Biology subjects

Voth, G. A.

Publications and source records attributed to Voth, G. A..

7 recordsLinked to original sources

bioRxiv2018

Ena/VASP processive elongation is modulated by avidity on actin filaments bundled by the filopodia crosslinker fascin

Ena/VASP are tetrameric assembly factors that bind F-actin barbed ends continuously while increasing their elongation rate within dynamic bundled networks such as filopodia. We used single-molecule TIRFM and developed a kinetic model to dissect Ena/VASPs processive mechanism on bundled filaments. Notably, Ena/VASPs processive run length increases with the number of both bundled filaments and Ena arms, revealing avidity facilitates enhanced processivity. Moreover, Ena tetramers form more filopodia than mutant dimer and trimers in Drosophila culture cells. Finally, enhanced processivity on trailing barbed ends of bundled filaments is an evolutionarily conserved property of Ena/VASP homologs and is specific to fascin-bundled filaments. These results demonstrate that Ena tetramers are tailored for enhanced processivity on fascin bundles and avidity of multiple arms associating with multiple filaments is critical for this process. Furthermore, we discovered a novel regulatory mechanism whereby bundle size and bundling protein specificity control activities of a processive assembly factor.

cell biology↗

bioRxiv2018

Insights into the cooperative nature of ATP hydrolysis in actin filaments

Actin filaments continually assemble and disassemble within a cell. Assembled filaments \"age\" as a bound nucleotide ATP within each actin subunit quickly hydrolyzes, followed by a slower release of the phosphate Pi, leaving behind a bound ADP. This subtle change in nucleotide state of actin subunits affects filament rigidity as well as its interactions with binding partners. We present here a systematic multiscale ultra-coarse-graining (UCG) approach that provides a computationally efficient way to simulate a long actin filament undergoing ATP hydrolysis and phosphate release reactions, while systematically taking into account available atomistic details. The slower conformational changes and their dependence on the chemical reactions are simulated with the UCG model by assigning internal states to the coarse-grained sites. Each state is represented by a unique potential surface of a local heterogeneous elastic network. Internal states undergo stochastic transitions that are coupled to conformations of the underlying molecular system. The UCG model reproduces mechanical properties of the filament and allows us to study whether fluctuations in actin subunits produce cooperative aging in the filament. Our model predicts that nucleotide state of neighboring subunit significantly modulates the reaction kinetics, implying cooperativity in ATP hydrolysis and Pi release. We further systematically coarse-grain the system into a Markov state model that incorporates assembly and disassembly, facilitating a direct comparison with previously published models. We find that cooperativity in ATP hydrolysis and Pi release significantly affects the filament growth dynamics only near the critical G-actin monomer concentration, while both cooperative and random mechanisms show similar growth dynamics far from the critical concentration. In contrast, filament composition in terms of the bound nucleotide distribution varies significantly at all monomer concentrations studied. These results provide new insights into the cooperative nature of ATP hydrolysis and Pi release and the implications it has for actin filament properties, providing novel predictions for future experimental studies.

biophysics↗

bioRxiv2018

Gating Mechanisms during Actin Filament Elongation by Formins

Formins play an important role in the polymerization of unbranched actin filaments, and particular formins slow elongation by 5-95%. We studied the interactions between actin and the FH2 domains of formins Cdc12, Bni1 and mDia1 to understand the factors underlying their different rates of polymerization. All-atom molecular dynamics simulations revealed two factors that influence actin filament elongation and correlate with the rates of elongation. First, FH2 domains can sterically block the addition of new actin subunits. Second, FH2 domains flatten the helical twist of the terminal actin subunits, making the end less favorable for subunit addition. Coarse-grained simulations over longer time scales support these conclusions. The simulations show that filaments spend time in states that either allow or block elongation. The rate of elongation is a time-average of the degree to which the formin compromises subunit addition rather than the formin-actin complex literally being in open or closed states.

biophysics↗

bioRxiv2018

Entropic Forces Drive Clustering and Spatial Localization of Influenza A M2 During Viral Budding

The influenza A matrix 2 (M2) transmembrane protein facilitates virion release from the infected host cell. In particular, M2 plays a role in the induction of membrane curvature and/or in the scission process whereby the envelope is cut upon virion release. Here we show using coarse-grained computer simulations that various M2 assembly geometries emerge due to an entropic driving force, resulting in compact clusters or linearly extended aggregates as a direct consequence of the lateral membrane stresses. Conditions under which these protein assemblies will cause the lipid membrane to curve are explored and we predict that a critical cluster size is required for this to happen. We go on to demonstrate that under the stress conditions taking place in the cellular membrane as it undergoes large-scale membrane remodeling, the M2 protein will in principle be able to both contribute to curvature induction and sense curvature in order to line up in manifolds where local membrane line tension is high. M2 is found to exhibit linactant behavior in liquid-disordered/liquid-ordered phase-separated lipid mixtures and to be excluded from the liquid-ordered phase, in near-quantitative agreement with experimental observations. Our findings support a role for M2 in membrane remodeling during influenza viral budding both as an inducer and a sensor of membrane curvature, and they suggest a mechanism by which localization of M2 can occur as the virion assembles and releases from the host cell, independent of how the membrane curvature is produced.\n\nSIGNIFICANCE STATEMENTFor influenza virus to release from the infected host cell, controlled viral budding must finalize with membrane scission of the viral envelope. Curiously, influenza carries its own protein, M2, which can sever the membrane of the constricted budding neck. Here we elucidate the physical mechanism of clustering and spatial localization of the M2 scission proteins through a combined computational and experimental approach. Our results provide fundamental insights into how M2 clustering and localization interplays with membrane curvature, membrane lateral stresses, and lipid bilayer phase behavior during viral budding in order to contribute to virion release.

