Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy.

Fluorescence resonance energy transfer (FRET) detects the proximity of fluorescently labeled molecules over distances >100 A. When performed in a fluorescence microscope, FRET can be used to map protein-protein interactions in vivo. We here describe a FRET microscopy method that can be used to determine whether proteins that are colocalized at the level of light microscopy interact with one another. This method can be implemented using digital microscopy systems such as a confocal microscope or a wide-field fluorescence microscope coupled to a charge-coupled device (CCD) camera. It is readily applied to samples prepared with standard immunofluorescence techniques using antibodies labeled with fluorescent dyes that act as a donor and acceptor pair for FRET. Energy transfer efficiencies are quantified based on the release of quenching of donor fluorescence due to FRET, measured by comparing the intensity of donor fluorescence before and after complete photobleaching of the acceptor. As described, this method uses Cy3 and Cy5 as the donor and acceptor fluorophores, but can be adapted for other FRET pairs including cyan fluorescent protein and yellow fluorescent protein.

Rapid cycling of lipid raft markers between the cell surface and Golgi complex.

The endocytic itineraries of lipid raft markers, such as glycosyl phosphatidylinositol (GPI)-anchored proteins and glycosphingolipids, are incompletely understood. Here we show that different GPI-anchored proteins have different intracellular distributions; some (such as the folate receptor) accumulate in transferrin-containing compartments, others (such as CD59 and GPI-linked green fluorescent protein [GFP]) accumulate in the Golgi apparatus. Selective photobleaching shows that the Golgi pool of both GPI-GFP and CD59-GFP constantly and rapidly exchanges with the pool of these proteins found on the plasma membrane (PM). We visualized intermediates carrying GPI-GFP from the Golgi apparatus to the PM and separate structures delivering GPI-GFP to the Golgi apparatus.GPI-GFP does not accumulate within endocytic compartments containing transferrin, although it is detected in intracellular structures which are endosomes by the criteria of accessibility to a fluid phase marker and to cholera and shiga toxin B subunits (CTxB and STxB, which are also found in rafts). GPI-GFP and a proportion of the total CTxB and STxB taken up into cells are endocytosed independently of clathrin-associated machinery and are delivered to the Golgi complex via indistinguishable mechanisms. Hence, they enter the Golgi complex in the same intermediates, get there independently of both clathrin and rab5 function, and are excluded from it at 20 degrees C and under conditions of cholesterol sequestration. The PM-Golgi cycling pathway followed by GPI-GFP could serve to regulate lipid raft distribution and function within cells.

High-resolution FRET microscopy of cholera toxin B-subunit and GPI-anchored proteins in cell plasma membranes.

"Lipid rafts" enriched in glycosphingolipids (GSL), GPI-anchored proteins, and cholesterol have been proposed as functional microdomains in cell membranes. However, evidence supporting their existence has been indirect and controversial. In the past year, two studies used fluorescence resonance energy transfer (FRET) microscopy to probe for the presence of lipid rafts; rafts here would be defined as membrane domains containing clustered GPI-anchored proteins at the cell surface. The results of these studies, each based on a single protein, gave conflicting views of rafts. To address the source of this discrepancy, we have now used FRET to study three different GPI-anchored proteins and a GSL endogenous to several different cell types. FRET was detected between molecules of the GSL GM1 labeled with cholera toxin B-subunit and between antibody-labeled GPI-anchored proteins, showing these raft markers are in submicrometer proximity in the plasma membrane. However, in most cases FRET correlated with the surface density of the lipid raft marker, a result inconsistent with significant clustering in microdomains. We conclude that in the plasma membrane, lipid rafts either exist only as transiently stabilized structures or, if stable, comprise at most a minor fraction of the cell surface.

Dynamics and retention of misfolded proteins in native ER membranes.

When co-translationally inserted into endoplasmic reticulum (ER) membranes, newly synthesized proteins encounter the lumenal environment of the ER, which contains chaperone proteins that facilitate the folding reactions necessary for protein oligomerization, maturation and export from the ER. Here we show, using a temperature-sensitive variant of vesicular stomatitis virus G protein tagged with green fluorescent protein (VSVG-GFP), and fluorescence recovery after photobleaching (FRAP), the dynamics of association of folded and misfolded VSVG complexes with ER chaperones. We also investigate the potential mechanisms underlying protein retention in the ER. Misfolded VSVG-GFP complexes at 40 degrees C are highly mobile in ER membranes and do not reside in post-ER compartments, indicating that they are not retained in the ER by immobilization or retrieval mechanisms. These complexes are immobilized in ATP-depleted or tunicamycin-treated cells, in which VSVG-chaperone interactions are no longer dynamic. These results provide insight into the mechanisms of protein retention in the ER and the dynamics of protein-folding complexes in native ER membranes.

