Rate Of Fluorescence Recovery Research Articles

Epileptiform activity in hippocampal slice cultures exposed chronically to bicuculline: increased gap junctional function and expression.

Chronic (18 h) exposure of cultured hippocampal slices to the type-A GABA receptor blocker, bicuculline methiodide (BMI) 10 micro m increased the levels of connexin 43 (Cx43) and connexin 32 (Cx32) mRNAs, but not connexin 26 and connexin 36, as demonstrated by RNase protection assays. The levels of Cx43 and Cx32 proteins in membrane fractions detected by western blotting were also significantly increased. Immunoblotting indicated that BMI also promoted a significant expression of the transcription protein c-fos. The rate of fluorescence recovery after photobleaching, an index of gap junctional coupling, was also significantly increased, whereas it was blocked by the gap junctional blocker, carbenoxolone (100 micro m). Extracellular recordings in CA1 stratum pyramidale, performed in BMI-free solution, demonstrated that BMI-exposed cultures possessed synaptic responses characteristic of epileptiform discharges: (i) significantly greater frequency of spontaneous epileptiform discharges, (ii) post-synaptic potentials with multiple population spikes, and (iii) significantly longer duration of primary afterdischarges. Carbenoxolone (100 micro m), but not its inactive analog, oleanolic acid (100 micro m), reversibly inhibited spontaneous and evoked epileptiform discharges. The findings of BMI-induced parallel increases in levels of gap junction expression and function, and the increase in epileptiform discharges, which were sensitive to gap junctional blockers, are consistent with the hypothesis that increased gap junctional communication plays an intrinsic role in the epileptogenic process.

Rac2 Regulation of Phospholipase C-β2 Activity and Mode of Membrane Interactions in Intact Cells

Phospholipase C-beta (PLCbeta) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCbeta(2) isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCbeta(2) with the plasma membrane. For either GFP-PLCbeta(2) or GFP-PLCbeta(2)Delta, a C-terminal deletion mutant lacking the region required for stimulation by Galpha(q), these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCbeta(2) and GFP-PLCbeta(2)Delta in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCbeta(2). Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCbeta(2), but had the opposite effect on GFP-PLCbeta(2)Delta, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCbeta isozyme by a GTP-binding protein and assign a novel function to the PLCbeta(2) C-terminal region, regulating its exchange between membrane-bound and cytosolic states.

Endothelial Cells Go with the Flow

Endothelial cell (EC) motility plays a critical role in angiogenesis and blood vessel repair. Angiogenic growth factors, such as vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), stimulate EC migration in vitro. Oxidized low density lipoprotein (LDL), which may contribute to the development of atherosclerotic lesions, inhibits EC motility in vitro. Ghosh et al. investigated the mechanisms by which lysophosphatidylcholine (LysoPC), the principle antimigratory agent in oxidized LDL, affects EC motility by treating cultured bovine aortic ECs with lipophilic molecules that affect membrane microviscosity, including α-tocopherol, cholesterol, and lysophospholipids. Using fluorescence anisotropy on plasma membrane-enriched cell fractions to measure intramembrane movement of 1,6-diphenyl-1,3,5-hexatriene as an indicator of membrane microviscosity, the authors determined that treatments that caused moderate increases in microviscosity enhanced cell migration, whereas treatments that increased microviscosity beyond a given threshold inhibited migration. Treatment with bFGF and VEGF increased microviscosity to an extent similar to promigratory concentrations of the lipophilic molecules, as determined by fluorescence anisotropy, and decreased the rate of fluorescence recovery after photobleaching a defined membrane region in living cells (another measure of membrane microviscosity). Promigratory concentrations of membrane-active agents and growth factors promoted translocation of Rac, a member of the Rho family of small guanosine triphosphatases implicated in the formation of lamellipodia, into the plasma membrane, whereas LysoPC inhibited Rac translocation. These data indicate that angiogenic growth factors and certain lipophilic molecules can influence cell motility through alterations in membrane microviscosity and the ensuing changes in the localization of key regulatory molecules. P. K. Ghosh, A. Vasanji, G. Murugesan, S. J. Eppell, L. M. Graham, P. L. Fox, Membrane microviscosity regulates endothelial cell motility. Nature Cell Biol. 4 , 894-900 (2002). [Online Journal]

A Functional Link between Glucokinase Binding to Insulin Granules and Conformational Alterations in Response to Glucose and Insulin

