasticity at Hippocampal Mossy Fibers in Male Mice.

Synapsins are highly abundant presynaptic proteins that play a crucial role in neurotransmission and plasticity via the clustering of synaptic vesicles. The synapsin III isoform is usually downregulated after development, but in hippocampal mossy fiber boutons, it persists in adulthood. Mossy fiber boutons express presynaptic forms of short- and long-term plasticity, which are thought to underlie different forms of learning. Previous research on synapsins at this synapse focused on synapsin isoforms I and II. Thus, a complete picture regarding the role of synapsins in mossy fiber plasticity is still missing. Here, we investigated presynaptic plasticity at hippocampal mossy fiber boutons by combining electrophysiological field recordings and transmission electron microscopy in a mouse model lacking all synapsin isoforms. We found decreased short-term plasticity, i.e., decreased facilitation and post-tetanic potentiation, but increased long-term potentiation in male synapsin triple knock-out (KO) mice. At the ultrastructural level, we observed more dispersed vesicles and a higher density of active zones in mossy fiber boutons from KO animals. Our results indicate that all synapsin isoforms are required for fine regulation of short- and long-term presynaptic plasticity at the mossy fiber synapse.

Live-Cell Quantification Reveals Viscoelastic Regulation of Synapsin Condensates by α-Synuclein.

Synapsin and α-synuclein represent a growing list of condensate-forming proteins where the material states of condensates are directly linked to cellular functions (e.g., neurotransmission) and pathology (e.g., neurodegeneration). However, quantifying condensate material properties in living systems has been a significant challenge. To address this, we develop MAPAC (micropipette aspiration and whole-cell patch clamp), a platform that allows direct material quantification of condensates in live cells. We find 10,000-fold variations in the viscoelasticity of synapsin condensates, regulated by the partitioning of α-synuclein, a marker for synucleinopathies. Through in vitro reconstitutions, we identify 4 molecular factors that distinctly regulate the viscosity and interfacial tension of synapsin condensates, verifying the cellular effects of α-synuclein. Overall, our study provides unprecedented quantitative insights into the material properties of neuronal condensates and reveals a crucial role of α-synuclein in regulating condensate viscoelasticity. Furthermore, we envision MAPAC applicable to study a broad range of condensates in vivo. .

Excess phosphoserine-129 α-synuclein induces synaptic vesicle trafficking and declustering defects at a vertebrate synapse.

α-Synuclein is a presynaptic protein that regulates synaptic vesicle (SV) trafficking. In Parkinson's disease (PD) and dementia with Lewy bodies (DLB), α-synuclein aberrantly accumulates throughout neurons, including at synapses. During neuronal activity, α-synuclein is reversibly phosphorylated at serine 129 (pS129). While pS129 comprises ∼4% of total α-synuclein under physiological conditions, it dramatically increases in PD and DLB brains. The impacts of excess pS129 on synaptic function are currently unknown. We show here that compared with wild-type (WT) α-synuclein, pS129 exhibits increased binding and oligomerization on synaptic membranes and enhanced vesicle "microclustering" in vitro. Moreover, when acutely injected into lamprey reticulospinal axons, excess pS129 α-synuclein robustly localized to synapses and disrupted SV trafficking in an activity-dependent manner, as assessed by ultrastructural analysis. Specifically, pS129 caused a declustering and dispersion of SVs away from the synaptic vicinity, leading to a significant loss of total synaptic membrane. Live imaging further revealed altered SV cycling, as well as microclusters of recently endocytosed SVs moving away from synapses. Thus, excess pS129 caused an activity-dependent inhibition of SV trafficking via altered vesicle clustering/reclustering. This work suggests that accumulation of pS129 at synapses in diseases like PD and DLB could have profound effects on SV dynamics.

Synapsin condensation is governed by sequence-encoded molecular grammars.

