Dual-view microscopy with a single camera: real-time imaging of molecular orientations and calcium.

A new microscope technique, termed "W" (double view video) microscopy, enables simultaneous observation of two different images of an object through a single video camera or by eye. The image pair may, for example, be transmission and fluorescence, fluorescence at different wavelengths, or mutually perpendicular components of polarized fluorescence. Any video microscope can be converted into a dual imager by simple insertion of a small optical device. The continuous appearance of the dual image assures the best time resolution in existing and future video microscopes. As an application, orientations of actin protomers in individual, moving actin filaments have been imaged at the video rate. Asymmetric calcium influxes into a cell exposed to an intense electric pulse have also been visualized.

Regulatory mechanisms of the acrosome reaction revealed by multiview microscopy of single starfish sperm.

The acrosome reaction in many animals is a coupled reaction involving an exocytotic step and a dramatic change in cell shape. It has been proposed that these morphological changes are regulated by intracellular ions such as Ca2+ and H+. We report here simultaneous visualization, under a multiview microscope, of intracellular free Ca2+ concentration ([Ca2+]i), intracellular pH (pHi), and morphological changes in a single starfish sperm (Asterina pectinifera). [Ca2+]i and pHi were monitored with the fluorescent probes indo-1 and SNARF-1, respectively. The acrosome reaction was induced with ionomycin. After the introduction of ionomycin in the medium, [Ca2+]i increased gradually and reached a plateau in approximately 30 s. The fusion of the acrosomal vacuole took place abruptly before the plateau, during the rising phase. Although the speed of the [Ca2+]i increase varied among the many sperm tested, exocytosis in all cases occurred at the same [Ca2+]i of approximately 2 microM (estimated using the dissociation constant of indo-1 for Ca2+ of 1.1 microM). This result suggests that the exocytotic mechanism in starfish sperm responds to [Ca2+]i rapidly, with a reaction time of the order of one second or less. Unlike the change in [Ca2+]i, an abrupt increase in pHi was observed immediately after exocytosis, suggesting the presence of a proton mobilizing system that is triggered by exocytosis. The rapid increase in pHi coincided with the formation of the acrosomal rod and the beginning of vigorous movement of the flagellum, both of which have been proposed to be pHi dependent. The exocytotic event itself was visualized with the fluorescent membrane probe RH292. The membrane of the acrosomal vacuole, concealed from the external medium in an unreacted sperm, was seen to fuse with the plasma membrane.

Degree of dissociation of apohemoglobin studied by nano-second fluorescence-polarization technique.

A fluorescent dye 1-anilino-8-naphthalene sulfonate was complexed with human apohemoglobin and sperm whale apomyoglobin. Nanosecond fluorescence-polarization kinetics were measured for each of these complexes in KC1 solutions to obtain their fluorescence lifetimes and rotational correlation times. The rotational correlation time of apohemoglobin-dye complex was found to be 21 ns, which was about twice that of apomyoglobin-dye complex, 11 ns. These values were constant over an ionic strength range from 0 to 1.7. Circular dichroism spectra (215-300 nm) and fluorescence lifetimes of the complexes were also found to be independent of the ionic strength, indicating that no gross conformational change occurs with the change in the salt concentration, These results suggest that apohemoglobin remains dimeric over the ionic-strength range examined.

Strength and lifetime of the bond between actin and skeletal muscle alpha-actinin studied with an optical trapping technique.

The force required to break the bond between skeletal muscle actin and alpha-actinin (unbinding force) was measured at the level of individual molecules with an optical trapping technique. An actin filament, to the barbed-end of which was attached a gelsolin-coated polystyrene bead, was bound to alpha-actinin molecules adsorbed to a nitrocellulose-coated glass surface (approximately equal to 1 alpha-actinin molecule per 1 micron actin filament). The filament-bound bead was held by the optical trap and the force was applied to break the bond by pulling the bead. The unbinding force ranged from 1.4 to 44 pN. The average magnitude of the force was approximately equal to 18 pN. As the probability of the bond breakage has been suggested to be governed by the magnitude of the external force, the relationship was studied between the magnitude of the unbinding force and the time required to break the bond (unbinding time). The unbinding time ranged from approximately equal to 0.1 to approximately equal to 20 seconds, and tended to become shorter as the unbinding force became larger. The unbinding time seemed to be classifiable into two major groups: one group having a time value of 1 sec or less and the other having a time value ranging from several to 20 seconds. This suggests the existence of at least two classes of the actin-actinin bonds.