Glucokinase (GK) activity is essential for the physiological regulation of insulin secretion by glucose. Because the enzyme exerts nearly total control over glucose metabolism in the beta-cell, even small changes in GK activity exert effects on glucose-stimulated insulin secretion and, consequently, the blood glucose concentration. Using quantitative imaging of multicolor fluorescent proteins fused to GK, we found that the association of GK with insulin granules is regulated by glucose in the beta-cell. Glucose stimulation increased the rate of fluorescence recovery after photobleaching of GK to insulin granules, indicating that GK is released into the cytoplasm after glucose stimulation. Changes in fluorescence resonance energy transfer between two different fluorescent protein variants inserted on opposing ends of GK were observed after glucose stimulation and correlated with increased enzyme activity. Furthermore, glucose-stimulated changes in GK regulation were blocked by two inhibitors of insulin secretion. Insulin treatment restored GK regulation in inhibited cells and stimulated GK translocation and activation by itself. Together, these data support a model for post-translational regulation of GK whereby insulin regulates both the association of GK with secretory granules and the activity of the enzyme within the pancreatic beta-cell.

Rebinding of IgE Fabs at haptenated planar membranes: measurement by total internal reflection with fluorescence photobleaching recovery.

In previous work, a general analytical theory for ligand rebinding at cell surfaces was developed for a reversible bimolecular reaction between ligands in solution and receptors on a membrane surface [Lagerholm, B. C., and Thompson, N. L. (1998) Biophys. J. 74, 1215-1228]. This theory can be used to predict theoretical forms for data obtained by using total internal reflection with fluorescence photobleaching recovery (TIR-FPR) [Thompson, N. L., Burghardt, T. P., and Axelrod, D. (1981) Biophys. J. 33, 435-454]. Thus, one method by which the rebinding theory can be tested is to use TIR-FPR. In the work described herein, the reversible kinetics of mouse monoclonal anti-dinitrophenyl (DNP) IgE Fabs at substrate-supported planar membranes composed of 25 mol % DNP-conjugated phosphatidylethanolamine and 75 mol % dipalmitoylphosphatidylcholine have been examined by using TIR-FPR. Data were obtained as a function of the Fab solution concentration. Higher Fab concentrations reduce rebinding (and increase the fluorescence recovery rate) because different Fab molecules compete for the same surface-binding sites. Data were also obtained for solutions containing different volume fractions of glycerol. In these measurements, higher glycerol concentrations increase rebinding (and decrease the fluorescence recovery rate) because the solution viscosity is increased and the Fab diffusion coefficient in solution is decreased. The TIR-FPR data were quantitatively compared with theoretical predictions which follow from the general theory for rebinding at the membrane surface. The data were consistent with the theoretical predictions and, therefore, provide experimental verification of the previously developed theory.

Exchangeability of actin in cardiac myocytes and fibroblasts as determined by fluorescence photobleaching recovery

Rhodamine (Rho)-labeled muscle and non-muscle actins were microinjected into cultured embryonic chicken cardiac myocytes and fibroblasts. After incorporation of the fluorescent actin analog into cellular structures, small areas of labeled structures were photobleached with a laser pulse, and fluorescence recovery (FR) was measured to determine the exchangeability of isoactins in these structures. With both Rho-muscle and Rho-non-muscle actins, the FR rate in any part of stress fibers was consistently faster than that observed in any part of myofibrils. Thus, although non-striated (proximal and terminal) portions of nascent myofibrils are similar in appearance and composition to stress fibers, our data clearly revealed differences in actin stability between these two structures. Further, although cardiomyocytes were incapable of discriminating between the incorporation of muscle and non-muscle actin isoforms into myofibrils, FR after photobleaching of Rho-muscle actin was faster than that of Rho-non-muscle actin in immature non-striated portions. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic non-muscle actin. Fluorescently labeled actin incorporated into non-striated (proximal and terminal) portions of myofibrils and terminal portions of stress fibers was found to be more stable than α-actinin. The relative stability of actin could facilitate the formation of nascent Z-bands of myofibrils and the reorganization of stress fibers at these portions.

Dynamics of actin in cardiac myofibrils and fibroblast stress fibers.

