Mobility Of Membrane Proteins Research Articles

Electron paramagnetic resonance studies of proteins in ozone-treated erythrocyte membranes.

The sulfhydryl group specific spin labels have been used to study the membrane proteins in the bovine and human erythrocyte membranes treated with ozone. The ratio hw/hs, determined from the respective peak amplitudes of the resulting EPR spectra of 3-maleimido-1-oxyl-2,2,5,5-tetramethylpyrrolidine bound to the ozone treated membranes, was shown to be elevated following an increased ozone concentration. The ratio of the central to high-field peak amplitude h0/h-1, a measure of the rotational correlation time, determined from EPR spectra of ozonized membranes covalently labeled with 4-(iodoacetamido)-1-oxyl-2,2,5, 5-tetramethylpyrrolidine was shown to be diminished. The changes in the erythrocyte membranes treated with ozone, observed for both spin labels, indicate a dose dependent increase in the mobility of membrane proteins.

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139

The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the pateint. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.

Diffusional mobility of Golgi proteins in membranes of living cells.

The mechanism by which Golgi membrane proteins are retained within the Golgi complex in the midst of a continuous flow of protein and lipid is not yet understood. The diffusional mobilities of mammalian Golgi membrane proteins fused with green fluorescent protein from Aequorea victoria were measured in living HeLa cells with the fluorescence photobleaching recovery technique. The diffusion coefficients ranged from 3 x 10(-9) square centimeters per second to 5 x 10(-9) square centimeters per second, with greater than 90 percent of the chimeric proteins mobile. Extensive lateral diffusion of the chimeric proteins occurred between Golgi stacks. Thus, the chimeras diffuse rapidly and freely in Golgi membranes, which suggests that Golgi targeting and retention of these molecules does not depend on protein immobilization.

Probing the mobility of membrane proteins inside the cell

Studies using a variety of microscopy-based approaches have led to a consensus that most cell-surface proteins are highly mobile and diffuse rapidly within fenced microdomains. Little attention, however, has so far been given to the analysis of the mobility of intracellular membrane proteins because of their comparative inaccessibility. Recent advances in microinjection, confocal microscopy and the construction of epitope-tagged proteins or of hybrids with an intrinsicallyfluorescent protein have allowed intracellular membrane proteins to be studied using approaches previously applied to characterize the mobility of cell-surface proteins. Confocal fluorescence recovery after photobleaching (c-FRAP) experiments show that intracellular membrane proteins may also be highly mobile.

Immune interferon gamma inhibits translational mobility of a plasma membrane protein in preimplantation stage mouse embryos: A T-helper 1 mechanism for immunologic reproductive failure

OBJECTIVE: Immune interferon gamma adversely affects mouse embryo development and has been proposed as a mediator of reproductive failure involving T-helper 1 immunity. We hypothesized that one mechanism by which interferon gamma could exert an adverse effect on embryos was by altering plasma membrane organization and transmembrane protein mobility. STUDY DESIGN: The fluorescence photobleaching recovery technique was used to measure the effect of the T-helper 1 cytokine interferon gamma on the translational mobility of a specific embryonic surface glycoprotein recognized by the monoclonal antibody S75. RESULTS: Compared with controls interferon gamma significantly decreased the fractional mobility of fluorescein isothiocyanate S75 in one- and two-cell mouse embryos. CONCLUSION: Interferon gamma may alter plasma membrane domains or cytoskeletal organization in early-stage embryos. By restricting plasma membrane protein mobility interferon gamma could effect T-helper 1 - mediated reproductive failure. (A M J O BSTET G YNECOL 1996;174:282-7.)