Voltage-induced pore formation and hemolysis of human erythrocytes.

Isotonic suspensions of human erythrocytes were exposed to single electric pulses of intensity at a few kV/cm and duration in microseconds. Upon pulsation, the cell membranes became permeable to Na+ and K+, and the erythrocytes eventually hemolysed through the colloid osmotic effect of hemoglobin. The enhanced permeability is attributed to the formation of pores in the cell membranes. These pores are formed within a fraction of a microsecond, once the transmembrane potential induced by the applied electric field reaches a critical value of 1.0 V. Increased field intensity and pulse duration, or pulsation at low ionic strengths all expand the pore size, leading to an accelerated hemolysis reaction. In contrast to this expansion process, the initial step of pore formatin is governed solely by the magnitude of the transmembrane potential: the critical value of the potential stays essentially constant in media of different ionic strengths, nor does it change appreciably with varying pulse duration. An abrupt increase in membrane permeability at a transmembrane potential adround 1 V has been observed in many cellular systems. It is suggested that a similar mechanism of pore formation may apply to these systems as well.

Dynamics of Z-form DNA.

The torsional and bending rigidities of Z-form DNA have been studied by nanosecond fluorescence anisotropy measurements of intercalated ethidium. The results suggested that Z-form DNA was considerably more flexible than B-form DNA. We have investigated the temperature dependence of the rigidity of B- and Z-form DNA and found that the temperature dependence of the torsional rigidity of Z-form DNA was remarkably lower than that of B-form DNA.

Reversible and irreversible modification of erythrocyte membrane permeability by electric field.

Electric fields of a few kV/cm and of duration in microseconds are known to implant pores of limited size in cell membranes. We report here a study of kinetics of pore formation and reversibility of pores. Loading of biologically active molecules was also attempted. For human erythrocytes in an isotonic saline, pores allowed passive Rb+ entry formed within 0.5 microsecond when a 4 kV/cm electric pulse was used. Pores that admitted oligosaccharides were introduced with an electric pulse of a longer duration in an isosmotic mixture of NaCl and sucrose. These pores were irreversible under most circumstances, but they could be resealed in an osmotically balanced medium. A complete resealing of pores that admitted Rb+ took approximately 40 min at 37 degrees C. Resealing of pores that admitted sucrose took much longer, 20 h, under similar conditions. In other cell types, resealing step may be omitted due to stronger membrane structures. Experimental protocols for loading small molecules into cells without losing cytoplasmic macromolecules are discussed.

Changes of the DNA packaging mode during boar sperm maturation.

Changes in the mode of DNA packaging in nuclei during spermatogenesis were studied by measuring of the fluorescence anisotropy decay of an ethidium dye intercalated in the DNA in whole nuclei. The nuclei were isolated from boar spermatid or sperm cells at three distinct stages of spermatogenesis: just before the completion of a maturation process in the testis (late spermatid), immediately after a subsequent transformation into spermatozoa (caput spermatozoon), and after full maturation (cauda spermatozoon). Although these three kinds of nucleus were morphologically indistinguishable from each other, the anisotropy decay detected a clear difference. In the late spermatid nuclei, in which the replacement of histones by protamine was still in progress, the anisotropy decayed extensively. The decay suggested that the DNA in the spermatid nuclei contained very flexible regions, in which the interaction of the DNA and proteins may be weak. The rapid and extensive anisotropy decay was absent in the caput and cauda nuclei. The flexible portions must have turned into very rigid structure during transformation from the late spermatid into the caput spermatozoon.

Fluorescence quenching measurements of the membrane bound lipid haptens with different length spacers.

Dansyl lipid haptens with three different length (short, intermediate and long) spacers have been incorporated into DMPC or DPPC liposomes. Anti-DNS-IgG bound most efficiently to these liposomes containing lipid haptens with an intermediate length spacer, although the binding efficiency became more increased when liposomes were made of a mixture of phospholipid (DMPC or DPPC) and cholesterol. To explain these results we have measured the accessibility of dansyl lipid haptens in liposomal membranes by the fluorescence quenching method. It was found that the dansyl haptens located on the surfaces of DMPC (or DPPC) membranes and fluorescence quenchers (iodide ions) possessed almost similar accessibility for the dansyl haptens with different length spacers. However, in the DMPC (or DPPC) membranes with 50% cholesterol, a part of the dansyl haptens became buried into the interior of the liposomal membranes depending on the length of the spacer and another part removed into the aqueous solution with greater affinity for antibody. These results were explained well by our recent model for antibody binding to the membrane-bound lipid haptens.

