In situ architecture of the ciliary base reveals the stepwise assembly of IFT trains

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

Preprint posted on 15 November 2021

All aboard! IFT trains assemble in a sequential manner at the ciliary base prior to entry

Selected by Nicola Stevenson

Categories: cell biology


Cilia are microtubule-based antenna-like organelles that extend out from the surface of the cell. Here they detect biochemical and mechanical cues and, in the case of motile cilia, beat to provide motility. The main structural component of a cilium is the axoneme; a cylindrical array of 9 microtubule doublets which extends from the triplet microtubules of the mother centriole to form a protrusion. This structure is enveloped by a specialised domain of the plasma membrane, the ciliary membrane, which wraps around the microtubules and anchors to the mother centriole at the base of the cilium. This anchorage point forms the transition zone, which is a selective gate formed of multiple protein complexes that controls the entry of proteins and lipids into the cilium.

The transition zone is vital for maintaining a compartment with a molecular identity distinct from that of the cytosol or plasma membrane. Its presence, however, poses a problem for proteins with a legitimate need to access the ciliary space. The exact mechanism of transition zone selectivity and function remains elusive, however one way to pass through is to hitch a ride on an intraflagellar transport (IFT) train. These trains are composed of long linear arrays of IFT particles which use the axoneme as a track to transport cargo into and out of the cilium. There are two types of IFT particle, IFTA and IFTB, which are themselves multi-subunit assemblies. Anterograde transport towards the ciliary tip is mediated by IFTB particles, which recruit the microtubule motor kinesin-2, whilst retrograde transport is mediated by IFTA particles and the dynein-2 (dynein-1b in Chlamydomonas reinhardtii) motor.

Whilst a lot is known about the structure of the assembled IFT train and its dynamics on the axoneme, the mechanisms of assembly, loading and unloading of trains remain enigmatic. The area around the ciliary base is a crowded place with centriolar and ciliary proteins all vying for space. Copious studies show that ciliary cargo, motors and IFT proteins all accumulate here waiting for their ticket through the gate. How then is order derived from this chaos to regulate ciliary entry? In this preprint, van den Hoek et al. make a big step towards understanding this process using cryo-electron tomography (ET) and expansion microscopy to visualise IFT train assembly.

Key findings

In this study, the authors use the model organism Chlamydomonas reinhardtii, which has a readily accessible flagellum (motile cilium), to investigate IFT assembly at the ciliary base. First, they look at the structure of the transition zone itself using cryo-ET and subtomogram averaging. Y-links are known to be a key structure within the transition zone, sitting between the axoneme and ciliary membrane. In this study, the authors show that membrane binding is not a pre-requisite for Y-link formation. Stellate fibres are also apparent, forming a cross-sectional 9-pointed star within the lumen of the axoneme. Distal to these structures a previously unseen helical ‘sleeve’ was observed decorating the microtubules, which the authors predict might designate sites of axoneme severing.

Intriguingly, filamentous strings of particles were also observed. These were tethered to the transition zone at one end, with the other end extending into the cytosol. Comparison with previous structures of mature axonemal trains confirmed that these strings were assembling IFT trains. Assembling trains were more flexible than their mature axonemal counterparts and contained an extra density on IFTB near the kinesin-2 binding site of unknown identity.

Comparison of the spatial arrangement of the various IFT components revealed that trains are assembled in a sequential manner. The most complete regions of the train are adjacent to the transition zone, then as it extends into the cytosol, first the dynein-1b and then the IFTA densities are missing. This suggests the IFTB backbone is built first, followed by IFTA and then dynein-2 recruitment. Kinesin-2 is relatively small and flexible and so could not be identified by cryo-ET. Instead, the authors use expansion microscopy to show that this is the final component recruited.

The transition zone and assembling IFT trains. Left: cryo-ET image taken from preprint figure 1. Right: Representative tomogram taken from preprint figure 2: yellow = IFTB; orange = IFTA; red = dynein1b; purple = stellate fibers; turquoise = Y-links; dark blue = MTD helical sleeve; grey = microtubules (added schematic).


