Thermally-induced aggregation and fusion of protein-free lipid vesicles

\u3cp\u3eMembrane fusion is an important phenomenon in cell biology and pathology. This phenomenon can be modeled using vesicles of defined size and lipid composition. Up to now fusion models typically required the use of chemical (polyethyleneglycol, cations) or enzymatic catalysts (phospholipases). We present here a model of lipid vesicle fusion induced by heat. Large unilamellar vesicles consisting of a phospholipid (dioleoylphosphatidylcholine), cholesterol and diacylglycerol in a 43:57:3mol ratio were employed. In this simple system, fusion was the result of thermal fluctuations, above 60°C. A similar system containing phospholipid and cholesterol but no diacylglycerol was observed to aggregate at and above 60°C, in the absence of fusion. Vesicle fusion occurred under our experimental conditions only when \u3csup\u3e31\u3c/sup\u3eP NMR and cryo-transmission electron microscopy of the lipid mixtures used in vesicle preparation showed non-lamellar lipid phase formation (hexagonal and cubic). Non-lamellar structures are probably the result of lipid reassembly of the products of individual fusion events, or of fusion intermediates. A temperature-triggered mechanism of lipid reassembly might have occurred at various stages of protocellular evolution.\u3c/p\u3

Controlling the size, shape and stability of supramolecular polymers in water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by p-p stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length > 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water

The role of the amorphous phase on the biomimetic mineralization of collagen

Bone is a hierarchically structured composite material whose basic building block is the mineralized collagen fibril, where the collagen is the scaffold into which the hydroxyapatite (HA) crystals nucleate and grow. Understanding the mechanisms of hydroxyapatite formation inside the collagen is key to unravelling osteogenesis. In this work, we employed a biomimetic in vitro mineralization system to investigate the role of the amorphous precursor calcium phosphate phase in the mineralization of collagen. We observed that the rate of collagen mineralization is highly dependent on the concentration of polyaspartic acid, an inhibitor of hydroxyapatite nucleation and inducer of intrafibrillar mineralization. The lower the concentration of the polymer, the faster the mineralization and crystallization. Addition of the non-collagenous protein C-DMP1, a nucleator of hydroxyapatite, substantially accelerates mineral infiltration as well as HA nucleation. We have also demonstrated that Cu ions interfere with the mineralization process first by inhibiting the entry of the calcium phosphate into the collagen, and secondly by stabilizing the ACP, such that it does not convert into HA. Interestingly, under these conditions mineralization happens preferentially in the overlap regions of the collagen fibril. Our results show that the interactions between the amorphous precursor phase and the collagen fibril play an important role in the control over mineralization

Controlling the Growth and Shape of Chiral Supramolecular Polymers in Water

A challenging target in the noncovalent synthesis of nanostructured functional materials is the formation of uniform features that exhibit well-defined properties, e.g., precise control over the aggregate shape, size, and stability. In particular, for aqueous-based one-dimensional supramolecular polymers, this is a daunting task. Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing out attractive noncovalent forces within the hydrophobic core of the polymerizing building blocks with electrostatic repulsive interactions on the hydrophilic rim we managed to switch from elongated, rod-like assemblies to small and discrete objects. Intriguingly this rod-to-sphere transition is expressed in a loss of cooperativity in the temperature-dependent self-assembly mechanism. The aggregates were characterized using circular dichroism, UV and 1H-NMR spectroscopy, small angle X-ray scattering, and cryotransmission electron microscopy. In analogy to many systems found in biology, mechanistic details of the self-assembly pathways emphasize the importance of cooperativity as a key feature that dictates the physical properties of the produced supramolecular polymers

Silicanin-1 is a conserved diatom membrane protein involved in silica biomineralization

\u3cp\u3eBackground: Biological mineral formation (biomineralization) proceeds in specialized compartments often bounded by a lipid bilayer membrane. Currently, the role of membranes in biomineralization is hardly understood. Results: Investigating biomineralization of SiO\u3csub\u3e2\u3c/sub\u3e (silica) in diatoms we identified Silicanin-1 (Sin1) as a conserved diatom membrane protein present in silica deposition vesicles (SDVs) of Thalassiosira pseudonana. Fluorescence microscopy of GFP-tagged Sin1 enabled, for the first time, to follow the intracellular locations of a biomineralization protein during silica biogenesis in vivo. The analysis revealed incorporation of the N-terminal domain of Sin1 into the biosilica via association with the organic matrix inside the SDVs. In vitro experiments showed that the recombinant N-terminal domain of Sin1 undergoes pH-triggered assembly into large clusters, and promotes silica formation by synergistic interaction with long-chain polyamines. Conclusions: Sin1 is the first identified SDV transmembrane protein, and is highly conserved throughout the diatom realm, which suggests a fundamental role in the biomineralization of diatom silica. Through interaction with long-chain polyamines, Sin1 could serve as a molecular link by which the SDV membrane exerts control on the assembly of biosilica-forming organic matrices in the SDV lumen.\u3c/p\u3

