Bottom-up approach to explore alpha-amylase assisted membrane remodelling.

Soluble alpha-amylases play an important role in the catabolism of polysaccharides. In this work, we show that the malt α -amylase can interact with the lipid membrane and further alter its mechanical properties. Vesicle fluctuation spectroscopy is used for quantitative measurement of the membrane bending rigidity of phosphatidylcholines lipid vesicles from the shape fluctuation based on the whole contour of Giant Unilamellar Vesicles (GUVs). The bending rigidity of the 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine lipid vesicles in water increases significantly with the presence of 0.14 micromolar alpha-amylase (AA) in the exterior solution. It appears that the enzyme present in the external solution interacts with the outer layer of the bilayer membrane, leading to an asymmetry of the solution on either side of the bilayer membrane and altering its elasticity. At AA concentration of 1.5 micromolars and above, changes in the morphology of the GUV membrane are observed. The interaction between AA in the external solution and the external leaflet causes the bilayer membrane to curve spontaneously, leading to the formation of outbuds, giving a positive spontaneous curvature of C0 ≤ 0.05 µm-1 at ≈ 1 mg / ml of the AA concentration. We validate and characterize its concentration-dependent role in stabilizing the membrane curvature. Our findings indicate that the involvement of the enzyme, depending on the concentration, can have a considerable effect on the mechanical characteristics of the membrane.

Mesoscale simulation of biomembranes with FreeDTS.

We present FreeDTS software for performing computational research on biomembranes at the mesoscale. In this software, a membrane is represented by a dynamically triangulated surface equipped with vertex-based inclusions to integrate the effects of integral and peripheral membrane proteins. Several algorithms are included in the software to simulate complex membranes at different conditions such as framed membranes with constant tension, vesicles and high-genus membranes with various fixed volumes or constant pressure differences and applying external forces to membrane regions. Furthermore, the software allows the user to turn off the shape evolution of the membrane and focus solely on the organization of proteins. As a result, we can take realistic membrane shapes obtained from, for example, cryo-electron tomography and backmap them into a finer simulation model. In addition to many biomembrane applications, this software brings us a step closer to simulating realistic biomembranes with molecular resolution. Here we provide several interesting showcases of the power of the software but leave a wide range of potential applications for interested users.

Macromolecular Crowding Is Surprisingly Unable to Deform the Structure of a Model Biomolecular Condensate.

The crowded interior of a living cell makes performing experiments on simpler in vitro systems attractive. Although these reveal interesting phenomena, their biological relevance can be questionable. A topical example is the phase separation of intrinsically disordered proteins into biomolecular condensates, which is proposed to underlie the membrane-less compartmentalization of many cellular functions. How a cell reliably controls biochemical reactions in compartments open to the compositionally-varying cytoplasm is an important question for understanding cellular homeostasis. Computer simulations are often used to study the phase behavior of model biomolecular condensates, but the number of relevant parameters increases as the number of protein components increases. It is unfeasible to exhaustively simulate such models for all parameter combinations, although interesting phenomena are almost certainly hidden in their high-dimensional parameter space. Here, we have studied the phase behavior of a model biomolecular condensate in the presence of a polymeric crowding agent. We used a novel compute framework to execute dozens of simultaneous simulations spanning the protein/crowder concentration space. We then combined the results into a graphical representation for human interpretation, which provided an efficient way to search the model's high-dimensional parameter space. We found that steric repulsion from the crowder drives a near-critical system across the phase boundary, but the molecular arrangement within the resulting biomolecular condensate is rather insensitive to the crowder concentration and molecular weight. We propose that a cell may use the local cytoplasmic concentration to assist the formation of biomolecular condensates, while relying on the dense phase to reliably provide a stable, structured, fluid milieu for cellular biochemistry despite being open to its changing environment.

Model biomolecular condensates have heterogeneous structure quantitatively dependent on the interaction profile of their constituent macromolecules.