Lateral mobility of FcγRIIa is reduced by protein kinase C activation

The lateral mobility of membrane proteins can reflect the extent of various protein-protein interactions. Using the fluorescence recovery after photobleaching technique, we have studied the lateral mobility of human FcγRIIa and some FcγRIIa mutants expressed in either P388D1 cells, a mouse macrophagelike cell line, or in Chinese hamster ovary (CHO) cells [1]. After treatment with phorbol myristate acetate (PMA), only the FcγRIIa molecules capable of mediating rapid endocytosis of immune complexes exhibited a reduced lateral diffusion coefficient with respect to untreated controls. Wild type FcγRIIa expressed in CHO cells, and nonfunctional FcγRIIa mutants expressed in P388D1 cells did not show any differences upon PMA treatment. This finding suggests that protein kinase C activation evokes additional protein-protein interactions with the cytoplasmic domain of functional FcγRIIa, which reduced receptor lateral mobility. The identity of these putative interacting proteins and the nature of the interactions remain to be elucidated.

Immobilization of nicotinic acetylcholine receptors in mouse C2 myotubes by agrin-induced protein tyrosine phosphorylation.

Agrin induces the formation of highly localized specializations on myotubes at which nicotinic acetylcholine receptors (AChRs) and many other components of the postsynaptic apparatus at the vertebrate skeletal neuromuscular junction accumulate. Agrin also induces AChR tyrosine phosphorylation. Treatments that inhibit tyrosine phosphorylation prevent AChR aggregation. To examine further the relationship between tyrosine phosphorylation and receptor aggregation, we have used the technique of fluorescence recovery after photobleaching to assess the lateral mobility of AChRs and other surface proteins in mouse C2 myotubes treated with agrin or with pervanadate, a protein tyrosine phosphatase inhibitor. Agrin induced the formation of patches in C2 myotubes that stained intensely with anti-phosphotyrosine antibodies and within which AChRs were relatively immobile. Pervanadate, on the other hand, increased protein tyrosine phosphorylation throughout the myotube and caused a reduction in the mobility of diffusely distributed AChRs, without affecting the mobility of other membrane proteins. Pervanadate, like agrin, caused an increase in AChR tyrosine phosphorylation and a decrease in the rate at which AChRs could be extracted from intact myotubes by mild detergent treatment, suggesting that immobilized receptors were phosphorylated and therefore less extractable. Indeed, phosphorylated receptors were extracted from agrin-treated myotubes more slowly than nonphosphorylated receptors. AChR aggregates at developing neuromuscular junctions in embryonic rat muscles also labeled with anti-phosphotyrosine antibodies, suggesting that tyrosine phosphorylation could mediate AChR aggregation in vivo as well. Thus, agrin appears to induce AChR aggregation by creating circumscribed domains of increased protein tyrosine phosphorylation within which receptors become phosphorylated and immobilized.

Carbohydrate-induced modulation of cell membrane—III. Interaction of sialic acid and mannose with hamster splenic lymphocytes: A spin label study

The role of different carbohydrates as ligands for adhesion molecules has been extensively studied. However, the physiological changes in the splenic lymphocytes on binding these ligands have never been studied. In this paper, we report that binding of sialic acid or mannose to hamster splenic lymphocytes restricts the mobility of membrane proteins and lipids as studied by EPR spectroscopy using spin probes. Binding of mucin and heparin totally restricts the mobility probably due to crosslinking of the surface lectins. Binding of these ligands also results in an increase in the viscosity of the cytoplasm.

Modification of fluid lipid and mobile protein fractions of reticulocyte plasma membranes affects agonist-stimulated adenylate cyclase. Application of the percolation theory

