1-2-oleoylphosphatidylcholine and tetramethylrhodamine

Membrane fusion is broadly envisioned as a two- or three-step process proceeding from contacting bilayers through one or two semistable, nonlamellar lipidic intermediate structures to a fusion pore. A true fusion event requires mixing of contents between compartments and is monitored by the movement of soluble molecules between trapped compartments. We have used poly(ethylene glycol) (PEG) to rapidly generate an ensemble aggregated state A that proceeds sequentially through intermediates (I₁ and/or I₂) to a final fusion pore state (FP) with rate constants k₁, k₂, and k₃. Movement of moderately sized solutes (e.g., Tb³⁺/dipicolinic acid) has been used to detect pores assigned to intermediate states as well as to the final state (FP). Analysis of ensemble kinetic data has required that mixing of contents occurs with defined probabilities (αi) in each ensemble state, although it is unclear whether pores that form in different states are different. We introduce here a simple new assay that employs fluorescence resonance energy transfer (FRET) between a 6-carboxyfluorescein (donor) and tetramethylrhodamine (acceptor), which are covalently attached to complementary sequences of 10 bp oligonucleotides. Complementary sequences of fluorophore-labeled oligonucleotides were incorporated in vesicles separately, and the level of FRET increased in a simple exponential fashion during PEG-mediated fusion. The resulting rate constant corresponded closely to the slow rate constant of FP formation (k₃) derived from small molecule assays. Additionally, the total extent of oligonucleotide mixing corresponded to the fraction of content mixing that occurred in state FP in the small molecule assay. The results show that both large "final pores" and small (presumably transient) pores can form between vesicles throughout the fusion process. The implications of this result for the mechanism of membrane fusion are discussed.

Topics: Cell Membrane Structures; Fluoresceins; Fluorescence Resonance Energy Transfer; Fluorescent Dyes; Indicators and Reagents; Kinetics; Membrane Fusion; Oligonucleotides; Phosphatidylcholines; Phosphatidylethanolamines; Picolinic Acids; Polyethylene Glycols; Porosity; Rhodamines; Solubility; Sphingomyelins; Surface Properties; Terbium

Previous studies indicate that binding of alpha-synuclein to membranes is critical for its physiological function and the development of Parkinson's disease (PD). Here, we have investigated the association of fluorescence-labeled alpha-synuclein variants with different types of giant unilamellar vesicles using confocal microscopy. We found that alpha-synuclein binds with high affinity to anionic phospholipids, when they are embedded in a liquid-disordered as opposed to a liquid-ordered environment. This indicates that not only electrostatic forces but also lipid packing and hydrophobic interactions are critical for the association of alpha-synuclein with membranes in vitro. When compared to wild-type alpha-synuclein, the disease-causing alpha-synuclein variant A30P bound less efficiently to anionic phospholipids, while the variant E46K showed enhanced binding. This suggests that the natural association of alpha-synuclein with membranes is altered in the inherited forms of Parkinson's disease.

Topics: alpha-Synuclein; Amino Acid Sequence; Anions; Binding Sites; Cell Membrane; Fatty Acids; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Lipids; Microscopy, Fluorescence; Molecular Sequence Data; Molecular Weight; Mutation; Parkinson Disease; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylglycerols; Phosphatidylinositol 4,5-Diphosphate; Phosphatidylserines; Phospholipids; Protein Binding; Protein Conformation; Protein Structure, Secondary; Protein Structure, Tertiary; Rhodamines; Static Electricity; Surface Properties; Unilamellar Liposomes