Spontaneous Membrane Fusion Research Articles

Mechanisms of SNARE proteins in membrane fusion.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of small conserved eukaryotic proteins that mediate membrane fusion between organelles and with the plasma membrane. SNAREs are directly or indirectly anchored to membranes. Prior to fusion, complementary SNAREs assemble between membranes with the aid of accessory proteins that provide a scaffold to initiate SNARE zippering, pulling the membranes together and mediating fusion. Recent advances have enabled the construction of detailed models describing bilayer transitions and energy barriers along the fusion pathway and have elucidated the structures of SNAREs complexed in various states with regulatory proteins. In this Review, we discuss how these advances are yielding an increasingly detailed picture of the SNARE-mediated fusion pathway, leading from first contact between the membranes via metastable non-bilayer intermediates towards the opening and expansion of a fusion pore. We describe how SNARE proteins assemble into complexes, how this assembly is regulated by accessory proteins and how SNARE complexes overcome the free energy barriers that prevent spontaneous membrane fusion.

MRI-FI-guided superimposed stimulus-responsive co-assembled liposomes for optimizing transmembrane drug delivery pathways and improving cancer efficacy

Cancer is a serious challenge for humanity and the key to advancing cancer therapy is to increase cellular uptake of drugs. This paper provides a folic acid GFC liposome that relies on magnetic resonance imaging-fluorescence imaging (MRI-FI) to precisely guide the multi-stimulus response at the tumor site. By accurately increasing the concentration of free drug at the tumor site, it promotes the efficient diffusion of small molecule drugs into cells and penetration into solid tumors. Furthermore, based on the stimulus response mechanism of GFC liposomes (dissociation-fusion-rearrangement-fragmentation), we demonstrated that GFC liposomes can optimize the high-energy unfavorable fusion behavior of liposomes with cell membranes. In this way, GFC liposomes can intelligently and efficiently deliver drugs into cells in the form of active endocytosis, spontaneous membrane fusion and diffusion, depending on the actual stimulus response effect. Finally, animal experiments demonstrated the potential of the highly efficient MRI-FI-based GFC carrier with a 9.13-fold tumor suppression efficiency. Herein, this paper highlights the advantages of MRI-FI-guided precise multi-stimulus-controlled release and high uptake rate strategies to provide ingenious and concise ideas for precise and efficient cancer therapy.

Spontaneous vesicle recycling in the synaptic bouton.

The trigger for synaptic vesicle exocytosis is Ca2+, which enters the synaptic bouton following action potential stimulation. However, spontaneous release of neurotransmitter also occurs in the absence of stimulation in virtually all synaptic boutons. It has long been thought that this represents exocytosis driven by fluctuations in local Ca2+ levels. The vesicles responding to these fluctuations are thought to be the same ones that release upon stimulation, albeit potentially triggered by different Ca2+ sensors. This view has been challenged by several recent works, which have suggested that spontaneous release is driven by a separate pool of synaptic vesicles. Numerous articles appeared during the last few years in support of each of these hypotheses, and it has been challenging to bring them into accord. We speculate here on the origins of this controversy, and propose a solution that is related to developmental effects. Constitutive membrane traffic, needed for the biogenesis of vesicles and synapses, is responsible for high levels of spontaneous membrane fusion in young neurons, probably independent of Ca2+. The vesicles releasing spontaneously in such neurons are not related to other synaptic vesicle pools and may represent constitutively releasing vesicles (CRVs) rather than bona fide synaptic vesicles. In mature neurons, constitutive traffic is much dampened, and the few remaining spontaneous release events probably represent bona fide spontaneously releasing synaptic vesicles (SRSVs) responding to Ca2+ fluctuations, along with a handful of CRVs that participate in synaptic vesicle turnover.

Versatile cellular uptake mediated by catanionic vesicles: simultaneous spontaneous membrane fusion and endocytosis.

