INTELLECTUAL MERIT: Lipid membrane fluidity is not well understood and membrane viscosity, the key property that describes fluid response, remains poorly quantified. This proposal describes a coordinated experimental and theoretical program that will illuminate lipid bilayer viscosity and viscoelastic response. Experimentally, lipid membranes will be probed using recently developed methods of two point microrheology, in which dynamic correlations between the trajectories of Brownian tracer particles provide insights into the physics of complex fluids. This approach has never before been applied to lipid membranes. The experiments will involve high-speed video microscopy of nanoparticles linked to planar, unsupported lipid bilayers, followed by image analysis and particle tracking. Theoretical work will analytically extend recent models of the response of ideal two-dimensional fluids to encompass elasticity, viscoelasticity, and tension ¨C key physical properties of consequence to membranes. The resulting theoretical framework will enable quantitative maps between experimentally observed tracer dynamics and the underlying collective dynamics of membranes. The studies target three important membrane systems: (1) Experiments will address homogenous lipid bilayer membranes to correlate viscosity with molecular structure and temperature. Structural studies will examine the dependence of viscosity on the lipids¡¯ acyl chain length, which will reveal whether the hydrophobic core or the hydrophilic headgroup dominates the fluid response. More fundamentally, the data together with new theoretical models will quantify the extent to which lipid membranes behave as viscous versus viscoelastic fluids. (2) The project will also study phase-separated membranes. Cellular membranes are not homogenous in composition, a feature that is believed to help govern protein©protein interactions. The mechanical consequences of heterogeneity remain poorly understood. Experiments will therefore probe viscosity in model bilayers that, below a critical temperature, exhibit cholesterol©dependent phase separation into coexisting fluid phases. (3) Finally, the PI will investigate asymmetric membranes. The two lipid leaflets of cellular membranes differ with respect to composition. By constructing asymmetric lipid bilayer membranes, the proposed studies will examine the consequences of asymmetry and the existence mechanical coupling across the leaflets.
BROADER IMPACTS: This project will advance the experimental methodology and the theory of viscous and viscoelastic flow in biological lipid membranes. A better understanding of membrane protein diffusion and assembly and of the mechanism of lipid domain formation should emerge. The PI has developed and will continue to deliver a general education course, The Stuff of Life, that explores biophysics for non-science members. Through readings, discussions, and hands©on exercises, students explore the physical properties of biological materials and the constraints these properties place on living organisms. The course includes discussions of the interplay between physics and biology, of how new modes of experimental analysis (e.g., particle tracking) enable new discoveries, and of the similarities between biomaterials (e.g. membranes) and non©iological soft matter (e.g. soap films). He will continue to lead efforts in his Department to organize weeklong Physics Day Camps for low economic status high school students. His lab will host undergraduate students participating in an REU program targeting female physical sciences students and students from another program that brings community college students to the campus for a summer research experience. As co-PI on an NSF-sponsored GK-12 project, he directs an activity that partners University of Oregon graduate students with schools in remote Oregon school districts to introduce inquiry-driven science curriculum elements, including science kits that remain with the participating schools.
Intellectual Merit Cellular membranes are remarkable materials: flexible, two-dimensional fluids. This fluidity is vital to their function, as the lipids and proteins that comprise membranes must be mobile in order to interact with one another. Viscosity is the key material property characterizing any fluid. For water, oils, and many other three-dimensional fluids, viscosity is well tabulated. For lipid bilayers, however, the two-molecule-thick liquids that form the basic structural element of all cellular membranes, viscosity remains poorly quantified, limiting our ability to understand basic membrane processes that underlie health as well as disease. Lipid membrane viscosity is very hard to measure, and therefore we sought to develop new approaches to characterize membrane fluidity. The primary method we devised hinges on Brownian motion: the random jiggling experienced by all objects due to ever-present thermal energy. As Einstein explained over a century ago, the magnitude of the random motion of a particle in a liquid is a function of the liquid’s viscosity. If one knows the size of the particle and the temperature, measuring the "diffusion coefficient" (which characterizes the random motion) is sufficient to extract the fluid’s viscosity. One can take this approach to measuring membrane viscosity, attaching particles to a membrane and watching their Brownian motion, but unfortunately the effective size of the diffusing object is not directly knowable. We realized that in addition to the "translational" Brownian motion of particles (i.e. their meandering position), one can examine their rotational motion: their orientation also shows diffusive behavior. Measuring both translational and rotational diffusion allows unambiguous determination of the fluid viscosity. We made elongated tracers out of 100-nanometer spherical particles, linked them membranes, and visualized their orientation. Using this approach, we were able to measure lipid bilayer viscosity. Moreover, we were able to study what happens when a protein that’s involved in intracellular cargo trafficking interacts with the lipid bilayer, discovering that it dramatically increases the two-dimensional viscosity, the first time such an effect has been reported. We suspect that this phenomenon may be very general, that proteins can alter the physical properties of the membranes they interact with, a finding made possible by our new experimental methods. In addition, we have also developed another technique for measuring membrane viscosity, one that examines correlations between the Brownian motions of different particles. Whether particles move coherently or not depends on the viscosity of the medium that transmits forces between them, and so correlation analysis offers a route to characterizing viscosity that is relatively independent on object size. We have performed the first ever correlation-based viscosity measurements of a lipid membrane. We have also examined other aspects of membrane mechanics beyond viscosity, developing and implementing new optical approaches to perform these studies. All of the above studies require computational image analysis, and we have devoted considerable effort to determining the position of point-like objects with high speed and accuracy. Notably, this task is crucial to a very wide range of scientific endeavors, including super-resolution microscopy, astronomy, single-molecule biophysics, and more. We devised a fundamentally new approach to particle tracking, in which one finds the point of maximal radial symmetry in a particle’s neighborhood. We derived an analytically exact mathematical expression for this symmetry center and showed that this approach to tracking is as accurate as state-of-the-art methods, but runs over a hundred times faster and has reduced sensitivity to the presence of nearby particles. Broader Impacts We performed outreach and educational activities that relate to the research themes of this project, focused especially on a new course for non-science-major undergraduates and a week-long camp for high school students from low socioeconomic backgrounds. A course ("The Physics of Life") created by the PI explores the physical properties of biological materials through readings, discussions, active learning activities, and demonstrations. This "biophysics for non-science-majors" course is unique, exploring subjects such as the physical properties of DNA and the biomechanics of animal locomotion that are rarely presented to non-scientists despite their relevance to everyday experience and contemporary technology. The class directly integrates the subject matter of this award via coursework on Brownian motion and discussion of the PI’s experiments on membrane-anchored particles. The course has been well-received, with evaluation scores considerably above the departmental mean. For low-income high school students, the PI has for each of the past several years been one of two co-organizers of a week-long Physics and Human Physiology Day Camp. About 15-20 students each year finishing 10th grade learn directly from college financial aid and admissions staff about university requirements, and directly from hands-on activities about topics spanning a wide swathe of physics, materials science, and physiology. A strong emphasis on biophysics and biomaterials is manifested, for example, in activities involving soap films, surfactants, and biomembranes led by the PI. Student evaluations have been extremely positive.
