Winchil L. C. Vaz

FAPP2, cilium formation, and compartmentalization of the apical membrane in polarized Madin–Darby canine kidney (MDCK) cells

We have analyzed the role of the phosphatidylinositol-4-phosphate adaptor protein-2 (FAPP2), a component of the apical transport machinery, in cilium formation in polarized Madin–Darby canine kidney (MDCK) cells. We show that ciliogenesis is defective in FAPP2 knockdown cells. Furthermore, by using fluorescence recovery after photobleaching studies of domain connectivity and the generalized polarization spectra of Laurdan, we demonstrate that FAPP2 depletion impairs the formation of condensed apical membrane domains. Laurdan staining also revealed that the ciliary membrane has a highly condensed bilayer domain at its base that could function as a fence to separate the ciliary membrane from the surrounding apical membrane. These results indicate that the compartmentalization of the apical membrane in MDCK cells into the ciliary membrane and the surrounding membrane depends on the balance of raft and nonraft domains.

Lateral diffusion of lipids and proteins in bilayer membranes

Lipid bilayers Lateral diffusion Membrane proteins Fluorescent probes Photobleaching

Determination of the critical micelle concentration of surfactants using the fluorescent probe N-phenyl-1-naphthylamine

The fluorescence quantum yield of N-phenyl-1-naphthylamine (NPN) increases about 10-fold and the wavelength of maximum fluorescence emission is blue-shifted when this molecule partitions into the apolar core of micellar structures from the aqueous phase. This property allowed the utilization of NPN as a fluorescent indicator of micelle formation by 14 different surfactants belonging to the families of alkyltrimethylammonium halides, alkylsulfates, alkylbetaines, alkylglucosides, and bile salts. The critical micelle concentrations (CMCs) determined with NPN agreed well with literature values. In this work NPN was used at a concentration of 10(-6) M which allowed determination of CMCs in the range between approximately 10(-5) and greater than 10(-2) M. With high-sensitivity instrumentation considerably lower NPN concentrations can be used and consequently considerably lower CMCs can be rapidly and accurately determined.

Translational diffusion and fluid domain connectivity in a two-component, two-phase phospholipid bilayer.

The two-dimensional connectivity is examined for mixed bilayers of dimyristoyl phosphatidylcholine (DMPC) and distearoyl phosphatidylcholine (DSPC) as a function of composition and temperature at constant pressure using the fluorescence recovery after photobleaching (FRAP) method. These phospholipid mixtures exhibit peritectic behavior with a large region in which both gel and liquid crystalline phases coexist. Dilauroyl phosphatidylethanolamine covalently linked through the amino function in its head group to the fluorescent nitrobenzodiazolyl group (NBD-DLPE) was used as the fluorescent probe in this study, because it was found to partition almost exclusively in the liquid crystalline phase. The results of these studies show the line of connectivity to be close to the liquidus line on the phase diagram over a rather broad range of concentrations. In this range, a gel phase comprising approximately 20% of the system disconnects a liquid crystalline phase comprising 80% of the system. The implications of this result are discussed for domain shape and the organization of biological membrane components.

Percolation and diffusion in three-component lipid bilayers: effect of cholesterol on an equimolar mixture of two phosphatidylcholines

The lateral diffusion of a phospholipid probe is studied in bilayers of binary mixtures of dimyristoylphosphatidylcholine (DMPC)/cholesterol and distearoylphosphatidylcholine (DSPC)/cholesterol and in the ternary system DMPC/DSPC/cholesterol using fluorescence recovery after photobleaching. An approximate phase diagram for the ternary system, as a function of temperature and cholesterol concentration, was obtained using differential scanning calorimetry and the phase diagrams of the binary systems. This phase diagram is similar to those of the phospholipid/cholesterol binary mixtures. In bilayers where solid and liquid phases coexist, the diffusion results are interpreted in terms of phase percolation. The size of the liquid-phase domains is estimated using percolation theory. In the ternary system, addition of cholesterol up to approximately 20 mol% shifts the percolation threshold to lower area fractions of liquid, but the size of the liquid-phase domains does not change. Above approximately 20 mol% cholesterol, the liquid phase is always connected. The size of solid-phase domains clusters is estimated using a model recently developed (Almeida, P.F.F., W.L.C. Vaz, and T.E. Thompson. 1992. Biochemistry. 31:7198-7210). For cholesterol concentrations up to 20 mol%, the size of solid-phase domain units does not change. Beyond 20 mol%, cholesterol causes the size of the solid units to decrease.