biophysics↗

bioRxiv2017

New Insights into the Conformational Activation of Full-Length Integrin

Integrin binding to extracellular matrix proteins is regulated by conformational transitions from closed, low affinity states to open, high affinity states. However, the pathways of integrin conformational activation remain incompletely understood. Here, by combining all-atom molecular dynamics simulation, coarse-graining, heterogeneous elastic network modeling, and experimental ligand binding measurements, we test the effect of integrin {beta} mutations that destabilize the closed conformation. Our results support a \"deadbolt\" model of integrin activation, where extension of the headpiece is not coupled to leg separation, consistent with recent cryo-EM reconstructions of integrin intermediates. Moreover, our results are inconsistent with a \"switchblade-like\" mechanism. The data show that locally correlated atomistic motions are likely responsible for extension of integrin headpiece before separation of transmembrane legs, without persistence of these correlations across the entire protein. By combining modeling and simulation with experiment, this study provides new insight into the structural basis of full-length integrin activation.

biophysics↗

bioRxiv2017

The lamellipodium is a myosin independent mechanosensor

The ability of adherent cells to sense changes in the mechanical properties of their extracellular environments is critical to numerous aspects of their physiology. It has been well documented that cell attachment and spreading are sensitive to substrate stiffness. Here we demonstrate that this behavior is actually biphasic, with a transition that occurs around a Youngs modulus of [~]7 kPa. Furthermore, we demonstrate that, contrary to established assumptions, this property is independent of myosin II activity. Rather, we find that cell spreading on soft substrates is inhibited due to reduced nascent adhesion formation within the lamellipodium. Cells on soft substrates display normal leading edge protrusion activity, but these protrusions are not stabilized due to impaired adhesion assembly. Enhancing integrin-ECM affinity through addition of Mn2+ recovers nascent adhesion assembly and cell spreading on soft substrates. Using a computational model to simulate nascent adhesion assembly, we find that biophysical properties of the integrin-ECM bond are optimized to stabilize interactions above a threshold matrix stiffness that is consistent with the experimentally observations. Together these results suggest that myosin II-independent forces in the lamellipodium are responsible for mechanosensation by regulating new adhesion assembly, which in turn, directly controls cell spreading. This myosin II-independent mechanism of substrate stiffness sensing could potentially regulate a number of other stiffness sensitive processes.\n\nSignificance StatementCell physiology can be regulated by the mechanics of the extracellular environment. Here, we demonstrate that cell spreading is a mechanosensitive process regulated by weak forces generated at the cell periphery and independent of motor activity. We show that stiffness sensing depends on the kinetics of the initial adhesion bonds that are subjected to forces driven by protein polymerization. This work demonstrates how the binding kinetics of adhesion molecules are sensitively tuned to a range of forces that enable mechanosensation.

biophysics↗

bioRxiv2017

Immature HIV-1 Lattice Assembly Dynamics are Regulated by Scaffolding from Nucleic Acid and the Plasma Membrane

The packaging and budding of Gag polyprotein and viral ribonucleic acid (RNA) is a critical step in the human immunodeficiency virus-1 (HIV-1) lifecycle. High-resolution structures of the Gag polyprotein have revealed that the capsid (CA) and spacer peptide 1 (SP1) domains contain important interfaces for Gag self-assembly. However, the molecular details of the multimerization process, especially in the presence of RNA and the cell membrane, have remained unclear. In this work, we investigate the mechanisms that work in concert between the polyproteins, RNA, and membrane to promote immature lattice growth. We develop a coarse-grained (CG) computational model that is derived from sub-nanometer resolution structural data. Our simulations recapitulate contiguous and hexameric lattice assembly driven only by weak anisotropic attractions at the helical CA-SP1 junction. Importantly, analysis from CG and single-particle tracking photoactivated localization (spt-PALM) trajectories indicates that viral RNA and the membrane are critical constituents that actively promote Gag multimerization through scaffolding, while over-expression of short competitor RNA can suppress assembly. We also find that the CA amino-terminal domain imparts intrinsic curvature to the Gag lattice. As a consequence, immature lattice growth appears to be coupled to the dynamics of spontaneous membrane deformation. Our findings elucidate a simple network of interactions that regulate the early stages of HIV-1 assembly and budding.\n\nSIGNIFICANCE STATEMENTIn order for human immunodeficiency virus to proliferate, viral proteins and genomic dimers are assembled at host cell membranes and released as immature virions. Disrupting this key intermediate step in viral replication is a potential target for treatment. However, a detailed molecular view of this process remains lacking. Here, we elucidate a network of constitutive interactions that regulate viral assembly dynamics through a combined computational and experimental approach. Specifically, our analysis reveals the active roles of nucleic acid and the membrane as scaffolds that promote the multimerization of Gag polyprotein which proceeds through multi-step and self-correcting nucleation. Our findings also illustrate the functional importance of the N-terminal, C-terminal, and spacer peptide 1 protein domains.

biophysics↗