Thanks To: We are very grateful for the work of our lab mates Dr. Tracy Bibelnieks, Alison Heussler, Dan Forseth, and Kyle Sontag. We also are thankful to our collaborators in Professor Xiaoyang Zhu’s lab at the University of Minnesota. We thank Dr. Sarah Veatch, Dr. Mike Halter, and Dr. Sarah Keller for helpful discussions. BJS acknowledges support from the Minnesota NASA Space Grant and Augsburg College’s office of Undergraduate Research and Graduate Opportunities. BLS acknowledges course release time provided by Augsburg College to complete this work. Background/Context 25 Hydroxycholesterol and other oxysterols play many important roles in life processes. Here are just some of the ways in which oxysterols impact cellular behavior. Oxysterols are: involved in the regulation of cholesterol synthesis. believed to inhibit cell growth. a major toxic component in oxLDL. believed to play a role in apoptosis, the controlled death of cells. known to participate in ion-uptake and alter the permeability of cell membranes. Summary of Results The addition of a hydroxyl group to the acyl chain of a cholesterol molecule has a dramatic effect on the phase behavior of phospholipid/sterol monolayers. Domain appearance is dependent on the path into the two phase region. Using fluorescence microscopy we observe a bimodal distribution of domain sizes. 25OH appears to have an expanding affect on the monolayer, in direct contrast to cholesterol. Preliminary evidence suggests domain growth occurs through collision and coalescence. Again, this is a contrast to cholesterol. Learn more about oxysterols: J.B. Massey. Membrane and protein interactions of oxysterols. Current Opinion in Lipidology, 2006, 17: T. Wielkoszynski, et al. Cellular toxicity of oxycholesterols. BioEssays 2006, 28: D. Larsson, et al. Oxysterol mixtures, in atheroma-relevant proportions, display synergistic proapoptotic effects. Free Radical Biology & Medicine 2006, 41: S.R. King et al. Oxysterols regulate expression of the steroidogenic acute regulatory protein. J. Mol. Endocrinol. 2004,32: M. Yehm et al. Role for sterol regulatory element-binding protein in activation of endothelial cells by phospholipid oxidation products. Circ. Res. 2004, 95: Fluorescence Microscopy Results Domain morphology depends on history Left: The two phase region was entered from above. Right: The two phase region was entered from below. The lipid monolayer is composed of 22(R)-DIOL and DLPC (1:1). All images taken at 6.5 mN/m. Two length scales? Summary of technique: Fluorescence microscopy is used to determine the miscibility phase behavior of mixed lipid systems. A trace amount of fluorescent probe is used to provide the contrast between immiscible phases. Images here were taken with an Olympus microscope and Pursuit Camera from Diagnostic Instruments. Increasing Surface Pressure 5.5 mN/m 6.5 mN/m 7.5 mN/m8.5 mN/m As the surface pressure increases so does the fraction of dark phase. In lipid/cholesterol monolayers the dark fraction is considered to be cholesterol rich. We are currently running experiments to confirm if this is true in hydroxycholesterol monolayers. Above: a 1:1 mixture of DMPC and 22(R)-DIOL in a lipid monolayer. Scale bar is 50 microns Histogram of Domain Size Area of Domain in Pixels Number of Domains Built-in ImageJ analysis tools quickly quantify the size distribution of domains. Here we set the lower size limit at 15 pixels to reduce the possibility of including noise into our distribution. The number of domains ~1000 pixels in size is just slightly less than the number of small domains ( pixels). Other Observations: We have preliminary evidence that domains observed in hydroxycholesterol monolayers grow by collision and coalescence. This suggests repulsion due to dipole densities is not as dominant as in monolayers containing cholesterol. Motivation for Oxysterol Studies What is exciting: Correlations between monolayer compressibility and miscibility phase transitions have long been predicted but have not been unambiguously verified. These results show liquid-liquid coexistence and miscibility transitions which are evident in the pressure-area isotherms. Additionally, the observation in lipid monolayers of a liquid-liquid immiscibility region bounded by both upper and lower critical points is novel. Two Hydrophilic Regions mole %: Small kinks observed in the pressure- area isotherms correspond to the