Lipid Nanotubes Research Articles

Mechanical characterization of freestanding lipid bilayers with temperature-controlled phase.

Coexistence of lipid domains in cell membranes is associated with vital biological processes. Here, we investigate two such membranes: a multi-component membrane composed of DOPC and DPPC lipids with gel and fluid separated domains, and a single component membrane composed of PMPC lipids forming ripples. We characterize their mechanical properties below their melting point, where ordered and disordered regions coexist, and above their melting point, where they are in fluid phase. To conduct these inquiries, we create lipid bilayers in a microfluidic chip interfaced with a heating system and optical tweezers. The chip features a bubble trap and enables high-throughput formation of planar bilayers. Optical tweezers experiments reveal interfacial hydrodynamics (fluid-slip) and elastic properties (membrane tension and bending rigidity) at various temperatures. For PMPC bilayers, we demonstrate a higher fluid slip at the interface in the fluid-phase compared to the ripple phase, while for the DOPC:DPPC mixture, similar fluid slip is measured below and above the transition point. Membrane tension for both compositions increases after thermal fluidization. Bending rigidity is also measured using the forces required to extend a lipid nanotube pushed out of the freestanding membranes. This novel temperature-controlled microfluidic platform opens numerous possibilities for thermomechanical studies on freestanding planar membranes.

SPOT-RASTR-A cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface.

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with β-galactosidase validates the method's ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.

Spontaneous nanotube formation of an asymmetric glycolipid

Over the past decades, advances in lipid nanotechnology have shown that self-assembled lipid structures providing ease of preparation, chemical stability, and biocompatibility represent a landmark on the development of multidisciplinary technologies. Lipid nanotubes (LNTs) are a unique class of lipid self-assembled structures, bearing unique properties such as high-aspect ratio, tunable diameter size, and precise molecular recognition. They can be obtained either by the action of external factors to already formed vesicles or spontaneously, the latter depending strongly on subtle molecular features. Here, we report on the spontaneous formation of supported lipid nanotubes of a particular type of glycolipid, ohmline, whose hydrophobic core displays remarkable asymmetry. The combination of bulk and surface-sensitive techniques indicates that below its main transition, ohmline displays an interdigitated gel phase, likely driven by the unique asymmetry in its hydrophobic core. Enhanced order packing by interdigitation favors the formation of ohmline nanotubes in agreement with chiral-based models of nanotube formation. The findings presented in this work call for additional studies to link lipid molecular structure-assembly relationships, whose understanding is relevant for the controlled design of lipid nanotubes networks in particular and controlled design of soft-matter nanomaterials in general.

Pt-Coated Lipid Nanotubes for Glucose Oxidation in a Neutral Medium

Pt-Coated Lipid Nanotubes for Glucose Oxidation in a Neutral Medium

Rotation of the c-Ring Promotes the Curvature Sorting of Monomeric ATP Synthases.

ATP synthases are proteins that catalyse the formation of ATP through the rotatory movement of their membrane-spanning subunit. In mitochondria, ATP synthases are found to arrange as dimers at the high-curved edges of cristae. Here, a direct link is explored between the rotatory movement of ATP synthases and their preference for curved membranes. An active curvature sorting of ATP synthases in lipid nanotubes pulled from giant vesicles is found. Coarse-grained simulations confirm the curvature-seeking behaviour of rotating ATP synthases, promoting reversible and frequent protein-protein contacts. The formation of transient protein dimers relies on the membrane-mediated attractive interaction of the order of 1.5kB T produced by a hydrophobic mismatch upon protein rotation. Transient dimers are sustained by a conic-like arrangement characterized by a wedge angle of θ ≈ 50°, producing a dynamic coupling between protein shape and membrane curvature. The results suggest a new role of the rotational movement of ATP synthases for their dynamic self-assembly in biological membranes.

Membrane activity of viral proteins.

COVID-19 infection, caused by the SARS-CoV-2 coronavirus, has led to the largest pandemic since the Spanish flu in 1918. In view of this, the development of vaccines and antiviral drugs that can stop the spread of this infection has become an acute issue. Currently, in the search for antiviral drugs against COVID-19, much attention is paid to the study of the structure of the receptor-binding domain of the surface protein S. However, the emergence of new SARS-CoV-2 coronavirus strains indicates its high variability, which reduces the effectiveness of vaccines and antiviral drugs. At the same time, the envelope protein E of this virus is membrane active and shows a rather high conservatism. Despite the critical importance of this protein in the coronavirus life cycle, the physicochemical mechanisms of its interaction with cell membranes still remain unclear. So, we investigated the membrane activity of protein E of the SARS-CoV-2 coronavirus on models of giant unilamellar vesicles and lipid nanotubes. As a result, it was found that the protein forms pores in the lipid bilayer, i.e., performs the main function of viroporin. In addition, protein E is able to deform lipid membranes and form double-membrane vesicles depending on the concentration.

