Phosphorylation of myosin regulatory light chain has minimal effect on kinetics and distribution of orientations of cross bridges of rabbit skeletal muscle

Abstract

Force production in muscle results from ATP-driven cyclic interactions of myosin with actin. A myosin cross bridge consists of a globular head domain, containing actin and ATP-binding sites, and a neck domain with the associated light chain 1 (LC1) and the regulatory light chain (RLC). The actin polymer serves as a "rail" over which myosin translates. Phosphorylation of the RLC is thought to play a significant role in the regulation of muscle relaxation by increasing the degree of skeletal cross-bridge disorder and increasing muscle ATPase activity. The effect of phosphorylation on skeletal cross-bridge kinetics and the distribution of orientations during steady-state contraction of rabbit muscle is investigated here. Because the kinetics and orientation of an assembly of cross bridges (XBs) can only be studied when an individual XB makes a significant contribution to the overall signal, the number of observed XBs was minimized to ∼20 by limiting the detection volume and concentration of fluorescent XBs. The autofluorescence and photobleaching from an ex vivo sample was reduced by choosing a dye that was excited in the red and observed in the far red. The interference from scattering was eliminated by gating the signal. These techniques decrease large uncertainties associated with determination of the effect of phosphorylation on a few molecules ex vivo with millisecond time resolution. In spite of the remaining uncertainties, we conclude that the state of phosphorylation of RLC had no effect on the rate of dissociation of cross bridges from thin filaments, on the rate of myosin head binding to thin filaments, and on the rate of power stroke. On the other hand, phosphorylation slightly increased the degree of disorder of active cross bridges.

Fig. 1.

Comparison of photobleaching of Alexa Fluor 647 (A) and SeTau (B). The photobleaching occurred in two phases: the rate of the first phase, lasting ∼0.5 s was 10.1 s⁻¹ for Alexa Fluor 647. It was 4.2 times slower for SeTau. The rate of the second phase was 0.036 s⁻¹ for Alexa Fluor 647 and ∼3 times slower for SeTau. Note that the mean count rate of SeTau was 4.2 times larger than that of Alexa Fluor 647. The laser (640 nm) power was 0.9 mW before the objective.

Fig. 2.

Fluorescence decay of parallel polarized light from rigor myofibril. A: no gate applied, i.e., the signal collection is started at the peak of the signal (violet line) and continued through the complete fluorescence decay. This includes scattered light (arrow). B: the residuals have significant dip at the time corresponding to scattering of light and are not well fitted by a straight horizontal line. C: gating applied. In TCSPC systems, photons are collected one-by-one. Here, photons detected within 8.8 ns of the start of the measurement (an arbitrarily determined point in time with relation to the excitation pulse) are eliminated from our measurements. All that remains are photons collected 8.8 ns or later from the start of the measurement (indicated by the violet line). D: residuals of the gated signal are quite flat. The same procedure is applied to perpendicular polarized light.

Fig. 3.

Images of contracting phosphorylated myofibril, effect of gating. A, B, E, and F: ungated images. C, D, G, and H: the same images after gating. The color scale (in FLIM images) and contrast scale (in intensity images) are much improved by gating. The green circle pointed to by the green arrow in A is a projection of the confocal aperture on the sample plane. Top row: images obtained with analyzer perpendicular to the myofibrillar axis. Bottom row: images obtained with analyzer parallel to the myofibrillar axis. Notice that perpendicular images are weaker than parallel images, indicating that a sample is anisotropic. A, C, E, G: FLIM images. B, D, F, H: intensity images. The fluorescent lifetime scales are in nanoseconds, with 1.5 ns corresponding to blue and 3.4 ns to red. The intensity scales are in counts with 30 corresponding to black and 150 to white. Native myofibrillar LC1 was exchanged with 10 nM SeTau-LC1. Scale bar = 5 μm, sarcomere length = 2.1 μm. Sarcomere length does not change during contraction because of cross-linking (30, 78). Images were acquired on a PicoQuant Micro Time 200 confocal lifetime microscope. The sample was excited with a 640-nm pulsed laser and observed through a LP 650 filter.

Fig. 4.

A: typical time course of intensity of contracting MLCK phosphorylated psoas muscle myofibril. Ch3 (red) and Ch2 (black) are the fluorescence intensities polarized parallel (I_‖) and perpendicular (I_⊥) to the myofibrillar axis, respectively. The direction of excitation polarization is ‖ to the myofibrillar axis. The gate time was set to 0 ns. B: same time course but with the gate set to 7.36 ns. Note the decrease of intensity due to elimination of light scattering. C: polarization of fluorescence of ungated (blue) and gated (green) at 7.36-ns signals. To emphasize the differences, the ungated signal is plotted on top of the gated signal in D. Laser intensity was 0.2–0.4 μW.

Fig. 5.

The three-state model of muscle contraction. M, myosin; D, ADP; P, inorganic phosphate; A, F-actin; T, ATP. The polarizations of fluorescence associated with different cross bridge states. a₁, a₂ and a₃ are amplitude of polarizations.

Fig. 6.

Representative traces of normalized autocorrelation functions of polarization of fluorescence of contracting MLCK phosphorylated psoas myofibril. Circles are experimental data, and a red line is the fit to Eq. 1. The fact that the correlation decays in time indicates that the orientation of absorption-emission dipoles change in time. The fact that ACF decays to a value >0 is due to the fact that mean polarization was nonzero (approximately −0.23). Delay time is in seconds.

Fig. 7.

Selected examples of the probability distributions of orientations of cross bridges (XBs) of contracting myofibrils containing dephosphorylated RLC. Dephosphorylation was affected by the addition of EGTA to WT myofibrils.

Fig. 8.

Schematic representation of the fact that phosphorylation of RLC has no effect on kinetics and minimal effect on distribution of XBs. Phosphorylated and dephosphorylated RLC are shown as blue and red spheres, respectively. The white sphere is LC1, and white arrows are the transition dipoles of the dye. The violet cone is the cone of angles within which the transition dipole fluctuates. The thick filament is shown in dark blue, actin filament in yellow, and myosin neck in green.