Investigation of human erythrocyte ghost membranes with intramolecular excimer probes

Human erythrocyte ghost membranes have been investigated using two intramolecular excimer probes, di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether. Values for the viscosity of the direct probe environment in the ghost membranes range from 76 cP at 37 degrees C to 570 cP at 5 degrees C, as reported for di(1-pyrenyl(propane, with liquid paraffin as the reference solvent. For the activation energy of the excimer formation process, determined here mainly by the viscosity of the medium, a value of 37 kJ/mol is obtained. The other probe molecule reports a higher local viscosity, 133 cP at 37 degrees C, as well as a higher activation energy of excimer formation, 54 kJ/mol. Neither thermotropic phase transitions nor temperature hysteresis effects are observed within the temperature range (0 to 40 degrees C) studied. From the vibrational structure of the fluorescence spectrum of di(1-pyrenylmethyl) ether, a polarity of the probe environment close to that of hexanol (epsilon - 13.3) results for the erythrocyte ghost membranes. The polarity measured in egg phosphatidylcholine membranes and in multibilayers of dimyristoylphosphatidylcholine is slightly larger, comparable to that of butanol (epsilon = 17.5), whereas a polarity comparable to that of methanol (epsilon = 32.7) is observed for aqueous micellar solutions of sodium dodecyl sulphate. Further, from the wavelength shifts in the absorption spectrum of di(1-pyrenyl)propane and di(1-pyrenylmethyl) ether, the polarizability of the probe surroundings can be determined, leading to a surprisingly high value for the apparent refractive index. This is attributed to a high local density of the direct environment of the probe, for which a location between the membrane/water interface and the unpolar bilayer mid-plane is deduced.

Phase topology and percolation in multi-phase lipid bilayers: is the biological membrane a domain mosaic?: Current Opinion in Structural Biology 1993, 3:482–488

Abstract The recent literature provides strong evidence that phase separations do occur in biological membranes. Phase co-existence, as microscopic domains, raises important questions with regard to domain connectivity, phase percolation, long-range translational diffusion, lateral segregation of membrane components and efficiency of bimolecular reactions that occur in membranes. It also provides a basis for understanding several documented structural and dynamic aspects of this important cellular structure. It is suggested that changes between different phase ‘landscapes’ in a membrane may function as switching mechanisms for physiologically important events in a cell.

Rotational and translational diffusion in membranes measured by fluorescence and phosphorescence methods.

Publisher Summary A lipid bilayer provides a compartmentalized matrix within which embedded proteins and other macromolecules are in constant states of motion and redistribution—the processes that are essential to cellular mechanisms, such as metabolism, endocytosis and secretion, differentiation, locomotion, and signal transduction. A lipid bilayer in natural and reconstituted membranes imposes constraints that restrict the larger segmental and global motions of constituent molecules to the microsecond to millisecond time domains. This chapter discusses methods for analyzing such rotational and translation displacements based on the time-resolved emission of light in the form of fluorescence and phosphorescence. These techniques offer the sensitivity and selectivity required for the studies of relatively sparse cell-surface receptors or other membrane components in preparations examined in a microscope and in a suspension.

REVERSIBLE ADSORPTION AND NONREVERSIBLE INSERTION OF ESCHERICHIA COLI ALPHA -HEMOLYSIN INTO LIPID BILAYERS

Alpha-Hemolysin is an extracellular protein toxin (107 kDa) produced by some pathogenic strains of Escherichia coli. Although stable in aqueous medium, it can bind to lipid bilayers and produce membrane disruption in model and cell membranes. Previous studies had shown that toxin binding to the bilayer did not always lead to membrane lysis. In this paper, we find that alpha-hemolysin may bind the membranes in at least two ways, a reversible adsorption and an irreversible insertion. Reversibility is detected by the ability of liposome-bound toxin to induce hemolysis of added horse erythrocytes; insertion is accompanied by an increase in the protein intrinsic fluorescence. Toxin insertion does not necessarily lead to membrane lysis. Studies of alpha-hemolysin insertion into bilayers formed from a variety of single phospholipids, or binary mixtures of phospholipids, or of phospholipid and cholesterol, reveal that irreversible insertion is favored by fluid over gel states, by low over high cholesterol concentrations, by disordered liquid phases over gel or ordered liquid phases, and by gel over ordered liquid phases. These results are relevant to the mechanism of action of alpha-hemolysin and provide new insights into the membrane insertion of large proteins.

Dehydration of the lipid-protein microinterface on binding of phospholipase A2 to lipid bilayers

A novel method is described to demonstrate inaccessibility to the bulk aqueous phase of the microinterface between pig pancreatic phospholipase A2 and lipid bilayers to which this protein is bound. The method is based on the fact that the fluorescence emission quantum yields of the tryptophan residue of the protein and of a 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) chromophore attached to a lipid are lower in water as compared to that in deuterated water. The fluorescence emission quantum yield of these chromophores is measured in water and in deuterated water under conditions where the protein is either bound or not bound to the surface of a lipid bilayer containing the dansyl chromophore. Under conditions where the protein is tightly bound to the surface of the bilayer, desolvation of both fluorophores abolishes the observed effect of deuterated water. The tryptophan residue in the bound phospholipase A2 also becomes inaccessible to fluorescence quenching by acrylamide or succinimide. Desolvation of the microinterface is observed only under conditions that are significant for the catalytic action of phospholipase A2 in the scooting mode and not in the hopping mode. Also, under similar conditions, binding of pro-phospholipase A2 to anionic vesicles does not cause dehydration of the microinterface. The mechanistic significance of these observations for lipid-protein interactions, in general, and for interfacial catalysis and interfacial activation, in particular, is discussed.