Polystyrene-modified carbon nanotubes: Promising carriers in targeted drug delivery

To design drug-delivery agents for therapeutic and diagnostic applications, understanding the mechanisms by which covalently functionalized carbon nanotubes penetrate and interact with cell membranes is of great importance. Here, we report all-atom molecular dynamics results from polystyrene and carboxyl-terminated polystyrene-modified carbon nanotubes and show their translocation behavior across a model lipid bilayer together with their potential to deliver a molecule of the drug ibuprofen into the cell. Our results indicate that functionalized carbon nanotubes are internalized by the membrane in hundreds of nanoseconds and that drug loading increases the internalization speed further. Both loaded and unloaded tubes cross the closest leaflet of the bilayer by nonendocytic pathways, and for the times studied, the drug molecule remains trapped inside the pristine tube while remaining attached at the end of polystyrene-modified tube. On the other hand, carboxyl-terminated polystyrene functionalization allows the drug to be completely released into the lower leaflet of the bilayer without imposing damage to the membrane. This study shows that polystyrene functionalization is a promising alternative and facilitates drug delivery as a benchmark case.     

Membrane-Mediated Aggregation of Curvature-Inducing Nematogens and?Membrane Tubulation

The shapes of cell membranes are largely regulated by membrane-associated, curvature-active proteins. Herein, we use a numerical model of the membrane, recently developed by us, with elongated membrane inclusions possessing spontaneous directional curvatures that could be different along, and perpendicular to, the membrane’s long axis. We show that, due to membrane-mediated interactions, these curvature-inducing membrane-nematogens can aggregate spontaneously, even at low concentrations, and change the local shape of the membrane. We demonstrate that for a large group of such inclusions, where the two spontaneous curvatures have equal sign, the tubular conformation and sometimes the sheet conformation of the membrane are the common equilibrium shapes. We elucidate the factors necessary for the formation of these protein lattices. Furthermore, the elastic properties of the tubes, such as their compressional stiffness and persistence length, are calculated. Finally, we discuss the possible role of nematic disclination in capping and branching of the tubular membranes.     

Fission of the bilayer lipid tube

The fusion of two black lipid membranes results in the formation of peculiar bilayer lipid tubes (‘cylindrical’) membranes (Neher, E. (1974) Biochim. Biophys. Acta 373, 328–336 and Melikyan, G.B., Abidor, L.G., Chernomordik, L.V. and Chailakhyan, L.M. (1983) Biochim. Biophys. Acta 730, 395–398). The mechanical stability of such tubes has been investigated experimentally and theoretically. With increasing hydrostatic pressure on the outside of the tube the radius of its middle part decreases. After this radius has reached a critical value, which constitutes 0.55 of the radius of the tube base, there occurs a collapse of the tube and its disintegration into two planar bilayers (fission). Expressions are obtained which relate the transmembrane difference of the hydrostatic pressure, causing the collapse, to the geometrical characteristics of the tube (its length and the radius of its base) and to the tension of the lipid bilayer. A method for measuring the membrane tension is proposed on the basis of the phenomenon considered.     

The regulation of vesicle trafficking by small GTPases and phospholipids during pollen tube growth

Polarized and directional growth of pollen tubes is the only means by which immotile sperm of flowering plants reach the deeply embedded female gametes for fertilization. Vesicle trafficking is among the most critical cellular activities for pollen tube growth. Vesicle trafficking maintains membrane homeostasis during rapid tube growth and provides polarity information by regulating protein/lipid compositions of different membrane compartments. In this review, we will focus on two classes of factors that orchestrate vesicle trafficking, small GTPases and phospholipids. We discuss the features of small GTPases and phospholipids that make them ideal components to regulate vesicle trafficking, review recent advances in understanding their involvement in vesicle trafficking, and propose directions for future research.     

Insights into eukaryotic evolution from transmembrane domain lengths

Biological membranes, comprised of proteins anchored by their trans-membrane domains (TMDs) creating a semi-permeable phase with lipid constituents, serve as ‘checkposts’ for not only intracellular trafficking in eukaryotic cells but also for material transactions of all living cells with external environments. Hydropathy (or hydrophobicity) plots of ‘bitopic’ proteins (i.e. having single alpha-helical TMDs) are routinely utilized in biochemistry texts for predicting their TMDs. The number of amino acids (i.e. TMD length) embedded as alpha-helices may serve as indicators of thickness of biological membranes in which they reside under assumptions that are universally applied for fixing window sizes for identifying TMDs using hydropathy plots. In this work we explore variations in thickness of different eukaryotic biological membranes (reflected by TMD lengths of their resident proteins) over evolutionary time scales. Rigorous in silico analyses of over 23,000 non-redundant membrane proteins residing in different subcellular locations from over 200 genomes of fungi, plants, non-mammalian vertebrates and mammals, reveal that differences in plasma membrane and organellar TMD lengths have decreased over time (scales) of eukaryotic cellular evolution. While earlier work has indicated decreasing differences in TMD lengths with increasing ‘perceived’ organismal complexity, this work is the first report on TMD length variations as a function of evolutionary time of eukaryotic cellular systems. We report that differences in TMD lengths of bitopic proteins residing in plasma membranes and other intra-cellular locations have decreased with evolutionary time, suggesting better/more avenues of intracellular trafficking in the emergence of eukaryotic organisms.     

