Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS

149

307

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 Ca 2þ 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 Ca 2þ injection, the system is in a state in which spontaneous fusion events between donor and acceptor vesicles are rare. Upon Ca 2þ injection, a rapid burst of complete fusion events emerges, followed by a biphasic decay. The present study focuses on neuronal SNAREs, the Ca 2þ sensor synaptotagmin 1, and the modulator complexin. However, other synaptic proteins could be added and their function examined. Ca 2þ 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.fast content mixing | single-vesicle fusion assay | membrane fusion | lipid mixing N euronal communication is made possible by the release of neurotransmitters, which in turn depends on the fusion of neurotransmitter-containing vesicles with the active zone in axonal terminals. Synaptic vesicle fusion is triggered by an influx of Ca 2þ ions into the neuron upon depolarization. Neurotransmitter release is quantized (1); that is, it involves a few to tens of individual synaptic fusion events. The process of individual synaptic vesicle fusion is in turn controlled by a set of relatively few proteins, such as the SNARE proteins (2-5), the Ca 2þ sensor for fast synchronous release synaptotagmin 1 (6-8), and the modulator complexin (9-11). Thus, neurotransmitter release is a macroscopic biological phenomenon that is ultimately controlled by a few individual molecules. The understanding of the underlying molecular mechanisms thus requires methods that are inherently capable of observing single vesicles and single molecules (12,13).Ideally, observations of single vesicles and single molecules would be performed in live neurons. Although progress for such studies has been made (14), they currently only provide limited information because the necessary genetic manipulations or labeling techniques may not provide the spatial and time resolution required for studying the dynamics of neurotransmitt...

Section: Methodsmentioning

confidence: 58%

In vitro system capable of differentiating fast Ca ²⁺ -triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release

Kyoung

Srivastava

Zhang

et al. 2011

149

307

“…To overcome this, SNARE complex formation can be enhanced by using the ΔN complex, which accelerates this process by at least 30-fold (26). In a dilute liposome regime (0.02-0.1 mM total lipid) we have previously found that with the ΔN complex the rate of docking is accelerated to an extent similar to the rate of fusion, a kinetic condition known as partial-rate limiting (8,30).…”

Section: Significancementioning

confidence: 99%

Variable cooperativity in SNARE-mediated membrane fusion

Hernandez

Kreutzberger

Kiessling

et al. 2014

Self Cite

101

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex drives the majority of intracellular and exocytic membrane fusion events. Whether and how SNAREs cooperate to mediate fusion has been a subject of intense study, with estimates ranging from a single SNARE complex to 15. Here we show that there is no universally conserved number of SNARE complexes involved as revealed by our observation that this varies greatly depending on membrane curvature. When docking rates of small (∼40 nm) and large (∼100 nm) liposomes reconstituted with different synaptobrevin (the SNARE present in synaptic vesicles) densities are taken into account, the lipid mixing efficiency was maximal with small liposomes with only one synaptobrevin, whereas 23-30 synaptobrevins were necessary for efficient lipid mixing in large liposomes. Our results can be rationalized in terms of strong and weak cooperative coupling of SNARE complex assembly where each mode implicates different intermediate states of fusion that have been recently identified by electron microscopy. We predict that even higher variability in cooperativity is present in different physiological scenarios of fusion, and we further hypothesize that plasticity of SNAREs to engage in different coupling modes is an important feature of the biologically ubiquitous SNARE-mediated fusion reactions.M embrane fusion is an essential reaction common to intracellular trafficking and exocytosis in eukaryotic cells. Although the process involves an intricate interplay of several proteins, the fusion of membranes is dependent on the conserved family of proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors, or SNAREs (1, 2). In the important case of the fusion of synaptic vesicles (SVs), the SNAREs responsible are vesicular synaptobrevin 2 (syb) and plasma membrane proteins SNAP-25A (SN25) and syntaxin-1A (syx). A critical intermediate seems to be an acceptor complex consisting of a threehelix bundle formed by a 1:1 syx:SN25 complex, which serves as a binding site for syb (3,4). According to the zipper hypothesis, the N termini of syb and the 1:1 syx:SN25 complex nucleate to form a parallel four-helix bundle called the SNARE complex. The directional assembly then proceeds toward the C termini, resulting in a pulling force between the membranes that leads to their fusion (4, 5). There is some consensus that the highly exergonic nature of the assembly of the SNARE complex provides the energy for overcoming the barrier for fusion (6, 7), although identification of putative fusion intermediates at molecular resolution as well as force measurement experiments suggest multiple energy barriers are present (7-10).The question of whether and how SNAREs cooperate to mediate fusion has received substantial attention. Although some studies have left open the possibility that the number of SNARE complexes that cooperate during fusion is variable (11, 12), much attention has been given to the notion of a preferred number of SNARE complexes, ...

“…Thus, spontaneous incorporation of lipids is unlikely. Before a protocell obtained an ability to synthesize lipids on its own, the vesicle size increase could be achieved by vesicle-to-vesicle fusion initiated by various external stimuli (19)(20)(21)(22) or surface functionalization (23)(24)(25)(26). Thus, it is conceivable that membrane fusion could be one of the primitive growth pathways for protocells composed of phospholipids.…”

mentioning

confidence: 99%

Coupling of the fusion and budding of giant phospholipid vesicles containing macromolecules

Terasawa

Nishimura

Suzuki

et al. 2012

156

166

Mechanisms that enabled primitive cell membranes to self-reproduce have been discussed based on the physicochemical properties of fatty acids; however, there must be a transition to modern cell membranes composed of phospholipids [Budin I, Szostak JW (2011) Proc Natl Acad Sci USA 108:5249–5254]. Thus, a growth-division mechanism of membranes that does not depend on the chemical nature of amphiphilic molecules must have existed. Here, we show that giant unilamellar vesicles composed of phospholipids can undergo the coupled process of fusion and budding transformation, which mimics cell growth and division. After gaining excess membrane by electrofusion, giant vesicles spontaneously transform into the budded shape only when they contain macromolecules (polymers) inside their aqueous core. This process is a result of the vesicle maximizing the translational entropy of the encapsulated polymers (depletion volume effect). Because the cell is a lipid membrane bag containing highly concentrated biopolymers, this coupling process that is induced by physical and nonspecific interactions may have a general importance in the self-reproduction of the early cellular compartments.