Mobility measurement by analysis of fluorescence photobleaching recovery kinetics.

Fluorescence photobleaching recovery (FPR) denotes a method for measuring two-dimensional lateral mobility of fluorescent particles, for example, the motion of fluorescently labeled molecules in approximately 10 mum2 regions of a single cell surface. A small spot on the fluorescent surface is photobleached by a brief exposure to an intense focused laser beam, and the subsequent recovery of the fluorescence is monitored by the same, but attenuated, laser beam. Recovery occurs by replenishment of intact fluorophore in the bleached spot by lateral transport from the surrounding surface. We present the theoretical basis and some practical guidelines for simple, rigorous analysis of FPR experiments. Information obtainable from FPR experiments includes: (a) identification of transport process type, i.e. the admixture of random diffusion and uniform directed flow; (b) determination of the absolute mobility coefficient, i.e. the diffusion constant and/or flow velocity; and (c) the fraction of total fluorophore which is mobile. To illustrate the experimental method and to verify the theory for diffusion, we describe some model experiments on aqueous solutions of rhodamine 6G.     

Direct imaging of dehydrogenase activity within living cells using enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP)

Reduced nicotine adenine dinucleotide (NADH) is a key metabolite involved in cellular energy conversion and many redox reactions. We describe the use of confocal microscopy in conjunction with enzyme-dependent fluorescence recovery after photobleaching (ED-FRAP) of NADH as a topological assay of NADH generation capacity within living cardiac myocytes. Quantitative validation of this approach was performed using a dehydrogenase system, in vitro. In intact cells the NADH ED-FRAP was sensitive to temperature (Q(10) of 2.5) and to dehydrogenase activation by dichloroacetate or cAMP (twofold increase for each). In addition, NADH ED-FRAP was correlated with flavin adenine dinucleotide (FAD(+)) fluorescence. These data, coupled with the cellular patterns of NADH ED-FRAP changes with dehydrogenase stimulation, suggest that NADH ED-FRAP is localized to the mitochondria. These results suggest that ED-FRAP enables measurement of regional dynamics of mitochondrial NADH production in intact cells, thus providing information regarding region-specific intracellular redox reactions and energy metabolism.     

Analysis of rhodamine and fluorescein-labeled F-actin diffusion in vitro by fluorescence photobleaching recovery.

Properties of filamentous acetamidofluorescein-labeled actin and acetamidotetramethylrhodamine-labeled actin (AF and ATR-actin, respectively) were examined to resolve discrepancies in the reported translational diffusion coefficients of F-actin measured in vitro by FPR and other techniques. Using falling-ball viscometry and two independent versions of fluorescence photobleaching recovery (FPR), the present data indicate that several factors are responsible for these discrepancies. Gel filtration chromatography profoundly affects the viscosity of actin solutions and filament diffusion coefficients. ATR-actin and, to a lesser degree, AF-actin show a reduction in viscosity in proportion to the fraction labeled, presumably due to filament shortening. Actin filaments containing AF-actin or ATR-actin are susceptible to photoinduced damage, including a covalent cross-linking of actin protomers within filaments and an apparent cleavage of filaments detected by a decrease of the measured viscosity and an increase in the measured filament diffusion coefficients. Quantum yields of the two photoinduced effects are quite different. Multiple cross-links are produced relative to each photobleaching event, whereas less than 1% filament cleavage occurs. Substantial differences in the filament diffusion coefficients measured by FPR are also the result of differences in illumination geometry and sampling time. However, under controlled conditions, FPR can be used as a quantitative tool for measuring the hydrodynamic properties of actin filaments. Incremented filament shortening caused by photoinduced cleavage or incremental addition of filament capping proteins produces a continuous and approximately linear increase of filament diffusion coefficients, indicating that filaments are not associated in solution. Our results indicate that actin filaments exhibit low mobilities and it is inferred that actin filaments formed in vitro by column-purified actin, under standard conditions, are much longer than has conventionally been presumed.     

Measurement of the lateral mobility of cell surface components in single living cells by fluorescence recovery after photobleaching

The use of fluorescence recovery after photobleaching (FRAP) techniques to monitor the lateral mobility of plant lectin-receptor complexes on the surface of single, living mammalian cells is described in detail. FRAP measurements indicate that over 75% of the wheat germ agglutinin receptor (WGA-receptor) complexes on the surface of human embryo fibroblasts are mobile. These WGA-receptor complexes diffuse laterally (as opposed to flow) on the cell surface with a diffusion coefficient in the range of 2 × 10^?11 to 2 × 10^?10 cm²/sec. Both the percentage of mobile WGA-receptor complexes and the mean diffusion coefficient of these complexes are higher than that obtained from earlier FRAP measurements of the mobility of concanavalin A-receptor (Con A-receptor) complexes in a variety of cell types. The possible reasons for the differing mobilities of WGA and Con A receptors are discussed.     

