Accelerating SNARE‐Mediated Membrane Fusion by DNA–Lipid Tethers

SNARE proteins are the core machinery to drive fusion of a vesicle with its target membrane. Inspired by the tethering proteins that bridge the membranes and thus prepare SNAREs for docking and fusion, we developed a lipid‐conjugated ssDNA mimic that is capable of regulating SNARE function, in situ. The DNA–lipid tethers consist of a 21 base pairs binding segment at the membrane distal end that can bridge two liposomes via specific base‐pair hybridization. A linker at the membrane proximal end is used to control the separation distance between the liposomes. In the presence of these artificial tethers, SNARE‐mediated lipid mixing is significantly accelerated, and the maximum fusion rate is obtained with the linker shorter than 40 nucleotides. As a programmable tool orthogonal to any native proteins, the DNA–lipid tethers can be further applied to regulate other biological processes where capturing and bridging of two membranes are the prerequisites for the subsequent protein function.     

Selective assembly of photosynthetic antenna proteins into a domain-structured lipid bilayer for the construction of artificial photosynthetic antenna systems: structural analysis of the assembly using surface plasmon resonance and atomic force microscopy

Two types of photosynthetic membrane proteins, the peripheral antenna complex (LH2) and the core antenna/reaction center complex (LH1-RC), play an essential role in the primary process of purple bacterial photosynthesis, that is, capturing light energy, transferring it to the RC where it is used in subsequent charge separation. Establishment of experimental platforms is required to understand the function of the supramolecular assembly of LH2 and LH1-RC molecules into arrays. In this study, we assembled LH2 and LH1-RC arrays into domain-structured planar lipid bilayers placed on a coverglass using stepwise combinations of vesicle-to-planar membrane formation and vesicle fusion methods. First, it was shown that assembly of LH2 and LH1-RC in planar lipid bilayers, through vesicle-to-planar membrane formation, could be confirmed by absorption spectroscopy and high resolution atomic force microscopy (AFM). Second, formation of a planar membrane incorporating LH2 molecules made by the vesicle fusion method was corroborated by AFM together with quantitative analysis by surface plasmon resonance (SPR). By combining planar membrane formation and vesicle fusion, in a stepwise manner, LH2 and LH1-RC were successfully organized in the domain-structured planar lipid membrane. This methodology for construction of LH2/LH1-RC assemblies will be a useful experimental platform with which to investigate energy transfer from LH2 to LH1-RC where the relative arrangement of these two complexes can be controlled.     

Coarse-grained transmembrane proteins: hydrophobic matching, aggregation, and their effect on fusion

Molecular transport between organelles is predominantly governed by vesicle fission and fusion. Unlike experimental vesicles, the fused vesicles in molecular dynamics simulations do not become spherical readily, because the lipid and water distribution is inappropriate for the fused state and spontaneous amendment is slow. Here, we study the hypothesis that enhanced transport across the membrane of water, lipids, or both is required to produce spherical vesicles. This is done by adding several kinds of model proteins to fusing vesicles. The results show that equilibration of both water and lipid content is a requirement for spherical vesicles. In addition, the effect of these transmembrane proteins is studied in bilayers and vesicles, including investigations into hydrophobic matching and aggregation. Our simulations show that the level of aggregation does not only depend on hydrophobic mismatch, but also on protein shape. Additionally, one of the proteins promotes fusion by inducing pore formation. Incorporation of these proteins allows even flat membranes to fuse spontaneously. Moreover, we encountered a novel spontaneous vesicle enlargement mechanism we call the engulfing lobe, which may explain how lipids added to a vesicle solution are quickly incorporated into the inner monolayer.     

Barcoding Biological Reactions with DNA‐Functionalized Vesicles

Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase‐segregated membranes, promote fusion between specific vesicle populations. Membrane phase‐segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA‐mediated fusion events. The orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. Vesicle fusion between DNA‐tethered vesicles can be used to initiate in vitro protein expression to produce model soluble and membrane proteins. Engineering orthogonal fusion events between DNA‐tethered vesicles provides a new strategy to control the spatiotemporal dynamics of cell‐free reactions, expanding opportunities to engineer artificial cellular systems.     