Scale-Dependent Thermomechanical-Forced Noncircular Torsional Vibration of Lipid Supramolecular Nanotubes via Timoshenko–Gere Theory

Dynamic modeling of lipid nanotubes as a drug carrier in the skin layer is important. The displacement fields of lipid nanotubes in the shunt path of the skin layer are considered twisting. The twisting of the lipid nanotube in the skin layer causes the warping of the structure and, as a result, causes normal strain. The normal strain in the strain fields is not considered in the torsional noncircular structures. Therefore, in this study, not only the effect of shear strains but also the effect of normal strain on the torsional vibration of lipid nanotubes are considered based on the Timoshenko–Gere theory for the first time. Also, the temperature can be considered in the modeling due to the normal strain in the torsional of warped structures. Then, the governing equations of the forced torsional vibrations of lipid nanotubes, by considering the general warping function of cross-section, are derived based on the nonlocal strain gradient theory. The governing equation is solved by utilizing the convolution integration, and the dynamic responses of lipid nanotubes in the presence of external nonlinear harmonic moving torque are obtained. In the results, dynamic and frequency responses in the presence of temperature for rectangular and elliptical lipid nanotubes have been analyzed. One of the methods of drug release in nanocarriers is stimulation with ultrasound waves. Therefore, stimulating the lipid nanotubes using ultrasound waves at the obtained frequencies makes it possible to release the drug from the lipid nanotubes. Also, the maximum dynamical response of Timoshenko–Gere torsion is less than typical torsion. Increasing the aspect ratio of cross-section dimensions of lipid nanotubes decreased the maximum dynamical response. Increasing the velocity parameter first increases the dynamical twist and then reduces it. Also, the effects of axial forces and temperature on the maximum dynamical responses and the dynamical twist of the lipid nanotubes are studied. For validation, the obtained results are compared with the results of previous research.

Structural and dynamical properties of Palmitoyl-Oleoyl phosphatidylserine lipid nanotubes containing cholesterols and PEGylated dioleoyl Phosphatidylethanolamine: A Coarse-Grained molecular dynamics simulation

Lipid nanotubes (LNTs) are a class of lipid-based drug delivery systems (LBDDSs). Reports indicate that LBDDSs are effective systems in drug delivery to deal with COVID-19. The rational design of LNTs requires a deeper understanding of their structural properties in various influential factors such as lipid composition and PEGylation. This work uses coarse-grained molecular dynamics simulation to study the structural/dynamical properties of palmitoyl-Oleoyl phosphatidylserine (POPS) LNTs. To this end, the density distribution profiles are examined to evaluate of diameters and thicknesses of LNTs. The results show that adding cholesterol to LNT leads to lipid tail regularity, increasing the bilayer thickness and LNT diameter. PEGylation tends to increase irregularities of lipid chains, reduce the molecular packing and increase lipids dynamics. Our calculations predicted that manipulating LNT composition can be used to obtain favorable structural/dynamical properties and desired dimensions.

Transport among protocells via tunneling nanotubes

We employ model protocell networks for evaluation of molecular transport through lipid nanotubes as potential means of communication among primitive cells on the early Earth. Network formation is initiated by deposition of multilamellar lipid reservoirs onto a silicon oxide surface in an aqueous environment. These reservoirs autonomously develop into surface-adhered protocells interconnected via lipid nanotubes, and encapsulate solutes from the ambient buffer in the process. We prepare networks in the presence of DNA and RNA and observe encapsulation of these molecules, and their diffusive transport between the lipid compartments via the interconnecting nanotubes.

Actomyosin-Assisted Pulling of Lipid Nanotubes from Lipid Vesicles and Cells.

Molecular motors are pivotal for intracellular transport as well as cell motility and have great potential to be put to use outside cells. Here, we exploit engineered motor proteins in combination with self-assembly of actin filaments to actively pull lipid nanotubes from giant unilamellar vesicles (GUVs). In particular, actin filaments are bound to the outer GUV membrane and the GUVs are seeded on a heavy meromyosin-coated substrate. Upon addition of ATP, hollow lipid nanotubes with a length of tens of micrometer are pulled from single GUVs due to the motor activity. We employ the same mechanism to pull lipid nanotubes from different types of cells. We find that the length and number of nanotubes critically depends on the cell type, whereby suspension cells form bigger networks than adherent cells. This suggests that molecular machines can be used to exert forces on living cells to probe membrane-to-cortex attachment.

