Effect of single chain lipids on phospholipase C-promoted vesicle fusion. A test for the stalk hypothesis of membrane fusion

The effect of low proportions (up to 5 mol %) of single-chain lipids on phospholipase C-promoted fusion of large unilamellar vesicles has been investigated with the aim of testing the so-called stalk model of membrane fusion. This model is known in two main versions, the one originally published by Kozlov and Markin [Kozlov, M. M. and Markin, V. S. (1983) Biofizika 28, 255-261] and what is known as the "modified stalk model" [Siegel, D. P. (1993) Biophys. J. 65, 2124-2140], that differ in a number of predictions. In the view of the latter author, hydrocarbons or other nonpolar lipids should help fusion by decreasing the interstitial energy of the stalk connecting the two apposed bilayers. We show that small amounts of hexadecane or squalene increase significantly the fusion rates in our system. Changes in monolayer curvature are the object of different predictions by the original and modified stalk theories. According to the original form, fusion would be promoted by lipids inducing a negative curvature in the closest (cis) monolayers of the fusing membranes and inhibited by the same lipids in the trans monolayers; the opposite would happen with lipids inducing a positive curvature. The modified stalk model predicts that fusion is helped by increasing the negative curvature of both monolayers. In our system, symmetrically distributed arachidonic acid, which increases the negative curvature, enhances lipid and content mixing, and the opposite is found with symmetrically distributed lysophosphatidylcholine or palmitoylcarnitine, which facilitate a positive monolayer curvature. In addition, fluorescence polarization and 31P NMR studies of the lamellar-to-isotropic (Q224 cubic) thermotropic transition of a lipid mixture corresponding to our liposomal composition reveal that all lipids that facilitate fusion decrease the transition temperature, while fusion inhibitors increase the transition temperature. Moreover, fusion (content mixing) rates show a maximum at the lamellar-to-isotropic transition temperature. These observations support the involvement of inverted lipid structures, as occurring in the inverted cubic phases, in membrane fusion. All these data are in full agreement with the stalk model of membrane fusion, particularly in its modified version.     

Organelle membrane fusion: a novel function for the syntaxin homolog Ufe1p in ER membrane fusion

The fusion of endoplasmic reticulum (ER) membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane. Instead, ER membranes require a NSF-related ATPase, Cdc48p. Since both vesicular and organelle fusion events use related ATPases, we investigated whether both fusion events are also SNARE mediated. We present evidence that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but no known v-SNAREs. We propose that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion as well as a t-SNARE for vesicular traffic.     

Phases and phase transitions of the phosphatidylcholines

LIPIDAT (http://www.lipidat.chemistry.ohio-state.edu) is an Internet accessible, computerized relational database providing access to the wealth of information scattered throughout the literature concerning synthetic and biologically derived polar lipid polymorphic and mesomorphic phase behavior and molecular structures. Here, a review of the data subset referring to phosphatidylcholines is presented together with an analysis of these data. This subset represents ca. 60% of all LIPIDAT records. It includes data collected over a 43-year period and consists of 12,208 records obtained from 1573 articles in 106 different journals. An analysis of the data in the subset identifies trends in phosphatidylcholine phase behavior reflecting changes in lipid chain length, unsaturation (number, isomeric type and position of double bonds), asymmetry and branching, type of chain-glycerol linkage (ester, ether, amide), position of chain attachment to the glycerol backbone (1,2- vs. 1,3-) and head group modification. Also included is a summary of the data concerning the effect of pressure, pH, stereochemical purity, and different additives such as salts, saccharides, amino acids and alcohols, on phosphatidylcholine phase behavior. Information on the phase behavior of biologically derived phosphatidylcholines is also presented. This review includes 651 references.     

Modulation of lipid polymorphism by the feline leukemia virus fusion peptide: implications for the fusion mechanism

The structural effects of the fusion peptide of feline leukemia virus (FeLV) on lipid polymorphism were studied, using differential scanning calorimetry (DSC), 31P nuclear magnetic resonance (NMR), and time-resolved X-ray diffraction. This peptide lowers the bilayer to inverted hexagonal phase transition temperature, TH, of dipalmitoleoylphosphatidylethanolamine (DiPoPE) at peptide mole fractions of up to 1.5 x 10(-3) at pH 5.0 and at pH 7.4. The temperature at which isotropic 31P NMR signals for monomethyldioleoylphosphatidylethanolamine (MeDOPE) first occurred is lowered by the FeLV peptide. The amount of isotropic signal seen at 40 degrees C is directly correlated to the peptide:lipid molar ratio. In the peptide-containing samples, more lipid remains in the isotropic state over the whole recorded temperature range. Isotropic 31P NMR signals were observed for DiPoPE in the presence of the FeLV peptide for the entire recorded temperature range of 35-50 degrees C, while pure DiPoPE showed no significant amount of isotropic signal. X-ray studies of DiPoPE show the formation of a new lipid phase with peptide, which is not seen in the pure lipid samples. Disordering of the Lalpha phase is evidenced by broadening of the diffraction peaks, and the hexagonal cell parameter is decreased with peptide present. Our results suggest that the FeLV peptide is increasing the negative curvature of the lipid system, which is thought to be crucial to the formation of highly bent, high-energy structural fusion intermediates, such as the "stalk" model. Fusion activity for this putative fusogenic peptide was also demonstrated, using a resonance energy transfer (RET) lipid mixing assay. To our knowledge, this work provides the first published experimental evidence of both fusogenic activity and effects on lipid polymorphism for the FeLV fusion peptide.     

