Jiajie Diao

ATG14 promotes membrane tethering and fusion of autophagosomes to endolysosomes

Autophagy, an important catabolic pathway implicated in a broad spectrum of human diseases, begins by forming double membrane autophagosomes that engulf cytosolic cargo and ends by fusing autophagosomes with lysosomes for degradation. Membrane fusion activity is required for early biogenesis of autophagosomes and late degradation in lysosomes. However, the key regulatory mechanisms of autophagic membrane tethering and fusion remain largely unknown. Here we report that ATG14 (also known as beclin-1-associated autophagy-related key regulator (Barkor) or ATG14L), an essential autophagy-specific regulator of the class III phosphatidylinositol 3-kinase complex, promotes membrane tethering of protein-free liposomes, and enhances hemifusion and full fusion of proteoliposomes reconstituted with the target (t)-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) syntaxin 17 (STX17) and SNAP29, and the vesicle (v)-SNARE VAMP8 (vesicle-associated membrane protein 8). ATG14 binds to the SNARE core domain of STX17 through its coiled-coil domain, and stabilizes the STX17–SNAP29 binary t-SNARE complex on autophagosomes. The STX17 binding, membrane tethering and fusion-enhancing activities of ATG14 require its homo-oligomerization by cysteine repeats. In ATG14 homo-oligomerization-defective cells, autophagosomes still efficiently form but their fusion with endolysosomes is blocked. Recombinant ATG14 homo-oligomerization mutants also completely lose their ability to promote membrane tethering and to enhance SNARE-mediated fusion in vitro. Taken together, our data suggest an autophagy-specific membrane fusion mechanism in which oligomeric ATG14 directly binds to STX17–SNAP29 binary t-SNARE complex on autophagosomes and primes it for VAMP8 interaction to promote autophagosome–endolysosome fusion.

In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release

Understanding the molecular principles of synaptic vesicle fusion is a long-sought goal. It requires the development of a synthetic system that allows manipulations and observations not possible in vivo. Here, we report an in vitro system with reconstituted synaptic proteins that meets the long-sought goal to produce fast content release in the millisecond time regime upon Ca2+ triggering. Our system simultaneously monitors both content and lipid exchange, and it starts from stable interacting pairs of donor and acceptor vesicles, mimicking the readily releasable pool of synaptic vesicles prior to an action potential. It differentiates between single-vesicle interaction, hemifusion, and complete fusion, the latter mimicking quantized neurotransmitter release upon exocytosis of synaptic vesicles. Prior to Ca2+ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca2+ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca2+ sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca2+ triggering is cooperative, requiring the presence of synaptotagmin, whereas SNAREs alone do not produce a fast fusion burst. Manipulations of the system mimic effects observed in vivo. These results also show that neuronal SNAREs alone do not efficiently produce complete fusion, that the combination of SNAREs with synaptotagmin lowers the activation barriers to full fusion, and that complexin enhances this kinetic control.

Native α-synuclein induces clustering of synaptic-vesicle mimics via binding to phospholipids and synaptobrevin-2/VAMP2

α-Synuclein is a presynaptic protein that is implicated in Parkinsons and other neurodegenerative diseases. Physiologically, native α-synuclein promotes presynaptic SNARE-complex assembly, but its molecular mechanism of action remains unknown. Here, we found that native α-synuclein promotes clustering of synaptic-vesicle mimics, using a single-vesicle optical microscopy system. This vesicle-clustering activity was observed for both recombinant and native α-synuclein purified from mouse brain. Clustering was dependent on specific interactions of native α-synuclein with both synaptobrevin-2/VAMP2 and anionic lipids. Out of the three familial Parkinsons disease-related point mutants of α-synuclein, only the lipid-binding deficient mutation A30P disrupted clustering, hinting at a possible loss of function phenotype for this mutant. α-Synuclein had little effect on Ca2+-triggered fusion in our reconstituted single-vesicle system, consistent with in vivo data. α-Synuclein may therefore lead to accumulation of synaptic vesicles at the active zone, providing a ‘buffer’ of synaptic vesicles, without affecting neurotransmitter release itself. DOI: http://dx.doi.org/10.7554/eLife.00592.001

Properties of native brain α-synuclein

Arising from T. Bartels, J. G. Choi & D. J. Selkoe. 477, 107–110 (2011).10.1038/nature10324α-Synuclein is an abundant presynaptic protein that binds to negatively charged phospholipids, functions as a SNARE-complex chaperone and contributes to Parkinson’s disease pathogenesis. Recombinant α-synuclein in solution is largely unfolded and devoid of tertiary structure, but Bartels et al. have proposed that native α-synuclein purified from human erythrocytes forms a stably folded, soluble tetramer that resists aggregation. By contrast, we show here that native α-synuclein purified from mouse brain consists of a largely unstructured monomer, exhibits no stable tetramer formation, and is prone to aggregation. The native state of α-synuclein is important for understanding its pathological effects as a stably folded protein would be much less prone to aggregation than a conformationally labile protein. There is a Reply to this Brief Communication Arising by Bartels, T. & Selkoe, D. J. Nature 498, http://dx.doi.org/10.1038/nature12126 (2013).

Architecture of the synaptotagmin-SNARE machinery for neuronal exocytosis.