Distribution of a glycosylphosphatidylinositol-anchored protein at the apical surface of MDCK cells examined at a resolution of <100 A using imaging fluorescence resonance energy transfer.

Membrane microdomains ("lipid rafts") enriched in glycosylphosphatidylinositol (GPI)-anchored proteins, glycosphingolipids, and cholesterol have been implicated in events ranging from membrane trafficking to signal transduction. Although there is biochemical evidence for such membrane microdomains, they have not been visualized by light or electron microscopy. To probe for microdomains enriched in GPI- anchored proteins in intact cell membranes, we used a novel form of digital microscopy, imaging fluorescence resonance energy transfer (FRET), which extends the resolution of fluorescence microscopy to the molecular level (<100 A). We detected significant energy transfer between donor- and acceptor-labeled antibodies against the GPI-anchored protein 5' nucleotidase (5' NT) at the apical membrane of MDCK cells. The efficiency of energy transfer correlated strongly with the surface density of the acceptor-labeled antibody. The FRET data conformed to theoretical predictions for two-dimensional FRET between randomly distributed molecules and were inconsistent with a model in which 5' NT is constitutively clustered. Though we cannot completely exclude the possibility that some 5' NT is in clusters, the data imply that most 5' NT molecules are randomly distributed across the apical surface of MDCK cells. These findings constrain current models for lipid rafts and the membrane organization of GPI-anchored proteins.

Range and magnitude of the steric pressure between bilayers containing phospholipids with covalently attached poly(ethylene glycol).

The interactive properties of liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are of interest because such liposomes are being developed as drug delivery vehicles and also are ideal model systems for measuring the properties of surface-grafted polymers. For bilayers containing PEG-lipids with PEG molecular weights of 350, 750, 2000, and 5000, pressure-distance relations have been measured by X-ray diffraction analysis of liposomes subjected to known applied osmotic pressures. The distance between apposing bilayers decreased monotonically with increasing applied pressure for each concentration of a given PEG-lipid. Although for bilayers containing PEG-350 and PEG-750 the contribution of electrostatic repulsion to interbilayer interactions was significant, for bilayers containing PEG-2000 and PEG-5000 the major repulsive pressure between bilayers was a steric pressure due to the attached PEG. The range and magnitude of this steric pressure increased both with increasing PEG-lipid concentration and PEG size, and the extension length of the PEG from the bilayer surface at maximum PEG-lipid concentration depended strongly on the size of the PEG, being less than 35 A for PEG-750, and about 65 A for PEG-2000 and 115 A for PEG-5000. The measured pressure-distance relations have been modeled in terms of current theories (deGennes, 1987; Milner et al., 1988b) for the steric pressure produced by surface-grafted polymers, as modified by us to take into account the effects of polymer polydispersity and the possibility that, at low grafting densities, polymers from apposing bilayers surfaces can interpenetrate or interdigitate. No one theoretical scheme is sufficient to account for all the experimental results. However, for a given pressure regime, PEG-lipid size, and PEG-lipid surface density, the appropriately modified theoretical treatment gives a reasonable fit to the pressure-distance data.

Structure and phase behavior of lipid suspensions containing phospholipids with covalently attached poly(ethylene glycol).

Liposomes containing phospholipids with covalently attached poly(ethylene glycol) (PEG-lipids) are being developed for in vivo drug delivery. In this paper we determine the structure and phase behavior of fully hydrated distearoylphosphatidylcholine (DSPC) suspensions containing PEG-lipids composed of distearoylphosphatidylethanolamine with attached PEGs of molecular weights ranging from 350 to 5000. For DSPC:PEG-lipid suspensions containing 0-60 mol % PEG-lipid, differential scanning calorimetry shows main endothermic transitions ranging from 55 to 64 degrees C, depending on the size of the PEG and concentration of PEG-lipid. The enthalpy of this main transition remains constant for all PEG-350 concentrations but decreases with increasing amounts of PEG-750, PEG-2000, or PEG-5000, ultimately disappearing at PEG-lipid concentrations greater than about 60 mol %. Low-angle and wide-angle x-ray diffraction show that tilted gel (L beta') phase bilayers are formed for all PEG-lipid molecular weights at concentrations of about 10 mol % or less, with the distance between bilayers depending on PEG molecular weight and PEG-lipid concentration. At PEG-lipid concentrations greater than 10 mol %, the lipid structure depends on the size of the PEG moiety. X-ray diffraction analysis shows that untilted interdigitated (L beta I) gel phase bilayers form with the incorporation of 40-100 mol % PEG-350 or 20-70 mol % PEG-750, and untilted gel (L beta) phase bilayers are formed in the presence of about 20-60 mol % PEG-2000 and PEG-5000. Light microscopy, turbidity measurements, x-ray diffraction, and 1H-NMR indicate that a pure micellar phase forms in the presence of greater than about 60% PEG-750, PEG-2000, or PEG-5000.

