Protein transport in growing and steady-state cilia

Abstract

Cilia and eukaryotic flagella are threadlike cell extensions with motile and sensory functions. Their assembly requires intraflagellar transport (IFT), a bidirectional motor-driven transport of protein carriers along the axonemal microtubules. IFT moves ample amounts of structural proteins including tubulin into growing cilia likely explaining its critical role for assembly. IFT continues in non-growing cilia contributing to a variety of processes ranging from axonemal maintenance and the export of non-ciliary proteins to cell locomotion and ciliary signaling. Here, we discuss recent data on cues regulating the type, amount and timing of cargo transported by IFT. A regulation of IFT-cargo interactions is critical to establish, maintain and adjust ciliary length, protein composition and function.

Keywords: diffusion; flagella; intraflagellar transport; microtubule.

Figure 1. The structure of cilia and flagella

A) Scanning micrograph showing the ciliated epithelium lining the ventricular system of the brain in mouse. B-D) Thin sections showing the proximal region of the basal body (B) with the attached basal foot (bf), a more distal section with the paddle-wheel like transitional fibers (C, arrowheads), and the transition zone (D) in cross-sections. In D, note the Y-shaped connectors (arrows) linking the doublet microtubules to the ciliary membrane. E) Micrograph showing airway cilia with typical 9+2 axonemes in cross-section. Bars = 10 μm (A) and 250 nm (E).

Figure 2. The intraflagellar transport machinery

A) Composition of IFT particles, IFT motors, and the BBSome. For the motors, the mammalian protein names are shown; the Chlamydomonas protein names are listed in the brackets. B) Schematic presentation of IFT. Ax, axoneme, TZ, transition zone, TF, transition fibers, BB, basal body. C) Schematic presentation and electron micrograph depicting IFT trains (open arrows). Bar = 200 nm. D) Still image (left) and kymogram (right) showing IFT54-NG inside a Chlamydomonas flagellum. In the kymogram, anterograde trains are indicated by trajectories running from the bottom left to the top tight (blue arrow); trajectories running from the top left to the bottom right represent retrograde trains (red arrow). Bars = 2s 2 μm. E) Kymograms depicting transport by IFT and unloading of the axonemal protein DRC4-GFP. IFT20-mCherry was expressed to visualize IFT. IFT trajectories are marked by open arrowheads. DRC4-GFP initially co-migrates with an IFT train but is then unloaded (white arrowhead) as indicated by the transition of the trajectory from a linear diagonal to a back-and-forth motion indicative for diffusion. Note that most cargoes are unloaded in the vicinity of the ciliary tip. Bar = 1s 2 μm.

Figure 3. Cargo transport by IFT during ciliary assembly and maintenance

A-D) Models for ciliary length control. A) The transport-limitation model suggests that cells will employ many IFT trains while cilia grow and reduce the number of trains in fully grown cilia. The cargo load per train is constant. B) In the balance-point model, the ciliary assembly rate will decrease with increasing ciliary length because the time the trains spend in transit will increase. Neither the number of IFT trains nor the cargo load/train are regulated. Chlamydomonas flagella grow at a rate of up to 350 nm/min. In addition to delivering the building blocks accounting for this gain in length, IFT would have to provide those lost by ongoing ciliary disassembly. Our simulations showed that it is not possible to assemble cilia of 10 - 12 μm length in ∼60 min when assuming that the continuous length-independent disassembly of cilia is large enough to balance the large amount of building blocks provided ceaselessly by anterograde IFT; rather cilia would need hours of slow growth to reach steady-state length. C) The supply-limitation model predicts that cilia will grow until the cell body pool of precursors is exhausted. IFT cargo load is regulated passively by the availability of cargoes. D) The differential-loading model suggests that cells alter the amount of cargo per IFT train in response to changes in ciliary length. The length of the arrows near the flagellar tip indicate the rates of material delivery and cilia disassembly. E-I) Models of IFT function in fully assembled cilia. E) Material delivery for cilia maintenance via a low but steady influx of ciliary proteins. F) Removal of non-ciliary proteins entering cilia via diffusion from the cell body. G) Back-and-forth movement of extracellular objects on the ciliary surface (left). In gliding motility, IFT dynein, immobilized via IFT particles and transmembrane proteins to the substrate, pulls the cell by moving toward the minus-end of the axonemal microtubules. H) Import and export of proteins to change ciliary protein composition; e.g. during adaptation. I) Conditional transport of activated signaling proteins. Signals such as ligand binding change the properties of a protein allowing it to adhere to IFT trains.