Ninefold Symmetry Research Articles

Centrosomal organization of Cep152 provides flexibility in Plk4 and procentriole positioning.

Centriole duplication is a high-fidelity process driven by Polo-like kinase 4 (Plk4) and a few conserved initiators. Dissecting how Plk4 and its receptors organize within centrosomes is critical to understand the centriole duplication process and biochemical and architectural differences between centrosomes of different species. Here, at nanoscale resolution, we dissect centrosomal localization of Plk4 in G1 and S phase in its catalytically active and inhibited state during centriole duplication and amplification. We build a precise distribution map of Plk4 and its receptor Cep152, as well as Cep44, Cep192, and Cep152-anchoring factors Cep57 and Cep63. We find that Cep57, Cep63, Cep44, and Cep192 localize in ninefold symmetry. However, during centriole maturation, Cep152, which we suggest is the major Plk4 receptor, develops a more complex pattern. We propose that the molecular arrangement of Cep152 creates flexibility for Plk4 and procentriole placement during centriole initiation. As a result, procentrioles form at variable positions in relation to the mother centriole microtubule triplets.

Single-molecule localization microscopy reveals the ultrastructural constitution of distal appendages in expanded mammalian centrioles

Distal appendages (DAPs) are vital in cilia formation, mediating vesicular and ciliary docking to the plasma membrane during early ciliogenesis. Although numerous DAP proteins arranging a nine-fold symmetry have been studied using superresolution microscopy analyses, the extensive ultrastructural understanding of the DAP structure developing from the centriole wall remains elusive owing to insufficient resolution. Here, we proposed a pragmatic imaging strategy for two-color single-molecule localization microscopy of expanded mammalian DAP. Importantly, our imaging workflow enables us to push the resolution limit of a light microscope well close to a molecular level, thus achieving an unprecedented mapping resolution inside intact cells. Upon this workflow, we unravel the ultra-resolved higher-order protein complexes of the DAP and its associated proteins. Intriguingly, our images show that C2CD3, microtubule triplet, MNR, CEP90, OFD1, and ODF2 jointly constitute a unique molecular configuration at the DAP base. Moreover, our finding suggests that ODF2 plays an auxiliary role in coordinating and maintaining DAP nine-fold symmetry. Together, we develop an organelle-based drift correction protocol and a two-color solution with minimum crosstalk, allowing a robust localization microscopy imaging of expanded DAP structures deep into the gel-specimen composites.

Grand canonical Brownian dynamics simulations of adsorption and self-assembly of SAS-6 rings on a surface.

The Spindle Assembly Abnormal Protein 6 (SAS-6) forms dimers, which then self-assemble into rings that are critical for the nine-fold symmetry of the centriole organelle. It has recently been shown experimentally that the self-assembly of SAS-6 rings is strongly facilitated on a surface, shifting the reaction equilibrium by four orders of magnitude compared to the bulk. Moreover, a fraction of non-canonical symmetries (i.e., different from nine) was observed. In order to understand which aspects of the system are relevant to ensure efficient self-assembly and selection of the nine-fold symmetry, we have performed Brownian dynamics computer simulation with patchy particles and then compared our results with the experimental ones. Adsorption onto the surface was simulated by a grand canonical Monte Carlo procedure and random sequential adsorption kinetics. Furthermore, self-assembly was described by Langevin equations with hydrodynamic mobility matrices. We find that as long as the interaction energies are weak, the assembly kinetics can be described well by coagulation-fragmentation equations in the reaction-limited approximation. By contrast, larger interaction energies lead to kinetic trapping and diffusion-limited assembly. We find that the selection of nine-fold symmetry requires a small value for the angular interaction range. These predictions are confirmed by the experimentally observed reaction constant and angle fluctuations. Overall, our simulations suggest that the SAS-6 system works at the crossover between a relatively weak binding energy that avoids kinetic trapping and a small angular range that favors the nine-fold symmetry.

Single-molecule localization microscopy of expanded mammalian centrioles unravels the ultrastructural constitution of distal appendages.

