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

Background

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).

Importance

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

doi: https://doi.org/10.1242/prelights.31030

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