Flow cytometric measurement of fluorescence resonance energy transfer on cell surfaces. Quantitative evaluation of the transfer efficiency on a cell-by-cell basis

A method has been developed for the determination of the efficiency (E) of the fluorescence resonance energy transfer between moieties on cell surfaces by use of a computer-controlled flow cytometer capable of dual wavelength excitation. The absolute value of E may be calculated on a single-cell basis. The analysis requires the measurement of samples stained with donor and acceptor conjugated ligands alone as well as together. In model experiments HK 22 murine lymphoma cells labeled with fluorescein-conjugated concanavalin A (Con A) and/or rhodamine conjugated Con A were used to determine energy transfer histograms. Using the analytic solution to energy transfer in two dimensions, a high surface density of Con A binding sites was found that suggests that the Con A receptor sites on the cell surface are to a degree preclustered . We call this technique flow cytometric energy transfer ( FCET ).     

Distribution of concanavalin A binding sites on the surface of dissociated rat submandibular gland acinar cells.

The submandibular glands of 4-week-old rats were dissociated by a procedure involving digestions with collagenase and hyaluronidase, chelation of divalent cations and mechanical force. A suspension of single cells was obtained in low yield by centrifugation in a Ficoll-containing medium. Immediately after dissociation and after a culture period of 16-18 hr the dissociated cells were tested for agglutinability by concanavalin A (Con A). Using ferritin (tfer)-conjugated Con A the lectin binding by the isolated acinar cells was also studied. The dissociated cells were agglutinated by low concentrations of Con A and bound Fer-Con A molecules on their entire surface without any indication of polarization of the cell membrane. There was a considerable cell to cell variation in the amount of Fer-Con A binding which was, in general, sparse and patchy. The contact surfaces between agglutinated cells revealed a dense binding of Fer-Con A molecules irrespective of the types of cells participating in the agglutination reaction. Cells cultured for 16-18 hr were no longer agglutinated by Con A. As compared to the freshly dissociated cells the cultured acinar cells revealed a more uniform and denser binding of Fer-Con A molecules. Furthermore, there were more lectin molecules bound to the cell surface corresponding to the basal part of the cell, where the nucleus and most of the rough surface endoplasmic reticulum were located, than to the apical cell surface. It is suggested that the higher density of lectin-binding sites on the cell surface in the vicinity of the cisternae of the rough endoplasmic reticulum indicates insertion sites of newly synthesized membrane glycoproteins.     

Effect of concanavalin A on the kinetics of ecto-5'-adenosine monophosphatase (5'-adenosine monophosphate phosphohydrolase) in the outer surface of intact neural cells in culture.

Oncogenic cultured rat C6 astroblastoma cells display strikingly high ecto-5'-adenosine monophosphatase (ecto-5'-AMPase) activity, 4.23 +/- 20 mumol of Pi liberated by intact cells from 3 mM extracellular 5'-AMP (mg of protein-1 h-1, as compared with 0.15 +/- 0.01 for nononcogenic cultured hamster astroblasts. A further rise in C6 cell ecto-5'-AMPase activity occurs with increase in cell density during growth. Less than 2 pg of the lectin, concanavalin A (Con A), bound per cell reversibly inhibits most of the cellular ecto-5'-AMPase activity. Inhibition by Con A binding is independent of cellular temperature. Con A binding suppresses phosphohydrolase activity of a pK=7.4 functional group on the cell surface. A direct proportionality is observed between quantity of Con A bound to the cell surface and simultaneous relative decreases both in Michaelis constant and maximum velocity of ecto-5'-AMPase in the intact cell. The findings suggest that a major consequence of the specific high affinity binding of Con A to the C6 cell surface is the inactivation of the enzyme--substrate complex of ecto-5'-AMPase.     

