Novel features of centriole polarity and cartwheel stacking revealed by cryo‐tomography
2020; Springer Nature; Volume: 39; Issue: 22 Linguagem: Inglês
10.15252/embj.2020106249
ISSN1460-2075
AutoresSergey Nazarov, Alexandra Bezler, Georgios N. Hatzopoulos, Veronika Nemčíková Villímová, Davide Demurtas, Maeva Le Guennec, Paul Guichard, Pierre Gönczy,
Tópico(s)Galectins and Cancer Biology
ResumoArticle20 September 2020Open Access Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography Sergey Nazarov Sergey Nazarov orcid.org/0000-0002-5240-5849 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Interdisciplinary Centre for Electron Microscopy (CIME), Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Alexandra Bezler Alexandra Bezler orcid.org/0000-0001-6443-8405 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Georgios N Hatzopoulos Georgios N Hatzopoulos orcid.org/0000-0003-4382-4459 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Veronika Nemčíková Villímová Veronika Nemčíková Villímová Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Davide Demurtas Davide Demurtas Interdisciplinary Centre for Electron Microscopy (CIME), Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Maeva Le Guennec Maeva Le Guennec orcid.org/0000-0002-3789-7988 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Search for more papers by this author Paul Guichard Paul Guichard orcid.org/0000-0002-0363-1049 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Search for more papers by this author Pierre Gönczy Corresponding Author Pierre Gönczy [email protected] orcid.org/0000-0002-6305-6883 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Sergey Nazarov Sergey Nazarov orcid.org/0000-0002-5240-5849 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Interdisciplinary Centre for Electron Microscopy (CIME), Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Alexandra Bezler Alexandra Bezler orcid.org/0000-0001-6443-8405 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Georgios N Hatzopoulos Georgios N Hatzopoulos orcid.org/0000-0003-4382-4459 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Veronika Nemčíková Villímová Veronika Nemčíková Villímová Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Davide Demurtas Davide Demurtas Interdisciplinary Centre for Electron Microscopy (CIME), Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Maeva Le Guennec Maeva Le Guennec orcid.org/0000-0002-3789-7988 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Search for more papers by this author Paul Guichard Paul Guichard orcid.org/0000-0002-0363-1049 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Search for more papers by this author Pierre Gönczy Corresponding Author Pierre Gönczy [email protected] orcid.org/0000-0002-6305-6883 Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland Search for more papers by this author Author Information Sergey Nazarov1,2,‡, Alexandra Bezler1,‡, Georgios N Hatzopoulos1,‡, Veronika Nemčíková Villímová1, Davide Demurtas2, Maeva Le Guennec3, Paul Guichard3 and Pierre Gönczy *,1 1Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland 2Interdisciplinary Centre for Electron Microscopy (CIME), Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland 3Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland ‡These authors contributed equally to this work *Corresponding author. Tel: +41 21 6930711; E-mail: [email protected] The EMBO Journal (2020)39:e106249https://doi.org/10.15252/embj.2020106249 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract 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 indicate 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. Synopsis Centrioles are microtubule-based organelles that are essential for cilia assembly across eukaryotes. Cryo-electron tomography of termite flagellates delineates the architecture of the centriole proximal region, which contains a SAS-6-based cartwheel, as well as peripheral pinhead, A-C linker, and microtubule triplets. Centriolar cartwheel inner densities (CID) are polarized in Trichonympha centrioles. A continuous filamentous CID (fCID) is present in Teranympha centrioles. SAS-6 rings can directly stack in vitro. SAS-6 rings stack in two ways in vivo: either with an offset, or in register. Introduction Centrioles are evolutionarily conserved microtubule-based organelles that seed the formation of primary cilia, as well as of motile cilia and flagella. Despite significant progress in recent years, the