Biomolecular condensates play numerous roles in cells by selectively concentrating client proteins while excluding others. These functions are likely to be sensitive to the spatial organization of the scaffold proteins forming the condensate. We use coarse-grained molecular simulations to show that model intrinsically-disordered proteins phase separate into a heterogeneous, structured fluid characterized by a well-defined length scale. The proteins are modelled as semi-flexible polymers with punctate, multifunctional binding sites in good solvent conditions. Their dense phase is highly solvated with a spatial structure that is more sensitive to the separation of the binding sites than their affinity. We introduce graph theoretic measures to quantify their heterogeneity, and find that it increases with increasing binding site number, and exhibits multi-timescale dynamics. The model proteins also swell on passing from the dilute solution to the dense phase. The simulations predict that the structure of the dense phase is modulated by the location and affinity of binding sites distant from the termini of the proteins, while sites near the termini more strongly affect its phase behaviour. The relations uncovered between the arrangement of weak interaction sites on disordered proteins and the material properties of their dense phase can be experimentally tested to give insight into the biophysical properties, pathological effects, and rational design of biomolecular condensates.

Computational Approaches to Explore Bacterial Toxin Entry into the Host Cell.

Many bacteria secrete toxic protein complexes that modify and disrupt essential processes in the infected cell that can lead to cell death. To conduct their action, these toxins often need to cross the cell membrane and reach a specific substrate inside the cell. The investigation of these protein complexes is essential not only for understanding their biological functions but also for the rational design of targeted drug delivery vehicles that must navigate across the cell membrane to deliver their therapeutic payload. Despite the immense advances in experimental techniques, the investigations of the toxin entry mechanism have remained challenging. Computer simulations are robust complementary tools that allow for the exploration of biological processes in exceptional detail. In this review, we first highlight the strength of computational methods, with a special focus on all-atom molecular dynamics, coarse-grained, and mesoscopic models, for exploring different stages of the toxin protein entry mechanism. We then summarize recent developments that are significantly advancing our understanding, notably of the glycolipid-lectin (GL-Lect) endocytosis of bacterial Shiga and cholera toxins. The methods discussed here are also applicable to the design of membrane-penetrating nanoparticles and the study of the phenomenon of protein phase separation at the surface of the membrane. Finally, we discuss other likely routes for future development.

Creasing of flexible membranes at vanishing tension.

The properties of freestanding tensionless interfaces and membranes at low bending rigidity κ are dominated by strong fluctuations and self-avoidance and are thus outside the range of standard perturbative analysis. We analyze this regime by a simple discretized, self-avoiding membrane model on a frame subject to periodic boundary conditions by use of Monte Carlo simulation and dynamically triangulated surface techniques. We find that at low bending rigidities, the membrane properties fall into three regimes: Below the collapse transition κ_{BP} it is subject to branched polymer instability where the framed surface is not defined, in a range below a threshold rigidity κ_{c} the conformational correlation function are characterized by power-law behavior with a continuously varying exponent α, 2<α≤4 and above κ_{c}, α=4 characteristic for linearized bending excitations. Response functions specific heat and area compressibility display pronounced peaks close to κ_{c}. The results may be important for the description of soft interface systems, such as microemulsions and membranes with in-plane cooperative phenomena.

Fluctuations and conformational stability of a membrane patch with curvature inducing inclusions.

Membranes with curvature inducing inclusions display a range of cooperative phenomena, which can be linked to biomembrane function, e.g. membrane tubulation, vesiculation, softening and spontaneous tension. We investigate how these phenomena are related for a fluctuating, framed membrane through analysis of a descretized membrane model by Monte Carlo simulation techniques. The membrane model is based on a dynamically triangulated surface equipped with non-interacting, up-down symmetry breaking inclusions where only terms coupled linearly to mean-curvature are maintained. We show that the lateral configurational entropy plays a key role for the mechanical properties of the semi-flexible membrane, e.g. a pronounced softening at intermediate inclusion coverages of the membrane and generation of membrane tension. Tensionless framed membranes will remain quasi-flat up to some threshold coverage, where a shape instability occurs with formation of pearling or tubular membranes, which below full coverage is associated with segregation of inclusions between the curved and flat membrane geometries. For inclusions with preference for highly curved membranes the instability appears at dilute inclusion coverages and is accompanied by strong configurational fluctuations.

A Multi-Scale Approach to Membrane Remodeling Processes.