Synaptotagmin-1 and neuronal SNARE proteins have central roles in evoked synchronous neurotransmitter release; however, it is unknown how they cooperate to trigger synaptic vesicle fusion. Here we report atomic-resolution crystal structures of Ca2+- and Mg2+-bound complexes between synaptotagmin-1 and the neuronal SNARE complex, one of which was determined with diffraction data from an X-ray free-electron laser, leading to an atomic-resolution structure with accurate rotamer assignments for many side chains. The structures reveal several interfaces, including a large, specific, Ca2+-independent and conserved interface. Tests of this interface by mutagenesis suggest that it is essential for Ca2+-triggered neurotransmitter release in mouse hippocampal neuronal synapses and for Ca2+-triggered vesicle fusion in a reconstituted system. We propose that this interface forms before Ca2+ triggering, moves en bloc as Ca2+ influx promotes the interactions between synaptotagmin-1 and the plasma membrane, and consequently remodels the membrane to promote fusion, possibly in conjunction with other interfaces.

Synaptic proteins promote calcium-triggered fast transition from point contact to full fusion.

The molecular underpinnings of synaptic vesicle fusion for fast neurotransmitter release are still unclear. Here, we used a single vesicle–vesicle system with reconstituted SNARE and synaptotagmin-1 proteoliposomes to decipher the temporal sequence of membrane states upon Ca2+-injection at 250–500 μM on a 100-ms timescale. Furthermore, detailed membrane morphologies were imaged with cryo-electron microscopy before and after Ca2+-injection. We discovered a heterogeneous network of immediate and delayed fusion pathways. Remarkably, all instances of Ca2+-triggered immediate fusion started from a membrane–membrane point-contact and proceeded to complete fusion without discernible hemifusion intermediates. In contrast, pathways that involved a stable hemifusion diaphragm only resulted in fusion after many seconds, if at all. When complexin was included, the Ca2+-triggered fusion network shifted towards the immediate pathway, effectively synchronizing fusion, especially at lower Ca2+-concentration. Synaptic proteins may have evolved to select this immediate pathway out of a heterogeneous network of possible membrane fusion pathways. DOI: http://dx.doi.org/10.7554/eLife.00109.001

Complexin and Ca2+ stimulate SNARE-mediated membrane fusion

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release.

A single vesicle-vesicle fusion assay for in vitro studies of SNAREs and accessory proteins

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins are a highly regulated class of membrane proteins that drive the efficient merger of two distinct lipid bilayers into one interconnected structure. This protocol describes our fluorescence resonance energy transfer (FRET)-based single vesicle-vesicle fusion assays for SNAREs and accessory proteins. Both lipid-mixing (with FRET pairs acting as lipophilic dyes in the membranes) and content-mixing assays (with FRET pairs present on a DNA hairpin that becomes linear via hybridization to a complementary DNA) are described. These assays can be used to detect substages such as docking, hemifusion, and pore expansion and full fusion. The details of flow cell preparation, protein-reconstituted vesicle preparation, data acquisition and analysis are described. These assays can be used to study the roles of various SNARE proteins, accessory proteins and effects of different lipid compositions on specific fusion steps. The total time required to finish one round of this protocol is 3–6 d.

Studying calcium-triggered vesicle fusion in a single vesicle-vesicle content and lipid-mixing system

This protocol describes a single vesicle-vesicle microscopy system to study Ca2+-triggered vesicle fusion. Donor vesicles contain reconstituted synaptobrevin and synaptotagmin-1. Acceptor vesicles contain reconstituted syntaxin and synaptosomal-associated protein 25 (SNAP-25), and they are tethered to a PEG-coated glass surface. Donor vesicles are mixed with the tethered acceptor vesicles and incubated for several minutes at a zero-Ca2+ concentration, resulting in a collection of single interacting vesicle pairs. The donor vesicles also contain two spectrally distinct fluorophores that allow simultaneous monitoring of temporal changes of the content and membrane. Upon Ca2+ injection into the sample chamber, our system therefore differentiates between hemifusion and complete fusion of interacting vesicle pairs and determines the temporal sequence of these events on a sub-100-millisecond time scale. Other factors such as complexin can be easily added. Our system is unique in that it monitors both content and lipid mixing and starts from a metastable state of interacting vesicle pairs before Ca2+ injection.

Fusion pore formation and expansion induced by Ca2+ and synaptotagmin 1

Fusion pore formation and expansion, crucial steps for neurotransmitter release and vesicle recycling in soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent vesicle fusion, have not been well studied in vitro due to the lack of a reliable content-mixing fusion assay. Using methods detecting the intervesicular mixing of small and large cargoes at a single-vesicle level, we found that the neuronal SNARE complexes have the capacity to drive membrane hemifusion. However, efficient fusion pore formation and expansion require synaptotagmin 1 and Ca2+. Real-time measurements show that pore expansion detected by content mixing of large DNA cargoes occurs much slower than initial pore formation that transmits small cargoes. Slow pore expansion perhaps provides a time window for vesicles to escape the full collapse fusion pathway via alternative mechanisms such as kiss-and-run. The results also show that complexin 1 stimulates pore expansion significantly, which could put bias between two pathways of vesicle recycling.