Architecture of the centriole cartwheel‐containing region revealed by cryo‐electron tomography
2020; Springer Nature; Volume: 39; Issue: 22 Linguagem: Inglês
10.15252/embj.2020106246
ISSN1460-2075
AutoresNikolai Klena, Maeva Le Guennec, Anne‐Marie Tassin, Hugo van den Hoek, Philipp S. Erdmann, Miroslava Schaffer, Stefan Geimer, Gabriel Aeschlimann, Ľubomír Kováčik, Yashar Sadian, Kenneth N. Goldie, Henning Stahlberg, Benjamin D. Engel, Virginie Hamel, Paul Guichard,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoArticle20 September 2020Open Access Architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography Nikolai Klena Nikolai Klena orcid.org/0000-0002-2631-2294 Department of Cell Biology, University of Geneva, Sciences III, Geneva, 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 Anne-Marie Tassin Anne-Marie Tassin Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, Gif sur Yvette, France Search for more papers by this author Hugo van den Hoek Hugo van den Hoek Helmholtz Pioneer Campus, Helmholtz Zentrum München, Neuherberg, Germany Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Philipp S Erdmann Philipp S Erdmann Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Miroslava Schaffer Miroslava Schaffer Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Stefan Geimer Stefan Geimer Department of Cell Biology and Electron Microscopy, Universität Bayreuth, Bayreuth, Germany Search for more papers by this author Gabriel Aeschlimann Gabriel Aeschlimann orcid.org/0000-0002-4630-6190 Ribosome Studio Aeschlimann, Oberrieden, Switzerland Search for more papers by this author Lubomir Kovacik Lubomir Kovacik Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Yashar Sadian Yashar Sadian Bioimaging and Cryogenic Center, University of Geneva, Geneva, Switzerland Search for more papers by this author Kenneth N Goldie Kenneth N Goldie orcid.org/0000-0002-7405-0049 Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Henning Stahlberg Henning Stahlberg orcid.org/0000-0002-1185-4592 Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Benjamin D Engel Corresponding Author Benjamin D Engel [email protected] orcid.org/0000-0002-0941-4387 Helmholtz Pioneer Campus, Helmholtz Zentrum München, Neuherberg, Germany Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Department of Chemistry, Technical University of Munich, Garching, Germany Search for more papers by this author Virginie Hamel Corresponding Author Virginie Hamel [email protected] orcid.org/0000-0001-5092-2343 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Search for more papers by this author Paul Guichard Corresponding Author Paul Guichard [email protected] orcid.org/0000-0002-0363-1049 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Search for more papers by this author Nikolai Klena Nikolai Klena orcid.org/0000-0002-2631-2294 Department of Cell Biology, University of Geneva, Sciences III, Geneva, 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 Anne-Marie Tassin Anne-Marie Tassin Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, Gif sur Yvette, France Search for more papers by this author Hugo van den Hoek Hugo van den Hoek Helmholtz Pioneer Campus, Helmholtz Zentrum München, Neuherberg, Germany Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Philipp S Erdmann Philipp S Erdmann Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Miroslava Schaffer Miroslava Schaffer Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Search for more papers by this author Stefan Geimer Stefan Geimer Department of Cell Biology and Electron Microscopy, Universität Bayreuth, Bayreuth, Germany Search for more papers by this author Gabriel Aeschlimann Gabriel Aeschlimann orcid.org/0000-0002-4630-6190 Ribosome Studio Aeschlimann, Oberrieden, Switzerland Search for more papers by this author Lubomir Kovacik Lubomir Kovacik Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Yashar Sadian Yashar Sadian Bioimaging and Cryogenic Center, University of Geneva, Geneva, Switzerland Search for more papers by this author Kenneth N Goldie Kenneth N Goldie orcid.org/0000-0002-7405-0049 Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Henning Stahlberg Henning Stahlberg orcid.org/0000-0002-1185-4592 Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland Search for more papers by this author Benjamin D Engel Corresponding Author Benjamin D Engel [email protected] orcid.org/0000-0002-0941-4387 Helmholtz Pioneer