Martin Margittai

Inhibition of SNARE Complex Assembly Differentially Affects Kinetic Components of Exocytosis

In chromaffin cells, an increase in intracellular Ca2+ leads to an exocytotic burst followed by sustained secretion. The burst can be further resolved into two kinetically distinct components, which suggests the presence of two separate pools of vesicles. To investigate how these components relate to SNARE complex formation, we introduced an antibody that blocks SNARE assembly but not disassembly. In the presence of the antibody, the sustained component was largely blocked, the burst was slightly reduced, and one of its kinetic components was eliminated. We conclude that SNARE complexes form before Ca(2+)-triggered membrane fusion and exist in a dynamic equilibrium between a loose and a tight state, both of which support exocytosis. Interaction of the antibody with preformed SNARE complexes favors the loose state.

MIXED AND NON-COGNATE SNARE COMPLEXES: CHARACTERIZATION OF ASSEMBLY AND BIOPHYSICAL PROPERTIES

Assembly of solubleN-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPaseN-ethylmaleimide-sensitive fusion protein and its cofactor α-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.

Small Misfolded Tau Species Are Internalized via Bulk Endocytosis and Anterogradely and Retrogradely Transported in Neurons

Background: Exogenous, misfolded Tau can be internalized, but details of the mechanism are unknown. Results: Small misfolded Tau species are internalized through endocytosis, anterogradely and retrogradely transported. Conclusion: Tau uptake is dependent on conformation and size of aggregates, and regulated through endocytosis. Significance: Understanding the mechanism by which pathological Tau is internalized provides a foundation for therapeutic approaches targeting uptake and propagation of tauopathy. The accumulation of Tau into aggregates is associated with key pathological events in frontotemporal lobe degeneration (FTD-Tau) and Alzheimer disease (AD). Recent data have shown that misfolded Tau can be internalized by cells in vitro (Frost, B., Jacks, R. L., and Diamond, M. I. (2009) J. Biol. Chem. 284, 12845–12852) and propagate pathology in vivo (Clavaguera, F., Bolmont, T., Crowther, R. A., Abramowski, D., Frank, S., Probst, A., Fraser, G., Stalder, A. K., Beibel, M., Staufenbiel, M., Jucker, M., Goedert, M., and Tolnay, M. (2009) Nat. Cell Biol. 11, 909–913; Lasagna-Reeves, C. A., Castillo-Carranza, D. L., Sengupta, U., Guerrero-Munoz, M. J., Kiritoshi, T., Neugebauer, V., Jackson, G. R., and Kayed, R. (2012) Sci. Rep. 2, 700). Here we show that recombinant Tau misfolds into low molecular weight (LMW) aggregates prior to assembly into fibrils, and both extracellular LMW Tau aggregates and short fibrils, but not monomers, long fibrils, nor long filaments purified from brain extract are taken up by neurons. Remarkably, misfolded Tau can be internalized at the somatodendritic compartment, or the axon terminals and it can be transported anterogradely, retrogradely, and can enhance tauopathy in vivo. The internalized Tau aggregates co-localize with dextran, a bulk-endocytosis marker, and with the endolysosomal compartments. Our findings demonstrate that exogenous Tau can be taken up by cells, uptake depends on both the conformation and size of the Tau aggregates and once inside cells, Tau can be transported. These data provide support for observations that tauopathy can spread trans-synaptically in vivo, via cell-to-cell transfer.

Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1

Protein conformational transitions form the molecular basis of many cellular processes, such as signal transduction and membrane traffic. However, in many cases, little is known about their structural dynamics. Here we have used dynamic single-molecule fluorescence to study at high time resolution, conformational transitions of syntaxin 1, a soluble N-ethylmaleimide-sensitive factor attachment protein receptors protein essential for exocytotic membrane fusion. Sets of syntaxin double mutants were randomly labeled with a mix of donor and acceptor dye and their fluorescence resonance energy transfer was measured. For each set, all fluorescence information was recorded simultaneously with high time resolution, providing detailed information on distances and dynamics that were used to create structural models. We found that free syntaxin switches between an inactive closed and an active open configuration with a relaxation time of 0.8 ms, explaining why regulatory proteins are needed to arrest the protein in one conformational state.