Surface functions during mitosis. III. Quantitative analysis of ligand- receptor movement into the cleavage furrow: diffusion vs. flow

The surface distribution of concanavalin A (Con A) bound to cell membrane receptors varies dramatically as a function of mitotic phase. The lectin is distributed diffusely on cells labeled and observed between mid-prophase and early anaphase, whereas cells observed in late anaphase or telophase demonstrate a marked accumulation of Con A- receptor complexes over the developing cleavage furrow (Berlin, Oliver, and Walter. 1978. Cell. 15:327-341). In this report, we first use a system based on video intensification fluorescence microscopy to describe the simultaneous changes in cell shape and in lectin-receptor complex topography during progression of single cells through the mitotic cycle. The video analysis establishes that fluorescein succinyl Con A (F-S Con A)-receptor complex redistribution begins coincident with the first appearance of the cleavage furrow and is essentially complete within 2-3 min. This remarkable redistribution of surface fluorescence occurs during only a modest change in cell shape from a sphere to a belted cylinder. It reflects the translocation of complexes and not the accumulation of excess labeled membrane in the cleavage furrow: first, bound fluorescent cholera toxin which faithfully outlines the plasma membrane is not accumulated in the cleavage furrow, and, second, electron microscopy of peroxidase-Con A labeled cells undergoing cleavage shows that there is a high linear density of lectin within the furrow while Con A is virtually eliminated from the poles. The rate of surface movement of F-S Con A was quantitated by photon counting during a repetitive series of laser-excited fluorescence scans across dividing cells. Results were analyzed in terms of two alternative models of movement: a flow model in which complexes moved unidirectionally at constant velocity, and a diffusion model in which complexes could diffuse freely but were trapped at the cleavage furrow. According to these models, the observed rates of accumulation were attainable at either an effective flow velocity of approximately 1 micron/min, or an effective diffusion coefficient of approximately 10(- 9) cm2/s. However, in separate experiments the lectin-receptor diffusion rate measured directly by the method of fluorescence recovery after photobleaching (FRAP) on metaphase cells was only approximately 10(-10) cm2/s. Most importantly, photobleaching experiments during the actual period of F-S Con A accumulation showed that lectin-receptor movement during cleavage occurs unidirectionally. These results rule out diffusion and make a process of oriented flow of ligand-receptor complexes the most likely mechanism for ligand-receptor accumulation in the cleavage furrow.     

Lectin binding to cell surfaces: comparisons between normal and migrating corneal epithelium

Comparisons were made between cell surfaces of normal and migrating corneal epithelium of the rat by localizing and/or quantifying concanavalin A (Con A) and wheat germ agglutinin (WGA) binding. Our results indicate that apical cell surfaces of the leading edge of a migrating sheet of epithelium differ from those of normal epithelium and that the various cell layers within the stratified normal epithelium have different lectin-binding characteristics. Three methods of monitoring lectin binding to cell surfaces were employed. Based on ferritin-conjugated Con A, ferritin-conjugated WGA, and [3H]Con A binding, apical cell membranes of migrating epithelia bind more Con A and WGA than do apical membranes of superficial cells of normal stratified epithelia. With both fluorescein isothiocyanate (FITC)-Con A and -WGA, membranes of all the cells of the leading edge of the migrating sheet fluoresce intensely. FITC-Con A binding of normal stratified epithelium is relatively uniform through all cell layers with no discernible staining of the apical membrane of superficial cells. With FITC-WGA, however, fluorescence is present only on basal cell layers but not on superficial cells. These data demonstrate that apical cell surface sugars on a sheet of epithelium migrating to cover a wound differ from the apical cell surface sugars of normal epithelium. As indicated by FITC-WGA binding, cells of the migrating sheet have cell surface characteristics similar to basal cells of normal epithelia. Perhaps, upon wounding, the leading edge of the migrating sheet is derived from the basal cell population of the normal stratified epithelium, or perhaps there is an alteration in cell surface glycoproteins as the cells become migratory.     

Flow microfluorometric studies of lectin binding to mammalian cells. I. General features

Flow microfluorometry has been used to quantitate cell-surface binding of fluorescein-conjugated lectins. Frequency distributions of total surface binding of Concanavalin A per cell were prepared for a variety of cultured cell populations, including established cell lines, virus-transformed lines and non-transformed parental lines. In the case of growing Chinese hamster cells (line CHO), much of the variability of Con A binding per cell could be related to variability of cell size. Experiments with cells synchronized by mitotic selection indicated that the modal surface density of binding sites was almost constant throughout the cell cycle. However, as indicated by inhibition of binding with α-methyl mannopyranoside and by the effect of trypsin, the sites on each cell were heterogeneous in chemical structure and/or exposure. Agglutinability of virus-transformed cell lines or trypsin-treated parental lines was demonstrated but could not be correlated closely with binding.     

Lectin receptor sites on rat liver cell nuclear membranes.

