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* Department of Molecular Biology and Immunology, University of North Texas, Fort Worth, Texas 76107; and Center for Fluorescence Spectroscopy, Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201
Correspondence: Address reprint requests to Julian Borejdo, Dept. of Molecular Biology and Immunology, University of North Texas, 3500 Camp Bowie Blvd., Fort Worth, TX 76107. Tel: 817-735-2106; E-mail: jborejdo{at}hsc.unt.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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600 by confocal microscopy, we were able to measure the rates of cross-bridge detachment and rebinding. However, we were unable to resolve the power stroke. We speculated that the reason for this was that the number of observed cross-bridges was too large. In an attempt to decrease this number, we used two-photon microscopy which permitted observation of 1/2 as many cross-bridges as before with the same signal/noise ratio. With the two-photon excitation, the number of cross-bridges was small enough to resolve the beginning of the power stroke. The results indicated that the power stroke begins 170 ms after the rigor cross-bridge first binds ATP. |
INTRODUCTION |
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; Goldman, 1998; Hopkins et al., 1998; Irving et al., 1995; Sabido-David et al., 1998). The same method was applied to measure anisotropy of fluorescence of cross-bridges residing in an extremely small observational volume defined by diffraction-limited laser beam and confocal aperture (Borejdo and Akopova, 2003; Borejdo et al., 2004).
If a small population of cross-bridges were acting in perfect synchrony, the lever arm anisotropy would be expected to change as illustrated in Fig. 1. Beginning with the heads in rigor, when they are immobilized by actin, the first phase after creation of ATP (arrow) is an increase in rotational mobility, reflecting dissociation of heads from thin filaments. At the end of this process, myosin heads rotate freely. The second phase is a partial immobilization, reflecting binding of the heads to thin filament in a short-lived, partially disordered, prepower stroke state (Warshaw et al., 1998). The final phase is the power stroke transition of the weakly bound, partially immobilized heads to a strongly bound, completely immobilized state triggered by product release. However, in the experiments using one-photon (1P) excitation in a confocal microscope, the power stroke was conspicuously absent. After synchronous activation, the cross-bridges started to rotate rapidly (indicating dissociation from actin) after which they were slowly immobilized (indicating binding to actin) (Borejdo and Akopova, 2003; Shepard and Borejdo, 2004). We speculated that the reason for the absence of a clearly defined power stroke was that the observed population of cross-bridges (600) was still too large. In an attempt to decrease this number, we use here two-photon (2P) microscopy.
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In conventional (1P) confocal microscopy, the thickness of the observational volume is defined by the diameter of the confocal aperture. In previous experiments, the aperture was 35 µm (2.37 Airy units). (It could not be further decreased, because closing it decreased the S/N ratio to the extent that measurements became impossible.) This made the depth of focus equal to 4 µm, the volume equal to 0.5 µm3, and the number of observed cross-bridges 600. On the other hand, in 2P microscopy, which is now possible because of the wide availability of ultrashort-pulsed near-infrared lasers, the signal originates only from the focal plane where the laser power density is high enough to produce 2P absorption (Pawley, 1995). In our experiments, this plane was 2 µm thick, allowing us to observe <300 cross-bridges.
Despite the fact that number of observed cross-bridges was <
, the S/N ratio was unchanged. This was due to the fact that 2P photobleaching was reduced and that absolute values of 2P anisotropy were larger. It is well known that out-of-focus photobleaching is reduced in 2P because out-of-focus planes are illuminated by less damaging infrared light (IR) light. We show here that, surprisingly, in the case of muscle exchanged with myosin light chains labeled with rhodamine, photobleaching in the plane of focus was also reduced. The anisotropy was larger because absorption/emission of two photons depends on the fourth power—not the second power as in 1P absorption—of cosine of the angle between dye dipole and the direction of polarization of exciting light.
We show here that although 1P and 2P spectra of rhodamine labeled myosin light chain are the same, the time courses of anisotropy during muscle contraction are sufficiently different to determine the onset of the power stroke. With 2P excitation, we were able to determine that the power stroke begins on the average 170 ms after a cross-bridge first binds ATP.
