RT Journal Article SR Electronic T1 In situ architecture of the ciliary base reveals the stepwise assembly of IFT trains JF bioRxiv FD Cold Spring Harbor Laboratory SP 2021.10.17.464685 DO 10.1101/2021.10.17.464685 A1 Hugo van den Hoek A1 Nikolai Klena A1 Mareike A. Jordan A1 Gonzalo Alvarez Viar A1 Miroslava Schaffer A1 Philipp S. Erdmann A1 William Wan A1 Jürgen M. Plitzko A1 Wolfgang Baumeister A1 Gaia Pigino A1 Virginie Hamel A1 Paul Guichard A1 Benjamin D. Engel YR 2021 UL http://biorxiv.org/content/early/2021/10/17/2021.10.17.464685.abstract AB The cilium is an antenna-like organelle that performs numerous cellular functions, including motility, sensing, and signaling. The base of the cilium contains a selective barrier that regulates the entry of large intraflagellar transport (IFT) trains, which carry cargo proteins required for ciliary assembly and maintenance. However, the native architecture of the ciliary base and the process of IFT train assembly remain unresolved. Here, we use in situ cryo-electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base. We combine this direct cellular visualization with ultrastructure expansion microscopy to describe the front-to-back stepwise assembly of IFT trains: IFT-B forms the backbone, onto which IFT-A, then dynein-1b, and finally kinesin-2 sequentially bind before entry into the cilium.Highlight Native molecular structure of the ciliary transition zone and hierarchical order of IFT assembly visualized within Chlamydomonas cells.Competing Interest StatementThe authors have declared no competing interest.