Lipopolysaccahride (LPS) is a unique lipoglycan, with two major functions: (i) as a major component of the outer membrane of Gram-negative bacteria and (ii) as a highly potent human toxin when released from cells into solu- tion ("endotoxin"). Divalent cations have long been known to neutralize and stabilize LPS in the outer membrane, whereas LPS in the presence of mono- valent cations forms highly mobile negatively-charged aggregates. We report fluorescence microscopy and atomic force microscopy analysis of the interaction between soluble LPS and a single component fluid-phase sLBA. Three remarkably different deformations are induced by LPS on the simple lipid membrane, dependent on cation availability. LPS is an amphiphile that spontaneously inserts into the outer leaflet of lipid bilayers to bury its hydrophobic lipidic domain and expose the hydrophilic polysac- charide chain to the exterior polar solvent. Net negative (LPS-Na þ ) induces membrane curvature due to electrostatic repulsive effects between clustered LPS. This leads to (1) the growth of 100mm-long flexible lipid tubules from surface associated lipid vesicles and (2) destabilization of the sLBA leading to micron-sized hole formation. In contrast, Ca 2þ promotes self-association and bridging of LPS, and (LPS-Ca 2þ ) induces (3) growth of 100mm-wide planar single- or multi-lamellar sheets of lipid and LPS from surface associ- ated lipid vesicles that exhibit 2-D membrane fluidity and represent a poten- tial means of organizing layer-by-layer membrane construction. Our findings have important implications about the physical interaction of LPS and lipids and the potential of using LPS and other amphiphilic materials as membrane soft-lithography tools. 2577-Pos Board B269 Lipid Tilt Regulates Ripple Phase Behavior in Lipid Bilayer Padmini Rangamani, Shachi Katira, Berend Smit, George Oster. UC Berkeley, Berkeley, CA, USA. Continuum modeling of lipid bilayers provides insight into the physics under- lying geometric changes to the shape of the membrane in response to biological processes. The Helfrich model has been the gold standard for many years and applies only to length scales larger than that of the thickness of the bilayer. For small length scale processes, orientation of the lipid, characterized by ‘lipid tilt’, is a suitable fundamental degree of freedom. In this work, we develop a continuum model with lipid tilt as the key degree of freedom. Using local force balance, we derive the equations of motion associated with the membrane. We use this model to study the characteristics of ripple phases in bilayers. Comparing the continuum model to coarse-grained simulations, we find that the tilt degree of freedom is important to allow for ripple formation in bilayer membranes. 2578-Pos Board B270 Macroscopic Phase Separation, Modulated Phases, and Microemulsions: a Unified Picture of Rafts Roie Shlomovitz 1 , Lutz Maibaum 2 , Michael Schick 1 . 1 Physics, University of Washington, Seattle, WA, USA, 2 Chemistry, University of Washington, Seattle, WA, USA. We are motivated by the observation that coupling of the height and the composition of fluctuating membranes can reduce the line tension between regions of different components. Consequently, instead of undergoing a tran- sition from a disordered fluid to liquid-ordered and liquid-disordered phases, the system undergoes a transition to a modulated phase. The disordered fluid is also affected, displaying the behavior of a microemulsion, one suggested as a model for rafts in the plasma membrane. We consider a model of a multi-component symmetric bilayer which highlights the competition be- tween the tendency to phase separate as the temperature is reduced, and to form modulated phases as the line tension is decreased. We simulate the model on a finite-size membrane and obtain its phase diagram. At low tem- perature, the system undergoes macroscopic phase separation. As tempera- ture increases, the system can evolve in two ways. If the line tension is sufficiently large, the system undergoes a transition to an ordinary disordered fluid. However if sufficiently small, the system undergoes a first-order tran- sition to a modulated phase. Only upon a further increase of temperature does the system undergo a transition to a fluid phase, and this fluid is a microemul- sion. In this disordered phase, the fluctuating regions rich in one component or the other are clearly seen in the simulation. The model provides a unified picture of the relationship between observations, in vitro and in vivo, of macroscopic phase separation and of modulated phases in bilayers and their relation to rafts. It lends support to the suggestion that rafts can be identified with a microemulsion whose characteristic length, the square root of the ratio of bending modulus to surface tension, is on the order of 100 nm in the plasma membrane. 