Tae-Young Yoon

Multiple intermediates in SNARE-induced membrane fusion

Membrane fusion in eukaryotic cells is thought to be mediated by a highly conserved family of proteins called SNAREs (soluble N-ethyl maleimide sensitive-factor attachment protein receptors). The vesicle-associated v-SNARE engages with its partner t-SNAREs on the target membrane to form a coiled coil that bridges two membranes and facilitates fusion. As demonstrated by recent findings on the hemifusion state, identifying intermediates of membrane fusion can help unveil the underlying fusion mechanism. Observation of SNARE-driven fusion at the single-liposome level has the potential to dissect and characterize fusion intermediates most directly. Here, we report on the real-time observation of lipid-mixing dynamics in a single fusion event between a pair of SNARE-reconstituted liposomes. The assay reveals multiple intermediate states characterized by discrete values of FRET between membrane-bound fluorophores. Hemifusion, flickering of fusion pores, and kinetic transitions between intermediates, which would be very difficult to detect in ensemble assays, are now identified. The ability to monitor the time course of fusion events between two proteoliposomes should be useful for addressing many important issues in SNARE-mediated membrane fusion.

Dynamic Ca2+-Dependent Stimulation of Vesicle Fusion by Membrane-Anchored Synaptotagmin 1

A Trick of the Tail The synaptic vesicle protein, synaptotagmin 1 (Syt1), acts as the main Ca2+-dependent switch for neurotransmitter release. In vitro studies of the truncated Syt1, which lacks the transmembrane domain, have unveiled the fusion-triggering mechanism of Syt1. However, in vitro approaches using the full-length, membrane-anchored Syt1 have not only failed to recapitulate Ca2+-triggered membrane fusion, but could even inhibit vesicle fusion. In contrast, the membrane anchor is conserved across the Syt family, suggesting a critical functional role for the membrane anchor. Now, using a single vesicle fusion assay, H.-K. Lee et al. (p. 760) show that the membrane anchor is indeed essential for Syt1 to induce physiological rates of Ca2+-induced vesicle fusion on a 100-millisecond time scale. A synaptic vesicle protein must be membrane-anchored to stimulate fusion in vitro at physiological Ca2+ concentrations. In neurons, synaptotagmin 1 (Syt1) is thought to mediate the fusion of synaptic vesicles with the plasma membrane when presynaptic Ca2+ levels rise. However, in vitro reconstitution experiments have failed to recapitulate key characteristics of Ca2+-triggered membrane fusion. Using an in vitro single-vesicle fusion assay, we found that membrane-anchored Syt1 enhanced Ca2+ sensitivity and fusion speed. This stimulatory activity of membrane-anchored Syt1 dropped as the Ca2+ level rose beyond physiological levels. Thus, Syt1 requires the membrane anchor to stimulate vesicle fusion at physiological Ca2+ levels and may function as a dynamic presynaptic Ca2+ sensor to control the probability of neurotransmitter release.

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.

MutS switches between two fundamentally distinct clamps during mismatch repair

Single-molecule trajectory analysis has suggested DNA repair proteins may carry out a one-dimensional (1D) search on naked DNA encompassing >10,000 nucleotides. Organized cellular DNA (chromatin) presents substantial barriers to such lengthy searches. Using dynamic single-molecule fluorescence resonance energy transfer, we determined that the mismatch repair (MMR) initiation protein MutS forms a transient clamp that scans duplex DNA for mismatched nucleotides by 1D diffusion for 1 s (~700 base pairs) while in continuous rotational contact with the DNA. Mismatch identification provokes ATP binding (3 s) that induces distinctly different MutS sliding clamps with unusual stability on DNA (~600 s), which may be released by adjacent single-stranded DNA (ssDNA). These observations suggest that ATP transforms short-lived MutS lesion scanning clamps into highly stable MMR signaling clamps that are capable of competing with chromatin and recruiting MMR machinery, yet are recycled by ssDNA excision tracts.

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.

