Day 1 – Wednesday, October 16th
8:30am-9:30am – Direct Imaging of Nanoscale Lipid Organization in Probe-Free Biomimetic Membranes – Fred Heberle, University of Tennessee
ABSTRACT: The three-dimensional architecture of biological membranes has functional consequences for living cells. In the outer leaflet of the plasma membrane (PM), lipids are thought to organize into ordered yet fluid domains, with diverse evidence supporting participation of these “rafts” in membrane processes including protein sorting and signaling. However, many details of PM domain structure remain elusive due to a lack of methods to directly probe membrane features at the nanoscale. In this talk, we report the first use of cryoEM to directly image coexisting nanoscopic domains in synthetic and bio-derived membranes without extrinsic probes. Analysis of images from vesicles composed of lipids ranging from 14 to 22 carbons in length show that cryoEM can resolve sub-angstrom differences in average bilayer thickness. Features in experimental images were reproduced in artificial images constructed from atomistic molecular dynamics simulations, which revealed a non-trivial relationship between the bilayer thickness measured with small-angle X-ray scattering and that calculated from cryoEM images. We further used simulations to predict two sources of contrast between coexisting phases within the same vesicle, namely 1) differences in membrane thickness, and 2) electron density arising from different lipid densities in the two phases. We observed both sources of contrast in vesicles composed of saturated lipids, unsaturated lipids, and cholesterol. We conclude that cryoEM imaging can in principle enable a quantitative analysis of membrane parameters such as thickness, domain size, and lipid packing, opening new avenues for direct investigation of nanoscopic membrane organization
9:30am-10:30am – Coarse-Grained Modeling of Morphological Heterogeneities in Biomembranes – Mohamed Laradji, University of Memphis
ABSTRACT: Many of the functions of living cells, including endocytosis, cytokinesis, cell motility, and apoptosis, require morphological deformations of the plasma membrane or organelles' membranes. While it is well established experimentally that morphological deformations of biomembranes are the result of their interactions with various proteins or external particles, the understanding of the mechanisms leading to these deformations is still in its infancy. In this talk, I will present two studies, based on an efficient coarse-grained implicit-solvent model, of biomembranes deformations which occur as a result of the interaction between lipid membranes and cytoskeletal proteins and as a result of the interaction between lipid membranes and nanoparticles.
11:00am-12:00pm – Membrane Protein Transport: Balancing Advection and Diffusion – Aurelia Honerkamp-Smith, Lehigh University
ABSTRACT: The fluidity of lipid membranes is essential to their biological function: cell membranes are required to be flexible, self-healing, and deformable. An additional consequence of membrane fluidity is that both lipids and proteins are highly mobile, which makes it possible for lipid-anchored proteins to travel long distances across the surface of cells. The significance of this lateral mobility for flow mechanosensing has not yet been determined. We study the mechanics of lateral membrane protein transport by flow in an in vitro model of the cell plasma membrane, lipid bilayers supported on glass. We prepare supported bilayers formed from individual GUVs inside microfluidic channels in order to study transport of membrane-anchored proteins. We observe and fit dynamic protein concentration gradients, which allow us to define protein mobility relative to a stationary lipid membrane.
1:00pm-2:00pm – The Biophysical Consequences of the Asymmetric Mammalian Lipidome – Ed Lyman, University of Delaware
ABSTRACT: It has been known for decades that the plasma membrane of mammalian cells is asymmetric in composition with respect to the two leaflets, but only at the most gross level of biochemical detail — negative charge on the inner leaflet, sphingomyelin in the outer leaflet. Very recently we (by we I mean Ilya Levental's group) have determined the detailed asymmetric composition of the human erythrocyte membrane by combining classic enzymatic digestion with modern shotgun lipidomics. The results confirm what was already suspected, but add substantial new information, including localization of plasmalogen to the inner leaflet and a dramatic enrichment of unsaturated chains on the inner leaflet, and raising new questions regarding the mechanism by which the cell senses and maintains leaflet asymmetry. Simulations of simplified mixtures comprising ca. 10 lipids for each leaflet predict distinct biophysical characteristics for the two leaflets, which are confirmed by leaflet specific in-cell assays of fluidity and lipid packing. Although the localization of cholesterol is not amenable to the protocol, the simulations suggest an enrichment of cholesterol in the outer leaflet at equilibrium, implying a new mechanism for maintaining cholesterol asymmetry.
