Phenotypic changes in the fluidity of the tonoplast membrane of crassulacean-acid-metabolism plants in response to temperature and salinity stress

Electron paramagnetic resonance-spectroscopic studies on spin-labeled purified tonoplast membranes showed that in the obligate crassulacean-acid-metabolism (CAM) plant Kalanchoë daigremontiana Hamet et Perr. the fluidity of the tonoplast decreased during acclimation to higher temperatures. This phenotypic change in tonoplast fluidity was paralleled by a decrease in the mobilization of malic acid from the vacuoles during CAM in the light. The shift from the C₃ to the CAM mode of photosynthesis in the facultative CAM plant Mesembryanthemum crystallinum L. also led to a decrease in the fluidity of the tonoplast membrane. The results are consistent with the hypothesis that the ability to store malic acid during CAM in the vacuoles depends largely on the actual fluidity of the tonoplast membrane.     

Metabolic control of the membrane fluidity in Bacillus subtilis during cold adaptation

Membrane fluidity adaptation to the low growth temperature in Bacillus subtilis involves two distinct mechanisms: (1) long-term adaptation accomplished by increasing the ratio of anteiso- to iso-branched fatty acids and (2) rapid desaturation of fatty acid chains in existing phospholipids by induction of fatty acid desaturase after cold shock. In this work we studied the effect of medium composition on cold adaptation of membrane fluidity. Bacillus subtilis was cultivated at optimum (40 °C) and low (20 °C) temperatures in complex medium with glucose or in mineral medium with either glucose or glycerol. Cold adaptation was characterized by fatty acid analysis and by measuring the midpoint of phospholipid phase transition T_m (differential scanning calorimetry) and membrane fluidity (DPH fluorescence polarization). Cells cultured and measured at 40 °C displayed the same membrane fluidity in all three media despite a markedly different fatty acid composition. The T_m was surprisingly the highest in the case of a culture grown in complex medium. On the contrary, cultivation at 20 °C in the complex medium gave rise to the highest membrane fluidity with concomitant decrease of T_m by 10.5 °C. In mineral media at 20 °C the corresponding changes of T_m were almost negligible. After a temperature shift from 40 to 20 °C, the cultures from all three media displayed the same adaptive induction of fatty acid desaturase despite their different membrane fluidity values immediately after cold shock.     

Modulation of serotonin binding in rat brain by membrane fluidity

Preincubation of rat brain homogenates with increasing concentrations of n-hexanol reduced specific serotonin (5-HT) binding and increased membrane fluidity as measured by fluorescence depolarization using 1,6-diphenyl-1,3,5-hexatriene (DPH) as a probe. At 5 mM ascorbate maximal reductions of both membrane fluidity and specific 5-HT binding were observed. Both effects were enhanced in the presence of ferrous sulphate and oxygen. In the presence of ascorbate (5.7 mM) only one 5-HT binding site was observed in contrast with high and low affinity binding sites (K_D1 = 0.08 ± 0.04 nM, K_D2 = 28.8 ± 1.3 nM) found in the absence of ascorbate. The ascorbate induced decrease of specific 5-HT binding may be explained by lipid peroxidation, which decreases membrane fluidity, and by ascorbate's reducing properties. Since different correlations were found between membrane fluidity and specific 5-HT binding depending upon the presence of ascorbate or n-hexanol, the results suggest that membrane fluidity is a critical factor, however, just one of several determinants in 5-HT binding studies.     

Viscosity Landscape of Phase-Separated Lipid Membrane Estimated from Fluid Velocity Field

In cell membranes, the functional constituents such as peptides, proteins, and polysaccharides diffuse in a sea of lipids as single molecules and molecular aggregates. Thus, the fluidity of the heterogeneous multicomponent membrane is important for understanding the roles of the membrane in cell functionality. Recently, Henle and Levine described the hydrodynamics of molecular diffusion in a spherical membrane. A tangential point force at the north pole induces a pair of vortices whose centers lie on a line perpendicular to the point force and are symmetrical with respect to the point force. The position of the vortex center depends on η_m/Rη_w, where R is the radius of the spherical membrane, and η_m and η_w are the viscosities of the membrane and the surrounding medium, respectively. Based on this theoretical prediction, we applied a point force to a phase-separated spherical vesicle composed of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine/cholesterol by means of a microinjection technique. The pathlines were visualized by trajectories of microdomains. We determined the position of the vortex center and estimated the membrane viscosity using the dependence of the position of the vortex center on η_m/Rη_w. The obtained apparent membrane viscosities for various compositions are mapped on the phase diagram. The membrane viscosity is almost constant in the range of 0 < ϕ_Lo ≤ 0.5 (ϕ_Lo: area fraction of the liquid ordered phase), whereas that in the range of 0.5 ≤ ϕ_Lo < 1.0 exponentially increases with increase of ϕ_Lo. The obtained viscosity landscape provides a basic understanding of the fluidity of heterogeneous multicomponent membranes.     

