Fluorescence Photobleaching Recovery Technique Research Articles

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.

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.)

A mechanistic role for polypeptide hormone receptor lateral mobility in signal transduction

Lateral diffusion of membrane-integral receptors within the plane of the membrane has been postulated to be mechanistically important for signal transduction. Direct measurement of polypeptide hormone receptor lateral mobility using fluorescence photobleaching recovery techniques indicates that tyrosine kinase receptors are largely immobile at physiological temperatures. This is presumably due to their signal transduction mechanism which requires intermolecular autophosphorylation through receptor dimerization and thus immobilization for activation. In contrast, G-protein coupled receptors must interact with other membrane components to effect signal transduction, and consistent with this, the phospholipase C-activating vasopressin V1- and adenylate cyclase activating V2-receptors are highly laterally mobile at 37°C. Modulation of the V2-receptor mobile fraction (f) has demonstrated a direct correlation between f and receptor-agonist-dependent maximal cAMP productionin vivo at 37°C. This indicates that f is a key parameter in hormone signal transduction especially at physiological hormone concentrations, consistent with mobile receptors being required to effect V2-agonist-dependent activation of G-proteins. Measurements using a V2-specific antagonist show that antagonist-occupied receptors are highly mobile at 37°C, indicating that receptor immobilization is not the basis of antagonism. In contrast to agonist-occupied receptor however, antagonistoccupied receptors are not immobilized prior to endocytosis and down-regulation. Receptors may thus be freely mobile in the absence of agonistic ligand; stimulation by hormone agonist results in receptor association with other proteins, probably including cytoskeletal components, and immobilization. Receptor immobilization may be one of the important steps of desensitization subsequent to agonistic stimulation, through terminating receptor lateral movement which is instrumental in generating and amplifying the initial stimulatory signal within the plane of the membrane.

Intracellular mediators regulate CD2 lateral diffusion and cytoplasmic Ca2+ mobilization upon CD2-mediated T cell activation

CD2 is a T cell surface glycoprotein that participates in T cell adhesion and activation. These processes are dynamically interrelated, in that T cell activation regulates the strength of CD2-mediated T cell adhesion. The lateral redistribution of CD2 and its ligand CD58 (LFA-3) in T cell and target membranes, respectively, has also been shown to affect cellular adhesion strength. We have used the fluorescence photobleaching recovery technique to measure the lateral mobility of CD2 in plasma membranes of resting and activated Jurkat T leukemia cells. CD2-mediated T cell activation caused lateral immobilization of 90% of cell surface CD2 molecules. Depleting cells of cytoplasmic Ca2+, loading cells with dibutyric cAMP, and disrupting cellular microfilaments each partially reversed the effect of CD2-mediated activation on the lateral mobility of CD2. These intracellular mediators apparently influence the same signal transduction pathways, because the effects of the mediators on CD2 lateral mobility were not additive. In separate experiments, activation-associated cytoplasmic Ca2+ mobilization was found to require microfilament integrity and to be negatively regulated by cAMP. By directly or indirectly controlling CD2 lateral diffusion and cell surface distribution, cytoplasmic Ca2+ mobilization may have an important regulatory role in CD2 mediated T cell adhesion.

Microtubule inhibitors differentially affect translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 cells

We used quantitative fluorescence microscopy and fluorescence photobleaching recovery techniques to investigate the translational movement, cell surface expression, and endocytosis of transferrin receptors in K562 human erythroleukemia cells. Receptors were labeled with fluorescein-conjugated transferrin (FITC-Tf). Coordinated decreases in surface fluorescence counts, the photobleaching parameter K, and transferrin receptor fractional mobility were observed as FITC-Tf was cleared from the cell surface by receptor-mediated endocytosis. Based on the kinetics of decrease in these parameters, first order rate constants for FITC-Tf uptake at 37 degrees C and 21 degrees C were calculated to be 0.10-0.15 min-1 and 0.02-0.03 min, respectively. K562 cells were treated with colchicine or vinblastine to investigate the role of microtubules in transferrin receptor movement and endocytosis. Treatment of cells for 1 hr with a microtubule inhibitor prevented transferrin receptor endocytosis but had no effect on the translational mobility of cell surface receptors. In contrast, drug treatment for 3 hr caused translational immobilization of cell surface receptors as well as inhibition of endocytosis. These effects were not produced by beta-lumicolchicine, an inactive colchicine analog, or by cytochalasin, a microfilament inhibitor. The effect of microtubule inhibitors on transferrin receptor mobility was reversed by pretreating cells with taxol, a microtubule-stabilizing agent. Microtubule inhibitors had no effect on the translational mobility of cell surface glycophorins or phospholipids, indicating that intact microtubules were not required for translational movement of these molecules. We conclude that the translational movement of cell surface transferrin receptors is directed by a subpopulation of relatively drug-resistant microtubules. In contrast, transferrin receptor endocytosis depends on a subpopulation of microtubules that is relatively sensitive to the action of inhibitors. These results appear to demonstrate at least two functional roles for microtubules in receptor-mediated transferrin uptake in K562 cells.

