Structural studies of ciliary components - PubMed (original) (raw)

Review

Structural studies of ciliary components

Naoko Mizuno et al. J Mol Biol. 2012.

Abstract

Cilia are organelles found on most eukaryotic cells, where they serve important functions in motility, sensory reception, and signaling. Recent advances in electron tomography have facilitated a number of ultrastructural studies of ciliary components that have significantly improved our knowledge of cilium architecture. These studies have produced nanometer-resolution structures of axonemal dynein complexes, microtubule doublets and triplets, basal bodies, radial spokes, and nexin complexes. In addition to these electron tomography studies, several recently published crystal structures provide insights into the architecture and mechanism of dynein as well as the centriolar protein SAS-6, important for establishing the 9-fold symmetry of centrioles. Ciliary assembly requires intraflagellar transport (IFT), a process that moves macromolecules between the tip of the cilium and the cell body. IFT relies on a large 20-subunit protein complex that is thought to mediate the contacts between ciliary motor and cargo proteins. Structural investigations of IFT complexes are starting to emerge, including the first three-dimensional models of IFT material in situ, revealing how IFT particles organize into larger train-like arrays, and the high-resolution structure of the IFT25/27 subcomplex. In this review, we cover recent advances in the structural and mechanistic understanding of ciliary components and IFT complexes.

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Figures

Graphical abstract

Fig. 1

Simplified representations of a cilium and various axonemal structures. (a) Longitudinal section through a motile cilium. The various regions of the cilium are indicated on the left (for detailed information on these regions, the reader is referred to a recent review by Fisch and Dupuis-Williams2). The A and B MTs of the basal body triplets are extended to form the outer doublets of the axoneme. Motility-related axonemal structures are attached to the A-tubule. IFT particles move between the B-tubule and the overlying ciliary membrane. A long anterograde IFT train (moving towards the ciliary tip) is shown on the left; a short retrograde IFT train (moving back to the base) is shown on the right. These two types of trains differ not only in their overall length but also in the length of their repeating units. Triangles marked with B and C indicate the planes of cross sections shown in (b) and (c), respectively. The dashed black rectangle indicates the region shown in detail in (d). (b) Cross section through the proximal part of the basal body. The MTs in the triplets are designated as A, B, and C, with the A-tubule being closest to the center. The ‘cartwheel’ structure can be found within the ring of triplets and connects to the A-tubules. (c) Cross section through the doublet zone of the ciliary axoneme. Nine MT doublets form a ring that is connected to the central pair complex by RSs. Outer dynein arms (ODAs) and inner dynein arms (IDAs) extend from the A-tubule towards the B-tubule of the adjacent doublet. Two IFT trains are shown, moving between the B-tubule and the overlying ciliary membrane. (d) Schematic illustration of data from ET on the structure of the 96‐nm axonemal repeat. ODAs, represented by the three dynein heavy chains (α, β, γ), are shown as red circles. IDA heavy chains are shown as green circles and the IDA intermediate and light chains (IDA IC/LC) are shown as a green oval. The gray oval indicates the location of the nexin/dynein regulatory complex (N-DRC). Two RSs (RS1 and RS2) are represented in purple and extend towards the central pair complex. An additional ‘stump’ (RS3 ‘stump’), as observed in C. reinhardtii flagella, is also indicated as a short purple structure. (e) View of the structure shown in (d) from distal to proximal. Colors and shapes of the various structures are the same as in (d). (f) The RS consists of three distinct regions (head, stalk, and neck). The RS proteins (RSPs) found in each region are indicated.

Fig. 2

SAS-6 crystal structures: molecular basis for the 9-fold symmetry of centrioles. (a) Schematic representation of the domain organization of SAS-6. The protein contains an N-terminal head domain, a central coiled-coil domain, and a C-terminal region that is not well conserved in sequence. (b) Crystal structure of the N-terminal head domain of SAS-6 from C. reinhardtii. Each monomer is shown in a different color and the phenylalanine (F145) crucial for dimer formation is labeled and shown in black stick representation. (c) Crystal structure of a longer N-terminal construct of SAS-6 from C. reinhardtii [compared to (b)] containing part of the central coiled-coil domain. This structure shows a second dimerization interface mediated by the coiled coil. (d) Model for 9-fold symmetrical multimerization of SAS-6. Modeling of the two dimer structures shown within the dashed rectangles of (b) and (c) results in a ring-like structure with exactly nine copies of SAS-6 dimers. Such an arrangement likely provides a molecular rationale for the 9-fold symmetry of centrioles. All structures shown in this figure were determined from C. reinhardtii proteins.

Fig. 3

Dynein motor domain crystal structure. (a) Crystal structure of the dynein motor domain from Dictyostelium discoideum shown in cartoon representation. The various structural elements such as stalk, strut, linker region, and AAA domains are shown in different colors. (b) Model for the dynein power stroke (drawn based on Fig. 5 from Kon _et al._74). The large conformation change in the linker region during power stroke is indicated by dark purple (pre-power stroke) and light purple (post-power stroke) colors. The AAA1–6 domains are labeled 1–6. MTBD, microtubule binding domain.

Fig. 4

EM structure of IFT trains in situ. Electron tomographic reconstruction of IFT trains using subtomographic averaging. The center image shows an IFT train (red) sandwiched between the flagellar membrane (gray) and an MT doublet (yellow). The top image is a 90° rotation (as indicated by the arrow) with the membrane removed. ‘X’ denotes points of contact between the IFT particles and the flagellar membrane. The bottom image is a 90° rotation (as indicated by the arrow) with the MT removed. ‘k?’ denotes points of contact between the MT and the IFT particles and may represent kinesin motors. From this image, it is clear that IFT trains are composed of strings of IFT particle dimers rather than monomers.

Fig. 5

Crystal structure of the IFT25/27 complex. Crystal structure at 2.6 Å resolution of the IFT25/27 complex from C. reinhardtii. IFT25 (purple) binds one divalent calcium ion (shown as an orange ball), and IFT27 (cyan) is a GTPase with the putative GTP-binding pocket indicated with the black circle. The GTP-binding site is located far from the IFT25/27 interface, indicating that IFT25 likely does not influence the GTP binding or GTPase activity of IFT27 directly.

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Structural studies of ciliary components - PubMed (original) (raw)