Towards understanding centriole elimination.

Centrioles are microtubule-based structures crucial for forming flagella, cilia and centrosomes. Through these roles, centrioles are critical notably for proper cell motility, signalling and division. Recent years have advanced significantly our understanding of the mechanisms governing centriole assembly and architecture. Although centrioles are typically very stable organelles, persisting over many cell cycles, they can also be eliminated in some cases. Here, we review instances of centriole elimination in a range of species and cell types. Moreover, we discuss potential mechanisms that enable the switch from a stable organelle to a vanishing one. Further work is expected to provide novel insights into centriole elimination mechanisms in health and disease, thereby also enabling scientists to readily manipulate organelle fate.

Centriole elimination during Caenorhabditis elegans oogenesis initiates with loss of the central tube protein SAS-1.

In most metazoans, centrioles are lost during oogenesis, ensuring that the zygote is endowed with the correct number of two centrioles, which are paternally contributed. How centriole architecture is dismantled during oogenesis is not understood. Here, we analyze with unprecedent detail the ultrastructural and molecular changes during oogenesis centriole elimination in Caenorhabditis elegans. Centriole elimination begins with loss of the so-called central tube and organelle widening, followed by microtubule disassembly. The resulting cluster of centriolar proteins then disappears gradually, usually moving in a microtubule- and dynein-dependent manner to the plasma membrane. Our analysis indicates that neither Polo-like kinases nor the PCM, which modulate oogenesis centriole elimination in Drosophila, do so in C. elegans. Furthermore, we demonstrate that the central tube protein SAS-1 normally departs initially from the organelle, which loses integrity earlier in sas-1 mutants. Overall, our work provides novel mechanistic insights regarding the fundamental process of oogenesis centriole elimination.

Chemical Probe for Imaging of Polo-like Kinase 4 and Centrioles.

Polo-like kinase (Plk4) is a serine/threonine-protein kinase that is essential for biogenesis of the centriole organelle and is enriched at centrioles. Herein, we introduce Cen-TCO, a chemical probe based on the Plk4 inhibitor centrinone, to image Plk4 and centrioles in live or fixed cultured human cells. Specifically, we established a bio-orthogonal two-step labeling system that enables the Cen-TCO-mediated imaging of Plk4 by STED super-resolution microscopy. Such direct labeling of Plk4 results in an increased resolution in STED imaging compared with using anti-Plk4 antibodies, underlining the importance of direct labeling strategies for super-resolution microscopy. We anticipate that Cen-TCO will become an important tool for investigating the biology of Plk4 and of centrioles.

Mapping of centriolar proteins onto the post-embryonic lineage of C. elegans.

Centrioles, together with the surrounding peri-centriolar material (PCM), constitute the centrosome, a major microtubule-organizing center of animal cells. Despite being critical in many cells for signaling, motility and division, centrioles can be eliminated in some systems, including in the vast majority of differentiating cells during embryogenesis in Caenorhabditis elegans. Whether the cells retaining centrioles in the resulting L1 larvae do so because they lack an activity that eliminates centrioles in the other cells is not known. Moreover, the extent to which centrioles and PCM remain present in later stages of worm development, when all cells but those of the germ line are terminally differentiated, is not known. Here, by fusing cells that lack centrioles with cells that retain them, we established that L1 larvae do not possess a diffusible elimination activity sufficient to remove centrioles. Moreover, analyzing PCM core proteins in L1 larval cells that retain centrioles, we found that some such proteins, but not all, are present as well. Furthermore, we uncovered that foci of centriolar proteins remain present in specific terminally differentiated cells of adult hermaphrodites and males, in particular in the somatic gonad. Correlating the time at which cells were born with the fate of their centrioles revealed that it is not cell age, but instead cell fate, that determines whether and when centrioles are eliminated. Overall, our work maps the localization of centriolar and PCM core proteins in the post-embryonic C. elegans lineage, thereby providing an essential blueprint for uncovering mechanisms modulating their presence and function.

Tubulin engineering by semi-synthesis reveals that polyglutamylation directs detyrosination.