Use of the Translational Mobility of a Plasma Membrane Protein to Assess Fertilization of Mouse Oocytes and Viability of Mouse Zygotes and Two-Cell Embryos1

The fluorescence photobleaching recovery (FPR) technique was used to measure the translational mobility of a glycoprotein recognized by the monoclonal antibody (mAb) S75 in plasma membranes of mouse oocytes, zygotes, and two-cell embryos. Glycoprotein fractional mobility (f) was significantly decreased in membranes of unfertilized oocytes compared to zygotes or two-cell embryos (f values, 46 +/- 2 and 65 +/- 2%, respectively; p < 0.0001). Reduced apparent glycoprotein mobility was also observed in morphologically degenerated zygotes and two-cell embryos compared to viable zygotes and two-cell embryos (f values, 8 +/- 1 and 60 +/- 3%, respectively; p < 0.0001). These results indicate that the FPR technique can be used to assess oocyte fertilization and preimplantation embryonic viability. This method may be useful in the evaluation of embryonic viability following in vitro fertilization and in the detection of toxic effects of novel compounds on embryonic development.

Deoxygenation affects fluorescence photobleaching recovery measurements of red cell membrane protein lateral mobility

We have used the fluorescence photobleaching recovery technique to study the dependence on oxygen tension of the lateral mobility of fluorescently labeled band 3, the phospholipid analogue fluorescein phosphatidylethanolamine, and glycophorins in normal red blood cell membranes. Band 3 protein and sialic acid moieties on glycophorins were labeled specifically with eosin maleimide and fluorescein thiosemicarbazide, respectively. The band 3 diffusion rate increased from 1.7 x 10(-11) cm2 s-1 to 6.0 x 10(-11) cm2 s-1 as oxygen tension was decreased from 156 to 2 torr, and a further increase to 17 x 10(-11) cm2 s-1 occurred as oxygen tension was decreased from 2 to 0 torr. The fractional mobility of band 3 decreased from 58 to 32% as oxygen tension was decreased from 156 to 0 torr. The phospholipid diffusion coefficient remained constant as oxygen tension was decreased from 156 to 20 torr, but increased from 2.3 x 10(-9) cm2 s-1 to 7.1 x 10(-9) cm2 s-1 as oxygen tension was decreased from 20 to 0 torr. Neither the diffusion coefficient nor the fractional mobility of glycophorins changed significantly at low oxygen tension. Under non-bleaching excitation conditions, intensities of fluorescence emission were identical for oxygenated and deoxygenated eosin-labeled RBCs. Deoxygenated eosin-labeled RBCs required 160-fold greater laser intensities than did oxygenated RBCs to achieve comparable extents of photobleaching, however. Oxygen seems to act as a facilitator of fluorophore photobleaching and may thereby protect the fluorescently labeled red cell membrane from photodamage. Removal of oxygen may allow excited state fluorophores in close proximity to the plasma membrane to react with neighboring proteins or lipids during photobleaching. This effect has important implications for the ability of the fluorescence photobleaching recovery technique to report accurate lateral mobilities of cell membrane molecules under hypoxic conditions.

Membrane changes in lipopolysaccharide-stimulated murine B lymphocytes associated with cell activation

