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. 2011 Oct 18;108(42):E845-53.
doi: 10.1073/pnas.1106178108. Epub 2011 Sep 19.

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella

Daniela Nicastro  1 , Xiaofeng Fu, Thomas Heuser, Alan Tso, Mary E Porter, Richard W Linck
Affiliations

Cryo-electron tomography reveals conserved features of doublet microtubules in flagella

Daniela Nicastro et al. Proc Natl Acad Sci U S A. .

Abstract

The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Cryo-ET provides an overview of the 3D structure of DMTs. Tomographic slices (A and B) and isosurface renderings (D–F) of averaged axonemal repeats from Chlamydomonas pseudo-WT (pWT; Table 1) show cross-sectional (A and D), longitudinal (B), and oblique (E and F) views of the DMT. The red lines in A indicate the cutting plane of the slice shown in B. In the surface renderings, only the DMT core is shown, whereas all peripheral structures [e.g., inner or outer dynein arm (IDA or ODA, respectively)] were removed but their positions are indicated in A (surface rendering overview with associated structures is shown in Fig. S1). PF numbers [according to Linck and Stephens (16)] are colored pink in the A-tubule (At) and dark blue in the B-tubule (Bt). In B, prominent left-handed helical lines with an 8-nm axial periodicity are apparent, probably corresponding to the helical lattice of tubulin subunits (28, 49). The IJ and trimeric outer junction (OJ) have distinct structures. Colored arrowheads point to MIP1 (light blue), MIP2 (red), MIP3 (yellow), and MIP4 (orange). DMT cross-sections are viewed from a proximal orientation (flagellar base) toward a distal (flagellar tip) orientation, and in the longitudinal view, the left side is proximal. The DMT orientations, labels, and colors shown here are used consistently in all subsequent figures unless otherwise noted and are valid for all panels. (C) Resolution of the DMT averages used in this study ranged from 3.3 to 3.9 nm (0.5 criterion of the Fourier shell correlation method). More details are provided in Table 1. (Scale bar: 10 nm.)
Fig. 2.
Fig. 2.
3D structure of DMTs reveals several MT-associated electron densities. Isosurface renderings of the averaged axonemal repeat from Chlamydomonas pseudo-WT show the DMT in cross-sectional views. (A) Crystal structures of the α/β-tubulin dimer were placed into the electron density of the DMT PFs, except for PF B1 (details are provided in the main text). No obvious differences were observed between the 13 A-tubule PFs; however, this does not exclude the possibility that one of them is composed of tektin instead of tubulin (69). Tubulin dimers and PF numbers are both colored pink in the A-tubule (At) and dark blue in the B-tubule (Bt). Note the channel that passes through the trimeric (PF A10-A11-B1) outer junction (OJ). The connecting density between PFs A1 and B10 at the IJ (purple-colored density) is relatively thin, suggesting that it is not a tubulin PF. Four additional features are present inside the DMT lumen (arrowheads and colored density): MIP1 (light blue), MIP2 (red), MIP3 (yellow), and MIP4 (orange). (Scale bar: 5 nm.) (B and B′) Stereo images provide a 3D view of the DMT and its associated inner structures.
Fig. 3.
Fig. 3.
Outer and inner AB-junctions are fundamentally different. Isosurface renderings (A, C, D, F, and H) and tomographic slices (B, E, and G) of averaged axonemal repeats from Chlamydomonas pseudo-WT (A–F) and drc mutants (details are provided in Table 1) that lack the hole in the IJ (G and H) are displayed in cross-sectional (A, C, and D) and longitudinal (B and E–H) views. Pink and dark blue numbers identify the PFs of the A- and B-tubules (At and Bt), respectively. Arrowheads point to MIP1 (light blue), MIP2 (red), MIP3 (yellow), and MIP4 (orange). Red lines in A and D indicate the orientation of the longitudinal slices in B, E, and G, respectively. Details of the outer junction (OJ; cyan outline) in an overview (A) and in a close-up (C) of the area indicated by the dashed box in A. PFs A10, A11, and B1 surround a channel (cyan arrow in B and C) that lacks significant EM density. The crystal structures of tubulin dimers were placed in the EM density and filtered to a resolution of ∼2 nm (pink and blue densities); note that the displayed orientation of tubulin-PF B1 (density highlighted in red) is not consistent with the observed empty channel. Details of the IJ (purple-colored density) that connects PFs A1 and B10 in an overview (D) and in a side-by-side comparison between DMTs with a WT phenotype (E and F) and drc mutants that lack major components of the nexin–dynein regulatory complex (N-DRC) (G and H). Longitudinal sections through the IJ-density reveal ladder rung-like densities with 8-nm periodicity (indicated by 3 white lines in E–H), instead of a continuous filamentous structure like the PFs (e.g., compare IJ with PFs A11–A13 in B). In DMTs with a WT phenotype (E and F), the IJ-rung closest to the N-DRC base plate is missing, leaving a hole within the IJ-density (red arrow in B, E, and F); when the N-DRC base plate is absent, however, as in the drc mutant pf3, the IJ-density is uninterrupted [i.e., no hole is observed (red dashed circle in G and H)]. (Scale bar: 20 nm.)
Fig. 4.
Fig. 4.
MIP1–4 and beak-MIP in Chlamydomonas axonemes show complex organization. Axonemal repeats from either all nine pseudo-WT DMTs (A–J) or only from DMT1, DMT5, and DMT6, which have the beak-MIP (K–N), were averaged. The MIP structures are shown in tomographic slices (A, C, E, G, I, K, and M) and isosurface renderings (B, D, F, H, J, L, and N) in cross-sectional (A, B, K, and L) and longitudinal views (C–J, M, and N). The blue, red, yellow, and orange lines in B and the green line in L indicate the cutting plane of the slices shown in C, E, G, I, and M, respectively. PFs of the A- and B-tubules (At and Bt) were numbered in pink and dark blue, respectively. In the isosurface renderings, the electron densities for MIP1–4 and the beak-MIP are color-coded (including the arrows and arrowheads pointing toward the MIPs): MIP1a and MIP1b (light and dark blue, respectively), MIP2a and MIP2b (light and dark red, respectively), MIP3a and MIP3b (yellow and olive, respectively), MIP4 (orange), and beak-MIP (green; also black and green brackets in L and M, respectively). The black arrows in E and H point to the hole in the IJ-density. (Scale bar: 20 nm).
Fig. P1.
Fig. P1.
Cryo-ET reveals the 3D structure of doublet microtubules. Tomographic slice (A) and isosurface renderings (B and C) of averaged axonemal repeats from Chlamydomonas flagella show cross-sectional views of the A- and B-tubules (At, Bt) and the following structures: MIP1 (blue arrowheads), MIP2 (red arrowheads), MIP3 (yellow arrowheads), MIP4 (orange arrowheads), inner junction (IJ), trimeric outer junction (OJ), At protofilaments (1–13), Bt protofilaments (1–10), and inner and outer dynein arms (IDA, ODA). In C, the crystal structures of the α/β-tubulin dimer were placed onto the electron density map of the doublet microtubule protofilaments. (Scale bars: A, 10 nm; C, 5 nm.)
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