Temperature- and pH-Induced Structural Changes in the Membrane of the Hyperthermophilic Archaeon Aeropyrum pernix K1

The influence of pH and temperature on the structural organization, fluidity and permeability of the hyperthermophilic archaeon membrane was investigated in situ by a combination of electron paramagnetic resonance (EPR) and fluorescence emission spectroscopy. For EPR measurements, Aeropyrum pernix cells, after growing at different pHs, were spin-labeled with the doxyl derivative of palmitic acid methylester (MeFASL[10,3]). From the EPR spectra maximal hyperfine splitting (2A _max) and empirical correlation time (τ_emp), which are related to mean membrane fluidity, were determined. The mean membrane fluidity increases with temperature and depends on the pH of the growth medium. Computer simulation of the EPR spectra shows that membrane of A. pernix is heterogeneous and consists of the regions characterized with three different types of motional characteristics, which define three types of membrane domains. Order parameter and proportion of the spin probes in the three types of domains define mean membrane fluidity. The fluidity changes of the membrane with pH and temperature correlate well with the ratio between the fluorescence emission intensity of the first and third bands in the vibronic spectra of pyrene, I₁/I₃. At pH 7.0 a decrease of I₁/I₃ from 2.0 to 1.2, due to the penetration of pyrene into the nonpolar membrane region, is achieved at temperatures above 65°C, the lower temperature limit of A. pernix growth.     

The fluidity of chloroplast thylakoid membranes and their constituent lipids: A comparative study by ESR

Comparative measurements were made of the fluidity of chloroplast thylakoids, total membrane lipids and polar lipids utilizing the order parameter and motion of spin labels.No significant differences were found in the fluidity of membranes or total membrane lipids from a wild type and a mutant barley (Hordeum vulgare chlorina f₂ mutant) which lacks chlorophyll $\text{b}$ and a 25 000 dalton thylakoid polypeptide. Redistribution of intrinsic, exoplasmic face (EF) membrane particles by unstacking thylakoid membranes in low salt medium also had no effect on membrane fluidity. However, heating of isolated thylakoids decreased membrane fluidity.The fluidity of vesicles composed of membrane lipids is much greater than that of the corresponding membranes. Fluidity of the membranes, however, increased during greening indicating that the rigidity of the membranes, compared with that of total membrane lipids, is not caused by chlorophyll or its associated peptides. It is concluded that the restriction of motion in the acyl chains in the thylakoids is not caused by chlorophyll or the major intrinsic polypeptide but by some other protein components.     

Spin label studies of microsomal membranes from Acanthamoeba castellanii in different states of differentiation

Two fatty acid spin labels—[I(1,14)], stearic acid bearing a paramagnetic nitroxide group on carbon 16, and [I(12,3)], stearic acid bearing a paramagnetic nitroxide group on carbon 5—have been used to compare the physical properties of lipid in rough and smooth microsomal membranes from trophozoites and cysts of Acanthamoeba castellanii. Arrhenius plots of rotational correlation times (τ_c) calculated from the spectra for I(1,14) showed an abrupt discontinuity in slope for membranes from both trophozoites and cysts. This occurred at temperatures ranging from ?3 to 1 °C for smooth microsomes and from 8 to 11 °C for rough microsomes for both cysts and amoebae. The value of τ_c at 29 °C, the culturing temperature, in effect scores fluidity of the membrane matrix, and did not show any significant difference for either rough or smooth microsomes during the transition from exponential to stationary phase growth. However, smooth microsomes from cysts showed a 14% increase in fluidity relative to trophozoites, and the fluidity of rough microsomes from cysts tended to be lower. An order parameter (S) calculated from spectra for I(12,3) did not change as a function of encystment for the smooth membranes and increased only slightly for rough microsomes. The activation energy (E_a) for Arrhenius plots of τ_c above the inflection temperature increased as a result of encystment, indicating a greater degree of molecular interaction within the cyst membranes. Moreover, the τ_c plots for both rough and smooth microsomal membranes from trophozoites tended to converge at 29 °C, the growth temperature, whereas plots for cyst membranes were virtually parallel, bracketing those for the trophozoite membranes. This suggests that the trophozoite is able to regulate its membrane fluidity and that cysts, which are resting cells, have lost this regulatory capacity.     