Colloid osmotic pressure of steer alpha- and beta-crystallins: possible functional roles for lens crystallin distribution and structural diversity.

This study addresses the general mechanisms whereby the major cytoplasmic proteins from the adult bovine lens contribute both to transparency and maintenance of the refractive index gradient across the lens. Colloid osmotic properties and quaternary structure were measured for alpha- and beta-crystallins isolated from the steer lens, including low-molecular-weight crystallins from the cortex (alpha Le and beta L) and nucleus (alpha Ln) and high-molecular-weight crystallins from the nucleus (alpha H and beta H). In electron microscopic images of rotary-shadowed preparations alpha Le appears as spherical particles 16 nm in diameter, alpha Ln appeared as individual spheres or small aggregates of spherical subunits, alpha H contained large irregular aggregates as large as 180 nm, and both beta L and beta H appeared as elliptical particles of 7-9 nm diameter. Secondary osmometry showed that for all these crystallins colloid osmotic pressure increased monotonically in a non-linear fashion with protein concentration. For the alpha-crystallins, osmotic pressure rose more steeply with concentration for alpha Le than for either alpha Ln or alpha H, so that at 0.3 g ml-1 at 0.1 M ionic strength, the colloid osmotic pressure of alpha Le, alpha Ln and alpha H were approximately 2.6 x 10(5) dyn cm-2, 1.6 x 10(5) dyn cm-2 and 1.0 x 10(5) dyn cm-2, respectively. In a similar manner, osmotic pressure rose more steeply with concentration of beta L than for beta H, so that at 0.3 g ml-1 at 0.1 M ionic strength the colloid osmotic pressures of beta L and beta H were 2.6 x 10(5) dyn cm-2 and 1.1 x 10(5) dyn cm-2, respectively. The osmotic pressure of alpha Le dropped as ionic strength was increased from 0.02 to 0.4 M. For beta L and beta H, osmotic pressure dropped as ionic strength was increased from 0.02 to 0.1 M but was nearly the same at 0.1 M and 0.4 M ionic strength. The data for steer alpha Ln and beta H were similar to previous reports for calf cortical alpha L and beta-crystallins, respectively. The osmotic pressure isotherms for alpha Le, beta L and that previously reported for steer cortical extract were nearly identical, whereas the nuclear crystallins (alpha Ln, alpha H or beta H) generated slightly higher pressures than those previously reported for steer nuclear crystallin extracts. In all cases, osmotic pressure rose more steeply with concentration for the cortical crystallins than for the nuclear crystallins.(ABSTRACT TRUNCATED AT 400 WORDS)

Intermolecular protein interactions in solutions of bovine lens beta L-crystallin. Results from 1/T1 nuclear magnetic relaxation dispersion profiles.

We report the magnetic field dependence of 1/T1 of solvent water protons and deuterons (nuclear magnetic relaxation dispersion, or NMRD, profiles) for solutions of steer lens beta L-crystallin. Such data allow the study of intermolecular protein interactions over a wide concentration range, here 1-34% vol/vol, by providing a measure of the rotational relaxation time of solute macromolecules. We conclude that, for approximately less than 5% protein, the solute particles are noncompact, with a rotationally averaged volume approximately three times that of a compact 60-kD sphere. (Earlier results for alpha-crystallin, approximately 1,000 kD, from optical and osmotic measurements (Vérétout and Tardieu, 1989. J. Mol. Biol. 205:713-728), show a similar, approximately twofold, effect). At intermediate concentrations, to approximately 20% protein, there is evidence for limited association or oligomerization, as found for the structurally related gamma II-crystallin (Koenig et al. 1990. Biophys. J. 57:461-469), to a limiting size about two-thirds that of alpha-crystallin. The difference in NMRD behavior of the three classes of crystallins is consonant with their differing osmotic properties (Vérétout and Tardieu. J. Mol. Biol. 1989, 205:713-728; Kenworthy, McIntosh, and Magid. Biophys. J. 1992. 61:A477; Tardieu et al. 1992. Eur. Biophys. J. 21:1-12). We indicate how the unusual structures and interactions of these three classes of proteins can be combined to optimize transparency and minimize colloid osmotic difficulties in eye lens.