Lactose-derived catanionic vesicles offer unique opportunities to overcome cellular barriers. These potential nanovectors, very easy to formulate as drug delivery systems, are able to encapsulate drugs of various hydrophilicity. This article highlights versatile interaction mechanisms between these catanionic vesicles, labeled with hydrophilic and amphiphilic fluorescent probes, and a mammalian cell line, Chinese Hamster Ovary. Confocal microscopy and flow cytometry techniques show that these vesicles are internalized by cells through cellular energy dependent processes, as endocytosis, but are simultaneously able to spontaneously fuse with cell plasma membranes and release their hydrophilic content directly inside the cytosol. Such innovative and polyvalent nanovectors, able to deliver their content via different internalization pathways, would positively be a great progress for the coadministration of drugs of complementary efficiency.

Complexin facilitates exocytosis and synchronizes vesicle release in two secretory model systems

Complexins (Cplxs) are small, SNARE-associated proteins believed to regulate fast, calcium-triggered exocytosis. However, studies have pointed to either an inhibitory and/or facilitatory role in exocytosis, and the role of Cplxs in synchronizing exocytosis is relatively unexplored. Here, we compare the function of two types of complexin, Cplx 1 and 2, in two model systems of calcium-dependent exocytosis. In mouse neuromuscular junctions (NMJs), we find that lack of Cplx 1 significantly reduces and desynchronizes calcium-triggered synaptic transmission; furthermore, high-frequency stimulation elicits synaptic facilitation, instead of normal synaptic depression, and the degree of facilitation is highly sensitive to the amount of cytoplasmic calcium buffering. In Cplx 2-null adrenal chromaffin cells, we also find decreased and desynchronized evoked release, and identify a significant reduction in the vesicle pool close to the calcium channels (immediately releasable pool, IRP). Viral transduction with either Cplx 1 or 2 rescues both the size of the evoked response and the synchronicity of release, and it restores the IRP size. Our findings in two model systems are mutually compatible and indicate a role of Cplx 1 and 2 in facilitating vesicle priming, and also lead to the new hypothesis that Cplxs may synchronize vesicle release by promoting coupling between secretory vesicles and calcium channels.

Juxtamembrane tryptophans of synaptobrevin 2 control the process of membrane fusion

Synaptobrevin 2 (Syb2), syntaxin (Sx1A), and SNAP-25, generate a force to induce fusion pore formation. The v-SNARE, Syb2, is anchored to the vesicle membrane by a single transmembrane domain. Here we show that 2 tryptophans (W89/W90) located in the juxtamembrane domain of Syb2, which stabilize the transmembrane (TM) domain position, control the ratio of spontaneous vs. stimulated membrane fusion events in chromaffin cells. Changing the 2 hydrophobic tryptophans to neutral alanines promotes spontaneous membrane fusion, faster transmitter release kinetics and complete release from individual vesicles. The results indicate that the two tryptophans act as a fusion clamp making fusion stimulus-dependent.

Interaction between GUVs and catanionic nanocontainers: new insight into spontaneous membrane fusion

Spontaneous receptor-free membrane fusion with pure lipid systems, used as a cell membrane model, is demonstrated with easy-to-handle lactose-derived catanionic vesicles. This fusion, mediated and controlled by phospholipids, emphasizes the great value of these nanovesicles for enhanced direct cytosolic drug delivery without the shortcomings linked with endocytic pathways.

Engineering Cell Surfaces via Liposome Fusion

In this study, we have rewired cell surfaces with ketone and oxyamine molecules based on liposome fusion for applications in cell-surface engineering. Lipid vesicles, functionalized with ketone and oxyamine molecules, display complementary chemistry and undergo recognition, docking, and subsequent fusion upon covalent oxime bond formation. Liposome fusion was characterized by several techniques including matrix-assisted laser-desorption/ionization mass spectrometry (MALDI-MS), light scattering, fluorescence resonance energy transfer (FRET), and transmission electron microscopy (TEM). When cultured with cells, ketone- and oxyamine-containing liposomes undergo spontaneous membrane fusion to present the respective molecules from cell surfaces. Ketone-functionalized cell surfaces serve as sites for chemoselective ligation with oxyamine-conjugated molecules. We tailored and fluorescently labeled cell surfaces with an oxyamine-conjugated rhodamine dye. As an application of this cell-surface engineering strategy, ketone- and oxyamine-functionalized cells were patterned on oxyamine- and ketone-presenting surfaces, respectively. Cells adhered, spread, and proliferated in the patterned regions via interfacial oxime linkage. The number of ketone molecules on the cell surface was also quantified by flow cytometry.

