The evolutionary conserved proteins CEP90, FOPNL, and OFD1 recruit centriolar distal appendage proteins to initiate their assembly.

In metazoa, cilia assembly is a cellular process that starts with centriole to basal body maturation, migration to the cell surface, and docking to the plasma membrane. Basal body docking involves the interaction of both the distal end of the basal body and the transition fibers/distal appendages, with the plasma membrane. Mutations in numerous genes involved in basal body docking and transition zone assembly are associated with the most severe ciliopathies, highlighting the importance of these events in cilium biogenesis. In this context, the ciliate Paramecium has been widely used as a model system to study basal body and cilia assembly. However, despite the evolutionary conservation of cilia assembly events across phyla, whether the same molecular players are functionally conserved, is not fully known. Here, we demonstrated that CEP90, FOPNL, and OFD1 are evolutionary conserved proteins crucial for ciliogenesis. Using ultrastructure expansion microscopy, we unveiled that these proteins localize at the distal end of both centrioles/basal bodies in Paramecium and mammalian cells. Moreover, we found that these proteins are recruited early during centriole duplication on the external surface of the procentriole. Functional analysis performed both in Paramecium and mammalian cells demonstrate the requirement of these proteins for distal appendage assembly and basal body docking. Finally, we show that mammalian centrioles require another component, Moonraker (MNR), to recruit OFD1, FOPNL, and CEP90, which will then recruit the distal appendage proteins CEP83, CEP89, and CEP164. Altogether, we propose that this OFD1, FOPNL, and CEP90 functional module is required to determine in mammalian cells the future position of distal appendage proteins.

Structures of SAS-6 coiled coil hold implications for the polarity of the centriolar cartwheel.

Centrioles are eukaryotic organelles that template the formation of cilia and flagella, as well as organize the microtubule network and the mitotic spindle in animal cells. Centrioles have proximal-distal polarity and a 9-fold radial symmetry imparted by a likewise symmetrical central scaffold, the cartwheel. The spindle assembly abnormal protein 6 (SAS-6) self-assembles into 9-fold radially symmetric ring-shaped oligomers that stack via an unknown mechanism to form the cartwheel. Here, we uncover a homo-oligomerization interaction mediated by the coiled-coil domain of SAS-6. Crystallographic structures of Chlamydomonas reinhardtii SAS-6 coiled-coil complexes suggest this interaction is asymmetric, thereby imparting polarity to the cartwheel. Using a cryoelectron microscopy (cryo-EM) reconstitution assay, we demonstrate that amino acid substitutions disrupting this asymmetric association also impair SAS-6 ring stacking. Our work raises the possibility that the asymmetric interaction inherent to SAS-6 coiled-coil provides a polar element for cartwheel assembly, which may assist the establishment of the centriolar proximal-distal axis.

Kinetic and structural roles for the surface in guiding SAS-6 self-assembly to direct centriole architecture.

Discovering mechanisms governing organelle assembly is a fundamental pursuit in biology. The centriole is an evolutionarily conserved organelle with a signature 9-fold symmetrical chiral arrangement of microtubules imparted onto the cilium it templates. The first structure in nascent centrioles is a cartwheel, which comprises stacked 9-fold symmetrical SAS-6 ring polymers emerging orthogonal to a surface surrounding each resident centriole. The mechanisms through which SAS-6 polymerization ensures centriole organelle architecture remain elusive. We deploy photothermally-actuated off-resonance tapping high-speed atomic force microscopy to decipher surface SAS-6 self-assembly mechanisms. We show that the surface shifts the reaction equilibrium by ~104 compared to solution. Moreover, coarse-grained molecular dynamics and atomic force microscopy reveal that the surface converts the inherent helical propensity of SAS-6 polymers into 9-fold rings with residual asymmetry, which may guide ring stacking and impart chiral features to centrioles and cilia. Overall, our work reveals fundamental design principles governing centriole assembly.

A first-takes-all model of centriole copy number control based on cartwheel elongation.

