Ryan Bradley

Exo70 Generates Membrane Curvature for Morphogenesis and Cell Migration

Dynamic shape changes of the plasma membrane are fundamental to many processes, ranging from morphogenesis and cell migration to phagocytosis and viral propagation. Here, we demonstrate that Exo70, a component of the exocyst complex, induces tubular membrane invaginations toward the lumen of synthetic vesicles in vitro and generates protrusions on the surface of cells. Biochemical analyses using Exo70 mutants and independent molecular dynamics simulations based on Exo70 structure demonstrate that Exo70 generates negative membrane curvature through an oligomerization-based mechanism. In cells, the membrane-deformation function of Exo70 is required for protrusion formation and directional cell migration. Exo70 thus represents a membrane-bending protein that may couple actin dynamics and plasma membrane remodeling for morphogenesis.

Multiscale Modeling of Functionalized Nanocarriers in Targeted Drug Delivery.

Targeted drug delivery using functionalized nanocarriers (NCs) is a strategy in therapeutic and diagnostic applications. In this paper we review the recent development of models at multiple length and time scales and their applications to targeting of antibody functionalized nanocarriers to antigens (receptors) on the endothelial cell (EC) surface. Our mesoscale (100 nm-1 μm) model is based on phenomenological interaction potentials for receptor-ligand interactions, receptor-flexure and resistance offered by glycocalyx. All free parameters are either directly determined from independent biophysical and cell biology experiments or estimated using molecular dynamics simulations. We employ a Metropolis Monte Carlo (MC) strategy in conjunction with the weighted histogram analysis method (WHAM) to compute the free energy landscape (potential of mean force or PMF) associated with the multivalent antigen-antibody interactions mediating the NC binding to EC. The binding affinities (association constants) are then derived from the PMF by computing absolute binding free energy of binding of NC to EC, taking into account the relevant translational and rotational entropy losses of NC and the receptors. We validate our model predictions by comparing the computed binding affinities and PMF to a wide range of experimental measurements, including in vitro cell culture, in vivo endothelial targeting, atomic force microscopy (AFM), and flow chamber experiments. The model predictions agree closely and quantitatively with all types experimental measurements. On this basis, we conclude that our computational protocol represents a quantitative and predictive approach for model driven design and optimization of functionalized NCs in targeted vascular drug delivery.

Coarse-Grained Models for Protein-Cell Membrane Interactions.

The physiological properties of biological soft matter are the product of collective interactions, which span many time and length scales. Recent computational modeling efforts have helped illuminate experiments that characterize the ways in which proteins modulate membrane physics. Linking these models across time and length scales in a multiscale model explains how atomistic information propagates to larger scales. This paper reviews continuum modeling and coarse-grained molecular dynamics methods, which connect atomistic simulations and single-molecule experiments with the observed microscopic or mesoscale properties of soft-matter systems essential to our understanding of cells, particularly those involved in sculpting and remodeling cell membranes.

Curvature–undulation coupling as a basis for curvature sensing and generation in bilayer membranes

Significance Most intracellular trafficking and many intercellular communications are orchestrated by curvature-driven or curvature-associated cellular processes. Hence, understanding how proteins sculpt lipid bilayers is vital to our understanding of how cell membranes modulate cell signaling pathways and consequent cell fate. In this study, we present coarse-grained molecular dynamics simulations of the epsin N-terminal homology domain interacting with a lipid bilayer and demonstrate a cooperative and fluctuation-mediated mechanism by which these proteins can generate curvature in bilayer membranes as well as sense curvature at large distances compared with their size. We present coarse-grained molecular dynamics simulations of the epsin N-terminal homology domain interacting with a lipid bilayer and demonstrate a rigorous theoretical formalism and analysis method for computing the induced curvature field in varying concentrations of the protein in the dilute limit. Our theory is based on the description of the height–height undulation spectrum in the presence of a curvature field. We formulated an objective function to compare the acquired undulation spectrum from the simulations to that of the theory. We recover the curvature field parameters by minimizing the objective function even in the limit where the protein-induced membrane curvature is of the same order as the amplitude due to thermal undulations. The coupling between curvature and undulations leads to significant predictions: (i) Under dilute conditions, the proteins can sense a site of spontaneous curvature at distances much larger than their size; (ii) as the density of proteins increases the coupling focuses and stabilizes the curvature field to the site of the proteins; and (iii) the mapping of the protein localization and the induction of a stable curvature is a cooperative process that can be described through a Hill function.

