Induction Of Curvature Research Articles

Nanoscale in silico and in vitro modeling of lipid bilayers for curvature induction and sensing

Cellular functions like motility, differentiation, or protein trafficking often require a change in membrane curvature and protein distribution. Defects associated with curvature sensitive proteins can cause several diseases. However, the mechanisms of the interactions between these proteins and lipid bilayers are not well known at the nanoscale. To study them, a combination of in-vitro and computational modeling of membranes is crucial. Here, we explore the curvature sensitivity and inducing properties of an F-BAR domain protein through in-vitro experiments with lipid coated nanobars in conjunction with all-atom molecular dynamics simulations. We simulated Formin Binding Protein 17 (FBP17) with different lipid compositions on flat and buckled bilayers. We determined the main residues involved in the interactions and proposed a mechanism. Our results, which suggest the range of local curvature induced and sensed by FBP17, give an insight into the role of BAR domain proteins in the context of membrane structural fluctuations.

Mechanistic Insights into Curvature Formation in Synthetic Vesicles.

The mimicking of natural lipid bilayers with synthetic amphiphilic systems is of great interest to researchers, as insights could lead to better understanding of the complexities of cell membranes, as well as new materials and healthcare technologies. Recapitulating natural lipid asymmetry across bilayer membranes has important implications for curvature in cell, vesicle, and organelle morphologies, but has been challenging to achieve with synthetic lipid combinations or standard amphiphilic block copolymers. In a recent article, Elizebath et al. report the synthesis of a new type of synthetic amphiphile able to induce asymmetry in an artificial bilayer membrane dynamically. The molecules were designed around an extended π-conjugated hydrophobic core with tertiary amine-terminated oxyalkylene side chains. Protonation of the tertiary amines on the bilayer exterior leads to curvature induction, bilayer fission, and vesicle formation as monitored by time-resolved spectroscopy techniques and microscopy. The results were further validated with density functional theory (DFT) calculations. The delicate balance between different molecular scale interactions in the supramolecular structures led to the dynamic transformation of the bilayer membranes. Insights described could be used to advance the assembly of hierarchical life-like materials.

Mechanistic Insights into Curvature Formation in Synthetic Vesicles

AbstractThe mimicking of natural lipid bilayers with synthetic amphiphilic systems is of great interest to researchers, as insights could lead to better understanding of the complexities of cell membranes, as well as new materials and healthcare technologies. Recapitulating natural lipid asymmetry across bilayer membranes has important implications for curvature in cell, vesicle, and organelle morphologies, but has been challenging to achieve with synthetic lipid combinations or standard amphiphilic block copolymers. In a recent article, Elizebath et al. report the synthesis of a new type of synthetic amphiphile able to induce asymmetry in an artificial bilayer membrane dynamically. The molecules were designed around an extended π‐conjugated hydrophobic core with tertiary amine‐terminated oxyalkylene side chains. Protonation of the tertiary amines on the bilayer exterior leads to curvature induction, bilayer fission, and vesicle formation as monitored by time‐resolved spectroscopy techniques and microscopy. The results were further validated with density functional theory (DFT) calculations. The delicate balance between different molecular scale interactions in the supramolecular structures led to the dynamic transformation of the bilayer membranes. Insights described could be used to advance the assembly of hierarchical life‐like materials.

Lysophospholipid headgroup size, and acyl chain length and saturation differentially affect vacuole acidification, Ca 2+ transport, and fusion.

SNARE-mediated membrane fusion is regulated by the lipid composition of the engaged bilayers. Lipid composition impacts fusion through direct protein lipid interactions or through modulating the physical properties of membranes at the site of contact, including the induction of positive curvature by lysophospholipids (LPLs). The degree of positive curvature induced is due to the length and saturation of the single acyl chain in addition to the size of the head group. Here we examined how yeast vacuole fusion and ion transport were differentially affected by changes in lysolipid properties. We found that lysophosphatidylcholine (LPC) with acyl chains containing 14-18 carbons all inhibited fusion with IC 50 values ranging from ∼40-120 µM. The monounsaturation of LPC-18:1 had no effect when compared to its saturated counterpart LPC-18:0. On the other hand, head group size played a more significant role in blocking fusion as lysophosphatidic acid (LPA)-18:1 failed to fully inhibit fusion. We also show that both Ca 2+ uptake and SNARE-dependent Ca 2+ efflux was sensitive to changes in the acyl chain length and saturation of LPCs, while LPA only affected Ca 2+ efflux. Finally, we tested these LPLs on vacuole acidification by the V-ATPase. This showed that LPC-18:0 could fully inhibit acidification whereas other LPCs had moderate effects. Again, LPA had no effect. Together these data suggest that the effects of LPLs were due to a combination of head group size and acyl chain length leading to a range in degree of positive curvature.

