Second harmonic generation microscopy probes different states of motor protein interaction in myofibrils

Abstract

The second harmonic generation (SHG) signal intensity sourced from skeletal muscle myosin II strongly depends on the polarization of the incident laser beam relative to the muscle fiber axis. This dependence is related to the second-order susceptibility χ((2)), which can be described by a single component ratio γ under generally assumed symmetries. We precisely extracted γ from SHG polarization dependence curves with an extended focal field model. In murine myofibrillar preparations, we have found two distinct polarization dependencies: With the actomyosin system in the rigor state, γ(rig) has a mean value of γ(rig) = 0.52 (SD = 0.04, n = 55); in a relaxed state where myosin is not bound to actin, γ(rel) has a mean value of γ(rel) = 0.24 (SD = 0.07, n = 70). We observed a similar value in an activated state where the myosin power stroke was pharmacologically inhibited using N-benzyl-p-toluene sulfonamide. In summary, different actomyosin states can be visualized noninvasively with SHG microscopy. Specifically, SHG even allows us to distinguish different actin-bound states of myosin II using γ as a parameter.

Figure 1

Second harmonic generation from skeletal muscle. SHG signals from muscle fibers and collagen in an entire murine extensor digitorum longus muscle (A and B), and from isolated myofibrils (C and D). The signals originate from the myosin filaments. The dip in signal intensity is located at the M-lines.

Figure 2

SHG orientation dependency in myofibrils. The SHG signal intensity depends on the angle of laser polarization relative to the myofibrillar axis. The minimum intensity is observed at α-ϕ = 0°, and the maximum intensities at ∼50° and ∼130° with a local minimum at 90°.

Figure 3

SHG laser polarization dependency. Myofibrils in relaxing and rigor solution show a different dependency on the angle of laser polarization relative to the myofibril axis. (A) The measured intensities of one rigor sample and one relaxed sample were normalized to a value of 100 for polarization perpendicular to the myofibril axis. (B) Data points from all samples, normalized as in panel A, shown in detail at the minimum intensity at α = 0°. The difference between the data sets for rigor and relaxed condition is clearly pronounced.

Figure 4

Distributions of parameter γ. (A) Simple model: There are two distinct distributions of the fitting parameter γ for the rigor state (mean 0.73, SD = 0.04, n = 55) and for the relaxed state (mean 0.50, SD = 0.05, n = 70). The parameter γ appears to be independent of the passively settled sarcomere lengths. (B) Extended model: parameter γ for the rigor state (mean 0.52, SD = 0.04, n = 55) and for the relaxed state (mean 0.24, SD = 0.07, n = 70).

Figure 5

Model of cross-bridge states. (A) The myosin molecule consists of a globular head domain (S1) and a rod-shaped tail domain (S2+LMM). The free space available to tilt motions between the thick filament and the actin filament is <26 nm (see text). (B) Simplified model geometry. Fractions p of the rod-shaped molecules are bent away by an angle β from the filament axis. (C) Predicted values for γ_eff within the model parameter space. Parameter sets that are compatible with the spatial constraints in the actin-myosin lattice (see text) are depicted in black.