Induce Membrane Curvature Research Articles

EXTENSION OF STICHOLYSINS N-TERMINAL α-HELIX SIGNALS MEMBRANE LIPIDS TO ACQUIRE CURVATURE FOR TOROIDAL PORE FORMATION

Sticholysin I and II (St I/II) belong to the actinoporins family; these proteins form pores in host cell membranes by binding their N-terminal segment to the membrane, leading to protein-lipid (toroidal) pores. Peptides derived from actinoporins pore-forming domains replicate their folding properties and permeabilizing effects. Despite the advances in understanding how these proteins and peptides mediate pore formation, the role of different N-terminal segments in inducing membrane curvature is still unclear. Here we combine circular dichroism, electron paramagnetic resonance, and small-angle X-ray scattering to investigate how synthetic peptides encompassing the N-terminal segments of St I and II (StI1-31, StII1-30, StI12-31, and StII11-30) interact with lipid bilayers and micelles as mimics of the topography of the initial membrane binding and of the subsequently formed positively curved pore. We investigate both the conformational changes and peptides' effects on membrane organization resulting from these interactions. According to the toroidal pore model, our results support that the actinoporins amphipathic α-helices rest at the membrane interface, forming pore walls with lipid head groups, while the 1-10 segment of St II penetrates the bilayer, acting as an anchor. We relate this ability to the higher hydrophobicity of this segment in St II, compared to St I. This unique feature of St II would contribute to enhanced pore formation, explaining St II's increased activity when compared to other actinoporins. Our results reinforce the notion that pore formation by actinoporins is a highly cooperative process where specific protein segments and the lipid bilayer mutually modulate their conformation and organization.

Coarse-Grained Simulations of Adeno-Associated Virus and Its Receptor Reveal Influences on Membrane Lipid Organization and Curvature.

Adeno-associated virus (AAV) is a well-known gene delivery tool with a wide range of applications, including as a vector for gene therapies. However, the molecular mechanism of its cell entry remains unknown. Here, we performed coarse-grained molecular dynamics simulations of the AAV serotype 2 (AAV2) capsid and the universal AAV receptor (AAVR) in a model plasma membrane environment. Our simulations show that binding of the AAV2 capsid to the membrane induces membrane curvature, along with the recruitment and clustering of GM3 lipids around the AAV2 capsid. We also found that the AAVR binds to the AAV2 capsid at the VR-I loops using its PKD2 and PKD3 domains, whose binding poses differs from previous structural studies. These first molecular-level insights into AAV2 membrane interactions suggest a complex process during the initial phase of AAV2 capsid internalization.

Modulation of Annexin-Induced Membrane Curvature by Cholesterol and the Anionic Lipid Headgroup during Plasma Membrane Repair.

Annexins (ANXAs), calcium-sensitive phospholipid-binding proteins, are pivotal for cellular membrane repair, which is crucial for eukaryotic cell survival under membrane stress. With their unique trimeric arrangements and crystalline arrays on the membrane surface, ANXA4 and ANXA5 induce membrane curvature and rapidly orchestrate plasma membrane resealing. However, the influence of cholesterol and anionic lipid headgroups on annexin-induced membrane curvature remains poorly understood at the molecular level. Using all-atom molecular dynamics simulations, we measured the local curvature-induced underneath ANXA4 and ANXA5 monomers and trimers when they bind to lipid bilayers of distinct lipid compositions: PSPC (20% POPS, 80% POPC), PAPC (20% POPA, 80% POPC), and PSPCCHL (14% POPS, 56% POPC, 30% cholesterol). Laser injury experiments were conducted on MCF7 cells transfected to transiently express fluorescently labeled ANXA4 or ANXA5 to facilitate the examination of protein and lipid accumulation at the damage site. Annexin trimers induce higher curvature than monomers, particularly with cholesterol present. Annexin trimers induce similar curvatures on both PAPC and PSPC membranes. Notably, among monomers, ANXA5 induces the highest curvature on PAPC, suggesting more efficient recruitment of ANXA5 compared with ANXA4 in the early stages of membrane repair near a lesion. Laser injury experiments confirm rapid coaccumulation of phosphatidic acid lipids with ANXA4 and ANXA5 at repair sites, potentially enhancing the accumulation of annexins in the early stages of membrane repair.

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.

A lever hypothesis for Synaptotagmin-1 action in neurotransmitter release.