Distal appendages (DAPs) are vital in ciliogenesis, mediating membrane docking and ciliary gating. Although the nine-fold symmetric constitution of DAP proteins has been investigated via superresolution microscopy analyses, the root structure of DAPs remains elusive owing to insufficient spatial optical resolution. Here, we proposed the in situ drift correction for optimized two-color imaging incorporating single-molecule localization microscopy with sample expansion approaches to achieve effective localization precision down to sub-5 nm. The robust workflow enables us to systematically perform the ultrastructural imaging study of protein mapping within cells, not limited to the proximity of coverslips. Notably, our findings unraveled the ultrastructural arrangement of the core DAP proteins (CEP83, CEP89, SCLT1, FBF1, and CEP164) and their spatial connection to microtubule triplets. Surprisingly, we found that C2CD3 was localized near a tubule of triplets guiding the DAP formation from microtubules to outer components of blades with nine-fold symmetry. We further identified the dual-layered distribution of ODF2. Intriguingly, the distal-layered ODF2 displayed a high spatial correlation with other DAP proteins, acting as a structural coordinator at the DAP root. Moreover, our results showed that the N- and C-terminus of ODF2 jointly encircled the microtubule triplets. Together, our studies suggest the unprecedented architectural framework of DAPs and provide a pragmatic roadmap for protein mapping with optical resolution at a molecular level.

Crystals of SctV from different species reveal variable symmetry for the cytosolic domain of the type III secretion system export gate.

Type III secretion systems (T3SSs) are proteinaceous devices employed by Gram-negative bacteria to directly transport proteins into a host cell. Substrate recognition and secretion are strictly regulated by the export apparatus of the so-called injectisome. The export gate SctV engages chaperone-bound substrates of the T3SS in its nonameric cytoplasmic domain. Here, the purification and crystallization of the cytoplasmic domains of SctV from Photorhabdus luminescens (LscVC) and Aeromonas hydrophila (AscVC) are reported. Self-rotation functions revealed that LscVC forms oligomers with either eightfold or ninefold symmetry in two different crystal forms. Similarly, AscVC was found to exhibit tenfold rotational symmetry. These are the first instances of SctV proteins forming non-nonameric oligomers.

Quantum chemical elucidation of a sevenfold symmetric bacterial antenna complex

The light-harvesting complex 2 (LH2) of purple bacteria is one of the most studied photosynthetic antenna complexes. Its symmetric structure and ring-like bacteriochlorophyll arrangement make it an ideal system for theoreticians and spectroscopists. LH2 complexes from most bacterial species are thought to have eightfold or ninefold symmetry, but recently a sevenfold symmetric LH2 structure from the bacterium Mch. purpuratum was solved by Cryo-Electron microscopy. This LH2 also possesses unique near-infrared absorption and circular dichroism (CD) spectral properties. Here we use an atomistic strategy to elucidate the spectral properties of Mch. purpuratum LH2 and understand the differences with the most commonly studied LH2 from Rbl. acidophilus. Our strategy exploits a combination of molecular dynamics simulations, multiscale polarizable quantum mechanics/molecular mechanics calculations, and lineshape simulations. Our calculations reveal that the spectral properties of LH2 complexes are tuned by site energies and exciton couplings, which in turn depend on the structural fluctuations of the bacteriochlorophylls. Our strategy proves effective in reproducing the absorption and CD spectra of the two LH2 complexes, and in uncovering the origin of their differences. This work proves that it is possible to obtain insight into the spectral tuning strategies of purple bacteria by quantitatively simulating the spectral properties of their antenna complexes.

Nine-fold symmetry of centriole: The joint efforts of its core proteins.

The centriole is a widely conserved organelle required for the assembly of centrosomes, cilia, and flagella. Its striking feature - the nine-fold symmetrical structure, was discovered over 70 years ago by transmission electron microscopy, and since elaborated mostly by cryo-electron microscopy and super-resolution microscopy. Here, we review the discoveries that led to the current understanding of how the nine-fold symmetrical structure is built. We focus on the recent findings of the centriole structure in high resolution, its assembly pathways, and its nine-fold distributed components. We propose a model that the assembly of the nine-fold symmetrical centriole depends on the concerted efforts of its core proteins. Also see the video abstract here: https://youtu.be/m4h_3II_WJo.

Superresolution characterization of core centriole architecture.

