Lipid Reorganization Research Articles

Toward understanding lipid reorganization in RNA lipid nanoparticles in acidic environments

The use of lipid nanoparticles (LNPs) for therapeutic RNA delivery has gained significant interest, particularly highlighted by recent milestones such as the approval of Onpattro and two mRNA-based SARS-CoV-2 vaccines. However, despite substantial advancements in this field, our understanding of the structure and internal organization of RNA-LNPs -and their relationship to efficacy, both in vitro and in vivo- remains limited. In this study, we present a coarse-grained molecular dynamics (MD) approach that allows for the simulations of full-size LNPs. By analyzing MD-derived structural characteristics in conjunction with cellular experiments, we investigate the effect of critical parameters, such as pH and composition, on LNP structure and potency. Additionally, we examine the mobility and chemical environment within LNPs at a molecular level. Our findings highlight the significant impact that LNP composition and internal molecular mobility can have on key stages of LNP-based intracellular RNA delivery.

Insertion and Anchoring of HIV-1 Fusion Peptide into Complex Membrane Mimicking Human T-cell.

A fundamental understanding of how HIV-1 envelope (Env) protein facilitates fusion is still lacking. The HIV-1 fusion peptide, consisting of 15 to 22 residues, is the N-terminus of the gp41 subunit of the Env protein. Further, this peptide, a promising vaccine candidate, initiates viral entry into target cells by inserting and anchoring into human immune cells. The influence of membrane lipid reorganization and the conformational changes of the fusion peptide during the membrane insertion and anchoring processes, which can significantly affect HIV-1 cell entry, remains largely unexplored due to the limitations of experimental measurements. In this work, we investigate the insertion of the fusion peptide into an immune cell membrane mimic through multiscale molecular dynamics simulations. We mimic the native T-cell by constructing a 9-lipid asymmetric membrane, along with geometrical restraints accounting for insertion in the context of gp41. To account for the slow timescale of lipid mixing while enabling conformational changes, we implement a protocol to go back and forth between atomistic and coarse-grained simulations. Our study provides a molecular understanding of the interactions between the HIV-1 fusion peptide and the T-cell membrane, highlighting the importance of conformational flexibility of fusion peptides and local lipid reorganization in stabilizing the anchoring of gp41 into the targeted host membrane during the early events of HIV-1 cell entry. Importantly, we identify a motif within the fusion peptide critical for fusion that can be further manipulated in future immunological studies.

Characterization of spatial lipidomic signatures in tick-bitten guinea pig skin as a model for host-vector-pathogen interaction profiling.

Here, we demonstrate the adaptability of spatial "omics" methods to identify interphylum processes regulated at the vector-host interface of ticks during a mammalian blood meal. This approach enables a better understanding of complex bipartite or tripartite molecular interactions between hosts, arthropod vectors and transmitted pathogens, and contributes toward the development of spatially aware therapeutic target discovery and description.

Lipid-driven interleaflet coupling of plasma membrane order regulates FcεRI signaling in mast cells

Interleaflet coupling—the influence of one leaflet on the properties of the opposing leaflet—is a fundamental plasma membrane organizational principle. This coupling is proposed to participate in maintaining steady-state biophysical properties of the plasma membrane, which in turn regulates some transmembrane signaling processes. A prominent example is antigen (Ag) stimulation of signaling by clustering transmembrane receptors for immunoglobulin E (IgE), FcεRI. This transmembrane signaling depends on the stabilization of ordered regions in the inner leaflet for sorting of intracellular signaling components. The resting inner leaflet has a lipid composition that is generally less ordered than the outer leaflet and that does not spontaneously phase separate in model membranes. We propose that interleaflet coupling can mediate ordering and disordering of the inner leaflet, which is poised in resting cells to reorganize upon stimulation. To test this in live cells, we first established a straightforward approach to evaluate induced changes in membrane order by measuring inner leaflet diffusion of lipid probes by imaging fluorescence correlation spectroscopy, by imaging fluorescence correlation spectroscopy (ImFCS), before and after methyl-α-cyclodexrin (mαCD)-catalyzed exchange of outer leaflet lipids (LEX) with exogenous order- or disorder-promoting phospholipids. We examined the functional impact of LEX by monitoring two Ag-stimulated responses: recruitment of cytoplasmic Syk kinase to the inner leaflet and exocytosis of secretory granules (degranulation). Based on the ImFCS data in resting cells, we observed global increase or decrease of inner leaflet order when outer leaflet is exchanged with order- or disorder-promoting lipids, respectively. We find that the degree of both stimulated Syk recruitment and degranulation correlates positively with LEX-mediated changes of inner leaflet order in resting cells. Overall, our results show that resting-state lipid ordering of the outer leaflet influences the ordering of the inner leaflet, likely via interleaflet coupling. This imposed lipid reorganization modulates transmembrane signaling stimulated by Ag clustering of IgE-FcεRI.

