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Biological membranes have a central role in mediating the organization of membrane-curving proteins, a dynamic process that has proven to be challenging to probe experimentally. Using atomic force microscopy, we capture the hierarchically organized assemblies of Bin/amphiphysin/Rvs (BAR) proteins on supported lipid membranes. Their structure reveals distinct long linear aggregates of proteins, regularly spaced by up to 300 nm. Employing accurate free-energy calculations from large-scale coarse-grained computer simulations, we found that the membrane mediates the interaction among protein filaments as a combination of short- and long-ranged interactions. The long-ranged component acts at strikingly long distances, giving rise to a variety of micron-sized ordered patterns. This mechanism may contribute to the long-ranged spatiotemporal control of membrane remodeling by proteins in the cell., High-resolution imaging and free-energy simulations reveal unexpected long-ranged interactions between filaments of membrane-curving proteins.

Breaking the anterior-posterior (AP) symmetry in mammals takes place at gastrulation. Much of the signaling network underlying this process has been elucidated in the mouse, however there is no direct molecular evidence of events driving axis formation in humans. Here, we use human embryonic stem cells to generate an in vitro 3D model of a human epiblast whose size, cell polarity, and gene expression are similar to a 10-day human epiblast. A defined dose of bone mor-phogenetic protein 4 (BMP4) spontaneously breaks axial symmetry, and induces markers of the primitive streak and epithelial to mesenchymal transition. By gene knockouts and live-cell imaging we show that, downstream of BMP4, WNT3 and its inhibitor DKK1 play key roles in this process. Our work demonstrates that a model human epiblast can break axial symmetry despite no asymmetry in the initial signal and in the absence of extraembryonic tissues or maternal cues. Our 3D model opens routes to capturing molecular events underlying axial symmetry breaking phenomena, which have largely been unexplored in model human systems.

Lipid membranes are key regulators of cellular function. An important step in membrane-related phenomena is the reshaping of the lipid bilayer, often induced by binding of macromolecules. Numerous experimental and simulation efforts have revealed that calcium, a ubiquitous cellular messenger, has a strong impact on the phase behavior, structural properties, and the stability of membranes. Yet, it is still unknown the way calcium and lipid interactions affect their macroscopic mechanical properties. In this work, we studied the interaction of calcium ions with membrane tethers pulled from giant unilamellar vesicles, to quantify the mechanical effect on the membrane. We found that calcium imposes a positive spontaneous curvature on negatively charged membranes, contrary to predictions we made based on the proposed atomic structure. Surprisingly, this effect vanishes in the presence of physiologically relevant concentrations of sodium chloride. Our work implies that calcium may be a trigger for membrane reshaping only at high concentrations, in a process that is robustly screened by sodium ions.

We present the Mesoscopic Membrane with Proteins (MesM-P) model, an extension of a previously developed elastic membrane model for mesoscale simulations of lipid membranes. MesM-P employs a discrete mesoscopic quasi-particle approach to model protein-facilitated shape and topology changes of the lipid membrane on length and time scales inaccessible to all-atom and quasimolecular coarse-grained molecular dynamics simulations. We investigate the ability of MesM-P to model the behavior of large lipid vesicles as a function of bound protein density. We find four distinct mechanisms for protein aggregation on the surface of the membrane, depending on membrane stiffness and protein spontaneous curvature. We also establish a connection between MesM-P and the results of higher resolution coarse-grained molecular dynamics simulations.

In vesicular transport pathways, membrane proteins and lipids are internalized, externalized or transported within cells, not by bulk diffusion of single molecules, but embedded in the membrane of small vesicles or thin tubules. The formation of these ‘transport carriers’ follows sequential events: membrane bending, fission from the donor compartment, transport and eventually fusion with the acceptor membrane. A similar sequence is involved during the internalization of drug or gene carriers inside cells. These membrane-shaping events are generally mediated by proteins binding to membranes. The mechanisms behind these biological processes are actively studied both in the context of cell biology and biophysics. Bin/amphiphysin/Rvs (BAR) domain proteins are ideally suited for illustrating how simple soft matter principles can account for membrane deformation by proteins. We review here some experimental methods and corresponding theoretical models to measure how these proteins affect the mechanics and the shape of membranes. In more detail, we show how an experimental method employing optical tweezers to pull a tube from a giant vesicle may give important quantitative insights into the mechanism by which proteins sense and generate membrane curvature and the mechanism of membrane scission. This article is part of the themed issue ‘Soft interfacial materials: from fundamentals to formulation’.