Neurotransmitter release is triggered in microseconds by Ca2+-binding to the Synaptotagmin-1 C2 domains and by SNARE complexes that form four-helix bundles between synaptic vesicles and plasma membranes, but the coupling mechanism between Ca2+-sensing and membrane fusion is unknown. Release requires extension of SNARE helices into juxtamembrane linkers that precede transmembrane regions (linker zippering) and binding of the Synaptotagmin-1 C2B domain to SNARE complexes through a 'primary interface' comprising two regions (I and II). The Synaptotagmin-1 Ca2+-binding loops were believed to accelerate membrane fusion by inducing membrane curvature, perturbing lipid bilayers or helping bridge the membranes, but SNARE complex binding orients the Ca2+-binding loops away from the fusion site, hindering these putative activities. Molecular dynamics simulations now suggest that Synaptotagmin-1 C2 domains near the site of fusion hinder SNARE action, providing an explanation for this paradox and arguing against previous models of Sytnaptotagmin-1 action. NMR experiments reveal that binding of C2B domain arginines to SNARE acidic residues at region II remains after disruption of region I. These results and fluorescence resonance energy transfer assays, together with previous data, suggest that Ca2+ causes reorientation of the C2B domain on the membrane and dissociation from the SNAREs at region I but not region II. Based on these results and molecular modeling, we propose that Synaptotagmin-1 acts as a lever that pulls the SNARE complex when Ca2+ causes reorientation of the C2B domain, facilitating linker zippering and fast membrane fusion. This hypothesis is supported by the electrophysiological data described in the accompanying paper.

Non-linear electro-rheological model of a membrane immersed in Tanner-Power law fluids applied to outer hair cells: Shear-thinning mechanisms

Flexoelectric actuation employs an applied electric field to induce membrane curvature, which is the mechanism utilized by the outer hair cells (OHC) present in the inner ear. The model developed for this study, representing the OHC, integrates two key components: (i) an approximation of the flexoelectric membrane shape equation for circular membranes attached to the inner surface of a circular capillary, and (ii) the coupled capillary flow of contacting liquid viscoelastic phases characterized by the Tanner-Power law rheological equation of state. A second-order non-linear differential equation for average curvature has been derived, and a robust numerical method has been programmed. This model simplifies to a linear model used previously. The main challenge involves identifying and describing the enhancement in curvature change rate. It was observed that low symmetry, low viscosity, and soft membrane and shear-thickening behavior of the phases enhance the curvature change rate. Additionally, there exists a critical electric field frequency value that maximizes the curvature change rate (resonance effect). The current theory, model, and computational simulations add to the ongoing development comprehension of how biological membrane shape actuation through electromechanical couplings.

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.

The consequence of ATP synthase dimer angle on mitochondrial morphology studied by cryo-electron tomography.

Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans and show that the dimer angle differs from previously determined structures. The consequences of this species-specific difference at the dimer interface were investigated by comparing C. elegans and S. cerevisiae mitochondrial morphology. We reveal that C. elegans has a larger ATP synthase dimer angle with more lamellar (flatter) cristae when compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling. A comparison of our C. elegans model to an existing S. cerevisiae structure reveals the presence of extensions and rearrangements in C. elegans subunits associated with maintaining the dimer interface. We speculate that increasing dimer angles could provide an advantage for species that inhabit variable-oxygen environments by forming flatter more energetically efficient cristae.

Topographical depth reveals contact guidance mechanism distinct from focal adhesion confinement.

Cellular response to the topography of their environment, known as contact guidance, is a crucial aspect to many biological processes yet remains poorly understood. A prevailing model to describe cellular contact guidance involves the lateral confinement of focal adhesions (FA) by topography as an underlying mechanism governing how cells can respond to topographical cues. However, it is not clear how this model is consistent with the well-documented depth-dependent contact guidance responses in the literature. To investigate this model, we fabricated a set of contact guidance chips with lateral dimensions capable of confining focal adhesions and relaxing that confinement at various depths. We find at the shallowest depth of 330 nm, the model of focal adhesion confinement is consistent with our observations. However, the cellular response at depths of 725 and 1000 nm is inadequately explained by this model. Instead, we observe a distinct reorganization of F-actin at greater depths in which topographically induced cell membrane deformation alters the structure of the cytoskeleton. These results are consistent with an alternative curvature-hypothesis to explain cellular response to topographical cues. Together, these results indicate a confluence of two molecular mechanisms operating at increased induced membrane curvature that govern how cells sense and respond to topography.

Investigating the mechanical properties and flexibility of N-BAR domains in PICK1 by molecular dynamics simulations.