The presence and localization of lectin receptor sites on rat liver cell nuclear and other endomembranes was studied by light and electron microscopy using fluorescein and ferritin-coupled lectin conjugates. Isolated nuclei labelled with fluorescein-conjugated Concanavalin A (Con A) or wheat germ agglutinin (WGA) often showed membrane staining, which sometimes was especially bright on small stretches of the nuclear surface. Unlabelled nuclei and nuclei with a complete ring fluorescence were also seen. The nuclear fluorescence corresponded in intensity to that seen on the surface of isolated rat liver cells. Con A-ferritin particles were seldom detected on the cytoplasmic surface of the intact nuclear envelope. However, at places where the 2 leaflets of the envelope were widely separated or where the outer nuclear membrane was partly torn away, heavy labelling was seen on the cisternal surface of both the inner and outer nuclear membranes. Labelling with Con A-ferritin was also found on the cisternal side of rough endoplasmic reticulum present in the specimens. No labelling was seen on the cytoplasmic surface of mitochondrial outer membrane. The results demonstrate the presence of binding sites for Con A and WGA in nuclei and an asymmetric localization of these sites on the cisternal side of ribosome-carrying endomembranes in rat liver cells.     

Alterations in the surface glycoproteins of chicken erythrocytes following transformation with erythroblastosis strain R virus

Summary Erythroblasts from marrows of chicks infected with RNA-virus (strain Rerythroblastosis virus) were found to possess a small but consistent increase in the number of concanavalin A binding sites per cell compared to erythroblasts derived from the marrows of phenylhydrazine-treated birds. Both types of erythroblast possessed more surface glycoproteins per cell accessible to concanavalin A (Con A) than marrow and peripheral blood erythrocytes. Employment of concanavalin A conjugated to ferritin showed marked differences in the spatial arrangement of the Con A receptors between phenylhydrazine and virus-induced erythroblasts but little difference was observed in the surface density of the Con A sites between erythrocytes and erythroblasts, a result which agrees with the amount of bound labeled Con A when this data is expressed in terms of the cell surface.The amount of labeled Con A bound to erythrocytes derived from the marrow was greater than that derived from the peripheral circulation, a result which is substantiated by the ferritin Con A studies which show an increase in the density of Con A sites on the marrow blood cells. Trypsinization increases the number of sites and the agglutininability of the marrow cells.The increase in the susceptibility of the cells to agglutinate with concanavalin A paralleled the observed increase in the number of binding sites per cell.     

Interaction of carbohydrate binding sites on concanavalin A-agarose with receptors on adipocytes studied by buoyant density method.

The interaction of concanavalin A (Con A) with isolated adipocytes was studied using Con A-Sepharose beads in the affinity binding buoyant density method previously used to study insulin receptors. Free Con A-Sepharose beads could be separated from the bound beads (cell-bead complexes) by sedimentation of the high density beads and floatation of the low density complexes. Sedimented and total beads could be determined by counting the radioactivity associated with [-125I]Con A coupled in tracer amounts to the beads. Various lines of evidence demonstrated the high specificity of binding. Soluble Con A, but neither insulin nor any of the other proteins tested, inhibited and reversed the binding of Con A-Sepharose to the cells. Whereas treatment of Con A- (and insulin-) derivatized beads with anti-insulin antiserum, and cells with trypsin, readily inhibited binding of insulin-Sepharose to cells, neither treatment inhibited Con A-Sepharose binding. According to the relative extents of inhibition and reversal of binding exhibited by 15 different carbohydrates, the saccharide binding sites on Con A-Sepharose appeared virtually identical with the known sites on free Con A. Protein-containing components of cell ghosts that were solubilized with Triton X-100 appeared to correspond to the Con A-Sepharose receptor sites on the basis of their ability to bind to Con A-Sepharose columns, be eluted with methyl alpha-D-mannopyranoside (MeMan) and be precipitated by the free lectin and redissolved by MeMan. According to (a) Normarski interference contrast microscopic examination of the topographical distribution of Con A-Sepharose beads and cells surrounding and bound to each other, and (b) absence of any apparent morphological changes in the cells due to binding, it is suggested that extensive clustering ("cap" or "macropatch" formation) of Con A receptors did not occur on the adipocyte as a consequence of the interaction of the cells with the Con A-Sepharose beads.     

Studies of the cell surface of Dictyostelium discoideum during differentiation. The binding of 125I-concanavalin A to the cell surface.