The exchangeability of actin in cardiac myofibrils and fibroblast stress fibers was investigated using fluorescent analogue cytochemistry in combination with fluorescence recovery (FR) after photobleaching. Living embryonic chicken cardiac myocytes and fibroblasts were microinjected with rhodamine (rh)-labeled muscle and nonmuscle actins. After incorporation of the fluorescent actin analogue into cellular structures, small areas of labeled structures were photobleached with a laser pulse. In cardiac myofibrils, FR in their proximal striated portions occurred at a slower rate than that in their proximal nonstriated and distal terminal portions with each rh-isoactin injected. Thus, nascent myofibrils at different developmental stages display different actin exchangeabilities. Further, in all portions of myofibrils, FR of rh-muscle actin was faster than that of rh-nonmuscle actin. This indicates that actin molecules in cardiac myofibrils cannot be readily exchanged by heterotypic nonmuscle actin. In fibroblasts, photobleaching of stress fibers yielded similar results in both their proximal mid-points and distal terminal portions, and the FR rate was consistently faster than that observed in any part of the myofibrils. This result seems to be related to the dynamic properties of actin filaments in stress fibers at all portions. Further, the fact that stress fibers possessed a similar exchange rate with muscle and nonmuscle actins appears to be related to a more primitive nature of stress fibers than myofibrils.

Fluorescence recovery after photobleaching of suspensions of vacuoles

In this work we derive theoretical expressions for the FRAP measured on a liquid suspension of vacuoles labelled by a fluorescent probe bound to the surface membrane of these vacuoles. The bleaching laser beam creates an inhomogeneity in the surface concentration of the probe molecules. We consider the case in which the randomization of these probe molecules on the vacuole surface occurs much faster than the fluorescent recovery due to the vacuole diffusion. For a given value of the bleaching parameter K, we found that the bleaching fraction of the fluorescent molecules and the fluorescence recovery rate are decreasing functions of the square ratio of the vacuole to the laser beam radius of the FRAP instrument.

Salt-induced [formula omitted] antiport in root plasma membrane of a glycophytic and halophytic species of tomato

Plasma membrane and tonoplast vesicles were isolated from roots of both the glycophytic cultivated tomato, Lycopersicon esculentum (Mill, cv. Heinz 1350) and its halophytic wild relative, Lycopersicon cheesmanii (Hook, C.H. Mull, ecotype 1401) grown under control and saline conditions. MgATP-dependent proton transport was measured by determining the rate of quench of quinacrine fluorescence. Rates of quench and rates of ATP hydrolysis were higher for both the plasma membrane and tonoplast from both species when grown under saline conditions. When ATPase activity was measured, the degree of stimulation of ATP hydrolysis in the presence of KCl, NaCl, and choline chloride was similar for the plasma membrane from control and salt grown plants. However, NaCl gave lower rates of proton transport than did KCl or choline chloride for the plasma membrane of both L. esculentum and L. cheesmanii grown under saline conditions. This may be interpreted as evidence of Na + H + antiport. A pH gradient (acid interior) was formed in vesicles by adding MgATP. After the establishment of a proton gradient, the effect of cations on proton efflux was estimated by adding EDTA to chelate the Mg 2+. Fluorescence recovery rate was used as an indication of the rate of proton efflux. The addition of Na + enhanced fluorescence recovery compared to K + in plasma membranes from both species grown under saline conditions. Addition of K + and valinomycin to the assay media did not affect Na + H + exchange, nor did addition of amiloride. No evidence was found for a Na + H + antiport mechanism in the tonoplast of either L. esculentum or L. cheesmanii regardless of growth conditions.

Further Evidence for Stachyose and Sucrose/H+ Antiporters on the Tonoplast of Japanese Artichoke (Stachys sieboldii) Tubers.

Vacuoles of Japanese artichoke (Stachys sieboldii) tubers accumulate up to 180 mM stachyose ([alpha]-galactose-[1->6]-[alpha]-galactose-[1->6]-[alpha]-glucose-[1 <->2]-[beta]-fructose) against a concentration gradient, probably by means of an active stachyose/H+ antiporter situated on the tonoplast. The goal of this study was to use isolated tonoplast vesicles to provide further evidence for the existence of such a transport mechanism. Therefore, vesicles were prepared from purified vacuoles of dormant tubers. ATP- and pyrophosphate (PPi)-dependent fluorescence quenching of the [delta]pH probe 9-amino-6-chloro-2-methoxyacridine (ACMA) indicated that these vesicles were capable of building up a pH gradient ([delta]pH, inside acid). The potent V-type H+-ATPase inhibitor bafilomycin prevented the formation of a [delta]pH in the vesicles. Bafilomycin (as well as nitrate, but not vanadate) also inhibited ATP hydrolysis, confirming the tonoplast origin of the isolated vesicles. Addition of stachyose (or sucrose, but not of mannitol) to energized vesicles caused a recovery of ACMA fluorescence, indicating a sugar-dependent dissipation of [delta]pH. The rate of fluorescence recovery was dependent on the external sugar concentration used. It displayed a single saturable response to increasing sugar concentrations. Apparent Km values of 52 and 25 mM were computed for stachyose and sucrose antiporter activities, respectively. It was also demonstrated that energized vesicles showed a much higher rate of [14C]stachyose (3 mM) and [14C]sucrose (1 mM) uptake than deenergized vesicles. The results obtained with isolated tonoplast vesicles were very similar to those obtained earlier with intact vacuoles and, therefore, confirm the existence of active stachyose and sucrose/H+ antiporters on the tonoplast of Stachys tuber vacuoles.