We present a multi-scale simulation procedure to describe membrane-related biological processes that span over a wide range of length scales. At macroscopic length-scale, a membrane is described as a flexible thin film modeled by a dynamic triangulated surface with its spatial conformations governed by an elastic energy containing only a few model parameters. An implicit protein model allows us to include complex effects of membrane-protein interactions in the macroscopic description. The gist of this multi-scale approach is a scheme to calibrate the implicit protein model using finer scale simulation techniques e.g., all atom and coarse grain molecular dynamics. We previously used this approach and properly described the formation of membrane tubular invaginations upon binding of B-subunit of Shiga toxin. Here, we provide a perspective of our multi-scale approach, summarizing its main features and sketching possible routes for future development.

The tension of framed membranes from computer simulations.

We have analyzed the behavior of a randomly triangulated, self-avoiding surface model of a flexible, fluid membrane subject to a circular boundary by Wang-Landau Monte Carlo computer simulation techniques. The dependence of the canonical free energy and frame tension on the frame area is obtained for flexible membranes. It is shown that for low bending rigidities the framed membrane is only stable above a threshold tension, suggesting a discontinuous transition from the collapsed (branched polymer) state to a finite tension extended state. In a tension range above this threshold tension the membranes display power-law characteristics for the equation of state, while higher tension levels includes both an extended linear (elastic) as well as a highly non-linear stretching regime. For semi-flexible membranes a transition from extended to buckled conformations takes place at negative frame tensions. Our analysis indicates that at zero frame tension the crumpling transition of fluid membranes show characteristics of both critical behavior and a discontinuous transition at low bending rigidities.

Clustering on Membranes: Fluctuations and More.

Clustering of extracellular ligands and proteins on the plasma membrane is required to perform specific cellular functions, such as signaling and endocytosis. Attractive forces that originate in perturbations of the membrane's physical properties contribute to this clustering, in addition to direct protein-protein interactions. However, these membrane-mediated forces have not all been equally considered, despite their importance. In this review, we describe how line tension, lipid depletion, and membrane curvature contribute to membrane-mediated clustering. Additional attractive forces that arise from protein-induced perturbation of a membrane's fluctuations are also described. This review aims to provide a survey of the current understanding of membrane-mediated clustering and how this supports precise biological functions.

Cholera toxin B subunit induces local curvature on lipid bilayers.

The B subunit of the bacterial cholera toxin (CTxB) is responsible for the toxin binding to the cell membrane and its intracellular trafficking. CTxB binds to the monosialotetrahexosyl ganglioside at the plasma membrane of the target cell and mediates toxin internalization by endocytosis. CTxB induces a local membrane curvature that is essential for its clathrin-independent uptake. Using all-atom molecular dynamics, we show that CTxB induces local curvature, with the radius of curvature around 36 nm. The main feature of the CTxB molecular structure that causes membrane bending is the protruding alpha helices in the middle of the protein. Our study points to a generic protein design principle for generating local membrane curvature through specific binding to their lipid anchors.

Membrane Tubulation in Lipid Vesicles Triggered by the Local Application of Calcium Ions.

Experimental and theoretical studies on ion-lipid interactions predict that binding of calcium ions to cell membranes leads to macroscopic mechanical effects and membrane remodeling. Herein, we provide experimental evidence that a point source of Ca2+ acting upon a negatively charged membrane generates spontaneous curvature and triggers the formation of tubular protrusions that point away from the ion source. This behavior is rationalized by strong binding of the divalent cations to the surface of the charged bilayer, which effectively neutralizes the surface charge density of outer leaflet of the bilayer. The mismatch in the surface charge density of the two leaflets leads to nonzero spontaneous curvature. We probe this mismatch through the use of molecular dynamics simulations and validate that calcium ion binding to a lipid membrane is sufficient to generate inward spontaneous curvature, bending the membrane. Additionally, we demonstrate that the formed tubular protrusions can be translated along the vesicle surface in a controlled manner by repositioning the site of localized Ca2+ exposure. The findings demonstrate lipid membrane remodeling in response to local chemical gradients and offer potential insights into the cell membrane behavior under conditions of varying calcium ion concentrations.

Capturing suboptical dynamic structures in lipid bilayer patches formed from free-standing giant unilamellar vesicles.