The Studies on Reaction Control and Development of New Practical Reagents Based on Characteristics of Reaction Field

Reaction rates and selectivities can be critically affected by the reaction field. Using a diverse set of reagents and reaction systems, the author reviews a variety of ways to control the reaction field. In the first example, we discuss 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-MM), which serves as an exceptionally convenient reagent for dehydrocondensation. In particular, formation of carboxamide by DMT-MM was found to take place even if water or alcohol were used as a reaction medium. Thus, chemical modification of carboxyl groups and/or amino groups of highly polar substrates, such as amino acid derivatives, peptides, glyco-chains, and nucleotides, can be simply effected by mixing them with DMT-MM in aqueous or alcoholic solvents. The author also found that a tertiary amine catalyzes the activation step of carboxylic acid with 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) via in situ generation of coupling reagents. Proceeding further in this direction, we determined that artificial acyltransferase and cyclotransferase could be formed by conjugation of a tertiary amine catalyst to host-molecules to mimic a substrate binding site. Finally, the micellar interface, well known for promoting hydrolysis, clearly provides a superior reaction field for dehydrocondensation. When a 1,3,5-triazine-type amphiphilic dehydrocondensing agent was used, bimolecular dehydrocondensation between amphiphilic carboxylate and amines was highly accelerated (2000-fold) in a micellar system. Spontaneous membrane fusion was induced by adoption of the micellar reaction in the ceramide synthesis at the surface of membranes. All together, these diverse findings strongly support the central importance of the reaction field in controlling reaction rates and selectivity.

Spontaneous Membrane Fusion Induced by Chemical Formation of Ceramides in a Lipid Bilayer

Mere chemical generation of ceramide and related double-chain lipids in the membrane of small unilamellar vesicles (SUVs) induces fusion of the vesicles. The lipids can be successfully prepared by dehydrocondensation between single-chain lipids (fatty acids and sphingosine or its analogues) in a lipid bilayer of the SUV by using a combination of 2-chloro-4,6-dimethoxy-1,3,5-triazine and amphiphilic tertiary amine catalysts, a process that can be compared to a successive enzyme model system for a fatty acyl-CoA synthetase followed by acyltransferase. The SUV spontaneously undergoes membrane fusion upon this internal chemical stimulation by the artificial enzyme system.

Regulated exocytosis and SNARE function (Review).

The pairing of cognate v- and t-SNAREs between two opposing lipid bilayers drives spontaneous membrane fusion and confers specificity to intracellular membrane trafficking. These fusion events are regulated by a cascade of protein-protein interactions that locally control SNARE activity and complex assembly, determining when and where fusion occurs with high efficiency in vivo. This basic regulation occurs at all transport steps and is mediated by conserved protein families such as Rab proteins and their effectors and Sec1/unc18 proteins. Regulated exocytosis employs auxiliary components that couple the signal (which triggers exocytosis) to the fusion machinery. At the neuronal synapse, munc13 as well as munc18 control SNARE complex assembly. Synaptotagmin and complexin ensure fast synchronous calcium-evoked neurotransmitter release.

Molecular Dynamics Simulation of Spontaneous Membrane Fusion during a Cubic-Hexagonal Phase Transition

We report a molecular dynamics simulation of the phase transition of monoolein from an inverted cubic phase to an inverted hexagonal phase. The transition proceeds via an intermediate structure consisting of water channels in a cubic geometry, in agreement with the predictions of the modified stalk theory ( Siegel, 1999). Two mechanisms are identified by which the topology changes during the transition. Bilayer fusion proceeds via the formation of trans-monolayer contacts, whereas bilayer rupture is observed as a gradual thinning of each monolayer.