The study of lipid rafts has attracted the attention of biological and physical scientists alike, and researchers have used model lipid systems as well as natural biological membranes to try to better understand these entities. It has been postulated that lipid rafts form due to complex formation between lipids and cholesterol, and elucidation of the interactions between lipids and cholesterol has formed the basis for this project. The project will use 3 different types of model system: monolayers, bilayers, and natural membranes of red blood cells, and a suite of biophysical techniques (surface-pressure measurements, fluorescence, beta-cyclodextrin desorption assays, grazing incidence x-ray diffraction, x-ray reflectivity as well as displacement assays in cells) to probe the nature of the interactions involved. The objectives of the research fall into three areas: (1) Nature of Condensed Complexes of Lipids and Cholesterol. What is their thermal stability? Can they only be formed between cholesterol and high melting point lipids? Are the structural and chemical activity data consistent with other models on lipid/cholesterol interactions besides the complex formation model? (2) Membrane Intercalators. What are the structural attributes of an effective membrane intercalator capable of disrupting the interactions between lipids and cholesterol? How would alterations in the hydrophobicity of the intercalator affect its displacement performance? Would other sterols be effective in displacing cholesterol? (3) Lipid Ordering Involving Gangliosides. How do gangliosides, thought to be enriched in lipid rafts, affect the organization of their surrounding lipids? Do they coordinate with high melting point lipids in a similar fashion as cholesterol? Or do they form other types of structures? The success in addressing these questions will help elucidate interactions between lipids and sterols as well as those involving lipids and gangliosides. It will also bridge the gap between model and natural membrane systems, and resolve some of the seeming inconsistencies found in various model systems. The knowledge gained from this work will greatly add to our fundamental understanding of lipid rafts.
The research activities will provide training opportunities for the researchers in a highly interdisciplinary area, and will help train the next generation of interdisciplinary research scientists. The project will further enhance the content of a graduate course in membrane biophysics, as well as the curriculum development of a new interdisciplinary degree-granting program in Biophysical Sciences. Apart from graduate students and postdocs, research opportunities will be extended to high school, undergraduate, summer research as well as international exchange students. The PI has spearheaded and will continue to participate in a 7-week summer research program, Summer Link, placing 10 high school seniors from the University of Chicago Laboratory Schools in laboratories across the Biological Sciences Division and the Physical Sciences Division at the University of Chicago. An early exposure to research can help entice these young students to consider a career in science. The PI will continue to host undergraduates and REU students for research in the laboratory. International research collaboration will be promoted via a summer exchange student program established by the PI with the Chinese University of Hong Kong, as well as the Chile Exchange Program of the University of Chicago MRSEC. Science, especially the concept of interdisciplinary science, will be brought to schoolchildren through outreach activities. The PI is committed to increasing the representation of women in academic science. Efforts will be made to address the opportunities for women in academic science through the PI's work with the Office of the Provost and her involvement as the Chair of the Women in Physical Sciences Committee of the Physical Sciences Division at the University of Chicago.
Intellectual Merit During the course of the grant, we have used monolayers and bilayers as model systems, and a suite of biophysical techniques (surface-pressure measurements, fluorescence, beta-cyclodextrin desorption assays, grazing incidence x-ray diffraction, x-ray reflectivity) to probe the nature of the interactions between cholesterol and lipids. We provided the first signature pointing to the ordering of structures consistent with the existence of lipid/cholesterol complexes. The time-resolved x-ray diffraction capability we developed allows, for the first time, in situ monitoring of changes in the composition of the surface film, due to cholesterol desorption by beta-cyclodextrin, affect its internal ordering. We have further correlated these structural data of mixed lipid/cholesterol systems with the stiffness and mechanical properties of lipid membrane using a peptide insertion assay. We confirm that the addition of cholesterol to lipid bilayers indeed tightens the membrane, rendering it different for cell penetrating peptides to insert. Membrane stiffening also occurs when small molecules, like glycerol, is present in the environment; such stiffening has a profound effect on the mechanical response of the membrane. We have further examined lipid ordering involving mixed lipid systems with gangliosides, instead of cholesterol, to determine the effects of ganglioside headgroup charge and geometry on its interactions with its neighboring zwitterionic lipids. Our work led to the observation that the complementary geometry of saturated, zwitterionic lipid, phosphatidylcholine, and gangliosides with different headgroup geometries affects their close molecular packing, inducing condensation of the layer at low ganglioside content, followed by an expansion of the average molecular area as ganglioside content increases beyond that which gives rise to minimal lipid packing. However, the mole fraction of ganglioside needed to give rise to the tightest average lipid packing varies for different gangliosides. This variations in critical packing mole ratios can be explained by global effects of headgroup charge and resultant dipole moments within the monolayer. Finally, by combining grazing incidence x-ray diffraction results with rheological data, we create the first experimental and theoretical connection between monolayer defects and interfacial rheology for phospholipid/cholesterol films. Broader Impacts The research activities have provided training opportunities for researchers in a highly interdisciplinary area, and help train the next generation of interdisciplinary research scientists. The project has served to enhance the content of a graduate course in membrane biophysics, as well as the curriculum development of a new interdisciplinary degree-granting program in Biophysical Sciences. Apart from graduate students and postdocs, research opportunities in the PI’s laboratory have been extended to qualified high school, undergraduate, summer research as well as international exchange students. The PI has run a 7-week summer research program, placing 10-15 rising high school seniors from the University of Chicago Laboratory Schools in laboratories across the Biological Sciences Division and the Physical Sciences Division at the University of Chicago to catalyze these young students’ interests in a career in science. Several of these students have been named semi-finalists and finalist in Intel Science Talent Search. The PI has also reached out to Lindblom Math and Science Academy, a minority-serving high school in the South Side of Chicago, and has established a program through which motivated 6-8 students from Lindblom are placed to carry out research in laboratories at the University of Chicago. The PI has hosted undergraduate and REU students for research in the laboratory. International research collaboration has been promoted via an exchange program with Chile. Science, especially the concept of interdisciplinary science, has been brought to school children through outreach activities, including participating in Physics with a Bang!, an annual Holiday Show and Open House, and performing science demonstrations to 1st through 4th grade students. The PI is committed to increasing the representation of women in academic science, and has worked with the Physical Science Division Dean’s Office and the Provost Office to put in place programs to improve the working environment on campus for women in science, to increase the number of women faculty, and to promote women to leadership positions.