miscibility phase transitions observed with fluorescence microscopy. β-region Hydroxy- cholesterol α-region Sterol Composition π (mN/m) Molecular Area (Å 2 ) :1 DPPC:25-hydroxycholesterol π (mN/m) Molecular Area (Å 2 ) :1 DPPC:25-hydroxycholesterol HO 25 Hydroxycholesterol OH HO 22 Hydroxycholesterol (R) In addition to the biophysical effects of hydroxycholesterol molecules on lipid membranes, the system presented here also allows us to investigate two dimensional coexisting liquids with properties different than the canonical phospholipid/cholesterol system. Pressure-area isotherms can be taken simultaneously to fluorescence microscopy images. If necessary the system can be surrounded by “glove bag” to provide an inert environment. Current Experimental Setup Experimental Notes We are using the fluorescently labeled lipid DiI C12 from Molecular Probes. Tests confirm that this dye partitions similarly to Texas Red. The majority of experiments presented here are done in an ambient environment. Transition pressures do not change more than +/- 1 mN/m over the course of an observation period, typically less than one hour. Preliminary experiments using β-cyclodextrin to extract 25OH from the air-water interface suggest that this process can occur extremely fast. Further work is warranted. All experiments were done at 24 +/- 1 degree Celsius. Isotherms were collected using a Nima 612 Langmuir trough. A Proposed Explanation Compression of monolayer Our current experimental set up can not reveal a reorientation (flip) of 25-OH within the monolayer which may be responsible for the kink in the pressure-area isotherm. If hydroxycholesterols have two possible vertical orientations this may explain the observations of two length scales in the size of domains (see results of fluorescence microcopy). OR Hydroxycholesterols within the monolayer Are both orientations possible within the monolayer? At low pressures the sterol molecule may lie flat against the air-water interface because of its two hydroxyl groups. As the molecular area decreases the sterol molecule stands up. This reorientation may coincide with the observed kink in the pressure-area isotherms. At this time we do not know if there are one or two possible orientations of the sterol molecule within the leaflet. Steroid ring structure Hydroxyl group Hydrocarbon chain The Miscibility Phase Behavior of Lipid Monolayers Containing 25-Hydroxycholesterol Benjamin L. Stottrup a) and Benjamin J. Sonquist b) a) Department of Physics, b) Department of Biology, Augsburg College, Minneapolis, MN Meeting of the Biophysical Society Pressure-Area Isotherm Results Unlike cholesterol, the addition of 25OH increases the molecular area at low surface pressures. Summary of technique: Langmuir film balance studies of lipid monolayers offer the opportunity to investigate lipid behavior over a wide range of molecular areas. Studies here were done using a Nima 612 Langmuir trough. Significance of Kink: The addition of a hydroxycholesterol to phospholipid monolayers has thus far resulted in a kink, change in slope, corresponding to entrance of a liquid-liquid region through a lower transition. We are currently investigating how the location of a second hydroxyl group affects monolayer pressure-area isotherms and domains observed using fluorescence microscopy. Other Observations: The presence of 25OH induced a two phase liquid-liquid coexistence region in phospholipid monolayers of DLPC, DMPC, and DPPC but did not in DSPC. Red: Expansion Black: Compression Molecular Area (Å 2 ) Surface Pressure (mN/m) 50:50 mixture of DLPC:22(R)-DIOL Hysteresis: We have preliminary evidence for hysteresis in the pressure-area isotherms. This may be the result of the hydroxycholesterol molecules preferring to stay vertical once they stand up in the monolayer. We are currently investigating how this hysteresis might also affect our fluorescence microscopy observations. Change in Molecular Area Mole Percent Composition Molecular Area (sq. Ang.) 3 mN/m 10 mN/m 20 mN/m Isobaric Cuts: Changes in molecular area at constant pressure can be observed and plotted. This allows us to determine the extent to which the monolayer is mixing ideally. In this figure we show isobaric cuts at three pressures for monolayers containing DMPC and 22(R)-DIOL. Pure 22(R)-DIOL Pressure-Area Isotherms of DMPC:22(R)-DIOL Molecular Area (sq. Angstroms) Surface Pressure (mN/m)