Activity modulation of the Escherichia coli F1FO ATP synthase by a designed antimicrobial peptide via cardiolipin sequestering

Most antimicrobial peptides (AMPs) exert their microbicidal activity through membrane permeabilization. The designed AMP EcDBS1R4 has a cryptic mechanism of action involving the membrane hyperpolarization of Escherichia coli, suggesting that EcDBS1R4 may hinder processes involved in membrane potential dissipation. We show that EcDBS1R4 can sequester cardiolipin, a phospholipid that interacts with several respiratory complexes of E.coli. Among these, F1FO ATP synthase uses membrane potential to fuel ATP synthesis. We found that EcDBS1R4 can modulate the activity of ATP synthase upon partition to membranes containing cardiolipin. Molecular dynamics simulations suggest that EcDBS1R4 alters the membrane environment of the transmembrane FO motor, impairing cardiolipin interactions with the cytoplasmic face of the peripheral stalk that binds the catalytic F1 domain to the FO domain. The proposed mechanism of action, targeting membrane protein function through lipid reorganization may open new venues of research on the mode of action and design of other AMPs.

Phase Transition and Phase Separation in Realistic Thylakoid Lipid Membrane of Marine Algae in All-Atom Simulations.

Thylakoid membranes are specialized membranes predominantly composed of uncommon galacto- and sulfolipids, having distinct roles in photosynthesis. Large acyl chain variety and richness in polyunsaturated fatty acid (PUFA) content of thylakoid lipids further add to the compositional complexity. The function of these membrane systems is intimately dependent on the fluidity of its lipid matrix, which is strongly modulated by the lipid composition and temperature. The present work, employing extensive atomistic simulations, provides the first atomistic view of the phase transition and domain coexistence in a model membrane composed of thylakoid lipids of a commercially important red alga Gracilaria corticata between 10 and 40 °C. The growth and photosynthetic activity of marine algae are greatly influenced by the seawater temperature. So far, little is known about the molecular organization of lipids in thylakoid membranes, in particular their adaptive arrangements under temperature stress. Our simulations show that the algal thylakoid membrane undergoes a transition from a gel-like phase at a low temperature, 10-15 °C, to a homogeneous liquid-crystalline phase at a high temperature, 40 °C. Clear evidence of spontaneous phase separation into coexisting nanoscale domains is detected at intermediate temperatures nearing the optimal growth temperature range. Particularly, at 25-30 °C, we identified the formation of a stable ripple phase, where the gel-like domains rich in saturated and nearly hexagonally packed lipids were separated from fluid-like domains enriched in lipids containing PUFA chains. The phase separation is driven by the spontaneous and preferential segregation of lipids into differentially ordered domains, mainly depending on the acyl chain types. Cholesterol impairs the phase transition and the emergence of domains and induces a fairly uniform liquid-ordered phase in the membrane over the temperatures studied. This work improves the understanding of the properties and reorganization of lipids in the thylakoid membrane in response to temperature variation.

Structural basis for the regulation mechanism of Ca2+-dependent activity of TMEM16 scramblase.

