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

Hoek, Hugo van den; Klena, Nikolai; Jordan, Mareike A; Viar, Gonzalo Alvarez; Righetto, Ricardo D.; Schaffer, Miroslava; Erdmann, Philipp; Wan, William; Geimer, Stefan; Plitzko, Jürgen M.; Baumeister, Wolfgang; Pigino, Gaia; Hamel, Virginie; Guichard, Paul; Engel, Benjamin D.

doi:10.1126/science.abm6704

Cited by 98 publications

(107 citation statements)

References 64 publications

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“…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).…”

Section: Resultsmentioning

confidence: 99%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Discussionmentioning

confidence: 99%

“…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).…”

Section: Ift-a Consists Of Two Flexibly Connected Modulesmentioning

confidence: 99%

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

Hesketh

Mukhopadhyay

Nakamura

et al. 2022

Preprint

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Intraflagellar transport (IFT) trains are molecular machines that traffic proteins between cilia and the cell body. With a molecular weight over 80 MDa, each IFT train is a dynamic polymer of two large complexes (IFT-A and -B) and motor proteins, posing a formidable challenge to mechanistic understanding. Here, we reconstituted the complete human IFT-A complex and obtained its structure using cryo-EM. Combined with AlphaFold prediction and genome-editing studies, our results illuminate how IFT-A polymerizes; interacts with IFT-B; and uses an array of beta-propeller and TPR domains to create "carriages" of the IFT train that engage TULP adaptor proteins. We show that IFT-A:TULP carriages are essential for cilia localization of diverse membrane proteins, as well as ICK - the key kinase regulating IFT train turnaround. These data establish a structural link between IFT-A's distinct functions, provide a blueprint for the IFT-A train, and shed light on how IFT evolved from a proto-coatomer ancestor.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Ift-a Consists Of Two Flexibly Connected Modulesmentioning

confidence: 99%

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

Hesketh

Mukhopadhyay

Nakamura

et al. 2022

Preprint

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“…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) .…”

Section: Introductionmentioning

confidence: 99%

The Molecular Structure of Anterograde Intraflagellar transport trains

Lacey

Foster

Pigino

2022

Preprint

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Anterograde intraflagellar transport trains are essential for cilia assembly and maintenance. These trains are formed of 22 IFTA and IFTB proteins that link structural and signalling cargoes to microtubule motors for import into cilia. It remains unknown how the IFTA/B proteins are arranged into complexes and how these complexes polymerise into functional trains. Here, we use in situ cryo-electron tomography and Alphafold2 protein structure predictions to generate the first molecular model of the entire anterograde train. We show how the conformation of both IFTA and IFTB is dependent on lateral interactions with neighbouring repeats, suggesting that polymerization is required to cooperatively stabilize the complexes. The retrograde dynein motor binding site is a composite surface involving multiple IFTB repeats, ensuring that dynein can only form a strong interaction with IFTB upon train assembly. Finally, we reveal how IFTB extends two flexible tethers to maintain a connection with IFTA that can withstand the mechanical stresses present in actively beating cilia. Overall, our findings provide a framework for understanding the fundamental processes that are involved in cilia assembly.

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Architecture of RabL2‐associated complexes at the ciliary base: A structural modeling perspective

Boegholm,

Petriman,

Tanvir

et al. 2024

BioEssays

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Cilia are slender, micrometer‐long organelles present on the surface of eukaryotic cells. They function in signaling and locomotion and are constructed by intraflagellar transport (IFT). The assembly of IFT complexes into so‐called IFT trains to initiate ciliary entry at the base of the cilium remains a matter of debate. Here, we use structural modeling to provide an architectural framework for how RabL2 is anchored at the ciliary base via CEP19 before being handed over to IFT trains for ciliary entry. Our models suggest that the N‐terminal domain of CEP43 forms a homo‐dimer to anchor at the subdistal appendages of cilia through a direct interaction with CEP350. A long linker region separates the N‐terminal domain of CEP43 from the C‐terminal domain, which captures CEP19 above the subdistal appendages and close to the distal appendages. Furthermore, we present a structural model for how RabL2‐CEP19 associates with the IFT‐B complex, providing insight into how RabL2 is handed over from CEP19 to the IFT complex. Interestingly, RabL2 association with the IFT‐B complex appears to induce a significant conformational change in the IFT complex via a kink in the coiled‐coils of the IFT81/74 proteins, which may prime the IFT machinery for entry into cilia.

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

Cited by 98 publications

References 64 publications

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

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

The Molecular Structure of Anterograde Intraflagellar transport trains

Architecture of RabL2‐associated complexes at the ciliary base: A structural modeling perspective

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