An X-ray diffraction study on a single rod outer segment from frog retina

Abstract

X-ray diffraction patterns were recorded from isolated single rod outer segments of frog. The outer segments in Ringer's solution were exposed to a 6 µm microbeam (15 keV) at the BL40XU beamline, SPring-8. The diffraction pattern demonstrated a remarkable regularity in the stacking and flatness of the disk membranes. The electron density profile calculated from the intensity of up to tenth-order reflections showed a pair of bilayers that comprise a disk membrane. The structure of the disk membrane and the changes in the profile on swelling generally agreed with previous reports. Radiation damage was significant with an irradiation of 5 × 10(5) Gy which is much lower than the known damaging dose on proteins at the liquid-nitrogen temperature.

Figure 1

(a) Image of the central section through a frog eye obtained by phase-contrast X-ray tomography using a Talbot grating interferometer (Hoshino et al., 2011 ▶). The rod is located at the outer rim of the retina and the outer segment is pointing away from the light. (b) Schematic drawing of the arrangement of the disk membranes in a rod outer segment. Each disk membrane comprises of a pair of bilayers. d is the inter-disk spacing, while a is the intra-disk distance, i.e. the distance between two bilayers in one disk. Since d is about 30 nm and the length of the rod outer segment is about 50 µm, the number of disk membranes is about 1500. The electron density profile across two disk membranes is represented schematically.

Figure 2

X-ray diffraction patterns from isolated ROSs of frog. The beam is passing through a ROS that lies vertically. The beam also passed through a part of another ROS that is horizontally oriented. The background is not subtracted in this image. The exposure time was 50 ms. The disk spacing of the vertical lamellar pattern is 30.4 nm.

Figure 3

Intensity distribution in the direction along the length of the rod outer segment obtained from Fig. 2 ▶. The black curve (red online) is the background-subtracted experimental data after the Lorentz correction and the grey curve (blue online) is the fitted intensity. The intensity is on an arbitrary scale. The disk spacing is 30.4 nm.

Figure 4

Dependence of the intensity of the lamellar reflections on the disk spacing. (a) The first, second-, third- and fourth-order reflections. (b) The sixth- and seventh-order reflections. (c) The eighth-, ninth- and tenth-order reflections. The fifth reflection was too weak to quantify. The integrated intensity of each order after correction with the Lorentz factor is given as a percentage of the sum of intensities of all ten reflections. The data from 13 ROSs are plotted.

Figure 5

Electron density profile calculated from the experimental data in Table 1 ▶. Profiles from the data with different disk membrane spacing are shown. For the profile at 29.3 nm, data from three ROSs were averaged. The center is that of the disk membrane. The fine ripples are due to truncation of the reflections used to calculate the density at the tenth order.