RESUMO
During mitotic entry of vertebrate cells, nuclear pore complexes (NPCs) are rapidly disintegrated. NPC disassembly is initiated by hyperphosphorylation of linker nucleoporins (Nups), which leads to the dissociation of FG repeat Nups and relaxation of the nuclear permeability barrier. However, less is known about disintegration of the huge nuclear and cytoplasmic rings, which are formed by annular assemblies of Y-complexes that are dissociated from NPCs as intact units. Surprisingly, we observe that Y-complex Nups display slower dissociation kinetics compared with other Nups during in vitro NPC disassembly, indicating a mechanistic difference in the disintegration of Y-based rings. Intriguingly, biochemical experiments reveal that a fraction of Y-complexes remains associated with mitotic ER membranes, supporting recent microscopic observations. Visualization of mitotic Y-complexes by super-resolution microscopy demonstrates that they form two classes of higher order assemblies: large clusters at kinetochores and small, focal ER-associated assemblies. These, however, lack features qualifying them as persisting ring-shaped subassemblies previously proposed to serve as structural templates for NPC reassembly during mitotic exit, which helps to refine current models of nuclear reassembly.
Assuntos
Microscopia , Mitose , Poro Nuclear , Núcleo Celular , Complexo de Proteínas Formadoras de Poros Nucleares/genéticaRESUMO
New strategies for visualizing self-assembly processes at the nanoscale give deep insights into the molecular origins of disease. An example is the self-assembly of misfolded proteins into amyloid fibrils, which is related to a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. Here, we probe the links between the mechanism of α-synuclein (AS) aggregation and its associated toxicity by using optical nanoscopy directly in a neuronal cell culture model of Parkinson's disease. Using superresolution microscopy, we show that protein fibrils are taken up by neuronal cells and act as prion-like seeds for elongation reactions that both consume endogenous AS and suppress its de novo aggregation. When AS is internalized in its monomeric form, however, it nucleates and triggers the aggregation of endogenous AS, leading to apoptosis, although there are no detectable cross-reactions between externally added and endogenous protein species. Monomer-induced apoptosis can be reduced by pretreatment with seed fibrils, suggesting that partial consumption of the externally added or excess soluble AS can be significantly neuroprotective.
Assuntos
Amiloide/metabolismo , Apoptose/fisiologia , Neurônios/metabolismo , Agregação Patológica de Proteínas/patologia , alfa-Sinucleína/metabolismo , alfa-Sinucleína/farmacologia , Doença de Alzheimer/patologia , Células Cultivadas , Humanos , Doença de Parkinson/patologia , Transporte Proteico , Deficiências na Proteostase/patologiaRESUMO
A dual-activatable, fluorogenic probe was developed to sense esterase activity with single-molecule resolution. Without enzymatic pre-activation, the diazoindanone-based probe has an electron-poor core and, upon irradiation, undergoes Wolff rearrangement to give a ring-expanded xanthene core that is nonemissive. If the probe is pre-activated by carboxylesterases, the tricyclic core becomes electron-rich, and the photoinduced Wolff rearrangement produces a highly emissive rhodol dye. Live-cell and solution studies confirmed the selectivity of the probe and revealed that the photoactivated dye does not diffuse away from the original location of activation because the intermediate ketene forms a covalent bond with surrounding macromolecules. Single-molecule localization microscopy was used to reconstruct a super-resolved image of esterase activity. These single-molecule images of enzymatic activity changed significantly upon treatment of the cells with inhibitors of human carboxylesterase I and II, both in terms of total number of signals and intracellular distribution. This proof-of-principle study introduces a sensing mechanism for single-molecule detection of enzymatic activity that could be applied to many other biologically relevant targets.