Synaptic vesicle proteins under conditions of rest and activation: analysis by 2-D difference gel electrophoresis

Synaptic vesicles are organelles of the nerve terminal that secrete neurotransmitters by fusion with the presynaptic plasma membrane. Vesicle fusion is tightly controlled by depolarization of the plasma membrane and a set of proteins that may undergo post-translational modifications such as phosphorylation. In order to identify proteins that undergo modifications as a result of synaptic activation, we induced massive exocytosis and analysed the synaptic vesicle compartment by benzyldimethyl-n-hexadecylammonium chloride (BAC)/SDS-PAGE and difference gel electrophoresis (DIGE) followed by MALDI-TOF-MS. We identified eight proteins that revealed significant changes in abundance following nerve terminal depolarization. Of these, six were increased and two were decreased in abundance. Three of these proteins were phosphorylated as detected by Western blot analysis. In addition, we identified an unknown synaptic vesicle protein whose abundance increased on synaptic activation. Our results demonstrate that depolarization of the presynaptic compartment induces changes in the abundance of synaptic vesicle proteins and post-translational protein modification.     

A novel approach to analyze membrane proteins by laser mass spectrometry: From protein subunits to the integral complex

A novel laser-based mass spectrometry method termed LILBID (laser-induced liquid bead ion desorption) is applied to analyze large integral membrane protein complexes and their subunits. In this method the ions are IR-laser desorbed from aqueous microdroplets containing the hydrophobic protein complexes solubilized by detergent. The method is highly sensitive, very efficient in sample handling, relatively tolerant to various buffers, and detects the ions in narrow, mainly low-charge state distributions. The crucial experimental parameter determining whether the integral complex or its subunits are observed is the laser intensity: At very low intensity level corresponding to an ultrasoft desorption, the intact complexes, together with few detergent molecules, are transferred into vacuum. Under these conditions the oligomerization state of the complex (i.e., its quaternary structure) may be analyzed. At higher laser intensity, complexes are thermolyzed into subunits, with any residual detergent being stripped off to yield the true mass of the polypeptides. The model complexes studied are derived from the respiratory chain of the soil bacterium Paracoccus denitrificans and include complexes III (cytochrome bc(1) complex) and IV (cytochrome c oxidase). These are well characterized multi-subunit membrane proteins, with the individual hydrophobic subunits being composed of up to 12 transmembrane helices.     

SNARE derived peptide mimic inducing membrane fusion

SNARE proteins mediate membrane fusion between synaptic vesicles and the plasma membrane. A minimized peptide SNARE model system with reduced complexity was introduced combining the native SNARE transmembrane (TMD) and linker domains with artificial coiled-coil forming peptides. Specific membrane fusion initiated by coiled-coil recognition was shown by lipid and content mixing vesicle assays.     

Self-assembly of solid-supported membranes using a triggered fusion of phospholipid-enriched proteoliposomes prepared from the inner mitochondrial membrane

A general procedure for the formation ofsolid-supported artificial membranes containing transmembrane proteins is reported. The main objective was to directly use the pool of proteins of the native biomembrane (here the inner membrane from mitochondria of human carcinogenic hepatic cells) and to avoid purification steps with detergent. Proteoliposomes of phospholipid-enriched inner membranes from mitochondria were tethered and fused onto a tailored surface via a streptavidin link. The failure of some preliminary experiments on membrane formation was attributed to strong nonspecific interactions between the solid surface and the protuberant hydrophilic parts of the transmembrane complexes. The correct loading of uniform membranes was performed after optimization of a tailored surface, covered with a grafted short-chain poly(ethylene glycol), so that nonspecific interactions are reduced. Step-by-step assembly of the structure and triggered fusion of the immobilized proteoliposomes were monitored by surface plasmon resonance and fluorescence photobleaching recovery, respectively. The long-range lateral diffusion coefficient (at 22 degrees C) for a fluorescent lipid varies from 2.5 x 10(-8) cm2 s(-1) for a tethered lipid bilayer without protein to 10(-9) cm2 s(-1) for a tethered membrane containing the transmembrane proteins of the respiratory chain at a protein area fraction of about 15%. The decrease in the diffusion coefficient in the tethered membrane with increase in protein area fraction was too pronounced to be fully explained by the theoretical models of obstructed lateral diffusion. Covalent tethering links with the solid are certainly involved in the decrease of the overall lateral mobility of the components in the supported membrane at the highest protein-to-lipid ratios.     