The collapse of the plasma membrane asymmetry by activated TMEM16 scramblases results in exposure of phosphatidylserine (PS), an essential trigger for multiple fundamental physiological processes. Functionally, Ca2+ binding to two conserved sites activates all TMEM16's scramblases. In contrast, Ca2+ binding leads to opening of a hydrophilic groove only in some homologues, whereas in others no opening is seen. Two questions thus arise: first, what are the structural determinants that enable groove opening in some TMEM16 homologues? Second, what are the conformational rearrangements that regulate Ca2+ dependent scrambling in the absence of groove opening? Here we used cryoEM to determine the structure of the fungal nhTMEM16 scramblase in nanodiscs in three states: Ca2+-free closed, Ca2+-bound closed and Ca2+-bound open. We find that the sequential appearance of two π-helical turns on the TM6 helix correlates with the stepwise conformational transitions from the Ca2+-free closed to Ca2+-bound closed and to the open state. Mutagenesis of three residues responsible for stabilizing the second π-helical turn prevented groove opening and trapped nhTMEM16 in the intermediate Ca2+-bound closed state, suggesting this is a necessary step in groove opening. The 2.6 Å resolution of the cryoEM map in the Ca2+-bound closed state enables us to visualize how lipid reorganization outside a closed groove results in a pronounced thinning of the membrane that could facilitate scrambling. In the absence of Ca2+ and in mutants displaying reduced scrambling activity we observe more pronounced bending of the TM6 and reduced thinning. We propose that the Ca2+ dependent straightening of the TM6 helix promotes scrambling by increasing membrane thinning. These findings provide the structural basis for the regulation of Ca2+-dependent activity of TMEM16 scramblase, which could be extended to other homologues with a similar fold.

Complex, convoluted, yet consistent: Protein-induced membrane remodeling around GPCRs.

Proteins embedded in cell membranes are the targets for ∼70% of all FDA-approved drugs. A single family of membrane proteins, called ‘GPCRs’ account for almost one third of all drug targets. These cell membranes are highly complicated environments, made up of many different types of constituent lipid molecules. Experimentally, the use of different types of lipids in the membrane that surrounds the protein is known to modulate the protein behavior. However, when designing or testing novel therapeutics, the influence of the membrane is often ignored; 50% of the environment is overlooked. Thus, studies carried out to optimize therapeutics can potentially be misleading - it is possible for the protein to respond differently depending on the properties of the membrane. Using novel multiscale simulations, and a sophisticated membrane model, we demonstrate just how complex and intricate the protein-lipid interaction are. Indeed, the property-altering interactions between the proteins and lipids are reciprocal. While lipids can induce changes in protein behavior, the protein can also drive either changes in lipid properties or reorganization of the lipids. The systems acclimatize and adjust to external stimuli to provide a more consistent, reliable environment for the protein to complete its function. The fine-tuning adaptation of the system further highlights the symbiotic nature of the membrane and the protein such that they should not be considered separate elements but treated as a single multicomponent drug target. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory under Contract DE-AC52-07NA27344 and was supported by the LLNL-LDRD Program under Project No. 21-ERD-047. LLNL-ABS-840667.

Surface-functionalized polystyrene nanoparticles alter transmembrane potential via ion-selective pores maintaining global bilayer integrity.

Although nanoplastics have well-known toxic effects towards the environment and living organisms, their molecular toxicity mechanisms, including the nature of nanoparticle-cell membrane interactions, are still under investigation. We employ dynamic light scattering (DLS), quartz crystal microbalance with dissipation monitoring (QCM-D) and electrophysiology to investigate the interaction between polystyrene nanoparticles (PS NPs) and phospholipid membranes. Our results show that PS NPs adsorb onto lipid bilayers creating soft inhomogeneous films that include disordered defects. PS NPs form an integral part of the generated channels so that the surface functionalization and charge of the NP determine the pore conductive properties. The large difference in size between nanoparticle diameter and lipid bilayer thickness (∼60 nm vs ∼5 nm) suggests a particular and complex lipid-NP assembly that is able to maintain overall membrane integrity. In view of that, we suggest that nanoparticle-induced toxicity in cells could operate in more subtle ways than membrane disintegration, such as inducing lipid reorganization and transmembrane ionic fluxes that disrupt the membrane potential.

Structural determinants of REMORIN nanodomain formation in anionic membranes

Remorins are a family of multigenic plasma membrane phosphoproteins involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nanosized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains; however, the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here, we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated phosphatidylcholine, phosphatidylinositol phosphates, and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nanosized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces.

Surface-Functionalized Polystyrene Nanoparticles Alter the Transmembrane Potential via Ion-Selective Pores Maintaining Global Bilayer Integrity.