The lateral diffusion of the fluorescent lipid analog 3,3′-dioctadecylindocarbocyanine iodide (DiI) was measured in the membranes of murine B lymphocytes treated with the B cell mitogen lipopolysaccharide (LPS). The mobility of DiI, as measured by fluorescence photobleaching recovery (FPR) techniques, was temperature-dependent with a value of 6.1 · 10 −9 cm 2 s −1 at 37°C. Untreated cells exhibited this diffusion coefficient over 72 h in culture. In contrast, DiI mobility decreased to 2.0 · 10 −9 cm 2s −1 at 37°C in membranes of LPS-stimulated lymphocytes 24 h following LPS exposure. Interestingly, this decreased lipid lateral diffusion was not accompanied by any change in surface immunoglobulin lateral diffusion which remained essentially unchanged at 3.6-4.3 · 10 −11 cm 2s −1 over 72 h. To determine whether LPS effects on lipid lateral diffusion were due to insertion of LPS into the cell plasma membrane, we examined TRITC-LPS diffusion in B lymphocytes from LPS-responsive Balb/c and C3Heb/FeJ mice and from hypo-responsive C3H/HeJ mice. DiI and TRITC-LPS mobility decreased more than 50% in LPS-stimulated Balb/c and C3Heb/FeJ cells by 72 h. On C3H/HeJ lymphocytes, there was no change in DiI or TRITC-LPS lateral diffusion throughout the incubation period. These data indicate that B lymphocyte membrane compositions is altered in LPS-activated lymphoblasts and that the decreased lateral diffusion of lipid probes does not result from membrane perturbation by LPS insertion into the lipid bilayer. Further, similarities between TRITC-LPS and DiI lateral diffusion suggest that most LPS molecules interact non-specifically with B cell membranes, presumably by acyl chain insertion of the lipid A moiety.

Fluorescent probes for dopamine receptors: synthesis and characterization of fluorescein and 7-nitrobenz-2-oxa-1,3-diazol-4-yl conjugates of D-1 and D-2 receptor ligands

Fluorescent probes have been designed and developed for dopamine D-1 and D-2 receptors. Fluorescein and/or NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) derivatives of PPHT (D-2 agonist), spiperone (D-2 antagonist), SKF 38393 (D-1 agonist), and SKF 83566 (D-1 antagonist) were synthesized via their amino-functionalized analogues and all ligands were pharmacologically evaluated by measuring their ability to displace [3H]SCH 23390 and [3H]spiperone from D-1 and D-2 receptor sites in caudate putamen of monkeys (Macaca fascicularis). The fluorescein derivatives of PPHT and SKF 83566 and the NBD derivatives of spiperone and SKF 83566 retained the high affinity and selectivity of the parent ligands. The NBD derivatives of PPHT showed higher D-2 receptor affinity and selectivity than their parent ligands. The enantiomers of the fluorescent derivatives of PPHT were also synthesized and were found to exhibit stereoselectivity in binding to the D-2 receptor, with the S enantiomers having a considerably higher affinity than their R analogues. In contrast to these results, the fluorescein derivative of SKF 38393 showed only a low affinity for the D-1 receptor. These fluorescein- and NBD-coupled D-1 and D-2 receptor ligands have considerable significance as potential probes in the study of distribution of the receptors at the cellular/subcellular level and of their mobility in membranes in normal/diseased states by use of fluorescence microscopic and fluorescence photobleaching recovery techniques, respectively. The development of these novel fluorescent probes should also provide new leads for the design and synthesis of additional fluorescent ligands with better fluorescent properties and/or higher affinity/selectivity for the DA receptors.

Flow rate measurements in isolated perfused kidney tubules by fluorescence photobleaching recovery

We have developed a new application of the fluorescence photobleaching recovery (FPR) technique for instantaneous measurement of volume flow rates at any axial position along isolated perfused kidney tubules. The method requires fast data acquisition of emitted fluorescence through a photomultiplier (time resolution, 0.5 ms) coupled with differential interference contrast microscopy to measure luminal diameters accurately. While the tubule is perfused in vitro with an impermeant fluorophore (fluorescein sulfonate), a 20-ms bleach pulse reduces the fluorescence in the observation region by 20-25%. Fluorescence recovery is a direct function of perfusate velocity; diffusion plays no significant role in the early phase of recovery. A fluid dynamics approach to data analysis shows that fractional recovery increases linearly with time until t = L/2vm, where L is the length of the observation window and vm is the mean axial velocity. Practically, a linear regression analysis of the early recovery phase allows measurement of vm of up to 0.14 cm/s, i.e., a 40-nl/min flow rate in a 25-microns-diameter tubule. Calibration experiments in small glass tubes perfused at predetermined flow rates demonstrated good accuracy (within 10%) and reproducibility (coefficient of variation, 8.7%). In rat inner medullary collecting ducts microperfused at 4-40 nl/min, the correlation with a standard fluid collection method was excellent (r2 greater than 0.97). The method should also be suitable for the direct measurement of fluid flow rate in kidney tubules or blood vessels microperfused in vivo.

