texas-red and 1-2-oleoylphosphatidylcholine

Fibrillar aggregates of the protein α-synuclein (αS) are one of the hallmarks of Parkinson's disease. Here, we show that measuring the fluorescence polarization (FP) of labels at several sites on αS allows one to monitor changes in the local dynamics of the protein after binding to micelles or vesicles, and during fibril formation. Most significantly, these site-specific FP measurements provide insight into structural remodeling of αS fibrils by small molecules and have the potential for use in moderate-throughput screens to identify small molecules that could be used to treat Parkinson's disease.

Topics: alpha-Synuclein; Amino Acid Sequence; Catechin; Dopamine; Fluorescence Polarization; Fluorescent Dyes; Humans; Masoprocol; Phosphatidylcholines; Protein Aggregates; Recombinant Proteins; Small Molecule Libraries; Sodium Dodecyl Sulfate; Unilamellar Liposomes; Xanthenes

Binding of amphiphilic molecules to supported lipid bilayers (SLBs) often results in lipid fibril extension from the SLBs. Previous studies proposed that amphiphiles with large and flexible hydrophilic regions trigger lipid fibril formation in SLBs by inducing membrane curvature via their hydrophilic regions. However, no experimental studies have verified this mechanism of fibril formation. In this work, we investigated the binding of lipopolysaccharide (LPS) and cholesterol-modified gelatin to SLBs using fluorescence microscopy. SLBs with restricted and unrestricted bilayer areas were employed to identify the mechanism of fibril generation. We show that the main cause of lipid fibril formation is an approximately 20% expansion in the bilayer area rather than increased membrane curvature. The data indicate that bilayer area confinement plays a critical role in morphological changes of SLBs even when bound amphiphilic molecules have a large hydrophilic domain. We also show that bilayer area change after LPS insertion is dependent on the patch shape of the SLB. When an SLB patch consists of a broad bilayer segment connected to a long thin streak, bilayer area expansion mainly occurs within the bilayer streak. The results indicate that LPS insertion causes net lipid flow from the broad bilayer region to the streak area. The differential increase in area is explained by the instability of planar bilayer streaks that originate from the large energetic contribution of line tension arising along the bilayer edge.

Topics: Biomimetic Materials; Cholesterol; Gelatin; Lipid Bilayers; Lipopolysaccharides; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Surface Tension; Unilamellar Liposomes; Xanthenes

The fusion of lipid membranes is a key process in biology. It enables cells and organelles to exchange molecules with their surroundings, which otherwise could not cross the membrane barrier. To study such complex processes we use simplified artificial model systems, i.e., an optical fusion assay based on membrane-coated glass spheres. We present a technique to analyze membrane-membrane interactions in a large ensemble of particles. Detailed information on the geometry of the fusion stalk of fully fused membranes is obtained by studying the diffusional lipid dynamics with fluorescence recovery after photobleaching experiments. A small contact zone is a strong obstruction for the particle exchange across the fusion spot. With the aid of computer simulations, fluorescence-recovery-after-photobleaching recovery times of both fused and single-membrane-coated beads allow us to estimate the size of the contact zones between two membrane-coated beads. Minimizing delamination and bending energy leads to minimal angles close to those geometrically allowed.

Topics: Algorithms; Cell Fusion; Computer Simulation; Diffusion; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Glass; Heterocyclic Compounds, 4 or More Rings; Lipopeptides; Membrane Fusion; Membranes, Artificial; Microscopy, Confocal; Models, Theoretical; Phosphatidylcholines; Phosphatidylethanolamines; Silicon Dioxide; Xanthenes

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 study the nonequilibrium shape fluctuations in fluorescence labeled phospholipid multibilayers composed of the model lipid DOPC and the well-known lipid dye Texas red, driven out of equilibrium by short laser pulses. The temporal evolution of the lipid bilayer undulations after excitation was recorded by time resolved x-ray diffraction. Already at moderate peak intensities (Pp≤10(5)  W/cm2), pulsed laser illumination leads to significant changes of the undulation modes in a well-defined lateral wavelength band. The observed phenomena evolve on nano- to microsecond time scales after optical excitation, and can be described in terms of a modulation instability in the lipid multilamellar stack.

Topics: Kinetics; Lasers; Membrane Lipids; Phosphatidylcholines; Phospholipids; Photoelectron Spectroscopy; X-Ray Diffraction; Xanthenes

Spontaneous changes in the morphology of cell-size liposomes (dioleoylphosphatidylcholine, DOPC and egg PC) as model cells were investigated in the presence of cholesterol. Tube structures and liposome networks connected by the tubes were observed in the presence of 5-30% cholesterol by dark-field and laser-scanning microscopy. Furthermore, in the presence of more than 40 mol% of cholesterol, the tubes disappeared and changed to small liposomes. Thus, cholesterol induced a morphological change in giant liposomes from tubes to small liposomes. These phenomena may be related to the role of cholesterol in the morphological changes in living cells such as neurons.

Topics: Cell Membrane; Cholesterol; Liposomes; Neurons; Phosphatidylcholines; Xanthenes

Real-time budding dynamics of multicomponent, tubular lipid vesicles was investigated. By using a fluorescence microscope, three typical growth modes of the buds were observed, corresponding, respectively, to bud growth through coalescence between flat patches, a bud and a patch, as well as that of two buds. The spatial and temporal scales measured in the observation were used to estimate the bending rigidity of the membrane. In the late stage, the continuing coalescence between the buds resulted in large shape deformation of the vesicles, from tubular to spherical vesicles, and the number of the buds decayed with time as N approximately t-2/3. This scaling relation was observed for the first time in experiment and confirmed early theoretical predictions. Our observation showed a difference between the diffusivity of the buds on the lipid membrane and that of the embedded membrane proteins.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Kinetics; Liposomes; Membrane Lipids; Microscopy, Fluorescence; Phosphatidylcholines; Rhodamines; Xanthenes