Bin/Amphiphysin/Rvs167 (BAR) domain superfamily proteins are considered to be able to induce membrane curvature. PICK1 is a unique protein consisting of both a PDZ and a BAR domain that has been associated with many diseases. PICK1 can force membrane curvature during receptor-mediated endocytosis. In addition to understanding how the N-BAR domain can force membrane curvature, it is also of particular interest to understand the hidden connections between structural and mechanical properties of the PICK1 BAR dimers. In this paper, steered molecular dynamics is used to investigate the mechanical properties that are associated with structural changes of the PICK1 BAR domains. Our results suggest that helix kinks may not only help generate curvature of BAR domains but may also provide the extra flexibility necessary to initiate the binding between BAR domains and the membrane. Interestingly, we observe a complicated interaction network both within the BAR monomer and at the binding interface of the two BAR monomers that is key to maintain the mechanical properties of the BAR dimer. Because of such an interaction network, the PICK1 BAR dimer demonstrated different responses to external forces in opposite directions.

Nuclear deformation regulates YAP dynamics in cancer associated fibroblasts

Cells cultured on stiff 2D substrates exert high intracellular force, resulting in mechanical deformation of their nuclei. This nuclear deformation (ND) plays a crucial role in the transport of Yes Associated Protein (YAP) from the cytoplasm to the nucleus. However, cells in vivo are in soft 3D environment with potentially much lower intracellular forces. Whether and how cells may deform their nuclei in 3D for YAP localization remains unclear. Here, by culturing human colon cancer associated fibroblasts (CAFs) on 2D, 2.5D, and 3D substrates, we differentiated the effects of stiffness, force, and ND on YAP localization. We found that nuclear translocation of YAP depends on the degree of ND irrespective of dimensionality, stiffness and total force. ND induced by the perinuclear force, not the total force, and nuclear membrane curvature correlate strongly with YAP activation. Immunostained slices of human tumors further supported the association between ND and YAP nuclear localization, suggesting ND as a potential biomarker for YAP activation in tumors. Additionally, we conducted quantitative analysis of the force dynamics of CAFs on 2D substrates to construct a stochastic model of YAP kinetics. This model revealed that the probability of YAP nuclear translocation, as well as the residence time in the nucleus follow a power law. This study provides valuable insights into the regulatory mechanisms governing YAP dynamics and highlights the significance of threshold activation in YAP localization. Statement of SignificanceYes Associated Protein (YAP), a transcription cofactor, has been identified as one of the drivers of cancer progression. High tumor stiffness is attributed to driving YAP to the nucleus, wherein it activates pro-metastatic genes. Here we show, using cancer associated fibroblasts, that YAP translocation to the nucleus depends on the degree of nuclear deformation, irrespective of stiffness. We also identified that perinuclear force induced membrane curvature correlates strongly with YAP nuclear transport. A novel stochastic model of YAP kinetics unveiled a power law relationship between the activation threshold and persistence time of YAP in the nucleus. Overall, this study provides novel insights into the regulatory mechanisms governing YAP dynamics and the probability of activation that is of immense clinical significance.

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.

The Knowns and Unknowns of Herpesvirus Nuclear Egress.

Nuclear egress of herpesvirus capsids across the intact nuclear envelope is an exceptional vesicle-mediated nucleocytoplasmic translocation resulting in the delivery of herpesvirus capsids into the cytosol. Budding of the (nucleo)capsid at and scission from the inner nuclear membrane (INM) is mediated by the viral nuclear egress complex (NEC) resulting in a transiently enveloped virus particle in the perinuclear space followed by fusion of the primary envelope with the outer nuclear membrane (ONM). The dimeric NEC oligomerizes into a honeycomb-shaped coat underlining the INM to induce membrane curvature and scission. Mutational analyses complemented structural data defining functionally important regions. Questions remain, including where and when the NEC is formed and how membrane curvature is mediated, vesicle formation is regulated, and directionality is secured. The composition of the primary enveloped virion and the machinery mediating fusion of the primary envelope with the ONM is still debated. While NEC-mediated budding apparently follows a highly conserved mechanism, species and/or cell type-specific differences complicate understanding of later steps.

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.