Effect of oxidative stress on plasma membrane fluidity of THP-1 induced macrophages

Plasma membrane is one of the preferential targets of reactive oxygen species which cause lipid peroxidation. This process modifies membrane properties such as membrane fluidity, a very important physical feature known to modulate membrane protein localization and function. The aim of this study is to evaluate the effect of oxidative stress on plasma membrane fluidity regionalization of single living THP-1 macrophages. These cells were oxidized with H₂O₂ at different concentrations, and plasma membrane fluidity was analyzed by two-photon microscopy in combination with the environment-sensitive probe Laurdan. Results show a significant H₂O₂ concentration dependent increase in the frequency of rigid lipid regions, mainly attributable to lipid rafts, at the expense of the intermediate fluidity regions. A novel statistical analysis evaluated changes in size and number of lipid raft domains under oxidative stress conditions, as lipid rafts are platforms aiding cell signaling and are thought to have relevant roles in macrophage functions. It is shown that H₂O₂ causes an increase in the number, but not the size, of raft domains. As macrophages are highly resistant to H₂O₂, these new raft domains might be involved in cell survival pathways.     

Electron spin resonance studies of changes in membrane fluidity of Chinese hamster ovary cells during the cell cycle

Electron spin resonance (ESR) spin-label methods were used with 5-doxyl-stearic acid as a probe to investigate membrane fluidity of Chinese hamster ovary (CHO) cells during the cell cycle. A 35 GHz ESR technique was developed to study membrane fluidity of intact cells. This technique requires only about $\text{1}\text{6}$ the amount of cells compared to the conventional spin-label techniques. With this technique we observed a cyclic change of membrane fluidity during the cell cycle of CHO cells: cells in mitosis had the highest membrane fluidity, whereas cells in G₁ and early S phases had the lowest membrane fluidity.     

Factors altering the membrane fluidity of spinach thylakoid as determined by fluorescence polarization

Alterations in fluidity of thylakoid membranes isolated from spinach chloroplasts in response to sodium bisulfite (NaHSO₃), hydrogen peroxide (H₂O₂), sodium dodecyl sulfate (SDS), bovine serum albumin (BSA), and free linoleic acid (LA) were investigated by means of a fluorescence polarization study with 1,6-diphenyl-1,3,5-hexatriene as the fluorescence probe. A decrease in fluidity and an increase in microviscosity of membrane were caused by NaHSO₃ and H₂O₂ treatment. In contrast, SDS and BSA were found to increase thylakoid membranes fluidity and decrease microviscosity, in which the corresponding correlation coefficients were −0.9995 to −0.9516 (SDS) and −0.9359 (BSA), respectively. No changes in thylakoid membranes fluidity induced by free LA were found until its concentration above 5 mM where the polarization value (P value) declined (increased fluidity). The results suggest that the changes in thylakoids membrane fluidity might depend on the characteristics, mechanism and extent of the interactions between membrane components and compounds added.     

Lipidomic adaptations of the Metarhizium robertsii strain in response to the presence of butyltin compounds

Metarhizium robertsii, a butyltin-resistant filamentous fungus, can rapid and complete biodegradation of di- (DBT) and tributyltin (TBT) under conditions of intensive aeration and ascorbic acid supplementation. In this paper, lipidomic investigations were performed to find the membrane adaptations necessary for effective butyltins degradation. HPLC-MS/MS analysis showed that the phospholipid profile was greatly modified during M. robertsii batch cultivation (pO₂?≥?20%), contributing to increased membrane fluidity and facilitated mass transfer, which could enhance butyltins biodegradation. Intensified biosynthesis of phospholipids, sphingolipids and ergosterol by the mycelia exposed to butyltins was noted. DIOC₆(3) fluorescence intensity for TBT-treated mycelium increased 9-fold pointing to membrane hyperpolarization. Fluorescent studies showed improved membrane rigidity and integrity in response to butyltins presence. Vitamin C supplementation restored membrane composition and dynamic properties, followed by supposed acceleration of transport of monobutyltin and its biodegradation thus protecting the M. robertsii cells against oxidative and nitrosative stress.     

