ECB-ART-42621
PLoS One
2012 Jan 01;710:e46494. doi: 10.1371/journal.pone.0046494.
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One of the nine doublet microtubules of eukaryotic flagella exhibits unique and partially conserved structures.
Lin J
,
Heuser T
,
Song K
,
Fu X
,
Nicastro D
.
???displayArticle.abstract???
The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1-2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella.
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Genes referenced: dnah3 ift52 LOC100887844 LOC100893907
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Figure 1. Schematic models of the axoneme organization in the sea urchin Strongylocentrotus and the green alga Chlamydomonas. (A) Two numbering systems for designating the DMTs in 9+2 cilia and flagella are currently in use (left [7], right [15]). The flagella from both organisms have a highly conserved cylindrical arrangement of nine DMTs (red boxes). Each DMT is built from many copies of a 96-nm long unit that repeats along the DMT length. The axonemes are shown in cross-sectional views from the flagellar base (proximal) towards the tip (distal). The locations of the previously described 5–6 bridge (left) and proximal 1–2 bridge (right) are indicated. (B) For both organisms, schematic representations of a 96-nm repeat are shown in longitudinal and cross-sectional views; orientations of the 96-nm repeat are maintained in all following figures unless stated otherwise. Other labels: A-tubule (At), B-tubule (Bt), inner dynein arms (IDA 1α, 1β, 2–6 and x; rose) [43], intermediate chain/light chain complex (ICLC), nexin-dynein regulatory complex (N-DRC, yellow) [37], outer dynein arm (ODA, purple), radial spoke (RS, gray) [13], [30]. |
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Figure 2. Distinct structural features in the distal 7/8th of Strongylocentrotus DMT5 revealed by cryo-ET.Isosurface renderings (A, B, I, and J) and tomographic slices (C–H) show unique structural features present only on DMT5 (left column) in cross-sectional (A–D) and longitudinal views from the front (E and F) and the bottom (G–J). The other eight DMTs are similar to each other but distinct from DMT5. For comparison, the averages of DMT6 are presented in the right column (see also Figure S1 and Movie S1). (A–D) Cross-sectional views of SUB5-6, which is present only on DMT5. SUB5-6 consists of two parts: the outer o-SUB5-6 (green) and the inner i-SUB5-6 (orange). The red dashed lines in (C and D) indicate the locations of the tomographic slices shown in (E–H). Note that the center of axonemal repeats shifts longitudinally between neighboring doublets; therefore the longitudinal positions of the slices shown in (A and C) and (B and D) are not identical. (E and F) Several IDAs (IA2, 3, 4, IAX) are missing from DMT5, and the ODAs are substituted by o-SUB5-6. The dotted circle in (E) outlines the i-SUB5-6 complex that is missing from other DMTs, as shown for DMT6 (F). (G–J) Details of the outer and inner SUB5-6 structures. The o-SUB5-6 complexes (green arrowheads) are morphologically distinct from the ODAs, but share the same 24-nm periodicity (compare G and H); i-SUB5-6 (orange) has a 96-nm periodicity and makes clear connections with I1 dynein and N-DRC while linking DMT5 to DMT6. Other labels: A-tubule (At), B-tubule (Bt), inner dynein arms (IDA 1α, 1β, 2–6, x; rose), nexin-dynein regulatory complex (N-DRC, yellow), outer dynein arm (ODA, purple), radial spoke (RS, dark-gray). Cross-sections are viewed from the proximal, and in longitudinal views proximal is on the left. The sea urchin DMT numbers are according to [7]. The protofilaments are numbered after [53]. Scale bar (H): 25 nm. |
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Figure 3. Distinct structural features in the distal three quarters of Chlamydomonas DMT1.Isosurface renderings (A, B, I, and J) and tomographic slices (C–H) show i-SUB5-6 and the missing IDAs on Chlamydomonas DMT1 (left column) in cross-sectional (A–D) and longitudinal views from the front (E and F) and the bottom (G–J). The structure of DMT1 is unique and different from DMTs 2–9, which have a similar structure; DMT2 is shown for comparison in the right column (see also Figure S1 and Movie S3). (A–D) Cross-sectional views of the i-SUB5-6 complex (orange), which is present on DMT1 only and links DMT1 to DMT2. The ODAs are missing from DMT1 but are present on all other DMTs. The red dashed lines in (C and D) indicate the locations of the tomographic slices shown in (E–H). Note that the beak structure in the B-tubule of DMT1 in (A and C) has previously been reported to be present in the proximal half of the flagellum [15], and is therefore still visible in this average of the distal three quarters of the axoneme. (E and F) Several IDAs (IA3, 4, and IAX) are absent on DMT1 (E) but present on DMT2 (F). The orange dotted circle in (E) outlines the i-SUB5-6 complex that is present on DMT1, but absent from DMT2 (F). (G–J) Details of the i-SUB5-6 structure (orange) demonstrate that it has a periodicity of 96 nm and makes clear connections with both I1 dynein and N-DRC while linking DMT1 to DMT2. Other labels: A-tubule (At), B-tubule (Bt), inner dynein arms (IDA 1α, 1β, 2–6 and x; rose), nexin-dynein regulatory complex (N-DRC, yellow), outer dynein arm (ODA, purple), radial spoke (RS, dark-gray). The Chlamydomonas DMT numbers are according to Hoops and Witman [15]. The protofilaments are numbered after Linck and Stephens [53]. Scale bar (H): 25 nm. |
