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.

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. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) proteins have emerged as the leading candidates for mediating membrane fusion. They comprise a superfamily of small membrane proteins distinguished by the SNARE motif, a conserved coiled-coil stretch of 60-70 amino acids. SNARE motifs spontaneously assemble into elongated four-helix bundles in which each helix is contributed by a SNARE motif belonging to a separate subclass. Complex formation is assumed to tie membranes together and to initiate membrane fusion along a reaction path involving so-farunknown conformational transitions (1-3).In most SNAREs, the SNARE motif is located adjacent to a C-terminal transmembrane domain. Furthermore, many SNAREs contain an independently folded domain at the N terminus that is connected to the SNARE motif by a linker region. In the syntaxin subfamily (also referred to as QaSNAREs), the N-terminal domains consist of antiparallel bundles of three ␣-helices that are structurally conserved despite high divergence in the primary structure. The N-terminal domains of several syntaxins interact reversibly with the SNARE motif, resulting in two distinct conformations; a closed conformation in which the SNARE motif is blocked (i.e., unable to form SNARE complexes), and an open conformation in which there is presumably no contact between these domains (2). Binding of munc-18, a regulatory protein essential for exocytosis, arrests syntaxin 1 in the closed conformation in which the N-terminal portion of the SNARE motif binds to a groove on the surface of the Habc domain (ref. 4 and Fig. 1). Mutations destabilizing the closed state of syntaxin have profound effects on exocytosis, suggesting that the conformational transition is a key element in the biological function of syntaxin 1 (4, 5).Conformational transitions such as those discussed above are difficult to observe directly due to limited temporal or spatial resolution. To overcome these limitations, we have recently developed a si...

Tau Filaments are found in >20 neurodegenerative diseases. Yet, because of their enormous molecular weights and poor tendency to form highly ordered 3D crystal lattices, they have evaded high-resolution structure determination. Here, we studied 25 derivatized tau mutants by using electron paramagnetic resonance and fluorescence spectroscopy to report structural details of tau filaments. Based on strong spin exchange and pyrene excimer formation of core residues, we find that individual tau proteins form single molecule layers along the fiber axis that perfectly stack on top of each other by in-register, parallel alignment of ␤-strands. We suggest a model of filament growth wherein the existing filament serves as a template for the incoming, unfolded tau molecule, resulting in a new structured layer with maximized hydrogen-bonded contact surface and side-chain stacking. In addition to its role in stabilizing microtubules in neuritic extensions, tau has gained prominence as the protein constituent of filamentous inclusions in numerous neurodegenerative diseases (1). These inclusions, together with extracellular fibril deposits of the amyloid-␤ (A␤)-peptide, constitute the pathological hallmarks of Alzheimer's disease.In the adult human CNS, six different tau isoforms, ranging in size from 352 to 441 aa, are produced by alternative mRNA splicing. These isoforms vary by the absence or presence of the second of four microtubule-binding repeats in the C-terminal half and two inserts in the near N-terminal half of the protein (Fig. 1). The tau inclusions in Alzheimer's disease contain all six isoforms (2) and consist of paired helical and straight filaments (3, 4). Viewed under an electron microscope, tau filaments have a fuzzy coat that can be cleaved off by pronase (5). Cleavage leaves a core that is comprised of three microtubule-binding repeats (6). The importance of these repeats in filament formation was underscored by the finding that tau fragments comprising only the repeat region aggregate in vitro (7). In contrast, recombinant full-length tau is remarkably unreactive and aggregates only when anionic cofactors such as heparin are present (8,9).A recent study involving x-ray and selected area electron diffraction of both straight and paired helical filaments (10) revealed a common cross-␤ structure, with ␤-strands running perpendicular to the fiber axis. These data agreed with earlier findings of diffraction patterns from filaments obtained from shorter tau fragments (11,12). Importantly, they resolved some controversy concerning the filament structure of full-length tau (12)(13)(14). Thus, with respect to the cross-␤ structure and a seeded growth mechanism (15), tau filaments share similarities with a whole range of amyloidogenic protein aggregates (16). Beyond this, however, little is known about the structure of tau filaments. For example, it is not known how individual ␤-strands are arranged with each other and whether tau molecules align along or across the fiber axis, (i.e., whether hydrogen bonding among...

The misfolding and fibril formation of ␣-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 ␣-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 ␣-synuclein fibrils. Such exchange narrowing requires the orbital overlap between multiple spin labels in close contact. The core region of ␣-synuclein fibrils must therefore be arranged in a parallel, inregister 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 ␤-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.

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.