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MATERIALS AND METHODS |
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65 mW. Fluorescent light (yellow) is collected by the objective, passed by the same scanner and reflected by the dichroic mirror M3 into photomultipliers 1 and 2, which detect orthogonally polarized light passed by crossed analyzers AN1 and AN2. Since the fluorescent light is scanned again on the way to the detectors, it is termed descanned detection. Alternatively, mirror M5 can be substituted by a dichroic filter to pass the fluorescent light to another set of photomultipliers 3 and 4. Since the fluorescent light does not pass through the scanner, it is termed nondescanned detection. The significant advantage of this mode of detection is that the distance between the detectors and the sample is shortened and that the fluorescent light does not enter the microscope at all, and hence does not pass through the scanner or is not attenuated by the internal optics. Unless otherwise stated, all the experiments were done in nondescanned mode. The 351 + 364 nm light from the ultraviolet (UV) laser (blue) (Enterprise, Coherent) is made collinear with the IR beam by the dichroic filter FT395. A fast-shutter SHT (Vincent Associates, Rochester, NY, model T132) is opened for 10 ms to admit UV light to a muscle fiber. The UV power impinging on muscle is 700 µW, giving a power flux of 9 x 10–4 mJ/µm2. The 1P excitation by Ar/Kr laser (light green) is achieved as previously described (Borejdo and Akopova, 2003).
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Muscle fibers
Single fibers were dissected from glycerinated rabbit psoas muscle bundles in the glycerinating solution. Fibers were mounted on a microscope slide containing aluminum clips glued 5 mm apart. Tautly stretched fibers were attached to clips and covered with a cover glass, which rested on an 2 mm layer of Vaseline. Mounted fibers were thoroughly washed with rigor solution. Muscle bundles were stored in glycerol for no more than 3 months. Opened bottles containing glycerinated bundles were discarded after a week.
Expressing and labeling of RLC
The regulatory light chain (RLC) containing a single cysteine at position 73 (Sabido-David et al., 1998) was prepared by expression of RLC in a pT7-7 plasmid in BL21(DE3) cells. The construct was a gift from Dr. S. Lowey (University of Vermont). The preparation and purification were done as described previously in Wolff-Long et al., (1993). Labeling was done as described earlier (Borejdo and Akopova, 2003), except that stock solution of IATR was not used, but the dye was dissolved in methanol just before the experiment. The Rh-RLC adduct contained 14% of rhodamine.
Expressing and labeling of LC1
The pQE60 vector and Escherichia coli M15[pREP4] cells (Qiagen, Valencia, CA) were used for the cloning and expression of essential light chain 1 (LC1). The human fast skeletal muscle essential light chain (ELC) (a gift from S. Lowey) was subcloned into the pQE60 vector using DNA polymerase chain reaction with the 3' end containing a tag of six histidines. The presence of the His tag at the N-terminus of ELC was confirmed by DNA sequencing. The expressed recombinant proteins were purified on Ni-NTA-agarose columns (Qiagen). Labeling was done as described earlier (Borejdo and Akopova, 2003), except that stock solution of IATR was not used, but the dye was dissolved in methanol just before the experiment; 3.5–7% of purified protein was fluorescently labeled.
Exchanging Rh-RLC and Rh-LC1 into muscle fibers
Labeled RLC was exchanged into fibers as described earlier (Ling et al., 1996), except that exchange was at room temperature; 0.5–1.1 mg/mL labeled ELCs were exchanged with endogenous light chains of myosin in muscle fibers at 30°C using the exchange solution described before (Borejdo et al., 2001). After labeling, the fibers were thoroughly washed with the rigor solution.
