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Theodore G. Wensel, Jared C. Gilliam, Juan T. Chang, Wah Chiu; Electron Cryo-Tomography of Cilia-Associated Structures of Rod Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2011;52(14):4403.
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© ARVO (1962-2015); The Authors (2016-present)
Vertebrate rod photoreceptors sense light with modified primary cilia known as rod outer segments. The regions of the cell surrounding the junction between the inner and outer segments, known as the connecting cilium or transition zone, are the sites of highly active biosynthetic activity and directional protein and membrane trafficking and sorting. Defects in the cellular machinery associated with these processes give rise to numerous blinding diseases, including the ciliopathies, but their structures and compositions are poorly understood. Our purpose was to determine the three-dimensional structures of the transition zone and adjacent structures in rods without fixation or stains, using electron cryo-tomography.
Rod outer segments with attached inner segments were isolated from mice. The samples were flash-frozen in liquid ethane and imaged in a thin layer of vitreous ice using a 200 kV electron microscope at a range of tilt angles. The images from the tilt series were aligned and used to generate 3D reconstructions. The 3D maps were analyzed voxel-by-voxel to identify continuous surfaces, which were segmented for visualization.
The tomograms reveal for the first time in quantitative detail the three-dimensional structure of cilia-associated machines in a mammalian neuron, without distortions caused by fixatives, stains and sectioning. In the inner segment, the basal body, the ciliary rootlet, ribosomes, rough endoplasmic reticulum, and mitochondria are all clearly observed, as are the microtubule doublets of the connecting cilium. Large globular non-vesicular structures are observed inside the 9 + 0 bundle of microtubules, and membrane complexes are observed associated with long tubes adjacent to the plasma membrane. Among the surprising features observed in the outer segments is the envelopment of basal disks by a non-invaginated plasma membrane.
A combination of vesicle-mediated, non-vesicle-mediated, and plasma membrane-associated mechanisms appear to contribute to trafficking to, from, and through the connecting cilium. Light regulates the size distribution of transport particles. Basal disks are enveloped by a plasma membrane that runs continuously from the inner segment, through the connecting cilium, and around the basal disks.
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