Bactericidal activity of amphipathic cationic antimicrobial peptides involves altering the membrane fluidity when interacting with the phospholipid bilayer

BackgroundAmphipathic cationic antimicrobial peptides (AMPs) TC19 and TC84, derived from the major AMPs of human blood platelets, thrombocidins, and Bactericidal Peptide 2 (BP2), a synthetic designer peptide showed to perturb the membrane of Bacillus subtilis. We aimed to determine the means by which the three AMPs cause membrane perturbation in vivo using B. subtilis and to evaluate whether the membrane alterations are dependent on the phospholipid composition of the membrane.MethodsPhysiological analysis was employed using Alexa Fluor 488 labelled TC84, various fluorescence dyes, fluorescent microscopy techniques and structured illumination microscopy.ResultsTC19, TC84 and BP2 created extensive fluidity domains in the membrane that are permeable, thus facilitating the entering of the peptides and the leakage of the cytosol. The direct interaction of the peptides with the bilayer create the fluid domains. The changes caused in the packing of the phospholipids lead to the delocalization of membrane bound proteins, thus contributing to the cell's destruction. The changes made to the membrane appeared to be not dependent on the composition of the phospholipid bilayer.ConclusionsThe distortion caused to the fluidity of the membrane by the AMPs is sufficient to facilitate the entering of the peptides and leakage of the cytosol.General significanceHere we show in vivo that cationic AMPs cause “membrane leaks” at the site of membrane insertion by altering the organization and fluidity of the membrane. Our findings thus contribute to the understanding of the membrane perturbation characteristic of cationic AMPs.     

Correlations between the thermal stability of chloroplast (thylakoid) membranes and the composition and fluidity of their polar lipids upon acclimation of the higher plant,Nerium oleander,to growth temperature

The thermal stability of excitation transfer from pigment proteins to the Photosystem II reaction center of Nerium oleander adjusts by 10 Celsius degrees when cloned plants grown at 20°C/15°C, day/night growth temperatures are shifted to 45°C/32°C growth temperature or vice versa. Concomitant with this adjustment is a decrease in the fluidity of thylakoid membrane polar lipids as determined by spin labeling. The results are consistent with the hypothesis that there is a limiting maximum fluidity compatible with maintenance of native membrane structure and function. This limiting fluidity was about the same as for a number of other species which exhibit a range of thermal stabilities. Inversely correlated shifts in lipid fluidity and thermal stability occurred during the time course of acclimation of N. oleander to new growth temperatures. Thus, the temperature at which the limiting fluidity was reached changed during acclimation while the limiting fluidity remained constant. Although the relative proportion of the major classes of membrane polar lipids remained constant during adjustments in fluidity, large changes occured in the abundance of specific fatty acids. These changes were different for the phospho- and galacto-lipids suggesting that the fatty acid composition of these two lipid classes is regulated by different mechanisms. Comparisons between membrane lipid fluidity and fatty acid composition indicate that fluidity is not a simple linear function of fatty acid composition.     

Effects of prostaglandins e(2) and f(2alpha) on the electrofusion of pea mesophyll protoplasts

The effects of prostaglandins E₂ and F_2α on the electrofusion of pea (Pisum sativum cv Ran 1) mesophyll protoplasts were examined. Prostaglandins E₂ and F_2α influenced electrofusion by lowering the threshold voltage necessary for fusion of dielectrophoretically arranged pairs of protoplasts. The direct current voltage threshold decreased with increasing Ca²⁺ concentration up to 0.1 millimolar CaCl₂ and the effects of prostaglandins E₂ and F_2α were more pronounced when CaCl₂ was present in the medium. Treatment with calcium channel blocker methoxy verapamil did not change the prostaglandin effects, while the addition of ethyleneglycol-bis (β-aminoethyl either)-N,N,N′,N′-tetraacetic acid, which binds free Ca²⁺, increased the threshold voltage. Influence of prostaglandins E₂ and F_2α and Ca²⁺ on the membrane fluidity was investigated by analysis of pyrene fluorescence spectra. The values of the ratio between the maximum fluorescence emission intensities of the excimer and the monomer forms (I_ex/I_mon) indicated that prostaglandins and Ca²⁺ decrease the membrane fluidity. It is proposed that electrically evoked displacement of plasmalemma components takes part in the fusion process (U Zimmermann 1982 Biochim Biophys Acta 694: 227-277). We suggest that prostaglandins E₂ and F_2α facilitate the electrofusion of pea mesophyll protoplasts by changing the fluidity of plasmalemma.     