The effect of cytochrome oxidase on lipid chain dynamics. A nanosecond fluorescence depolarization study.

Molecular motions in membranes composed of purified cytochrome oxidase (EC 1.9.3.1) and synthetic lipid (L-alpha-dimyristoylphosphatidylcholine or L-alpha-dioleoylphosphatidylcholine) at various ratios were investigated with a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene. Nanosecond fluorescence depolarization kinetics of the probe showed that the rod-shaped probe molecules perform a fast wobbling motion (restricted rotation) in all membranes studied, presumably reflecting the motion of lipid acyl chains. At temperatures where the pure lipid was in the liquid-crystalline phase, presence of cytochrome oxidase reduced the angular range of the wobbling motion, whereas its rate, the wobbling diffusion constant, was unaffected. On the other hand, incorporation of the protein into lipid in the gel phase resulted in the increase in the wobbling diffusion constant while the range of the wobbling motion remained the same. A time-dependent view of lipid dynamics that accounts for the above findings, as well as the results of recent electron spin resonance and nuclear spin resonance studies of protein-lipid interactions, is proposed.

Nanosecond time-resolved fluorescence investigations of temperature-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicles and solubilized systems.

Intrinsic and lipid phase transition-induced conformational changes in cytochrome oxidase in phosphatidylcholine vesicle and solubilized systems were examined by the fluorescence lifetime of N-(1-anilinonaphthyl-4)-maleimide conjugated with the enzyme. The time-dependent fluorescence intensity of N-(1-anilinonaphthyl-4)-maleimide attached to cytochrome oxidase was described as a triple exponential decay. Both the intrinsic and lipid phase transition-induced conformational changes were detectable in plots of the average lifetime against temperature. In most cases a peak occurred at the temperature of the conformational change. The time-dependent emission anisotropy showed that N-(1-anilinonaphthyl-4)-maleimide embedded in cytochrome oxidase in phosphatidylcholine vesicles underwent a rapid restricted wobbling within a cone. The half-angle of the cone was around 30 degrees for cytochrome oxidase in dimyristoyl phosphatidylcholine vesicles.

The dynamics of lipid motion in sarcoplasmic reticulum membranes determined by steady-state and time-resolved fluorescence measurements on 1,6-diphenyl-1,3,5-hexatriene and related molecules.

Steady-state and time-resolved fluorescence anisotropy measurements were made on 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene (TMA-DPH) and 1-acyl-2-(DPH)-phosphatidylcholine (DPH-PC) incorporated into sarcoplasmic reticulum membranes. The results were analysed in terms of the 'wobbling-in-cone' model. Considerable differences in the fluorescence parameters were found. In particular TMA-DPH and DPH-PC showed a smaller cone angle, relating to the range of acyl chain motion, compared to DPH, taken to be a reflection of a difference in probe locations. The influence of the protein component was also found to restrict DPH motion more than TMA-DPH and DPH-PC. Effectiveness in assessment of perturbation of the membrane by the non-esterified fatty acid, oleic acid again revealed differences. The steady-state anisotropy decreased on addition of oleic acid; a recovery to control values was observed with DPH but not with the other probes. Time-resolved parameters followed the same pattern. The results of this work demonstrated the effectiveness of these three probes in revealing differences in membrane properties, such as protein and fatty acid perturbation of membrane lipid structure and dynamics.

Rotational dynamics of immunoglobulins with fluorescent haptens on a membrane surface.

The rotational dynamics of rabbit immunoglobulin G with fluorescent lipid haptens on a membrane surface has been studied by nanosecond fluorescence emission anisotropic spectroscopy. It has been found that the rotational angles of the antibody are very restricted on the membrane, but that the rotation rate itself is not appreciably lower than that in solution, and is independent of the membrane fluidity.

Direct measurement of the torsional rigidity of single actin filaments.