Surface binding kinetics of prothrombin fragment 1 on planar membranes measured by total internal reflection fluorescence microscopy

Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca(2+)-dependent membrane-binding characteristics of fluorescein-labeled bovine prothrombin-fragment 1 (F-BF1). Light scattering measurements demonstrated that F-BF1 bound to small unilamellar phosphatidylserine/phosphatidylcholine (25/75, mol/mol) vesicles with an apparent dissociation constant (1.5 +/- 0.2 microM) similar to that of unlabeled protein (1.1 +/- 0.1 microM). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF1 bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.2 microM) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF1 on 25 mol% PS planar surfaces were diffusion-influenced at F-BF1 solution concentrations less than or equal to 5 microM. Fluorescence recovery rates from samples of high F-BF1 concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF1 concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF1 solution concentrations greater than or equal to 10 microM gave average association and dissociation kinetic rates of approximately 10(5) M-1 s-1 and approximately 0.1 s-1, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.

Lateral mobility of integral proteins in red blood cell tethers

The red blood cell membrane is a complex material that exhibits both solid- and liquidlike behavior. It is distinguished from a simple lipid bilayer capsule by its mechanical properties, particularly its shear viscoelastic behavior and by the long-range mobility of integral proteins on the membrane surface. Subject to sufficiently large extension, the membrane loses its shear rigidity and flows as a two-dimensional fluid. These experiments examine the change in integral protein mobility that accompanies the mechanical phenomenon of extensional failure and liquidlike flow. A flow channel apparatus is used to create red cell tethers, hollow cylinders of greatly deformed membrane, up to 36-microns long. The diffusion of proteins within the surface of the membrane is measured by the technique of fluorescence redistribution after photobleaching (FRAP). Integral membrane proteins are labeled directly with a fluorescein dye (DTAF). Mobility in normal membrane is measured by photobleaching half of the cell and measuring the rate of fluorescence recovery. Protein mobility in tether membrane is calculated from the fluorescence recovery rate after the entire tether has been bleached. Fluorescence recovery rates for normal membrane indicate that more than half the labeled proteins are mobile with a diffusion coefficient of approximately 4 x 10(-11) cm2/s, in agreement with results from other studies. The diffusion coefficient for proteins in tether membrane is greater than 1.5 x 10(-9) cm2/s. This dramatic increase in diffusion coefficient indicates that extensional failure involves the uncoupling of the lipid bilayer from the membrane skeleton.

Contact-Dependent Intercellular Communication of Bovine Luteal Cells in Culture*

Mammalian gap junctions permit exchange of nutrients, ions, and regulatory molecules of less than 1.5 kDa among contacting communication-competent cells and may be important for regulation of luteal function and maintenance of luteal homeostasis. The present studies were designed to evaluate gap junction-mediated intercellular communication between bovine luteal cells in culture. Using a dye-coupling technique along with interactive laser cytometry, selected luteal cells were studied for the rate of contact-dependent fluorescence redistribution after photobleaching. The rate of communication, reported as the rate of fluorescence recovery (percentage per min), was determined for steroidogenic cells as follows: 1) small luteal cells contacting only small luteal cells, 2) large luteal cells contacting only small luteal cells, and 3) large luteal cells contacting only large luteal cells. In addition, the effects of known regulators of luteal function [LH, prostaglandin F2 alpha (PGF), and forskolin] on the rate of intercellular communication were determined. Small luteal cells communicated rapidly with each other, exhibiting an initial rate of fluorescence recovery of 4.1 +/- 0.1%/min (n = 187). The rate of small cell-small cell communication was unaffected by LH and PGF. For large luteal cells contacting small luteal cells, however, LH and PGF stimulated (P less than 0.02) the rate of communication compared with no hormone [1.6 +/- 0.2 (n = 18) and 1.5 +/- 0.6 (n = 20) vs. 0.8 +/- 0.3%/min (n = 27), respectively]. LH and PGF in combination, however, did not enhance the rate (0.6 +/- 0.2%/min; n = 19) of large cell-small cell communication. In contrast, forskolin significantly stimulated both small cell-small cell and large cell-small cell communication rates compared with no forskolin [34% increase (n = 48) and 50% increase (n = 23), respectively]. Large luteal cells did not communicate with each other under any condition tested. Transmission electron microscopy revealed the presence of numerous gap junction-like structures in bovine luteal cells in culture. These data suggest that luteal cells are capable of intercellular communication and that the rate of communication may be influenced by hormones. Contact-dependent intercellular communication among luteal cells may, therefore, play a significant role in the regulation of luteal function.