Microfluidic Synthesis of Hybrid Nanoparticles with Controlled Lipid Layers: Understanding Flexibility-Regulated Cell–Nanoparticle Interaction

The functionalized lipid shell of hybrid nanoparticles plays an important role for improving their biocompatibility and in vivo stability. Yet few efforts have been made to critically examine the shell structure of nanoparticles and its effect on cell-particle interaction. Here we develop a microfluidic chip allowing for the synthesis of structurally well-defined lipid-polymer nanoparticles of the same sizes, but covered with either lipid-monolayer-shell (MPs, monolayer nanoparticles) or lipid-bilayer-shell (BPs, bilayer nanoparticles). Atomic force microscope and atomistic simulations reveal that MPs have a lower flexibility than BPs, resulting in a more efficient cellular uptake and thus anticancer effect than BPs do. This flexibility-regulated cell-particle interaction may have important implications for designing drug nanocarriers.

Mechanical unzipping and rezipping of a single SNARE complex reveals hysteresis as a force-generating mechanism.

Formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex provides mechanical thrust for membrane fusion, but its molecular mechanism is still unclear. Here using magnetic tweezers, we observe mechanical responses of a single neuronal SNARE complex under constant pulling force. Single SNARE complexes may be unzipped with 34 pN force. When rezipping is induced by lowering the force to 11 pN, only a partially assembled state results, with the C-terminal half of the SNARE complex remaining disassembled. Reassembly of the C-terminal half occurs only when the force is further lowered below 11 pN. Thus, mechanical hysteresis, characterized by the unzipping and rezipping cycle of a single SNARE complex, produces the partially assembled state. In this metastable state, unzipping toward the N-terminus is suppressed while zippering toward the C-terminus is initiated as a steep function of force. This ensures the directionality of SNARE-complex formation, making the SNARE complex a robust force-generating machine.

Low-power nano-optical vortex trapping via plasmonic diabolo nanoantennas

Optical vortex trapping can allow the capture and manipulation of micro- and nanometre-sized objects such as damageable biological particles or particles with a refractive index lower than the surrounding material. However, the quest for nanometric optical vortex trapping that overcomes the diffraction limit remains. Here we demonstrate the first experimental implementation of low-power nano-optical vortex trapping using plasmonic resonance in gold diabolo nanoantennas. The vortex trapping potential was formed with a minimum at 170 nm from the central local maximum, and allowed polystyrene nanoparticles in water to be trapped strongly at the boundary of the nanoantenna. Furthermore, a large radial trapping stiffness, ~0.69 pN nm(-1) W(-1), was measured at the position of the minimum potential, showing good agreement with numerical simulations. This subwavelength-scale nanoantenna system capable of low-power trapping represents a significant step toward versatile, efficient nano-optical manipulations in lab-on-a-chip devices.

Real-time single-molecule co-immunoprecipitation analyses reveal cancer-specific Ras signalling dynamics

The conventional co-immunoprecipitation provides static and qualitative information about protein-protein interactions. Lee et al. report real-time imaging of co-immunoprecipitation process with single-molecule resolution, allowing for characterization of the native Ras proteins derived from individual cancers.

Single-Vesicle Fusion Assay Reveals Munc18-1 Binding to the SNARE Core Is Sufficient for Stimulating Membrane Fusion

Munc18, an essential regulatory protein for intracellular membrane fusion mediated by SNAREs, is known for stabilizing the closed conformation of syntaxin through the interaction with the N-terminal Habc domain (amino acids 28−146) of syntaxin. In addition, Munc18 accelerates membrane fusion and its interaction with SNARE core and the N-peptide (amino acids 1−24) of syntaxin is thought to be necessary for this function. Using the recently developed fluorescence resonance energy transfer assay to detect the fusion between two individual vesicles harboring cognate SNARE proteins, we studied the effect of Munc18 on the fusion induced by neuronal SNARE proteins by following the mixing of lipid molecules between the two vesicles. We found that Munc18-1 stimulates neuronal SNARE-mediated fusion not only with full-length syntaxin 1A but also with a truncated syntaxin 1A that is missing both the Habc domain and the N-peptide. The electron paramagnetic resonance analysis indicates that the SNARE core/Munc18 interaction is responsible for this stimulatory function and the membrane plays a role for establishing this interaction.