2:00pm-3:00pm – Brief Calcium Influx through the Plasma Membrane Transiently Clusters PIP2 in the Inner Plasma Membrane Leaflet of Intact Cells – Arnd Pralle, University at Buffalo
ABSTRACT: Calcium entering the cell through ion channels acts as fast diffusion signal molecule. However, local calcium levels near the membrane can reach several hundred micromoles, sufficiently high to act electrostatically. The inner membrane leaflet contains various negatively charged lipids, such as phosphatidylinositol 4,5-bisphosphates (PIP2) and Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). These modulate protein function and recruit specific proteins to the plasma membrane. In model membrane systems it has been shown that Calcium ions induce clusters of PIP2. Hence, we have investigated how Calcium influx modulates the lateral PIP3 and PIP3 distribution in intact cells. The plasma membrane is a complex lipid protein mixture in which small changes in lateral interaction energy may induce transient lateral order. This permits quick rearrangements during cell signaling but creates a challenge to image the membrane faithfully. In addition, temporal resolution is required to capture transient effect. We employed fluorescent correlation spectroscopy (FCS) measuring diffusion on multiple length scales simultaneously, to show that the transient calcium in flux leads to a transient clustering of PIP2 and PIP3 on the inner membrane leaflet. To study the formation of PIP2 clusters in the plasma membrane we use GFP-PHPLCdelta and Halo-PHPLCdelta labelled with the JaneliaFluor647; as marker for PIP3 PH-AKT-GFP; and for cholesterol-stabilized nano-domains Lck-mGFP. We observe that opening TRPV1 channels leads to a transient rise in calcium as imaged using GCaMP5G, transiently stabilizes PIP2 and PIP3 with PIP3 following a slower kinetics. It also increases the interaction between the Lck-mGFP and cholesterol domains. Using an ionophore to clamp the calcium level to a fixed value, we determine the threshold for these effects. We find that vinculin in rest cells stabilizes PIP2 clusters. In vinculin knockdown cells the calcium induced clustering and redispersion are accelerated. These results suggest a concentration dependence of calcium-induced PIP2 clusters and cholesterolstabilized nano-domains in the PM at calcium levels, which may be reached in intact cells locally by opening of ion channels.
3:30pm-4:30pm – Modeling Membrane Heterogeneities at Small and Large Length Scales – Lutz Maibaum, University of Washington
ABSTRACT: Heterogeneities in multicomponent lipid bilayer systems emerge from molecular interactions but can alter the surface morphologies of entire vesicles and cells. This discrepancy in relevant length scales between cause and effect poses a difficult challenge for analytical or simulation approaches that aim to model membrane heterogeneities in biologically and experimentally relevant systems. I discuss two complementary approaches to obtain detailed information about the thermodynamic phases and their structures of mixed lipid bilayers. The first employs extensive coarse-grained molecular dynamics computer simulations, which reveal the locations of phase boundaries and coexistence regions when properly analyzed. The second is based on the Landau-Ginzburg theory of phase transitions, extended to allow for microemulsion and modulated phases. I present results of Monte Carlo simulations of this model and emphasize the importance of finite size and topological effects that become apparent in the experimentally relevant geometry of small, spherical vesicles.