Delineating the shape of COat Protein complex-II coated membrane bud.

Curvature-generating proteins that direct membrane trafficking assemble on the surface of lipid bilayers to bud transport intermediates, which move protein and lipid cargoes from one cellular compartment to another. However, it remains unclear what controls the overall shape of the membrane bud once curvature induction has begun. In vitro experiments showed that excessive concentrations of the COPII protein Sar1 promoted the formation of membrane tubules from synthetic vesicles, while COPII-coated transport intermediates in cells are generally more spherical or lobed in shape. To understand the origin of these morphological differences, we employ atomistic, coarse-grained (CG), and continuum mesoscopic simulations of membranes in the presence of multiple curvature-generating proteins. We first characterize the membrane-bending ability of amphipathic peptides derived from the amino terminus of Sar1, as a function of interpeptide angle and concentration using an atomistic bicelle simulation protocol. Then, we employ CG simulations to reveal that Sec23 and Sec24 control the relative spacing between Sar1 protomers and form the inner-coat unit through an attachment with Sar1. Finally, using dynamical triangulated surface simulations based on the Helfrich Hamiltonian, we demonstrate that the uniform distribution of spacer molecules among curvature-generating proteins is crucial to the spherical budding of the membrane. Overall, our analyses suggest a new role for Sec23, Sec24, and cargo proteins in COPII-mediated membrane budding process in which they act as spacers to preserve a dispersed arrangement of Sar1 protomers and help determine the overall shape of the membrane bud.

Structure of the flotillin complex in a native membrane environment

In this study, we used cryoelectron microscopy to determine the structures of the Flotillin protein complex, part of the Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) superfamily, from cell-derived vesicles without detergents. It forms a right-handed helical barrel consisting of 22 pairs of Flotillin1 and Flotillin2 subunits, with a diameter of 32 nm at its wider end and 19 nm at its narrower end. Oligomerization is stabilized by the C terminus, which forms two helical layers linked by a β-strand, and coiled-coil domains that enable strong charge-charge intersubunit interactions. Flotillin interacts with membranes at both ends; through its SPFH1 domains at the wide end and the C terminus at the narrow end, facilitated by hydrophobic interactions and lipidation. The inward tilting of the SPFH domain, likely triggered by phosphorylation, suggests its role in membrane curvature induction, which could be connected to its proposed role in clathrin-independent endocytosis. The structure suggests a shared architecture across the family of SPFH proteins and will promote further research into Flotillin's roles in cell biology.

Shape of the membrane neck around a hole during plasma membrane repair

Plasma membrane damage and rupture occurs frequently in cells, and holes must be sealed rapidly to ensure homeostasis and cell survival. The membrane repair machinery is known to involve recruitment of curvature-inducing annexin proteins, but the connection between membrane remodeling and hole closure is poorly described. The induction of curvature by repair proteins leads to the possible formation of a membrane neck around the hole as a key intermediate structure before sealing. We formulate a theoretical model of equilibrium neck shapes to examine the potential connection to a repair mechanism. Using variational calculus, the shape equations for the membrane near a hole are formulated and solved numerically. The system is described under a condition of fixed area, and a shooting approach is applied to fulfill the boundary conditions at the free membrane edge. A state diagram of neck shapes is produced describing the variation in neck morphology with respect to the membrane area. Two distinct types of necks are predicted, one with conformations curved beyond π existing at positive excess area, whereas flat neck conformations (curved below π) have negative excess area. The results indicate that in cells, the supply of additional membrane area and a change in edge tension is linked to the formation of narrow and curved necks. Such necks may be susceptible to passive or actively induced membrane fission as a possible mechanism for hole sealing during membrane repair in cells.

Uneven Corneal Epithelial Redistribution After Femtosecond Laser-Assisted Lenticule Intrastromal Keratoplasty in Correcting Moderate-to-High Hyperopia.