Real-time membrane fusion of giant polymer vesicles

Membrane fusion is very important for the formation of many complex organs in metazoans throughout evolution, such as muscles, bones, and placentae. Lipid vesicles (liposomes) are frequently used as model membranes to study the fusion process. This work demonstrates for the first time the real-time membrane fusion of giant polymer vesicles by directly displaying a series of high-resolution and real-time transformation images of individual vesicles. The fusion process includes the sequential steps of membrane contact, forming the center wall, symmetric expansion of fusion pore and complete fusion, undergoing the intermediates of "8" shape with a protruding rim at the contact site, peanut (pear) shape, and oblate sphere. The vesicle swells during fusion, and the fusing vesicle only deforms in the neck domain around the fusion pore in the lateral direction, which verifies the importance of the lateral tension on the fusion pore at the vesicle deformation level. The successful fusion of the synthetic and protein-free polymer vesicles reported here also supports that vesicle proximity combined with membrane perturbation suffices to induce membrane fusion, and that the protein is not necessary for the fusion process.     

Dissipative particle dynamic simulation study of lipid membrane

The lipid membrane plays crucial roles in countless biologic processes, ranging from cell motility, endo- and exocytosis, and cell division to protein aggregation and trafficking. To gain a molecular insight in these biologic processes, the recently developed mesoscale simulation technique, dissipative particle dynamics (DPD) simulation, has become an invaluable tool. By providing a brief survey of existing atomistic and popular coarse-grained models used today in studying the dynamics (including vesicle formation and (protein-mediated) vesicle fusion) and phase behavior of lipid bilayers, this review illustrates how mesoscopic DPD models can be used to obtain a better understanding of these biologic processes currently inaccessible to atomistic and most coarse-grained models.     

Evidence for Multiple Binding Modes in the Initial Contact Between SARS-CoV-2 Spike S1 Protein and Cell Surface Glycans**

Infection of host cells by SARS-CoV-2 begins with recognition by the virus S (spike) protein of cell surface heparan sulfate (HS), tethering the virus to the extracellular matrix environment, and causing the subunit S1-RBD to undergo a conformational change into the ‘open’ conformation. These two events promote the binding of S1-RBD to the angiotensin converting enzyme 2 (ACE2) receptor, a preliminary step toward viral-cell membrane fusion. Combining ligand-based NMR spectroscopy with molecular dynamics, oligosaccharide analogues were used to explore the interactions between S1-RBD of SARS CoV-2 and HS, revealing several low-specificity binding modes and previously unidentified potential sites for the binding of extended HS polysaccharide chains. The evidence for multiple binding modes also suggest that highly specific inhibitors will not be optimal against protein S but, rather, diverse HS-based structures, characterized by high affinity and including multi-valent compounds, may be required.     

Bioinspired vesicle restraint and mobilization using a biopolymer scaffold

Biology employs vesicles to package molecules (e.g., neurotransmitters) for their targeted delivery in response to specific spatiotemporal stimuli. Biology is also capable of employing localized stimuli to exert an additional control on vesicle trafficking; intact vesicles can be restrained (or mobilized) by association with (or release from) a cytoskeletal scaffold. We mimic these capabilities by tethering vesicles to a biopolymer scaffold that can undergo (i) stimuli-responsive network formation (for vesicle restraint) and (ii) enzyme-catalyzed network cleavage (for vesicle mobilization). Specifically, we use the aminopolysaccharide chitosan as our scaffold and graft a small number of hydrophobic moieties onto its backbone. These grafted hydrophobes can insert into the bilayer to tether vesicles to the scaffold. Under acidic conditions, the vesicles are not restrained by the hydrophobically modified chitosan (hm-chitosan) because this scaffold is soluble. Increasing the pH to neutral or basic conditions allows chitosan to form interpolymer associations that yield a strong, insoluble restraining network. Enzymatic hydrolysis of this scaffold by chitosanase cleaves the network and mobilizes intact vesicles. Potentially, this approach will provide a controllable means to store and liberate vesicle-based reagents/therapeutics for microfluidic/medical applications.     