Role of curvature and phase transition in lipid sorting and fission of membrane tubules

We have recently developed a minimal system for generating long tubular nanostructures that resemble tubes observed in vivo with biological membranes. Here, we studied membrane tube pulling in ternary mixtures of sphingomyelin, phosphatidylcholine and cholesterol. Two salient results emerged: the lipid composition is significantly different in the tubes and in the vesicles; tube fission is observed when phase separation is generated in the tubes. This shows that lipid sorting may depend critically on both membrane curvature and phase separation. Phase separation also appears to be important for membrane fission in tubes pulled out of giant liposomes or purified Golgi membranes.     

Continuum simulations of biomembrane dynamics and the importance of hydrodynamic effects

Traditional particle-based simulation strategies are impractical for the study of lipid bilayers and biological membranes over the longest length and time scales (microns, seconds and longer) relevant to cellular biology. Continuum-based models developed within the frameworks of elasticity theory, fluid dynamics and statistical mechanics provide a framework for studying membrane biophysics over a range of mesoscopic to macroscopic length and time regimes, but the application of such ideas to simulation studies has occurred only relatively recently. We review some of our efforts in this direction with emphasis on the dynamics in model membrane systems. Several examples are presented that highlight the prominent role of hydrodynamics in membrane dynamics and we argue that careful consideration of fluid dynamics is key to understanding membrane biophysics at the cellular scale.     

Modeling of RNA nanotubes using molecular dynamics simulation

In this study, we construct novel RNA nanoclusters, RNA nanotubes made of several nanorings up to the size of 20 nm, utilizing the molecular dynamics simulation, and study their structural properties [i.e., the root mean square deviation, the radius of gyration and the radial distribution function (RDF)] in physiological solutions that can be used for drug delivery into the human body. The patterns of energy and temperature variations of the systems are also discussed. Furthermore, we study the concentration of ions around the tube as a function of time at a particular temperature. We have found that when the temperature increases, the number of ions increases within a certain distance of the tube. We report that the number of ions within this distance around the tubes decreases in quenched runs. This indicates that some ions evaporate with decrease in temperature, as has been observed in the case of the nanoring. RDF plots also demonstrate a similar trend with temperature, as was found in the case of RNA nanorings.     

生物膜细管形成过程的研究*

我们都知道磷脂双亲分子在水或者油溶液中会形成各种不同的形状。现在我们探讨的是一种细的管状生物膜泡。它通常是指在磷脂双亲分子形成膜泡后,我们对它在显微镜下进行的一系列使用光镊施加拉力操作而形成的细的管状结构。实验显示,膜泡形成的管状结构在生物学中是普遍存在的。本文主要研究怎样应用数值计算的方法进行模拟细的管状膜泡,从而得到一些长短,粗细不同的管状生物膜泡。我们从极小曲面悬链面的角度出发,研究管状膜泡的形成结构特点。最后我们认为管状膜泡的形成不同形状是与对膜泡施加拉力、细管的半径、以及管子的长度等因素有着密切的联系。     

ArfGAP1 generates an Arf1 gradient on continuous lipid membranes displaying flat and curved regions

ArfGAP1, which promotes GTP hydrolysis on the small G protein Arf1 on Golgi membranes, interacts preferentially with positively curved membranes through its amphipathic lipid packing sensor (ALPS) motifs. This should influence the distribution of Arf1‐GTP when flat and curved regions coexist on a continuous membrane, notably during COPI vesicle budding. To test this, we pulled tubes from giant vesicles using molecular motors or optical tweezers. Arf1‐GTP distributed on the giant vesicles and on the tubes, whereas ArfGAP1 bound exclusively to the tubes. Decreasing the tube radius revealed a threshold of R≈35 nm for the binding of ArfGAP1 ALPS motifs. Mixing catalytic amounts of ArfGAP1 with Arf1‐GTP induced a smooth Arf1 gradient along the tube. This reflects that Arf1 molecules leaving the tube on GTP hydrolysis are replaced by new Arf1‐GTP molecules diffusing from the giant vesicle. The characteristic length of the gradient is two orders of magnitude larger than a COPI bud, suggesting that Arf1‐GTP diffusion can readily compensate for the localized loss of Arf1 during budding and contribute to the stability of the coat until fission.     