Campus, Helmholtz Zentrum München, Neuherberg, Germany Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany Department of Chemistry, Technical University of Munich, Garching, Germany Search for more papers by this author Virginie Hamel Corresponding Author Virginie Hamel [email protected] orcid.org/0000-0001-5092-2343 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Search for more papers by this author Paul Guichard Corresponding Author Paul Guichard [email protected] orcid.org/0000-0002-0363-1049 Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) Search for more papers by this author Author Information Nikolai Klena1,‡, Maeva Le Guennec1,‡, Anne-Marie Tassin2, Hugo van den Hoek3,4, Philipp S Erdmann4, Miroslava Schaffer4, Stefan Geimer5, Gabriel Aeschlimann6, Lubomir Kovacik7, Yashar Sadian8, Kenneth N Goldie7, Henning Stahlberg7, Benjamin D Engel *,3,4,9, Virginie Hamel *,1 and Paul Guichard *,1,10 1Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland 2Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Univ. Paris Sud, Université Paris-Saclay, Gif sur Yvette, France 3Helmholtz Pioneer Campus, Helmholtz Zentrum München, Neuherberg, Germany 4Department of Molecular Structural Biology, Max Planck Institute of Biochemistry, Martinsried, Germany 5Department of Cell Biology and Electron Microscopy, Universität Bayreuth, Bayreuth, Germany 6Ribosome Studio Aeschlimann, Oberrieden, Switzerland 7Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland 8Bioimaging and Cryogenic Center, University of Geneva, Geneva, Switzerland 9Department of Chemistry, Technical University of Munich, Garching, Germany 10Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) ‡These authors contributed equally to this work. ‡[Correction added on January 15th 2021, after first online publication: Helmholtz Zentrum München - Deutsches Forschungszentrum für Gesundheit und Umwelt (GmbH) was added for Paul Guichard.] *Corresponding author. Tel: +49 089 3187 1822; E-mail: [email protected] *Corresponding author. Tel: +41 22 379 6735; E-mail: [email protected] *Corresponding author. Tel: +41 22 379 6750; E-mail: [email protected] The EMBO Journal (2020)39:e106246https://doi.org/10.15252/embj.2020106246 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 evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis. Synopsis The centriole's intricate architecture underlies its crucial role in assembly of centrosomes and cilia/flagellae. Here, in situ and ex vivo cryo-electron tomography of centrioles from Chlamydomonas, Paramecium, Naegleria and humans provides architectural maps of the centriole's proximal region and reveals evolutionarily-conserved structures as well as differences between species. The cartwheel central hub is an evolutionarily-conserved stack of ring pairs. Cartwheel inner densities (CID) are present in Trichonympha, human, Paramecium, Chlamydomonas and Naegleria centrioles. Cartwheel radial spokes are polarized, with spoke architecture diverging between species. The A-C linker is connected to the pinhead through the triplet base. Structural elements in the proximal region are specifically positioned relative to each other, with clear boundaries and overlaps. Introduction Centrioles and basal bodies (hereafter referred to as centrioles for simplicity) are cytoskeletal organelles, typically 450–550 nm in length and ~250 nm in outer diameter, which are present in most eukaryotic cells and play organizing roles in the assembly of cilia, flagella, and centrosomes (Nigg & Raff, 2009; Gönczy, 2012; Winey & O'Toole, 2014). Centrioles are characterized by a near-universal ninefold radial arrangement of microtubule triplets that contain a complete 13-protofilaments A-microtubule and incomplete B- and C-microtubules, each composed of 10 protofilaments (Guichard et al, 2013). Centrioles are polarized along their proximal-to-distal axis, with distinct structural features along their length. The proximal region is defined by the presence of the cartwheel structure, which serves as a seed for centriole formation and is thought to impart ninefold symmetry to the entire organelle (Nakazawa et al, 2007; Strnad & Gönczy, 2008; Gönczy, 2012; Hirono, 2014; Hilbert et al, 2016). In most species, the cartwheel stays within the centriole after maturation; however, it is not present in mature human centrioles (Azimzadeh & Bornens, 2007). The native architecture of the proximal region, and in