Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead and A-C-linker. Moreover, we report ∼25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicates that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.


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Centrioles are evolutionarily conserved microtubule-based organelles that seed the 50 formation of primary cilia, as well as of motile cilia and flagella. Despite significant 51 progress in recent years, the mechanisms orchestrating centriole assembly remain 52 incompletely understood, in part because the detailed architecture of the organelle has 53 not been fully unraveled.

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The centriole is a 9-fold radially symmetric cylindrical organelle typically ~500 55 nm in length and ~250 nm in diameter, which is polarized along a proximal-distal axis

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The SAS-6 family of proteins is thought to constitute the principal building block 63 of the cartwheel and is essential for its formation across systems (Culver et al, 2009;

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We also analyzed transverse sections of resin-embedded T. agilis centrioles 150 using TEM. As shown in Fig. 1C, we found the characteristic features of the cartwheel-151 bearing region, including a central hub from which emanate 9 spokes that extend 152 towards peripheral microtubule triplets. In addition, we noted the presence of the 153 pinhead and the A-C linker, as well as of the triplet base connecting these two elements 154 (Gibbons and Grimstone, 1960;Vorobjev and Chentsov, 1980), which is more 155 apparent in the circularized and symmetrized image (Fig. 1C).

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Overall, given the presence of a long cartwheel-bearing region, we conclude 157 that T. agilis also provides a suitable system to investigate the architecture of the 158 proximal part of the centriole using cryo-ET and STA.

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We also investigated peripheral elements in the proximal region of T. spp. and T. agilis 217 centrioles. To this end, we extracted peripheral sub-volumes from the tomograms and 218 generated for each species three maps using STA centered either on the microtubule 219 triplet, the pinhead or the A-C linker (Fig. 3A, 3E).

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For the microtubule triplet, the resulting analysis revealed a characteristic 221 centriolar architecture. Thus, the A-microtubule bears 13 protofilaments, the B-222 microtubule 10 protofilaments proper, with 3 extra ones shared with the A-microtubule,

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whereas the C-microtubule exhibits a similar organization as the B-microtubule ( Fig.   224 3B, 3F). Moreover, we detected prominent densities corresponding to microtubule 225 inner proteins (MIPs). In both species, we found a MIP located along protofilament A9, 226 close to protofilament A10 (Fig. 3B, 3F, empty arrowhead). A MIP was discovered at 227 this location in ciliary axonemes (Nicastro et al, 2006), and was also observed in

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For both T. spp. and T. agilis, we next conducted STA centered on the pinhead 238 or the A-C linker to uncover features in these peripheral elements. We thus found that 239 the pinhead connects to the A3 protofilament in both species (Fig. 3C, 3G (Guichard et al, 2013(Guichard et al, , 2020. We found also that the spacing between PinF1 and PinF2 245 elements is ~8.6 nm and ~7.9 nm in T. spp., whereas it is ~8.4 nm and ~8.4 nm in T. 246 agilis (Fig. 3C, 3G), compatible with power spectra of 2D class averages considering 247 the standard deviation of the measurements (Fig. S2I, S3L).

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Similarities between the two species are also apparent in the A-C linker that 249 bridges neighboring MT triplets. Thus, we found that the T. spp. A-C linker connects 250 protofilament A8/A9 from one triplet with protofilaments C9/C10 of the adjacent triplet 251 (Fig. 3D), furthering the mapping of these connections compared to previous work 252 (Guichard et al, 2013). As shown in Fig. 3H, we found that the A-C linker in T. agilis 253 has a similar architecture. To generate an overview of the entire proximal region of the 254 T. agilis centriole, comprising hub, spoke, pinhead and A-microtubule, we conducted 255 STA centered on the spokes for both 55% and 45% classes (

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As shown in Figure 4D-G, the two central cartwheel T. mirabilis classes differ 294 in hub architecture, as revealed by vertical line profile intensity measurements. In the 295 64 % class, a periodicity of ~8.4 nm is apparent between hub densities, each consisting 296 seemingly of a single vertically elongated unit (Fig. 4D, 4F). By contrast, in the 36 % 297 class, each hub density comprises a double unit (Fig. 4E, 4G). Such double hub units 298 exhibit a peak-to-peak distance of ~3.2 nm and are separated from the adjacent double 299 hub unit by ~5.2 nm (Fig. 4E). The sum of the two distances, namely 8.4 nm is 300 equivalent to that observed in the 64 % class. Moreover, the periodicity at the level of 301 emerging spoke densities is likewise equivalent in the two classes (Fig. 4F, 4G).