The technique of fluorescence recovery after photobleaching was used to measure the lateral mobility of membrane integral proteins in reticulocyte plasma membranes which were treated to modify the ‘fluid’ lipid or immobilized protein fractions, hence increasing the relative prevalence of obstacles to protein lateral motion. This was achieved by either: (1) treating the plasma membranes with phospholipase A 2 followed by extraction of the hydrolysis products using fatty-acid-free bovine serum albumin, resulting in a decrease in the membrane ‘fluid’ lipid portion; or (2) preincubating the plasma membranes with polylysines, resulting in plasma membrane protein aggregation and immobilization. As the prevalence of obstacles to lateral motion increased in plasma membranes through the treatments described above, the mobility of the membrane integral proteins diminished. Experimental results for the dependence of protein mobility on the prevalence of obstacles to lateral motion were compared to theoretical data in order to verify the applicability of the percolation theory to reticulocyte plasma membranes. The influence of a decrease in the ‘fluid’ lipid and an increase in the immobilized membrane protein fractions upon the hormone-stimulated adenylate cyclase activity has been studied as well. As the ‘solid’ lipid and immobilized membrane protein fractions decreased, both the hormone-stimulated adenylate cyclase activity and the fraction of β-adrenergic receptors with high affinity to hormone diminished. It was shown that this correlation can be caused by a decrease in membrane fraction accessible to the movement of the interacting proteins of the adenylate cyclase complex. Hormonal stimulation of adenylate cyclase is discussed in terms of the percolation theory.

Lateral mobility of plasma membrane proteins in dividing eggs of the loach (Misgurnus fossilis): Regional differences and changes during the cell cycle

Regional differences in lateral diffusion rates of fluorescence-labeled proteins have been studied in the plasma membrane of dividing eggs of the loach (Misgurnus fossilis) by fluorescence recovery after photobleaching (FRAP). Apparent animal-vegetal differences in fluorescence intensity, lateral diffusion coefficients, and fractions of mobile proteins have been found, with all these quantities being higher in the animal pole region than in the yolk region. Cyclic changes in protein diffusion coefficients and mobile fractions during the first few cell cycles have also been recorded. Soon after the end of a cleavage, the diffusion coefficient reaches its minimal value and increases rapidly before the next cleavage.

Evidence for a relationship between oxidative stress and insulin action in non-insulin-dependent (type II) diabetic patients

Ten healthy subjects and 30 non-insulin-dependent (type II) diabetic patients matched for age, gender ratio, body mass index, lean body mass (LBM), waist to hip ratio, and arterial blood pressure volunteered for the study. In all subjects, fasting plasma free radical (O 2 −) levels and basal membrane lipid fluidity (MLF) and protein mobility (MPM) were determined. The whole group of subjects underwent a euglycemic hyperinsulinemic glucose clamp with simultaneous indirect calorimetry for substrate oxidation determination. Diabetic patients versus controls displayed higher fasting plasma glucose (8.3 ± 0.4 v 5.1 ± 0.4 mmol/L, P ± .001), O 2 − (0.48 ± 0.02 v 0.16 ± 0.02 μmol/L × min), and hemoglobin A 1c ([HbA 1c] 7.9% ± 0.4% v 5.7% ± 0.3%, P < .03) levels and a stronger reduction in basal MLF (0.243 ± 0.006 v 0.318 ± 0.009, P < .003) and basal MPM (0.348 ± 0.003 v 0.518 ± 0.010, P < .002). Whole-body glucose disposal (WBGD) and oxidative and nonoxidative glucose metabolism were also significantly lower in diabetics than in controls. In diabetic patients (n = 30), plasma O 2 − levels correlated with basal MLF ( r = −.59, P < .005), basal MPM ( r = −.84, P < .001), fasting plasma insulin level ( r = .51, P < .004), WBGD ( r = −.53, P < .002), and nonoxidative ( r = −.45, P < .01) glucose metabolism. In conclusion, our results demonstrate that a relationship between plasma O 2 − levels and insulin action occurs in non-insulin-dependent diabetics.

Cytosolic calcium ions regulate lipid mobility in the plasma membrane of the human megakaryoblastic cell line MEG-01.