Effect of Richinus communis lectins on the membrane fluidity of human peripheral lymphocytes

The mobility of Ricinus communis lectins bound to lymphocyte cell surface was determined by fluorescence polarization of fluorescein-labeled lectins. R. communis hemagglutinin and R. communis toxin have high mobility. Furthermore, the change of membrane fluidity upon binding of the lectins to lymphocytes was measured by fluorescence polarization of fluorescent hydrocarbon embedded in the membrane. The hemagglutinin, the toxin and its binding subunit apparently increased the membrane fluidity. The hemagglutinin was also found to have mitogenic activity against human peripheral lymphocytes.     

Alterations in Photosystem II Organization by Cobalt Treatment in the Cyanobacterium Spirulina piatensis

The presence of low concentrations of cobalt ions (200 μMCoCl₂) retarded the growth of the cyanobacterium Spirulina platensis. However, cells grown in the presence of CoCl₂ exhibited an enhancement in the levels of phycocyanin and carotenoids, while showed a decrease in the content of chlorophyll a, when compared to the untreated control cells. Thylakoid membranes isolated from CoCl₂ treated cells demonstrated enhanced rates of both photosystem II (H₂O → pBQ) and photosystem I (DCPIPH₂ → MV) mediated electorn transport activities. The stimulation in the rates of photosynthetic electron transport activities in the CoCl ₂ grown cells are attributed to the alterations occurring in the thylakoid membrane organization, as the thylakoids from CoCl₂ grown cells possessed more membrane fluidity. The decrease in the chlorophyll a content may have occurred due to modifications in the chlorophyll a complexes in this cynaobacterium. The enhancement in the phycocyanin levels in the CoCl₂ grown cells appears to be because of the stimulated induction of heme oxygenase. Thus, the cobalt ion treatment appears to be useful in studying the thylakoid membrane and antennae organizations in cyanobacteria.     

Fluidity of natural membranes and phosphatidylserine and ganglioside dispersions: Effects of local anesthetics,cholesterol and protein

The microviscosity of artificial lipid membranes and natural membranes was measured by the fluorescence polarization technique employing perylene as the probe. Lipid dispersions composed of brain gangliosides exhibited greater microviscosity than phosphatidylserine (268 cP vs 173 cP, at 25 °C). Incorporation of cholesterol (30–50%) increased the microviscosity of lipid phases by 200–500 cP. Cholesterol's effect on membrane fluidity was completely reversed by digitonin but not by amphotericin B. Incorporation of membrane proteins into lipid vesicles gave varying results. Cytochrome b₅ did not alter membrane fluidity. However, myelin proteolipid produced an apparent increase in microviscosity, but this effect might be due to partitioning of perylene between lipid and protein binding sites since the latter have a higher fluorescence anisotropy than the lipid. The local anesthetics tetracaine and butacaine increased the fluidity of lipid dispersions, natural membranes and intact ascites tumor cell membranes. The effect of the anesthetics appears to be due to an increased disordering of lipid structure. The fluidity of natural membranes at the 25 °C varied as follows:polymorphonuclear leukocytes, 335 cP; bovine brain myelin, 270 cP; human erytherocyte, 180 cP; rat liver microsomes, 95 cP; rat liver mitochondria, 90 cP. In most cases the microviscosity of natural membranes reflects their cholesterol : phospholipid ratio. The natural variations in fluidity of cellular membranes probably reflect important fuctional requirements. Similarly, the effects of some drugs which alter membrane permeability may be the result of their effects on membrane fluidity.     

