Barry R. Lentz

Membrane “fluidity” as detected by diphenylhexatriene probes

Abstract This article reviews the properties and uses of one class of popular membrane “fluidity probe”, namely molecules containing the fluorophore 1,6-diphenyl-1,3,5-hexatriene (DPH). In the first two sections, necessary concepts and basic principles of fluorescence polarization and the meaning of membrane “fluidity” are discussed. The primary purpose has been to evaluate the usefulness of different probes through a consideration of probe motion and location within the bilayer, probe photophysical properties, probe partitioning between bilayers or bilayer domains, and probe perturbation of bilayer structure. Finally, the review concludes with a description of the different types of information about membrane order that are provided by DPH and its two common derivatives, DPHpPC and TMA-DPH.

Use of fluorescent probes to monitor molecular order and motions within liposome bilayers

This article reviews the use of fluorescent probes to monitor the order and dynamics within the acyl chain region of liposome lipid bilayers. Fluorescence anisotropy is first defined and the theoretical framework that allows interpretation of steady-state or dynamic measurements in terms of molecular details is reviewed. The general advantages and/or limitations of fluorescent versus other methods of monitoring membrane order and dynamics are discussed. The properties of two classes of fluorescence probes are then described. The linear probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and parinaric acid (PA) and their derivatives are seen as particularly useful when quantitative interpretation of observations in terms of details of bilayer dynamics and order are critical. Of these, DPH is the more widely and easily used, although parinaric acid has advantages for certain applications. The non-linear probes considered include the anthroyloxyl fatty acids and the recently introduced fluorenyl fatty acid probes. While the geometry and electronic configurations of these probes do not allow for detailed molecular interpretations, these probes can provide unique qualitative information about the state of the lipid bilayer at various positions along the acyl chains.

Light-scattering effects in the measurement of membrane microviscosity with diphenylhexatriene

Data from several membrane systems are presented to confirm an empirical means of correcting diphenylhexatriene fluorescence for depolarization caused by sample turbidity. The depolarization proportionally constants obtained are not equal, but are shown to vary with (a) the physical state of the membrane, (b) the cholesterol content of the membrane, (c) the protein content of the membrane, and (d) the method of membrane preparation or isolation. It is concluded that depolarization corrections must always be considered when diphenylhexatriene fluorescence anisotropy is used to compare the fluidities within different membrane bilayers.

Protein machines and lipid assemblies: current views of cell membrane fusion

Protein machines and lipid bilayers both play central roles in cell membrane fusion, a process crucial to life. Recent results provide clues to how both components function in fusion. Recent observations suggest a common mechanism by which very different fusion machines (from lipid-enveloped viruses and synaptic vesicles) may function to produce compartment-joining pores. This mechanism presumes that fusion proteins act as machines that use stored conformational energy to assemble closely juxtaposed lipid bilayers, bend these to form fusion-competent structures, stabilize unfavorable lipid structures and destabilize a committed intermediate to drive fusion pore formation.

PEG as a tool to gain insight into membrane fusion

Thirty years ago, Klaus Arnold and others showed that the action of PEG in promoting cell–cell fusion was not due to such effects as surface absorption, cross-linking, solubilization, etc. Instead PEG acted simply by volume exclusion, resulting in an osmotic force driving membranes into close contact in a dehydrated region. This simple observation, based on a number of physical measurements and the use of PEG-based detergents that insert into membranes, spawned several important areas of research. One such area is the use of PEG to bring membranes into contact so that the role of different lipids and fusion proteins in membrane fusion can be examined in detail. We have summarized here insights into the fusion mechanism that have been obtained by this approach. This evidence indicates that fusion of model membranes (and probably cell membranes) occurs via severely bent lipidic structures formed at the point of sufficiently close contact between membranes of appropriate lipid composition. This line of research has also suggested that fusion proteins seem to catalyze fusion in part by reducing the free energy of hydrophobic interstices inherent to the lipidic fusion intermediate structures.

Polymer-induced membrane fusion: potential mechanism and relation to cell fusion events

Poly(ethylene glycol) (PEG) is used widely to mediate cell-cell fusion in the production of somatic cell hybrids and in the fusion injection of macromolecules into cultured cells from erythrocytes or liposomes. However, little is known about the mechanisms by which PEG induces fusion of cell membranes, making its use much more an art than a science. This article considers possible molecular events involved in biomembrane fusion and summarizes what we have learned about these in recent years from studies of fusion of well-defined model membranes. In addition, it recounts observations made over the past several years about the process of PEG-mediated fusion of model membranes. These observations have defined the process to an extent sufficient to allow us to propose a model for the molecular events involved in the process. It is suggested that dehydration leads to asymmetry in the lipid packing pressure in the two leaflets of the membrane bilayer leading to formation of a single bilayer septum at a point of close apposition of two membranes. The single bilayer septum then decays during formation of the initial fusion pore. Agents that enhance or alleviate the dehydration-induced asymmetric packing stress will favor or inhibit fusion. Although the proposed picture is consistent with much accumulated data, it is not yet proven; experiments must now be devised to test its details. Finally, the proposed model is discussed in terms of potential implications for the mechanisms available to a cell in controlling more complex in vivo cell fusion processes such as endocytosis, exocytosis, protein sorting/transport, and viral budding/infection.