particular of the cartwheel, was revealed by cryo-electron tomography (cryo-ET) of the Trichonympha centriole. Owing to its exceptionally long proximal region, many structural repeats could be sampled for subtomogram averaging, revealing the overall 3D structure of the cartwheel for the first time (Guichard et al, 2012, 2013). The Trichonympha cartwheel was observed to be built from a hub of stacked rings spaced every 8.5 nm. Radial spokes, emanating from two adjacent rings, merged at the pinhead near the microtubule triplet to form a repeating structural unit with a periodicity of 17 nm. Moreover, this study demonstrated that each Trichonympha hub ring could accommodate nine homodimers of SAS-6, a protein that is essential for cartwheel assembly across eukaryotes (van Breugel et al, 2011, 2014; Kitagawa et al, 2011). Unexpectedly, a cartwheel inner density (CID), was also identified at the center of the hub ring. This CID contacts the hub ring at nine locations and has been hypothesized to be Trichonympha-specific, as CIDs have never been observed in other species, possibly due to lack of resolution. In this respect, the CIDs have been proposed to facilitate TaSAS-6 oligomerization or confer additional mechanical stability to these exceptional long centrioles, which are subjected to strong forces inside the intestine of the host termite (Guichard et al, 2013, 2018). Note that in Guichard et al, (2013), the abbreviation CID was defined as a connected circle of nine "cartwheel inner densities", but here we define this whole structure as a single CID to allow a clear description of our data. In the proximal region, the cartwheel is connected to the pinhead, which bridges the cartwheel to the A-microtubule of the microtubule triplet (Dippell, 1968; Hirono, 2014). This connection is thought to be partially composed of Bld10p/Cep135 proteins, which can interact with both SAS-6 and tubulin (Hiraki et al, 2007; Carvalho-Santos et al, 2012; Kraatz et al, 2016; Guichard et al, 2017). In addition to the cartwheel/pinhead ensemble, adjacent microtubule triplets in the proximal region are also connected by the A-C linker. Cryo-ET combined with subtomogram averaging has revealed distinct structures of the A-C linker in Trichonympha and Chlamydomonas reinhardtii (Guichard et al, 2013; Li et al, 2019). In Trichonympha, the structure consists of the A-link, which is laterally inclined and contacts the A-tubule at the A8 protofilament, and the C-link, which connects to the C-tubule at the C9 protofilament. Overall, the Trichonympha A-C linker displays a longitudinal periodicity of 8.5 nm. In contrast, the A-C linker in C. reinhardtii is a crisscross-shaped structure composed of a central trunk region from which two arms and two legs extend to contact the A- and C-tubules (Li et al, 2019). Whereas these two studies provide major advances in our understanding of A-C linker organization, they also clearly highlight structural divergence between Trichonympha and C. reinhardtii centrioles. The question thus arises as to the evolutionary conservation of the centriole's proximal region, including characteristic structures such as the A-C linker and the cartwheel's hub, CID, and radial spokes. In particular, the structure of the cartwheel remains unexplored beyond Trichonympha. A more universal description of the proximal region is important for understanding of how these structures direct centriole biogenesis. Here, we use cryo-ET to tackle this fundamental question using four evolutionarily distant species: Chlamydomonas reinhardtii, Paramecium tetraurelia, Naegleria gruberi, and humans. Results In situ structural features of the cartwheel in Chlamydomonas centrioles The power of biodiversity proved extremely useful for resolving the first 3D architecture of the cartwheel within the exceptionally long proximal region of Trichonympha centrioles (Guichard et al, 2012). This study identified the CID as well as an 8.5 nm longitudinal periodicity along the central hub of the cartwheel. Whether these structural features hold true in other species is an open question that we address here by analyzing the cartwheel of the green algae C. reinhardtii, a canonical model for centriole biology with similar centriole structure and protein composition to humans (Keller et al, 2005; Keller & Marshall, 2008; Li et al, 2011; Hamel et al, 2017). However, extracting centrioles from cells can limit the analysis of these