Membrane fatty acid composition and fluidity are involved in the resistance to freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175

Determinations of membrane fatty acid composition and fluidity were used together with acidification activity and viability measurements to characterize the physiological state after freezing of Lactobacillus buchneri R1102 and Bifidobacterium longum R0175 cells harvested in the exponential and stationary growth phases. For both strains, lower membrane fluidity was achieved in cells harvested in the stationary growth phase. This change was linked to a lower unsaturated-to-saturated fatty acid ratio for both strains and a higher cyclic-to-saturated fatty acid ratio for L. buchneri R1102 alone. These membrane properties were linked to survival and to maintenance of acidification activity of the cells after freezing, which differed according to the strain and the growth phase. Survival of B. longum R0175 was increased by 10% in cells with low membrane fluidity and high relative saturated fatty acid contents, without any change in acidification activity. Acidification activity was more degraded (70 min) in L. buchneri R1102 cells displaying low membrane fluidity and high saturated and cyclic fatty acid levels. Finally, this study showed that membrane modifications induced by the growth phase differed among bacterial strains in terms of composition. By lowering membrane fluidity, these modifications could be beneficial for survival of B. longum R0175 during the freezing process but detrimental for maintenance of acidification activity of L. buchneri R1102.     

Eubacterium cellulosolvens Alters its Membrane Protein,Lipoprotein, and Fatty Acid Composition in Response to Growth on Cellulose

The cellulolytic bacterium, Eubacterium cellulosolvens, altered its cytoplasmic membrane protein composition in response to growth on specific energy substrates. Electrophoresis profiles obtained from membrane protein fractions of cellulose-grown cells were different from that obtained from cells cultivated with other carbohydrates, such as cellobiose or glucose. In addition, [³H]palmitic acid labelling of cellulose-grown E. cellulosolvens revealed two lipoproteins that were not detected in glucose- or cellobiose-grown cultures. These lipoproteins partitioned with the membrane fraction, indicating their association with the cytoplasmic membrane. Proteinase K treatment of whole cells further suggested that these lipoproteins were exposed to the surface of the cell envelope. These membrane proteins and lipoproteins appear to be under some substrate-specific regulatory control with distinct, but as yet undetermined, roles in cellulose utilization. In addition, cellulose-grown E. cellulosolvens was found to posses a higher ratio of oleic acid (C_18:1) to palmitic acid (C_16:0) than cells cultivated on soluble carbohydrates. This change in the ratio of unsaturated to saturated fatty acids was consistent with a comparative increase of membrane fluidity. Further analysis of this shift in the fatty acid profile revealed a correlation with the appearance of protruberances on the cell surface. Such a shift of fatty acid composition may indicate that the assembly and function of proteins for cellulose utilization necessitates an increase of the membrane fluidity.     

Membrane association of the PTEN tumor suppressor: molecular details of the protein-membrane complex from SPR binding studies and neutron reflection

The structure and function of the PTEN phosphatase is investigated by studying its membrane affinity and localization on in-plane fluid, thermally disordered synthetic membrane models. The membrane association of the protein depends strongly on membrane composition, where phosphatidylserine (PS) and phosphatidylinositol diphosphate (PI(4,5)P₂) act pronouncedly synergistic in pulling the enzyme to the membrane surface. The equilibrium dissociation constants for the binding of wild type (wt) PTEN to PS and PI(4,5)P₂ were determined to be K_d∼12 µM and 0.4 µM, respectively, and K_d∼50 nM if both lipids are present. Membrane affinities depend critically on membrane fluidity, which suggests multiple binding sites on the protein for PI(4,5)P₂. The PTEN mutations C124S and H93R show binding affinities that deviate strongly from those measured for the wt protein. Both mutants bind PS more strongly than wt PTEN. While C124S PTEN has at least the same affinity to PI(4,5)P₂ and an increased apparent affinity to PI(3,4,5)P₃, due to its lack of catalytic activity, H93R PTEN shows a decreased affinity to PI(4,5)P₂ and no synergy in its binding with PS and PI(4,5)P₂. Neutron reflection measurements show that the PTEN phosphatase “scoots" along the membrane surface (penetration <5 Å) but binds the membrane tightly with its two major domains, the C2 and phosphatase domains, as suggested by the crystal structure. The regulatory C-terminal tail is most likely displaced from the membrane and organized on the far side of the protein, ∼60 Å away from the bilayer surface, in a rather compact structure. The combination of binding studies and neutron reflection allows us to distinguish between PTEN mutant proteins and ultimately may identify the structural features required for membrane binding and activation of PTEN.