The fluidity of the plasma membrane is thought to play a role in the activation of blood platelets. We investigated the lateral diffusion of the lipophilic probe 1,1'-ditetradecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiIC14) and derivatives in the plasma membrane of the megakaryoblast MEG-01 by fluorescence recovery after photobleaching. The lateral diffusion coefficient (D) of DiIC14 in an unstimulated cell was (3.53 +/- 0.06) x 10(-9) cm2/s with a mobile fraction of 75%. Similar data were found with DiIC12 and DiIC18, but lipophilic probes specific for the outer leaflet showed a slower diffusion with a D value of (2.99 +/- 0.31) x 10(-9) cm2/s and a mobile fraction of 58%. Stimulation with platelet-activating agents decreased the diffusion of DiIC14 within 2 min, but left the mobile fraction unchanged. Signal processing was required for the decrease in D as D-Phenylalanyl-L-prolyl-L-arginyl-chloromethane-treated thrombin, which binds normally to the thrombin receptor but fails to activate the cell, had no effect. The decrease in D was accompanied by an increase in cytosolic Ca2+ content, [Ca2+]i, and studies using different concentrations of thrombin, the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethylester and the Ca2+ ionophore ionomycin revealed that lipid mobilty in the plasma membrane is regulated by Ca2+. In contrast, treatments thought to interfere with the mobility of membrane proteins had little effect. We conclude that the rigidification of the plasma membrane during cell activation is caused by an increase in [Ca2+]i and is therefore a late event and might only contribute to signal transduction at steps downstream of the mobilization/influx of Ca2+.

Aging and the liver: fucntional aspects

The drastic decline in the function of the hepatic microsomal cytochrome monooxygenase system, initially reported in male rat livers, was shown to be due to a feminization of male rat livers with aging. In female rat livers as well as in mouse livers, this system was found to stay unchanged with age. Phase II reactions which showed some decline with aging in male rat livers again stayed fairly stable with age in female rat and mouse livers. Glutathione S-transferase (GST) enzyme activities, which are very stable with age in female rat and mouse livers, demonstrated highly age-dependent changes when dietary conditions were manipulated, suggesting a potential age difference in the homeostatic regulation of this enzyme system. Using the fluorescence recovery after photobleaching (FRAP) technique, unique studies revealed an age-dependent decline in the lateral mobility of proteins in hepatocyte surface membranes. The protease inhibitor model of aging, initially proposed by Ivy for brain cells, has been validated in hepatocytes, demonstrating an accumulation of lipofuscin-like granules in young animals treated with i.p. infusion of leupeptin for only 2 weeks. Antioxidant enzyme activities such as superoxide dimutase (SOD) and catalase (CAT) in the liver were clearly demonstrated no to be reduced in general terms with aging. Rather, a clear increase in CAT enzyme activities with age was demonstrated in female rat livers, thus challenging the concept that intracellular enzyme activities generally decline with aging. In this paper, studies performed in Japan on aging and the liver over the past 30 years, with a focus on its functional aspects, are critically reviewed in terms of the clinical implications of these studies as well as on theories of aging in general.

The intracellular mobility of a viral membrane glycoprotein measured by confocal microscope fluorescence recovery after photobleaching.

Fluorescence recovery after photobleaching (FRAP) has been a powerful tool for characterizing the mobility of cell surface membrane proteins. However, the application of FRAP to the study of intracellular membrane proteins has been hampered by the lack of specific probes and their physical inaccessibility in the cytoplasm. We have measured the mobility of a model transmembrane protein, the temperature-sensitive vesicular stomatitis viral membrane glycoprotein (ts-O45-G), in transit from the endoplasmic reticulum (ER) to the Golgi complex. ts-O45-G accumulates in the ER at nonpermissive temperature (39.5 degrees C) and is transported via the Golgi complex to the surface upon shifting cells to the permissive temperature (31 degrees C). Rhodamine-labeled Fab fragments against a cytoplasmic epitope of ts-O45-G (rh-P5D4-Fabs) were microinjected into cells to visualize the intracellular viral membrane protein and to determine its mobility by FRAP with a confocal microscope. Moreover, we have measured the effects of microinjected antibodies against beta-COP on the mobility of ts-O45-G following release of the temperature block. FRAP was essentially complete when rh-P5D4-Fab-injected cells were bleached either following release of labeled ts-O45-G from the ER or upon its accumulation at 20 degrees C in the trans-Golgi network (TGN). In contrast, recovery was reduced by about one third when infected cells had been injected with antibodies that bind to beta-COP in vivo. The diffusion constant of mobile ts-O45-G under all conditions was approximately 10 x 10(-10) cm2/s. These results validate the feasibility of FRAP for the study of an intracellular transmembrane protein and provide the first evidence that such a protein is highly mobile.

