Three-dimensional architecture of murine rod outer segments determined by cryoelectron tomography

Abstract

The rod outer segment (ROS) of photoreceptor cells houses all components necessary for phototransduction, a set of biochemical reactions that amplify and propagate a light signal. Theoretical approaches to quantify this process require precise information about the physical boundaries of the ROS. Dimensions of internal structures within the ROS of mammalian species have yet to be determined with the precision required for quantitative considerations. Cryoelectron tomography was utilized to obtain reliable three-dimensional morphological information about this important structure from murine retina. Vitrification of samples permitted imaging of the ROS in a minimally perturbed manner and the preservation of substructures. Tomograms revealed the characteristic highly organized arrangement of disc membranes stacked on top of one another with a surrounding plasma membrane. Distances among the various membrane components of the ROS were measured to define the space available for phototransduction to occur. Reconstruction of segments of the ROS from single-axis tilt series images provided a glimpse into the three-dimensional architecture of this highly differentiated neuron. The reconstructions revealed spacers that likely maintain the proper distance between adjacent discs and between discs and the plasma membrane. Spacers were found distributed throughout the discs, including regions that are distant from the rim region of discs.

Figure 1.

Cryoelectron micrograph of a vitrified ROS. Photoreceptor cells, schematically shown in panel b, are highly differentiated cells with a cylindrical ROS. (a) Montage of five cryoelectron micrographs of a single ROS. The reduced thickness of the ROS on the right side allows for a clear image of stacked discs and the plasma membrane. (c and d) Other components of photoreceptor cells such as connecting cilia (c) and mitochondria (d) are also found on EM grids. Bars (a), 500 nm; (c and d) 400 nm.

Figure 2.

Highly ordered disc membranes in vitrified ROS. (a) Cryoelectron micrograph of stacked disc membranes. A single transmission projection was obtained at a defocus value of −10 μm. The diffraction spectrum was obtained from the region highlighted by the boxed area. (b) The high order of discs is reflected in the diffraction spectrum. Up to five maxima are distinguishable. Bar, 100 nm.

Figure 3.

Electron tomogram of vitrified ROS. The electron tomogram is represented in three orthogonal slices through the volume of the ROS. (a and b) An x-y slice (a) and a y-z slice (b) display the high order and regular arrangement of stacked discs. (c) A x-z slice shows the high concentration of rhodopsin found in disc membranes. The reconstruction was not binned, and the pixel size is 1.1 nm. The boxed areas are visualized by isosurface representation in Fig. 4. Bar, 200 nm.

Figure 4.

Isosurface representation of a ROS. (a) A subvolume containing 10 discs (yellow) and the plasma membrane (blue) are shown. Areas of high density (dark yellow), presumably rhodopsin, can be differentiated from less dense areas (light yellow). (b) A top view of a single disc is shown. Spacers are shown that connect adjacent discs to each other (red) and the rim region of discs to the plasma membrane (orange).

Figure 5.

Measured distances between ROS membrane components. A schematic of a plasma membrane and two discs is shown. The measured distances between membrane components listed in Table I are illustrated on the schematic.

Figure 6.

Histogram of the gray values of disc membranes. (a) The gray value for each voxel (three-dimensional pixel) in the disc membrane volume was computed for 10 disc samples. Areas of low density (gray value < 0.33; light gray bars) represented 29% of the disc volume, and areas of high density (gray value > 0.33; dark gray bars) represented 71% of the disc volume. (b) The distribution of high (dark gray) and low (light gray) density regions is shown in a top view of a single disc.