Transport among protocells via tunneling nanotubes.

We employ model protocell networks for evaluation of molecular transport through lipid nanotubes as potential means of communication among primitive cells on the early Earth. Network formation is initiated by deposition of lipid reservoirs onto a SiO2 surface in an aqueous environment. These reservoirs autonomously develop into surface-adhered protocells interconnected via lipid nanotubes while encapsulating solutes from the ambient buffer. We observe the uptake of DNA and RNA, and their diffusive transport between the lipid compartments via the interconnecting nanotubes. By means of an analytical model we determine key physical parameters affecting the transport, such as nanotube diameter and compartment size. We conclude that nanotube-mediated transport could have been a possible pathway of communication between primitive cells on the early Earth, circumventing the necessity for crossing the membrane barrier. We suggest this transport as a feasible means of RNA and DNA exchange under primitive prebiotic conditions, possibly facilitating early replication.

Single-compartment abiotic direct glucose fuel cell using Pd nanoparticles supported on phospholipid nanotubes

• Phospholipid nanotube was synthesized for catalyst support. • Lipid nanotubes were decorated with Pd nanoparticles by two-step reduction. • Pd-decorated lipid nanotubes showed strong catalytic activity for glucose oxidation. • Glucose fuel cell was built using the Pd-decorated lipid nanotubes. Lipid nanotubes (LNTs) synthesized by polymerization of 1,2-bis(10,12-tricosadiynoyl)- sn - glycero -3-phosphocholine are used as a catalyst support. The LNTs are decorated with Pd nanoparticles (PdNPs) by a two-step reduction of PdCl 4 2− to provide uniform coverage. The PdNP-decorated LNTs exhibit a strong catalytic activity towards non-enzymatic oxidation of glucose in 0.5 M KOH. A direct glucose fuel cell is built using the PdNP-decorated LNT anode and activated carbon cathode. The fuel cell produces an open circuit potential of 1.2 V and the highest power density of 1.9 mW cm −2 with a glucose concentration of 1.0 M and KOH concentration of 2.0 M. It is also demonstrated that the fuel cell can be built into a multiple-stack to attain the required power output.

Fabricating Multi-Component Lipid Nanotube Networks Using the Gliding Kinesin Motility Assay.

Lipid nanotube (LNT) networks represent an in vitro model system for studying molecular transport and lipid biophysics with relevance to the ubiquitous lipid tubules found in eukaryotic cells. However, in vivo LNTs are highly non-equilibrium structures that require chemical energy and molecular motors to be assembled, maintained, and reorganized. Furthermore, the composition of in vivo LNTs is complex, comprising of multiple different lipid species. Typical methods to extrude LNTs are both time- and labor-intensive, and they require optical tweezers, microbeads, and micropipettes to forcibly pull nanotubes from giant lipid vesicles. Presented here is a protocol for the gliding motility assay (GMA), in which large scale LNT networks are rapidly generated from giant unilamellar vesicles (GUVs) using kinesin-powered microtubule motility. Using this method, LNT networks are formed from a wide array of lipid formulations that mimic the complexity of biological LNTs, making them increasingly useful for in vitro studies of lipid biophysics and membrane-associated transport. Additionally, this method is capable of reliably producing LNT networks in a short time (<30 min) using commonly used laboratory equipment. LNT network characteristics such as length, width, and lipid partitioning are also tunable by altering the lipid composition of the GUVs used for fabricating the networks.

Surface-Assisted Formation of Model Protocells from Fatty Acid and Phospholipid Mixtures

Solid substrates have been shown to enhance the formation of prebiotic containers by supporting a spontaneous, multi-step transformation of surface-adhered phospholipid reservoirs to giant unilamellar primitive compartments connected via lipid nanotube networks. It has been previously shown that membranes from mixtures of fatty acids and phospholipids have a selective advantage over the pure phospholipid membranes. Here we show the solid surface-assisted formation of primitive compartments, i.e.

Lipid nanotubes as an organic template for the fabrication of carbon nanostructures by pyrolysis.

We demonstrate the fabrication of carbon nanoribbons with a width of 40 nm based on fixation and pyrolysis of an organic template, lipid nanotubes. To our best knowledge, this is the smallest feature size achieved by pyrolysis of surface-patterned organic templates. Such a pyrolytic carbon nanostructure can be used for electronics and sensing applications in future.