2579-Pos Board B271 The Structural Basis of Cholesterol Accessibility in Membranes Brett N. Olsen 1 , Agata A. Bielska 1 , Tiffany Lee 1 , Michael D. Daily 2 , Douglas F. Covey 3 , Paul H. Schlesinger 4 , Nathan A. Baker 2 , Daniel S. Ory 1 . 1 Medicine, Washington University, St. Louis, MO, USA, 2 Knowledge Discovery and Informatics, Pacific Northwest National Laboratory, Richland, WA, USA, 3 Developmental Biology, Washington University, St. Louis, MO, USA, 4 Cell Biology, Washington University, St. Louis, MO, USA. While the majority of free cellular cholesterol is present in the plasma mem- brane, cholesterol homeostasis is principally regulated through sterol-sensing proteins that reside in the cholesterol-poor endoplasmic reticulum. In response to acute cholesterol loading or depletion, there is rapid equilibration between the ER and plasma membrane cholesterol pools, suggesting a biophysical model in which the availability of plasma membrane cholesterol for trafficking to internal membranes modulates ER membrane behavior. Previous studies have predominantly examined cholesterol availability in terms of binding to extra-membrane acceptors, but have provided limited insight into the structural changes underlying cholesterol activation. In the present study we use both mo- lecular dynamics simulations and experimental membrane systems to examine the behavior of cholesterol in membrane bilayers. We find that cholesterol depth within the bilayer provides a reasonable structural metric for cholesterol availability and that this is correlated with cholesterol-acceptor binding. Further, the distribution of cholesterol availability in our simulations is contin- uous rather than divided into distinct available and unavailable pools. This data provides support for a revised cholesterol activation model in which activation is driven not by saturation of membrane-cholesterol interactions but rather by bulk membrane remodeling that reduces membrane-cholesterol affinity. 2580-Pos Board B272 Keeping Order While Moving Fast: Ergosterol Pairs Lead to Dynamic Networks in Lipid Membranes Juan M. Vanegas 1 , Roland Faller 2 , Marjorie L. Longo 2 . 1 Sandia National Lab, Albuquerque, NM, USA, 2 UC Davis, Davis, CA, USA. We analyze the dynamic structure in lipid-ergosterol membranes by means of time-dependent pair-correlation functions obtained from molecular dynamics simulations. We observe that ergosterol molecules form transient pairs with lifetimes in the nanosecond-microsecond range. These sterol pairs are suffi- ciently long-lived to form linear sterol clusters (> 4 molecules) as the ergosterol concentration increases, and at high enough concentrations (> 30 mol %) these linear clusters turn into larger networks. Because of the high mobility of the sterols, as well as the dynamic nature of their pair interaction, these sterol networks are constantly reshaping. 2581-Pos Board B273 Multi-Color, Live Super-Resolution Microscopy Reveals the Timescale and Potential of Mean Force for Co-Clustering Between the B cell Receptor and Lyn Kinase Matthew B. Stone, Sarah L. Veatch. Biophysics, University of Michigan, Ann Arbor, MI, USA. The super-resolution techniques STORM and PALM allow the nanometer scale localization of fluorescent probes in both live and fixed cells. In fixed cells, robust quantification of interactions between proteins is accomplished by cross correlating the reconstructed images of spectrally separated probes. Here, we extend this technique to live cells using simultaneous STORM and PALM measurements quantified using time resolved cross correlation with 20ms temporal resolution. We demonstrate that the potential of mean force be- tween two labeled proteins can be determined even when objects have signif- icant diffusion. We apply this technique to the activation of B cell receptor (BCR) and investigate the timescale and magnitude of co-clustering between the BCR and the Src kinase Lyn. We simultaneously compare quantitative measurements of receptor clustering, receptor-Lyn co-clustering, and protein mobility. We find that Lyn is recruited to BCR clusters with a potential of mean force greater than 1kT after stimulation with multivalent antigen, con- current with BCR self-clustering and slowdown. We correlate two distinct lipid modified PALM probes with the B cell receptor in live cells. A saturated lipid moiety is recruited to BCR clusters with a potential of mean force of greater than 0.5kT, while a branched and unsaturated lipid moiety is not re- cruited to BCR clusters. These observations confirm that BCR clustering leads to the formation of a stable lipid domain enriched in saturated components, in quantitative agreement with our observations in chemically fixed cells. In ongoing work, we are using these experimental and analytical methods to quantify how lipids mediate interactions between proteins involved in early stages of BCR cell signaling. Tuesday, February 18, 2014 509a