There is accumulating evidence that the small-scale lateral organization of biological membranes has a crucial role in signaling and trafficking in cells. However, it has been difficult to characterize these features with existing methods for preparing and analyzing freestanding membranes, because the dynamics occurs below the optical resolution possible with these protocols. We have developed a protocol that permits the imaging of lipid nanodomains and lateral protein organization in membranes of giant unilamellar vesicles (GUVs). Freestanding GUVs are transferred onto a mica support, and after treatment with magnesium chloride, they collapse to form planar lipid bilayer (PLB) patches. Rapid GUV collapse onto the mica preserves the lateral organization of freestanding membranes and thus makes it possible to image 'snapshots' of GUVs up to nanometer resolution by high-resolution microscopy. The method has been applied to classical lipid raft mixtures in which suboptical domain fluctuations have been imaged in both the liquid-ordered and liquid-disordered membrane phases. High-resolution scanning by atomic force microscopy (AFM) of membranes composed of binary and ternary lipid mixtures reconstituted with Na+/K+-ATPase (NKA) has revealed the spatial distribution and orientations of individual proteins, as well as details of membrane lateral structure. Immunolabeling followed by confocal microscopy can also provide information about the spatial distribution of proteins. The protocol opens up a new avenue for quantitative biophysical studies of suboptical dynamic structures in biomembranes, which are local and short-lived. Preparation of GUVs, PLB patches and their imaging takes <24 h.

Effects of seaweed sterols fucosterol and desmosterol on lipid membranes.

Higher sterols are universally present in large amounts (20-30%) in the plasma membranes of all eukaryotes whereas they are universally absent in prokaryotes. It is remarkable that each kingdom of the eukaryotes has chosen, during the course of evolution, its preferred sterol: cholesterol in animals, ergosterol in fungi and yeast, phytosterols in higher plants, and e.g., fucosterol and desmosterol in algae. The question arises as to which specific properties do sterols impart to membranes and to which extent do these properties differ among the different sterols. Using a range of biophysical techniques, including calorimetry, fluorescence microscopy, vesicle-fluctuation analysis, and atomic force microscopy, we have found that fucosterol and desmosterol, found in red and brown macroalgae (seaweeds), similar to cholesterol support liquid-ordered membrane phases and induce coexistence between liquid-ordered and liquid-disordered domains in lipid bilayers. Fucosterol and desmosterol induce acyl-chain order in liquid membranes, but less effectively than cholesterol and ergosterol in the order: cholesterol>ergosterol>desmosterol>fucosterol, possibly reflecting the different molecular structure of the sterols at the hydrocarbon tail.

Spatial distribution and activity of Na(+)/K(+)-ATPase in lipid bilayer membranes with phase boundaries.

We have reconstituted functional Na(+)/K(+)-ATPase (NKA) into giant unilamellar vesicles (GUVs) of well-defined binary and ternary lipid composition including cholesterol. The activity of the membrane system can be turned on and off by ATP. The hydrolytic activity of NKA is found to depend on membrane phase, and the water relaxation in the membrane on the presence of NKA. By collapsing and fixating the GUVs onto a solid support and using high-resolution atomic-force microscopy (AFM) imaging we determine the protein orientation and spatial distribution at the single-molecule level and find that NKA is preferentially located at lo/ld interfaces in two-phase GUVs and homogeneously distributed in single-phase GUVs. When turned active, the membrane is found to unbind from the support suggesting that the protein function leads to softening of the membrane.

Curvature inducing macroion condensation driven shape changes of fluid vesicles.

We study the effect of curvature inducing macroion condensation on the shapes of charged deformable fluid interfaces using dynamically triangulated Monte Carlo simulations. In the weak electrostatic coupling regime, surface charges are weakly screened and the conformations of a vesicle, with fixed spherical topology, depend on the charge-charge interaction on the surface. While in the strong coupling regime, condensation driven curvature induction plays a dominant role in determining the conformations of these surfaces. Condensation itself is observed to be dependent on the induced curvature, with larger induced curvatures favoring increased condensation. We show that both curvature generation and curvature sensing, induced by the interplay of electrostatics and curvature energy, contribute to determination of the vesicle configurations.

Preparing giant unilamellar vesicles (GUVs) of complex lipid mixtures on demand: Mixing small unilamellar vesicles of compositionally heterogeneous mixtures.