To evaluate the characteristic of corrective epithelial thickness after femtosecond laser-assisted lenticule intrastromal keratoplasty (LIKE) to correct moderate-to-high hyperopia. The prospective case series study of the LIKE procedure was performed to correct moderate-to-high hyperopia. The epithelial thickness map was generated by anterior segment optical coherence tomography (AS-OCT) in the corneal central 9-mm zone. Keratometry and corneal higher order aberrations were analyzed by Pentacam (Oculus Optikgeräte GmbH) preoperatively and postoperatively. In the 26 eyes of 13 participants who underwent the LIKE procedure for moderate-to-high hyperopia, the attempted spherical equivalence (SEQ) was +6.50 ± 1.09 diopters (D). Compared to the preoperative epithelial thickness maps, the postoperative epithelial thickness had become significantly thinner in the central 5-mm zone; the difference was 6 to 7 µm. The paracentral epithelium performed nonuniform remodeling; the thinnest epithelial thickness was located in the inferotemporal section, which has the greatest difference from the superonasal; the difference between these two was approximately 3 µm. Through correlation analysis, it was found that the sections with thinner epithelium were significantly related to corneal curvature and corneal vertical coma. The LIKE procedure can be used to correct moderate-to-high hyperopia. This study further indicated the epithelial remodeling characteristic after the LIKE procedure: the central and paracentral corneal epithelial thickness becomes thinner, and the epithelial thickness distributes non-uniformly, which may be the important factor of the postoperative curvature asymmetric distribution and induction of corneal vertical coma. [J Refract Surg. 2024;40(5):e321-e327.].

Spontaneous Curvature Induction in an Artificial Bilayer Membrane.

Maintaining lipid asymmetry across membrane leaflets is critical for functions like vesicular traffic and organelle homeostasis. However, a lack of molecular-level understanding of the mechanisms underlying membrane fission and fusion processes in synthetic systems precludes their development as artificial analogs. Here, we report asymmetry induction of a bilayer membrane formed by an extended π-conjugated molecule with oxyalkylene side chains bearing terminal tertiary amine moieties (BA1) in water. Autogenous protonation of the tertiary amines in the periphery of the bilayer by water induces anisotropic curvature, resulting in membrane fission to form vesicles and can be monitored using time-dependent spectroscopy and microscopy. Interestingly, upon loss of the induced asymmetry by extensive protonation using an organic acid restored bilayer membrane. The mechanism leading to the compositional asymmetry in the leaflet and curvature induction in the membrane is validated by density functional theory (DFT) calculations. Studies extended to control molecules having changes in hydrophilic (BA2) and hydrophobic (BA3) segments provide insight into the delicate nature of molecular scale interactions in the dynamic transformation of supramolecular structures. The synergic effect of hydrophobic interaction and the hydrated state of BA1 aggregates provide dynamicity and unusual stability. Our study unveils mechanistic insight into the dynamic transformation of bilayer membranes into vesicles.

Spontaneous Curvature Induction in an Artificial Bilayer Membrane

AbstractMaintaining lipid asymmetry across membrane leaflets is critical for functions like vesicular traffic and organelle homeostasis. However, a lack of molecular‐level understanding of the mechanisms underlying membrane fission and fusion processes in synthetic systems precludes their development as artificial analogs. Here, we report asymmetry induction of a bilayer membrane formed by an extended π‐conjugated molecule with oxyalkylene side chains bearing terminal tertiary amine moieties (BA1) in water. Autogenous protonation of the tertiary amines in the periphery of the bilayer by water induces anisotropic curvature, resulting in membrane fission to form vesicles and can be monitored using time‐dependent spectroscopy and microscopy. Interestingly, upon loss of the induced asymmetry by extensive protonation using an organic acid restored bilayer membrane. The mechanism leading to the compositional asymmetry in the leaflet and curvature induction in the membrane is validated by density functional theory (DFT) calculations. Studies extended to control molecules having changes in hydrophilic (BA2) and hydrophobic (BA3) segments provide insight into the delicate nature of molecular scale interactions in the dynamic transformation of supramolecular structures. The synergic effect of hydrophobic interaction and the hydrated state of BA1 aggregates provide dynamicity and unusual stability. Our study unveils mechanistic insight into the dynamic transformation of bilayer membranes into vesicles.

Synthesis, insertion, and characterization of SARS-CoV-2 membrane protein within lipid bilayers.

Throughout history, coronaviruses have posed challenges to both public health and the global economy; nevertheless, methods to combat them remain rudimentary, primarily due to the absence of experiments to understand the function of various viral components. Among these, membrane (M) proteins are one of the most elusive because of their small size and challenges with expression. Here, we report the development of an expression system to produce tens to hundreds of milligrams of M protein per liter of Escherichia coli culture. These large yields render many previously inaccessible structural and biophysical experiments feasible. Using cryo-electron microscopy and atomic force microscopy, we image and characterize individual membrane-incorporated M protein dimers and discover membrane thinning in the vicinity, which we validated with molecular dynamics simulations. Our results suggest that the resulting line tension, along with predicted induction of local membrane curvature, could ultimately drive viral assembly and budding.

Mechanism of Membrane Curvature Induced by SNX1: Insights from Molecular Dynamics Simulations.