Flexural and torsional rigidities of actin filaments are important factors in cell motility and muscle contraction, where actin filaments serve as mechanical elements. The flexural rigidity has already been determined by directly observing the bending of individual filaments under a microscope, but measurement of the torsional rigidity has been relatively scarce and indirect, because torsion of an actin filament is difficult to visualize. This paper shows that the torsional rigidity can be measured directly by visualizing the torsional Brownian motion of a single actin filament with a novel methodology based on an optical trapping technique. Actin filaments (F-actin) were prepared by polymerizing actin monomers binding Ca2+ ion or Mg2+ ion at the high affinity site. The torsional rigidity of F-Ca(2+)-actin ((8.5(+/- 1.3)) x 10(-26) N m2) was about three times as large as that of F-Mg(2+)-actin ((2.8(+/- 0.3)) x 10(-26) N m2), whereas the flexural rigidity ((6.0(+/- 0.2)) x 10(-26) N m2) was almost independent of the kind of the bound cation. The dynamic structure of F-actin is regulated by the bound metal in an anisotropic manner. The torsional rigidities above, whether of F-Ca(2+)-actin or F-Mg(2+)-actin, are one to two orders of magnitude greater than previous experimental estimates.

Fluorescence energy transfer studies of transmembrane location of retinal in purple membrane.

A diffusion-enhanced energy transfer technique was employed for the determination of transmembrane location of the retinal chromophore in the purple membrane. Theoretical considerations showed that the rate of energy transfer from an energy donor embedded within a membrane to acceptors dissolved in solvent could be described by an analytical function of the distance a of closest approach between the donor and acceptor, if the "rapid-diffusion limit" was attained. The criterion for this limit was given by the relation: (RO)6 much less than 20D tau Da4, where RO is the characteristic distance of energy transfer, D is the diffusion coefficient of the acceptor and tau D is the fluorescence lifetime of the donor in the absence of acceptor. By photo-reduction of the purple membrane with sodium borohydride, the retinal chromophore was converted to a highly fluorescent derivative, which showed a broad emission band in the visible region. From analysis of the fluorescence decay curves of the photo-reduced purple membrane in the presence of various concentrations of cobalt-ethylenediamine tetraacetate (Co-EDTA: energy acceptor), the depth of the chromophore from the membrane surface was estimated to be 8 (+/-3) A. This result was supported by investigations of energy transfer processes in a system where the native purple membranes and the photo-reduced membranes were stacked in parallel: the energy acceptor in this system was the native retinal chromophore.

Torsional motion of eosin-labeled F-actin as detected in the time-resolved anisotropy decay of the probe in the sub-millisecond time range.

The internal motion of F-actin in the time range from 10(-6) to 10(-3) second has been explored by measuring the transient absorption anisotropy of eosin-labeled F-actin using laser flash photolysis. The transient absorption anisotropy of eosin-F-actin at 20 degrees C has a component that decays in the submicrosecond time scale to an anisotropy of about 0.3. This anisotropy then decays with a relaxation time of about 450 microseconds to a residual anisotropy of about 0.1 after 2 ms. When the concentration of eosin-F-actin was varied in the range from 7 to 28 microM, the transient absorption anisotropy curves obtained were almost indistinguishable from each other. These results show that the anisotropy decay arises from internal motion of eosin-F-actin. Analysis of the transient absorption anisotropy curves indicates that the internal motion detected by the decay in anisotropy is primarily a twisting of actin protomers in the F-actin helix; bending of the actin filament makes a minor contribution only to the measured decay. The torsional rigidity calculated from the transient absorption anisotropy is 0.2 X 10(-17) dyn cm2 at 20 degrees C, which is about an order of magnitude smaller than the flexural rigidity determined from previous studies. Thus, we conclude that F-actin is more flexible in twisting than in bending. The calculated root-mean-square fluctuation of the torsional angle between adjacent actin protomers in the actin helix is about 4 degrees at 20 degrees C. We also found that the torsional rigidity is approximately constant in the temperature range from 5 to approximately 35 degrees C, and that the binding of phalloidin does not appreciably affect the torsional motion of F-actin.

F1-ATPase: a highly efficient rotary ATP machine.

A single molecule of F1-ATPase is by itself a rotary motor in which a central subunit, gamma, rotates against a surrounding stator cylinder made of alpha 3 beta 3 hexamer. Driven by the three beta subunits that hydrolyse ATP sequentially, the motor runs with discrete 120 degrees steps at low ATP concentrations. Over broad ranges of load and speed, the motor produces a constant torque of 40 pN.nm. The mechanical work the motor does in the 120 degrees step, or the work per ATP hydrolysed, is also constant and amounts to 80-90 pN.nm, which is close to the free energy of ATP hydrolysis. Thus this motor can work at near 100% efficiency.

