SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs

During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane–bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events

Conformational Basis for Asymmetric Seeding Barrier in Filaments of Three- and Four-Repeat Tau

*S Supporting Information ABSTRACT: Tau pathology in Alzheimer’s disease is intimately linked to the deposition of proteinacious filaments, which akin to infectious prions, have been proposed to spread via seeded conversion. Here we use double electron−electron resonance (DEER) spectroscopy in combination with extensive computational analysis to show that filaments of three- (3R) and four-repeat (4R) tau are conformationally distinct. Distance measurements between spin labels in the third repeat, reveal tau amyloid filaments as ensembles of known β-strand−turn−β-strand U-turn motifs. Whereas filaments seeded with 3R tau are structurally homogeneous, filaments seeded with 4R tau are heterogeneous, composed of at least three distinct conformers. These findings establish a molecular basis for the seeding barrier between different tau isoforms and offer a new powerful approach for investigating the composition and dynamics of amyloid fibril ensembles

Structural Heterogeneity and Quantitative FRET Efficiency Distributions of Polyprolines through a Hybrid Atomistic Simulation and Monte Carlo Approach

Förster Resonance Energy Transfer (FRET) experiments probe molecular distances via distance dependent energy transfer from an excited donor dye to an acceptor dye. Single molecule experiments not only probe average distances, but also distance distributions or even fluctuations, and thus provide a powerful tool to study biomolecular structure and dynamics. However, the measured energy transfer efficiency depends not only on the distance between the dyes, but also on their mutual orientation, which is typically inaccessible to experiments. Thus, assumptions on the orientation distributions and averages are usually made, limiting the accuracy of the distance distributions extracted from FRET experiments. Here, we demonstrate that by combining single molecule FRET experiments with the mutual dye orientation statistics obtained from Molecular Dynamics (MD) simulations, improved estimates of distances and distributions are obtained. From the simulated time-dependent mutual orientations, FRET efficiencies are calculated and the full statistics of individual photon absorption, energy transfer, and photon emission events is obtained from subsequent Monte Carlo (MC) simulations of the FRET kinetics. All recorded emission events are collected to bursts from which efficiency distributions are calculated in close resemblance to the actual FRET experiment, taking shot noise fully into account. Using polyproline chains with attached Alexa 488 and Alexa 594 dyes as a test system, we demonstrate the feasibility of this approach by direct comparison to experimental data. We identified cis-isomers and different static local environments as sources of the experimentally observed heterogeneity. Reconstructions of distance distributions from experimental data at different levels of theory demonstrate how the respective underlying assumptions and approximations affect the obtained accuracy. Our results show that dye fluctuations obtained from MD simulations, combined with MC single photon kinetics, provide a versatile tool to improve the accuracy of distance distributions that can be extracted from measured single molecule FRET efficiencies

Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)

In 2008 we published the first set of guidelines for standardizing research in autophagy. Since then, research on this topic has continued to accelerate, and many new scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Accordingly, it is important to update these guidelines for monitoring autophagy in different organisms. Various reviews have described the range of assays that have been used for this purpose. Nevertheless, there continues to be confusion regarding acceptable methods to measure autophagy, especially in multicellular eukaryotes. For example, a key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers or volume of autophagic elements (e.g., autophagosomes or autolysosomes) at any stage of the autophagic process versus those that measure fl ux through the autophagy pathway (i.e., the complete process including the amount and rate of cargo sequestered and degraded). In particular, a block in macroautophagy that results in autophagosome accumulation must be differentiated from stimuli that increase autophagic activity, defi ned as increased autophagy induction coupled with increased delivery to, and degradation within, lysosomes (inmost higher eukaryotes and some protists such as Dictyostelium ) or the vacuole (in plants and fungi). In other words, it is especially important that investigators new to the fi eld understand that the appearance of more autophagosomes does not necessarily equate with more autophagy. In fact, in many cases, autophagosomes accumulate because of a block in trafficking to lysosomes without a concomitant change in autophagosome biogenesis, whereas an increase in autolysosomes may reflect a reduction in degradative activity. It is worth emphasizing here that lysosomal digestion is a stage of autophagy and evaluating its competence is a crucial part of the evaluation of autophagic flux, or complete autophagy. Here, we present a set of guidelines for the selection and interpretation of methods for use by investigators who aim to examine macroautophagy and related processes, as well as for reviewers who need to provide realistic and reasonable critiques of papers that are focused on these processes. These guidelines are not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to monitor autophagy. Along these lines, because of the potential for pleiotropic effects due to blocking autophagy through genetic manipulation it is imperative to delete or knock down more than one autophagy-related gene. In addition, some individual Atg proteins, or groups of proteins, are involved in other cellular pathways so not all Atg proteins can be used as a specific marker for an autophagic process. In these guidelines, we consider these various methods of assessing autophagy and what information can, or cannot, be obtained from them. Finally, by discussing the merits and limits of particular autophagy assays, we hope to encourage technical innovation in the field