The centrosome is the main microtubule-organizing center in animal cells. It comprises of two centrioles and the surrounding pericentriolar material. Protein organization at the outer layer of the centriole and outward has been studied extensively; however, an overall picture of the protein architecture at the centriole core has been missing. Here we report a direct view of Drosophila centriolar proteins at ∼50-nm resolution. This reveals a Sas6 ring at the C-terminus, where it overlaps with the C-terminus of Cep135. The ninefold symmetrical pattern of Cep135 is further conveyed through Ana1-Asterless axes that extend past the microtubule wall from between the blades. Ana3 and Rcd4, whose termini are close to Cep135, are arranged in ninefold symmetry that does not match the above axes. During centriole biogenesis, Ana3 and Rcd4 are sequentially loaded on the newly formed centriole and are required for centriole-to-centrosome conversion through recruiting the Cep135-Ana1-Asterless complex. Together, our results provide a spatiotemporal map of the centriole core and implications of how the structure might be built.

A centriole's subdistal appendages: contributions to cell division, ciliogenesis and differentiation.

The centrosome is a highly conserved structure composed of two centrioles surrounded by pericentriolar material. The mother, and inherently older, centriole has distal and subdistal appendages, whereas the daughter centriole is devoid of these appendage structures. Both appendages have been primarily linked to functions in cilia formation. However, subdistal appendages present with a variety of potential functions that include spindle placement, chromosome alignment, the final stage of cell division (abscission) and potentially cell differentiation. Subdistal appendages are particularly interesting in that they do not always display a conserved ninefold symmetry in appendage organization on the mother centriole across eukaryotic species, unlike distal appendages. In this review, we aim to differentiate both the morphology and role of the distal and subdistal appendages, with a particular focus on subdistal appendages.

If You Split Something into Two Parts, You Will Get Three Pieces: The Bilateral Binomial Theorem and its Consequences

If a mathematical quantity is taken apart, the binomial expansion will result in three Pascal triangles. Thus a split into two parts generates a mathematical pattern of three pieces. This binomial expansion can now be generalized into a bilateral picture, resulting in an expansion of positive and negative powers as well. In so doing the Binomial Theorem will be generalized into the Bilateral Binomial Theorem, applying the intriguing mathematics of bilateral hypergeometric functions.And if a mathematical quantity is taken apart into two anti-commuting parts, the threefold pattern will triple again. Thus a ninefold symmetry appears. As an identical ninefold symmetry will appear if complex numbers and their conjugates are multiplied, complex conjugation must be seen as a strange, brutal, and illegitimate mathematical trick to model non-commutative structures by using commuting quantities.

Optimal sidestepping of intraflagellar transport kinesins regulates structure and function of sensory cilia.

Cytoskeletal-based molecular motors produce force perpendicular to their direction of movement. However, it remains unknown whether and why motor proteins generate sidesteps movement along their filamentous tracks invivo. Using Hessian structured illumination microscopy, we located green fluorescent protein (GFP)-labeled intraflagellar transport (IFT) particles inside sensory cilia of live Caenorhabditis elegans with 3-6-nanometer accuracy and 3.4-ms resolution. We found that IFT particles took sidesteps along axoneme microtubules, demonstrating that IFT motors generate torque in a living animal. Kinesin-II and OSM-3-kinesin collaboratively drive anterograde IFT. We showed that the deletion of kinesin-II, a torque-generating motor protein, reduced sidesteps, whereas the increase of neck flexibility of OSM-3-kinesin upregulated sidesteps. Either increase or decrease of sidesteps of IFT kinesins allowed ciliogenesis to the regular length, but changed IFT speeds, disrupted axonemal ninefold symmetry, and inhibited sensory cilia-dependent animal behaviors. Thus, an optimum level of IFT kinesin sidestepping is associated with the structural and functional fidelity of cilia.

Mechanism and Regulation of Centriole and Cilium Biogenesis.

The centriole is an ancient microtubule-based organelle with a conserved nine-fold symmetry. Centrioles form the core of centrosomes, which organize the interphase microtubule cytoskeleton of most animal cells and form the poles of the mitotic spindle. Centrioles can also be modified to form basal bodies, which template the formation of cilia and play central roles in cellular signaling, fluid movement, and locomotion. In this review, we discuss developments in our understanding of the biogenesis of centrioles and cilia and the regulatory controls that govern their structure and number. We also discuss how defects in these processes contribute to a spectrum of human diseases and how new technologies have expanded our understanding of centriole and cilium biology, revealing exciting avenues for future exploration.

Rapid Evolution of Sperm Produces Diverse Centriole Structures that Reveal the Most Rudimentary Structure Needed for Function.

Centrioles are ancient subcellular protein-based organelles that maintain a conserved number and structure across many groups of eukaryotes. Centriole number (two per cells) is tightly regulated; each pre-existing centriole nucleates only one centriole as the cell prepares for division. The structure of centrioles is barrel-shaped, with a nine-fold symmetry of microtubules. This organization of microtubules is essential for the ancestral function of centriole–cilium nucleation. In animal cells, centrioles have gained an additional role: recruiting pericentriolar material (PCM) to form a centrosome. Therefore, it is striking that in animal spermatozoa, the centrioles have a remarkable diversity of structures, where some are so anomalous that they are referred to as atypical centrioles and are barely recognizable. The atypical centriole maintains the ability to form a centrosome and nucleate a new centriole, and therefore reveals the most rudimentary structure that is needed for centriole function. However, the atypical centriole appears to be incapable of forming a cilium. Here, we propose that the diversity in sperm centriole structure is due to rapid evolution in the shape of the spermatozoa head and neck. The enhanced diversity may be driven by a combination of direct selection for novel centriole functions and pleiotropy, which eliminates centriole properties that are dispensable in the spermatozoa function.

Gorab is a Golgi protein required for structure and duplication of Drosophila centrioles.

We demonstrate that a Drosophila Golgi protein, Gorab, is present not only in the trans-Golgi but also in the centriole cartwheel where, complexed to Sas6, it is required for centriole duplication. In addition to centriole defects, flies lacking Gorab are uncoordinated due to defects in sensory cilia, which lose their 9-fold symmetry. We demonstrate the separation of centriole and Golgi functions of Drosophila Gorab in two ways: First, we have created Gorab variants that are unable to localize to trans-Golgi but can still rescue the centriole and cilia defects of gorab null flies. Second, we show that expression of C-terminally tagged Gorab disrupts Golgi functions in cytokinesis of male meiosis, a dominant phenotype overcome by mutations preventing Golgi targeting. Our findings suggest that during metazoan evolution, a Golgi protein has arisen with a second, apparently independent, role in centriole duplication.

Atypical centrioles are present in Tribolium sperm.

Typical centrioles are made of microtubules organized in ninefold symmetry. Most animal somatic cells have two centrioles for normal cell division and function. These centrioles originate from the zygote, but because the oocyte does not provide any centrioles, it is surprising that the zygotes of many animals are thought to inherit only one centriole from the sperm. Recently, in the sperm of Drosophila melanogaster, we discovered a second centriolar structure, the proximal centriole-like structure (PCL), which functions in the zygote. Whether the sperm of other insects has a second centriolar structure is unknown. Here, we characterized spermiogenesis in the red flour beetle, Tribolium castaneum. Electron microscopy suggests that Tribolium has one microtubule-based centriole at the tip of the axoneme and a structure similar to the PCL, which lacks microtubules and lies in a cytoplasmic invagination of the nucleus. Immunostaining against the orthologue of the centriole/PCL protein, Ana1, also recognizes two centrioles near the nucleus during spermiogenesis: one that is microtubule-based at the tip of the axoneme, suggesting it is the centriole; and another that is more proximal and appears during early spermiogenesis, suggesting it is the PCL. Together, these findings suggest that Tribolium sperm has one microtubule-based centriole and one microtubule-lacking centriole.

Computational support for a scaffolding mechanism of centriole assembly.

Centrioles are essential for forming cilia, flagella and centrosomes. Successful centriole assembly requires proteins of the SAS-6 family, which can form oligomeric ring structures with ninefold symmetry in vitro. While important progress has been made in understanding SAS-6 protein biophysics, the mechanisms enabling ring formation in vivo remain elusive. Likewise, the mechanisms by which a nascent centriole forms near-orthogonal to an existing one are not known. Here, we investigate possible mechanisms of centriole assembly using coarse-grained Brownian dynamics computer simulations in combination with a rate equation approach. Our results suggest that without any external factors, strong stabilization associated with ring closure would be needed to enable efficient ring formation. Strikingly, our simulations reveal that a scaffold-assisted assembly mechanism can trigger robust ring formation owing to local cooperativity, and that this mechanism can also impart orthogonalilty to centriole assembly. Overall, our findings provide novel insights into the organizing principles governing the assembly of this important organelle.

