The molecular structure of primary cilia revealed by cryo-electron tomography

Abstract

Primary cilia are microtubule-based organelles involved in key signaling and sensing processes in eukaryotic cells. Unlike motile cilia, which have been thoroughly studied, the structure and the composition of primary cilia remain largely unexplored despite their fundamental role in development and homeostasis. They have for long been falsely regarded as simplified versions of motile cilia because they lack distinctive elements such as dynein arms, radial spokes, and central pair complex. However, revealing the detailed molecular composition and 3D structure of primary cilia is necessary in order to understand the mechanisms that govern their functions. Such structural investigations are so far being precluded by the challenging preparation of primary cilia for cryo-electron microscopy. Here, we developed an enabling method for investigating the structure of primary cilia at molecular resolution by cryo-electron tomography. We show that the well-known "9+0" arrangement of microtubule doublets is present only at the base of the primary cilium. A few microns away from the base the ciliary architecture changes into an unstructured bundle of EB1-decorated microtubule singlets and some actin filaments. Our results suggest the existence of a previously unobserved crosstalk between actin filaments and microtubules in the primary cilium. Our work provides unprecedented insights into the molecular structure of primary cilia and a general framework for uncovering their molecular composition and function in health and disease. This opens up new possibilities to study aspects of this important organelle that have so far been out of reach.We would like to thank the Electron Microscopy Facility (in particular Tobias Fürstenhaupt, Weihua Leng, Michaela Wilsch-Bräuninger) and the Light Microscope Facility from the Services and Facilities of the MPI-CBG for their support. We are thankful to Helin Rägel and Cécilie Martin-Lemaitre for their tips on MDCKII cell culture, Noreen Walker for the Imaris tutorial, and Tim-Oliver Buchholz for denoising cryo-tomography data. We thank Pavel Tomancak, Florian Jug, Dennis Diener, and Jan Brugues for the fruitful discussions and suggestions to the manuscript. We thank Oscar Gonzales for IT support. . developed the cryo-peel-off method, prepared samples for FM and EM imaging, acquired and reconstructed room temperature and cryo-tomograms, contributed to FM data acquisition, analysed EM and FM data, prepared figures, interpreted results, and contributed to writing and revising the manuscript. G.A.V. prepared samples and contributed to data acquisition of the room temperature tomography, analyzed cryo-EM data with subtomogram averaging and tomogram segmentation, analysed EM and FM data, prepared figures, interpreted results, and contributed to writing and revising the manuscript. N.T. analyzed cryo-ET data to average the microtubule singlets, contributed to supplementary figure preparation, contributed to the interpretation of data, and contributed to writing and revising the manuscript. R.M. pre...

Microtubule‐associated proteins and emerging links to primary cilium structure, assembly, maintenance, and disassembly

“…Although early cross‐sectional electron microscopy studies have challenged the 9 + 0 paradigm of the ciliary axoneme, its three‐dimensional high‐resolution structural map has remained unknown until recently. The application of serial section electron tomography and cryo‐electron microscopy combined 'cryo‐peel‐off' sample preparation method in kidney epithelial cells together revealed unprecedented insight into the near‐native 3D structure of the primary cilia and confirmed that its axonemal organization does not conform to the canonical 9 + 0 configuration [18,19] (Fig. 1B).…”

“…In contrast to the periodic localization of the MIPs in the motile cilia axonemes, the MIPs localize sparsely along the axoneme of the primary cilium, which raises questions that pertain to how the stability of the primary cilium is maintained. The identities of the densities identified in the 3D structural maps are not known except for microtubule (+) end‐tracking protein EB1 that decorates the axoneme and actin filament bundles that underlie the ciliary membrane [18]. A recent study identified a helical inner scaffold for the centrioles and showed that it is required for maintaining the structural integrity of the centrioles [26].…”

“…Of note, CCDC66 and TOGARAM‐1 decorate the axoneme in a punctate pattern with irregular spacing and the axonemal pool of CCDC66 in steady‐state cilium is immobile [55,75]. Analogously, the recent molecular architecture of primary cilium revealed axonemal localization of EB1, which was unexpected due to its well‐described interaction with the microtubule plus ends [18]. Based on the periodicity of EB1 densities along the axoneme, EB1 was proposed to contribute to stabilizing the configuration of axonemal microtubules.…”

Intraflagellar transport trains and motors: Insights from structure

“…Anterograde train movement to the ciliary tip is powered by kinesin-II (stage 3) [ 12 , 35 , 36 ]. It occurs on the B-tubule of microtubule doublets [ 37 ], and can also occur on the singlet A-tubules found in the distal regions of primary cilia [ [38] , [39] , [40] ]. At the ciliary tip, anterograde trains remodel into retrograde trains (stage 4) [ 28 , 37 , [41] , [42] , [43] , [44] ], which return to the ciliary base by moving along the A-tubule under the power of dynein-2 (stage 5) [ 37 , [45] , [46] , [47] ].…”

A Proximity Mapping Journey into the Biology of the Mammalian Centrosome/Cilium Complex

“…The basal body is anchored to the plasma membrane through its distal appendages. Recent electron tomography studies of the cilium generated its 3D structural maps, which significantly changed the widely accepted 9+0 paradigm for the axonemal organization of ciliary microtubules [51,52]. According to the revised model, the nine radially arranged microtubule doublets at the proximal end of the axoneme transitions into an unstructured bundle of microtubule singlets of variable lengths towards the distal end of the cilium.…”