125I-concanavalin A (125I-Con A) was found to be equally effective as native Con A in binding to and agglutinating cells of Dictyostelium discoideum, suggesting that iodination of the molecule had no effect on the interaction of the protein with the cell surface. Almost all of the 125I-Con A binding to the cells was inhibited by alpha-methyl glucoside. The binding of 125I-Con A to the cells was extremely rapid, and once bound, the molecule was not readily displaced by prolonged incubation or by the addition of excess native concanavalin A (Con A). In contrast, the 125I-Con A was displaced rapidly from the cell surface by alpha-methyl glucoside. The binding of 125I-Con A to D. discoideum was identical at 22 degrees and 4 degrees, and was unaffected by metabolic inhibitors, suggesting that the protein was not subject to endocytosis. The cell surface Con A binding sites became saturated at high 125I-Con A concentrations. Scatchard plots of the data indicated that growing cells possessed 4 X 10(7) sites/cell, all of equal affinity. Similar plots for "aggregation phase" cells indicated at least two classes of binding sites. A small proportion of the sites had an affinity close to that for the sites on growing cells, but the majority of the sites had a markedly decreased affinity. The total number of binding sites increased only slightly during aggregation to 5.6 X 10(7) sites/cell.     

Con A Binds to the Membranellar and Somatic Cilia of Stentor and to the Developing Oral Primordium During Oral Regeneration1

Binding sites for Concanavalin A have been located in the ciliate Stentor coeruleus by utilizing FITC-Con A and fluorescence microscopy. When both nonregenerating and regenerating Stentor are fixed prior to FITC-Con A exposure, FITC-Con A binds intensely to the cilia of the membranellar band and to the somatic cilia that cover much of the cell surface. No binding is observed between the ciliary rows. The FITC-Con A also binds to the developing oral primordia of regenerating cells. Binding of FITC-Con A in the early stages of regeneration (prior to stage 4) appears to be less intense than that in the later stages. Additional FITC-Con A binding appeared as a granular fluorescence in the area of the developing buccal cavity beginning at about stage 4 and disappearing around stages 6–7. The presence of α-D-methyl mannoside prevented the binding of FITC-Con A to either regenerating or nonregenerating cells. If nonregenerating Stentor are exposed to FITC-Con A prior to fixation, the binding pattern is entirely different with the fluorescence primarily in the form of random, granular patches spread over much of the cell but with no binding to either type of cilia. These results demonstrate that membrane glycoproteins capable of binding Con A are located primarily in the membranellar and somatic cilia and in the developing oral primordia during oral regeneration in Stentor. Concanavalin A binding to these sites may be involved in the Con A-induced inhibition of oral regeneration observed in earlier studies.     

Differences in density of Concanavalin A-binding sites due to differences in surface morphology of suspended normal and transformed 3T3 fibroblasts.

Calculations of the density of Concanavalin A (Con A)-binding sites on normal and transformed fibroblasts have, as yet, been based on the unproven assumption that suspended cells are smooth spheres. We studied the surface morphology of suspended normal and transformed fibroblasts with scanning and transmission electron microscopes, and found a large difference in surface morphology between suspended normal and transformed 3T3 cells. When this difference in surface morphology was taken into account, the estimated cell surface area of normal 3T3 cells was approximately seven times larger than that of transformed 3T3 cells. Since equal numbers of 3H-Con A molecules are bound on normal and transformed cells, the density of Con A-binding sites is approximately seven times greater on transformed than on normal 3T3 cells. The difference in density of Con A-binding sites between normal and transformed fibroblasts might be sufficient to explain the difference in agglutination response, as originally suggested by Burger, and may also be the cause of the different degrees of clustering of Con A-binding sites on the plasma membrane of these cells.     

Fluorophore-conjugated lectin labeling of the cell surface of isolated male and female gametes,central cells and synergids before and after fertilization in maize