fragile structures, as exemplified by the loss of the cartwheel during a study of isolated C. reinhardtii centrioles (Li et al, 2011). In addition, the > 300 nm thick vitreous ice surrounding uncompressed centrioles on an EM grid reduces the signal and contrast of cryo-ET (Kudryashev et al, 2012), making it difficult to resolve fine details in the relatively small cartwheel structure (Guichard et al, 2018). We therefore decided to analyze the C. reinhardtii cartwheel in situ using a cryo-focused ion beam (cryo-FIB) milling approach, which creates thin 100–150 nm sections of the native cellular environment in a vitreous state (Schaffer et al, 2017). Combining this approach with new direct electron detector cameras (Grigorieff, 2013), it was possible for us to visualize the centriole and cartwheel with unprecedented clarity and structural preservation. As shown in Fig 1A and B, in situ cryo-ET clearly revealed both mature centrioles and procentrioles, providing the first observation of the centriole's cartwheel-bearing region in its native environment. The cartwheel's structural features were analyzed in both types of centrioles (Figs 1C–H and EV1 and Appendix Fig S1). Strikingly, we found that the cartwheel's central hub has an average longitudinal periodicity of 4.0 nm in both mature centrioles and procentrioles, distinct from the 8.5 nm periodicity originally described in Trichonympha (Guichard et al, 2012) (Figs 1H and EV1A, D and G). Moreover, we noticed pronounced densities inside the central hub that were reminiscent of the CIDs originally described in Trichonympha, suggesting that this structure is not Trichonympha-specific but rather is a conserved feature of the cartwheel (Fig 1). Several CIDs in C. reinhardtii are spaced along the lumen of the central hub, forming an 8.7 nm periodicity on average, in mature centrioles and procentrioles (Figs 1H–J and EV1B and E), similar to Trichonympha. Figure 1. In situ cryo-ET reveals the native cartwheel structure in C. reinhardtii centrioles A, B. In situ cryo-electron tomogram displaying the proximal region of a mature mother centriole (A) and procentriole (B). Mature centriole, MC; procentriole, PC; mitochondria, mito; vacuole, vac; white dashed line, lamella edge. Scale bars, 100 nm. C. Side view z-projection of cartwheels containing the central hub and several CIDs from a mature centriole. Central hub, CH; cartwheel inner density, CID. Scale bar, 20 nm. D. Cross section of the cartwheel-containing region from a mature centriole. Microtubule triplet, MTT; spokes, SP. Scale bar, 200 nm. F. Side view z-projection of a cartwheel containing the central hub and several CIDs from a procentriole. Scale bar, 20 nm. G. Cross section of the cartwheel-containing region from a procentriole. Scale bar, 200 nm. H. Ninefold symmetrized cross sections of the cartwheel-containing region from a mature centriole (left side) and a procentriole (right side). Dashed white circle, central hub. Scale bars, 100 nm. J. Longitudinal periodicity measurements of the central hub and CIDs. Central hub, blue; CID, red. Mean values are displayed above the data range. Blue data points are measured distance between individual units of the central hub, and red data points are measured distances between individual units of the cartwheel inner density. Mature centriole, central hub, n = 30, mean = 4.1 ± 0.67 (SD); mature centriole, cartwheel inner density, n = 18, mean = 8.7 ± 1.6 (SD); procentriole, central hub, n = 10, mean = 4.0 ± 0.66 (SD); procentriole, cartwheel inner density, n = 5, mean = 8.6 ± 1.2 (SD). I, J. Ninefold symmetrized central hub z-projections, starting at the proximal end of the cartwheel and continuing distally along the cartwheel by 5.4 nm steps in a mature centriole (I) and a procentriole (J). Red arrow, CID; blue arrow; central hub. Scale bar: 20 nm. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Periodicity along the central hub, cartwheel inner densities, and A-C linker in C. reinhardtii in situ centrioles A–C. Cryo-ET sections depicting representative central hub (CH) (A), several cartwheel inner densities (CIDs) (B), and A-C linker (C). Dashed white line denotes region from which plot profiles were generated. Microtubule triplet, MTT. Scale bar, 25 nm. D–F. Plot profiles with their associated mean periodicity displayed below. G. Top and side views of Trichonympha cartwheel and associated periodicities from (Guichard et al, 2013). Download figure Download PowerPoint To investigate whether the discrepancy we observed in central hub periodicity was accompanied by other differences in cartwheel structure, we measured features of the cartwheel such as the central hub diameter and the distances from the hub to D1 and D2, two densities previously described on the cartwheel spokes of C. reinhardtii centrioles (Guichard et al, 2017) in both mature centrioles and procentrioles (Appendix Fig S2A–F). Similar to previous measurements, we found that the central hub is ~21 nm in diameter (peak-to-peak from the intensity plot profile through the hub), and the D1 and D2 densities are positioned ~36 nm and ~47 nm from the external edge of the cartwheel hub, respectively. These measurements suggest that only the longitudinal periodicity of the central hub differs in the in situ C. reinhardtii centrioles. While most of the cartwheel's structural features, including the CIDs, are conserved between Trichonympha and C. reinhardtii, the periodicity of the central hub appears to diverge. This discrepancy poses the important question of how conserved the architecture of the cartwheel-containing region is between species. Moreover, as cartwheel periodicity was previously only measured in isolated centrioles, this raises the possibility that cartwheel periodicity may be affected during purification. Conservation of the cartwheel's structural features in Paramecium, Naegleria, and humans To address these questions, we analyzed the proximal region of isolated centrioles from three different species. Centrioles were purified from P. tetraurelia, N. gruberi and human KE37 leukemia acute lymphoblastic T cells, vitreously frozen onto EM grids, and then imaged by cryo-ET (Figs 2A–I and EV2). Despite the high level of noise expected in cryo-ET of isolated centrioles, as well as the previously observed strong compression of N. gruberi and human centrioles (Guichard et al, 2010; Greenan et al, 2018; Le Guennec et al, 2020) that affects cartwheel integrity, we could reliably measure the central hub periodicity in each of these species. Strikingly, we found that the longitudinal periodicity of the central hub is similar to the C. reinhardtii in situ cartwheel, with average periodicities of 4.3 ± 0.38 nm, 4.4 ± 0.53 nm, and 4.2 ± 0.68 nm in P. tetraurelia, N. gruberi, and human, respectively (Figs 2J and EV2). Moreover, we observed that CID structures are present in every species, forming a periodicity along the central hub of 8.4 ± 1.25 nm, 8.3 ± 1.83 nm, and 8.1 ± 2.46 nm (Fig 2A–J and Appendix Fig S2G–O). These results indicate that structural features of the C. reinhardtii cartwheel seem to be conserved, including the central hub's ~4.2 nm periodicity, as well as the presence of CIDs every ~8.4 nm. Moreover, these measurements demonstrate that the discrepancy between Trichonympha and C. reinhardtii is probably not due to purification artifacts, as the other isolated centrioles also display ~4 nm periodicities along their central hubs. Figure 2. Cryo-ET of isolated centrioles from P. tetraurelia, N. gruberi, and H. sapiens reveals novel cartwheel periodicities A–C. Cryo-electron tomograms of the proximal regions of a P. tetraurelia centriole (A), a N. gruberi centriole (B), and a H. sapiens procentriole (C). White arrows denote a broken cartwheel; procentriole, PC; mature centriole, MC; Scale bar, 100 nm. Note that most N. gruberi and H. sapiens centrioles were heavily compressed during the cryo-EM preparation, as previously described (Guichard et al, 2010). The displayed N. gruberi centriole illustrates the damage caused by compression. The periodicities of N. gruberi and H. sapiens cartwheels were measured only on regions that were not damaged (see Fig EV2). D. Cross section from cartwheel-containing region of a P. tetraurelia centriole. Scale bar, 50 nm. E. Zoomed side view of cartwheel from P. tetraurelia, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow. Scale bar, 25 nm. F. Cross section from cartwheel-containing region of a N. gruberi centriole. Same scale bar as in (D). G. Zoomed side view of cartwheel from N. gruberi, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow. Same scale bar as in (E). H. Cross section from cartwheel-containing region of a H. sapiens centriole. Same scale bar as in (D). I. Zoomed side view of cartwheel from H. sapiens, displaying the central hub (CH) and several cartwheel inner densities (CIDs), white arrow. Same scale bar as in (E). J. Longitudinal periodicity of the central hub and CIDs in P. tetraurelia, N. gruberi, and H. sapiens. Mean values are displayed above data range. Black lines indicate the mean and the standard deviation. Blue data points are measured distance between individual units of the central hub, and red data points are measured distances between adjacent cartwheel inner densities. P. tetraurelia, CH, n = 10, mean = 4.3 ± 0.38 (SD); N. gruberi, CH, n = 10, mean = 4.4 ± 0.53 (S.D); H. sapiens, CH, n = 10, mean = 4.2 ± 0.68 (S.D); P. tetraurelia, CID, n = 8, mean = 8.4 ± 1.3 (SD); N. gruberi, CID, n = 8, mean = 8.3 ± 1.8 (SD); H. sapiens, CID, n = 8, mean = 8.1 ± 2.5 (SD). K, L. Proximal protrusion length of the cartwheel beyond the microtubule triplets in C. reinhardtii, P. tetraurelia, N. gruberi, and H. sapiens. Internal cartwheel inside the microtubule barrel, dark blue (INT); external cartwheel beyond the microtubule wall, light blue (EXT). Mean values are displayed above each bar plot, with the black lines indicating the standard deviation (K). The start of each microtubule wall is delineated by a dashed white line (L). Mature C. reinhardtii, n = 4, external cartwheel length = 37.6 ± 3.4, internal cartwheel length = 74.2 ± 16.6; C. reinhardtii procentriole, n = 2, external cartwheel length = 52.8 ± 22.0, internal cartwheel length = 26.5 ± 29.7; P. tetraurelia, n = 23, external cartwheel length = 11.5 ± 9.2, internal cartwheel length = 66.6 ± 15.7; N. gruberi, n = 19, external cartwheel length = 41.0 ± 22.5, internal cartwheel length = 259.0 ± 87.2; human procentriole, n = 7, external cartwheel length = 23.1 ± 10.3, internal cartwheel length = 154.7 ± 47.7. Reported values are mean and errors are standard deviation. Scale bar, 50 nm. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Additional examples of the cartwheel periodicities in N. gruberi and H. sapiens A–F. Side views of cartwheels in N. gruberi (A) and H. sapiens (B) with corresponding insets (C-F) highlighting the hub periodicities (blue) and CIDs (red). Scale bar, 50 nm. G, H. Plot profiles of the boxed regions in (C-F) depicting an average periodicity of the hub (blue) of 4.4 nm in N. gruberi and 4.2 nm in H. sapiens as well as for the CIDs with an average periodicity of 8.3 nm in N. gruberi and 8.1 nm in H. sapiens (see Fig 2). I, J. Top views of representative, compressed N. gruberi (I) and H. sapiens (J) centrioles displaying that the central hub of the cartwheel is off-centered (white arrows). Scale bars, 100 nm. Download figure Download PowerPoint Interestingly, in tomograms of both in situ and isolated centrioles, we observed that the position of the cartwheel did not fully correlate with the position of the microtubule triplets. In all four species, the cartwheels protruded proximally 10–40 nm beyond the microtubule wall (Figs 1A and B, and 2K and L). In C. reinhardtii, which enabled observations of assembling and mature centrioles within the same cells, the cartwheel extension was more prominent in procentrioles, with 67% of the cartwheel protruding in contrast to 34% in mature centrioles (Fig 2K). Until now, this proximal extension of the cartwheel has only been reported in isolated C. reinhardtii procentrioles (Geimer & Melkonian, 2004; Guichard et al, 2017). Our in situ C. reinhardtii tomograms demonstrate that the cartwheel extension is not an artifact of purifying centrioles, but rather occurs within the native cellular environment. We further corroborated this conclusion with serial sections of resin-embedded N. gruberi cells, which show the cartwheel protruding beyond the proximal end of the microtubule triplets in both assembling and mature centrioles (Appendix Fig S3). Interestingly, by applying a ninefold circularization on the cryo-tomograms of P. tetraurelia and C. reinhardtii (Fig EV3), we observed that the spokes emanating from the cartwheel proximal extension are organized similarly to the cartwheel region surrounded by microtubules. Moreover, we could identify that the extremities of the spokes are connected together vertically via the D2 densities (which we name the D2-rod) without any pinhead density visible, both in procentrioles and mature centrioles (Fig EV3). Click here to expand this figure. Figure EV3. Native architecture of the proximal cartwheel extension Side view of a symmetri
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