Driving Tau into Phase-separated Liquid Droplets

Liquid–liquid phase separation of tau protein has been implicated in normal biological function as well as neurodegenerative diseases, including Alzheimer\u27s. However, knowledge about these links is still scant, and the mechanisms driving tau into liquid droplets are poorly understood. A simplified in vitro system that uses unmodified human tau protein now suggests electrostatic interactions provide the basic instructions underlying liquid droplet formation

Abbruch und Wiederaufnahme der diplomatischen Beziehungen 1965 bis 1972

Title and Contents 1 Introduction 1 2 Materials and Methods 15 3 Results 27 4 Discussion 65 5 Summary 79 6 Zusammenfassung 81 7 References 83 8 Appendix 96SNARE proteins are thought to be key mediators of all intracellular fusion reactions ranging from yeast to man. The assembly of ternary SNARE complexes between the synaptic vesicle-protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP-25 initiates membrane fusion at the nerve terminal. The interaction between SNARE proteins is mediated by a stretch of approximately 60 amino acids referred to as the SNARE motif. The crystal structure of the synaptic SNARE complex revealed an extended four helix bundle with all helices aligned in parallel (two from SNAP-25, one from syntaxin, and one from synaptobrevin). Syntaxin contains an additional independently folded domain known as the Habc domain. An intramolecular interaction between this domain and the SNARE motif arrests syntaxin in a closed conformation. Only when there is no interaction between the two domains (open conformation) does the SNARE complex assemble. In this study, the conformational switching of syntaxin and the structures of SNARE intermediates and fully assembled SNARE complexes are investigated. Fluorescence anisotropy measurements reveal that the Habc domain slows down SNARE complex formation by a factor of 5-7, implying a weak interaction between the Habc domain and the SNARE motif (ΔG ≈1 kcal/mol) . The intramolecular distances between the two domains were measured by single molecule fluorescence resonance energy transfer. Only 15-30% of all syntaxin molecules are in a closed conformation. Conformational fluctuations between the open and closed states occur in a time range of ~0.7 ms. In order to arrest syntaxin in a closed conformation an additional protein (munc-18) is required. Binary complexes between syntaxin and SNAP-25 (2:1 stoichiometry) are potential intermediates in SNARE complex assembly. Electron paramagnetic resonance data indicate that the binary complex is composed of a bundle of four Α-helices with the syntaxin helices being in register. Furthermore, binary complexes are loosely structured at the very N-and C-terminal ends of syntaxin and the C-terminal ends of both SNAP-25 helices. Binding of synaptobrevin displaces one of the syntaxin molecules and thus completes SNARE complex formation. Assembly of binary and ternary complexes from individual SNAREs is accompanied by large structural changes. Isolated synaptobrevin and SNAP-25 are unstructured. Syntaxin, too, is unstructured but oligomerizes at higher µM concentrations. Oligomerization is mediated by the SNARE motif of syntaxin and may compete with intramolecular binding of the Habc domain. To find out whether helices in the SNARE complex require juxtaposing helices, a C-terminally truncated synaptobrevin was used. Helices remain completely intact upstream from the truncated site; helices downstream from the truncated site collapse. Interacting layers in the four-helix bundle of SNARE complexes therefore appear to be independent from layers in nearby positions. This agrees with the proposed "zippering mechanism" of complex formation and provides a structural basis according to which partially assembled complexes form intermediates in the progression toward membrane fusion. To test whether SNARE proteins interact via their transmembrane regions syntaxin and synaptobrevin were studied in proteoliposomes. Syntaxin formed specific dimers that might be precursors in binary complex formation. In addition a new interaction between the transmembrane domains of syntaxin and synaptobrevin was observed which might be important for a late stage in membrane fusion. An extension of the four-helix bundle into the transmembrane region might contribute to the formation of membrane continuity between proximal