Assuntos
Hidrolases de Éster Carboxílico/metabolismo , Corantes Fluorescentes/química , Imagem Óptica , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Estrutura MolecularRESUMO
Amyloid fibrils have been shown to have peculiar optical properties since they can exhibit fluorescence in the absence of aromatic residues. In a recent study, we have shown that proton transfer (PT) events along hydrogen bonds (HBs) are coupled to absorption in the near UV range. Here, we gain more insights into the different types of hydrogen bonding interactions that occur in our model systems and the molecular factors that control the susceptibility of the protons to undergo PT and how this couples to the optical properties. In the case of the strong N-C termini interactions, a nearby methionine residue stabilizes the non-zwitterionic NH2-COOH pair, while zwitterionic NH3+-COO- is stabilized by the proximity of nearby crystallographic water molecules. Proton motion along the hydrogen bonds in the fibril is intimately coupled to the compression of the heavier atoms, similar to what is observed in bulk water. Small changes in the compression of the hydrogen bonds in the protein can lead to significant changes in both the ground and excited state potential energy surfaces associated with PT. Finally, we also reinforce the importance of nuclear quantum fluctuations of protons in the HBs of the amyloid proteins.
Assuntos
Amiloide/química , Modelos Químicos , Fenômenos Ópticos , Amiloide/metabolismo , Cristalização , Ligação de Hidrogênio , Modelos Moleculares , PrótonsRESUMO
Protein structures which form fibrils have recently been shown to absorb light at energies in the near UV range and to exhibit a structure-specific fluorescence in the visible range even in the absence of aromatic amino acids. However, the molecular origin of this phenomenon has so far remained elusive. Here, we combine ab initio molecular dynamics simulations and fluorescence spectroscopy to demonstrate that these intrinsically fluorescent protein fibrils are permissive to proton transfer across hydrogen bonds which can lower electron excitation energies and thereby decrease the likelihood of energy dissipation associated with conventional hydrogen bonds. The importance of proton transfer on the intrinsic fluorescence observed in protein fibrils is signified by large reductions in the fluorescence intensity upon either fully protonating, or deprotonating, the fibrils at pH = 0 or 14, respectively. Thus, our results point to the existence of a structure-specific fluorophore that does not require the presence of aromatic residues or multiple bond conjugation that characterize conventional fluorescent systems. The phenomenon may have a wide range of implications in biological systems and in the design of self-assembled functional materials.
Assuntos
Amiloide/química , Proteínas/química , Amiloide/metabolismo , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Ligação de Hidrogênio , Microscopia de Força Atômica , Microscopia de Fluorescência , Simulação de Dinâmica Molecular , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas/metabolismo , Prótons , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
Understanding the formation and propagation of aggregates of the Alzheimer disease-associated Tau protein in vivo is vital for the development of therapeutics for this devastating disorder. Using our recently developed live-cell aggregation sensor in neuron-like cells, we demonstrate that different variants of exogenous monomeric Tau, namely full-length Tau (hTau40) and the Tau-derived construct K18 comprising the repeat domain, initially accumulate in endosomal compartments, where they form fibrillar seeds that subsequently induce the aggregation of endogenous Tau. Using superresolution imaging, we confirm that fibrils consisting of endogenous and exogenous Tau are released from cells and demonstrate their potential to spread Tau pathology. Our data indicate a greater pathological risk and potential toxicity than hitherto suspected for extracellular soluble Tau.