The effect of caloric restriction on the lateral diffusion constant of hepatocyte membrane proteins in C57BL/6 male mice of different ages: FRAP studies on liver smears

The lateral mobility of proteins in hepatocyte plasma membranes was compared in calorically restricted and ad libitum (AL)-fed C57BL/6 male mice in age groups from 7 to 28 months. Caloric restriction was achieved by means of the every-other-day (EOD) feeding regimen, maintained for various periods from 1 to 15 months. Protein lateral diffusion constant (D) in hepatocyte membranes was measured by means of fluorescence recovery after photobleaching (FRAP) in liver smears. The peroxide-induced autofluorescence (PIAF) was utilized as a fluorescent label. A mild (1 mM for 10 min) H 2O 2 treatment of liver smears produces oxidation of riboflavin that is bound to all proteins of the cell membrane. Using this technique, the average lateral diffusion constant (D) and the fractional recovery (FR) of these proteins can be measured. EOD feeding resulted in a significant decrease in body weights and also a significant increase in the values of D in all age groups after 1 month of EOD feeding. After 3.5 months of EOD feeding a further increase of D was observed (up to about 15%). Nevertheless no further change in D occurred if the EOD feeding was maintained for 6.5 or even 15 months. The negative linear age correlation of D observed in the AL-fed animals was present also under the EOD feeding; however, the whole regression equation shifted towards higher values. These experiments indicate that caloric restriction influences the lateral diffusion constant of membrane proteins in hepatocytes. The results are interpreted as a result of an increased protein turnover caused by the caloric restriction.

Protein lateral mobility as a reflection of membrane microstructure

The lateral mobility of membrane lipids and proteins is presumed to play an important functional role in biomembranes. Photobleaching studies have shown that many proteins in the plasma membrane have diffusion coefficients at least an order of magnitude lower than those obtained when the same proteins are reconstituted in artificial bilayer membranes. Depending on the protein, it has been shown that either the cytoplasmic domain or the ectodomain is the key determinant of its lateral mobility. Single particle tracking microscopy, which allows the motions of single or small groups of membrane molecules to be followed, promises not only to reveal new features of membrane dynamics, but also to help explain longstanding puzzles presented by the photobleaching studies, particularly the so-called immobile fraction. The combination of the two complementary technologies should measurably enhance our understanding of membrane microstructure.

Nitroxides as metabolic and EPR imaging probes in biological model systems

Nitroxides are unusually stable free radicals and sensitive “reporters” of the chemico-physical environment which surround them. The term “reporter group” was used to indicate this property, later the term spin label or spin probe was preferred. The facile synthesis and the multiplicity of compounds which have been coupled to the nitroxide moiety has allowed the development of a number of applications. Nitroxides have been used to study molecular mobility of proteins and lipids in membranes, enzyme active sites, DNA synthesis, and measure of electrical potential, pH, temperature as well as oxygen gradients across membranes; more recently, as contrast agents in magnetic resonance imaging and electron paramagnetic resonance imaging (EPRI). Aim of this work is to review and comment on the use of various nitroxides as metabolic probes, and in EPRI in studies of biomedical interest.