Although nanoplastics have well-known toxic effects toward the environment and living organisms, their molecular toxicity mechanisms, including the nature of nanoparticle-cell membrane interactions, are still under investigation. Here, we employ dynamic light scattering, quartz crystal microbalance with dissipation monitoring, and electrophysiology to investigate the interaction between polystyrene nanoparticles (PS NPs) and phospholipid membranes. Our results show that PS NPs adsorb onto lipid bilayers creating soft inhomogeneous films that include disordered defects. PS NPs form an integral part of the generated channels so that the surface functionalization and charge of the NP determine the pore conductive properties. The large difference in size between the NP diameter and the lipid bilayer thickness (∼60 vs ∼5 nm) suggests a particular and complex lipid-NP assembly that is able to maintain overall membrane integrity. In view of this, we suggest that NP-induced toxicity in cells could operate in more subtle ways than membrane disintegration, such as inducing lipid reorganization and transmembrane ionic fluxes that disrupt the membrane potential.

Enhanced Expansion and Reduced Kiss-and-Run Events in Fusion Pores Steered by Synaptotagmin-1 C2B Domains

The fusion pore controls the release of exocytotic vesicle contents through a precise orchestration of lipids from the fusing membranes and proteins. There is a major lipid reorganization during the different stages in life of the fusion pore (membrane fusion, nucleation, and expansion) that can be scrutinized thermodynamically. In this work, using umbrella sampling simulations we describe the expansion of the fusion pore. We have calculated free energy profiles to drive a nascent, just nucleated, fusion pore to its expanded configuration. We have quantified the effects on the free energy of one and two Synaptotagmin-1 C2B domains in the cytosolic space. We show that C2B domains cumulatively reduce the cost for expansion, favoring the system to evolve toward full fusion. Finally, by conducting thousands of unbiased molecular dynamics simulations, we show that C2B domains significantly decrease the probability of kiss-and-run events.

Developmental Competence of equine MI oocytes following in vitro maturation culture

With an increasing demand for the in vitro production (IVP) of equine embryos over the past 10 years, the value of each oocyte recovered has also increased. Intracytoplasmic sperm injection (ICSI) is performed to fertilize the oocytes reaching MII stage of nuclear maturation, which is determined by microscopic evidence of an extruded polar body (PB). Cytoplasmic maturation, during which replication of organelles, proteins and RNA's necessary for postfertilization development occurs, is more difficult to evaluate. However, equine oocytes contain a large amount of cytoplasmic lipids and the migration and polarization of lipid granules can be microscopically visualized in mature oocytes. Furthermore, the distribution of lipids may reflect a level of cytoplasmic capacitation and developmental competence, although the cleavage rate of equine oocytes fertilized following maturation culture does not appear to be significantly different based on lipid polarization (Ambruosi et al. Therio 2009; 71:1093-1104). The present study evaluated the migration and polarization of cytoplasmic lipids (granularity) following in vitro maturation to determine if blastocyst development can occur in the absence of PB extrusion. Cumulus-oocyte-complexes (COC; n=411) obtained from ovarian follicles by transvaginal aspiration were stored in a holding medium overnight at 20°C before undergoing incubation at 38.5°C in 50ul drops of equilibrated maturation media as described by Foss. et al (EVJ Supp 2013;45:39-43). After 28.5 hours, oocytes were denuded followingexposure to 80IU hyaluronidase and evaluated for the presence of an extruded PB as well as lipid reorganization. The cytoplasm of MI oocytes that did not exhibit PB extrusion was graded according to the percentage with a dark granular appearance. All oocytes were fertilized by conventional ICSI with thawed spermatozoa from ICSI-fertile stallions. Fertilized oocytes were cultured as described by Foss et al (EVJ Suppl 2013;45:39-43). When examined under an inverted 1000X light microscope, 66% (N=273/411) of the denuded oocytes exhibited an intact membrane with an extruded polar body and were considered mature (MII), while 26% (N=106/411) of the oocytes without a visible polar body were classified as immature (MI). Of the MI oocytes injected, 23% (N=24/106) were deemed to have attained some level of cytoplasmic capacitation (Cc=40-70% granularity). A cleavage rate of 25.5% was recorded for all injected MI oocytes regardless of Cc status. Of the 24 MI oocytes with some Cc, nine (37%) cleaved and continued to develop, and seven of those developed to the blastocyst stage. Of the 82 MI oocytes that did not demonstrate Cc, 18 (22%) cleaved but no blastocysts (0%) were obtained. This study found a blastocyst developmental potential of 6.6% per total MI's injected and 29% per MI oocytes with at least a 40-70% Cc. Therefore, injecting oocytes that do not show nuclear maturation, yet have reached a level of cytoplasmic capacitation, may still result in blastocyst development.

