(331d) Investigation of pH-Dependent Formation of Lipid Heterogeneities On Giant Unilamellar Vesicles
AIChE Annual Meeting
2009
2009 Annual Meeting
Engineering Sciences and Fundamentals
Transport at Interfaces
Tuesday, November 10, 2009 - 4:20pm to 4:40pm
There is growing evidence that nature uses lipid membranes as a universal wrap around cells to control critical functions by dynamically reorganizing lipids into rafts. These are nanometer- to micron-sized lipid domains of laterally phase separated lipids occurring at the sites of action during cell-to-cell communication. Understanding the biophysical forces among lipids that play a role in these phenomena, and characterizing the potential changes of collective physical properties of membranes caused by these phenomena may potentially impact the control of related cell functions and associated diseases.
We study the kinetics of lipid phase separation, and domain formation and growth in phospholipid bilayers in the form of giant unilamellar vesicles (GUV's) using fluorescence microscopy. The bilayers are composed of: a first lipid type with a zwitterionic phosphatidylcholine (PC) headgroup, a second lipid type with a titratable headgroup such as phosphatidic acid (PA) or phosphatidyl serine (PS), and cholesterol. Phase separation vs. pH is evaluated in these studies. In all preparations GUV's were labeled with C20:0-DiI and (16:0,Bodipy)-PC, both at 0.1 mol %, and observed at 25 °C.
For GUV's composed of DOPC:DSPA:Chol (at 1:1:1 mole ratio) at pH 7.0, the membrane appears homogenously mixed. At the lower pH of 5.0, phase separated domains are observed with complex shapes that could be the result of aggregation and ripening of smaller circular domains. When DSPA is replaced by DSPS, small circular phase separated domains are formed at pH 7.0. At the lower pH values of 6.0 and 5.0, larger phase separated domains with complex shapes are also observed. Phase separated domains should be rich in protonated PA or PS lipid. Domain formation is attributed to decrease in electrostatic repulsion among charged lipids and formation of hydrogen bonding between protonated lipids. We are currently investigating the extent to which heterogeneous short-range lipid order within the domains and diffusion-limited domain ripening affect the formation of the observed complex shapes.
To understand the effect of lipid heterogeneities on altering long range membrane structure, optical observations are compared to membrane characterization using differential scanning calorimetry.
Abbreviations: dioleoyl phosphocholine (DOPC), distearoyl phosphatidic acid (DSPA), distearoyl phosphatidyl serine (DSPS), cholesterol (Chol), 1,1′-dieicosanyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (C20:0-DiI), 1-hexadecanoyl-2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3-phosphocholine (16:0,Bodipy-PC).