Microtubules, a critical component of the cytoskeleton, carry post-translational modifications (PTMs) that are important for the regulation of key cellular processes. Long-lived microtubules, in neurons particularly, exhibit both detyrosination of α-tubulin and polyglutamylation. Dysregulation of these PTMs can result in developmental defects and neurodegeneration. Owing to a lack of tools to study the regulation and function of these PTMs, the mechanisms that govern such PTM patterns are not well understood. Here we produce fully functional tubulin carrying precisely defined PTMs within its C-terminal tail. We ligate synthetic α-tubulin tails-which are site-specifically glutamylated-to recombinant human tubulin heterodimers by applying a sortase- and intein-mediated tandem transamidation strategy. Using microtubules reconstituted with these designer tubulins, we find that α-tubulin polyglutamylation promotes its detyrosination by enhancing the activity of the tubulin tyrosine carboxypeptidase vasohibin/small vasohibin-binding protein in a manner dependent on the length of polyglutamyl chains. We also find that modulating polyglutamylation levels in cells results in corresponding changes in detyrosination, corroborating the link between the detyrosination cycle to polyglutamylation.

Extensive programmed centriole elimination unveiled in C. elegans embryos.

Centrioles are critical for fundamental cellular processes, including signaling, motility, and division. The extent to which centrioles are present after cell cycle exit in a developing organism is not known. The stereotypical lineage of Caenorhabditis elegans makes it uniquely well-suited to investigate this question. Using notably lattice light-sheet microscopy, correlative light electron microscopy, and lineage assignment, we found that ~88% of cells lose centrioles during embryogenesis. Our analysis reveals that centriole elimination is stereotyped, occurring invariably at a given time in a given cell type. Moreover, we established that experimentally altering cell fate results in corresponding changes in centriole fate. Overall, we uncovered the existence of an extensive centriole elimination program, which we anticipate to be paradigmatic for a broad understanding of centriole fate regulation.

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.

Sperm-contributed centrioles segregate stochastically into blastomeres of 4-cell stage Caenorhabditis elegans embryos.

Whereas both sperm and egg contribute nuclear genetic material to the zygote in metazoan organisms, the inheritance of other cellular constituents is unequal between the 2 gametes. Thus, 2 copies of the centriole are contributed solely by the sperm to the zygote in most species. Centrioles can have a stereotyped distribution in some asymmetric divisions, but whether sperm-contributed centrioles are distributed in a stereotyped manner in the resulting embryo is not known. Here, we address this question in Caenorhabditis elegans using marked mating experiments, whereby the presence of the 2 sperm-contributed centrioles is monitored in the embryo using the stable centriolar component SAS-4::GFP, as well as GFP::SAS-7. Our analysis demonstrates that the distribution of sperm-contributed centrioles is stochastic in 4-cell stage embryos. Moreover, using sperm from zyg-1 mutant males that harbor a single centriole, we show that the older sperm-contributed centriole is likewise distributed stochastically in the resulting embryo. Overall, we conclude that, in contrast to the situation during some asymmetric cell divisions, centrioles contributed by the male germ line are distributed stochastically in embryos of C. elegans.

CenFind: a deep-learning pipeline for efficient centriole detection in microscopy datasets.

BACKGROUND: High-throughput and selective detection of organelles in immunofluorescence images is an important but demanding task in cell biology. The centriole organelle is critical for fundamental cellular processes, and its accurate detection is key for analysing centriole function in health and disease. Centriole detection in human tissue culture cells has been achieved typically by manual determination of organelle number per cell. However, manual cell scoring of centrioles has a low throughput and is not reproducible. Published semi-automated methods tally the centrosome surrounding centrioles and not centrioles themselves. Furthermore, such methods rely on hard-coded parameters or require a multichannel input for cross-correlation. Therefore, there is a need for developing an efficient and versatile pipeline for the automatic detection of centrioles in single channel immunofluorescence datasets. RESULTS: We developed a deep-learning pipeline termed CenFind that automatically scores cells for centriole numbers in immunofluorescence images of human cells. CenFind relies on the multi-scale convolution neural network SpotNet, which allows the accurate detection of sparse and minute foci in high resolution images. We built a dataset using different experimental settings and used it to train the model and evaluate existing detection methods. The resulting average F1-score achieved by CenFind is > 90% across the test set, demonstrating the robustness of the pipeline. Moreover, using the StarDist-based nucleus detector, we link the centrioles and procentrioles detected with CenFind to the cell containing them, overall enabling automatic scoring of centriole numbers per cell. CONCLUSIONS: Efficient, accurate, channel-intrinsic and reproducible detection of centrioles is an important unmet need in the field. Existing methods are either not discriminative enough or focus on a fixed multi-channel input. To fill this methodological gap, we developed CenFind, a command line interface pipeline that automates cell scoring of centrioles, thereby enabling channel-intrinsic, accurate and reproducible detection across experimental modalities. Moreover, the modular nature of CenFind enables its integration in other pipelines. Overall, we anticipate CenFind to prove critical for accelerating discoveries in the field.