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Figure 4. Distinct structural features in specific regions of Chlamydomonas DMT1 and Strongylocentrotus DMT5.(A–E) The 1–2 bridge in the proximal quarter of Chlamydomonas DMT1. Tomographic slices show the proximal 1–2 bridge between DMTs 1 and 2 in Chlamydomonas pWT in cross-sectional (A and B) and longitudinal views from the front (C and E) and the bottom (D). Red dashed lines in (A) indicate the locations of tomographic slices shown in (C–E). The 1–2 bridge consists of two parts that link DMTs 1 and 2; each part consists of a row of periodic densities (blue circles in A, and C–E). The connection of the top row to DMT2 is highlighted (yellow arrowheads in A and D). The red circles in (A and C) indicate an additional row of periodic but shorter densities that do not reach DMT2. Note that the I1 dynein is absent from the proximal quarter of DMT1 and only a small neighboring density, the I1 tether [34], is visible (C and E). (F) A tomographic slice from the distal part of DMT1 is shown for direct comparison with the proximal slice in (E). Note that the distal region of Chlamydomonas DMT1 lacks the 1–2 bridge, while the I1 dynein is present. (G and H) Tomographic slices in front view show a comparison between the proximal 1/8th (G) and distal 7/8th (H) of the Strongylocentrotus DMT5. While in the very proximal region regular ODAs are present, they are substituted by o-SUB5-6 structures in the distal 7/8th of the flagellum. Note that along the entire length of DMT5 i-SUB5-6 is consistently present, while several IDAs (IA 2–4, IAX) are lacking. Scale bars are 25 nm (scale bar in B valid for A and B; scale bar in F valid for C–H). |
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Figure 5. i-SUB5-6 is present in Chlamydomonas I1 mutants and N-DRC mutants.(A–H) A comparison of tomographic slices from averages of the distal part of DMT1 from Chlamydomonas pWT (A and B), I1-dynein-lacking mutant pf9-3 (C and D), a combined average of the N-DRC mutants sup-pf-3 and pf2 (E and F), and the dynein-IA4-lacking mutant ida6 (G–I) indicates that the lack of I1 dynein or IA4 (dynein e) or a dramatic reduction in the N-DRC has little effect on the i-SUB5-6 structure (orange arrowheads). The tomographic slices were viewed from the front (left column) and the bottom (right column) and show the same locations of the axonemal repeat as shown in Figure 3E and 3G, respectively. The orientation of the tomographic slice shown in (B) is indicated by red dashed lines in (A). (I and J) Tomographic slices from combined averages of DMT2-9 show the absence of IA4 in the ida6 axoneme (I) compared to pWT (J). Scale bar (B): 10 nm. |
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Figure 6. Schematic models of the axonemes and doublets D-V from Strongylocentrotus and Chlamydomonas.(A and C) Simplified diagrams of the axonemes viewed in cross-section from proximal correlate the DMTs from Strongylocentrotus and Chlamydomonas. A unifying numbering convention for the DMTs in eukaryotic 9+2 axonemes is used, based on the observed conserved structural features. This convention facilitates comparisons of DMTs among species and assigns the same doublet number to corresponding DMTs. The previously used numbering systems (Arabic numerals in B-tubules) are also depicted for comparison. Under the new numbering system, the conserved inter-doublet linkers of both species described in this study, are present only on doublet D-V (some of these features connect to D-VI). Double-headed arrows indicate the flagellar beating plane that runs through doublet D-I and between D-V/D-VI that are connected by i-SUB5-6. (B and D) The diagrams of D-V show longitudinal front views that summarize the conserved structural features. The i-SUB5-6 complex (orange) is present in every 96-nm repeat along the unique DMT D-V and links to the neighboring DMT D-VI. ODAs are absent from the distal 7/8th of Strongylocentrotus DMT D-V, and almost from the entire length of Chlamydomonas DMT D-V. However, additional inter-doublet links such as the Strongylocentrotus o-SUB5-6 complexes (green) or the Chlamydomonas proximal 1–2 bridge (blue) are present on DMT D-V, while several IDAs (IA2, 3, 4, and IAX in Strongylocentrotus; IA3, 4, and IAX in Chlamydomonas) are missing from this DMT. |
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