Assembly of Acanthamoeba actin in the presence of Acanthamoeba profilin measured by fluorescence photobleaching recovery

Assembly of Acanthamoeba actin, of which trace quantities had been labeled with 5-(iodoacetamido)-fluorescein, was quantified using the modulation detection method of fluorescence photobleaching recovery (FPR). This technique permits explicit determination of the fraction of labeled actin incorporated into filaments and the translational diffusion coefficients of the filaments, from which filament length can be calculated. Addition of Acanthamoeba profilin in molar ratios to actin of about 1.1:1 and 2.3:1 retarded the initial kinetics of assembly (induced by addition of 2mM Mg+2) and reduced the fraction of actin incorporated into filaments. The diffusion coefficients of filaments formed were greatly changed by the presence of profilin at short times, but the differences became increasingly smaller at longer times. After 26 hr. the filaments formed in 1.1:1 profilin were about 12% shorter and in 2.3:1 profilin were about 20% shorter than filaments formed by actin alone under the same conditions.     

Turnover of actin in Chlamydomonas flagella detected by fluorescence recovery after photobleaching (FRAP)

Recent indirect observations have suggested that various axonemal proteins in cilia and flagella of live cells undergo turnover independently of shortening or elongation of the axoneme. To gain direct evidence, here we examined using a FRAP (fluorescence recovery after photobleaching) technique whether actin, a subunit of inner arm dynein, is being turned over in Chlamydomonas flagella. Fluorescently labeled rabbit actin was introduced by electroporation into the cells of ida5oda1, a double mutant between oda1 lacking outer arm dynein and ida5 lacking several species of inner arm dyneins due to the absence of a conventional-type actin. In actin-loaded cells, flagella became motile and fluorescent due to incorporation of inner-arm dyneins containing the labeled actin. Cells were sandwiched between an agar layer and a coverslip so as to restrict flagellar movement. After a small portion of a flagellum was photobleached, the fluorescence intensity in the bleached area was monitored with a sensitive video camera. The fluorescence intensity in the photobleached region was found to recover 10-40% of the original level over several tens of minutes without changing its position. The time course and extent of the recovery varied greatly from one cell to another, suggesting that the turnover depends on cellular conditions. Western blot analysis indicated that 70-80% of flagellar actin was associated with the axoneme. Hence this experiment provides direct evidence that an axonemal component undergoes dynamic exchange in stationary flagella.     

Mobility of late endosomal and lysosomal markers on phagosomes analyzed by fluorescence recovery after photobleaching

During phagosome maturation, the late endosomal marker Rab7 and the lysosomal marker LAMP1 localize to the phagosomes. We investigated the mobility of Rab7 and LAMP1 on the phagosomes in macrophages by fluorescence recovery after photobleaching (FRAP) analysis. Rab7 was mobile between the phagosomal membrane and the cytosol in macrophages that ingested latex beads during phagosome maturation. The addition of interferon-γ (IFN-γ) restricted this mobility, suggesting that Rab7 is forced to bind to the phagosomal membrane by IFN-γ-mediated activation. Immobilization of LAMP1 on the phagosomes was observed irrespective of IFN-γ-activation. We further examined the mobility of Rab7 on the phagosomes containing Mycobacterium bovis BCG by FRAP analysis. The rate of fluorescence recovery for Rab7 on mycobacterial phagosomes was lower than that on the phagosomes containing latex beads, suggesting that mycobacteria impaired the mobility of Rab7 and arrested phagosome maturation.     

Molecular diffusion within sol-gel derived matrices viewed via fluorescence recovery after photobleaching.

A suitable matrix to host enzymes for biosensor applications should encage and retain the bioactive species, while allowing it to be accessed to exploit its catalytic properties. Sol-gel derived monoliths are promising in this aspect. Molecular diffusion was monitored using fluorescence labelled proteins and unbound fluorescence dye molecules (representative of enzyme substrates) and their interaction with and mobility within the host assessed using time-resolved fluorescence anisotropy and fluorescence recovery after photobleaching observed via confocal microscopy.     

Lateral diffusion of rhodopsin in photoreceptor cells measured by fluorescence photobleaching and recovery.