Fabrication of highly insulating tethered bilayer lipid membrane using yeast cell membrane fractions for measuring ion channel activity

A tethered bilayer lipid membrane (tBLM) was fabricated on a gold electrode using 1,2-dipalmitoyl-sn-glycero-phosphothioethanol as a tethering lipid and the membrane fractions of Saccharomyces pombe yeast cells to deposit the upper leaflet. The membrane fractions were characterized using transmission electron microscopy and dynamic light scattering and found to be similar in size to small unilamellar vesicles of synthetic lipids. The dynamics of membrane-fraction deposition and rupture on the tethering-lipid layer were measured using quartz crystal microgravimetry. The electrochemical properties of the resulting tBLM were characterized using electrical impedance spectroscopy and cyclic voltammetry. The tBLM's electrical resistance was greater than 1 MOmegacm(2), suggesting a defect-free membrane. The suitability of tBLM produced using membrane fractions for measuring ion-channel activities was shown by a decrease in membrane resistance from 1.6 to 0.43 MOmegacm(2) following addition of gramicidin. The use of membrane fractions to form high-quality tBLM on gold electrodes suggests a new approach to characterize membrane proteins, in which the upper leaflet of the tBLM is deposited, and overexpressed membrane proteins are incorporated, in a single step. This approach would be especially useful for proteins whose activity is lost or altered during extraction, purification, and reconstitution, or whose activities are strongly influenced by the lipid composition of the bilayer.     

Determinants for membrane fusion induced by cholesterol-modified DNA zippers

Intracellular membrane fusion is coordinated by membrane-anchored fusion proteins. The cytosolic domains of these proteins form a specific complex that pulls the membranes into close proximity. Although some results indicate that membrane merger can be accomplished solely on the basis of proximity, others emphasize the importance of bilayer stress exerted by transmembrane peptides. In a reductionist approach, we recently introduced a fusion machinery built from cholesterol-modified DNA zippers to mimic fusion protein function. Aiming to further optimize DNA-mediated fusion, we varied in this work length and number of DNA strands and used either one or two cholesterol groups for membrane anchoring of DNA. The results reveal that the use of two cholesterol anchors is essential to prevent cDNA strands from shuttling to the same membrane, which leads to vesicle release instead of membrane merger. A surface coverage of 6-13 DNA strands was a precondition for efficient fusion, whereas fusion was insensitive to DNA length within the tested range. Besides lipid mixing, we also demonstrate DNA-induced content mixing of large unilamellar vesicles composed of the most abundant cellular lipids phosphatidylcholine, phosphatidylethanolamine, cholesterol, and sphingomyelin. Taken together, DNA-mediated fusion emerges as a promising tool for the functionalization of artificial and biological membranes and may help to dissect the functional role of fusion proteins.     

Single-cell FRET imaging of transferrin receptor trafficking dynamics by Sfp-catalyzed, site-specific protein labeling

Fluorescence imaging of living cells depends on an efficient and specific method for labeling the target cellular protein with fluorophores. Here we show that Sfp phosphopantetheinyl transferase-catalyzed protein labeling is suitable for fluorescence imaging of membrane proteins that spend at least part of their membrane trafficking cycle at the cell surface. In this study, transferrin receptor 1 (TfR1) was fused to peptide carrier protein (PCP), and the TfR1-PCP fusion protein was specifically labeled with fluorophore Alexa 488 by Sfp. The trafficking of transferrin-TfR1-PCP complex during the process of transferrin-mediated iron uptake was imaged by fluorescence resonance energy transfer between the fluorescently labeled transferrin ligand and TfR1 receptor. We thus demonstrated that Sfp-catalyzed small molecule labeling of the PCP tag represents a practical and efficient tool for molecular imaging studies in living cells.     

