4-4-difluoro-4-bora-3a-4a-diaza-s-indacene and 1-2-dipalmitoyl-3-phosphatidylethanolamine

Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir-Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10(-8) mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Boron Compounds; Chickens; Cholesterol; Fluorescent Dyes; Microscopy, Fluorescence; Microscopy, Polarization; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins; Xanthenes

We report a new method for measuring the partitioning kinetics of membrane biomolecules to different lipid phases using a patterned supported lipid bilayer (SLB) platform composed of liquid-ordered (lipid raft) and liquid-disordered (unsaturated lipid-rich) coexistent phases. This new approach removes the challenges in measuring partitioning kinetics using current in vitro methods due to their lack of spatial and temporal control of where phase separation occurs and when target biomolecules meet those phases. The laminar flow configuration inside a microfluidic channel allows us to pattern SLBs with coexistent phases in predetermined locations and thus eliminates the need for additional components to label the phases. Using a hydrodynamic force provided by the bulk flow in the microchannel, target membrane-bound species to be assayed can be transported in the bilayers. The predefined location of stably coexistent phases, in addition to the controllable movement of the target species, allows us to control and monitor when and where the target molecules approach or leave different lipid phases. Using this approach with appropriate experimental designs, we obtain the association and dissociation kinetic parameters for three membrane-bound species, including the glycolipid G(M1), an important cell signaling molecule. We examine two different versions of G(M1) and conclude that structural differences between them impact the kinetics of association of these molecules to raft-like phases. We also discuss the possibilities and limitations for this method. One possible extension is measuring the partitioning kinetics of other glycolipids or lipid-linked proteins with posttranslational modifications to provide insight into how structural factors, membrane compositions, and environmental factors influence dynamic partitioning.

Topics: Boron Compounds; G(M1) Ganglioside; Kinetics; Lipid Bilayers; Membrane Microdomains; Phosphatidylethanolamines

The effects of annexin A5 on the lateral diffusion of single-molecule lipids and single-molecule proteins were studied in an artificial lipid bilayer membrane. Annexin A5 is a member of the annexin superfamily, which binds preferentially to anionic phospholipids in a Ca2+-dependent manner. In this report, we were able to directly monitor single BODIPY 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (DHPE) and ryanodine receptor type 2 (RyR2) labeled with Cy5 molecules in lipid bilayers containing phosphatidylserine (PS) by using fluorescence microscopy. The diffusion coefficients were calculated at various annexin A5 concentrations. The diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 in the absence of annexin A5 were 4.81 x 10(-8) cm(2)/s and 2.13 x 10(-8) cm(2)/s, respectively. In the presence of 1 microM annexin A5, the diffusion coefficients of BODIPY-DHPE and Cy5-RyR2 were 2.2 x 10(-10) cm(2)/s and 9.5 x 10(-11) cm(2)/s, respectively. Overall, 1 microM of annexin A5 was sufficient to induce a 200-fold decrease in the lateral diffusion coefficient. Additionally, we performed electrophysiological examinations and determined that annexin A5 has little effect on the function of RyR2. This means that annexin A5 can be used to immobilize RyR2 in a lipid bilayer when imaging and analyzing RyR2.

Topics: Animals; Annexin A5; Boron Compounds; Carbocyanines; Dogs; Lipid Bilayers; Microscopy, Fluorescence; Phosphatidylethanolamines; Ryanodine Receptor Calcium Release Channel