Fluorescence microscopy: A tool for studying the physical chemistry of interfaces

The technique of fluorescence microscopy, a long-standing, powerful, and still evolving research tool in molecular biology, has recently found application to the study of Langmuir monolayers and to multilayers and vesicles. In this review, the basic instrumentation of fluorescence microscopy will be reviewed and a number of powerful modifications of the technique will be surveyed. Resolution limits of the techniques will be discussed. The technique of fluorescence photobleaching recovery is used to study diffusion rates. Fluorescence polarization microscopy measures orientational distributions and the rotational diffusion of molecules. Total internal reflection fluorescence microscopy can be used to selectively excite fluorophores near an aqueous-glass interface. Spectral resolution is employed in the techniques of fluorescence ratio imaging and multiparameter studies using more than one kind of probe molecule. Improved two-dimensional sectioning is achieved by confocal techniques using laser scanning leading to the realization of three-dimensional fluorescence imaging. After a concise survey of the most prominent biophysical applications, recent work on Langmuir monolayers will be described.

Corticosteroid effects on lipid lateral diffusion in CEM-C1 and CEM-C7 acute lymphoblastic leukemia cells

We have examined glucocorticoid effects on CEM-C7 and CEM-C1 subclones of a leukemic human T-cell line using fluorescence photobleaching recovery techniques. Incubation with 10 −5 M triamcinolone acetonide (TA) increased lipid lateral diffusion on steroid-sensitive CEM-C7 cells but had no effect on steroid-resistant CEM-C1 cells. CEM-C7 cells incubated in serum-free medium responded only to TA but, when fetal calf serum was added to the incubation medium, would also respond to 10 −5 M dexamethasone and hydrocortisone. Thus, glucocorticoids can cause increased lipid lateral diffusion in CEM-C7 cells, while having no effect on steroid-resistant CEM-C1 cells.

Transforming growth factor-beta enhances the extent of intercellular communication between normal rat kidney cells

Normal rat kidney (NRK) cells cultured in the presence of epidermal growth factor are contact-inhibited at confluent densities. In the additional presence of transforming growth factor (TGF)-beta, however, cells undergo phenotypic transformation which is accompanied by a loss of contact inhibition. In this study, we show by means of the fluorescence photobleaching recovery technique and a scrape-loading dye transfer technique that quiescent confluent cultures of NRK cells do not show extensive gap junction-mediated intercellular communication. Cells contact-inhibited in the presence of epidermal growth factor also show only limited intercellular communication, although with an enhanced permeability coefficient. Cells phenotypically transformed upon addition of TGF beta, however, show extensive intercellular communication, with a similarly enhanced permeability coefficient. This enhanced intercellular communication induced by TGF beta is paralleled by an increase in intracellular pH. It is concluded that in contrast to what has been observed during tumorigenic transformation, phenotypic transformation of NRK cells induced by TGF beta results in an enhancement of the extent of gap junction-mediated intercellular communication.

Self-diffusion and probe diffusion in dilute and semidilute aqueous solutions of dextran

Tracer diffusion processes in polymeric solutions of dextran in both the dilute and semidilute regimes were investigated by the fluorescence photobleaching recovery technique. The molecular weight of the matrices covered the range from 40 kDa to 2 MDa. The tracer species consisted of fluorescein-labeled dextrans of molecular weights 40 and 150 kD, fluorescein-labeled polystyrene spheres of 38-nm diameter, and fluorescein (molecular weight of 332 Da). The viscosity of selected solutions was measured with capillary viscosimeters. We report here an extensive set of experimental data on diffusion processes and viscosity, obtained from a consistent set of samples. Comparison of our data with some of the current models is presented.

Evidence that Folliculo-Stellate Cells do not Impede the Permeability of Intercellular Spaces to Molecular Diffusion in Three-Dimensional Aggregate Cell Cultures of Rat Anterior Pituitary*