Expression of coagulant activity in human platelets: release of membranous vesicles providing platelet factor 1 and platelet factor 3.

The relationship between the appearance of membrane-associated factor V-like activity (platelet factor 1, PF1) and phospholipid-like catalytic activity (platelet factor 3, PF3) has been examined, in vitro, in collagen-stimulated, human platelets. Both activities increased 7 fold upon collagen treatment relative to stirred controls. After sedimentation of stimulated platelets, 31% of total PF1 and 41% of PF3 remained in the supernatant fraction. PF1 eluted from a Sepharose CL-4B column in the same void volume fractions as PF3, phospholipid, and vesicular particles. These fractions had roughly 100 fold (lipid basis) or 1000 fold (protein basis) enhanced specific activity when compared to the stimulated platelet suspension. Freeze-fracture electron microscopy demonstrated that these void volume fractions contain two populations of membranous vesicles (80-200 nm and 400-600 nm in diameter). Upon centrifugation of the void volume fractions, PF1 and PF3 activities, phosphate-containing material, and ultraviolet-absorbing material all sedimented at the same rate, indicating that PF1 and PF3 are activities associated with one or both of the platelet-derived vesicle populations. Finally, we examined the effects of inhibitors on the appearance of PF1, PF3, platelet factor 4, total intrinsic factor V activity, and serotonin as well as on platelet aggregation. These studies suggest that the collagen-stimulated release of PF1 and PF3 is not coupled to either platelet aggregation or PF4 release but is probably a separate phase of the release reaction.

Large vesicle contamination in small, unilamellar vesicles

Small, unilamellar phospholipid vesicles have been prepared using a new, high-powdered cup sonifier that avoids contact of the sample with a titanium probe. These vesicles have been characterized by gel filtration chromatography both before and after fractionation by high-speed centrifugation. Plots of the turbidity of centrifuged vesicles between 300 and 650 nm against the reciprocal fourth power of the scattering wavelength were linear with zero intercepts (extrapolated to infinite wavelength). In the presence of minute quantities of large, multilamellar vesicles, these plots remained linear but had intercepts quantitatively proportional to the amount of contaminating large vesicles. Since this measurement requires only a standard spectrophotometer and very small quantities of lipid, this method is suggested as a useful assay for determining contamination of small vesicle preparations by large vesicles. Two applications of this method as well as a practical limitation are discussed.

Association of factor V activity with membranous vesicles released from human platelets: Requirement for platelet stimulation

The membrane-associated factor V-like activity (platelet factor 1, PF1) and the phospholipid-like catalytic surface activity (platelet factor 3, PF3) were studied in human platelets from normal and two factor V-deficient donors. Collagen stimulation or mechanical disruption of gel-filtered platelets was necessary for the expression of significant amounts of PF1 and PF3. Stimulation was also necessary for the uptake of factor V or Va by PF1-deficient platelets from the factor V-deficient donors. The activity of PF1 was also generated by association of factor V or Va with membrane-rich fractions obtained by gel filtration of the supernatant from collagen-stimulated or frozen-thawed PF1-deficient platelets. The amount of PF1 obtained by such all-or-none binding experiments was directly proportional to the amount of PF3 already expressed in the platelet preparation. These data have been summarized in terms of a hypothesis which views PF1 and PF3 to be activities associated with membranous vesicles released from platelets only after stimulation.

Soluble Phosphatidylserine Triggers Assembly in Solution of a Prothrombin-activating Complex in the Absence of a Membrane Surface

Factor Xa (FXa) binding to factor Va (FVa) on platelet-derived membranes containing surface-exposed phosphatidylserine (PS) forms the “prothrombinase complex” that is essential for efficient thrombin generation during blood coagulation. There are two naturally occurring isoforms of FVa, FVa1 and FVa2. These two isoforms differ by a 3-kDa polysaccharide chain (at Asn2181 in human FVa1 (Kim, S. W., Ortel, T. L., Quinn-Allen, M. A., Yoo, L., Worfolk, L., Zhai, X., Lentz, B. R., and Kane, W. H. (1999)Biochemistry 38, 11448–11454)) and have different coagulant activities. We examined the interaction of the two bovine isoforms with active site-labeled FXa, finding no significant difference. A soluble form of PS (C6PS) bound to FVa1 and FVa2 with comparable affinities (K d = 11–12 μm) and changes in FVa intrinsic fluorescence. At concentrations well below its critical micelle concentration, C6PS binding to bovine FVa2 enhanced its affinity for FXa in solution by nearly 3 orders of magnitude (K d eff = 40–2 nm over a C6PS range of 30–400 μm) but had no effect on the affinity of FVa1for FXa (K d = 1 μm). This results in a soluble complex between FXa and FVa2, whose expected molecular weight was confirmed by calibrated native gel electrophoresis. This complex behaved as a normal Michaelis-Menten enzyme in its ability to produce thrombin from meizothrombin (apparentk cat/K m ≅ 109 m −1 s−1). The ability of soluble PS to trigger formation of a soluble prothrombinase complex suggests that exposure of PS molecules during platelet activation is likely the key event responsible for the assembly of an active membrane-bound complex.