SNX proteins have been found to induce membrane remodeling to facilitate the generation of transport carriers in endosomal pathways. However, the molecular mechanism of membrane bending and the role of lipids in the bending process remain elusive. Here, we conducted coarse-grained molecular dynamics simulations to investigate the role of the three structural modules (PX, BAR, and AH) of SNX1 and the PI3P lipids in membrane deformation. We observed that the presence of all three domains is essential for SNX1 to achieve a stable membrane deformation. BAR is capable of remodeling the membrane through the charged residues on its concave surface, but it requires PX and AH to establish stable membrane binding. AH penetrates into the lipid membrane, thereby promoting the induction of membrane curvature; however, it is inadequate on its own to maintain membrane bending. PI3P lipids are also indispensable for membrane remodeling, as they play a dominant role in the interactions of lipids with the BAR domain. Our results enhance the comprehension of the molecular mechanism underlying SNX1-induced membrane curvature and help future studies of curvature-inducing proteins.

Ubiquitination regulates ER-phagy and remodelling of endoplasmic reticulum

The endoplasmic reticulum (ER) undergoes continuous remodelling via a selective autophagy pathway, known as ER-phagy1. ER-phagy receptors have a central role in this process2, but the regulatory mechanism remains largely unknown. Here we report that ubiquitination of the ER-phagy receptor FAM134B within its reticulon homology domain (RHD) promotes receptor clustering and binding to lipidated LC3B, thereby stimulating ER-phagy. Molecular dynamics (MD) simulations showed how ubiquitination perturbs the RHD structure in model bilayers and enhances membrane curvature induction. Ubiquitin molecules on RHDs mediate interactions between neighbouring RHDs to form dense receptor clusters that facilitate the large-scale remodelling of lipid bilayers. Membrane remodelling was reconstituted in vitro with liposomes and ubiquitinated FAM134B. Using super-resolution microscopy, we discovered FAM134B nanoclusters and microclusters in cells. Quantitative image analysis revealed a ubiquitin-mediated increase in FAM134B oligomerization and cluster size. We found that the E3 ligase AMFR, within multimeric ER-phagy receptor clusters, catalyses FAM134B ubiquitination and regulates the dynamic flux of ER-phagy. Our results show that ubiquitination enhances RHD functions via receptor clustering, facilitates ER-phagy and controls ER remodelling in response to cellular demands.

Engineering a membrane-binding protein to trimerize and induce high membrane curvature

The annexins are a family of Ca2+-dependent peripheral membrane proteins. Several annexins are implicated in plasma membrane repair and are overexpressed in cancer cells. Annexin A4 (ANXA4) and annexin A5 (ANXA5) form trimers that induce high curvature on a membrane surface, a phenomenon deemed to accelerate membrane repair. Despite being highly homologous to ANXA4, annexin A3 (ANXA3) does not form trimers on the membrane surface. Using molecular dynamics simulations, we have reverse engineered an ANXA3-mutant to trimerize on the surface of the membrane and induce high curvature reminiscent of ANXA4. In addition, atomic force microscopy images show that, like ANXA4, the engineered protein forms crystalline arrays on a supported lipid membrane. Despite the trimer-forming and curvature-inducing properties of the engineered ANXA3, it does not accumulate near a membrane lesion in laser-punctured cells and is unable to repair the lesion. Our investigation provides insights into the factors that drive annexin-mediated membrane repair and shows that the membrane-repairing property of trimer-forming annexins also necessitates high membrane binding affinity, other than trimer formation and induction of negative membrane curvature.

Structural basis of mitochondrial membrane bending by the I–II–III2–IV2 supercomplex

Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane1. Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I–II–III2–IV2 supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization.

Membrane mechanics regulate the cooperativity of proteins in curvature sensing.

Recruitment of proteins to a membrane surface is critical for many biological processes, such as clathrin-mediated endocytosis and signaling. When interacting with the lipid membrane, many proteins exhibit different affinities to membranes with different curvature, known as curvature sensing. They also generate membrane curvature (curvature induction) by a variety of mechanisms, including the insertion of amphipathic helices. Here we use a continuum membrane model to study how helix insertions can alter the membrane energetics and how the mechanical feedback of the membrane regulates the helix insertions. Our membrane model has two coupled layers of triangular mesh to represent the two monolayers of the bilayer membrane, capturing membrane thickness and tilt. We analyzed the membrane deformation around one helix insertion and compared it to molecular dynamic simulations to optimally define the lipid tilt energies. We then investigate the interactions between insertions and membranes by considering the copy number of insertions and their interval distance, membrane tension or stress before the insertions, membrane curvatures, and lipid composition. Our results showed that multiple insertions exhibit a mechanism of cooperativity; each binding event is not independent of previous events. Our results show that this cooperativity is dependent on membrane tension and curvature. In comparison to experimental data, we validated our simulation results and analyzed the role of lipid flip-flop across the bilayers. Quantifying how protein and membrane interactions feedback on one another is critical for understanding the dynamics and control of proteins that remodel membranes through localization and self-assembly in various cellular processes.