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Analysis of the peripheral STA microtubule triplet map of the T. mirabilis 306 centriole uncovered the canonical protofilament configuration for A-, B-and C-307 microtubules (Fig. 4H, 4I). As in Trichonympha, we detected additional densities on 308 the external side of the microtubules between A-B and B-C inner junctions (Fig. 4I,   309 arrowheads). The previously described C-stretch that extends from protofilament C1 310 in T. spp. (Guichard et al, 2013) is also observed in T. mirabilis (Fig. 4I,   The map generated by centering on the pinhead revealed a connection with 313 the A3 protofilament, with PinF1 and PinF2 being separated by 8.4 nm (Fig. 4J), as 314 suggested also by power spectra of 2D class averages (Fig. S4L). Moreover, we found 315 that the T. mirabilis A-C linker architecture is similar to that in the two Trichonympha 316 species, with anchoring to microtubules at protofilaments A9 and C9/C10 (Fig. 4K).

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Overall, these findings indicate that T. mirabilis peripheral elements share     (Table S2). However, coiled-coil moieties extend slightly outside 341 the spoke densities in this case, potentially because of coiled-coil bending in vivo or 342 species-specific features. Moreover, rigid body fitting of CrSAS-6[6HR] homodimers 343 confirmed that two directly superimposed dimers provide a better fit for the T. mirabilis 344 36 % class, a fit that also revealed a slight offset between them (Fig. S9A). Next, we

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Overall, these findings lead us to propose that pairs of SAS-6 rings directly 384 stack in the cellular context and can do so with an offset (see Discussion).

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We investigated further the possibility that SAS-6 rings directly stack onto one

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In the absence of fossil record or complete sequence information, it is not 429 possible to assess sequence divergence between centriolar proteins in the species 430 analyzed here, nor to date with precision the evolutionary times separating them.

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However, an approximation is provided in the case of T. spp. and T. agilis by the 432 phylogenetic divergence between their respective hosts Zootermopsis spp. and

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The CID exhibits a 9-fold radial symmetry and connects with the hub approximately 446 where neighboring SAS-6 homodimers interact with one another (Fig. 6B), a 447 suggestive location raising the possibility that the CID imparts or maintains the 9-fold 448 symmetrical SAS-6 ring structure (Guichard et al, 2013). We discovered here that the 449 CID is polarized along the proximal-distal centriole axis, so that may also play a role in 450 imparting or maintaining organelle polarity (Fig. 6C). Furthermore, the CID exhibits an ~8.4 nm periodicity along the proximal-distal centriole axis, with no apparent continuity 452 between two superimposed CID elements. This is in contrast to the fCID, which runs 453 throughout the center of the cartwheel-bearing portion of the T. mirabilis centriole.

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Although the fCID seems disconnected from the hub, small elements linking the two 455 can be discerned in the T. mirabilis 36% class (Fig. 6D, see Fig. S7N). Moreover, the 456 fCID might be linked with the hub through other protein segments that are small,

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Our findings taken together lead us to propose the following working model of 531 SAS-6 stacking in the cellular context (Fig. 6E, 6F). This model entails two modes of

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Tilt series alignments using gold fiducials were performed with IMOD v4.9 630 (Kremer et al, 1996). The contrast transfer function (CTF) was estimated with

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The authors declare that they have no competing interests.

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The average distance between spokes is 8.4 ± 0.6 nm (N=8) in the 64 % class and 8.2 916 ± 1.5 nm (N=7) in the 36 % class. Note in both cases the continuous ~7 nm fCID inside 917 the hub. Note also densities bridging successive hubs vertically (arrows). Asterisks