How cells control the numbers of subcellular components is a fundamental question in biology. Given that biosynthetic processes are fundamentally stochastic it is utterly puzzling that some structures display no copy number variation within a cell population. Centriole biogenesis, with each centriole being duplicated once and only once per cell cycle, stands out due to its remarkable fidelity. This is a highly controlled process, which depends on low-abundance rate-limiting factors. How can exactly one centriole copy be produced given the variation in the concentration of these key factors? Hitherto, tentative explanations of this control evoked lateral inhibition- or phase separation-like mechanisms emerging from the dynamics of these rate-limiting factors but how strict centriole number is regulated remains unsolved. Here, a novel solution to centriole copy number control is proposed based on the assembly of a centriolar scaffold, the cartwheel. We assume that cartwheel building blocks accumulate around the mother centriole at supercritical concentrations, sufficient to assemble one or more cartwheels. Our key postulate is that once the first cartwheel is formed it continues to elongate by stacking the intermediate building blocks that would otherwise form supernumerary cartwheels. Using stochastic models and simulations, we show that this mechanism may ensure formation of one and only one cartwheel robustly over a wide range of parameter values. By comparison to alternative models, we conclude that the distinctive signatures of this novel mechanism are an increasing assembly time with cartwheel numbers and the translation of stochasticity in building block concentrations into variation in cartwheel numbers or length.

With Age Comes Maturity: Biochemical and Structural Transformation of a Human Centriole in the Making.

Centrioles are microtubule-based cellular structures present in most human cells that build centrosomes and cilia. Proliferating cells have only two centrosomes and this number is stringently maintained through the temporally and spatially controlled processes of centriole assembly and segregation. The assembly of new centrioles begins in early S phase and ends in the third G1 phase from their initiation. This lengthy process of centriole assembly from their initiation to their maturation is characterized by numerous structural and still poorly understood biochemical changes, which occur in synchrony with the progression of cells through three consecutive cell cycles. As a result, proliferating cells contain three structurally, biochemically, and functionally distinct types of centrioles: procentrioles, daughter centrioles, and mother centrioles. This age difference is critical for proper centrosome and cilia function. Here we discuss the centriole assembly process as it occurs in somatic cycling human cells with a focus on the structural, biochemical, and functional characteristics of centrioles of different ages.

A helical inner scaffold provides a structural basis for centriole cohesion.

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.

Regulated protein stabilization underpins the functional interplay among basal body components in Trypanosoma brucei.

The basal body in the human parasite Trypanosoma brucei is structurally equivalent to the centriole in animals and functions in the nucleation of axonemal microtubules in the flagellum. T. brucei lacks many evolutionarily conserved centriolar protein homologs and constructs the basal body through unknown mechanisms. Two evolutionarily conserved centriole/basal body cartwheel proteins, TbSAS-6 and TbBLD10, and a trypanosome-specific protein, BBP65, play essential roles in basal body biogenesis in T. brucei, but how they cooperate in the regulation of basal body assembly remains elusive. Here using RNAi, endogenous epitope tagging, immunofluorescence microscopy, and 3D-structured illumination super-resolution microscopy, we identified a new trypanosome-specific protein named BBP164 and found that it has an essential role in basal body biogenesis in T. brucei Further investigation of the functional interplay among BBP164 and the other three regulators of basal body assembly revealed that BBP164 and BBP65 are interdependent for maintaining their stability and depend on TbSAS-6 and TbBLD10 for their stabilization in the basal body. Additionally, TbSAS-6 and TbBLD10 are independent from each other and from BBP164 and BBP65 for maintaining their stability in the basal body. These findings demonstrate that basal body cartwheel proteins are required for stabilizing other basal body components and uncover that regulation of protein stability is an unusual control mechanism for assembly of the basal body in T. brucei.

Studying Centriole Duplication and Elongation in Human Cells.

Centrioles assemble centrosomes and cilia/flagella, which are microtubule-based structures with key roles in cell division, polarity, motility, and signaling. Centriole biogenesis is a tightly regulated process, and deregulation of centriole numbers and structure can have dramatic consequences for cellular function and integrity. However, their small size poses a challenge to study them. Here, we describe protocols that allow the identification and assessment of true centrioles and that provide straightforward strategies to study the role of new candidate proteins in centriole duplication and elongation.

