Three-Dimensional Structures Associated with Photoreceptor Cilia by Cryo-Electron Tomography

Abstract

The outer segment of rod and cone photoreceptor cells is a modified primary cilium. Specialized machinery is needed to assemble and maintain its structure, and to carry out the transport and sorting of molecular components between the inner segment, where most biosynthesis occurs, and the outer segment, where those components are utilized for phototransduction. The narrow connection between the inner and outer segment, known as the connecting cilium, is the location of proteins encoded by genes implicated in inherited diseases known as ciliopathies. Its structural core is a nine + zero microtubule-based axoneme that emerges from a centriole termed the basal body complex. Conserved features of this organelle include the transition fibers and transition zone with a ciliary necklace studding the ciliary membrane. We used cryo-electron tomography to determine the structures of the second basal body (daughter centriole), the connecting cilium and associated machinery in wildtype mice. Recently we used computational sub-tomogram averaging to determine the three-dimensional structure of the basal bodies. The 3D density map reveals a tapered cylindrical structure composed of nine MT bundle from the plus (triplet) end to the minus (doublet) end of the centriole. Additional densities that represent non-tubulin proteins attached to the triplet were revealed, which presumably stabilize the entire basal body. In addition, we have observed transition fibers, located at the ciliary base, connecting to microtubule triplets. The transition zone connects the microtubule doublets of the axoneme to the membrane near the basal body and extends into the connecting cilium toward the outer segment. These structural features in wildtype cilia will help us to understand the normal mechanisms and functions of the many proteins essential for cilium function and photoreceptor survival, and shed light on the ciliopathies that arise from defects in those proteins.