Structural insights into the architecture and assembly of eukaryotic flagella

Abstract

Cilia and flagella are slender projections found on most eukaryotic cells including unicellular organisms such as Chlamydomonas, Trypanosoma and Tetrahymena, where they serve motility and signaling functions. The cilium is a large molecular machine consisting of hundreds of different proteins that are trafficked into the organelle to organize a repetitive microtubule-based axoneme. Several recent studies took advantage of improved cryo-EM methodology to unravel the high-resolution structures of ciliary complexes. These include the recently reported purification and structure determination of axonemal doublet microtubules from the green algae Chlamydomonas reinhardtii, which allows for the modeling of more than 30 associated protein factors to provide deep molecular insight into the architecture and repetitive nature of doublet microtubules. In addition, we will review several recent contributions that dissect the structure and function of ciliary trafficking complexes that ferry structural and signaling components between the cell body and the cilium organelle.

Keywords: BBSome; cilia; intraflagellar transport (IFT); microtubule doublets.

Figure 1. FIGURE 1: Schematic representation of a cilium from Chlamydomonas reinhardtii.

(a) Architecture of a cilium with a simplified scheme of the bi-directional intraflagellar transport (IFT) system trafficking proteins between the cilium and the cell body. IFT complexes (green) assemble into train-like polymers powered by heterotrimeric kinesin-II (purple) in the anterograde direction (base->tip). Inactive dynein-1b (yellow) is loaded as a cargo onto anterograde IFT ‘trains'. Once assembled and loaded with ciliary cargo, these trains are driven across the transition zone and Y-links (depicted as grey connections spanning from the ciliary membrane to the axoneme at the ciliary base) to reach the ciliary tip. Upon arrival at the ciliary tip, the IFT ‘trains' are re-modelled, turnover products are picked up and moved back again to the ciliary base by retrograde IFT powered by the now activated dynein-1b motor. (b) Schematic representation of an axoneme cross section of a motile cilium, which depicts the nine peripheral doublet microtubules (DMTs; PDB entry: 6u42) that surround a central microtubule pair (CP, shown in grey). The DMTs are connected though the nexin dynein regulatory complex (N-DRC, shown in green). Complexes responsible for ciliary beating are the inner- and outer- dynein arms (IDAs and ODAs; orange and magenta, respectively). The central pair is connected to the nine DMTs by radial spokes (RS; blue).

Figure 2. FIGURE 2: Structure of the 48nm ciliary doublet microtubule repeat (pdb entry: 6u42).

(a) Cross section through a ciliary DMT. The outer dynein arms docking complexes 2 and 3 serve as docking sites for ODAs on the exterior of the DMTs and are labelled ODA-DC2 and ODA-DC3. Inner microtubule proteins (MIPs) are highlighted in different colours and labelled according to protein name. (b-d) Depiction of microtubule inner proteins, their interaction network and periodicity visualized within the confinements of the doublet microtubule from the minus (-) to the plus (+) end. (b) Lateral view of MIPs decorating the lumen of B-tubule as seen after a 10 nm deep slice facing the viewer was removed. The black arrow in the transparent region on the left representation indicates the region that has been removed for better clarity. The ODA-DC complex is left out as part of the removed section. (c) A lateral view displaying MIPs decorating both the A- and B-tubules. A 10 nm deep slice facing the viewer was removed as shown by the black arrow in the transparent region on the left representation. MIPs are visualized after rotation of the DMTs of 120° along the longitudinal axis relative to (b). (d) Visualisation of MIPs decorating the lumen of the A-tubule after removal of a 10 nm deep slice facing the viewer and rotation of the doublet microtubule with 120° along its longitudinal axis relative to (c). In this representation the alternating PCRG/FAP20 complex is observed from the exterior of the B-tubule.