A cryo-electron tomography study of ciliary rootlet organization.

Peer reviewed: True ; Acknowledgements: We thank D Morado for training and support in cryo-ET and subtomogram averaging and S Scheres for early access to Relion 4.0 alpha. We acknowledge K Qu, H Foster, and S Lacey for technical advice. We thank G Manigrasso for feedback on the manuscript; C Ventura, F Abid Ali, S Chaaban, and members of the Carter laboratory for discussion and T Dendooven and A Burt for support with subtomogram averaging. We thank the MRC Laboratory of Molecular Biology Electron Microscopy Facility for access and support of electron microscopy sample preparation and data collection; J Grimmett and T Darling for providing scientific computing resources; The Mouse Facility for providing mouse eyes. This work was supported by Wellcome [210711/Z/18/Z], the Medical Research Council, as part of United Kingdom Research and Innovation (also known as UK Research and Innovation) [MRC file reference number MC_UP_A025_1011], and C van Hoorn was funded by a Gates Cambridge Scholarship. ; Funder: Gates Cambridge Trust; FundRef: http://dx.doi.org/10.13039/501100005370 ; Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the mechanisms underlying these functions remain unknown, in part due to a lack of structural information of rootlet organization. In this study, we obtain 3D reconstructions of membrane-associated and purified rootlets from mouse retina using cryo-electron tomography. We show that flexible protrusions on the rootlet surface, which emanate from the cross-striations, connect to intracellular membranes. In purified rootlets, the striations were classified into amorphous (A)-bands, associated with accumulations on the rootlet surface, and discrete (D)-bands corresponding to punctate lines of density that run through the rootlet. These striations connect a flexible network of longitudinal filaments. Subtomogram averaging suggests the filaments consist of two ...