Three fluorescein isothiocyanate (FITC)-conjugated lectins, Canavalia ensiformis agglutinin (Con A), Triticum vulgaris agglutinin (WGA) and Phaseolus vulgaris erythroagglutinin (PHA-E), were used as probes to localize sugar moieties of glycoconjugates on the cell surface of isolated maize sperm, egg, central, antipodal cells, synergids, and in vitro- and in vivo-fertilized zygotes. Fluorescence signals on the surface of the cells were due to specific binding. Calcium was necessary for WGA and PHA-E binding and enhanced Con A labeling. Differences in glycoconjugate composition of the membranes of gametes and other embryo sac component cells were found. FITC-Con A strongly labeled egg and central cells, but labeled sperm only weakly. FITC-WGA binding sites were detected on egg, but not sperm cells. Con A and WGA binding sites were equally distributed around egg and central cell protoplasts. FITC-PHA-E binding sites were not found on sperm and egg cells before fertilization. Binding sites of these lectins were located on synergids, especially on their filiform apparatus. Interestingly, WGA binding to egg cells was enhanced after fertilization, whereas PHA-E binding to egg cell membranes could only be detected after fertilization. These results suggest the occurrence of fertilization-induced changes in glycoconjugate composition of the maize egg cell membrane. An increase in the number of WGA and PHA-E binding sites was also observed on newly formed cell walls of cultured two-celled embryos derived from in vitro-produced zygotes.     

Spreading of B16 F1 cells on laminin and its proteolytic fragments P1 and E8: involvement of laminin carbohydrate chains

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.     

Mechanism of cell-mediated cytotoxicity at the single cell level. VI. Direct assessment of the cytotoxic potential of human peripheral blood non-lytic effector-target cell conjugates

Single cell cytotoxicity assays reveal that a large percentage of lymphocytes are unable to kill attached targets in a 4- to 18-hr assay. Additional signals (in the form of lectin or anti-target antibody) delivered to target-bound lymphocytes enable these previously non-lytic lymphocytes to kill attached target cells. This finding was obtained by using a modification of the single cell assay, in which lectin or target cell antibody is incorporated into agarose with preformed lymphocyte-target conjugates. Human peripheral blood lymphocytes (PBL) or Percoll density gradient-enriched large granular lymphocytes (LGL) were used as effector cells in natural killer (NK), antibody-dependent cellular cytotoxicity (LDCC) assay systems. The targets used were NK-sensitive K562 and Molt-4 and NK-insensitive Raji. Several findings were made in the modified single cell assay, namely a) the frequency of cytotoxic NK or ADCC effector cells was not augmented, suggesting that the initial trigger was sufficient for lytic expression in these instances. Furthermore, these results showed that the NK-sensitive targets used do not bind nonspecifically to the LDCC effector cells. K562 coated with Con A, however, serve as LDCC targets. b) The frequency of two target conjugate lysis by NK/K effectors was not augmented by Con A. These results suggest that Con A does not potentiate the killing of multiple targets bound to a single cytotoxic lymphocyte. c) Although conjugates formed between LGL or PBL and NK-insensitive Raji are non-lethal, significant lysis was observed when these conjugates were suspended in Con A or antibody agarose. These results demonstrate that Raji bind to cytotoxic NK, K, and LDCC effector cells, but are lysed only when the appropriate trigger is provided. d) The cytotoxic potential of non-lytic conjugates appears to lie within the low density Percoll fraction, although the high density lymphocytes are able to nonlethally bind to targets. Altogether the results demonstrate that target recognition and/or binding by the effector cells is a distinct event from the trigger or lytic process. The implications of these findings are discussed.     

Concanavalin A-Induced Morphological Changes and Its Binding Sites in Starfish Follicle Cells as Revealed by Electron Microscopy

Concanavalin A (Con A) stimulates the production in starfish follicle cells of 1-methyladenine, a hormone which induces oocyte maturation. We have therefore investigated Con A-induced morphological changes and Con A-binding sites in the follicle cell using native Con A and horseradish peroxidase- or ferritin-labeled Con A (HRP-Con A, Fer-Con A). After isolated follicle cells were incubated with Con A (1 mg/ml), vacuoles, the Golgi complex and multivesicular body-like organelles (MVBs) became prominent in most of the cells. After follicle cells were prefixed and then incubated with Fer-Con A for 60 min, tagged ferritin was diffusely and randomly distributed as single or small clustered particles on the cell surface. The incubation of isolated follicle cells with Fer-Con A for 10 min before fixation resulted in numerous ferritin particles localized along the internalized membrane, and also in vacuoles, MVBs and small lysosome-like structures. After 60 min incubation with Fer-Con A, ferritin was further located in large lysosome-like structures and in vesicles near and in the Golgi area as well as in the organelles described above. HRP-Con A binding sites were also observed in vacuoles and MVBs of the intact cells.
These results suggest that Con A binds at first to the cell surface and causes rapid internalization and that membrane-bound Con A is easily endocytosed into vacuoles, MVBs and lysosome-like structures, and is later incorporated in some vesicles in the Golgi area.     