The relationship between compositional phase separation and vesicle morphology: implications for the regulation of phospholipase A2 by membrane structure

The action of phospholipase A2 (PLA2) on bilayer substrates causes the accumulation of reaction products, lyso-phospholipid and fatty acid. These reaction products and the phospholipid substrate generate compositional heterogeneities and then apparently phase separate when a critical mole fraction of reaction product accumulates in the membrane. This putative phase separation drives an abrupt morphologic rearrangement of the vesicle, which may be in turn responsible for modulating the activity of PLA2. Here we examine the thermotropic properties of the phase-separated lipid system formed upon hydrating colyophilized reaction products (1:1 palmitic acid:1-palmitoyl-2-lyso-phosphatidylcholine) and substrate, dipalmitoylphosphatidylcholine. The mixture forms structures which are not canonical spherical vesicles and appear to be disks in the gel-state. The main gel-liquid transition of these structures is hysteretic. This hysteresis is apparent using several techniques, each selected for its sensitivity to different aspects of a lipid aggregate's structure. The thermotropic hysteresis reflects the coupling between phase separation and changes in vesicle morphology.     

Hong Kong nurses' health-related behaviours: implications for nurses' role in health promotion

The health-related behaviors of a random sample (n = 92) of Hong Kong nurses were assessed by a questionnaire written either in English or in English and Chinese. Hong Kong nurses reported negligible smoking or alcohol use, low levels of breast self-examination, cervical screening behaviour and regular exercising, seat belt use and driving within the speed limit. The sample reported high levels of making efforts to avoid foods high in cholesterol, eating foods high in fibre and eating fruit daily. Dental hygiene was reported to be high. Just over half the sample reported sleeping 7-8 hours each night and eating breakfast daily. Most nurses reported maintaining their body weight at a healthy level and eating snacks between meals. The English language version of the questionnaire produced a slightly better response rate than the bilingual questionnaire. The results are discussed with reference to previous studies of females' health-related behaviours in Hong Kong and elsewhere. The implications for Hong Kong nurses' role in health promotion is discussed.     

On the kinematics of incoherent phase transitions

Incoherent phase transitions are far more difficult to treat than their coherent counterparts. The interface, which appears as a single surface in the deformed configuration, is represented in its undeformed state by a separate surface in each phase. This leads to a rich but detailed kinematics, one in which defects such as vacancies and dislocations are generated by the moving interface. We introduce an incoherency tensor that measures the stretching and twisting of one phase relative to the other. We show that incoherency is completely characterized by the incoherency tensor, the vacancy production, and the slip between phases.     

SNAREs and membrane fusion in the Golgi apparatus

Soluble factors, NSF and SNAPs, are required at many membrane fusion events within the cell. They interact with a class of type II integral membrane proteins termed SNAP receptors, or SNAREs. Interaction between cognate SNAREs on opposing membranes is a prerequisite for NSF dependent membrane fusion. NSF is an ATPase which will disrupt complexes composed of different SNAREs. However, there is increasingly abundant evidence that the SNARE complex recognised by NSF does not bridge the two fusing membranes, but rather is composed of SNAREs in the same membrane. The essential role of NSF may be to prime SNAREs for a direct role during fusion. The best characterised SNAREs in the Golgi are Sed5p in yeast and its mammalian homologue syntaxin 5, both of which are predominantly localised to the cis Golgi. The SNARE-SNARE interactions in which these two proteins are involved are strikingly similar. Sed5p and syntaxin 5 may mediate three distinct pathways for membrane flow into the cis Golgi, one from the ER, one from later Golgi cisternae, and possibly a third from endosomes. Syntaxin 5 is itself likely to cycle through the ER, and thus may be involved in homotypic fusion of ER derived transport vesicles. In all well characterised SNARE dependent membrane fusion events one of the interacting SNAREs is a syntaxin homologue. There are only eight members of the syntaxin family in yeast. Besides Sed5p two others, Tlg1p and Tlg2p, are found in the Golgi complex. They are present in a late Golgi compartment, but neither is required for transit of secreted proteins through the Golgi. We suggest that these observations are most compatible with a model for transit through the Golgi in which anterograde cargo is carried in cisternae, the enzymatic composition of which changes with time as Golgi resident enzymes are delivered in retrograde transport vesicles.     

Annexins and ATP in membrane traffic: a comparison with membrane fusion machinery

A rabbit antiserum (anti-EP), induced against a synthetic peptide corresponding to residues 68 to 86 of guinea pig myelin basic protein, powerfully immunostained abnormal-appearing oligodendrocytic processes and cell bodies in demyelinating areas associated with multiple system atrophy (MSA). However, as we reported previously, the antiserum, which is highly specific for the sequence QDENPVV corresponding to human myelin basic protein residues 82 to 88, failed to recognize any structures in normal human brain. QD-9, a mouse monoclonal antibody raised against human myelin basic protein residues 69 to 88, which also recognizes specifically the epitope QDENPVV, gave the same results as did anti-EP. The unusual epitope recognized by anti-EP/QD-9 antibodies appears to be accessible in areas of myelin degeneration, and the antibodies have been shown to detect such areas in multiple sclerosis and infarcted brains. These antibodies detect myelin degeneration more widely than previous conventional methods. The present study emphasizes the importance of myelin degeneration in the pathogenesis of multiple system atrophy.