Molecular Transport and Growth of Lipid Vesicles Exposed to Antimicrobial Peptides.

It is well-known that lipids constituting the cytoplasmic membrane undergo continuous reorganization to maintain the appropriate composition important for the integrity of the cell. The transport of lipids is controlled by mainly membrane proteins, but also spontaneous lipid transport between leaflets, lipid “flip–flop”, occurs. These processes do not only occur spontaneously under equilibrium, but also promote structural rearrangements, morphological transitions, and growth processes. It has previously been shown that intravesicular lipid “flip–flop” and intervesicular lipid exchange under equilibrium can be deduced indirectly from contrast variation time-resolved small-angle neutron scattering (TR-SANS) where the molecules are “tagged” using hydrogen/deuterium (H/D) substitution. In this work, we show that this technique can be extended to simultaneously detect changes in the growth and the lipid “flip–flop” and exchange rates induced by a peptide additive on lipid vesicles consisting of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine), d-DMPC (1,2-dimyristoyl-d54-sn-glycero-3-phosphocholine), DMPG (1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol)), and small amounts of DMPE-PEG (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000]). Changes in the overall size were independently monitored using dynamic light scattering (DLS). We find that the antimicrobial peptide, indolicidin, accelerates lipid transport and additionally induces limited vesicular growth. Moreover, in TR-SANS experiments using partially labeled lipid mixtures to separately study the kinetics of the lipid components, we show that, whereas peptide addition affects both lipids similarly, DMPG exhibits faster kinetics. We find that vesicular growth is mainly associated with peptide-mediated lipid reorganization that only slightly affects the overall exchange kinetics. This is confirmed by a TR-SANS experiment of vesicles preincubated with peptide showing that after pre-equilibration the kinetics are only slightly slower.

Rafting through the palms: S-acylation of SARS-CoV-2 spike protein induces lipid reorganization.

The spike protein of SARS-CoV-2 directs binding to the viral receptor, stimulating a membrane fusion reaction that allows virus entry. In this issue of Developmental Cell, Mesquita et al. (2021) demonstrate that S-acylation of spike protein protects it from degradation and triggers cholesterol/sphingolipid-rich raft-like domains to form, enabling membrane fusion.

Pore Forming Protein Induced Biomembrane Reorganization and Dynamics: A Focused Review.

Pore forming proteins are a broad class of pathogenic proteins secreted by organisms as virulence factors due to their ability to form pores on the target cell membrane. Bacterial pore forming toxins (PFTs) belong to a subclass of pore forming proteins widely implicated in bacterial infections. Although the action of PFTs on target cells have been widely investigated, the underlying membrane response of lipids during membrane binding and pore formation has received less attention. With the advent of superresolution microscopy as well as the ability to carry out molecular dynamics (MD) simulations of the large protein membrane assemblies, novel microscopic insights on the pore forming mechanism have emerged over the last decade. In this review, we focus primarily on results collated in our laboratory which probe dynamic lipid reorganization induced in the plasma membrane during various stages of pore formation by two archetypal bacterial PFTs, cytolysin A (ClyA), an α-toxin and listeriolysin O (LLO), a β-toxin. The extent of lipid perturbation is dependent on both the secondary structure of the membrane inserted motifs of pore complex as well as the topological variations of the pore complex. Using confocal and superresolution stimulated emission depletion (STED) fluorescence correlation spectroscopy (FCS) and MD simulations, lipid diffusion, cholesterol reorganization and deviations from Brownian diffusion are correlated with the oligomeric state of the membrane bound protein as well as the underlying membrane composition. Deviations from free diffusion are typically observed at length scales below ∼130 nm to reveal the presence of local dynamical heterogeneities that emerge at the nanoscale—driven in part by preferential protein binding to cholesterol and domains present in the lipid membrane. Interrogating the lipid dynamics at the nanoscale allows us further differentiate between binding and pore formation of β- and α-PFTs to specific domains in the membrane. The molecular insights gained from the intricate coupling that occurs between proteins and membrane lipids and receptors during pore formation are expected to improve our understanding of the virulent action of PFTs.