Investigation of α-Synuclein Fibril Structure by Site-directed Spin Labeling

The misfolding and fibril formation of α-synuclein plays an important role in neurodegenerative diseases such as Parkinson disease. Here we used electron paramagnetic resonance spectroscopy, together with site-directed spin labeling, to investigate the structural features of α-synuclein fibrils. We generated fibrils from a total of 83 different spin-labeled derivatives and observed single-line, exchange-narrowed EPR spectra for the majority of all sites located within the core region of α-synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of α-synuclein fibrils must therefore be arranged in a parallel, in-register structure wherein same residues from different molecules are stacked on top of each other. This parallel, in-register core region extends from residue 36 to residue 98 and is tightly packed. Only a few sites within the core region, such as residues 62–67 located at the beginning of the NAC region, as well as the N- and C-terminal regions outside the core region, are significantly less ordered. Together with the accessibility measurements that suggest the location of potential β-sheet regions within the fibril, the data provide significant structural constraints for generating three-dimensional models. Furthermore, the data support the emerging view that parallel, in-register structure is a common feature shared by a number of naturally occurring amyloid fibrils.

A Transient N-terminal Interaction of SNAP-25 and Syntaxin Nucleates SNARE Assembly

The SNARE proteins syntaxin, SNAP-25, and synaptobrevin play a central role during Ca2+-dependent exocytosis at the nerve terminal. Whereas syntaxin and SNAP-25 are located in the plasma membrane, synaptobrevin resides in the membrane of synaptic vesicles. It is thought that gradual assembly of these proteins into a membrane-bridging ternary SNARE complex ultimately leads to membrane fusion. According to this model, syntaxin and SNAP-25 constitute an acceptor complex for synaptobrevin. In vitro, however, syntaxin and SNAP-25 form a stable complex that contains two syntaxin molecules, one of which is occupying and possibly obstructing the binding site of synaptobrevin. To elucidate the assembly pathway of the synaptic SNAREs, we have now applied a combination of fluorescence and CD spectroscopy. We found that SNARE assembly begins with the slow and rate-limiting interaction of syntaxin and SNAP-25. Their interaction was prevented by N-terminal but not by C-terminal truncations, suggesting that for productive assembly all three participating helices must come together simultaneously. This suggests a complicated nucleation process that might be the reason for the observed slow assembly rate. N-terminal truncations of SNAP-25 and syntaxin also prevented the formation of the ternary complex, whereas neither N- nor C-terminal shortened synaptobrevin helices lost their ability to interact. This suggests that binding of synaptobrevin occurs after the establishment of the syntaxin-SNAP-25 interaction. Moreover, binding of synaptobrevin was inhibited by an excess of syntaxin, suggesting that a 1:1 interaction of syntaxin and SNAP-25 serves as the on-pathway SNARE assembly intermediate.

Fibrils with parallel in-register structure constitute a major class of amyloid fibrils: molecular insights from electron paramagnetic resonance spectroscopy.

The deposition of amyloid- and amyloid-like fibrils is the main pathological hallmark of numerous protein misfolding diseases including Alzheimers disease, transmissible spongiform encephalopathy, and type 2 diabetes. Besides the well-established role in disease, recent work on a variety of organisms ranging from bacteria to humans suggests that amyloid fibrils can also convey biological functions. To better understand the molecular mechanisms by which amyloidogenic proteins misfold in disease or perform biological functions, structural information is essential. Although high-resolution structural analysis of amyloid fibrils has been challenging, a combination of biophysical approaches is beginning to unravel the various structural features of amyloid fibrils. Here we review these recent developments with particular emphasis on amyloid fibrils that have been studied using site-directed spin labeling and electron paramagnetic resonance spectroscopy. This approach has been used to define the precise location of fibril-forming core regions and identify local secondary structures within such core regions. Perhaps one of the most remarkable findings arrived at by site-directed spin labeling was that most fibrils that contain an extensive core region of 20 amino acids or more share a common parallel in-register arrangement of beta strands. The preference for this arrangement can be explained on topological grounds and may be rationalized by the maximization of hydrophobic contact surface.

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.

A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains

The proteins synaptobrevin (VAMP), SNAP‐25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co‐reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.

Variations in filament conformation dictate seeding barrier between three- and four-repeat tau.

Tau filaments are the pathological hallmark of >20 neurodegenerative diseases including Alzheimers disease. Six tau isoforms exist that can be grouped into 4-repeat (4R) tau and 3-repeat (3R) tau based on the presence or absence of the second of four microtubule binding repeats. Recent evidence suggests that tau filaments can transfer between cells and spread through the brain. Here we demonstrate in vitro that seeded filament growth, a prerequisite for tau spreading, is crucially dependent on the isoform composition of individual seeds. Seeds of 3R tau and 3R/4R tau recruit both types of isoforms. Seeds of 4R tau recruit 4R tau, but not 3R tau, establishing an asymmetric barrier. Conformational templating of 4R tau onto 3R tau seeds eliminates this barrier, giving rise to a new type of tau filament. These findings provide fundamental mechanistic insights into the seeding, propagation, and diversification of tau filaments.