Assuntos
Endocitose , Emaranhados Neurofibrilares/metabolismo , Neurônios/metabolismo , Proteínas tau/metabolismo , Animais , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Endossomos/metabolismo , Exocitose , Espaço Extracelular/metabolismo , Humanos , Lisossomos/metabolismo , Microscopia Confocal , Microscopia Eletrônica , Modelos Biológicos , Emaranhados Neurofibrilares/ultraestrutura , Neurônios/patologia , Tauopatias/metabolismo , Vesículas Transportadoras/metabolismoRESUMO
The soluble fraction of brain samples from patients with Alzheimer's disease contains highly biologically active amyloid-ß seeds. In this study, we sought to assess the potency of soluble amyloid-ß seeds derived from the brain and cerebrospinal fluid. Soluble Alzheimer's disease brain extracts were serially diluted and then injected into the hippocampus of young, APP transgenic mice. Eight months later, seeded amyloid-ß deposition was evident even when the hippocampus received subattomole amounts of brain-derived amyloid-ß. In contrast, cerebrospinal fluid from patients with Alzheimer's disease, which contained more than 10-fold higher levels of amyloid-ß peptide than the most concentrated soluble brain extracts, did not induce detectable seeding activity in vivo. Similarly, cerebrospinal fluid from aged APP-transgenic donor mice failed to induce cerebral amyloid-ß deposition. In comparison to the soluble brain fraction, cerebrospinal fluid largely lacked N-terminally truncated amyloid-ß species and exhibited smaller amyloid-ß-positive particles, features that may contribute to the lack of in vivo seeding by cerebrospinal fluid. Interestingly, the same cerebrospinal fluid showed at least some seeding activity in an in vitro assay. The present results indicate that the biological seeding activity of soluble amyloid-ß species is orders of magnitude greater in brain extracts than in the cerebrospinal fluid.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Lobo Frontal/metabolismo , Hipocampo/metabolismo , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Peptídeos beta-Amiloides/química , Precursor de Proteína beta-Amiloide/genética , Animais , Feminino , Lobo Frontal/patologia , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fragmentos de Peptídeos/química , Distribuição Aleatória , SolubilidadeRESUMO
The self-assembly of normally soluble proteins into fibrillar amyloid structures is associated with a range of neurodegenerative disorders, such as Parkinson's and Alzheimer's diseases. In the present study, we show that specific events in the kinetics of the complex, multistep aggregation process of one such protein, α-synuclein, whose aggregation is a characteristic hallmark of Parkinson's disease, can be followed at the molecular level using optical super-resolution microscopy. We have explored in particular the elongation of preformed α-synuclein fibrils; using two-color single-molecule localization microscopy we are able to provide conclusive evidence that the elongation proceeds from both ends of the fibril seeds. Furthermore, the technique reveals a large heterogeneity in the growth rates of individual fibrils; some fibrils exhibit no detectable growth, whereas others extend to more than ten times their original length within hours. These large variations in the growth kinetics can be attributed to fibril structural polymorphism. Our technique offers new capabilities in the study of amyloid growth dynamics at the molecular level and is readily translated to the study of the self-assembly of other nanostructures.
Assuntos
Amiloide/química , Amiloide/ultraestrutura , Cristalização/métodos , Aumento da Imagem/métodos , Microscopia de Fluorescência por Excitação Multifotônica/métodos , alfa-Sinucleína/química , alfa-Sinucleína/ultraestrutura , Teste de Materiais/métodos , Conformação MolecularRESUMO
Several RNA binding proteins involved in membraneless organelles can form pathological amyloids associated with neurodegenerative diseases, but the mechanisms of how this aggregation is modulated remain elusive. Here we investigate how heterotypic protein-RNA interactions modulate the condensation and the liquid to amyloid transition of hnRNPA1A, a protein involved in amyothropic lateral sclerosis. In the absence of RNA, formation of condensates promotes hnRNPA1A aggregation and fibrils are localized at the interface of the condensates. Addition of RNA modulates the soluble to amyloid transition of hnRNPA1A according to different pathways depending on RNA/protein stoichiometry. At low RNA concentrations, RNA promotes both condensation and amyloid formation, and the catalytic effect of RNA adds to the role of the interface between the dense and dilute phases. At higher RNA concentrations, condensation is suppressed according to re-entrant phase behaviour but formation of hnRNPA1A amyloids is observed over longer incubation times. Our findings show how heterotypic nucleic acid-protein interactions affect the kinetics and molecular pathways of amyloid formation.
Assuntos
Amiloide , Ribonucleoproteína Nuclear Heterogênea A1 , RNA , Ribonucleoproteína Nuclear Heterogênea A1/química , Ribonucleoproteína Nuclear Heterogênea A1/metabolismo , Amiloide/química , Amiloide/metabolismo , RNA/química , RNA/metabolismo , Humanos , Condensados Biomoleculares/química , Condensados Biomoleculares/metabolismo , CinéticaRESUMO
Kinetic assay of seeded growth: The graph shows the variation in intrinsic fluorescence intensity of amyloid fibrils. Fluorescence increases during the seeded aggregation of α-synuclein seeds with α-synuclein monomeric protein (blue curve) but not when α-synuclein seeds are incubated with ß-synuclein monomeric protein (black curve), thus showing that no seeded growth occurred in this case.