Colloid osmotic pressure of steer crystallins: implications for the origin of the refractive index gradient and transparency of the lens.

The osmotic behavior of soluble cortical and nuclear steer lens crystallins was characterized by secondary osmometry for several ionic strength and pH conditions. Osmotic pressure versus protein concentration relationships were measured for pressures up to 1.15 x 10(6) dyn cm-2. At low concentrations (< 0.2 g ml-1), the osmotic pressure increased linearly with pressure, whereas for concentrations above 0.2 g ml-1, the pressure rose more sharply, giving progressively larger changes in osmotic pressure with increasing crystallin concentration. At a given ionic strength and applied osmotic pressure, the nuclear proteins attained a higher protein concentration than did the cortical proteins. For example, at the highest osmotic pressure of 1.15 x 10(6) dyn cm-2 at pH 7.6 and 0.1 M ionic strength, the observed protein concentrations were 0.43 g ml-1 for the cortical proteins and 0.52 g ml-1 for the nuclear proteins. For both cortical and nuclear steer crystallins, the pressure rose more steeply with concentration than do pressures for calf crystallins described in the literature. The impact of these developmental differences in osmotic pressure on lens transparency is discussed. Both the nuclear and cortical crystallins exhibited ionic strength-dependent shifts in their pressure-concentration behavior. At 0.02 M ionic strength, higher pressures were observed, whereas at 0.4 M ionic strength lower pressures were observed for a given protein concentration. The crystallins were also found to equilibrate to different protein concentrations at a constant osmotic pressure and 0.1 M ionic strength over a pH range of 4-9, with a maximum concentration around pH 5 for the cortical crystallins and pH 6 for the nuclear crystallins. Thus, the adult bovine cortical and nuclear soluble lens extracts are different in their osmotic properties, reflecting underlying differences in protein composition. The results of the ionic strength and pH experiments suggest that hard-sphere, electrostatic, and Donnan forces contribute to the total colloid osmotic pressure of the lens crystallins. However, near physiologic pH and ionic strength the charges of the proteins are screened to the extent that the colloid osmotic pressure exhibits only minor changes for large changes in ionic conditions. The differences in the osmotic behavior of the cortical and nuclear proteins are consistent with a model where regional variations in the colloid osmotic properties of the proteins across the lens help support the radial refractive index gradient that is present in vertebrate lenses. The importance of a radial concentration gradient of metabolites is also discussed.

The hydration pressure between lipid bilayers. Comparison of measurements using x-ray diffraction and calorimetry.

The hydration pressure between dipalmitoyl phosphatidyl-N,N-dimethylethanolamine (DPPE-Me2) bilayers has been analyzed by both x-ray diffraction measurements of osmotically stressed liposomes and by differential scanning calorimetry. By the x-ray method, we obtain a magnitude (Po) and decay length (lambda) for the hydration pressure which are both quite similar to those found for bilayers of other zwitterionic lipids, such as phosphatidylcholines. That is, x-ray analysis of DPPE-Me2 in the gel phase gives lambda = 1.3 A, the same as that previously measured for the analogous gel phase lipid dipalmitoylphosphatidylcholine (DPPC), and Po = 3.9 x 10(9) dyn/cm2, which is in excellent agreement with the value of 3.6 x 10(9) dyn/cm2 calculated from the measured Volta potential of DPPE-Me2 monolayers in equilibrium with liposomes. These results indicate that the removal of one methyl group to convert DPPC to DPPE-Me2 does not markedly alter the range or magnitude of the hydration pressure. Calorimetry shows that the main gel to liquid-crystalline phase transition temperature of DPPE-Me2 is approximately constant for water contents ranging from 80 to 10 water molecules per lipid molecule, but increases monotonically with decreasing water content below 10 waters per lipid. A theoretical fit to these temperature vs. water content data predicts lambda = 6.7 A. The difference in observed values of lambda for x-ray and calorimetry measurements can be explained by effects on the thermograms of additional intra- and intermolecular interactions which occur at low water contents where apposing bilayers are in contact. We conclude that, although calorimetry provides important data on the energetics of bilayer hydration, it is difficult to obtain quantitative information on the hydration pressure from this technique.