4:30pm-5:30pm – Insights into Solvent-Microbial Stress in Biofuel Production from Small-Angle Scattering and Complementary Molecular Dynamics Simulations – Micholas Smith, ORNL/University of Tennessee
ABSTRACT: The production of next-generation liquid fuels (and chemical precursors) from the microbial fermentation of lignocellulose is limited by the inherent sensitivity of microbes to their fermentation products. One route to overcoming this limitation is to engineer microbial membranes to become more resilient to solvent-induced (fermentation product) stressors; however, the pathway to achieve this is unclear as the molecular mechanisms that disrupt microbial membranes is not yet well understood. Here I present, as a first step in elucidating the solvent-induced disruption of microbial membranes, the results of a Small-Angle Scattering and All-Atom Molecular Dynamics Study of a model bacterial membrane under aqueous cosolvents of 1-butanol and tetrahydrofuran (THF).
Day 2 – Thursday, October 17th
8:30am-9:30am – Functional Roles for Lipid-Encoded Properties in Engineered Cell Membranes – Itay Budin, UC San Diego
ABSTRACT: Lipid composition controls the physical and material properties of biological membranes but has traditionally been challenging to manipulate and study in living cells. I will present our work using metabolic engineering approaches to modulate lipid composition in the membranes of genetically-tractable model organisms. Engineered bacterial and yeast strains allow for experimental control of phospholipid acyl chains and sterols, the primary determinants of membrane viscosity and phase separation in model membranes. I will show how these systems have allowed us to characterize functions for membrane physical properties in cellular respiration, gene regulation, and transport processes. I will then briefly discuss the potential for investigating membrane and lipid heterogeneity in complex cells and whole animal models.
9:30am-10:30am – All-Atom Modeling Nanometer-Scale Lateral Compositional Heterogeneity of the Liquid Ordered Phase Agrees with Small Angle Neutron Scattering Experiments – Alexander Sodt, National Institute of Health
ABSTRACT: Our lab has gathered simulation evidence that the nanometer scale molecular structure of lipid bilayers can be connected to curvature stresses. This in turn leads to the energy required to reshape membranes with complex lipid distributions, like those in the cell. No single technique can be used to show high resolution structure of a dynamical ensemble of configurations, like those in a fluid bilayer. Molecular simulation has long been used to model the collapsed, ensemble averaged signal of scattering and so provide molecular configurational information. Typically this has been applied to the transverse chemical structure along the bilayer normal. Here we extend this by using simulation to compute the lateral scattering from the nanometer-scale heterogeneity of a moderately complex mixture: the liquid ordered phase. We have developed new simulation software and methodology to compute the full intensity from the simulation. With simulation and experiment working together, we then designed a set of samples and deuteration schemes that can best report this signal, which is relatively weak compared with the transverse scattering. Showing that a specific scattering feature is due to a specific molecular feature is challenging, and requires comparing the many weak features that are overlaid on the major transverse features. Our conclusion is that the good agreement shown by theory, simulation, and experiment validates the nanometer-scale lateral feature observed in the simulation.
11:00am-12:00pm – Using 13C and 15N Isotope Tracers to Monitor Membrane Dynamics in C. Elegans – Carissa Perez Olsen, Worcester Polytechnic Institute
ABSTRACT: The membranes within an animal are comprised of phospholipids, cholesterol and proteins that together form a dynamic barrier. The types of lipids that are found within a membrane bilayer impact its biophysical properties including its fluidity, permeability and susceptibility to damage. While membrane composition is very stable in healthy adults, aberrant membrane structure is seen in a wide and varied array of diseases. The composition of a membrane impacts the function of that membrane and affects many cellular processes including cell signaling, energy production and membrane protein function. Despite the wide-reaching impacts of membrane composition, there is relatively little known about how membrane composition is established and maintained over time. In vivo biochemical modeling of the membrane lipids is needed to understand how these molecules interact in their natural configurations. There are challenges to the study of membrane biology, namely the tremendous diversity and the dynamic nature of the phospholipid population. Here, we have described analytical methods that increase our capacity to map the dynamics of the individual membrane phospholipids using mass spectrometry. Specifically, we have developed novel stable isotope (13C and 15N) strategies to quantify the turnover of dozens of fatty acid tails and intact phospholipids simultaneously. We saw that different lipid populations have distinct rates of replacement suggesting sophisticated regulatory mechanisms. In fact, we identified an unexpected and profound role for Stearoyl-CoA desaturases (SCDs) in regulating the rates of membrane replacement as inhibition of SCDs leads to reduced turnover of nearly all phospholipid species measured. Thus, we have identified the first example of a major regulator of membrane lipid turnover which will be critical for generating tools to understand the role of membrane maintenance.