Frog rod outer segments were labeled with the sulfhydryl-reactive label iodoacetamido tetramethylrhodamine. The bulk of the label reacted with the major disk membrane protein, rhodopsin. Fluorescence photobleaching and recovery (FPR) experiments on labeled rods showed that the labeled proteins diffused rapidly in the disk membranes. In these FPR experiments we observed both the recovery of fluorescence in the bleached spot and the loss of fluorescence from nearby, unbleached regions of the photoreceptor. These and previous experiments show that the redistribution of the fluorescent labeled proteins after bleaching was due to diffusion. The diffusion constant, D, was (3.0 +/- 10(-9) cm2 s-1 if estimated from the rate of recovery of fluorescence in the bleached spot, and (5.3 +/- 2.4) x 10(-9) cm2 s-1 if estimated from the rate of depletion of fluorescence from nearby regions. The temperature coefficient, Q10, for diffusion was 1.7 +/- 0.5 over the range 10 degrees--29 degrees C. These values obtained by FPR are in good agreement with those previously obtained by photobleaching rhodopsin in fresh, unlabeled rods. This agreement indicates that the labeling and bleaching procedures required by the FPR method did not significantly alter the diffusion rate of rhodopsin. Moreover, the magnitude of the diffusion constant for rhodopsin is that to be expected for an object of its diameter diffusing in a bilayer with the viscosity of the disk membrane. In contrast to the case of rhodopsin, FPR methods applied to other membrane proteins have yielded much smaller diffusion constants. The present results help indicate that these smaller diffusion constants are not artifacts of the method but may instead be due to interactions the diffusing proteins have with other components of the membrane in addition to the viscous drag imposed by the lipid bilayer.     

A new simple method of preparing actin from chicken gizzard

A simple method for preparing actin from chicken gizzard was described. This method takes advantage of a property of gizzard tropomyosin, that is, that it does not form Mg paracrystals readily.     

Detection and characterization of actin monomers, oligomers, and filaments in solution by measurement of fluorescence photobleaching recovery

Fluorescence photobleaching recovery (FPR) was measured to determine the diffusion coefficient of fluorescein-labeled G-actin in low-salt buffer. The result obtained, 7.15 +/- 0.35 X 10(-7) cm2/s, is in good agreement with that computed from the molecular weight, partial specific volume, and sedimentation coefficient, but is higher than previously obtained values. It is demonstrated from theory that at low ionic strength, the electrostatic contribution to the intrinsic viscosity leads to an overestimate of the hydrodynamic eccentricity of G-actin. Data from FPR, sedimentation, and fluorescence polarization experiments all indicate that the true low-salt form of the actin monomer has an axial ratio less than or equal to 3.0. The G-F transformation of actin was also observed by measurement of FPR during the assembly phase, in the steady state, and in the presence of ligands such as cytochalasin and aldolase. Each FPR record in general yields three data: relative proportion of rapidly and slowly diffusing actin, diffusion coefficient for the high-mobility fraction, and a mean diffusion coefficient for the low-mobility fraction. A relation between the mean low-mobility diffusion coefficient and the number-average filament length is derived and applied to the analysis of FPR data. Under typical conditions, the average filament length was much greater than 10 micron in the steady state. Cytochalasin D was found to decrease filament length and total amount of filament proportionally; total filament number was not greatly affected. In all polymerizations of G-actin, the high-mobility material observed in situ was found to be essentially monomeric actin. Relatively stable oligomers of actin were separated by fractionating G-AF-actin by gel filtration in 50 microM MgCl2 at 4 degrees C. On the basis of the diffusion coefficient, we conclude that monomer and dimer constitute the major particle types present under these conditions. Sedimentation of labeled actin polymerized in 1.0 mM MgCl2 yielded a graded supernatant that contained actin oligomers significantly larger than the monomer.     

Fluorescence photobleaching recovery in solutions of labeled actin.

We have demonstrated that the technique of fluorescence photobleaching recovery (FPR) can be used to examine the state of a single component in complex self-assembling macromolecular systems. Polymerization of actin, initiated by addition of salt or Mg+2 to a low-ionic-strength solution of G-actin, has been observed by sequential measurement of FPR with the aid of fluorescein-labeled actin. Solutions of actin which had been labeled using 5-iodoacetamido fluorescein (5-IAF) showed anomalous recovery of fluorescence above the initial value, which indicates a photoinduced increase in local polymerization. No such anomaly was observed with actin that had been labeled with fluorescein isothiocyanate (FITC). The FPR data are directly interpretable in terms of the fraction of labeled protein that is immobilized in the supramolecular assembly and in terms of the average diffusion coefficient of the mobile fraction. Our data are consistent with the "treadmill" model of actin polymerization, in that they show that actin is present under polymerizing conditions either as a high polymer or as monomer or low oligomer. We believe that the FPR technique can be applied to the study of many types of reconstituted motile or cytoskeletal systems in vitro or in vivo.     