The role of prenucleation clusters in surface-induced calcium phosphate crystallization

Unravelling the processes of calcium phosphate formation is important in our understanding of both bone and tooth formation, and also of pathological mineralization, for example in cardiovascular disease. Serum is a metastable solution from which calcium phosphate precipitates in the presence of calcifiable templates such as collagen, elastin and cell debris. A pathological deficiency of inhibitors leads to the uncontrolled deposition of calcium phosphate. In bone and teeth the formation of apatite crystals is preceded by an amorphous calcium phosphate (ACP) precursor phase. ACP formation is thought to proceed through prenucleation clusters—stable clusters that are present in solution already before nucleation—as was recently demonstrated for CaCO3. However, the role of such nanometre-sized clusters as building blocks for ACP has been debated for many years. Here we demonstrate that the surface-induced formation of apatite from simulated body fluid starts with the aggregation of prenucleation clusters leading to the nucleation of ACP before the development of oriented apatite crystals

Understanding the formation mechanism of magnetic mesocrystals with (cryo-)electron microscopy

\u3cp\u3eMagnetite (Fe\u3csub\u3e3\u3c/sub\u3eO\u3csub\u3e4\u3c/sub\u3e) nanoaggregates with a flower-like morphology are considered promising materials in the field of magnetically induced hyperthermia in cancer therapy due to their good heating efficiency at low applied alternating magnetic fields. Although the structure and the magnetic state of such flower-like aggregates have been investigated previously, the mechanism that leads to the hierarchical morphology is still poorly understood. Here, we study the formation mechanism of Fe\u3csub\u3e3\u3c/sub\u3eO\u3csub\u3e4\u3c/sub\u3e aggregates synthesized through the partial oxidation of ferrous hydroxide in the presence of poly(acrylic acid) by using cryogenic electron microscopy. The aggregates are formed through a multistep process involving first the conversion of ferrous hydroxide precursors in ∼5 nm primary particles that aggregate into ∼10 nm primary Fe\u3csub\u3e3\u3c/sub\u3eO\u3csub\u3e4\u3c/sub\u3e crystals that finally arrange into the secondary mesocrystal structure. High-resolution electron tomography is used to show that the Fe\u3csub\u3e3\u3c/sub\u3eO\u3csub\u3e4\u3c/sub\u3e mesocrystals are composed of ∼10 nm subunits, often showing a uniform crystallographic orientation resulting in single-crystal-like diffraction patterns. Furthermore, electron holography reveals that mesocrystals have a single magnetic domain despite polymeric interfaces between subunits being present throughout the mesocrystal. Our findings could be used to design materials with specific properties by modulating the morphology and/or magnetic state that is suitable for biomedical application.\u3c/p\u3

Coiled coil driven membrane fusion between cyclodextrin vesicles and liposomes

\u3cp\u3eControlled fusion events between natural membranes composed of phospholipids with synthetic unnatural membranes will yield valuable fundamental information on the mechanism of membrane fusion. Here, fusion between vastly different phospholipid liposomes and cyclodextrin amphiphile based vesicles (CDVs) controlled by a pair of coiled coil forming lipidated peptides was investigated. Fusion events were characterized using lipid and content mixing assays and the resulting hybrid assemblies were characterized with cryo-TEM imaging. The secondary/quaternary structure of the lipidated peptides at the membrane interface was studied using circular dichroism spectroscopy. This is the first example of targeted fusion between natural and non-natural bilayer membranes and the in situ formation of hybrid CDV-liposome structures is of interest as it yields fundamental information about the mechanism through which fusion proceeds.\u3c/p\u3

Cryo-TEM and electron tomography reveal leaching-induced pore formation in ZSM-5 zeolite

\u3cp\u3eZeolites are the catalytic workhorses of the refinery and chemicals production industry. Their inherent micropores lead to remarkable shape-selectivity, but also present diffusion limitations on reactions. One scalable approach to further their functionality is to introduce mesopores into the individual zeolite crystal by base leaching. Using cryogenic transmission electron microscopy (cryo-TEM), we are able to capture the pore formation process in ZSM-5 zeolites in the solution state, first forming mesopores, to eventually yielding hollow crystals. Electron tomography indicates that the larger pores in the initial leaching stages tend to exist at the boundary between the aluminum-rich shell and the aluminum-poor core, while multiple small pores are also present within the aluminum-poor core. This indicates that pore formation is based on crystalline and chemical inhomogeneities at the single crystal level.\u3c/p\u3

Peptide functionalised discotic amphiphiles and their self-assembly into supramolecular nanofibres

The multicomponent co-assembly of discotic amphiphiles provides a modular and versatile approach to prepare RGDS- and PHSRN-peptide functionalised supramolecular nanofibres, bearing pendant paramagnetic Gd(III)-chelates