leaflets of the fusing membranes. In conclusion, syntaxin participates in many molecular interactions and undergoes multiple conformational transitions until it is bound in a fully assembled SNARE complex.SNARE Proteine spielen eine wichtige Rolle in allen intrazellulären Fusionsreaktionen von der Hefe bis zum Menschen. Es wird vermutet, daß die Bildung von ternären SNARE Komplexen zwischen dem synaptischen Vesikelprotein Synaptobrevin und den Plasmamembranproteinen Syntaxin und SNAP-25 zur Membranfusion in der Nervenendigung führt. Die Interaktion zwischen SNARE Proteinen erfolgt über einen 60 Aminosäuren umfassenden Abschnitt, dem SNARE Motiv. Die Kristallstruktur des synaptischen SNARE Komplexes ergab ein langgestrecktes Vierhelixbündel, in dem alle Helizes parallel angeordnet sind (zwei SNAP-25 Helizes und je eine Syntaxin und eine Synaptobrevin Helix). Syntaxin besitzt eine zusätzliche, unabhängig gefaltete Domäne, die Habc Domäne. Eine intramolekulare Interaktion zwischen der Habc Domäne und dem SNARE Motiv arretiert Syntaxin in einer geschlossenen Konformation. Nur wenn es keine Interaktion zwischen diesen Domänen gibt (offene Konformation) kann sich der SNARE Komplex ausbilden. In dieser Arbeit wurden der konformationelle Schaltmechanismus Syntaxins und die Strukturen von SNARE Intermediaten und vollständig ausgebildeten SNARE Komplexen untersucht. Fluoreszenz Anisotropie Untersuchungen ergaben, daß die Habc Domäne die SNARE Komplexbildung um das fünf bis siebenfache verlangsamt. Dies spricht für eine schwache Interaktion zwischen der Habc Domäne und dem SNARE Motiv (ΔG ≈ 1 kcal/mol). Die intramolekularen Abstände zwischen diesen Domänen wurden durch Einzelmolekülfluoreszenz Resonanz Energietransfermessungen bestimmt. Nur 15-30% aller Syntaxin Moleküle sind geschlossen. Konformationelle Fluktuationen zwischen den offenen und geschlossenen Zuständen erfolgen in einem Zeitraum von ~0.7 ms. Um Syntaxin in eine geschlossene Konformation zu drängen, wird ein zusätzliches Protein (Munc-18) benötigt. Binäre Komplexe zwischen Syntaxin und SNAP-25 (2:1 Stöchiometrie) sind potentielle Intermediate in der Ausbildung von SNARE Komplexen. Elektronen paramagnetische Resonanz Daten ergaben, daß der binäre Komplex ein Vierhelixbündel ist, in dem die beiden Syntaxinhelizes parallel angeordnet sind. Die N- und C-termini Syntaxins und die C-termini beider SNAP-25 Helizes sind in diesem Komplex unstrukturiert. Die Bindung Synaptobrevins führt zu einer Verdrängung eines Syntaxinmoleküls und damit zur Bildung eines ternären Komplexes. Bei der Bildung binärer und ternärer Komplexe aus individuellen SNAREs erfolgen große strukturelle Veränderungen. Synaptobrevin und SNAP-25 sind alleine völlig unstrukturiert. Syntaxin ist auch unstrukturiert, oligomerisiert aber bei höheren mikromolekularen Konzentrationen. Die Oligomerisierung wird vom SNARE Motiv vermittelt und kann mit einer intramolekularen Interaktion mit der Habc Domäne konkurrieren. Um herauszufinden, ob die Struktur von einzelnen Helizes im SNARE Komplex von dem Vorhandensein gegenüberliegender Helizes abhängt, wurde ein C-terminal verkürztes Synaptobrevin eingesetzt. N-terminal von der Verkürzung waren die Komplexe strukturell intakt, wohingegen C-terminale Helizes, kollabierten. Die Interaktionsebenen im Vierhelixbündel von SNARE Komplexen scheinen somit unabhängig von benachbarten Ebenen zu sein. Dieser Befund stimmt mit dem vorgeschlagenen "Reissverschlussprinzip der Komplexbildung" überein und liefert eine strukturelle Basis für die Ausbildung von SNARE Komplex Intermediaten im Verlauf der Membranfusion. Um zu prüfen, ob SNARE Proteine über ihre Transmembrandomänen interagieren und somit die SNARE Komplexbildung beeinflussen, wurden die Interaktionen zwischen Syntaxin und Synaptobrevin in Proteoliposomen untersucht. Syntaxin bildete spezifische Dimere die Vorläufer von binären Komplexen sein könnten. Zusätzlich wurde eine neue Interaktion zwischen den Transmembranregionen von Syntaxin und Synaptobrevin beobachtet, die wichtig für eine späte Phase der Fusion sein könnte. Eine Verlängerung des Vierhelixbündels bis in die Membranregion könnte zur Ausbildung eines Membrankontinuums zwischen proximalen Schichten fusionierender Membranen sorgen. Syntaxin ist an vielen molekularen Interaktionen beteiligt und durchläuft multiple Konformationsänderungen bevor es im vollständig ausgebildeten SNARE Komplex gebunden ist