Abstract:An assay for vesicle--vesicle fusion involving resonance energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl), the energy donor, and rhodamine, the energy acceptor, has been developed. The two fluorophores are coupled to the free amino group of phosphatidylethanolamine to provide analogues which can be incorporated into a lipid vesicle bilayer. When both fluorescent lipids are in phosphatidylserine vesicles at appropriate surface densities (ratio of fluorescent lipid to total lipid), efficient energy t… Show more
“…It is also worth noting that cholesterol, even at 50% per mole, does not significantly affect the overall transition pressure 47, 87. Finally, the presence of charged lipids can modify the short‐range repulsion; for instance, when there are charged lipids, the addition of Ca 2+ (at concentrations > 1 m m which are unlikely to be physiologically relevant) can significantly reduce the transition pressure and facilitate fusion 88, 89.Figure A1(A) Short range repulsion between two DOPC / DOPE (60/40) lipid bilayers. The values are based on published data [47–49].…”
Neural networks are optimized to detect temporal coincidence on the millisecond timescale. Here, we offer a synthetic hypothesis based on recent structural insights into SNAREs and the C2 domain proteins to explain how synaptic transmission can keep this pace. We suggest that an outer ring of up to six curved Munc13 ‘MUN’ domains transiently anchored to the plasma membrane via its flanking domains surrounds a stable inner ring comprised of synaptotagmin C2 domains to serve as a work‐bench on which SNAREpins are templated. This ‘buttressed‐ring hypothesis’ affords straightforward answers to many principal and long‐standing questions concerning how SNAREpins can be assembled, clamped, and then released synchronously with an action potential.
“…It is also worth noting that cholesterol, even at 50% per mole, does not significantly affect the overall transition pressure 47, 87. Finally, the presence of charged lipids can modify the short‐range repulsion; for instance, when there are charged lipids, the addition of Ca 2+ (at concentrations > 1 m m which are unlikely to be physiologically relevant) can significantly reduce the transition pressure and facilitate fusion 88, 89.Figure A1(A) Short range repulsion between two DOPC / DOPE (60/40) lipid bilayers. The values are based on published data [47–49].…”
Neural networks are optimized to detect temporal coincidence on the millisecond timescale. Here, we offer a synthetic hypothesis based on recent structural insights into SNAREs and the C2 domain proteins to explain how synaptic transmission can keep this pace. We suggest that an outer ring of up to six curved Munc13 ‘MUN’ domains transiently anchored to the plasma membrane via its flanking domains surrounds a stable inner ring comprised of synaptotagmin C2 domains to serve as a work‐bench on which SNAREpins are templated. This ‘buttressed‐ring hypothesis’ affords straightforward answers to many principal and long‐standing questions concerning how SNAREpins can be assembled, clamped, and then released synchronously with an action potential.
“…The polymer-modified liposomes containing NBD-PE and Rh-PE were prepared to detect fusion of the liposomes with intracellular membranes [6,25]. Fusion of the labeled liposomes with endosomal membranes causes dilution of these fluorescent lipids in the membrane, resulting in decrease of energy transfer efficiency between these fluorescent lipids.…”
Section: Fusion Of Polymer-modified Liposomes Within Cellmentioning
Previous reports by the authors described intracellular delivery using liposomes modified with various carboxylated poly(glycidol) derivatives. These linear polymer-modified liposomes exhibited pH-dependent membrane fusion behavior in cellular acidic compartments. However, the effect of the backbone structure on membrane fusion activity remains unknown. Therefore, this study specifically investigated the backbone structure to obtain pH-sensitive polymers with much higher fusogenic activity and to reveal the effect of the polymer backbone structure on interaction with the membrane.Hyperbranched poly(glycidol) (HPG) derivatives were prepared as a new type of pH-sensitive polymers and modified liposomes using these polymers. The resultant HPG derivatives exhibited high hydrophobicity and intensive interaction with the membrane concomitantly with the increasing degree of polymerization. Furthermore, HPG derivatives showed stronger interaction with the membrane than linear polymers show. Liposomes modified with HPG derivatives of high DP delivered contents into cytosol of DC2.4 cells, a dendritic cell line, more effectively than the linear polymer-modified liposomes do. Results show that the backbone structure of pH-sensitive polymers affected their pH-sensitivity and interaction with liposomal and cellular membranes.
Defensins comprise a family of broad-spectrum antimicrobial peptides that are stored in the cytoplasmic granules of mammalian neutrophils and Paneth cells of the small intestine. Neutrophil defensins are known to permeabilize cell membranes of susceptible microorganisms, but the mechanism of permeabilization is uncertain. We report here the results of an investigation of the mechanism by which HNP-2, one of 4 human neutrophil defensins, permeabilizes large unilamellar vesicles formed from the anionic lipid palmitoyloleoylphosphatidylglycerol (POPG). As observed by others, we find that HNP-2 (net charge = +3) cannot bind to vesicles formed from neutral lipids. The binding of HNP-2 to vesicles containing varying amounts of POPG and neutral (zwitterionic) palmitoyloleoylphosphatidylcholine (POPC) demonstrates that binding is initiated through electrostatic interactions.Because vesicle aggregation and fusion can confound studies of the interaction of HNP-2 with vesicles, those processes were explored systematically by varying the concentrations of vesicles and HNP-2, and the P0PG:POPC ratio. Vesicles (300 pM POPG) readily aggregated at HNP-2 concentrations above 1 pM, but no mixing of vesicle contents could be detected for concentrations as high as 2 pM despite the fact that intervesicular lipid mixing could be demonstrated. This indicates that if fusion of vesicles occurs, it is hemi-fusion, in which only the outer monolayers mix at bilayer contact sites. Under conditions of limited aggregation and intervesicular lipid mixing, the fractional leakage of small solutes is a sigmoidal function of peptide concentration. For 300 pM POPG vesicles, 50% of entrapped solute is released by 0.7 p M HNP-2. We introduce a simple method for determining whether leakage from vesicles is graded or all-or-none. We show by means of this fluorescence "requenching" method that native HNP-2 induces vesicle leakage in an all-or-none manner, whereas reduced HNP-2 induces partial, or graded, leakage of vesicle contents. At HNP-2 concentrations that release 100% of small (-400 Da) markers, a fluorescent dextran of 4,400 Da is partially retained in the vesicles, and a 18,900-Da dextran is mostly retained. These results suggest that HNP-2 can form pores that have a maximum diameter of approximately 25 A. A speculative multimeric model of the pore is presented based on these results and on the crystal structure of a human defensin.
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