Mobility of filamentous actin in living cytoplasm

Filamentous actin in living cultured cells was labeled by microinjecting trace amounts of rhodamine-phalloidin (rh-pha) as a specific, high-affinity probe. The microinjection caused no detectable effect on cell morphology or cell division. The distribution of rh-pha- labeled filaments was then examined in dividing cells using image- intensified fluorescence microscopy, and the exchangeability of labeled filaments along stress fibers was studied during interphase using fluorescence recovery after photobleaching. rh-pha showed a rapid concentration at the contractile ring during cell division. In addition, recovery of fluorescence after photobleaching occurred along stress fibers with a halftime as short as 8 min. These observations suggest that at least some actin filaments undergo continuous movement and reorganization in living cells. This dynamic process may play an important role in various cellular functions.     

Theoretical analysis of fluorescence photobleaching recovery experiments.

We derive an exact closed formula for the fluorescence recovery curve measured in fluorescence photobleaching recovery experiments employing uniform circular laser beams. In contrast to the expression used currently, this result is very simple and free of mathematical drawbacks, thus facilitating the quantitative analysis of experimental data.     

Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery

Multiphoton fluorescence photobleaching recovery (MP-FPR) is a technique for measuring the three-dimensional (3D) mobility of fluorescent molecules with 3D spatial resolution of a few microns. A brief, intense flash of mode-locked laser light pulses excites fluorescent molecules via multiphoton excitation in an ellipsoidal focal volume and photobleaches a fraction. Because multiphoton excitation of fluorophores is intrinsically confined to the high-intensity focal volume of the illuminating beam, the bleached region is restricted to a known, three-dimensionally defined volume. Fluorescence in this focal volume is measured with multiphoton excitation, using the attenuated laser beam to measure fluorescence recovery as fresh unbleached dye diffuses in. The time course of the fluorescence recovery signal after photobleaching can be analyzed to determine the diffusion coefficient of the fluorescent species. The mathematical formulas used to fit MP-FPR recovery curves and the techniques needed to properly utilize them to acquire the diffusion coefficients of fluorescently labeled molecules within cells are presented here. MP-FPR is demonstrated on calcein in RBL-2H3 cells, using an anomalous subdiffusion model, as well as in aqueous solutions of wild-type green fluorescent protein, yielding a diffusion coefficient of 8.7 x 10(-7) cm(2)s(-1) in excellent agreement with the results of other techniques.     

Photocrosslinking of calmodulin and/or actin to chicken gizzard caldesmon

The two sulfhydryl groups of chicken gizzard caldesmon were specifically labeled with a photoreactive crosslinker, benzophenone-maleimide, to study its interactions with calmodulin and/or actin. When incubated with F-actin caldesmon crosslinks to a single actin monomer; it can, however, crosslink to up to two calmodulin molecules in the presence, but not in the absence, of Ca2+. Thus caldesmon may have two calmodulin-binding sites, each containing, or being near, one of the two thiol residues. One of these two sites may also be adjacent to the actin-binding site. A calmodulin-binding fragment of caldesmon resulting from cyanogen bromide digestion crosslinks to a single calmodulin molecule, also in a Ca2+-dependent manner. Crosslinking of calmodulin to caldesmon does not prevent the latter from binding F-actin, suggesting that calmodulin and actin do not compete with each other for the same binding site(s) on the caldesmon molecule.     

Characterization of the actin polymerization-inhibiting protein from chicken gizzard smooth muscle

An actin polymerization-inhibiting protein, occurring in crude preparations of vinculin from chicken gizzard, has been found to be heterogeneous. The molecular masses of the polymerization-inhibiting peptides have been reported to range from 20 kDa to 80 kDa [Schr?er, E. & Wegner, A (1985) Eur. J. Biochem. 153, 515-520]. In this paper, a 21-kDa peptide was isolated from the bulk of the other peptides by gel chromatography. The 21-kDa peptide was identified as a polymerization-inhibiting peptide by its ability to retard nucleated actin polymerization and to bind polymeric actin when it was blotted onto nitrocellulose. Antiserum raised to the 21-kDa peptide was found to react with almost all peptides of the blotted heterogeneous polymerization-inhibiting protein. The same peptides which reacted with antiserum cosedimented with polymeric actin. The major peptides of the blotted polymerization-inhibiting protein bound polymeric actin. The largest peptide which reacted with antiserum and cosedimented with polymeric actin had a molecular mass of 85 kDa. The results suggest that the preparation of polymerization-inhibiting protein contains mainly polymerization-inhibiting peptides and only some contaminants, and that all the polymerization-inhibiting peptides are proteolytic fragments stemming from a common precursor.