Molecular architecture of the C. elegans centriole.

Uncovering organizing principles of organelle assembly is a fundamental pursuit in the life sciences. Caenorhabditis elegans was key in identifying evolutionary conserved components governing assembly of the centriole organelle. However, localizing these components with high precision has been hampered by the minute size of the worm centriole, thus impeding understanding of underlying assembly mechanisms. Here, we used Ultrastructure Expansion coupled with STimulated Emission Depletion (U-Ex-STED) microscopy, as well as electron microscopy (EM) and electron tomography (ET), to decipher the molecular architecture of the worm centriole. Achieving an effective lateral resolution of approximately 14 nm, we localize centriolar and PeriCentriolar Material (PCM) components in a comprehensive manner with utmost spatial precision. We found that all 12 components analysed exhibit a ring-like distribution with distinct diameters and often with a 9-fold radial symmetry. Moreover, we uncovered that the procentriole assembles at a location on the centriole margin where SPD-2 and ZYG-1 also accumulate. Moreover, SAS-6 and SAS-5 were found to be present in the nascent procentriole, with SAS-4 and microtubules recruited thereafter. We registered U-Ex-STED and EM data using the radial array of microtubules, thus allowing us to map each centriolar and PCM protein to a specific ultrastructural compartment. Importantly, we discovered that SAS-6 and SAS-4 exhibit a radial symmetry that is offset relative to microtubules, leading to a chiral centriole ensemble. Furthermore, we established that the centriole is surrounded by a region from which ribosomes are excluded and to which SAS-7 localizes. Overall, our work uncovers the molecular architecture of the C. elegans centriole in unprecedented detail and establishes a comprehensive framework for understanding mechanisms of organelle biogenesis and function.

Atypical and distinct microtubule radial symmetries in the centriole and the axoneme of Lecudina tuzetae.

The centriole is a minute cylindrical organelle present in a wide range of eukaryotic species. Most centrioles have a signature ninefold radial symmetry of microtubules that is imparted to the axonemes of the cilia and flagella they template, with nine centriolar microtubule doublets growing into nine axonemal microtubule doublets. There are exceptions to the ninefold symmetrical arrangement of axonemal microtubules in some species, with lower or higher fold symmetries. In the few cases where this has been examined, such alterations in axonemal symmetries are grounded in similar alterations in centriolar symmetries. Here, we examine the question of microtubule number continuity between centriole and axoneme in flagellated gametes of the gregarine Lecudina tuzetae, which have been reported to exhibit a sixfold radial symmetry of axonemal microtubules. We used time-lapse differential interference microscopy to identify the stage at which flagellated gametes are present. Thereafter, using electron microscopy and ultrastructure-expansion microscopy coupled to stimulated emission depletion superresolution imaging, we uncover that a six- or fivefold radial symmetry in the axoneme is accompanied by an eightfold radial symmetry in the centriole. We conclude that the transition between centriolar and axonemal microtubules can be characterized by unexpected plasticity.

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.

TRIM37: a critical orchestrator of centrosome function.

Loss of function mutations in the E3 ubiquitin ligase TRIM37 result in MULIBREY nanism, a disease characterized by impaired organ growth and a high propensity to develop different tumor types. Additionally, increased copy number of TRIM37 is a feature of some breast cancers and neuroblastomas. The molecular role played by TRIM37 in such loss and gain of function conditions has been a focus of research in the last decade, which led notably to the identification of critical roles of TRIM37 in centrosome biology. Specifically, deletion of TRIM37 results in the formation of aberrant centrosomal proteins assemblies, including Centrobin-PLK4 assemblies, which can act as extra MTOCs, thus resulting in defective chromosome segregation. Additionally, TRIM37 overexpression targets the centrosomal protein CEP192 for degradation, thereby preventing centrosome maturation and increasing the frequency of mitotic errors. Interestingly, increased TRIM37 protein levels sensitize cells to the PLK4 inhibitor centrinone. In this review, we cover the emerging roles of TRIM37 in centrosome biology and discuss how this knowledge may lead to new therapeutic strategies to target specific cancer cells.