Multiple biomolecular condensates coexist at the pre- and post- synapse to enable vesicle dynamics and controlled neurotransmitter release in the brain. In pre-synapses, intrinsically disordered regions (IDRs) of synaptic proteins are drivers of condensation that enable clustering of synaptic vesicles (SVs). Using computational analysis, we show that the IDRs of SV proteins feature evolutionarily conserved non-random compositional biases and sequence patterns. Synapsin-1 is essential for condensation of SVs, and its C-terminal IDR has been shown to be a key driver of condensation. Focusing on this IDR, we dissected the contributions of two conserved features namely the segregation of polar and proline residues along the linear sequence, and the compositional preference for arginine over lysine. Scrambling the blocks of polar and proline residues weakens the driving forces for forming micron-scale condensates. However, the extent of clustering in subsaturated solutions remains equivalent to that of the wild-type synapsin-1. In contrast, substituting arginine with lysine significantly weakens both the driving forces for condensation and the extent of clustering in subsaturated solutions. Co-expression of the scrambled variant of synapsin-1 with synaptophysin results in a gain-of-function phenotype in cells, whereas arginine to lysine substitutions eliminate condensation. We report an emergent consequence of synapsin-1 condensation, which is the generation of interphase pH gradients realized via differential partitioning of protons between coexisting phases. This pH gradient is likely to be directly relevant for vesicular ATPase functions and the loading of neurotransmitters. Our study highlights how conserved IDR grammars serve as drivers of synapsin-1 condensation.

Actin-nucleation promoting factor N-WASP influences alpha-synuclein condensates and pathology.

Abnormal intraneuronal accumulation of soluble and insoluble α-synuclein (α-Syn) is one of the main pathological hallmarks of synucleinopathies, such as Parkinson's disease (PD). It has been well documented that the reversible liquid-liquid phase separation of α-Syn can modulate synaptic vesicle condensates at the presynaptic terminals. However, α-Syn can also form liquid-like droplets that may convert into amyloid-enriched hydrogels or fibrillar polymorphs under stressful conditions. To advance our understanding on the mechanisms underlying α-Syn phase transition, we employed a series of unbiased proteomic analyses and found that actin and actin regulators are part of the α-Syn interactome. We focused on Neural Wiskott-Aldrich syndrome protein (N-WASP) because of its association with a rare early-onset familial form of PD. In cultured cells, we demonstrate that N-WASP undergoes phase separation and can be recruited to synapsin 1 liquid-like droplets, whereas it is excluded from α-Syn/synapsin 1 condensates. Consistently, we provide evidence that wsp-1/WASL loss of function alters the number and dynamics of α-Syn inclusions in the nematode Caenorhabditis elegans. Together, our findings indicate that N-WASP expression may create permissive conditions that promote α-Syn condensates and their potentially deleterious conversion into toxic species.

Condensate biology of synaptic vesicle clusters.

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.

Intersectin and Endophilin condensates prime synaptic vesicles for release site replenishment.

Neurotransmitter is released from dedicated sites of synaptic vesicle fusion within a synapse. Following fusion, the vacated sites are replenished immediately by new vesicles for subsequent neurotransmission. These replacement vesicles are assumed to be located near release sites and used by chance. Here, we find that replacement vesicles are clustered around this region by Intersectin-1. Specifically, Intersectin-1 forms dynamic molecular condensates with Endophilin A1 near release sites and sequesters vesicles around this region. In the absence of Intersectin-1, vesicles within 20 nm of the plasma membrane are reduced, and consequently, vacated sites cannot be replenished rapidly, leading to depression of synaptic transmission. Similarly, mutations in Intersectin-1 that disrupt Endophilin A1 binding result in similar phenotypes. However, in the absence of Endophilin, this replacement pool of vesicles is available but cannot be accessed, suggesting that Endophilin A1 is needed to mobilize these vesicles. Thus, our work describes a distinct physical region within a synapse where replacement vesicles are harbored for release site replenishment.