The Evolution of Centriole Structure: Heterochrony, Neoteny, and Hypermorphosis.

Centrioles are subcellular organelles that were present in the last eukaryotic common ancestor, where the centriole's ancestral role was to form cilia. Centrioles have maintained a remarkably conserved structure in eukaryotes that have cilia, while groups that lack cilia have lost their centrioles, highlighting the structure-function relationship that exists between the centriole and the cilium. In contrast, animal sperm cells, a ciliated cell, exhibit remarkable structural diversity in the centriole. Understanding how this structural diversity evolved may provide insight into centriole assembly and function, as well as their unique role in sperm. Here, we apply concepts used in the study of the evolution of animal morphology to gain insight into the evolution of centriole structure. We propose that centrioles with an atypical structure form because of changes in the timing of centriole assembly events, which can be described as centriolar "heterochrony." Atypical centrioles of insects and mammals appear to have evolved through different types of heterochrony. Here, we discuss two particular types of heterochrony: neoteny and hypermorphosis. The centriole assembly of insect sperm cells exhibits the retention of "juvenile" centriole structure, which can be described as centriolar "neoteny." Mammalian sperm cells have an extended centriole assembly program through the addition of novel steps such as centrosome reduction and centriole remodeling to form atypical centrioles, a form of centriole "hypermorphosis." Overall, centriole heterochrony appears to be a common mechanism for the development of the atypical centriole during the evolution of centriole assembly of various animals' sperm.

The Cep57-pericentrin module organizes PCM expansion and centriole engagement.

Centriole duplication occurs once per cell cycle to ensure robust formation of bipolar spindles and chromosome segregation. Each newly-formed daughter centriole remains connected to its mother centriole until late mitosis. The disengagement of the centriole pair is required for centriole duplication. However, the mechanisms underlying centriole engagement remain poorly understood. Here, we show that Cep57 is required for pericentriolar material (PCM) organization that regulates centriole engagement. Depletion of Cep57 causes PCM disorganization and precocious centriole disengagement during mitosis. The disengaged daughter centrioles acquire ectopic microtubule-organizing-center activity, which results in chromosome mis-segregation. Similar defects are observed in mosaic variegated aneuploidy syndrome patient cells with cep57 mutations. We also find that Cep57 binds to the well-conserved PACT domain of pericentrin. Microcephaly osteodysplastic primordial dwarfism disease pericentrin mutations impair the Cep57-pericentrin interaction and lead to PCM disorganization. Together, our work demonstrates that Cep57 provides a critical interface between the centriole core and PCM.

Insights into centriole geometry revealed by cryotomography of doublet and triplet centrioles.

Centrioles are cylindrical assemblies comprised of 9 singlet, doublet, or triplet microtubules, essential for the formation of motile and sensory cilia. While the structure of the cilium is being defined at increasing resolution, centriolar structure remains poorly understood. Here, we used electron cryo-tomography to determine the structure of mammalian (triplet) and Drosophila (doublet) centrioles. Mammalian centrioles have two distinct domains: a 200 nm proximal core region connected by A-C linkers, and a distal domain where the C-tubule is incomplete and a pair of novel linkages stabilize the assembly producing a geometry more closely resembling the ciliary axoneme. Drosophila centrioles resemble the mammalian core, but with their doublet microtubules linked through the A tubules. The commonality of core-region length, and the abrupt transition in mammalian centrioles, suggests a conserved length-setting mechanism. The unexpected linker diversity suggests how unique centriolar architectures arise in different tissues and organisms.

High-speed photothermal off-resonance atomic force microscopy reveals assembly routes of centriolar scaffold protein SAS-6.

The self-assembly of protein complexes is at the core of many fundamental biological processes1, ranging from the polymerization of cytoskeletal elements, such as microtubules2, to viral capsid formation and organelle assembly3. To reach a comprehensive understanding of the underlying mechanisms of self-assembly, high spatial and temporal resolutions must be attained. This is complicated by the need to not interfere with the reaction during the measurement. As self-assemblies are often governed by weak interactions, they are especially difficult to monitor with high-speed atomic force microscopy (HS-AFM) due to the non-negligible tip-sample interaction forces involved in current methods. We have developed a HS-AFM technique, photothermal off-resonance tapping (PORT), which is gentle enough to monitor self-assembly reactions driven by weak interactions. We apply PORT to dissect the self-assembly reaction of SAS-6 proteins, which form a nine-fold radially symmetric ring-containing structure that seeds the formation of the centriole organelle. Our analysis reveals the kinetics of SAS-6 ring formation and demonstrates that distinct biogenesis routes can be followed to assemble a nine-fold symmetrical structure.

Reconstruction From Multiple Particles for 3D Isotropic Resolution in Fluorescence Microscopy.

The imaging of proteins within macromolecular complexes has been limited by the low axial resolution of optical microscopes. To overcome this problem, we propose a novel computational reconstruction method that yields isotropic resolution in fluorescence imaging. The guiding principle is to reconstruct a single volume from the observations of multiple rotated particles. Our new operational framework detects particles, estimates their orientation, and reconstructs the final volume. The main challenge comes from the absence of initial template and a priori knowledge about the orientations. We formulate the estimation as a blind inverse problem, and propose a block-coordinate stochastic approach to solve the associated non-convex optimization problem. The reconstruction is performed jointly in multiple channels. We demonstrate that our method is able to reconstruct volumes with 3D isotropic resolution on simulated data. We also perform isotropic reconstructions from real experimental data of doubly labeled purified human centrioles. Our approach revealed the precise localization of the centriolar protein Cep63 around the centriole microtubule barrel. Overall, our method offers new perspectives for applications in biology that require the isotropic mapping of proteins within macromolecular assemblies.

STED nanoscopy of the centrosome linker reveals a CEP68-organized, periodic rootletin network anchored to a C-Nap1 ring at centrioles.

The centrosome linker proteins C-Nap1, rootletin, and CEP68 connect the two centrosomes of a cell during interphase into one microtubule-organizing center. This coupling is important for cell migration, cilia formation, and timing of mitotic spindle formation. Very little is known about the structure of the centrosome linker. Here, we used stimulated emission depletion (STED) microscopy to show that each C-Nap1 ring at the proximal end of the two centrioles organizes a rootletin ring and, in addition, multiple rootletin/CEP68 fibers. Rootletin/CEP68 fibers originating from the two centrosomes form a web-like, interdigitating network, explaining the flexible nature of the centrosome linker. The rootletin/CEP68 filaments are repetitive and highly ordered. Staggered rootletin molecules (N-to-N and C-to-C) within the filaments are 75 nm apart. Rootletin binds CEP68 via its C-terminal spectrin repeat-containing region in 75-nm intervals. The N-to-C distance of two rootletin molecules is ∼35 to 40 nm, leading to an estimated minimal rootletin length of ∼110 nm. CEP68 is important in forming rootletin filaments that branch off centrioles and to modulate the thickness of rootletin fibers. Thus, the centrosome linker consists of a vast network of repeating rootletin units with C-Nap1 as ring organizer and CEP68 as filament modulator.

Ultrastructural diversity between centrioles of eukaryotes.

Several decades of centriole research have revealed the beautiful symmetry present in these microtubule-based organelles, which are required to form centrosomes, cilia and flagella in many eukaryotes. Centriole architecture is largely conserved across most organisms; however, individual centriolar features such as the central cartwheel or microtubule walls exhibit considerable variability when examined with finer resolution. In this paper, we review the ultrastructural characteristics of centrioles in commonly studied organisms, highlighting the subtle and not-so-subtle differences between specific structural components of these centrioles. In addition, we survey some non-canonical centriole structures that have been discovered in various species, from the coaxial bicentrioles of protists and lower land plants to the giant irregular centrioles of the fungus gnat Sciara. Finally, we speculate on the functional significance of these differences between centrioles, and the contribution of individual structural elements such as the cartwheel or microtubules towards the stability of centrioles.

Human microcephaly protein RTTN interacts with STIL and is required to build full-length centrioles.