Giant unilamellar vesicles (GUVs) are simple model membrane systems of cell-size, which are instrumental to study the function of more complex biological membranes involving heterogeneities in lipid composition, shape, mechanical properties, and chemical properties. We have devised a method that makes it possible to prepare a uniform sample of ternary GUVs of a prescribed composition and heterogeneity by mixing different populations of small unilamellar vesicles (SUVs). The validity of the protocol has been demonstrated by applying it to ternary lipid mixture of DOPC, DPPC, and cholesterol by mixing small unilamellar vesicles (SUVs) of two different populations and with different lipid compositions. The compositional homogeneity among GUVs resulting from SUV mixing is quantified by measuring the area fraction of the liquid ordered-liquid disordered phases in giant vesicles and is found to be comparable to that in GUVs of the prescribed composition produced from hydration of dried lipids mixed in organic solvent. Our method opens up the possibility to quickly increase and manipulate the complexity of GUV membranes in a controlled manner at physiological buffer and temperature conditions. The new protocol will permit quantitative biophysical studies of a whole new class of well-defined model membrane systems of a complexity that resembles biological membranes with rafts.

Monte Carlo simulations of fluid vesicles.

Lipid vesicles are closed two dimensional fluid surfaces that are studied extensively as model systems for understanding the physical properties of biological membranes. Here we review the recent developments in the Monte Carlo techniques for simulating fluid vesicles and discuss some of their applications. The technique, which treats the membrane as an elastic sheet, is most suitable for the study of large scale conformations of membranes. The model can be used to study vesicles with fixed and varying topologies. Here we focus on the case of multi-component membranes with the local lipid and protein composition coupled to the membrane curvature leading to a variety of shapes. The phase diagram is more intriguing in the case of fluid vesicles having an in-plane orientational order that induce anisotropic directional curvatures. Methods to explore the steady state morphological structures due to active flux of materials have also been described in the context of Monte Carlo simulations.

Membrane properties of Enchytraeus albidus originating from contrasting environments: a comparative analysis.

Ectothermic animals adapted to different environmental temperatures are hypothesized to have biological membranes with different chemical and physical properties such that membrane properties are optimized for their particular thermal environments. To test this hypothesis we analyzed the composition of phospholipid fatty acids (PLFAs) in seven different populations of Enchytraeus albidus originating from different thermal environments. The seven populations differ markedly in origin (polar-temperate) and are also characterized by marked difference in cold tolerance. The dominant PLFAs of E. albidus were C20:5, C20:4 and C20:2 (53-61% of total PLFA) followed by C18:0, C20:1 and C22:2 (17-20% of total PLFA). As hypothesized the PLFA composition varied significantly between populations and molar percentage of several of the PLFAs (particularly C18:2) correlated with the lower lethal temperature (LT50) of the seven populations. Unsaturation ratio (UFA/SFA) and average PLFA chain length also correlated significantly with LT50, such that cold sensitive populations had a shorter chain length and a lower UFA/SFA compared to cold tolerant populations. Reconstituted membranes of the least and most cold tolerant populations were used to compare membranes' physical properties by fluorescence anisotropy and bending rigidity. Measurements of anisotropy did not show any overall difference between populations with different cold tolerance. This could be interpreted as if E. albidus populations have achieved a similar "optimal" fluidity of the membrane with a somewhat different PLFA composition. Our study suggests that membrane lipid composition could be important for the cold tolerance of E. albidus; however, these differences are not easily differentiated in the measurements of the membranes' physical properties. Other parameters such as accumulation of glucose for cryoprotection and energy supply may also be important components of enchytraeid freeze tolerance.

Organelle morphogenesis by active membrane remodeling.

Intracellular organelles are subject to a steady flux of lipids and proteins through active, energy consuming transport processes. Active fission and fusion are promoted by GTPases, e.g., Arf-Coatamer and the Rab-Snare complexes, which both sense and generate local membrane curvature. Here we investigate, through Dynamical Triangulation Monte Carlo simulations, the role that these active processes play in determining the morphology and composition segregation in closed membranes. We find that the steady state shapes obtained as a result of such active processes, bear a striking resemblance to the ramified morphologies of organelles in vivo, pointing to the relevance of nonequilibrium fission-fusion in organelle morphogenesis.