BrILL2 is catalytically the most efficient auxin amidohydrolase from Brassica rapa, playing a key role in auxin metabolism by catalyzing its release from amino acid conjugates. Auxins, with the most abundant representative indole-acetic acid ([1H-indol-3-yl]-acetic acid, IAA), are a group of plant hormones that in very small concentrations regulate ubiquitin-mediated degradation of transcription regulators. Kinetic studies on BrILL2 showed that it hydrolyzes alanine conjugates of IAA and of its larger analogues, indole-propionic acid (3-[1H-indol-3-yl]-propionic acid, IPA) and indole-butyric acid (4-[1H-indol-3-yl]-butyric acid, IBA). Structurally, BrILL2 belongs to the largest known family of metallopeptidases (M20) that share a recognizable 3D structure, characterized by two perpendicular domains. Its members have been implicated in numerous biochemical processes and have been found across all species sequenced to date. Here, molecular dynamics simulations were carried out to study structural and thermodynamic properties of ligand binding to BrILL2. A conformational change was captured in multiple copies of 10 ns long simulations, described by a rigid body movement of the two domains, and its associated key interactions between residues were examined. For the three substrates, complexes in two possible binding modes were recreated, along with a single binding mode for the putative substrate tryptophanyl–alanine (Trp–Ala), which were subsequently simulated in multiple copies of 10 ns long simulations. Thermodynamic calculations were used to assess their binding affinities and explain the selectivity toward the longer ligands. Based on the results, a possible route for the reaction is proposed. Copyright © 2011 John Wiley & Sons, Ltd.

In cells, membranes are highly bent in many places in order to fulfill different functions. This is the case of membrane trafficking, which is accompanied by the formation of tubes or vesicles with diameter below 100 nm that transport proteins and lipids throughout the cell. Moreover, during vesicle budding, curvature at the neck becomes even higher before final scission. Structures at the cell periphery, such as lamellipodia’ edge or filopodia used for migration and environment sensing, also exhibit high membrane curvatures. Many peripheral or integral proteins have an intrinsic shape that produces membrane bending or can deform membrane upon binding. In membrane physics, this property is described by a phenomenological parameter, the protein spontaneous curvature Cp, which represents the capability of the protein to produce membrane spontaneous bending. It is expected that proteins that deform membranes can reciprocally be enriched in curved areas. We have addressed the question of the role of membrane curvature in protein sorting both experimentally and theoretically. We have tested the sorting hypothesis using in vitro membrane system (GUV) and different proteins with positive Cp (BAR-domain), negative Cp (I-BAR-domain) and with integral proteins reconstituted in the GUVs (a potassium channel KvAP). Membrane nanotubes with a controlled diameter (15-500 nm) are pulled out of the GUV with optical tweezers; the relative protein sorting is measured as a function of curvature (inverse of tube radius) from fluorescence measurements with a confocal microscope. We show that our mechanical model based on spontaneous curvature induction is in good agreement with these experiments. All together, this demonstrates that membrane shape is an important parameter that might drive the lateral distribution proteins in membranes, independently of more specific sorting mechanisms.