Comparing elastic and molecular models of membrane bending local to SARS-CoV-2 envelope protein for mechanistic understanding.

The Envelope (E) protein of SARS-CoV-2 is an ion channel that plays several roles in the viral lifecycle, including the induction of membrane curvature during the budding process. While its function as a viroporin has been explored by several labs, the precise mechanism through which the E protein controls membrane bending is not presently understood. In the present study, we show that the E protein induces membrane bending in silico in coarse-grain molecular dynamics (CG-MD) simulations, suggesting that the computational microscope is a promising means for exploring this phenomenon further. We quantify the extent and type of membrane bending present in systems with varying membrane thickness, lipid saturation, protein structure, and protein concentration using our CG-MD analysis suite nougat. We then compare those results with theoretical predictions from a continuum membrane model that treats each leaflet as an elastic surface with bulk properties. These comparisons allow us to gain insight into the bending mechanism of the E protein. The results have implications for membrane biophysics and lays the groundwork for further research on the efficacy of drug candidates that target the SARS-CoV-2 E protein.

Atomistic insights into the role of Sar1 GTPase in COPII mediated protein secretary pathway.

In the protein secretory pathway of the cell, the Coat Protein Complex II (COPII) is a crucial molecular machine that transports protein payloads from the Endoplasmic Reticulum (ER) to the Golgi Body. Sar1, a COPII component protein, initiates the protein transport mechanism by budding vesicles on the subdomains of ER after being activated by GDP/GTP exchange. Based on crystallographic and cryo-EM study, the conformational change of Sar1's amino terminal amphipathic helix following GTP binding is considered to be a critical factor in the initiation of the vesicle budding step. However, such GTP triggered conformational switch mechanism remains incomplete without the explicit molecular details of the penetration of the amino terminus of GDP and GTP bound state into the membrane. We perform all-atom, explicit solvent Molecular Dynamics simulation to capture atomistic insights into the differential membrane binding and bending ability of GDP and GTP bound Sar1. We demonstrate that in the GDP-bound state, Sar1 partially inserts its amino terminus into the membrane (residues 1-12), whereas in the GTP-bound state, the whole amino terminus stays horizontally buried inside the membrane (residues 1-23). Thus, GTP bound Sar1 triggers strong inter-leaflet stress due to excess volume insertion in the membrane leading to ∼10-20 fold enhancement in positive curvature induction compared to that due to GDP bound form. We reveal that the magnitude of membrane curvature formation is further amplified by the dimerization of the GTP bound state. Although amphipathic helices are known to generate positive curvature on membranes our simulations reinforce the dominance of the amount of volume inclusion over its penetration depth inside the membrane in this process. Together, we propose a thorough molecular basis for the activity of Sar1-mediated membrane remodeling activity.

Dynamic Interaction of Peptides with Membrane

Induction of curvature in lipid bilayers may lead to increased exposure of hydrophobic membrane core to cell surfaces and the eventual hydrophobic interaction with amphiphilic peptides. Not only physicochemical driving force, the membrane curvature and exposure of the hydrophobic core can be elicited by physiological cellular actions including movement of the cell membrane driven by cytoskeletal F–actin restructuring. This review highlights our findings during our studies on developing curvature–inducing peptides and intracellular IgG delivery peptides.

Interplay of membrane crosslinking and curvature induction by annexins

Efficient plasma membrane repair (PMR) is required to repair damage sustained in the cellular life cycle. The annexin family of proteins, involved in PMR, are activated by Ca2+ influx from extracellular media at the site of injury. Mechanistic studies of the annexins have been overwhelmingly performed using a single annexin, despite the recruitment of multiple annexins to membrane damage sites in living cells. Hence, we investigate the effect of the presence of the crosslinking annexins, annexin A1, A2 and A6 (ANXA1, ANXA2 and ANXA6) on the membrane curvature induction of annexin A4 (ANXA4) in model membrane systems. Our data support a mechanistic model of PMR where ANXA4 induced membrane curvature and ANXA6 crosslinking promotes wound closure. The model now can be expanded to include ANXA1 and ANXA2 as specialist free edge membrane crosslinkers that act in concert with ANXA4 induced curvature and ANXA6 crosslinking.