Observations of rotation within the F(o)F(1)-ATP synthase: deciding between rotation of the F(o)c subunit ring and artifact.

F(o)F(1)-ATP synthase mediates coupling of proton flow in F(o) and ATP synthesis/hydrolysis in F(1) through rotation of central rotor subunits. A ring structure of F(o)c subunits is widely believed to be a part of the rotor. Using an attached actin filament as a probe, we have observed the rotation of the F(o)c subunit ring in detergent-solubilized F(o)F(1)-ATP synthase purified from Escherichia coli. Similar studies have been performed and reported recently [Sambongi et al. (1999) Science 286, 1722-1724]. However, in our hands this rotation has been observed only for the preparations which show poor sensitivity to dicyclohexylcarbodiimde, an F(o) inhibitor. We have found that detergents which adequately disperse the enzyme for the rotation assay also tend to transform F(o)F(1)-ATP synthase into an F(o) inhibitor-insensitive state in which F(1) can hydrolyze ATP regardless of the state of the F(o). Our results raise the important issue of whether rotation of the F(o)c ring in isolated F(o)F(1)-ATP synthase can be demonstrated unequivocally with the approach adopted here and also used by Sambongi et al.

Cooperative association of actin protomers and crosslinked actin oligomers in filaments at low ionic strength.

F-Actin treated with a half molar equivalent of the heterobifunctional cross-linker, 4-bromomethyl-3-nitrobenzoic acid succinimide ester (BNBA-SE), produced a population of actin oligomers containing 2-14 or more protomers and a significant amount of uncrosslinked actin protomers. The crosslinking reaction is presumed to occur between Cys 374 of one actin protomer with Lys 191 of an adjacent protomer on the genetic helix of F-actin, in a similar manner to N-maleimidobenzoic acid succinimidyl ester, which shares similar reactive groups and crosslinking dimensions. Crosslinked oligomers and uncrosslinked protomers were found to form filaments that sediment after high-speed centrifugation, even in a buffer of low ionic strength [G-buffer: 2 mM tris-hydroxymethylaminomethane (pH 8.0), 0.2 mM CaCl2, 0.2 mM ATP, 0.3 mM NaN3, 5 mM 2-mercaptoethanol] where affinity between actin protomers usually becomes weak, leading to the depolymerization of native F-actin. By performing the crosslinking reaction in the presence of an environment-sensitive fluorescent label, 6-acryloyl-2-(dimethylamino)naphthalene (acrylodan), which competes with BNBA-SE for Cys 374 of actin protomers, fluorescent, crosslinked F-actin filaments were obtained which were also stable in G-buffer. Fluorometric analysis of actin labeled with acrylodan (prodan-actin) in these depolymerization-resistant filaments suggested that the molecular environment around Cys 374 of actin protomers is similar to that of actin protomers in native F-actin in F-buffer (G-buffer plus 0.1 M KCl and 1 mM MgCl2). When G-actin labeled with acrylodan was co-polymerized with non-fluorescent crosslinked F-actin, some of the labeled actin was incorporated into filaments that were resistant to depolymerization in G-buffer. The emission spectrum of the prodan-actin in these filaments measured in G-buffer was almost identical to that of prodan-actin in native F-actin in F-buffer. Our interpretation of this result is that actin protomers are locked into an F-actin-like conformation in the depolymerization-resistant filament by the subunit interactions they make with crosslinked oligomers. We also present evidence which suggests that the depolymerization rate in G-buffer of filaments made with crosslinked oligomers is much slower than that of native actin because the ends of the depolymerization-resistant filaments are capped with crosslinked oligomers.

Nanosecond fluorometric investigation of hydrodynamic properties of adenosine triphosphatase from thermophilic bacterium PS3.

The soluble portion (TF1) of proton-translocating ATPase from thermophilic bacterium PS3 was labeled with a fluorescent dye N-(1-pyrene)maleimide. The decay of fluorescence anisotropy of the adduct showed that TF1 in aqueous solution was characterized by a volume of equivalent sphere of 1,120 nm3. This value is 2.4 times the volume calculated from the molecular weight and partial specific volume, indicating a non-spherical shape and/or extensive hydration. A prolate ellipsoid with an axial ratio of 2 to 3 is suggested as a first approximation of the shape of hydrated TF1. The presence or absence of ATP, ADP, or Mg2+ did not alter the volume of the equivalent sphere appreciably; the probable conformational change of TF1 induced by these ligands does not lead to a gross alteration of its hydrodynamic properties.