Docking of photosystem I subunit C using a constrained geometric simulation

The elucidation of assembly pathways of multi-subunit protein complexes is a problem of great interest in structural biology and biomolecular modeling. In this study, we use a new computer algorithm for the simulation of large-scale motion in proteins to dock the subunit PsaC onto Photosystem I. We find that a complicated docking pathway involving multiple conformational changes can be quickly simulated by actively targeting only a few residues at a time to their target positions. Simulations for two possible docking scenarios are explored, and experimental approaches to distinguish between them are discussed.     

Functional redundancy between <Emphasis Type="Italic">trans</Emphasis>-Golgi network SNARE family members in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background

Vesicle fusion is an essential process for maintaining the structure and function of the endomembrane system. Fusion is mediated by t-SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) fusion proteins on the target membrane and v-SNAREs on the vesicle membrane; v-and t-SNAREs interact with each other, driving vesicle fusion with the target membrane. The Arabidopsis thaliana trans-Golgi network resident SNAREs SYP41 and VTI12, along with YKT61/62, have been shown to function in vesicle fusion in vitro, consistent with immunoprecipitation results showing their interaction in Arabidopsis cell extracts. Conflicting published results have indicated that SYP4 family members are either functionally redundant or have distinct and essential functions; the reason for this discrepancy is unclear.

Results

Here we used a proteoliposome fusion assay to demonstrate that SYP42 and SYP43 can substitute for SYP41 in driving lipid mixing, providing support for functional overlap between family members. Previous reports have also suggested that VTI11 and VTI12 SNAREs show partial overlap in function, despite having mostly distinct localizations and binding partners. We show that VTI11 can substitute for VTI12 in in vitro lipid mixing reactions, providing molecular support for the genetic evidence for partial functional redundancy in vivo.

Conclusions

Our data provide biochemical evidence for functional overlap in membrane fusion between members of the SYP4 or VTI1 SNARE groups, supporting previous genetic data suggesting redundancy.

     

Directed aggregation and fusion of lipid vesicles induced by DNA-surfactants

We investigated DNA-directed aggregation of vesicles using DNA-surfactants. Following tethering of single-stranded DNA oligonucleotides to vesicles using DNA-surfactant, the tethered vesicles were assembled with other vesicles bearing complementary strands. The vesicle aggregation was strongly affected by the salt concentration and by temperature according to the characteristics of DNA hybridization. Restriction enzyme, which can hydrolyze the double-stranded DNA used in the present study, dissociated the vesicle aggregates. Exploration using fluorescently labeled vesicles suggested that the DNA-directed vesicle aggregation took place in a sequence-specific manner through DNA-duplex formation. Interestingly, the DNA-directed aggregation using short DNA-surfactant induced the fusion of vesicles to produce giant vesicles, resulting in an enzymatic reaction in the giant vesicle.     

Model cell membranes: Techniques to form complex biomimetic supported lipid bilayers via vesicle fusion

Vesicle fusion has long provided an easy and reliable method to form supported lipid bilayers (SLBs) from simple, zwitterionic vesicles on siliceous substrates. However, for complex compositions, such as vesicles with high cholesterol content and multiple lipid types, the energy barrier for the vesicle-to-bilayer transition is increased or the required vesicle–vesicle and vesicle–substrate interactions are insufficient for vesicle fusion. Thus, for vesicle compositions that more accurately mimic native membranes, vesicle fusion often fails to form SLBs. In this paper, we review three approaches to overcome these barriers to form complex, biomimetic SLBs via vesicle fusion: (i) optimization of experimental conditions (e.g., temperature, buffer ionic strength, osmotic stress, cation valency, and buffer pH), (ii) α-helical (AH) peptide-induced vesicle fusion, and (iii) bilayer edge-induced vesicle fusion. AH peptide-induced vesicle fusion can form complex SLBs on multiple substrate types without the use of additional equipment. Bilayer edge-induced vesicle fusion uses microfluidics to form SLBs from vesicles with complex composition, including vesicles derived from native cell membranes. Collectively, this review introduces vesicle fusion techniques that can be generalized for many biomimetic vesicle compositions and many substrate types, and thus will aid efforts to reliably create complex SLB platforms on a range of substrates.