Concanavalin A inhibition of alpha-bungarotoxin binding to a nonfusing muscle cell line.

Incubation of a nonfusing muscle cell line, BC3H1, with concanavalin A (Con A) results in a maximum decrease of 35% in the cell's ability to bind alpha-bungarotoxin (alpha-BuTx). The Con A-induced inhibition of 125I-alpha-BuTx binding is reversible and the degree of inhibition parallels the degree of saturation of Con A binding sites on the cell surface. The maximum level of Con A-induced inhibition of 125I-alpha-BuTx binding is not affected by increasing the time of incubation in Con A, using higher concentrations of Con A or by increasing the time of incubation in the presence of 125I-alpha-BuTx. In addition, all BC3H1 cells in culture are sensitive to the Con A-induced inhibition of 125I-alpha-BuTx binding. A comparison of the pseudo-first order rate constants for 125I-alpha-BuTx binding to untreated (8.6 x 10(4) M-1 S-1) and Con A-treated (5.4 x 10(4) M-1 S-1) BC3H1 cells, however, shows that those acetylcholine receptors in Con A-treated cells which bind 125I-alpha-BuTx do so with a lowered apparent affinity. Partial inhibition of toxin-binding capacity is not a consequence of two classes of acetylcholine receptors on the cell surface. Furthermore, individual receptors experience partial inhibition of their binding capacity by Con A, resulting in receptors with at least one binding site blocked and at least one site available for alpha-BuTx binding.     

Flow cytometric measurements of fluorescence energy transfer using single laser excitation

Flow cytometric energy transfer (FCET) measurements between labeled specific sites of cell surface elements (Sz?llosi et al., Cytometry, 5:210-216, 1984) have been extended in a simplified form using a flow cytometer equipped with single excitation beam. This versatile and easily applicable method has several advantages over any nonflow cytometric (i.e., spectrofluorimetric) energy transfer measurements on cell surfaces: The labeled ligands can be applied in excess, without washing, thereby enabling the investigation of relatively labile receptor-ligand complexes. Contributions of signals from cell debris, from cell aggregates, or from nonviable cells can be avoided by gating the data collection on the light scatter signal. The heterogeneity of the cell population with respect to the proximity of the labeled binding sites can be studied. In the cases of homologous ligands or of ligands binding to the same molecule but at different epitopes, the determination of fluorescence resonance energy transfer efficiency values can be carried out on a cell-by-cell basis, offering data on intramolecular conformational changes. This modified FCET method enabled us to demonstrate the uniform density of glycoproteins, specific for Con A binding, in the plasma membrane of normal and Gross virus leukemic mouse cells of different sizes. The utility of this procedure has also been demonstrated by using the mean fluorescence intensities of the distribution curves in the calculation of the fluorescence energy transfer efficiency.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Summary Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair. The 16-day fetal tissue is mitotically active, does not exhibit a well defined monolayer, and demonstrates weak fluorescence binding for WGA, Con A and RCA. Conversely, SBA binding is readily detected on many cell surfaces. By 19 days in utero, the endothelial monolayers becomes organized and cell proliferation greatly diminishes. WGA, Con A and RCA now exhibit binding similar to that seen in the adult tissue. SBA binding is not detected at this time. Thus, changes in lectin binding during wound repair of the adult rat corneal endothelium mimic changes in lectin binding seen during the development of the tissue.Supported by grant EY-06435 from The National Institutes of Health     

Flow cytometry as a new method for the measurement of electrophoretic mobility of erythrocytes using membrane charge staining by fluoresceinated polycations.

The binding of FITC-labeled poly-L-ornithine and poly-L-lysine to fresh or neuraminidase treated human, rat or rabbit erythrocytes was investigated by simultaneous cell volume and cell membrane fluorescence measurements in a flow cytometer. The cell volume was converted into cell surface and the distribution curve of the fluorescence/micrometer2 cell surface was calculated from all histogram classes by a computer program. The mean fluorescence/micrometer2 cell surface as a measure of the density of the negative charges on the cell surface was directly proportional to the elctrophoretic mobility of the erythrocytes, showing that polycation binding can effectively be used for the measurement of the electrophoretic mobility of erythrocytes. The computer fitting of the experimental two parameter histograms by two dimensional Gaussian normal distributions was found to be a very efficient way of data reduction, and a good separation of overlapping cell clusters was possible even in the case of low total numbers of cells in the histogram.