In situ architecture of the ciliary base reveals the stepwise assembly of intraflagellar transport trains

Abstract

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. In this work, we used in situ cryo–electron tomography to reveal native structures of the transition zone region and assembling IFT trains at the ciliary base in Chlamydomonas . We combined 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 bind IFT-A, dynein-1b, and finally kinesin-2 before entry into the cilium.

IFT-A Structure Reveals Carriages for Membrane Protein Transport into Cilia

“…Cryo-ET has revealed the morphology of the C. reinhardtii IFT-A polymer within anterograde IFT trains at 24–33 Å resolution in situ (van den Hoek et al, 2022; Jordan et al, 2018). To understand where individual proteins are located, we docked our atomic models of human IFT-A1 and -A2 into the cryo-ET map (EMDB-4304) (Jordan et al, 2018) (Figure 5A).…”

“…We did not observe spontaneous polymerization of IFT-A in solution, suggesting that IFT-A polymers may be stabilized by binding to IFT-B or the membrane. Striking views of assembling IFT trains show strings of IFT-B polymers extending from the cilia base into the cytoplasm, which could form a scaffold for IFT-A self-assembly (van den Hoek et al, 2022). In these flexible assembling trains, IFT-A carriages would be exposed on the surface, where they could latch onto cargoes in the nearby peri-ciliary membrane, which is a hotspot for vesicular fusion (Nachury et al, 2010).…”

“…Once IFT-A is coupled to IFT-B and train assembly is complete, kinesin-2 would move the IFT train through the ciliary diffusion barrier (the transition zone) and into the cilium (van den Hoek et al, 2022; Lechtreck, 2015). Membranous cargoes would be dragged laterally from the peri-ciliary membrane through the transition zone (Figure 1A), which has both transmembrane and soluble components (Garcia-Gonzalo and Reiter, 2017).…”

“…The IFT-A1 module flexes around three principal axes with respect to IFT-A2, moving by up to 115° and 285 Å about the flexible linker (Figure 1D-F; Movie S1). This striking flexibility may enable IFT-A to adopt the different curvatures observed for IFT trains queuing at the ciliary base and traveling along the ciliary shaft (van den Hoek et al, 2022;Jordan et al, 2018). (C) Cryo-EM structure of IFT-A complex, which consists of two modules (IFT-A1 and IFT-A2).…”

The Molecular Structure of Anterograde Intraflagellar transport trains

“…Together these assemble into ordered and repetitive IFT “trains” that link the microtubule motors to hundreds of different IFT cargoes. The IFT process is initiated at the base of the cilium, where IFTB complexes start to polymerise on their own 3 . This nascent train acts as a platform for IFTA polymerisation, and recruits kinesin-2 motors ( Figure 1A) .…”