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Nanoscale Electrical and Coarse-grained Molecular Dynamics Studies of Influenza Hemagglutinin-mediated Membrane Fusion Pores
Fusion of viral and host membranes is a key step during infection by membrane-enclosed viruses. The fusion pore plays a critical role, and must dilate to release the viral genome. Prior studies of fusion mediated by influenza A hemagglutinin (HA) revealed ~2-5 nm pores that flickered before dilating to >10 nm. The mechanisms involved are unknown.
Here we studied HA-mediated fusion pore dynamics using a novel single-pore assay (supported by a novel, robust, single-cell optical assay for fusion between HA-expressing cells and nanodiscs), combined with computational simulations accessing extraordinarily long (ms) timescales. We measured pores between HA-expressing fibroblasts and bilayer nanodiscs. From pore currents we infer pore size with millisecond time resolution. Unlike previous in vitro studies, the use of nanodiscs limited the membrane contact areas and maximum pore sizes, better mimicking the initial phases of virus-endosome fusion. In wild-type (WT) HA-mediated fusion pores, pores flickered about a mean pore size ~1.7 nm. In contrast, fusion pores formed by GPI-anchored HA nucleated at less than half the WT rate; results were consistent with earlier findings that showed that while GPI-HA pores stabilize at larger initial conductances than WT, they were not able to enlarge beyond their initial size.
We developed radically coarse-grained, explicit lipid molecular dynamics simulations of the fusion pore reconstituted with post-fusion, trans HA hairpins. With WT HA, fusion pores were small, similar to experiment. Over time hairpins gradually converted from trans to cis. With lipid-anchored HA, the trans â cis transition was much accelerated. Once most hairpins had converted to cis, because apposing membranes were released, the fusion pore was able to dilate to sizes close to protein-free. Additionally, in crowded simulations with HA densities approximating those found in HA clusters, we found that HA aggregation, promoted by TMD-TMD interactions, delayed fusion pore dilation by inhibiting the trans â cis transition.
Our results suggest that pore dilation requires the trans â cis transition. We hypothesize that this transition is accelerated in GPI-HA by the more mobile lipid anchor, and may explain the larger observed nascent fusion pores
The role of influenza neuraminidase transmembrane domain on budding and virus morphology
Influenza A virus neuraminidase (NA), a type II transmembrane glycoprotein plays a role in the cleavage of sialic acids and facilitating the release of mature virions from the surface of infected cells. NA has also previously been shown to play a role in virion formation during influenza A virus budding, although the exact mechanisms by which NA contributes to influenza virion formation and morphology is currently unknown. Previous research has shown that mutations within the transmembrane domain (TMD) of NA can result in alteration in virion morphology, particularly in the production of filament like influenza virions. In this research project we examined if the TMD does indeed play a role in influenza virus budding and morphology. We utilised both full and partial mutations of the TMD of NA from A/WSN/33, a primarily spherical lab adapted influenza strain, with the TMD of a primarily filamentous strain A/California/09. To evaluate the effects of TMD on the morphology of a primarily spherical strain with that of filamentous strain. This study used a transfection based virus like particle (VLP) system to examine the effects of TMD alterations on morphology, utilising various biochemical and microscopy methods. Our findings show that as previously indicated mutations within the TMD do result in alterations to virion morphology, as well as showing that despite previous theories both NA and NAâs TMD may play a more active role in in budding and morphology than previously though
Visualizing Biological Membrane Organization and Dynamics
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Rafts: scale-dependent, active lipid organization at the cell surface
Rafts have been conceptualized as lateral heterogeneities in the organization of cholesterol and sphingolipids, endowed with sorting and signaling functions. In this review we critically examine evidence for the main tenet of the 'raft hypothesis', namely lipid-dependent segregation of specific membrane components in the plasma membrane. We suggest that conventional approaches to studying raft organization wherein membranes are treated as passive, thermally equilibrated systems are unlikely to provide an adequate framework to understand the mechanisms of raft-organization in vivo. An emerging view of raft organization is that it is spatio-temporally regulated at different scales by the cell. This argues that rafts must be defined by simultaneous observation of components involved in particular functions. Recent evidence from the study of glycosylphosphatidyl inositol-anchored proteins, a common raft-marker, supports this picture in which larger scale, more stable rafts are induced from preexisting small-scale lipid-dependent structures actively maintained by cellular processes
Molecular mechanism for bidirectional regulation of CD44 for lipid raft affiliation by palmitoylations and PIP2