Functionality of exchanged fibers
Tension development was studied by an MKB force transducer (Scientific Instruments, Heidelberg, Germany) coupled to an analog counter (model 6024E, National Instruments, Austin, TX). Control (unlabeled) fibers developed normal 0.94 ± 0.05 mN/fiber (mean ± SE, n = 32) maximum isometric tension. Fibers exchanged with fluorescently labeled RLC developed 0.96 ± 0.03 mN/fiber tension. Fibers exchanged with fluorescently labeled LC1 also developed normal tension. We recognize, however, that the degree of exchange with LC1 was too small to make tension measurements a reliable test of functionality. Effect of exchange of LC1 on muscle was assessed, therefore, by measuring static polarization of fluorescence as described earlier (Borejdo et al., 2001). Exchange had no effect on polarization of fluorescence, suggesting that it also had no effect on the functionality of muscle.
Photogeneration of ATP
ATP was photogenerated from a caged precursor by perfusing fibers with 2 mM of 5-dimethyoxy-2-nitrobenzyl-caged ATP in rigor solution. The UV beam was focused by the objective to a Gaussian spot with a width, length, and depth of 0.2 x 0.2 x 3 µm; 3 s after beginning the scan, a shutter admitting the UV light was opened for exactly 10 ms. The energy flux through the illuminated area during the time ATP stayed in the experimental volume (300 µs) was 9 x 10–4 mJ/µm2. The amount of released ATP was enough for a single turnover of ATP.
Anisotropy of solutions
Isolated myosin, prepared according to Tonomura et al. (1966) was exchanged with Rh-RLC as in Ling et al. (1996). Fluorescence anisotropy was measured in an ISS K2 spectrofluorometer (ISS, Champaign, Illinois). The commercial ISS fluorometer chamber was modified to accept a Ti:Sapphire laser beam, and the spectra were collected using a single photon counting fluorometer SLM8000. The two-photon excited volume was 0.3 µm3. To check the mode of excitation, fluorescence intensity of myosin (excited at 822 nm) was monitored as a function of exciting light power. The intensity of the fluorescence induced by two photons must be proportional to the square of instantaneous photon flux. In experiments with myosin subfragment-1, the correlation factor between the square of the incident intensity and observed fluorescence was 1.97. Time-domain lifetime measurements were carried out using compact fluorescence lifetime spectrometer FluoTime 100 (PicoQuant, Berlin, Germany). For excitation (442 nm, 10 MHz, 60 ps width) the diode laser system (PTD 800B with LDH PC 440, PicoQuant) was used. To avoid scattered light, a cutoff filter (500 nm) was used in the emission channel. All measurements were done using magic-angle conditions. Data analysis was performed using multiexponential fluorescence decay fitting software FluoroFit version 3.2.0 (PicoQuant). Experiments were done at 20°C in the rigor solution. Experiments on immobilized proteins were performed at 0°C in 90% glycerol. All samples used in fluorescence measurements had absorption of <0.1.
Absolute value of anisotropy
The static anisotropy is higher in the 2P mode. This is a consequence of the fact that that the absorption/emission of a single photon by a dye molecule depends on the square of the cosine of the angle between dye dipole and the direction of polarization of exciting light. The absorption/emission of two photons, therefore, depends on the fourth power of this angle (Lakowicz. 1986). A complete description of the anisotropy with 2P is complex (Wan and Johnson, 1994) and requires consideration of the tensor properties of the transitions. However, in the case of rhodamine, the two-photon transition appears to be colinear and have the same orientation as the one-photon transition. In this case, the expected anisotropy can be easily predicted based on the usual orientation dependence of electronic transitions. Theoretically, for 1P and 2P, the maximal values of anisotropy are 0.4 and 0.57, respectively (the multiphoton anisotropy values are given in (Gryczynski et al., 1995). The steady-state anisotropy of Rh-RLC incorporated into myosin was 0.370 at 822 nm and 0.261 at 550 nm. The ratio, 1.42, was close to the theoretically predicted ratio of 1.43. Probably, the relative orientation of excitation/emission dipoles is similar in 1P and 2P excitation (Malak et al., 1997).