Assuntos
Peptídeos beta-Amiloides/análise , Fluorescência , Fragmentos de Peptídeos/análise , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Benzotiazóis , Humanos , Cinética , Tamanho da Partícula , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Tiazóis/químicaRESUMO
Proteins in solution tend to coat solid surfaces upon exposure. Depending on the nature of the surface, the environmental conditions, and the nature of the protein these adsorbed proteins may self-assemble into ordered, fibre-like structures called amyloids. Nanoparticulate surfaces, with their high surface to volume ratio, are particularly favourable to amyloid formation. Most prior research has focussed on either inorganic or organic nanoparticles in solution. In this research, we instead focus on aerogels created from TEMPO-oxidized cellulose nanofibers (TO-CNF) to serve as bio-based, three-dimensional amyloid templates with a tuneable surface chemistry. Previous research on the use of cellulose as a protein adsorption template has shown no evidence of a change in the secondary protein structure. Herein, however, with the aid of the reducing agent TCEP, we were able to induce the formation of amyloid-like 'worms' on the surface of TO-CNF aerogels. Furthermore, we demonstrate that the addition of the TO-CNF aerogel can also induce bulk aggregation under conditions where it previously did not exist. Finally, we show that the addition of the aerogel increases the rate of 'worm' formation in conditions where previous research has found a long lag-phase. Therefore, TO-CNF aerogels are shown to be excellent templates for inducing ordered protein aggregation.
Assuntos
Nanofibras , Géis/química , Nanofibras/química , Celulose/química , Proteínas Amiloidogênicas , AdsorçãoRESUMO
The maturation of liquid-like protein condensates into amyloid fibrils has been associated with several neurodegenerative diseases. However, the molecular mechanisms underlying this liquid-to-solid transition have remained largely unclear. Here we analyse the amyloid formation mediated by condensation of the low-complexity domain of hnRNPA1, a protein involved in amyotrophic lateral sclerosis. We show that phase separation and fibrillization are connected but distinct processes that are modulated by different regions of the protein sequence. By monitoring the spatial and temporal evolution of amyloid formation we demonstrate that the formation of fibrils does not occur homogeneously inside the droplets but is promoted at the interface of the condensates. We further show that coating the interface of the droplets with surfactant molecules inhibits fibril formation. Our results reveal that the interface of biomolecular condensates of hnRNPA1 promotes fibril formation, therefore suggesting interfaces as a potential novel therapeutic target against the formation of aberrant amyloids mediated by condensation.
RESUMO
5 nanometer sized detonation nanodiamonds (DNDs) are studied as potential single-particle labels for distance measurements in biomolecules. Nitrogen-vacancy (NV) defects in the crystal lattice can be addressed through their fluorescence and optically-detected magnetic resonance (ODMR) of a single particle can be recorded. To achieve single-particle distance measurements, we propose two complementary approaches based on spin-spin coupling or optical super-resolution imaging. As a first approach, we try to measure the mutual magnetic dipole-dipole coupling between two NV centers in close DNDs using a pulse ODMR sequence (DEER). The electron spin coherence time, a key parameter to reach long distance DEER measurements, was prolonged using dynamical decoupling reaching T 2,DD ≈ 20 µs, extending the Hahn echo decay time T 2 by one order of magnitude. Nevertheless, an inter-particle NV-NV dipole coupling could not be measured. As a second approach, we successfully localize the NV centers in DNDs using STORM super-resolution imaging, achieving a localization precision of down to 15 nm, enabling optical nanometer-scale single-particle distance measurements.