The co-localization of Cluster-of-Differentiation-44 protein (CD44) and cytoplasmic adaptors in specific membrane environments is crucial for cell adhesion and migration. The process is controlled by two different pathways: On the one hand palmitoylation keeps CD44 in lipid raft domains and disables the linking to the cytoplasmic adaptor, whereas on the other hand, the presence of phosphatidylinositol-4,5-biphosphate (PIP2) lipids accelerates the formation of the CD44-adaptor complex. The molecular mechanism explaining how CD44 is migrating into and out of the lipid raft domains and its dependence on both palmitoylations and the presence of PIP2 remains, however, elusive. In this study, we performed extensive molecular dynamics simulations to study the raft affinity and translocation of CD44 in phase separated model membranes as well as more realistic plasma membrane environments. We observe a delicate balance between the influence of the palmitoylations and the presence of PIP2 lipids: whereas the palmitoylations of CD44 increases the affinity for raft domains, PIP2 lipids have the opposite effect. Additionally, we studied the association between CD44 and the membrane adaptor FERM in dependence of these factors. We find that the presence of PIP2 lipids allows CD44 and FERM to associate in an experimentally observed binding mode whereas the highly palmitoylated species shows no binding affinity. Together, our results shed light on the sophisticated mechanism on how membrane translocation and peripheral protein association can be controlled by both protein modifications and membrane composition
Characterization of Phosphorylated G Protein Function and Membrane Culstering by Super Resolution Imaging
Heterotrimeric G proteins play crucial roles in various signal transduction pathways, where they act as molecular switches in transducing a signal from G protein coupled receptors (GPCRs) at the plasma membrane to downstream effectors. Although their mechanism of action is mostly concentrated at the plasma membrane, their dynamic membrane organization and how it is regulated are not understood. Due to the diffraction limited resolution of fluorescence microscopy, studying the precise organization of membrane proteins can be challenging. In this study, we took advantage of super-resolution fluorescence photoactivation localization microscopy (FPALM) to overcome this challenge. Dictyostelium discoideum was used as a cellular model to study G protein function and membrane organization. These cells rely on chemotaxis toward a secreted chemoattractant, cyclic adenosine monophosphate (cAMP) during the development phase of their life cycle. The Gα2 subunit of D. discoideum is required for the chemotactic response. Once activation occurs, Gα2 is known to be phosphorylated on serine 113; however, the role of this phosphorylation remains poorly defined. Exchange of serine residue 113 to alanine causes starved cells to begin the aggregation phase several hours sooner when compared to wild type, while exchanging this serine to aspartic acid (phosphorylation mimic) shows a dramatic decrease in plasma membrane surface localization. At the nanoscale level, images using FPALM show that activation and phosphorylation cause significant changes to Gα2 cluster density in the plasma membrane. Getting these first nanoscale images of G protein provided robust information, which adds to our understanding of the ligand-dependent reorganization and clustering of Gα2 required for precise signaling. Cell fractionation experiments supported this result. In addition, phosphorylation-dependent interaction between phosphorylated Gα2 and D. discoideum 14-3-3 protein was detected
Biomolecular simulations: From dynamics and mechanisms to computational assays of biological activity
Biomolecular simulation is increasingly central to understanding and designing biological molecules and their interactions. Detailed, physicsâbased simulation methods are demonstrating rapidly growing impact in areas as diverse as biocatalysis, drug delivery, biomaterials, biotechnology, and drug design. Simulations offer the potential of uniquely detailed, atomicâlevel insight into mechanisms, dynamics, and processes, as well as increasingly accurate predictions of molecular properties. Simulations can now be used as computational assays of biological activity, for example, in predictions of drug resistance. Methodological and algorithmic developments, combined with advances in computational hardware, are transforming the scope and range of calculations. Different types of methods are required for different types of problem. Accurate methods and extensive simulations promise quantitative comparison with experiments across biochemistry. Atomistic simulations can now access experimentally relevant timescales for large systems, leading to a fertile interplay of experiment and theory and offering unprecedented opportunities for validating and developing models. Coarseâgrained methods allow studies on larger lengthâ and timescales, and theoretical developments are bringing electronic structure calculations into new regimes. Multiscale methods are another key focus for development, combining different levels of theory to increase accuracy, aiming to connect chemical and molecular changes to macroscopic observables. In this review, we outline biomolecular simulation methods and highlight examples of its application to investigate questions in biology.
This article is categorized under:
Molecular and Statistical Mechanics > Molecular Dynamics and MonteâCarlo Methods
Structure and Mechanism > Computational Biochemistry and Biophysics
Molecular and Statistical Mechanics > Free Energy Method
Computational Modeling of Realistic Cell Membranes
Cell membranes contain a large variety of lipid types and are crowded with proteins, endowing them with the plasticity needed to fulfill their key roles in cell functioning. The compositional complexity of cellular membranes gives rise to a heterogeneous lateral organization, which is still poorly understood. Computational models, in particular molecular dynamics simulations and related techniques, have provided important insight into the organizational principles of cell membranes over the past decades. Now, we are witnessing a transition from simulations of simpler membrane models to multicomponent systems, culminating in realistic models of an increasing variety of cell types and organelles. Here, we review the state of the art in the field of realistic membrane simulations and discuss the current limitations and challenges ahead
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