mechanisms orchestrating centriole assembly remain incompletely understood, in part because the detailed architecture of the organelle has not been fully unraveled. The centriole is a 9-fold radially symmetric cylindrical organelle typically ~500 nm in length and ~250 nm in diameter, which is polarized along a proximal–distal axis (reviewed in Azimzadeh & Marshall, 2010; Gönczy & Hatzopoulos, 2019). In the proximal region lies a likewise symmetrical cartwheel usually ~100 nm in length, which is critical for scaffolding the onset of centriole assembly (reviewed in Guichard et al, 2018; Hirono, 2014). In transverse view, the cartwheel is characterized by a central hub from which emanates 9 spokes that extend toward peripherally located microtubule triplets. The SAS-6 family of proteins is thought to constitute the principal building block of the cartwheel and is essential for its formation across systems (Dammermann et al, 2004; Leidel et al, 2005; Kilburn et al, 2007; Kleylein-Sohn et al, 2007; Nakazawa et al, 2007; Rodrigues-Martins et al, 2007; Strnad et al, 2007; Yabe et al, 2007; Culver et al, 2009; Jerka-Dziadosz et al, 2010). SAS-6 proteins contain an N-terminal globular head domain, followed by a ~45 nm long coiled-coil and a C-terminal region predicted to be unstructured (Dammermann et al, 2004; Leidel et al, 2005; van Breugel et al, 2011; Kitagawa et al, 2011). In vitro, SAS-6 proteins readily homodimerize through their coiled-coil moiety; such homodimers can undergo higher order oligomerization through an interaction between neighboring head domains (van Breugel et al, 2011; Kitagawa et al, 2011; Nievergelt et al, 2018). Ultimately, this results in the formation of a SAS-6 ring with a central hub harboring 18 juxtaposed head domains, from which emanate 9 paired coiled-coils that extend peripherally. Such ring oligomers are ~23 nm in diameter and bear striking resemblance with a transverse section of the cartwheel observed in cells. Moreover, recombinant Chlamydomonas reinhardtii SAS-6 (CrSAS-6) possesses the ability not only to self-assemble into ring oligomers, but also to undergo stacking of such entities, together generating a structure akin to the cartwheel present in the cellular context (Guichard et al, 2017). Additional features of cartwheel architecture have been unveiled through cryo-electron tomography (cryo-ET) of centrioles purified from Trichonympha (Guichard et al, 2012, 2013). Three closely related species of these unicellular symbiotic flagellates, T. campanula, T. collaris, and T. sphaerica, referred to collectively as T. spp., populate the gut of Zootermopsis damp wood termites (Tai et al, 2013). T. spp. centrioles are particularly well suited for sub-tomogram averaging (STA) of cryo-ET specimens because they harbor an exceptionally long cartwheel-bearing region, reaching several microns (Gibbons & Grimstone, 1960; Guichard et al, 2012, 2013). STA of purified T. spp. centrioles yielded a ~34 Å map (Fourier Shell Correlation (FSC) criterion 0.143), which established that the cartwheel comprises stacks of ring-containing elements bearing a central hub from which emanate spokes. Suggestively, a 9-fold symmetrical SAS-6 ring generated computationally from the crystal structure of the CrSAS-6 head domain plus the first 6 heptad repeats of the coiled-coil (CrSAS-6[6HR]) could be fitted in the hub of this STA map (Guichard et al, 2012). However, some vertical hub densities remained unaccounted for upon such fitting, raising the possibility that additional components are present. In addition, the T. spp. STA map revealed 9-fold symmetrical cartwheel inner densities (CID) inside the hub proper, with contacts between hub and CID occurring where the fitted CrSAS-6[6HR] head domains interact with one another (Guichard et al, 2013). The T. spp. STA map uncovered a vertical periodicity of ~8.5 nm between spoke densities emanating from the hub (Guichard et al, 2012, 2013). Two such emanating densities merge with one another as they extend toward the periphery, where the vertical spacing between merged entities is hence of ~17 nm. There, spokes abut a pinhead structure that bridges the central cartwheel with peripheral microtubule triplets. The STA map also revealed the architecture of the A-C linker, which connects the A-microtubule from a given triplet with the C-microtubule of the adjacent one. Interestingly, both pinhead and A-C linker are polarized along the proximal–distal centriole axis (Guichard et al, 2013, 2020). Given that the centrally located hub and CID were not noted at the time as being polarized, this led to the suggestion that the pinhead and the A-C linker might be critical for imparting polarity to the entire organelle (Guichard et al, 2013). Further cryo-ET analysis of procentrioles from Chlamydomonas and mammalian cells established that aspects of A-C linker architecture are evolutionarily conserved, including the attachment points on the A- and C-microtubules; moreover, novel features were revealed, such as a vertical crisscross pattern for the A-C linker in Chlamydomonas (Greenan et al, 2018; Li et al, 2019). Whether the central elements of the cartwheel, including the CID, are likewise conserved beyond T. spp. is unclear. Considering that the earlier work in T. spp. was conducted without direct electron detector and that software improvements have occurred since, we sought to achieve a higher resolution map of the T. spp. cartwheel-bearing region. Moreover, to explore the evolutionarily conservation of cartwheel architecture, we investigated two other flagellates living in the gut of termites that might likewise harbor long cartwheels well suited for STA. Results Exceptionally long cartwheel region in Trichonympha agilis We set out to obtain a high-resolution STA map of the native cartwheel in T. spp. Moreover, we likewise aimed at investigating Trichonympha agilis, a symbiotic flagellate that lives in the gut of the Japanese termite Reticulitermes speratus (Ohkuma & Kudo, 1998). This choice was guided by the fact that transcriptomic and genomic information is being assembled in T. agilis (Yuichi Hongoh, Tokyo Institute of Technology, Japan, personal communication), which will be instrumental to map proteins onto the STA map should sub-nanometer resolution be reached in the future. As shown in Fig 1A, T. agilis bears a large number of flagella, which stem from similarly numerous centrioles inserted below the plasma membrane (Kubai, 1973). Many of these flagella are tightly packed in a region called the rostrum located at the cell anterior (Fig 1A, arrow). To determine the length of the cartwheel-bearing region of T. agilis centrioles, cells were resin-embedded and analyzed by transmission electron microscopy (TEM), focusing on the rostral region. Longitudinal views established that the cartwheel-bearing region is ~2.3 μm in length on average (Fig 1B, pink line; SD = 0.36 μm, N = 8). This is less than the ~4 μm observed in T. spp. (Guichard et al, 2013), yet over 20 times the size of the ~100 nm cartwheel in centrioles of most systems, including Chlamydomonas reinhardtii and Homo sapiens (Guichard et al, 2010, 2017; O'Toole & Dutcher, 2014). In addition, we found that the centriole distal region devoid of cartwheel is ~0.4 μm in T. agilis (Fig 1B, white line; SD = 0.07 μm, N = 6), similar to its dimensions in other systems (Guichard et al, 2013; Le Guennec et al, 2020). Figure 1. Exceptionally long centriolar cartwheel in T. agilis Differential interference contrast micrograph of live T. agilis cell. The arrow points to the cell anterior, where the rostrum is located; arrowheads point to some of the flagella. Transmission electron micrographs of T. agilis centriole embedded in resin—longitudinal view; the hub (arrowhead) is visible in the cartwheel-bearing region (pink line), but not in the distal region (white line). Transmission electron micrographs of T. agilis centriole embedded in resin in transverse view from distal end (left) and corresponding image circularized and symmetrized with the CentrioleJ plugin (middle), with schematic indicating principal architectural elements (right). Download figure Download PowerPoint We also analyzed transverse sections of resin-embedded T. agilis centrioles using TEM. As shown in Fig 1C, we found the characteristic features of the cartwheel-bearing region, including a central hub from which emanate 9 spokes that extend toward peripheral microtubule triplets. In addition, we noted the presence of the pinhead and the A-C linker, as well as of the triplet base connecting these two elements (Gibbons & Grimstone, 1960; Vorobjev & Chentsov, 1980), which is more apparent in the circularized and symmetrized image (Fig 1C). Overall, given the presence of a long cartwheel-bearing region, we conclude that T. agilis also provides a suitable system to investigate the architecture of the proximal part of the centriole using cryo-ET and STA. Novel features