Variation of lipid membrane composition caused by strong bending

Dynamic coupling between the morphology and molecular composition of cellular membranes is crucial for formation of the intracellular organelles and transport vesicles. Most of the membrane proteins and lipids discriminate membrane curvatures. However, it remains unclear whether the curvature alone is sufficient to support heterogeneous distribution of lipids. Here we demonstrate that the curvature-driven redistribution of phospholipids, such as dioleoylphosphatidylethanolamine (DOPE), requires strong membrane bending. We used cylindrical lipid nanotubes (NTs) pulled from planar lipid membranes with lateral tension of ∼1 dyn/cm. Such high tensions forced extreme curvatures of the NT membrane, with luminal radius approaching the thickness of the lipid bilayer, 5nm. When the NT contained lipid species with high spontaneous curvature (SC), such as DOPE, we observed slow reduction of its radius. This reduction indicated the redistribution of DOPE between the inner and outer monolayers of the NT. Accordingly, the SC of DOPE was recovered from the measured changes in the radii: the SC value, calculated under the assumption that the DOPE content is coupled to the monolayer curvature, was ∼0.4 nm⁻¹, consistent with the published data. Thus, redistribution of lipids should be taken into account in calculations of composition and material properties of strongly deformed membrane structures, such as intermediate structures arising in the processes of membrane fusion and fission.     

Spatial Control of Epsin-induced Clathrin Assembly by Membrane Curvature

Epsins belong to the family of highly conserved clathrin-associated sorting proteins that are indispensable for clathrin-mediated endocytosis, but their precise functions remain unclear. We have developed an assay system of budded supported membrane tubes displaying planar and highly curved membrane surfaces to analyze intrinsic membrane curvature preference shown by clathrin-associated sorting proteins. Using real-time fluorescence microscopy, we find that epsin preferentially partitions to and assembles clathrin on highly curved membrane surfaces. Sorting of epsin to regions of high curvature strictly depends on binding to phosphatidylinositol 4,5-bisphosphate. Fluorescently labeled clathrins rapidly assemble as foci, which in turn cluster epsin, while maintaining tube integrity. Clathrin foci grow in intensity with a typical time constant of ∼75 s, similar to the time scales for coated pit formation seen in cells. Epsin therefore effectively senses membrane curvature to spatially control clathrin assembly. Our results highlight the potential role of membrane curvature in orchestrating the myriad molecular interactions necessary for the success of clathrin-mediated membrane budding.     

Simulations of electrophoretic RNA transport through transmembrane carbon nanotubes

The study of interactions between carbon nanotubes and cellular components, such as membranes and biomolecules, is fundamental for the rational design of nanodevices interfacing with biological systems. In this work, we use molecular dynamics simulations to study the electrophoretic transport of RNA through carbon nanotubes embedded in membranes. Decorated and naked carbon nanotubes are inserted into a dodecane membrane and a dimyristoylphosphatidylcholine lipid bilayer, and the system is subjected to electrostatic potential differences. The transport properties of this artificial pore are determined by the structural modifications of the membrane in the vicinity of the nanotube openings and they are quantified by the nonuniform electrostatic potential maps at the entrance and inside the nanotube. The pore is used to transport electrophoretically a short RNA segment and we find that the speed of translocation exhibits an exponential dependence on the applied potential differences. The RNA is transported while undergoing a repeated stacking and unstacking process, affected by steric interactions with the membrane headgroups and by hydrophobic interaction with the walls of the nanotube. The RNA is structurally reorganized inside the nanotube, with its backbone solvated by water molecules near the axis of the tube and its bases aligned with the nanotube walls. Upon exiting the pore, the RNA interacts with the membrane headgroups and remains attached to the dodecane membrane while it is expelled into the solvent in the case of the lipid bilayer. The results of the simulations detail processes of molecular transport into cellular compartments through manufactured nanopores and they are discussed in the context of applications in biotechnology and nanomedicine.     

Lipid polymorphisms and membrane shape

Morphological plasticity of biological membrane is critical for cellular life, as cells need to quickly rearrange their membranes. Yet, these rearrangements are constrained in two ways. First, membrane transformations may not lead to undesirable mixing of, or leakage from, the participating cellular compartments. Second, membrane systems should be metastable at large length scales, ensuring the correct function of the particular organelle and its turnover during cellular division. Lipids, through their ability to exist with many shapes (polymorphism), provide an adequate construction material for cellular membranes. They can self-assemble into shells that are very flexible, albeit hardly stretchable, which allows for their far-reaching morphological and topological behaviors. In this article, we will discuss the importance of lipid polymorphisms in the shaping of membranes and its role in controlling cellular membrane morphology.     