RESUMO
Monomeric alpha-synuclein (aSyn) is a well characterised protein that importantly binds to lipids. aSyn monomers assemble into amyloid fibrils which are localised to lipids and organelles in insoluble structures found in Parkinson's disease patient's brains. Previous work to address pathological aSyn-lipid interactions has focused on using synthetic lipid membranes, which lack the complexity of physiological lipid membranes. Here, we use physiological membranes in the form of synaptic vesicles (SV) isolated from rodent brain to demonstrate that lipid-associated aSyn fibrils are more easily taken up into iPSC-derived cortical i3Neurons. Lipid-associated aSyn fibril characterisation reveals that SV lipids are an integrated part of the fibrils and while their fibril morphology differs from aSyn fibrils alone, the core fibril structure remains the same, suggesting the lipids lead to the increase in fibril uptake. Furthermore, SV enhance the aggregation rate of aSyn, yet increasing the SV:aSyn ratio causes a reduction in aggregation propensity. We finally show that aSyn fibrils disintegrate SV, whereas aSyn monomers cause clustering of SV using small angle neutron scattering and high-resolution imaging. Disease burden on neurons may be impacted by an increased uptake of lipid-associated aSyn which could enhance stress and pathology, which in turn may have fatal consequences for neurons.
Assuntos
Células-Tronco Pluripotentes Induzidas , alfa-Sinucleína , Animais , alfa-Sinucleína/metabolismo , Vesículas Sinápticas/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Roedores/metabolismo , LipídeosRESUMO
Photoactivatable dyes enable single-molecule imaging and tracking in biology. Despite progress in the development of new fluorophores and labeling strategies, many intracellular compartments remain difficult to image beyond the limit of diffraction in living cells. For example, lipid domains, e.g., membranes and droplets, remain difficult to image with nanometric resolution. To visualize these challenging subcellular targets, it is necessary to develop new fluorescent molecular devices beyond simple on/off switches. Here, we report a fluorogenic molecular logic gate that can be used to image single molecules associated with lipid domains, most notably droplets, with excellent specificity. This probe requires the subsequent action of light, a lipophilic environment, and a competent nucleophile to produce a fluorescent product. The combination of these inputs results in a probe that can be used to image the boundary of lipid droplets in three dimensions with resolution beyond the limit of diffraction. Moreover, this probe enables single-molecule tracking of lipid trafficking between droplets and the endoplasmic reticulum.
Assuntos
Corantes Fluorescentes/química , Gotículas Lipídicas/metabolismo , Lipídeos de Membrana/metabolismo , Corantes Fluorescentes/efeitos da radiação , Células HeLa , Humanos , Indanos/química , Indanos/efeitos da radiação , Luz , Gotículas Lipídicas/química , Lógica , Microdomínios da Membrana , Microscopia Confocal , Microscopia de Fluorescência , Compostos de Organossilício/química , Compostos de Organossilício/efeitos da radiação , Rodaminas/química , Rodaminas/efeitos da radiação , Imagem Individual de Molécula/métodosRESUMO
A number of new Correlative Light and Electron Microscopy approaches have been developed over the past years, offering the opportunity to combine the specificity and bio-compatibility of light microscopy with the high resolution achieved in electron microscopy. More recently, these approaches have taken one step further and also super-resolution light microscopy was combined with transmission or scanning electron microscopy. This combination usually requires moving the specimen between different imaging systems, an expensive set-up and relatively complicated imaging workflows. Here we present a way to overcome these difficulties by exploiting a commercially available wide-field fluorescence microscope integrated in the specimen chamber of a Scanning Electron Microscope (SEM) to perform correlative LM/EM studies. Super-resolution light microscopy was achieved by using a recently developed algorithm - the Super-Resolution Radial Fluctuations (SRRF) - to improve the resolution of diffraction limited fluorescent images. With this combination of hardware/software it is possible to obtain correlative super-resolution light and scanning electron microscopy images in an easy and fast way. The imaging workflow is described and demonstrated on fluorescently labelled amyloid fibrils, fibrillar protein aggregates linked to the onset of multiple neurodegenerative diseases, revealing information about their polymorphism.
RESUMO
Photoswitchable molecules have multiple applications in the physical and life sciences because their properties can be modulated with light. Fluxional molecules, which undergo rapid degenerate rearrangements in the electronic ground state, also exhibit switching behavior. The stochastic nature of fluxional switching, however, has hampered its application in the development of functional molecules and materials. Here we combine photoswitching and fluxionality to develop a fluorophore that enables very long (>30 min) time-lapse single-molecule localization microscopy in living cells with minimal phototoxicity and no apparent photobleaching. These long time-lapse experiments allow us to track intracellular organelles with unprecedented spatiotemporal resolution, revealing new information of the three-dimensional compartmentalization of synaptic vesicle trafficking in live human neurons.