revealed by improved STA of Trichonympha centrioles Using a direct electron detector, we acquired tilt series of purified T. spp. centrioles, focusing on the proximal cartwheel-bearing region (Appendix Fig S1A and B), followed by tomogram reconstruction and STA (Fig 2A–E; for all datasets, see Appendix Fig S1C–F for raw tomograms, as well as Appendix Fig S1G–J and Appendix Table S1 for processing pipeline). For the central cartwheel, we achieved a local resolution ranging from ~16 Å to ~40 Å (Appendix Fig S2A), with a global resolution of ~24 Å (FSC criterion 0.143; Appendix Fig S2B; see Appendix Figs S3–S5 for resolution of all other STA maps, which have been deposited in EMDB). Figure 2. Conserved architecture and polarity in T. spp. and T. agilis cartwheel Transverse 2D slices through T. spp. central cartwheel STA at indicated height from proximal (0 nm) to distal (8.7 nm). The dashed box indicates corresponding region shown in (C). Schematic on top center illustrates the area used to generate the 3D maps of the central cartwheel. Transverse view of T. spp. central cartwheel STA 3D map; 9 spoke densities emanate from the hub, and the CID is present within the hub. The diameter of the hub is 23.0 ± 0 nm (N = 3; here and thereafter in the figure legends, N corresponds to 2D measurements from STA). An electron-dense structure is present inside the CID (gray), which is also visible to some extent before symmetrizing. Note that the coloring of elements in this and other figure panels is merely illustrative and not meant to reflect underlying molecular boundaries. 2D longitudinal view of T. spp. central cartwheel STA delineated by a dashed box in (A). Arrowheads denote position of line scans along the vertical axis at the level of the CID (purple) and hub (blue), with corresponding pixel intensities in arbitrary units (AU). Plot profiles are shifted horizontally relative to each other for better visibility. Some maxima are highlighted with dashed lines; the average distance between two CID elements is 8.5 ± 0.2 nm (N = 4). Note that hub densities are elongated in the vertical direction, resulting in broad peak profiles where two maxima can be discerned (dashed blue lines). Longitudinal view of T. spp. central cartwheel STA. The average distance between emanating spokes is 8.0 ± 1.5 nm (N = 2); these are measurements on STA and are within the error of the CID periodicities reported in (C); the same applies for other measurements hereafter. Discontinuous densities in the center of the CID (gray) are visible, as well as densities that vertically bridge successive hub elements (arrows). The dashed box is shown magnified in (E). Proximal is down in this and all other figure panels showing STA longitudinal views. The CID axis is located distal relative to the axis of spoke densities, corresponding to an average shift of 0.9 ± 0.7 nm (N = 3). Transverse 2D slice through T. agilis central cartwheel STA at indicated height from proximal (0 nm) to distal (12 nm). The dashed box indicates corresponding region shown in (H). Transverse view of T. agilis central cartwheel STA 3D map; 9 spoke densities emanate from the hub, and the CID is present within the hub, which has a diameter of 22.7 ± 0.2 nm (N = 3). 2D longitudinal view of T. agilis central cartwheel STA delineated by a dashed box in (F). Arrowheads denote position of line scans along the vertical axis at the level of the CID (dark pink) and hub (light pink), with corresponding pixel intensities in arbitrary units (AU). Plot profiles are shifted horizontally relative to each other for better visibility. Some maxima are highlighted with dashed lines; the average distance between two CID elements is 8.3 ± 0.5 nm (N = 9). Note that hub densities are elongated in the vertical direction, resulting in broad peak profiles where two maxima can be discerned (dashed light pink lines). Longitudinal view of T. agilis central cartwheel STA. The average distance between emanating spokes is 8.2 ± 0.5 nm (N = 9). Discontinuous densities in the center of the CID (gray) are visible at this lower contour level compared to (G). Note densities vertically bridging successive hub elements (arrows). Boxed area is shown magnified in (J). The CID axis is located distal relative to the axis of spoke densities, corresponding to an average shift of 0.8 ± 0.3 nm (N = 9). Download figure Download PowerPoint Using line scans on 2D projections of the STA map, we determined the T. spp. hub diameter