The permeability of intercellular spaces within the anterior pituitary (AP) and the influence of folliculo-stellate (FS) cells on compartmentalization within this tissue, has become a matter of debate. In reaggregated pituitary cell cultures as well as in the AP in situ the intercellular gaps and follicle-like structures remain accessible to molecular diffusion, whereas in some studies FS cells were reported to form tight epithelia that impede macromolecular transport through the spaces between the epithelial cells. In the present study the permeability of AP cell reaggregates was examined using fluorescent BSA as a tracer. Using confocal scanning laser microscopy a direct visualization of the permeation process was achieved. Quantitative estimation of the effective diffusion coefficient (Deff) for fluorescein-BSA within the aggregates was obtained using the fluorescence photobleaching recovery technique. Deff was 1.33 +/- 0.31 x 10(-7) cm2/sec (mean +/- SD) in aggregates from 14-day-old female rats and 2.45 +/- 0.55 x 10(-7) cm2/sec in aggregates from adult female rats. These values are about three times lower than in free solution. Calculation of the time-dependent concentration distribution inside the aggregate for a Deff = 2 x 10(-7) cm2/sec revealed that the concentration of the fluorescent tracer in the center of the aggregate reaches 90% of the concentration outside the aggregate after 0.5 min for aggregates with a radius of 50 microns and 6 min for aggregates with a radius of 150 microns. Aggregates enriched in FS cells, in which we previously showed a sustained inhibition of secretory responses to stimulatory and inhibitory agents as compared to total population aggregates, showed a diffusion coefficient (Deff = 1.85 +/- 0.77 x 10(-7) cm2/sec) which was not significantly different from that in the total population aggregates. The present study shows that AP cell aggregates are fully permeable to diffusing molecules within minutes and that in a three-dimensional tissue configuration FS cells, which were reported to tonically inhibit AP hormone release in response to various secretagogues, do not impede molecular diffusion to an extent which would account for sustained inhibition of hormone release.

A monoclonal antibody to LFA‐3, the CD2 ligand, specifically immobilizes major histocompatibility complex proteins

T cells are activated when the antigen-specific T cell receptor recognizes antigen in association with major histocompatibility complex (MHC) proteins. The T cell surface protein CD2 (T11, LFA-2, the T erythrocyte receptor) and its target or stimulator cell ligand, lymphocyte function-associated antigen-3 (LFA-3), are also involved in T cell adhesion and activation. The molecular mechanisms by which the CD2/LFA-3 interaction affects T cell adhesion and activation are unclear. The CD2/LFA-3 interaction may be modeled by the interaction between LFA-3 and anti-LFA-3 monoclonal antibody (mAb). We used the fluorescence photobleaching recovery technique to investigate the effect of anti-LFA-3 mAb on the lateral mobility of MHC proteins in plasma membranes of JY, a human Epstein-Barr virus-transformed B cell line. Anti-LFA-3 mAb induced immobilization of class I MHC proteins labeled with bivalent but not monovalent fluorescein-conjugated W6/32 mAb. Anti-LFA-3 mAb also caused immobilization of class II MHC proteins labeled with bivalent fluoresceinated LB3.1 mAb. In contrast, anti-LFA-3 mAb did not affect the mobilities of either a B cell membrane protein labeled with bivalent fluoresceinated anti-CD45 (human leukocyte antigen) mAb or a membrane lipid analogue. Unlike anti-LFA-3 mAb, anti-LFA-1 mAb did not affect class I MHC protein mobility. These results suggest that CD2 binding to LFA-3 may trigger a physiological response in which target cell MHC proteins, cross-linked by receptors on the T cell surface, are immobilized at and thereby localized to the T cell-target cell interface.

Rheology of G-actin solutions.

Recent reports that dilute solutions of G-actin form viscoelastic gels have been investigated using several experimental variations of three distinct physical techniques in independent laboratories. Direct measurement of storage and loss moduli were made using two rheometers of different design. Measurements of the diffusion of G-actin and of tracer particles added to G-actin solutions were carried out using dynamic light scattering and fluorescence photobleaching recovery techniques. Measurements were performed over a period of many hours, on actin solutions prepared under conditions for which anomalous gelation had been reported. All data from this investigation are consistent with the conclusion that dilute G-actin solutions behave as newtonian liquids.

Class I major histocompatibility complex proteins diffuse isotropically on immune interferon-activated endothelial cells despite anisotropic cell shape and cytoskeletal organization: application of fluorescence photobleaching recovery with an elliptical beam.