Activity and Structural Dynamics of Human ABCA1 in a Lipid Membrane

The human ATP-binding cassette (ABC) transporter ABCA1 plays a critical role in lipid homeostasis as it extracts sterols and phospholipids from the plasma membrane for excretion to the extracellular apolipoprotein A-I and subsequent formation of high-density lipoprotein (HDL) particles. Deleterious mutations of ABCA1 lead to sterol accumulation and are associated with atherosclerosis, poor cardiovascular outcomes, cancer, and Alzheimer’s disease. The mechanism by which ABCA1 drives lipid movement is poorly understood, and a unified platform to produce active ABCA1 protein for both functional and structural studies has been missing. In this work, we established a stable expression system for both a human cell-based sterol export assay and protein purification for in vitro biochemical and structural studies. ABCA1 produced in this system was active in sterol export and displayed enhanced ATPase activity after reconstitution into a lipid bilayer. Our single-particle cryo-EM study of ABCA1 in nanodiscs showed protein induced membrane curvature, revealed multiple distinct conformations, and generated a structure of nanodisc-embedded ABCA1 at 4.0-Å resolution representing a previously unknown conformation. Comparison of different ABCA1 structures and molecular dynamics simulations demonstrates both concerted domain movements and conformational variations within each domain. Taken together, our platform for producing and characterizing ABCA1 in a lipid membrane enabled us to gain important mechanistic and structural insights and paves the way for investigating modulators that target the functions of ABCA1.

The effect of tailing lipidation on the bioactivity of antimicrobial peptides and their aggregation tendency

AbstractAntimicrobial peptides (AMPs) are potentially powerful alternatives to conventional antibiotics in combating multidrug resistance, given their broad spectrum of activity. They mainly interact with cell membranes through surface electrostatic potentials and the formation of secondary structures, resulting in permeability and destruction of target microorganism membranes. Our earlier work showed that two leading AMPs, MSI‐78 (4–20) and pardaxin (1–22), had potent antimicrobial activity against a range of bacteria. It is known that the attachment of moderate‐length lipid carbon chains to cationic peptides can further improve the functionality of these peptides through enhanced interactions with the membrane lipid bilayer, inducing membrane curvature, destabilization, and potential leakage. Thus, in this work, we aimed to investigate the antimicrobial activity, oligomerization propensity, and lipid‐membrane binding interactions of a range of N‐terminal lipidated analogs of MSI‐78 (4–20) and pardaxin (1–22). Molecular modeling results suggest that aggregation of the N‐lipidated AMPs may impart greater structural stability to the peptides in solution and a greater depth of lipid bilayer insertion for the N‐lipidated AMPs over the parental peptide. Our experimental and computational findings provide insights into how N‐terminal lipidation of AMPs may alter their conformations, with subsequent effects on their functional properties in regard to their self‐aggregation behavior, membrane interactions, and antimicrobial activity.

Counting AP2 using quantitative correlative light and electron microscopy (quant-CLEM).

Clathrin-mediated-endocytosis (CME) constitutes a ubiquitous and essential mechanism for the internalization of membrane segments or extracellular cargo in eukaryotic cells. Together with the pathway's namesake protein clathrin, dozens of other proteins contribute to the binding of specific cargo, coating the plasma membrane, inducing membrane curvature and eventual budding of vesicles. Facilitated by their characteristic honeycomb structure, clathrin-coated membranes have proven an ideal subject for correlative light and electron microscopy (CLEM) imaging. Combination of this approach with super-resolution light microscopy allowed mapping the precise locations of numerous key proteins during different stages of vesicle formation.

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.

Modeling the mechanisms through which protein phase separation induces membrane curvature.

Membrane curvature is an essential component of life. Many cellular processes, such as endocytosis, exocytosis, and cell motility induce curvature in membranes to carry out their functions. Though it is traditionally thought that structured proteins drive curvature, recent work has shown that disordered proteins can also be potent drivers of curvature. In particular, liquid-like condensation of disordered proteins has recently been shown to produce concave membrane curvature, resulting in protein-lined buds and tubules of the membrane. However, the mechanism behind this phenomenon remains poorly understood. Here we seek to understand the protein-protein and protein-membrane interactions that give rise to these behaviors. We begin by using coarse grained molecular dynamics simulations to investigate the conformational preferences of disordered proteins under different conditions: in varying salt concentrations, in solution or tethered to a lipid membrane. Specifically, we examine the deviation of disordered protein behavior from that of a simple random walk, both in solution and on membrane surfaces. By comparing modeling results to experimental data, we separate the role of protein-protein and protein-membrane interactions to shed light on the role of each in both phase separation and curvature generation. Our work will provide insight on the role that disordered regions have in membrane complexes, particularly those which facilitate membrane curvature.

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.