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.

Pulchelloid A, a sesquiterpene lactone from the Canadian prairie plant Gaillardia aristata inhibits mitosis in human cells.

BACKGROUND: The Canadian prairie ecosystem presents a rich source of natural products from plants that are subjected to herbivory by grazing mammals. This type of ecological competition may contribute to the production of natural products of interest in cell biology and medical research. We provide the first biological description of the sesquiterpene lactone, pulchelloid A, which we isolated from the prairie plant, Gaillardia aristata (Asteraceae) and report that it inhibits mitosis in human cells. METHODS AND RESULTS: We found that G. aristata (Blanket flower) extracts were cytotoxic to human cell lines and used phenotypic assays to characterize the bioactivity of extracts. Before dying, cells were characterized by a rounded morphology, phospho-histone H3 signals, mitotic spindles, and active Cdk1. By biology-guided fractionation of Gaillardia extracts, we isolated a sesquiterpene lactone named pulchelloid A. We used immunofluorescence microscopy and observed that cells treated with pulchelloid A have phospho-histone H3 positive chromosomes and a mitotic spindle, confirming that they were in mitosis. Treated cells arrest with an unusual phenotype; they enter a prolonged mitotic arrest in which the spindles become multipolar and the chromosomes acquire histone γH2AX foci, a hallmark of damaged DNA. CONCLUSIONS: We propose that pulchelloid A, a natural product present in the prairie plant Gaillardia aristata, delays cells in mitosis. There is a growing body of evidence that a small number of members of the sesquiterpene lactone chemical family may target proteins that regulate mitosis.

Tuning SAS-6 architecture with monobodies impairs distinct steps of centriole assembly.

Centrioles are evolutionarily conserved multi-protein organelles essential for forming cilia and centrosomes. Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. The importance of this architecture has been difficult to decipher notably because of the lack of precise tools to modulate the underlying assembly reaction. Here, we developed monobodies against Chlamydomonas reinhardtii SAS-6, characterizing three in detail with X-ray crystallography, atomic force microscopy and cryo-electron microscopy. This revealed distinct monobody-target interaction modes, as well as specific consequences on ring assembly and stacking. Of particular interest, monobody MBCRS6-15 induces a conformational change in CrSAS-6, resulting in the formation of a helix instead of a ring. Furthermore, we show that this alteration impairs centriole biogenesis in human cells. Overall, our findings identify monobodies as powerful molecular levers to alter the architecture of multi-protein complexes and tune centriole assembly.

Physically asymmetric division of the C. elegans zygote ensures invariably successful embryogenesis.

Asymmetric divisions that yield daughter cells of different sizes are frequent during early embryogenesis, but the importance of such a physical difference for successful development remains poorly understood. Here, we investigated this question using the first division of Caenorhabditis elegans embryos, which yields a large AB cell and a small P1 cell. We equalized AB and P1 sizes using acute genetic inactivation or optogenetic manipulation of the spindle positioning protein LIN-5. We uncovered that only some embryos tolerated equalization, and that there was a size asymmetry threshold for viability. Cell lineage analysis of equalized embryos revealed an array of defects, including faster cell cycle progression in P1 descendants, as well as defects in cell positioning, division orientation, and cell fate. Moreover, equalized embryos were more susceptible to external compression. Overall, we conclude that unequal first cleavage is essential for invariably successful embryonic development of C. elegans.

TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin.

TRIM37 is an E3 ubiquitin ligase mutated in Mulibrey nanism, a disease with impaired organ growth and increased tumor formation. TRIM37 depletion from tissue culture cells results in supernumerary foci bearing the centriolar protein Centrin. Here, we characterize these centriolar protein assemblies (Cenpas) to uncover the mechanism of action of TRIM37. We find that an atypical de novo assembly pathway can generate Cenpas that act as microtubule-organizing centers (MTOCs), including in Mulibrey patient cells. Correlative light electron microscopy reveals that Cenpas are centriole-related or electron-dense structures with stripes. TRIM37 regulates the stability and solubility of Centrobin, which accumulates in elongated entities resembling the striped electron dense structures upon TRIM37 depletion. Furthermore, Cenpas formation upon TRIM37 depletion requires PLK4, as well as two parallel pathways relying respectively on Centrobin and PLK1. Overall, our work uncovers how TRIM37 prevents Cenpas formation, which would otherwise threaten genome integrity.

Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography.

Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report ~25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms.