CryoEM structure of a post-assembly MS-ring reveals plasticity in stoichiometry and conformation
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The flagellar motor supports bacterial chemotaxis, a process that allows bacteria to move in response to their environment. A central feature of this motor is the MS-ring, which is composed entirely of repeats of the FliF subunit. This MS-ring is critical for the assembly and stability of the flagellar switch and the entire flagellum. Despite multiple independent cryoEM structures of the MS-ring, there remains a debate about the stoichiometry and organization of the ring-building motifs (RBMs). Here, we report the cryoEM structure of a Salmonella MS-ring that was purified from the assembled flagellar switch complex (MSC-ring). We term this the 'post-assembly' state. Using 2D class averages, we show that under these conditions, the post-assembly MS-ring can contain 32, 33, or 34 FliF subunits, with 33 being the most common. RBM3 has a single location with C32, C33, or C34 symmetry. RBM2 is found in two locations with RBM2inner having C21 or C22 symmetry and an RBM2outer-RBM1 having C11 symmetry. Comparison to previously reported structures identifies several differences. Most strikingly, we find that the membrane domain forms 11 regions of discrete density at the base of the structure rather than a contiguous ring, although density could not be unambiguously interpreted. We further find density in some previously unresolved areas, and we assigned amino acids to those regions. Finally, we find differences in interdomain angles in RBM3 that affect the diameter of the ring. Together, these investigations support a model of the flagellum with structural plasticity, which may be important for flagellar assembly and function.Plant phytochemicals can act as natural "medicines" for animals against parasites [1Huffman M.A. Animal self-medication and ethno-medicine: exploration and exploitation of the medicinal properties of plants.Proc. Nutr. 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Springer Science+Business Media, 1993Crossref Google Scholar] reduces the availability of natural bee "medicine" and could exacerbate the contribution of diseases to pollinator declines.Video AbstracteyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiIyZDgwNDk2NDk4NGFlYmRkMzVhMWM1MmRlNzBkODg1YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjc4MTk3MjYzfQ.Lcl4UKihoA0dcGxxI20wsJbeB6aLzEEGl5lFr-SLsXi1oRnWihfUvdsgF2HbctZNA-fmjXJvokUfTEXzvhKtPhTtgjfzFtettoGm_ZlezB_bOZ2tSWN8iI_Mi0Yxgl3uLnhBQZnbiskQblLVl5rD8V_HGS6za8OG7usqIN5jHp1pKed_4GsN144gQwc5Tz66IoGgRWA6Ns522YnFPkLRE20Qh5-QauSOSV0BsBa3W6oHSakTFxoSgOvJNYj9IBO6B-iDQKZJMhSFfZDX6Seu9jSfXn5s1i4LVlW79qqSlY_Qq0LKkrYYx0R-GmR66psuLsfB191c87-DmnOKyUDXxg(mp4, (401.21 MB) Download video
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African Trypanosomes are flagellated protozoan parasites that cause sleeping sickness in humans and Nagana in cattle. During its life cycle, Trypanosoma brucei alternates between an insect vector (tsetse fly) and a mammalian host. Within each of these, the parasite proliferates and undergoes
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Article10 March 2021Open Access Transparent process The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility Miguel Ricardo Leung Miguel Ricardo Leung orcid.org/0000-0002-3348-1096 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands The Division of Structural Biology, Wellcome Centre for Human Genetics, The University of Oxford, Oxford, UK Search for more papers by this author Marc C Roelofs Marc C Roelofs orcid.org/0000-0002-2342-3474 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Ravi Teja Ravi Ravi Teja Ravi orcid.org/0000-0002-0360-4307 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Paula Maitan Paula Maitan orcid.org/0000-0001-6677-9609 Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Veterinary Department, Universidade Federal de Viçosa, Viçosa, Brazil Search for more papers by this author Heiko Henning Heiko Henning orcid.org/0000-0003-4064-7792 Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Min Zhang Min Zhang Department of Farm & Animal Health and Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Elizabeth G Bromfield Elizabeth G Bromfield orcid.org/0000-0001-7256-1403 Department of Farm & Animal Health and Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Priority Research Centre for Reproductive Science, Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia Search for more papers by this author Stuart C Howes Stuart C Howes orcid.org/0000-0001-6129-1882 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Bart M Gadella Bart M Gadella Department of Farm & Animal Health and Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Hermes Bloomfield-Gadêlha Hermes Bloomfield-Gadêlha orcid.org/0000-0001-8053-9249 Department of Engineering Mathematics, University of Bristol, Bristol, UK Search for more papers by this