Building a ninefold symmetrical barrel: structural dissections of centriole assembly.

Centrioles are short microtubule-based organelles with a conserved ninefold symmetry. They are essential for both centrosome formation and cilium biogenesis in most eukaryotes. A core set of five centriolar proteins has been identified and their sequential recruitment to procentrioles has been established. However, structures at atomic resolution for most of the centriolar components were scarce, and the underlying molecular mechanisms for centriole assembly had been a mystery—until recently. In this review, I briefly summarize recent advancements in high-resolution structural characterization of the core centriolar components and discuss perspectives in the field.

Recruitment of FOR20 and OFD1 onto pericentriolar satellites and centrosomes depends on the formation of a ternary complex with KIAA0753

Stable microtubules organized on the basis of a nine-fold rotational symmetry constitute the structural basis of centrioles and basal bodies. A number of proteins associate with these structures to drive the process of centrosomes and cilia formation and to participate in their function. Pericentriolar satellites are small round-shaped particles closely associated with microtubules with PCM1 as a major component. To date they have been described only in vertebrates where they participate in the recruitment of proteins to the centrosomes and cilia. We identified the poorly characterized KIAA0753 protein as a direct interactor of FOR20, a pericentriolar satellite protein required for basal body integrity and ciliogenesis. This binary association allows the recruitment of OFD1, a centriole distal-end protein and regulator of centriole length, to form a ternary complex associated with pericentriolar satellites. Inhibiting expression of KIAA0753 by small inhibitory RNA limits the recruitment of FOR20 and OFD1 onto pericentriolar satellites and centrosomes, and decreases cilia length in serum-starved RPE1 cells. We also show that KIAA0753 has a microtubule-stabilizing activity. We propose that pericentriolar satellite and associated proteins such as PCM1 and KIAA0753 appeared in the course of early metazoan evolution to regulate ancestral eukaryotic centriole/basal body proteins such as FOR20 and OFD1.

The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies.

Sas-6 and Ana2/STIL proteins are required for centriole duplication and the homo-oligomerisation properties of Sas-6 help establish the ninefold symmetry of the central cartwheel that initiates centriole assembly. Ana2/STIL proteins are poorly conserved, but they all contain a predicted Central Coiled-Coil Domain (CCCD). Here we show that the Drosophila Ana2 CCCD forms a tetramer, and we solve its structure to 0.8 Å, revealing that it adopts an unusual parallel-coil topology. We also solve the structure of the Drosophila Sas-6 N-terminal domain to 2.9 Å revealing that it forms higher-order oligomers through canonical interactions. Point mutations that perturb Sas-6 or Ana2 homo-oligomerisation in vitro strongly perturb centriole assembly in vivo. Thus, efficient centriole duplication in flies requires the homo-oligomerisation of both Sas-6 and Ana2, and the Ana2 CCCD tetramer structure provides important information on how these proteins might cooperate to form a cartwheel structure.

Isolation, cryotomography, and three-dimensional reconstruction of centrioles.

Centrioles and basal bodies (referred to hereafter as centrioles for simplicity) are microtubule-based cylindrical organelles that are typically ∼450-nm long and ∼250nm in diameter. The centriole is composed of three distinct regions: the distal part characterized by microtubule doublets, the central core that harbors microtubule triplets, which are also present in the proximal part that also contains the cartwheel, a structure crucial for centriole assembly. The cartwheel was initially revealed by conventional electron microscopy of resin-embedded samples and is thought to impart the near universal ninefold symmetry of centrioles. Deciphering the native architecture of the cartwheel has proven challenging owing to its small dimensions and the difficulties in isolating it. Here, we present a method to purify and analyze the structure of the exceptionally long Trichonympha centriole by cryotomography and subtomogram averaging. Using this method, we revealed the native architecture of the proximal cartwheel-containing region at ∼40Å-resolution. This method can be applied as a general strategy for uncovering the structure of centrioles in other species.