Fluorescence anisotropy of fibers
Fluorescence was measured with a high-aperture lens (C-Apo, Zeiss, 40x, NA = 1.2). Calculations showed that high NA of the objective causes minimal distortion to the polarized intensities (Burghardt et al., 2001). The subscripts after the intensity indicate the direction of polarization of emitted light relative to the axis of the muscle fiber. The excitation light was always parallel to the axis of fiber. The muscle axis was oriented horizontally on a stage of a microscope. I
and I|| are recorded by photomultipliers 1 and 2 in descanned mode, and photomultipliers 3 and 4 in nondescanned mode, respectively.
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RESULTS |
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). Significantly, addition of F-actin makes no difference either to the shape or the position of the spectral peak, consistent with no effect of actin on steady-state anisotropy (see below). As shown in Fig. 3, bottom, the two-photon induced fluorescence signals of Rh-RLC (blue, green, red) are only approximately twice lower than the signal of rhodamine B (RhB) in MeOH, which is considered a very good two-photon absorber with one of the highest known two-photon cross sections of 120 GM (Xu and Webb, 1996).
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0.5 µm3 = 0.5 x 10–15 L and V2P = 0.25 x 10–15 L, and the numbers of observed myosins carrying LC1 were 600 and 300 in 1P and 2P experiments, respectively. Labeling with RLC at room temperature was approximately twice as efficient as labeling with ELC at 30°C. The numbers of observed myosins carrying RLC were 1200 and 600 in 1P and 2P experiments, respectively.
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), but the intensities shown in the figure originate from the focal plane. Perhaps the effect is due to less toxic environment within the muscle fiber caused by the absence of out-of-plane photobleaching.
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), the anisotropy (black) changed rapidly and later recovered slowly to the original level. (Note that I and I|| changed in the same direction. Whether they change in the same or opposite direction depends on cross-bridge angle (Burghardt et al., 2001). It is the orthogonal anisotropies, not the intensities, that must change in the opposite direction.) Fig. 7 B shows the time course of anisotropy change of 2P signal from the adjacent area of the same muscle fiber. The polarization of the IR laser illuminating the muscle was parallel to the fiber axis. The perpendicular (dark shaded) intensity bleached at the rate of 0.39 s–1. The parallel (light shaded) intensity was very stable, bleaching at a negligible rate of 0.082 s–1. The resulting anisotropy (black) bleached therefore at a rate primarily determined by the rate of perpendicular intensity. The UV pulse was applied at the time indicated by the arrow (it is much more prominent in the 2P than in the 1P experiment because no confocal aperture was used). The anisotropy changed rapidly and recovered slowly to the original level. The rate of rapid change was too fast to measure. The rate of the slow recovery was 1.57 s–1 and 0.72 s–1 for 1P and 2P experiments. The 1P rate is in good agreement with previous results (Borejdo and Akopova, 2003). In analogy with 1P experiments (Borejdo and Akopova, 2003), we think that the rapid phase of 2P anisotropy change represents cross-bridge dissociation from thin filaments, and the slow recovery corresponds to rebinding of cross-bridges to actin in rigor conformation after photocreated ATP has been depleted.
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17°. The 1P and 2P anisotropies of myosin-Rh-RLC at 540 and 822 nm are decreased to 0.253 and 0.370, respectively (red squares). The ratio of 1P/2P anisotropy is close to the predicted value (Gryczynski et al., 1995). Since myosin at low ionic strength is filamentous and does not execute any nanosecond-timescale motions, this decrease indicates collective motion of the probe and of the myosin-bound light chain. The 1P and 2P anisotropies of free Rh-RLC (blue triangles) are only slightly lower than corresponding anisotropies of light chain bound to myosin, suggesting that rotation of RLC on myosin is restricted by the interactions with the heavy chain. The residual 1P and 2P anisotropies of free rhodamine (pink diamonds) at 540 and 822 nm are 0.021 and 0.033, respectively.
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DISCUSSION |
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170 ms after the beginning of the cross-bridge cycle.