Assuntos
Corantes Fluorescentes/química , Microscopia Intravital/métodos , Sondas Moleculares/química , Neurônios/química , Vesículas Sinápticas/química , Corantes Fluorescentes/efeitos da radiação , Células HeLa , Humanos , Isomerismo , Luz , Microscopia de Fluorescência/métodos , Sondas Moleculares/efeitos da radiação , Neurônios/citologia , Neurônios/metabolismo , Fotodegradação , Imagem Individual de Molécula/métodos , Espectrometria de Fluorescência , Vesículas Sinápticas/metabolismo , Imagem com Lapso de Tempo/métodosRESUMO
Molecular self-assembly of short peptide building blocks leads to the formation of various material architectures that may possess unique physical properties. Recent studies had confirmed the key role of biaromaticity in peptide self-assembly, with the diphenylalanine (FF) structural family as an archetypal model. Another significant direction in the molecular engineering of peptide building blocks is the use of fluorenylmethoxycarbonyl (Fmoc) modification, which promotes the assembly process and may result in nanostructures with distinctive features and macroscopic hydrogel with supramolecular features and nanoscale order. Here, we explored the self-assembly of the protected, noncoded fluorenylmethoxycarbonyl-ß,ß-diphenyl-Ala-OH (Fmoc-Dip) amino acid. This process results in the formation of elongated needle-like crystals with notable aromatic continuity. By altering the assembly conditions, arrays of spherical particles were formed that exhibit strong light scattering. These arrays display vivid coloration, strongly resembling the appearance of opal gemstones. However, unlike the Rayleigh scattering effect produced by the arrangement of opal, the described optical phenomenon is attributed to Mie scattering. Moreover, by controlling the solution evaporation rate, i.e., the assembly kinetics, we were able to manipulate the resulting coloration. This work demonstrates a bottom-up approach, utilizing self-assembly of a protected amino acid minimal building block, to create arrays of organic, light-scattering colorful surfaces.
RESUMO
Native silk fibroin (NSF) is a unique biomaterial with extraordinary mechanical and biochemical properties. These key characteristics are directly associated with the physical transformation of unstructured, soluble NSF into highly organized nano- and microscale fibrils rich in ß-sheet content. Here, it is shown that this NSF fibrillation process is accompanied by the development of intrinsic fluorescence in the visible range, upon near-UV excitation, a phenomenon that has not been investigated in detail to date. Here, the optical and fluorescence characteristics of NSF fibrils are probed and a route for potential applications in the field of self-assembled optically active biomaterials and systems is explored. In particular, it is demonstrated that NSF can be structured into autofluorescent microcapsules with a controllable level of ß-sheet content and fluorescence properties. Furthermore, a facile and efficient fabrication route that permits arbitrary patterns of NSF microcapsules to be deposited on substrates under ambient conditions is shown. The resulting fluorescent NSF patterns display a high level of photostability. These results demonstrate the potential of using native silk as a new class of biocompatible photonic material.
Assuntos
Materiais Biocompatíveis/química , Fibroínas/química , Seda/química , Animais , Bombyx/química , Cápsulas/química , Fluorescência , Conformação Proteica em Folha betaRESUMO
Alpha-synuclein is known to bind to small unilamellar vesicles (SUVs) via its N terminus, which forms an amphipathic alpha-helix upon membrane interaction. Here we show that calcium binds to the C terminus of alpha-synuclein, therewith increasing its lipid-binding capacity. Using CEST-NMR, we reveal that alpha-synuclein interacts with isolated synaptic vesicles with two regions, the N terminus, already known from studies on SUVs, and additionally via its C terminus, which is regulated by the binding of calcium. Indeed, dSTORM on synaptosomes shows that calcium mediates the localization of alpha-synuclein at the pre-synaptic terminal, and an imbalance in calcium or alpha-synuclein can cause synaptic vesicle clustering, as seen ex vivo and in vitro. This study provides a new view on the binding of alpha-synuclein to synaptic vesicles, which might also affect our understanding of synucleinopathies.