Figure 4. Elongation of the axial pitch... | Rockefeller University Press

Figure 4.

$Figure 4. Elongation of the axial pitch of GDP microtubule measured by x-ray fiber diffraction. (A) X-ray fiber diffraction patterns of GDP microtubules (94 µM) without KIF5C, with 94 µM KIF5C, and with 94 µM KIF5C plus 1 mM AMPPNP and those of GMPCPP microtubules without KIF5C (from left to right panels). The bottom black circle in each panel is an x-ray beam stop placed around the image center. The arrowheads on the left side of the first panel indicate the first to fourth diffraction peaks (from bottom to top of the image) corresponding the axial tubulin periodicity (∼4 nm). The arrowhead on the right side of the third panel indicates the first diffraction peak corresponding to the bound kinesin periodicity (∼8 nm). Without KIF5C, the diffraction peaks of both the GDP and GMPCPP microtubules fit well with the previously reported tubulin axial periodicity (4.1 nm for GDP microtubules and 4.2 nm for GMPCPP microtubules). (B) The images of the fourth diffraction peak of GDP microtubules. As the KIF5C concentration increased, the peak shifted toward the center; however, the peak returned to the initial position by addition of 1 mM AMPPNP. (C) The relation of KIF5C concentration and axial tubulin periodicity of GDP microtubules in the presence of 1 mM ATP and 1 mM AMPPNP. The Hill coefficient of this reaction was estimated to be 6.4 ± 1.5 by fitting the data to Hill equation (gray line). (D) Time course of the axial tubulin periodicity after adding 19 µM KIF5C to 94 µM GDP microtubule (the microtubule concentration is expressed as tubulin dimer concentration) and 1 mM AMPPNP. (C and D) Error bars indicate the diffraction peak FWHM. Among the four individual trials, the SDs of the axial tubulin periodicity of each data point were below 0.1 Å. (E and F) The time course of the layer line positions after the addition of mutant KIF5C (K11C, the L11-α4 junction was replaced with the corresponding sequence of KIF1A). K11C was added at 18.7 µM, which corresponds to the 20% (molar ratio) of tubulin in the assay chamber. The lattice of the GDP microtubule was elongated both in magnitude and duration similarly to wild-type KIF5C (Fig. 4 D). Error bars indicate the diffraction peak FWHM. SD of the three independent experiments was below 0.1 Å. (G and H) The time course after the addition of excess (1 mM) AMPPNP to the K11C–GDP microtubule complex. The microtubule pitch was not shortened. These results suggest that the L11-α4 junction would be involved in the ATP-dependent shortening of the microtubule lattice, but not in the elongation of the microtubule lattice upon binding of KIF5C. Error bars represent FWHM. SD of three individual experiments was below 0.1 Å. Note the presence of the peak at 8 nm−1 (arrowheads at 1/8 in E and G), which demonstrates the binding of K11C to the microtubules in these experiments.$

Elongation of the axial pitch of GDP microtubule measured by x-ray fiber diffraction. (A) X-ray fiber diffraction patterns of GDP microtubules (94 µM) without KIF5C, with 94 µM KIF5C, and with 94 µM KIF5C plus 1 mM AMPPNP and those of GMPCPP microtubules without KIF5C (from left to right panels). The bottom black circle in each panel is an x-ray beam stop placed around the image center. The arrowheads on the left side of the first panel indicate the first to fourth diffraction peaks (from bottom to top of the image) corresponding the axial tubulin periodicity (∼4 nm). The arrowhead on the right side of the third panel indicates the first diffraction peak corresponding to the bound kinesin periodicity (∼8 nm). Without KIF5C, the diffraction peaks of both the GDP and GMPCPP microtubules fit well with the previously reported tubulin axial periodicity (4.1 nm for GDP microtubules and 4.2 nm for GMPCPP microtubules). (B) The images of the fourth diffraction peak of GDP microtubules. As the KIF5C concentration increased, the peak shifted toward the center; however, the peak returned to the initial position by addition of 1 mM AMPPNP. (C) The relation of KIF5C concentration and axial tubulin periodicity of GDP microtubules in the presence of 1 mM ATP and 1 mM AMPPNP. The Hill coefficient of this reaction was estimated to be 6.4 ± 1.5 by fitting the data to Hill equation (gray line). (D) Time course of the axial tubulin periodicity after adding 19 µM KIF5C to 94 µM GDP microtubule (the microtubule concentration is expressed as tubulin dimer concentration) and 1 mM AMPPNP. (C and D) Error bars indicate the diffraction peak FWHM. Among the four individual trials, the SDs of the axial tubulin periodicity of each data point were below 0.1 Å. (E and F) The time course of the layer line positions after the addition of mutant KIF5C (K11C, the L11-α4 junction was replaced with the corresponding sequence of KIF1A). K11C was added at 18.7 µM, which corresponds to the 20% (molar ratio) of tubulin in the assay chamber. The lattice of the GDP microtubule was elongated both in magnitude and duration similarly to wild-type KIF5C (Fig. 4 D). Error bars indicate the diffraction peak FWHM. SD of the three independent experiments was below 0.1 Å. (G and H) The time course after the addition of excess (1 mM) AMPPNP to the K11C–GDP microtubule complex. The microtubule pitch was not shortened. These results suggest that the L11-α4 junction would be involved in the ATP-dependent shortening of the microtubule lattice, but not in the elongation of the microtubule lattice upon binding of KIF5C. Error bars represent FWHM. SD of three individual experiments was below 0.1 Å. Note the presence of the peak at 8 nm⁻¹ (arrowheads at 1/8 in E and G), which demonstrates the binding of K11C to the microtubules in these experiments.