Actin is not required for nanotubular protrusions of primary astrocytes grown on metal nano-lawn

We used sub-micron metal rod decorated surfaces, 'nano-lawn' structures, as a substrate to study cell-to-cell and cell-to-surface interactions of primary murine astrocytes. These cells form thin membranous tubes with diameters of less than 100 nm and a length of several microns, which make contact to neighboring cells and the substrate during differentiation. While membrane protrusions grow on top of the nano-lawn pillars, nuclei sink to the bottom of the substrate. We observed gondola-like structures along those tubes, suggestive of their function as transport vehicles. Elements of the cytoskeleton such as actin fibers are commonly believed to be essential for triggering the onset and growth of tubular membrane protrusions. A rope-pulling mechanism along actin fibers has recently been proposed to account for the transport or exchange of cellular material between cells. We present evidence for a complementary mechanism that promotes growth and stabilization of the observed tubular protrusions of cell membranes. This mechanism does not require active involvement of actin fibers as the formation of membrane protrusions could not be prevented by suppressing polymerization of actin by latrunculin B. Also theoretically, actin fibers are not essential for the growing and stability of nanotubes since curvature-driven self-assembly of interacting anisotropic raft elements is sufficient for the spontaneous formation of thin nano-tubular membrane protrusions.     

Composition Based Strategies for Controlling Radii in Lipid Nanotubes

Nature routinely carries out small-scale chemistry within lipid bound cells and organelles. Liposome–lipid nanotube networks are being developed by many researchers in attempt to imitate these membrane enclosed environments, with the goal to perform small-scale chemical studies. These systems are well characterized in terms of the diameter of the giant unilamellar vesicles they are constructed from and the length of the nanotubes connecting them. Here we evaluate two methods based on intrinsic curvature for adjusting the diameter of the nanotube, an aspect of the network that has not previously been controllable. This was done by altering the lipid composition of the network membrane with two different approaches. In the first, the composition of the membrane was altered via lipid incubation of exogenous lipids; either with the addition of the low intrinsic curvature lipid soy phosphatidylcholine (soy-PC) or the high intrinsic curvature lipid soy phosphatidylethanolamine (soy-PE). In the second approach, exogenous lipids were added to the total lipid composition during liposome formation. Here we show that for both lipid augmentation methods, we observed a decrease in nanotube diameter following soy-PE additions but no significant change in size following the addition of soy-PC. Our results demonstrate that the effect of soy-PE on nanotube diameter is independent of the method of addition and suggests that high curvature soy-PE molecules facilitate tube membrane curvature.     

Insertion of short amino-functionalized single-walled carbon nanotubes into phospholipid bilayer occurs by passive diffusion

Carbon nanotubes have been proposed to be efficient nanovectors able to deliver genetic or therapeutic cargo into living cells. However, a direct evidence of the molecular mechanism of their translocation across cell membranes is still needed. Here, we report on an extensive computational study of short (5 nm length) pristine and functionalized single-walled carbon nanotubes uptake by phospholipid bilayer models using all-atom molecular dynamics simulations. Our data support the hypothesis of a direct translocation of the nanotubes through the phospholipid membrane. We find that insertion of neat nanotubes within the bilayer is a "nanoneedle" like process, which can often be divided in three consecutive steps: landing and floating, penetration of the lipid headgroup area and finally sliding into the membrane core. The presence of functional groups at moderate concentrations does not modify the overall scheme of diffusion mechanism, provided that their deprotonated state favors translocation through the lipid bilayer.     

Tubular Membrane Formation of Binary Giant Unilamellar Vesicles Composed of Cylinder and Inverse-Cone-Shaped Lipids

We have succeeded in controlling tubular membrane formations in binary giant unilamellar vesicles (GUVs) using a simple temperature changing between the homogeneous one-phase region and the two-phase coexistence region. The binary GUV is composed of inverse-cone (bulky hydrocarbon chains and a small headgroup) and cylinder-shaped lipids. When the temperature was set in the two-phase coexistence region, the binary GUV had a spherical shape with solidlike domains. By increasing the temperature to the homogeneous one-phase region, the excess area created by the chain melting of the lipid produced tubes inside the GUV. The tubes had a radius on the micrometer scale and were stable in the one-phase region. When we again decreased the temperature to the two-phase coexisting region, the tubes regressed and the GUVs recovered their phase-separated spherical shape. We infer that the tubular formation was based on the mechanical balance of the vesicle membrane (spontaneous tension) coupled with the asymmetric distribution of the inverse-cone-shaped lipids between the inner and outer leaflets of the vesicle (lipid sorting).