I chose this preprint because it beautifully visualises the mysterious early stages of IFT train assembly. The events occurring at the basal body prior to cilium entry are essential in regulating cilium behaviour, both when it comes to building its structure and in determining its signalling capabilities, yet we know so little about them. Elucidating the mechanism of IFT train assembly helps shed light on how IFT is regulated to balance trafficking and ensure that only functional trains can take up valuable space on the axonemal highway. It is also a key step towards unravelling how the transition zone can function as a selective gate. The importance of all of these processes is evidenced by the devastating consequences of mutations in genes encoding IFT and transition zone proteins, which lead to a set of pleiotropic diseases termed ‘ciliopathies’.

Tags: cilium, dynein-2, flagella, ift, kinesin-2, protein complex assembly, structural biology, transport

Posted on: 15 November 2021


Read preprint (1 votes)

Author's response

Benjamin Engel shared

Questions to authors

One of the outstanding questions that comes to mind is how IFT trains can pass through the transition zone given their size. Did you see any relaxing or restructuring of the transition zone proteins at the points of train entry that would permit their passing? Or alternatively can you tell from their alignment and relative size if they could pass between structures such as the Y-links?


Interesting question! We did not observe evidence for transition zone relaxation. As seen in the raw tomogram slice in Fig. 1A (left image in this preLight), there is sufficient space between the microtubules and the membrane for IFT trains to pass through the transition zone. The Fig. 2A composite structure of a transition zone with bound IFT trains (right image in this preLight) shows how the trains run along the transition zone by tracking between the Y-links of two neighboring doublet microtubules. Furthermore, Fig. 3A-B shows the assembly state and position of every train in our dataset relative to the transition zone. We observed the front ends of many trains bound along the Y-links, but only a couple further distal along the MTD sleeve. As we estimate that each assembling train stays at the ciliary base for ~9 seconds prior to entry (see Discussion), we interpret this to mean that the sleeve is the point of no return, where a train will continue traveling into the cilium.


It would be difficult to determine from structural studies, but has your work thrown up any clues as to how IFT train length could be regulated during this assembly process?


IFT train length is a topic that Gaia Pigino and I have been thinking about for a long time. Will Ludington and Wallace Marshall observed that the size of an IFT train is correlated with the amount of time it spends at the ciliary base (Ludington et al., 2013; Our cryo-ET measurements show that both assembling and mature trains have a continuum of lengths (Fig. 3A and C), which is consistent with the idea of a relatively constant rate of IFT assembly at the base combined with stochastic entry into the cilium. However, this is speculative, and the precise molecular mechanisms that regulate IFT assembly and entry require further study. In my first paper as a student in the Marshall Lab, Will and I found that IFT trains are larger during the early stages of ciliary regrowth following pH-shock abscission (Engel et al., 2009; It would be very interesting to examine assembling IFT trains under these conditions, but we would need much better targeting of the ciliary base by cryo-FIB milling, as the modest cryo-ET dataset in this paper already took over 5 years to collect.


Did you see any evidence of disassembling trains and is this something you could look at in the future?


We haven’t spotted disassembling retrograde trains. We interpret this to mean that trains are rapidly disassembled at the ciliary base, similar to the rapid remodelling that occurs at the ciliary tip. We cannot say whether retrograde trains are disassembled before or after passing the MTD sleeve, but this microtubule decoration could potentially stop retrograde transport or detach the dynein motors. The reason we can see the assembling anterograde trains is that they hang around for several seconds before entry, and indeed every doublet microtubule of the transition zone is bound by an assembling train (see Fig. 4D-E).

Have your say

Your email address will not be published. Required fields are marked *

This site uses Akismet to reduce spam. Learn how your comment data is processed.

Sign up to customise the site to your preferences and to receive alerts

Register here

preLists in the cell biology category:

The Society for Developmental Biology 82nd Annual Meeting

This preList is made up of the preprints discussed during the Society for Developmental Biology 82nd Annual Meeting that took place in Chicago in July 2023.


List by Joyce Yu, Katherine Brown

CSHL 87th Symposium: Stem Cells

Preprints mentioned by speakers at the #CSHLsymp23


List by Alex Eve

Journal of Cell Science meeting ‘Imaging Cell Dynamics’

This preList highlights the preprints discussed at the JCS meeting 'Imaging Cell Dynamics'. The meeting was held from 14 - 17 May 2023 in Lisbon, Portugal and was organised by Erika Holzbaur, Jennifer Lippincott-Schwartz, Rob Parton and Michael Way.