1:00pm-2:00pm – Modeling Lipid Composition Dynamics on the Surface of Membranes – Frank L. H. Brown, UC Santa Barbara
ABSTRACT: A continuum-level approach for simulating the dynamics of lipids and proteins on membrane surfaces will be presented. Applications to the thermal motion of domains and phase-separation in model membrane systems will be discussed
2:00pm-3:00pm – The Interplay Between Lipid Cross-Linking, Phase Separation, and Diffusion with Nanoscale Membrane Curvature – Christopher V. Kelly, Wayne State University
ABSTRACT: Many essential biological processes depend on the interaction of lipids, proteins, and carbohydrates at length scales that are unresolvable by conventional optical microscopes. We are developing and implementing sub-diffraction-limited optical methods to elucidate the molecular-scale details of membrane curvature. We employ single-molecule localization microscopy and single-lipid tracking to detect membrane curvature and to monitor the effects of curvature on single-molecule behavior. We developed Polarized Localization Microscopy to measure membrane curvature as small as 20 nm radii and correlated with molecular reorganization. We discovered that lipid phases couple with membrane curvature to jointly affect single-lipid diffusion. Additionally, we discovered that lipid cross-linking is essential for cholera toxin subunit B to form nanoscale membrane buds in quasi-one component bilayers.
3:30pm-4:30pm – Stimuli-Responsive Liposomes Through Modulation of Membrane Properties Using Synthetic Lipid Switches – Michael Best, University of Tennessee
ABSTRACT: Liposomes are effective medicinal nanocarriers due to their ability to encapsulate and deliver a range of therapeutic cargo. While liposome formulations have been approved for clinical use, delivery properties would be improved by enhancing control over the release of contents. Toward this end, we have developed stimuli-responsive liposome platforms containing synthetic lipid switches for which membrane properties are modulated in the presence of specific disease-related stimuli. Multiple approaches to tuning membrane properties will be discussed. In one strategy, molecular recognition events involving biomolecules that are overabundant in diseased tissues are exploited to trigger liposome delivery and content release based on the chemical makeup of diseased cells. This is achieved by designing synthetic lipids that undergo conformational changes upon binding that alter membrane mixing properties and thereby trigger release of contents. Additionally, we are developing enzyme-responsive liposomes in which the removal of a trigger in the form of a substrate mimic within the lipid switch causes the release of non-bilayer lipids that destabilize the liposomal membrane and promote content release. A general strategy for targeting multiple enzymes that are overexpressed in diseased cells will be discussed. For these approaches, the design and synthesis of lipid switches, liposome release assays, analysis of changes in liposome properties upon target binding, and cellular delivery studies will be presented.
4:30pm-5:30pm – Neutron vs. X-ray Scattering to Address Structures of Single Model Lipid Mono- and Bi-layers – Jaroslaw Majewski, NSF
ABSTRACT: X-ray and neutron surface scattering methodologies (reflectivity and x-ray grazing incidence diffraction (GIXD)) will be discussed to address the structure of model lipid membranes in different environments including monolayers and single bilayers at the air-liquid at the solid-liquid interfaces, respectively. Comparison of the two methods will be given to argue about to their relative advantages and shortcomings. The first GIXD measurements of single phospholipid membranes, composed of two lipid monolayers, at the solid-liquid interface will be provided and the structural coupling between the lipid leaflets will be discussed. The x-ray and neutron scattering results of interactions of single lipid membranes with bacterial cholera and shiga toxins will conclude the presentation.