author Tzviya Zeev-Ben-Mordehai Corresponding Author Tzviya Zeev-Ben-Mordehai [email protected] orcid.org/0000-0002-2571-550X Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands The Division of Structural Biology, Wellcome Centre for Human Genetics, The University of Oxford, Oxford, UK Search for more papers by this author Miguel Ricardo Leung Miguel Ricardo Leung orcid.org/0000-0002-3348-1096 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands The Division of Structural Biology, Wellcome Centre for Human Genetics, The University of Oxford, Oxford, UK Search for more papers by this author Marc C Roelofs Marc C Roelofs orcid.org/0000-0002-2342-3474 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Ravi Teja Ravi Ravi Teja Ravi orcid.org/0000-0002-0360-4307 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Paula Maitan Paula Maitan orcid.org/0000-0001-6677-9609 Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Veterinary Department, Universidade Federal de Viçosa, Viçosa, Brazil Search for more papers by this author Heiko Henning Heiko Henning orcid.org/0000-0003-4064-7792 Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Min Zhang Min Zhang Department of Farm & Animal Health and Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Elizabeth G Bromfield Elizabeth G Bromfield orcid.org/0000-0001-7256-1403 Department of Farm & Animal Health and Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Priority Research Centre for Reproductive Science, Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia Search for more papers by this author Stuart C Howes Stuart C Howes orcid.org/0000-0001-6129-1882 Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Bart M Gadella Bart M Gadella Department of Farm & Animal Health and Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands Search for more papers by this author Hermes Bloomfield-Gadêlha Hermes Bloomfield-Gadêlha orcid.org/0000-0001-8053-9249 Department of Engineering Mathematics, University of Bristol, Bristol, UK Search for more papers by this author Tzviya Zeev-Ben-Mordehai Corresponding Author Tzviya Zeev-Ben-Mordehai [email protected] orcid.org/0000-0002-2571-550X Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands The Division of Structural Biology, Wellcome Centre for Human Genetics, The University of Oxford, Oxford, UK Search for more papers by this author Author Information Miguel Ricardo Leung1,2, Marc C Roelofs1, Ravi Teja Ravi1, Paula Maitan3,4, Heiko Henning3, Min Zhang5, Elizabeth G Bromfield5,6, Stuart C Howes1, Bart M Gadella5, Hermes Bloomfield-Gadêlha7 and Tzviya Zeev-Ben-Mordehai *,1,2 1Cryo-Electron Microscopy, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands 2The Division of Structural Biology, Wellcome Centre for Human Genetics, The University of Oxford, Oxford, UK 3Department of Equine Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 4Veterinary Department, Universidade Federal de Viçosa, Viçosa, Brazil 5Department of Farm & Animal Health and Biomolecular Health Sciences, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands 6Priority Research Centre for Reproductive Science, Faculty of Science, The University of Newcastle, Callaghan, NSW, Australia 7Department of Engineering Mathematics, University of Bristol, Bristol, UK *Corresponding author. Tel: +31 30 253 3178; E-mail: [email protected] The EMBO Journal (2021)40:e107410https://doi.org/10.15252/embj.2020107410 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 Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures reinforcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution. SYNOPSIS Diverse organisms and cell types rely on evolutionarily ancient and structurally conserved motile cilia to swim through a broad range of fluid environments. Here, in situ cryo-electron tomography uncovers structural specialisations of sperm flagella in several mammalian species. Mammalian sperm flagella are reinforced by micron-scale accessory structures and nano-scale microtubule inner proteins. The connecting piece at the base of the flagellum forms an intricate, asymmetric chamber around the sperm centrioles. The atypical distal centriole is composed of doublet microtubules splayed out around two singlet microtubules. Outer dense fibers are directly coupled to axonemal doublet microtubules in the principal piece, but not in the midpiece. Introduction Cilia, also called flagella, are evolutionarily ancient organelles used by diverse eukaryotic cell types and organisms to propel themselves through fluid environments or to move fluid across their surfaces (Mitchell, 2017; Wan, 2018). These intricate molecular machines are paragons of self-organization built from an extensive array of active and passive structural elements that, together, are able to spontaneously generate oscillatory wave-like motion (Gaffney et al, 2011). The basic architecture of motile cilia is conserved across broad swaths of the eukaryotic tree, providing information on the minimal structures needed for spontaneous undulation (Brokaw, 2009). However, because they operate in a wide range of environments, cilia from different cell types generate different waveforms (Khan & Scholey, 2018) that are modulated by fluid viscosity (Smith et al, 2009). The motile cilium is a continuous assembly of compound microtubules (Ishikawa, 2017). The base of the cilium is the centriole or basal body, which is typically a cylinder of triplet microtubules. The centriole transitions into the axoneme, which consists of nine doublet microtubules arrayed around a central pair of singlet microtubules. Axonemal microtubules anchor hundreds of dynein motors and accessory proteins to power and regulate movement. Axoneme structure has been studied extensively by cryo-electron tomography (cryo-ET) in Chlamydomonas, Tetrahymena, and sea urchin sperm (Nicastro et al, 2006, 2011; Pigino et al, 2012; Owa et al, 2019). Recent studies have begun to shed light on species- and cell type-specific specializations (Lin et al, 2014a; Yamaguchi et al, 2018; Imhof et al, 2019; Greenan et al, 2020), motivating efforts to expand the pantheon of organisms and cell types used in axoneme research. Perhaps the most striking example of ciliary diversity across species is in sperm, which are highly specialized for a defined function—to find and fuse with the egg. Sperm consist of a head, which contains the genetic payload, and a tail, which is a modified motile cilium. Despite their streamlined structure, sperm are simultaneously the most diverse eukaryotic cell type (Gage, 2012; Lüpold & Pitnick, 2018), reflecting the sheer range of reproductive modes and fertilization arenas, from watery media for marine invertebrates and freshwater species to the viscous fluids of the female reproductive tract for mammals. Because motility is crucial to sperm function, the natural variation of sperm form presents a unique opportunity to understand the structural diversification of motile cilia. Mammalian sperm flagella are characterized by accessory structures that surround and dwarf the axoneme (Fawcett, 1975), unlike marine invertebrates whose sperm tails consist essentially of the axoneme (Fawcett, 1970). In mammalian sperm, axonemal doublets are associated with filamentous cytoskeletal elements called outer dense fibers (ODFs) for most of their lengths. The ODFs are further surrounded by a sheath of mitochondria in the midpiece and by a reticular structure called the fibrous sheath in the principal piece. These accessory structures are proposed to stabilize beating of the long flagella of mammalian sperm (Lindemann, 1996; Lindemann & Lesich, 2016). The accessory structures also facilitate movement through the viscous fluids of the female reproductive tract by suppressing buckling instabilities that would otherwise cause sperm to swim in circles (Gadêlha & Gaffney, 2019). Indeed, many cases of male infertility are linked to defects in these accessory elements (Serres et al, 1986; Haidl et al, 1991; Zhao et al, 2018). However, we still do not fully understand how these accessory structures modulate the flagellar beat since there is very little structural information on how they interact with the axoneme proper. Another distinguishing feature of mammalian sperm flagella is that they are not anchored by a basal body (Avidor-Reiss, 2018). Instead, the base of the mammalian sperm flagellum is surrounded by a large cytoskeletal scaffold called the connecting piece. The isolated bovine sperm connecting piece was characterized by cryo-ET, revealing a complex asymmetric assembly (Ounjai et al, 2012). However, the purification process resulted in loss of the centrioles. Thus, there is still a paucity of structural information on the flagellar base in intact cells and on how it varies across species that often have very different head shapes. Sperm have two centrioles that are located in the neck, where the nucleus attaches to the flagellum. The centriole closer to the nucleus is referred to as the proximal centriole (PC) and the one at the base of the flagellum the distal centriole (DC). During spermiogenesis in mammals, the DC is remodeled to the point that it no longer resembles a canonical centriole. This was thought to represent a process of degeneration (Manandhar et al, 2000), but recent work showed that the DC is in fact a functional centriole that participates in orchestrating the first zygotic division (Fishman et al, 2018). Such drastic deviations from canonical centriole structure have not been investigated in detail. Here, we combine cryo-focused ion beam (cryo-FIB) milling-enabled cryo-ET (Marko et al, 2007; Rigort et al, 2012) with subtomogram averaging to image mature sperm from three mammalian species—the pig (Sus scrofa), the horse (Equus caballus), and the mouse (Mus musculus)—that differ in terms of gross morphology, motility, and metabolism (Fig 1A–C). We leverage the uniquely multi-scale capabilities of cryo-ET to define the molecular architecture of microtubule-based assemblies and their critical interactions with accessory structures. We take advantage of the highly streamlined shape of sperm in order to define how these structures and relationships change throughout the flagellum. Figure 1. The proximal centriole (PC) in mammalian sperm is asymmetric and contains novel microtubule inner proteins A–C. Low-magnification cryo-EM projection images of pig (A), horse (B), and mouse (C) sperm. Different regions of the flagellum discussed in this paper are annotated as follows: green—neck, yellow—midpiece, coral—principal piece, pink—endpiece. D–F. Tomographic slices through cryo-FIB-milled lamellae of pig (D), horse (E), and mouse (F) sperm. Transverse slices (D'–F') show complete triplets in the pig (D') and the horse (E'), but not in the mouse (F'). Complete triplets are indicated by green arrowheads with black outlines, while degenerated triplets are indicated by white arrowheads with green outlines. Note the electron-dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece (asterisks in D and D'). G. In situ structure of triplet microtubules from the proximal region of the pig sperm PC with the tubulin backbone in gray and microtubule inner protein densities colored individually. A-tubule MIPs are colored: MIP1—green, MIP2—yellow, MIP3—orange, MIP4—red, MIP5—purple, MIP6—blue. B-tubule MIPs are colored: MIP7—magenta. C-tubule MIPs are colored: MIP8—light pink, MIP9—pink. The A-C linker is colored gold and the putative A-link is colored olive green. H. Reconstruction of the proximal region of the pig sperm PC generated by plotting the average back into the original particle positions and orientations in the tomogram. This plotback only contains four triplets as only part of the centriole was captured in the lamella. The A–C linker is colored in gold and the putative A-link in olive green. Data information: Labels: nuc—nucleus, bp—baseplate. Scale bars: (A–C) 20 µm; (D–F) 250 nm; (D'–F') 100 nm. Download figure Download PowerPoint We define the architecture of the flagellar base and show that the ODFs are anchored through an intricate structure that forms a large, asymmetric chamber around the centrioles. We show that the ODFs are directly coupled to axonemal microtubules in the principal piece, but not in the midpiece. We find that mammalian sperm microtubules are additionally decorated throughout by large, protofilament-bridging microtubule inner protein densities. Thus, mammalian sperm flagella are modified across scales—from large accessory structures that increase the effective size and rigidity of the entire assembly to extensive microtubule inner proteins that likely reinforce the microtubules themselves. We further discuss the implications of these accessory structures to ciliary motility. Results The base of the flagellum is anchored through a large, asymmetric chamber around the centrioles The neck region containing the PC and DC is too thick (~600–700 nm) for direct imaging by cryo-ET, so in order to image sperm centrioles in their native subcellular milieu, we used cryo-FIB milling to generate thin lamellae suitable for high-resolution imaging (Fig 1). Cryo-ET of lamellae containing the PC confirmed that it is indeed composed of triplet microtubules in pig and in horse sperm (Fig 1D–F). Unexpectedly, we found that triplets of the pig sperm PC are not all the same length (Figs 1, EV1). Shorter triplets are grouped on one side of the centriole, giving the PC a striking dorsoventral asymmetry (Fig EV1A). Consistent with previous reports that the PC degenerates in rodents (Woolley & Fawcett, 1973; Manandhar et al, 1998), the PC was not prominent in mouse sperm. However, cryo-ET showed unequivocally that some centriolar microtubules remain (Fig 1F), demonstrating that degeneration is incomplete. We observed complete triplets as well as triplets in various stages of degeneration, including triplets in which only the B-tubule had degraded (Fig 1F'). Click here to expand this figure. Figure EV1. Structural features of the pig sperm proximal centriole (PC) PC triplets have unequal lengths (top panel), and shorter triplets are grouped on one side, giving the PC dorsoventral asymmetry (bottom panel). Many of the microtubule inner proteins (MIPs) in the pig sperm PC are not found in other mammalian centriole structures. Details of the MIP densities in the pig sperm PC. Download figure Download PowerPoint We determined the in situ structure of the pig sperm PC by subtomogram averaging (Fig 1G). Because only parts of the PC were captured in cryo-FIB-milled lamellae, our average includes only particles from the proximal ~400 nm of the centriole. While the overall structure of the PC triplet is similar to other centriole structures (Li et al, 2012; Guichard et al, 2013; Greenan et al, 2018, 2020; Le Guennec et al, 2020), it differs in terms of the microtubule inner protein densities (MIPs; Fig EV1B). We observed nine MIPs, six in