to be ~23 nm (Fig 2A and B), in line with previous work (Guichard et al, 2012, 2013). Importantly, the improved resolution achieved here enabled us to uncover novel features in the central cartwheel of the T. spp. centriole. Of particular interest, we discovered that the position of the CID is polarized along the proximal–distal centriole axis with respect to the hub and the spokes that emanate from it (Fig 2C and D). This is apparent from vertical intensity profiles of longitudinal views, which show that the CID is positioned distal to the center of the hub density, which itself appears to be elongated in the vertical direction (Fig 2C). Occasionally, two units can be discerned within one hub density (Fig 2C, double peaks in blue intensity profile and corresponding dashed lines), a point that will be considered further below. Such double units were not recognized previously, presumably owing to the lower resolution map (Guichard et al, 2012, 2013). Moreover, we found densities that vertically bridge successive hub elements (Fig 2D, arrows). The polarized location of the CID unveiled here is also apparent with respect to where spoke densities emerge from the hub (Fig 2E, arrow). In addition, we identified discontinuous densities in the center of the CID (Fig 2B and D). We likewise analyzed the central cartwheel in T. agilis, observing variations along the centriole axis in 2D longitudinal views (Appendix Fig S1D). Focused 3D classification of sub-volumes indeed uncovered two classes (Fig 2F–J; Fig EV1), corresponding to 55 and 45% of sub-volumes, which can occur within the same centriole (Fig EV1F). We found that the central cartwheel STA map of both T. agilis classes exhibits many similarities with that of T. spp. Thus, the CID is present and spoke densities emanate from a hub ~23 nm in diameter (Figs 2F and G, and EV1A and B). Moreover, vertical densities bridging successive hub elements are also present in both T. agilis classes (Figs 2I and EV1D, arrows). Furthermore, we found that the CID is also polarized along the proximal–distal centriole axis, being distal with respect to the hub in both T. agilis classes, as evidenced from vertical intensity profiles (Figs 2H and I and EV1C and D), as well as from the location of the CID relative to where spoke densities emerge from the hub (Fig 2J, arrow; Fig EV1D, arrowhead). In the T. agilis 55% class, like in T. spp., hub densities are elongated in the vertical direction and can be sometimes discerned as two units (Fig 2H, double peaks in light pink intensity profile and corresponding dashed lines). The presence of such double hub units next to the CID is more apparent in the 45% class, where they also exhibit a slight offset relative to the vertical axis (Fig EV1C, white dashed line), a point considered further below. In addition, we found in this class that double hub units alternate with single hub units that do not have a CID in their vicinity (Fig EV1C, dashed pink arrow, Fig EV1D), an absence noticeable also in raw tomograms (Appendix Fig S1D, empty arrowheads) and verified in 3D top views (Fig EV1A and B, full circle). Moreover, we found that spoke densities emanating from single hub units are thinner than those stemming from double hub units (Fig EV1D). The plausible origin of alternating double and single hub units in the 45% T. agilis class will be considered below. We noted also that the 45% sub-volumes exhibit slight variations in the spacing between double and single hub units (Fig EV1E, 25 and 20% sub-classes). Click here to expand this figure. Figure EV1. Variation in hub architecture in some T. agilis sub-volumes Transverse 2D slices through central cartwheel STA of T. agilis 45% class, which comprises 25 and 20% sub-classes (see E), at indicated height from proximal (0 nm) to distal (18.0 nm). The pink circles mark spokes with CID (12.0 nm, dashed line) or without CID (2.0 nm, solid line), as represented in (B); dashed box in the 4.0 nm slice indicates longitudinal section shown in (C). Schematic on top illustrates the area used to generate the 3D maps of the central cartwheel. Transverse views of central cartwheel STA 3D map of T. agilis 45% class. The hub diameter is 22.7 nm ± 0.2 nm (N = 3) with 9 emanating spoke densities, either at a level where the CID is present (dashed circle) or absent (solid circle), as indicated in (A). 