List by Helen Zenner

9th International Symposium on the Biology of Vertebrate Sex Determination

This preList contains preprints discussed during the 9th International Symposium on the Biology of Vertebrate Sex Determination. This conference was held in Kona, Hawaii from April 17th to 21st 2023.


List by Martin Estermann

Alumni picks – preLights 5th Birthday

This preList contains preprints that were picked and highlighted by preLights Alumni - an initiative that was set up to mark preLights 5th birthday. More entries will follow throughout February and March 2023.


List by Sergio Menchero et al.

CellBio 2022 – An ASCB/EMBO Meeting

This preLists features preprints that were discussed and presented during the CellBio 2022 meeting in Washington, DC in December 2022.


List by Nadja Hümpfer et al.


The advances in fibroblast biology preList explores the recent discoveries and preprints of the fibroblast world. Get ready to immerse yourself with this list created for fibroblasts aficionados and lovers, and beyond. Here, my goal is to include preprints of fibroblast biology, heterogeneity, fate, extracellular matrix, behavior, topography, single-cell atlases, spatial transcriptomics, and their matrix!


List by Osvaldo Contreras

EMBL Synthetic Morphogenesis: From Gene Circuits to Tissue Architecture (2021)

A list of preprints mentioned at the #EESmorphoG virtual meeting in 2021.


List by Alex Eve

FENS 2020

A collection of preprints presented during the virtual meeting of the Federation of European Neuroscience Societies (FENS) in 2020


List by Ana Dorrego-Rivas

Planar Cell Polarity – PCP

This preList contains preprints about the latest findings on Planar Cell Polarity (PCP) in various model organisms at the molecular, cellular and tissue levels.


List by Ana Dorrego-Rivas

BioMalPar XVI: Biology and Pathology of the Malaria Parasite

[under construction] Preprints presented at the (fully virtual) EMBL BioMalPar XVI, 17-18 May 2020 #emblmalaria


List by Dey Lab, Samantha Seah


Cell Polarity

Recent research from the field of cell polarity is summarized in this list of preprints. It comprises of studies focusing on various forms of cell polarity ranging from epithelial polarity, planar cell polarity to front-to-rear polarity.


List by Yamini Ravichandran

TAGC 2020

Preprints recently presented at the virtual Allied Genetics Conference, April 22-26, 2020. #TAGC20


List by Maiko Kitaoka et al.

3D Gastruloids

A curated list of preprints related to Gastruloids (in vitro models of early development obtained by 3D aggregation of embryonic cells). Updated until July 2021.


List by Paul Gerald L. Sanchez and Stefano Vianello

ECFG15 – Fungal biology

Preprints presented at 15th European Conference on Fungal Genetics 17-20 February 2020 Rome


List by Hiral Shah

ASCB EMBO Annual Meeting 2019

A collection of preprints presented at the 2019 ASCB EMBO Meeting in Washington, DC (December 7-11)


List by Madhuja Samaddar et al.

EMBL Seeing is Believing – Imaging the Molecular Processes of Life

Preprints discussed at the 2019 edition of Seeing is Believing, at EMBL Heidelberg from the 9th-12th October 2019


List by Dey Lab


Preprints on autophagy and lysosomal degradation and its role in neurodegeneration and disease. Includes molecular mechanisms, upstream signalling and regulation as well as studies on pharmaceutical interventions to upregulate the process.


List by Sandra Malmgren Hill

Lung Disease and Regeneration

This preprint list compiles highlights from the field of lung biology.


List by Rob Hynds

Cellular metabolism

A curated list of preprints related to cellular metabolism at Biorxiv by Pablo Ranea Robles from the Prelights community. Special interest on lipid metabolism, peroxisomes and mitochondria.


List by Pablo Ranea Robles

BSCB/BSDB Annual Meeting 2019

Preprints presented at the BSCB/BSDB Annual Meeting 2019


List by Dey Lab


This list of preprints is focused on work expanding our knowledge on mitochondria in any organism, tissue or cell type, from the normal biology to the pathology.


List by Sandra Franco Iborra

Biophysical Society Annual Meeting 2019

Few of the preprints that were discussed in the recent BPS annual meeting at Baltimore, USA


List by Joseph Jose Thottacherry

ASCB/EMBO Annual Meeting 2018

This list relates to preprints that were discussed at the recent ASCB conference.


List by Dey Lab, Amanda Haage