Mutations in many centriolar protein-encoding genes cause primary microcephaly. Using super-resolution and electron microscopy, we find that the human microcephaly protein, RTTN, is recruited to the proximal end of the procentriole at early S phase, and is located at the inner luminal walls of centrioles. Further studies demonstrate that RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly. CRISPR/Cas9-mediated RTTN gene knockout in p53-deficient cells induce amplification of primitive procentriole bodies that lack the distal-half centriolar proteins, POC5 and POC1B. Additional analyses show that RTTN serves as an upstream effector of CEP295, which mediates the loading of POC1B and POC5 to the distal-half centrioles. Interestingly, the naturally occurring microcephaly-associated mutant, RTTN (A578P), shows a low affinity for STIL binding and blocks centriole assembly. These findings reveal that RTTN contributes to building full-length centrioles and illuminate the molecular mechanism through which the RTTN (A578P) mutation causes primary microcephaly.Mutations in many centriolar protein-encoding genes cause primary microcephaly. Here the authors show that human microcephaly protein RTTN directly interacts with STIL and acts downstream of STIL-mediated centriole assembly, contributing to building full-length centrioles.

Biophysical and Structural Characterization of the Centriolar Protein Cep104 Interaction Network.

Dysfunction of cilia is associated with common genetic disorders termed ciliopathies. Knowledge on the interaction networks of ciliary proteins is therefore key for understanding the processes that are underlying these severe diseases and the mechanisms of ciliogenesis in general. Cep104 has recently been identified as a key player in the regulation of cilia formation. Using a combination of sequence analysis, biophysics, and x-ray crystallography, we obtained new insights into the domain architecture and interaction network of the Cep104 protein. We solved the crystal structure of the tumor overexpressed gene (TOG) domain, identified Cep104 as a novel tubulin-binding protein, and biophysically characterized the interaction of Cep104 with CP110, Cep97, end-binding (EB) protein, and tubulin. Our results represent a solid platform for the further investigation of the microtubule-EB-Cep104-tubulin-CP110-Cep97 network of proteins. Ultimately, such studies should be of importance for understanding the process of cilia formation and the mechanisms underlying different ciliopathies.

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.

SAS-6 engineering reveals interdependence between cartwheel and microtubules in determining centriole architecture.

Centrioles are critical for the formation of centrosomes, cilia and flagella in eukaryotes. They are thought to assemble around a nine-fold symmetric cartwheel structure established by SAS-6 proteins. Here, we have engineered Chlamydomonas reinhardtii SAS-6-based oligomers with symmetries ranging from five- to ten-fold. Expression of a SAS-6 mutant that forms six-fold symmetric cartwheel structures in vitro resulted in cartwheels and centrioles with eight- or nine-fold symmetries in vivo. In combination with Bld10 mutants that weaken cartwheel-microtubule interactions, this SAS-6 mutant produced six- to eight-fold symmetric cartwheels. Concurrently, the microtubule wall maintained eight- and nine-fold symmetries. Expressing SAS-6 with analogous mutations in human cells resulted in nine-fold symmetric centrioles that exhibited impaired length and organization. Together, our data suggest that the self-assembly properties of SAS-6 instruct cartwheel symmetry, and lead us to propose a model in which the cartwheel and the microtubule wall assemble in an interdependent manner to establish the native architecture of centrioles.

The centriolar protein CPAP G-box: an amyloid fibril in a single domain.

Centrioles are evolutionarily conserved cylindrical cell organelles with characteristic radial symmetry. Despite their considerable size (400 nm × 200 nm, in humans), genetic studies suggest that relatively few protein components are involved in their assembly. We recently characterized the molecular architecture of the centrosomal P4.1-associated protein (CPAP), which is crucial for controlling the centriolar cylinder length. Here, we review the remarkable architecture of the C-terminal domain of CPAP, termed the G-box, which comprises a single, entirely solvent exposed, antiparallel ß-sheet. Molecular dynamics simulations support the stability of the G-box domain even in the face of truncations or amino acid substitutions. The similarity of the G-box domain to amyloids (or amyloid precursors) is strengthened by its oligomeric arrangement to form continuous fibrils. G-box fibrils were observed in crystals as well as in solution and are also supported by simulations. We conclude that the G-box domain may well represent the best analogue currently available for studies of exposed ß-sheets, unencumbered by additional structural elements or severe aggregations problems.