Normal band 3-cytoskeletal interactions are maintained on tanktreading erythrocytes

Normal nonnucleated erythrocytes subjected to continuous hydrodynamic shear exhibit membrane deformation or "tanktreading," a process important for reduction of the bulk viscosity of circulating blood. To characterize the effect of this unique process on the erythrocyte membrane we have measured the lateral diffusion of band 3 during tanktreading. Band 3 is normally constrained through interactions with the spectrin-actin cytoskeleton, therefore, any significant disruption of these interactions would result in alterations in band 3 dynamics. Band 3 of human erythrocytes was labeled with dichlorotriazinyl amino fluorescein. After laser photobleaching of an equatorial stripe, fluorescence images were recorded from cells in the presence or absence of shear. The amplitude of induced nonuniformity in the surface distribution of fluorescence was calculated directly from images of unsheared cells. In shear the bleached line rotated with the tanktreading motion of the cells. The surface integral of fluorescence oscillated with this motion. For this case, the amplitude of photobleaching-induced nonuniformity was defined as the amplitude at the fundamental frequency of fast Fourier transforms in time of the oscillations. Shear stress-induced membrane flow did not interrupt the linkage of band 3 with the erythrocyte cytoskeleton. Diffusion coefficient and mobile fraction (1.5 +/- 0.5 x 10(-10) cm2/s and 54 +/- 11%, respectively) were unaffected by shear. The rate of fluorescence recovery of cells in shear was also similar at the centers and at the edges, where in-plane shear forces are maximal.

A fluorescence photobleaching assay of gap junction-mediated communication between human cells.

Gap junction-mediated communication between contiguous cells has been implicated in the regulation of cell proliferation and differentiation. This report describes a new technique to measure cell-cell communication, gap fluorescence redistribution after photobleaching, which is based on the diffusion-dependent return of 6-carboxyfluorescein-mediated fluorescence in a photobleached cell that is in contact with other fluorescently labeled cells. Fluorescence recovery rates are interpreted as dye transport across gap junctions. Results of experiments on normal human fibroblasts and human teratocarcinoma cells show that this technique can measure rapid dye transfer and detect inhibition of communication (between teratocarcinoma cells) by the tumor promoters 12-O-tetradecanoyl-phorbol-13-acetate and the pesticide dieldrin.

Lateral diffusion of wheat germ agglutinin-labeled glycoconjugates in the membrane of differentiating HL-60 and U-937 cells assessed with fluorescence recovery after photobleaching (FRAP).

The promyelocytic leukemia cell line HL-60 and the histiocytic cell line U-937 were grown in suspension culture. They were induced to differentiate during 5-d cultivation in the presence of dimethylsulfoxide (DMSO; 1.3% w/v) or phorbol-12-myristate-acetate (PMA; 10(-7) M), which yields granulocyte- and macrophage-like cells, respectively. Differentiation was evidenced by increased capacity to recognize and phagocytize IgG- or complement-coated yeast particles. Aliquots taken from the cultures with and without DMSO (or PMA) were spun down directly on glass microscope slides, washed, labeled with fluoresceinated wheat germ agglutinin (WGA), and directly examined at room temperature for the rate of fluorescence recovery after photobleaching (FRAP). It was found that cultivation of the HL-60 and the U-937 cells in the presence of DMSO, which yields granulocyte-like cells, reduced the average value of lateral diffusion coefficient D (X 10(10] from 1.72 +/- 0.13 cm2s-1 to 0.97 +/- 0.13 cm2s-1 and from 1.77 +/- 0.11 cm2s-1 to 0.82 +/- 0.13 cm2s-1, respectively. U-937 cells grown with PMA also showed a reduction of D(X 10(10] to 0.88 +/- 0.10 cm2s-1. There was a larger immobile fraction of fluorescence in the HL-60 cells than in the U-937 cells, viz., 70-80% compared to 10-50%. The total number of binding sites for WGA was not altered, but the surface density changed, since the HL-60 and the U-937 cells became smaller and larger, respectively, when grown in the presence of DMSO. It is concluded that differentiation reduces the average lateral mobility of the WGA-binding membrane component by a factor around 2.