the A-tubule, one in the B-tubule, and two in the C-tubule (Fig 1G and EV1C). In the A-tubule, most of the MIPs are unique, including MIP2 (yellow) that binds to protofilament A12, MIP3 (orange) bridging protofilaments A13 and A1, MIP4 (red) that binds to A2, MIP5 (purple) that binds to A5, and MIP6 (blue) bridging A6 and A7. MIP1 (green), a prominent MIP associated with protofilament A9, was also reported in centrioles isolated from CHO cells (Greenan et al, 2018), Trichonympha (Guichard et al, 2013), Chlamydomonas, and Paramecium (Le Guennec et al, 2020), and in basal bodies from bovine respiratory epithelia (Greenan et al, 2020). The seam is located between protofilaments A9 and A10 (Ichikawa et al, 2017; Ma et al, 2019), which suggests that MIP1 is a highly conserved seam-stabilizing or seam-recognizing structure. In the B-tubule, we observed a large helical MIP7 (magenta) bridging protofilaments B3-9. We observed two groups of unique MIPs in the C-tubule, MIP8 (light pink) associated with C2-C4 and MIP9 (pink) with C5–C7. The inner junctions between A- and B-tubules (cyan) and between B- and C-tubules (turquoise) are non-tubulin proteins that repeat every 8 nm and are staggered relative to each other when viewed from the luminal side of the triplet (Fig EV1C). We resolved density for the A–C linker (gold), which is associated with protofilaments C9 and C10, and possibly for the A-link (olive green), associated with protofilament A8/A9 (Fig 1G and H). The B- and C-tubule MIPs we observed are not present in other centriole structures. However, helical MIPs have been observed in the transition zone of bovine respiratory cilia (Greenan et al, 2020). Unlike other mammalian centriole structures, we do not observe MIPs that bridge B1-B2 or C1-C2 (Fig EV1B). It is difficult to tell whether these differences in MIP patterns are due to differences in cell type or species. As this is the first in situ structure of any mammalian centriole, these differences may also be because previous structures were of isolated centrioles. Nonetheless, it is clear that there is great diversity in how core centriolar microtubules are accessorized, which raises questions about the functions of these MIPs. We next determined the organization of the atypical DC by tracing microtubules through Volta phase plate (VPP; Danev et al, 2014) cryo-tomograms of whole sperm (Fig 2). The DC consists of doublet microtubules, with a pair of singlets extending through the lumen (Fig 2A–F). In pig and in horse sperm (Fig 2A–D), doublets extend almost as far proximally as the central pair. In a further departure from canonical centriole structure, DC doublets are splayed open and arranged asymmetrically around the singlets. The central singlets themselves are spaced inconsistently, suggesting that they lack the projections characteristic of the central pair apparatus (CPA). Mouse sperm lack the splayed doublets, but they also have a pair of singlets extending beyond the axoneme (Fig 2E and F). Figure 2. The distal centriole (DC) in mammalian sperm is composed of doublet microtubules arrayed asymmetrically around a pair of singlet microtubules A–F. Microtubules in the DC of pig (A, B), horse (C, D), and mouse (E, F) sperm traced from Volta phase plate cryo-tomograms of intact sperm. Doublets are colored blue (A-tubule in light blue, B-tubule in dark blue), while singlets are pink. G–I. Tomographic slices through cryo-FIB-milled lamellae of the DC-to-axoneme transition in pig sperm show how the change in geometry (G, white arrows and white dashed lines) coincides with the appearance of axoneme accessory structures (H, white arrows) and with density in the A-tubule (I, compare insets in white and black boxes). In (I), the white and black arrows indicate where the cross-sections in white and black boxes were taken from. J. In situ structure of the pig sperm DC microtubule doublet with the tubulin backbone in gray and microtubule inner protein densities colored individually. The gold and turquoise densities on the luminal side of the doublet are consistent with the positions of parts of the inner scaffold. This structure represents the DC doublets closest to the axoneme (the area shown in (G–I)). Data information: Labels: RSs—radial spokes, cpa—central pair apparatus, At—A-tubule, Bt—B-tubule. Scale bars: 250 nm. Download figure Download PowerPoint To more precisely define the DC-to-axoneme transition, we imaged cryo-FIB-milled sperm (Fig 2G–J). We directly observed this transition in situ in pig sperm, defined by the appearance of axonemal accessory proteins such as the radial spokes and the projections of the CPA (Fig 2H). The onset of the axoneme coincides with a change in microtubule geometry (Fig 2G), suggesting that the splayed-open doublets are indeed characteristic of the DC. The transition zone also coincides with an increase in density in the A-tubule (Fig 2I), suggesting that binding of axonemal accessory structures is related to the