Custom-made lipid nanotubes as a tissue and hydrogel adhesive

Recently, nanomaterial-based adhesives have arised as alternatives to be used in the industrial field or to provide the next generation of biomedical adhesives with their enhanced mechanical properties and adhesion strengths. In this work, we developed a method for achieving adhesion between two PDMA (Poly dimethylacrylamide) hydrogel surfaces by using different nanomaterial-based adhesive dispersions with different sizes and shapes that have high mechanical adhesion strength. We obtained a new adhesive consisting of a lipid-based nanotube formed by the special custom-made molecule AQua (C25H29NO4) and measured the adhesion force between the hydrogel surfaces. Our results revealed that lipid AQua nanotube dispersions, which also has a high potential as drug delivery agents, showed efficient adhesion by strongly binding the hydrogel strips together and exhibit better adhesive effects in comparison with other nanomaterials that are used for this purpose. This high adhesion capability is also valid for various hydrogels that have different chemical properties or rigidities and soft tissues such as calf livers. So, the system developed here is believed to have a usage potential in different industrial and biomedical areas.

Protocells: Rapid Growth and Fusion of Protocells in Surface‐Adhered Membrane Networks (Small 38/2020)

In article number 2002529, Irep Gozen and co‐workers present experimental evidence that nucleation and growth of protocell‐like membrane compartments from surface‐adhered lipid nanotube networks are significantly enhanced at temperatures between 40 and 70 °C, and fusion can be initiated at ≈90 °C. They show that the microcontainers (5–15 μm) formed in this manner encapsulate and redistribute RNA, and corroborate that lipid nanotube–interconnected neighboring vesicles join and fuse more rapidly than in bulk suspensions.

Generation of interconnected vesicles in a liposomal cell model

We introduce an experimental method based upon a glass micropipette microinjection technique for generating a multitude of interconnected vesicles (IVs) in the interior of a single giant unilamellar phospholipid vesicle (GUV) serving as a cell model system. The GUV membrane, consisting of a mixture of soybean polar lipid extract and anionic phosphatidylserine, is adhered to a multilamellar lipid vesicle that functions as a lipid reservoir. Continuous IV formation was achieved by bringing a micropipette in direct contact with the outer GUV surface and subjecting it to a localized stream of a Ca2+ solution from the micropipette tip. IVs are rapidly and sequentially generated and inserted into the GUV interior and encapsulate portions of the micropipette fluid content. The IVs remain connected to the GUV membrane and are interlinked by short lipid nanotubes and resemble beads on a string. The vesicle chain-growth from the GUV membrane is maintained for as long as there is the supply of membrane material and Ca2+ solution, and the size of the individual IVs is controlled by the diameter of the micropipette tip. We also demonstrate that the IVs can be co-loaded with high concentrations of neurotransmitter and protein molecules and displaying a steep calcium ion concentration gradient across the membrane. These characteristics are analogous to native secretory vesicles and could, therefore, serve as a model system for studying secretory mechanisms in biological systems.

Reconstitution and real-time quantification of membrane remodeling by single proteins and protein complexes.

Cellular membrane processes, from signal transduction to membrane fusion and fission, depend on acute membrane deformations produced by small and short-lived protein complexes working in conditions far from equilibrium. Real-time monitoring and quantitative assessment of such deformations are challenging; hence, mechanistic analyses of the protein action are commonly based on ensemble averaging, which masks important mechanistic details of the action. In this protocol, we describe how to reconstruct and quantify membrane remodeling by individual proteins and small protein complexes in vitro, using an ultra-short (80- to 400-nm) lipid nanotube (usNT) template. We use the luminal conductance of the usNT as the real-time reporter of the protein interaction(s) with the usNT. We explain how to make and calibrate the usNT template to achieve subnanometer precision in the geometrical assessment of the molecular footprints on the nanotube membrane. We next demonstrate how membrane deformations driven by purified proteins implicated in cellular membrane remodeling can be analyzed at a single-molecule level. The preparation of one usNT takes ~1 h, and the shortest procedure yielding the basic geometrical parameters of a small protein complex takes 10 h.

Lipid Nanotubes as an Organic Template for an Electrically Conductive Gold Nanostructure Network

We demonstrate an approach to fabricate a gold nanowire network that presents a macroscopic electrical conductivity based on a lipid nanotube (LNT) template with attached gold nanoparticles. The poor electrical conductivity that we have previously faced was overcome by centrifugation and resuspension of gold nanoparticle solution for removing stabilizing agents, which increased the density of gold nanoparticles on the LNTs. An additional electroless metal plating further enhanced their contacts at nanoscale. Thanks to these procedures, the sheet resistance was improved by 11 orders of magnitude. As a proof of principle, transparent conductive films were fabricated with these gold nanowires, which exhibited sheet resistance of maximum 70 Ω/□ and transmittance of 50-75% in visible light.