Relationship between membrane lipid mobility and spectrin distribution in lymphocytes

We have previously established that T and B lymphocytes in situ are remarkably heterogeneous with respect to the cytoskeletal protein spectrin. Since in erythrocytes spectrin is known to play an important role in the regulation of membrane fluidity, lipid organization and lateral mobility of membrane proteins, we have sought to determine if the heterogeneous patterns of spectrin distribution that we have observed are related to possible differences in membrane lipid organization in these various subsets. To this end, we have utilized a fluorescent pyrene-labelled phospholipid as a probe orthe lipid lateral mobility and have examined two related T cell systems maintained in vitro, DO.11.10 cells and a spontaneously arising variant, DO.11.10V. In these (and other cloned in vitro systems) we have previously observed that the cells homogeneously express one of the kinds of spectrin distribution patterns observed in situ. Thus the uniformity of staining of these systems permits us to address whether the various patterns of spectrin distribution may be predictive of differences in membrane lipid properties. Here we show that in cells in which there is little or no spectrin at the plasma membrane (DO.11.10) that the lipids in the plasma membrane are considerably less mobile than in its related variant in which spectrin is diffusely distributed within the cell and at the plasma membrane. From this and previous results, we conclude that differences in the distribution of the cytoskeletal protein spectrin among lymphocytes may be a useful parameter in helping to predict the status of membrane lipid organization.

Cytoskeletal elements regulate the distribution of nerve growth factor receptors in PC12 cells.

Nerve growth factor receptor (NGFR)-like immunoreactivity (IR) was studied in PC12 cells treated for 96 hr with NGF (40 ng/ml), using immunogold labeling and electron microscopic morphometric analysis. The cells were exposed to the anti-NGFR antibody 192-IgG, followed by immunoglobulin (IgG) conjugated with colloidal gold. PC12 cells exhibited occasional gold label (positive NGFR-IR) on all surfaces. Cells treated with colcemid (0.05 micrograms/ml) or cytochalasin D (2 micrograms/ml), which limit microtubule (MT) and microfilament (MF) formation, respectively, displayed an increased NGFR-IR in terms of gold labeling. NGFR-IR was also seen on taxol (0.85 micrograms/ml)-exposed cells, an agent that promotes MT assembly. Cells treated simultaneously with cytochalasin D and taxol had a dramatically augmented NGFR-IR on their surfaces, which exceeded levels obtained with either agent alone. Prominent NGFR-IR was localized frequently in coated endocytotic vesicles, in smooth endoplasmic reticulum, and in secondary multivesicular lysosomes, in both treated and untreated cells. The results suggest that a large number of NGFRs (positive NGFR-IR) in PC12 cells are cryptic and not available for ligand binding. Changes in cytoskeletal organization that may affect mobility of integral membrane proteins can modulate the distribution of NGFR-IR on neuronal surfaces.

Effect of leupeptin on the lateral mobility of proteins in the plasma membrane of hepatocytes of C57BL/6 mice: FRAP studies on liver smears

Four groups of male C57BL/6 mice (ages 2, 3.8, 11.8–12.5 and 24 months) were administered leupeptin at 5 mg/100 g body weight/day via intraperitoneally implanted osmotic minipumps. Both untreated and saline-treated mice served as controls. The time and dose-dependence of the effects of leupeptin on the lateral mobility of proteins in hepatocyte surface membranes were analyzed first in mice treated for 2, 5, 8, 11 and 14 days, respectively, by using the fluorescence recovery after photobleaching (FRAP) technique. The age dependence of the response to this treatment was also studied after 14 days of treatment in mice of various ages. The average lateral diffusion constant (D) and the fractional recovery (FR) were measured, and D × FR calculated. Leupeptin treatment at a daily dose of 5 mg/100g for 2 weeks increased plasma glutamic-pyruvlc transaminase levels 2-fold in all age groups. Leupeptin treatment caused a linear increase of D and a decrease of FR with respect to the duration of the treatment in adult mice. In all age groups about 30% of the membrane proteins became immobile after 14 days of treatment, whereas the still mobile fraction displayed a large increase of D. The values of D × FR in the leupeptin-treated groups were slightly higher than those in the untreated mice until the age of 15 months but decreased after this age. The results are interpreted in terms of the known inhibitory effect of leupeptin on cytoplasmic and lysosomal thiol proteases, causing a general slowing down of the protein turnover and, specifically, of proteins in the hepatocyte membrane.