regulated binding of A-tubule MIPs. We then determined the structure of doublet microtubules from the region of the DC closest to the axoneme by subtomogram averaging, revealing the presence of MIPs distinct from those in the PC (Fig 2J). We resolved some density for structures on the luminal side of the doublet (gold and turquoise), which may correspond to parts of the inner scaffold (Li et al, 2012; Le Guennec et al, 2020). The flagellar waveform depends greatly on the properties of the base (Riedel-Kruse & Hilfinger, 2007), but there is very little information on how this region is organized in three dimensions in any cell type. In order to capture the full three-dimensional complexity of the flagellar base, we took advantage of enhanced contrast provided by the VPP, which allowed us to trace microtubules while retaining the context of the surrounding connecting piece (Fig 3A–C, Movies EV1–EV3). Semi-automated neural network-based segmentation (Chen et al, 2017) revealed that the connecting piece forms a large chamber enclosing the sperm centrioles. Although precise dimensions and shapes of the connecting piece differ across species (Fig 3D–F), its general architecture appears to be conserved across mammalian species. Figure 3. The connecting piece forms a large, asymmetric chamber around the sperm centrioles A–C. Slices through Volta phase plate cryo-tomograms of the neck region in intact pig (A), horse (B), and mouse (C) sperm. Proximal centriole triplets are shown in green, distal centriole doublets in blue (A-tubule in light blue, B-tubule in dark blue) and singlets in pink, and electron-dense bars in yellow. Note the electron-dense structures flanking the connecting piece in the mouse (asterisks in C, F). D–F. Three-dimensional architecture of the flagellar base, with the connecting piece in gray, the proximal centriole in green, distal centriole doublets in blue (A-tubule in light blue, B-tubule in dark blue) and singlets in pink, and electron-dense bars in yellow. The connecting piece was segmented semi-automatically with a neural network, while microtubules were traced manually. Data information: Labels: nuc—nucleus, bp—baseplate, sc—striated columns, odf—outer dense fibers, mito—mitochondria. Scale bars: 250 nm. Download figure Download PowerPoint The proximal region of the connecting piece consists of striated columns (SCs), called such because of their banded appearance. Following the numbering scheme laid out in (Ounjai et al, 2012), we found that the SCs follow a conserved pattern of grouping and splitting. The proximal connecting piece can be grossly divided into left and right regions. The right region forms the proximal centriolar vault where columns 8, 9, 1, 2, and 3 merge, whereas the left region comprises columns 4, 5, 6, and 7 (Fig 3D–F, panels iv). The columns gradually separate, eventually splitting into nine separate columns that fuse distally with the ODFs (Fig 3D–F, panels v). The connecting piece displays both marked left–right asymmetry and dorsoventral asymmetry in all three species. The PC is embedded within the proximal region of the connecting piece, and always on the same side. In pig sperm, one side of the proximal centriolar vault is formed by the Y-shaped SC 9, which also gives the entire connecting piece dorsoventral asymmetry (Fig 3D, panel ii). The material of the connecting piece extends through the interstices of the PC triplets (Fig 1D–F) and is continuous with electron-dense material within the proximal lumen of the PC (asterisks in Fig 1D and D'). Intriguingly, the dorsoventral asymmetry of the pig sperm PC is defined relative to the connecting piece, with the side of the shorter triplets always facing the Y-shaped segmented column 9 (Fig 3D). The pig sperm connecting piece also has two electron-dense bars associated with the central singlets of the DC (Fig 3A and D, yellow and goldenrod), which resemble the bars observed in the bovine sperm connecting piece (Ounjai et al, 2012). These bars are conspicuously absent from horse and from mouse sperm. Instead, mouse sperm have two electron-dense structures flanking the SCs (asterisks in Fig 3C and F), an arrangement reminiscent of the distribution pattern of the centrosomal protein speriolin (Goto et al, 2010; Ito et al, 2019). The mammalian sperm axoneme anchors unique accessory structures and species-specific microtubule inner proteins To gain insight into the molecular architecture of the axoneme, we determined in situ structures of the central pair apparatus (Fig 4) and of the 96-nm axonemal doublet repeat (Fig 5). Our structures of the CPA are the first from any mammalian system, and our structures of the doublets are the first from any mammalian sperm, thus filling crucial gaps in the gallery of axoneme structures. The overall architecture of the mammalian CPA projection network is similar across the three species we examined (Fig EV2) and resembles that of
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