Fig. 11 explains why the appearance of the inflection is indicative of the power stroke. If a single cross-bridge were observed, the anisotropy of lever arm would be expected to change in three steps as illustrated in Fig. 11 A (same as Fig. 1). If the number is >1, the steps are obscured. Due to synchronization imposed by photogeneration of ATP, all the cross-bridges dissociate from actin at the same time (because the rate of dissociation is fast), but they rebind to actin at different times. The amount of time a cross-bridge remains free depends on the its position relative to the actin "target site"(Huxley, 1957). This effect is illustrated by a simple calculation in which the observed population is assumed to consist of 50 (Fig. 11 B), 100 (C), or 1000 (D) cross-bridges, each spending different amounts of time in an unbound state. For simplicity, we assume that these times differ by 1 ms. When the number of observed cross-bridges is small, the step indicating beginning of the power stroke is preserved (Fig. 11 B). In the actual experiments, this step is indicated by the inflection. But when the number of cross-bridges is large enough, stepwise character is lost (C and D). The exact number of cross-bridges necessary for loss of steps is model-dependent, but can be as small as factor of 2. This explains why the mode of detection makes a difference, i.e., that the effect may be unobservable in 1P experiments but present in 2P experiments. But it does not mean that it is impossible to see the inflection in 1P experiments—in 1P, a fiber has to be more lightly labeled; this decreases S/N and makes observations more difficult.
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). It is therefore likely that in steady-state isometric contraction, the onset of the power stroke lags the beginning of the cross-bridge cycle by 11 ms.
We recognize the fact that the power stroke is expected to be small in isometric contraction. However, this probably does not alter our results. Since the cross-bridges executed only a single cycle, they had no opportunity to be restrained by series elasticity. It is also unlikely that the rotational motion of the lever arm occurs exclusively in the azimuthal plane, and that the polar component is too small to be detected. There are theoretical indications that there exists significant polar component (Burghardt and Ajtai, 1994).
It is interesting to note that 5'-IATR, whether free or coupled to the protein, always has two lifetimes. This is consistent with other measurements (Harley et al., 2002) and may be due to sample relaxation, sample heterogeneity, or conformational effects. It is impossible that it is due to the experimental artifact, because the lifetime of rhodamine B in water, measured under exactly the same conditions, gives expected monoexponential decay (
= 1.706 ns, amplitude 1.0, 
2 = 1.225. The fit to two exponentials gives the same result: = 1.704 ns with amplitude 1.0, and = 0.09 ns with amplitude 0.0, 2 = 1.271). It is unlikely that the observed rotational relaxation involves transition between local conformations, because the relative contribution of the two lifetimes did not change with the addition of actin (Table 1).
An important advantage of 2P excitation that cannot be overemphasized is that it is possible to measure signal through the side port of a conventional microscope. This avoids attenuation by microscope optics and reduces the length of the emission light path, thus increasing S/N ratio.
A resolution of each step in cross-bridge cycle in vivo must wait until a single cross-bridge within contracting muscle fiber can be followed.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Institutes of Health (R21CA9732 and RO1AR048622), by grant 000130-0008-2001 from the Texas Higher Education Coordinating Board, and by the National Center for Research Resources (RR-08119).
Submitted on May 4, 2004; accepted for publication September 7, 2004.
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REFERENCES |
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Borejdo, J., D. Ushakov, R. Moreland, I. Akopova, Y. Reshetnyak, L. D. Saraswat, K. Kamm, and S. Lowey. 2001. The power stroke causes changes in orientation and mobility of the termini of essential light chain 1 of myosin. Biochemistry. 40:3796–3803.[CrossRef][Medline]
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Ling, N., C. Shrimpton, J. Sleep, J. Kendrick-Jones, and M. Irving. 1996. Fluorescent probes of the orientation of myosin regulatory light chains in relaxed, rigor, and contracting muscle. Biophys. J. 70:1836–1846.
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ex = 568 nm, LP 590 nm emission filter. The bright peak at right is the light reflected by a coverslip. The microsphere profile is at left. HWHM of the profile = 2.2 µm. (B) 2P scan. No pinhole. 