Proteins containing a Bin/Amphiphysin/Rvs (BAR) domain regulate membrane curvature in the cell. Recent simulations have revealed that BAR proteins assemble into linear aggregates, strongly affecting membrane curvature and its in-plane stress profile. Here, we explore the opposite question: do mechanical properties of the membrane impact protein association? By using coarse-grained molecular dynamics simulations, we show that increased surface tension significantly impacts the dynamics of protein assembly. While tensionless membranes promote a rapid formation of long-living linear aggregates of N-BAR proteins, increase in tension alters the geometry of protein association. At high tension, protein interactions are strongly inhibited. Increasing surface density of proteins leads to a wider range of protein association geometries, promoting the formation of meshes, which can be broken apart with membrane tension. Our work indicates that surface tension may play a key role in recruiting proteins to membrane-remodelling sites in the cell., BAR domain proteins are known to reshape cell membranes. Using coarse-grained molecular dynamics simulations, Simunovic and Voth demonstrate that membrane tension strongly affects the association of BAR proteins, in turn controlling their recruitment to membrane-remodelling sites.

Lipid membranes are highly dynamic. Over several decades, physicists and biologists have uncovered a number of ways they can change the shape of membranes or alter their phase behavior. In cells, the intricate action of membrane proteins drives these processes. Considering the highly complex ways proteins interact with biological membranes, molecular mechanisms of membrane remodeling still remain unclear. When studying membrane remodeling phenomena, researchers often observe different results, leading them to disparate conclusions on the physiological course of such processes. Here we discuss how combining research methodologies and various experimental conditions contributes to the understanding of the entire phase space of membrane-protein interactions. Using the example of clathrin-mediated endocytosis we try to distinguish the question ‘how can proteins remodel the membrane?’ from ‘how do proteins remodel the membrane in the cell?’ In particular, we consider how altering physical parameters may affect the way membrane is remodeled. Uncovering the full range of physical conditions under which membrane phenomena take place is key in understanding the way cells take advantage of membrane properties in carrying out their vital tasks.

Cell plasma membranes are highly deformable and are strongly curved, even cut, upon membrane trafficking or during cell motility. BAR-domain proteins with their intrinsically curved shape and their interaction with the actin cytoskeleton are involved in many of these processes, including membrane scission in some cases. Inspired by in vivo experiments, we have used in vitro experiments to study the interaction of BAR-domain proteins with curved membranes for understanding the mechanical action of BAR-domain proteins on biological membranes. Our nanotube assay consists in pulling membrane nanotubes of controlled curvature (diameter ranging between 15 to a few hundreds of nm) from Giant Unilamellar Vesicles (GUVs) using optical tweezers and micropipette aspiration. From force and quantitative fluorescence measurements, we analyze how proteins bound to these nanotubes affect their tube diameter and conditions required for their destabilization and scission. We compare our results to theoretical models based on membrane mechanics. Eventually, we propose new mechanisms: a) for the initiation of membrane deformations (protrusions or endocytic buds) by weakly bent BAR-domains b) for membrane scission when membrane tubules scaffolded by BAR-domains are extended by an external force.

The novel urea primaquine derivatives 3 were prepared by aminolysis of primaquine benzotriazolide 2 with several hydroxyamines and ethylendiamine, while carbamates 4 were synthesized from the same precursor 2 and alcohols. All compounds are fully chemically characterized and evaluated for their cytostatic and antioxidant activities. The most prominent antiproliferative activity was obtained by compounds 3c, 3d, 3g, and 5b (IC(50)=9-40 microM). 1-(5-Hydroxypentyl)-3-[4-(6-methoxy-quinolin-8-ylamino)-pentyl]urea (3c) showed extreme selectivity toward SW 620 colon cancer cells (IC(50)=0.2 microM) and a bit less toward lung cancer cells H 460. Hydroxyurea 3h showed the highest interaction with DPPH. Primaquine twin drug 3g showed very significant inhibition on LOX soybean (IC(50)=62 microM). Almost all the tested derivatives highly inhibited lipid peroxidation, significantly stronger than primaquine phosphate.

