Results: 5

1.
Fig. 5.

Fig. 5. From: Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

Fraction of attached heads during isometric contraction. The gray circles show γact versus SL (same as in Fig. 3D). The colored lines show the trends of γ calculated for different fractions of attached myosin molecules, as indicated.

V. Nucciotti, et al. Proc Natl Acad Sci U S A. 2010 April 27;107(17):7763-7768.
2.
Fig. 2.

Fig. 2. From: Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

SPA sensitivity to the fraction of attached myosin cross-bridges. (A) Measured γ versus SL in a representative demembranated rabbit fiber. The data in relaxing solution and in rigor are shown by green and red circles, respectively. (B) Measured γ versus PPi concentration. Each point represents the average ± SD of three fibers.

V. Nucciotti, et al. Proc Natl Acad Sci U S A. 2010 April 27;107(17):7763-7768.
3.
Fig. 4.

Fig. 4. From: Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

SPA modeling. (A) Scheme of the myosin molecule (S2 in light blue and S1 in dark blue, thick filament as black continuous line) detached from actin (black dotted line). In the scheme, the Z line is on the right side. The two S1 heads are indicated by S1 and S1′. For clarity, only the angle of one head is indicated (θS1). (B) Calculation of γ (surface color plot) as a function of θS1 and θS1 for different values of θS2. The value of γrest on the surface plot is pointed by the arrow. The black iso-γ curves (average, dashed line; one SD interval, solid lines) report the measured value of γrest. (C) Scheme of the myosin molecule attached to actin. The catalytic domains (dark blue trapezoids) are rigidly fixed to actin. For clarity, only the lever-arm angle of one head is indicated (θLA). (D) Calculation of γ as a function of θLA and θLA for different values of θS2. The black iso-γ curves report the measured value of γrig. The asterisks show the geometry of rigor heads according to cryo-EM.

V. Nucciotti, et al. Proc Natl Acad Sci U S A. 2010 April 27;107(17):7763-7768.
4.
Fig. 3.

Fig. 3. From: Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

SPA in intact muscle fibers. AC show force (A, blue line), line scan raw data (B, for detailed explanation see legend of Fig. 1B), and SPA curve (C, black line) measured in a single intact frog fiber at SL = 2.2 μm. The common horizontal axis is mapped in terms of polarization rotation with the scale of C. The red bar in A indicates the period of tetanic stimulation. During tetanic contraction, some sliding of the fiber segment monitored by line scan occurs, giving rise to the tilt of the sarcomere pattern in the kymograph (B). The red lines in C show the best fits of Eq. 1 to the data at rest (from -70° to 155°, yielding as best fit parameter γ = 0.35) and during contraction (from 200° to 385°, yielding as best fit parameter γ = 0.69). (D) Measurements of γ versus SL in seven intact fibers at rest (green circles) and during contraction (blue circles). The black squares and line represent the normalized isometric force (right axis) of a representative fiber.

V. Nucciotti, et al. Proc Natl Acad Sci U S A. 2010 April 27;107(17):7763-7768.
5.
Fig. 1.

Fig. 1. From: Probing myosin structural conformation in vivo by second-harmonic generation microscopy.

SPA in demembranated fiber. (A) SHG image of a single demembranated rabbit psoas fiber in rigor (SL = 2.4 μm). The bright bands in the image correspond to sarcomeric A bands. The inset shows, at 2.5× magnification, the area highlighted in yellow. The green line corresponds to the line scanned for SPA measurement. (B) Rigor fiber line scan raw data, horizontally varying in brightness with changing polarizer angle for 1,000 successive axial 1D scans along the green line in panel A Inset. The data are represented as a kymograph of the scanned line, i.e., the ISHG from each scan trace is displaced one pixel from the previous scan trace along the horizontal time axis, incrementing from left to right the accumulating array of repeated SHG 1D scans, each scan recording the sarcomeric cross-striation pattern of A and I bands in six axially successive sarcomeres. The temporal horizontal axis is mapped in terms of polarization rotation with the same scale as in C. (C) SPA curve in rigor. Each ISHG data point (in black) reports the intensity integrated by summing the 100 pixel values that form the corresponding vertical line in B. The red line shows the best fit of Eq. 1 to the data, yielding, as best fit parameter, γ = 0.68. D and E are the same as B and C but measured on a relaxed fiber. The best fit parameter in E is γ = 0.36.

V. Nucciotti, et al. Proc Natl Acad Sci U S A. 2010 April 27;107(17):7763-7768.

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