Multiscale computational models in physical systems biology of intracellular trafficking

In intracellular trafficking, a definitive understanding of the interplay between protein binding and membrane morphology remains incomplete. The authors describe a computational approach by integrating coarse-grained molecular dynamics (CGMD) simulations with continuum Monte Carlo (CM) simulations of the membrane to study protein-membrane interactions and the ensuing membrane curvature. They relate the curvature field strength discerned from the molecular level to its effect at the cellular length-scale. They perform thermodynamic integration on the CM model to describe the free energy landscape of vesiculation in clathrin-mediated endocytosis. The method presented here delineates membrane morphologies and maps out the free energy changes associated with membrane remodeling due to varying coat sizes, coat curvature strengths, membrane bending rigidities, and tensions; furthermore several constraints on mechanisms underlying clathrin-mediated endocytosis have also been identified, Their CGMD simulations have revealed the importance of PIP2 for stable binding of proteins essential for curvature induction in the bilayer and have provided a molecular basis for the positive curvature induction by the epsin N-terminal homology (EIMTH) domain. Calculation of the free energy landscape for vesicle budding has identified the critical size and curvature strength of a clathrin coat required for nucleation and stabilisation of a mature vesicle.

Curvature-Undulation Coupling as a Basis for Curvature Sensing and Generation in Bilayer Membranes

Cell membranes play an important role in modulating cell signaling pathways by hosting the assembly of protein structures which are capable of initiating downstream signaling events. Understanding how proteins both sense and generate membrane curvature during these processes is therefore necessary to relate specific protein-membrane interactions with larger mesoscale membrane shape changes observed in experiments. We employ coarse-grained molecular dynamics simulations of the epsin N-terminal homology (ENTH) domain bound to phosphatidylinositol 4,5-bisphosphate (PIP2) in a lipid bilayer to demonstrate a formalism for computing the induced curvature field. We formulate an objective function for matching the fluctuations observed in simulations to the deformation fields defined by the theory. This method allows us to recover the curvature fields induced by proteins at varying concentrations near the dilute limit, even when the protein-induced curvature is the same order as the amplitude of thermal undulations. We predict that proteins at dilute concentrations can sense a site of spontaneous curvature at distances much larger than their size. This suggests that increasing protein concentrations can localize or focus the dynamic curvature field to the site of the proteins, and that the protein-induced stabilization of membrane curvature is a cooperative process. This method may be used to predict the influence of protein concentration, assembly, and membrane binding on endocytosis and exocytosis events in cells.

Quantum and All-Atom Molecular Dynamics Simulations of Protonation and Divalent Ion Binding to Phosphatidylinositol 4,5-Bisphosphate (PIP2)

Molecular dynamics calculations have been used to determine the structure of phosphatidylinositol 4,5 bisphosphate (PIP2) at the quantum level and to quantify the propensity for PIP2 to bind two physiologically relevant divalent cations, Mg2+ and Ca2+. We performed a geometry optimization at the Hartree–Fock 6-31+G(d) level of theory in vacuum and with a polarized continuum dielectric to determine the conformation of the phospholipid headgroup in the presence of water and its partial charge distribution. The angle between the headgroup and the acyl chains is nearly perpendicular, suggesting that in the absence of other interactions the inositol ring would lie flat along the cytoplasmic surface of the plasma membrane. Next, we employed hybrid quantum mechanics/molecular mechanics (QM/MM) simulations to investigate the protonation state of PIP2 and its interactions with magnesium or calcium. We test the hypothesis suggested by prior experiments that binding of magnesium to PIP2 is mediated by a water molecu...

Molecular modeling of membrane curvature driven by epsin

A molecular description of membrane curvature induction by the ENTH domain of the protein epsin has the potential to shed light on clathrin mediated endocytosis and its effect on pathological cell signaling processes. To that end, we are performing coarse-grained and all-atom molecular dynamics simulations of membrane remodeling by epsin. Coarse-grained simulations are used to characterize the local stress distribution throughout the membrane, yielding a microscopic picture of the induced curvature field, the additive effects of multiple epsins, and the role of surface tension in mediating epsin-membrane interactions.