Interferon gamma induces striking phenotypic alterations in confluent cultures of human vascular endothelial cells (HEC), including cell shape change from polygonal to elongated and cytoskeletal actin rearrangement from dense peripheral bands to longitudinal bundles of stress fibers. Since many transmembrane proteins, including class I major histocompatibility complex (MHC) proteins, interact with cytoskeletal actin, an interferon-gamma-induced anisotropic arrangement of stress fibers might cause anisotropic lateral diffusion of HEC class I MHC proteins. To test this hypothesis, we adapted the fluorescence photobleaching recovery technique to allow measurement of anisotropic diffusion of fluorescently labeled molecules on two-dimensional surfaces. A highly eccentric elliptical Gaussian laser beam was used to photobleach the sample and to monitor fluorescence recovery. In this technique, named "line fluorescence photobleaching recovery," lateral diffusion is measured along that axis of the sample that is perpendicular to the major axis of the elliptical beam. The lateral diffusion coefficient and fractional mobility are obtained by fitting the experimental data to a theoretical recovery curve, the form of which is determined by the solution to a modified version of the diffusion equation in which a tensor is used to describe diffusion in two orthogonal directions. Fluorescein-conjugated murine monoclonal antibodies were used to label class I MHC proteins on interferon-gamma-treated HEC and human dermal fibroblasts. These two cultured human cell types were found to be similar in their elongated shape and anisotropic stress fiber organization. Class I MHC protein lateral mobility was compared to that of fluorescein-labeled phosphatidyl-ethanolamine, a membrane phospholipid probe. Class I MHC proteins diffused anisotropically on human dermal fibroblasts, whereas fluorescein-labeled phosphatidylethanolamine diffused isotropically on this cell type. In contrast, both class I MHC proteins and fluorescein-labeled phosphatidylethanolamine diffused isotropically on interferon-gamma-treated HEC. These data suggest that neither elongated shape nor anisotropic stress fiber arrangement is sufficient to induce anisotropic diffusion of proteins on the HEC plasma membrane.

Micrometer-scale domains in fibroblast plasma membranes.

We have used the technique of fluorescence photobleaching recovery to measure the lateral diffusion coefficients and the mobile fractions of a fluorescent lipid probe, 1-acyl-2-(12-[(7-nitro-2-1, 3-benzoxadiazol-4-yl)aminododecanoyl]) phosphatidylcholine (NBD-PC), and of labeled membrane proteins of human fibroblasts. Values for mobile fractions decrease monotonically with increasing size of the laser spot used for the measurements, over a range of 0.35-5.0 microns. Values for NBD-PC diffusion coefficients increase in part of this range to reach a plateau at larger laser spots. This variation is not an artifact of the measuring system, since the effects are not seen if diffusion of the probe is measured in liposomes. We also find that the distribution of diffusion coefficients measured with small laser spots is heterogeneous indicating that these small spots can sample different regions of the membrane. These regions appear to differ in protein concentration. Our data strongly indicate that fibroblast surface membranes consist of protein-rich domains approximately 1 micron in diameter, embedded in a relatively protein-poor lipid continuum. These features appear in photographs of labeled cell surfaces illuminated by the expanded laser beam.

Restriction of the lateral motion of band 3 in the erythrocyte membrane by the cytoskeletal network: dependence on spectrin association state

The effects of incubation of erythrocyte ghosts under various conditions (ionic strength or addition of ankyrin, diamines, or ATP) on the lateral motion of band 3 in the membranes were studied by using the fluorescence photobleaching recovery technique. Incubation of ghosts with exogenous ankyrin increased the immobile fraction of band 3, from 0.6 in intact ghosts to 0.8-0.9 when an average of 0.2 mol of extra ankyrin was bound per mole of band 3. Ankyrin-free band 3 proteins were mobile, but their mobility was governed by the spectrin association state in the cytoskeletal network. The diffusion constant was 5.3 X 10(-11) cm2 s-1 at a spectrin tetramer mole fraction of 0.3-0.4 in 10 mM NaCl/5 mM sodium phosphate, pH 7.8, and decreased 1 order of magnitude when the tetramer fraction increased to 0.5 in higher NaCl concentration (150 mM NaCl). A similar decrease was observed when the spectrin tetramer fraction was increased by 0.2 mM spermine in 10 mM NaCl/10 mM tris(hydroxymethyl)aminomethane hydrochloride, pH 7.6. On the other hand, the rotational motion of band 3 in the membranes was not affected by the spectrin association state. Trypsin treatment of ghosts cleaved off the cytoplasmic domain of band 3 and caused a marked (8-fold) increase in the lateral mobility, D = 4.0 X 10(-10) cm2 s-1. These results indicate that the lateral mobility of ankyrin-free band 3 protein is restricted by interactions of their cytoplasmic domain with the cytoskeletal network. A model is presented that band 3 can pass the network when spectrins are in dissociated dimers and cannot pass when they are tetramers. The lateral diffusion constant is thus determined by the spectrin dimer population in the network.