Bacterial protein listeriolysin O induces nonmonotonic dynamics because of lipid ejection and crowding

Membrane-bound protein complexes involving pore forming toxins (PFTs) released by virulent bacteria are known to form transmembrane pores leading to host cell lysis. Developing alternative strategies against PFT mediated bacterial virulence factors requires an understanding of the cellular membrane response. However, membrane disruption and related lipid reorganization events during attack by PFTs remain largely unexplored. We report counterintuitive and nonmonotonic variations in lipid diffusion, measured using confocal fluorescence correlation spectroscopy, due to interplay of lipid ejection and crowding by membrane-bound oligomers of a prototypical cholesterol-dependent cytolysin, listeriolysin O (LLO). The observed dynamical crossover is correlated with concentration dependent transitions of LLO oligomeric state populations from rings to arc-like pore complexes, predicted using a proposed two-state free area-based diffusion model. At low PFT concentrations, a hitherto unexplored regime of increased lipid diffusivity is attributed to lipid ejection events because of a preponderance of ring-like pore states. At higher protein concentrations in which membrane-inserted arc-like pores dominate, lipid ejection is less efficient and the ensuing crowding results in a lowering of lipid diffusion. These variations in lipid dynamics are corroborated by macroscopic rheological response measurements of PFT bound vesicles. Our study correlates PFT oligomeric state transitions, membrane remodeling, and mechanical property variations, providing unique insights into the pore forming mechanisms of cholesterol-dependent cytolysins.

Time-resolved SANS reveals pore-forming peptides cause rapid lipid reorganization

Time-resolved SANS showed alamethicin and melittin promote DMPC lipid vesicle mixing and perturb DMPC kinetics in similar ways.

Effects of Candelilla and Carnauba Wax Incorporation on the Functional Properties of Edible Sodium Caseinate Films.

The aim of this study was to evaluate the effects of candelilla (CAN) or carnauba wax (CAR) incorporation on functional properties of edible sodium caseinate (CAS) films. Glycerol and Tween-80 were used as the plasticizer and the emulsifier, respectively. The results showed that the incorporation of waxes increased film opacity, total color differences (∆E), and mechanical resistance and reduced film lightness, water vapor permeability (WVP), and elongation at break. Scanning electron microscopy showed heterogeneous structure of emulsion films with regular distribution of lipid particles. A different internal arrangement was observed as a function of the film composition with both layered and incorporated film structure. Films containing candelilla wax exhibited more regular lipid reorganization, which resulted in better water vapor barrier efficacy and mechanical resistance in comparison to control films. The presence of Tween-80 resulted in better dispersion of lipid particles in film-forming solutions and lower water solubility, lightness, film opacity, and water vapor permeability, whereas the total color differences (∆E) were significantly larger and the improvement in mechanical properties was also achieved.

The Synaptotagmin-1 C2B Domain Is a Key Regulator in the Stabilization of the Fusion Pore.

Fusion pores serve as an effective mechanism to connect intracellular organelles and release vesicle contents during exocytosis. A complex lipid rearrangement takes place as membranes approximate, bend, fuse, and establish a traversing water channel to define the fusion pore, linking initially isolated chambers. Thermodynamically, the process is unfavorable and thought to be mediated by specialized proteins. In this work, we have developed a reaction coordinate to induce fusion pores from initially flat and parallel lipid bilayers and we have used it to describe the effects of the synaptotagmin-1 C2B domain during the process. We have obtained free-energy profiles of the whole lipid reorganization in biologically realistic membranes, going from planar and parallel bilayers through stalk hemifusion to water channel formation. Our results point to a lysine-rich polybasic region on synaptotagmin-1 C2B as the key to lipid reorganization control through the formation of phosphatidylinositol bisphosphate clusters that stabilize the fusion pore.