Theoretical approaches to studying biological problems have seen appreciable advancements in the past decade, allowing detailed structural and thermodynamic description of fundamental cellular processes, such as protein folding, lipid self-assembly, large-scale structural rearrangements of macromolecules, etc. Nevertheless, a full atomic description of even simple microscopic biological systems requires billions of atoms, beyond the reach of current computational methods. Membrane remodeling induced by members of the BAR domain protein family, is an innately multiscale problem, in which molecular interactions between the BAR protein and the lipids induces local curvature, and ultimately leads to large-scale reticulations of liposomes. In our study, we start with a discretized field-theoretical description of a liposome and carry out continuum mechanics simulations that generate reticulated topologies, quite similar to the ones seen in experiments. The obtained configurations are used as templates for mapping coarse-grained lipids and BARs over the continuum model. Subsequently, we use the coarse-grained representation of the liposome to run massive molecular dynamics simulations, with the aim of capturing the underlying molecular mechanisms that direct the liposome to undergo such large-scale biological restructuring and detecting the preferred localization of BARs at high resolution.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The most abundant cellular protein, Hsp90, partakes in many biological pathways, not only in times of induced stress, but also under normal physiological conditions. Its role in controlling proteostasis, by assisting in protein folding and reducing aggregation, together with its direct involvement in cancer cell survival and neurological disorders, makes this protein an attractive drug target. In solution, Hsp90 is a homodimer, with each subunit composed of three dynamically independent domains, contributing to the great structural flexibility and the ability to accommodate a large number of clients. Our molecular dynamic simulations of the E. coli homologue gave us a unique insight into observing the conformational transitions at atomic resolution. We observed dramatic structural rearrangements, independent of the initial state or the presence of nucleotides. The apo state was free to shift between the compact and fully stretched states, separated by a free energy barrier that was very close to the crystal open conformation. ATP binding stabilized the extended closed state, similar to the closed crystal state of HtpG. In the ADP-bound state the dynamics was limited to local motions in the N-terminal region, although the outward twisting of middle domains indicated the transitioning into an open state. Release of the nucleotides led to the formation of a compact conformation, guided by interactions between Asp287 and Asp292 from the middle domain and Lys103 from the N-terminal domain. The electrostatic interactions between opposite subunits appear to be key in directing the conformational change and making Hsp90 amenable to evolutionary fine-tuning that may regulate the populations of different conformations between homologues.

Hsp90, the most abundant cellular protein, has been implicated in numerous physiological and pathological processes. It controls protein folding and prevents aggregation, but it also plays a role in cancer and neurological disorders, making it an attractive drug target. Experimental efforts have demonstrated its remarkable structural flexibility and conformational complexity, which enable it to accommodate a variety of clients, but have not been able to provide a detailed molecular description of the conformational transitions. In our molecular dynamics simulations, Hsp90 underwent dramatic structural rearrangements into energetically favorable stretched and compact states. The transitions were guided by key electrostatic interactions between specific residues of opposite subunits. Nucleotide-bound structures showed the same conformational flexibility, although ADP and ATP seemed to potentiate these interactions by stabilizing two different closed conformations. Our observations may explain the difference in dynamic behavior observed among Hsp90 homologs, and the atomic resolution of the conformational transitions helps elucidate the complex chaperone machinery.

Many biological phenomena require the membrane to change its shape. This process is often mediated by curvature-generating proteins, such as those containing one of many BAR domains. At the same time, membrane curvature controls the way proteins interact with one another and so it acts as a vital signaling mechanism in the cell. We combine theoretical modeling with experimental biophysical methods to study the driving force underlying the reshaping of biological membranes induced by BAR proteins. In particular, we employ a combination of coarse-grained molecular dynamics with field-theoretical simulation methods to study the assembly of proteins on the membrane at molecular resolution. This approach allows us to study how protein-lipid interactions couple with membrane's large-scale morphology. It also lets us identify a surprising sensitivity of protein-protein attractions on key physical membrane properties. On the other hand, by using quantitative fluorescence and atomic force microscopies, we elucidate the curvature-function relationship of membranes at much larger time and length scales. We study the recruitment of BAR proteins on highly curved membranes and how this type of curvature drives the formation of protein scaffolds. We measure the way protein assemblies impact the mechanical properties of membranes and how such effect may lead either to stabilization of complex geometries or the breakage of membrane topology, i.e. scission. Our combined theoretical and experimental approach gives vital clues on the two-way relationship between the assembly of proteins and membrane's mechanical properties and how it may regulate the dynamics in living cells.