2D longitudinal view of central cartwheel STA of T. agilis 45% class delineated by a dashed box in (A). Arrowheads denote position of line scans along the vertical axis at the level of the CID (red) and the hub (pink), with corresponding normalized pixel intensities in arbitrary units (AU). The plot profiles are shifted horizontally relative to each other for better visibility. The distance between hub densities alternates between 3.2 nm (N = 1) and 6.0 ± 0.3 nm (N = 2); maxima are indicated by dashed pink lines. Dashed white line indicates offset of two superimposed hub units. The average distance between two CID elements is 15.2 nm (N = 1; dashed red line). The middle hub density that comprises only one unit and lacks a neighboring CID is indicated by a dashed arrow. Longitudinal view of central cartwheel STA of T. agilis 45% class at lower contour level than in (B). Note densities bridging successive hubs vertically (arrows), as well as proximal location of CID relative to the spoke density axis, resulting in a vertical offset of 1.2 ± 0 nm (N = 2; arrowhead). Note also absence of CID in middle hub element comprising a single unit. The spacing between the spoke density emanating from a double hub unit and the spoke density emanating from a single hub unit distal to it is 8.0 nm in the 25% class and 7.1 nm in the 20% class (both N = 1). By contrast, the spacing between the spoke density emanating from a double hub unit and the spoke density emanating from a single hub unit proximal to it is 8.7 nm in the 25% class and 7.6 nm in the 20% class (both N = 1). Distribution of sub-volumes of the 55% (black circle) and 45% (white circle) classes along five T. agilis centrioles; areas with neither black nor white circle could not be clearly assigned to either class. Note individual centrioles constituted of mostly one of the two classes, and others where the two classes are mixed without an apparent pattern, indicating that the distribution of 55% and 45% classes is not stereotyped along the centriole. Download figure Download PowerPoint Taken together, our findings establish that T. spp. and T. agilis central cartwheel architecture shares many features, including a polarized CID position. Comparative STA of peripheral centriole elements in Trichonympha We also investigated peripheral elements in the proximal region of T. spp. and T. agilis centrioles. To this end, we extracted peripheral sub-volumes from the tomograms and generated for each species three maps using STA centered either on the microtubule triplet, the pinhead, or the A-C linker (Fig 3A and E). Figure 3. Architecture of peripheral elements in T. spp. and T. agilis (Top) 2D slice through STA transverse view of T. spp. microtubule triplet, with insets showing position of pinhead (dashed green box) and A-C linker (dashed red box). (Bottom) Longitudinal 2D slice of STA centered on the pinhead (left) or A-C linker (right). Schematic on top center illustrates the different areas used to generate maps of the microtubule triplets (B and F), pinhead (C and G), and A-C linker (D and H). Transverse view of T. spp. microtubule triplet STA. Microtubule protofilament numbers are indicated, as are the pinhead and A-C linker (only the C-link is visible; the A-link lies on the edge of the volume and is thus less well resolved in this STA—for better view, see STA centered on A-C linker in (D)). Prominent microtubule inner densities within the A-microtubule are highlighted (empty arrowhead next to A9, chevron next to A5), as are additional external densities at the A-B and B-C inner junctions (black arrowheads). Double arrowheads point to viewing direction in indicated panels. Longitudinal view of T. spp. STA centered on the pinhead from the viewing point indicated in (B). The pinfeet (PinF1 and PinF2) and pinbody (PinB) are indicated, as are microtubule protofilaments A3 and A4. The average distance between pinfeet elements is 8.6 ± 0.4 nm and 7.9 ± 0.4 nm (N = 3 each). Corresponding transverse views are shown below, illustrating the connection of PinF2 with protofilament A3. Longitudinal view of T. spp. STA centered on the A-C linker from the viewing point indicated in (B). Microtubule protofilaments A8/9 and C9/C10 of two adjacent triplets are indicated, as are the connected A- and C-links. The average distance between A- and C-links is 8.4 ± 0.4 nm and 8.4 ± 0.3 nm (both N = 6). Corresponding transverse views are shown below; chevrons point to connection. (Top) 2D slice through STA transverse view of T. agilis microtubule tripl
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