A hemifused complex is the hub in a network of pathways to membrane fusion

Membrane fusion is required for essential processes from neurotransmission to fertilization. For over 40 years protein-free fusion driven by calcium or other cationic species has provided a simplified model of biological fusion, but the mechanisms remain poorly understood. Cation-mediated membrane fusion and permeation are essential in their own right to drug delivery strategies based on cell-penetrating peptides or cation-bearing lipid nanoparticles. Experimental studies suggest cations drive anionic membranes to a hemifused intermediate which serves as a hub for a network of pathways, but the pathway selection mechanism is unknown. Here we develop a mathematical model that identifies the network hub as a highly dynamical hemifusion complex. We find multivalent cations drive expansion of a high tension hemifusion interface between interacting vesicles during a brief transient. During this window, rupture of the interface competes with vesicle membrane rupture to determine the outcome, either fusion, dead-end hemifusion or vesicle lysis. The model reproduces the unexplained finding that fusion of vesicles with planar membranes typically stalls at hemifusion, and we show that the equilibrated hemifused state is a novel lens-shaped complex. Thus, membrane fusion kinetics follow a stochastic trajectory within a network of pathways, with outcome weightings set by fusogen concentration, vesicle size, lipid composition and geometry.

Fibril Growth Behavior of Amyloid β on Polymer-Based Planar Membranes: Implications for the Entanglement and Hydration of Polymers

The design of biosensors and artificial organs using biocompatible materials with a low affinity for amyloid β peptide (Aβ) would contribute to the inhibition of fibril growth causing Alzheimer’s disease. We systematically studied the amyloidogenicity of Aβ on various planar membranes. The planar membranes were prepared using biocompatible polymers, viz., poly(methyl methacrylate) (PMMA), polysulfone (PSf), poly(L-lactic acid) (PLLA), and polyvinylpyrrolidone (PVP). Phospholipids from biomembranes, viz., 1,2-dioleoyl-phosphatidylcholine (DOPC), 1,2-dipalmitoyl-phosphatidylcholine (DPPC), and polyethylene glycol-graft-phosphatidyl ethanolamine (PEG-PE) were used as controls. Phospholipid- and polymer-based membranes were prepared to determine the kinetics of Aβ fibril formation. Rates of Aβ nucleation on the PSf- and DPPC-based membranes were significantly higher than those on the other membranes. Aβ accumulation, calculated by the change in frequency of a quartz crystal microbalance (QCM), followed the order: PSf > PLLA > DOPC > PMMA, PVP, DPPC, and PEG-PE. Nucleation rates exhibited a positive correlation with the corresponding accumulation (except for the DPPC-based membrane) and a negative correlation with the molecular weight of the polymers. Strong hydration along the polymer backbone and polymer–Aβ entanglement might contribute to the accumulation of Aβ and subsequent fibrillation.

How does curvature affect the free-energy barrier of stalk formation? Small vesicles vs apposing, planar membranes

Using molecular simulations of POPC lipids in conjunction with the calculation of the Minimum Free-Energy Path (MFEP), we study the effect of strong membrane curvature on the formation of the first fusion intermediate—the stalk between a vesicle and its periodic image. We find that the thermodynamic stability of this hourglass-shaped, hydrophobic connection between two vesicles is largely increased by the strong curvature of small vesicles, whereas the intrinsic barrier to form a stalk, i.e., associated with dimple formation and lipid tails protrusions, is similar to the case of two, apposing, planar membranes. A significant reduction of the barrier of stalk formation, however, stems from the lower dehydration free energy that is required to bring highly curved vesicle into a distance, at which stalk formation may occur, compared to the case of apposing, planar membranes.

Controlling nanochannel orientation and dimensions in graphene-based nanofluidic membranes

There is great interest in exploiting van der Waals gaps in layered materials as nanofluidic channels. Graphene oxide (GO) nanosheets are known to spontaneously assemble into stacked planar membranes with transport properties that are highly selective to molecular structure. Use of conventional GO membranes in liquid-phase applications is often limited by low flux values, due to intersheet nanochannel alignment perpendicular to the desired Z-directional transport, which leads to circuitous fluid pathways that are orders of magnitude longer than the membrane thickness. Here we demonstrate an approach that uses compressive instability in Zr-doped GO thin films to create wrinkle patterns that rotate nanosheets to high angles. Capturing this structure in polymer matrices and thin sectioning produce fully dense membranes with arrays of near-vertically aligned nanochannels. These robust nanofluidic devices offer pronounced reduction in fluid path-length, while retaining the high selectivity for water over non-polar molecules characteristic of GO interlayer nanochannels.

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

A major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. In the bacterial cell cycle, the assembly of cytoskeletal proteins into a ring defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane-recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the liposome, generating elongated membrane necks and budding vesicles. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

Cell-free biogenesis of bacterial division proto-rings that can constrict liposomes

ABSTRACTA major challenge towards the realization of an autonomous synthetic cell resides in the encoding of a division machinery in a genetic programme. A key event in the bacterial cell cycle is the assembly of cytoskeletal proteins into a ring that defines the division site. At the onset of the formation of the Escherichia coli divisome, a proto-ring consisting of FtsZ and its membrane recruiting proteins takes place. Here, we show that FtsA-FtsZ ring-like structures driven by cell-free gene expression can be reconstituted on planar membranes and inside liposome compartments. Such cytoskeletal structures are found to constrict the membrane and generate budding vesicles, a phenotype that has not been reported before. Additional expression of the FtsZ cross-linker protein ZapA yields more rigid FtsZ bundles that attach to the membrane but fail to produce budding spots or necks in liposomes. These results provide new insights on the self-organization of basic cytoskeletal elements involved in bacterial division. Moreover, they demonstrate that gene-directed protein synthesis and assembly of membrane-constricting FtsZ-rings can be combined in a liposome-based artificial cell.

Effects of surfaces and macromolecular crowding on bimolecular reaction rates

Biological cells are complex environments that are densely packed with macromolecules and subdivided by membranes, both of which affect the rates of chemical reactions. It is well known that crowding reduces the volume available to reactants, which increases reaction rates, and also inhibits reactant diffusion, which decreases reaction rates. This work investigates these effects quantitatively using analytical theory and particle-based simulations. A reaction rate equation based on only these two processes turned out to be inconsistent with simulation results. However, accounting for diffusion inhibition by the surfaces of nearby obstacles, which affects access to reactants, led to perfect agreement for reactions near impermeable planar membranes and improved agreement for reactions in crowded spaces. A separate model that quantified reactant occlusion by crowders, and extensions to a thermodynamic “cavity” model proposed by Berezhkovskii and Szabo (J. Phys. Chem. B 120:5998, 2016), were comparably successful. These results help elucidate reaction dynamics in confined spaces and improve prediction of in vivo reaction rates from in vitro ones.