Publications by authors named "Pierre Gönczy"

111 Publications

Physically asymmetric division of the zygote ensures invariably successful embryogenesis.

Elife 2021 Feb 23;10. Epub 2021 Feb 23.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.

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 embryos, which yields a large AB cell and a small P cell. We equalized AB and P 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 P 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 .
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http://dx.doi.org/10.7554/eLife.61714DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7972452PMC
February 2021

TRIM37 prevents formation of centriolar protein assemblies by regulating Centrobin.

Elife 2021 Jan 25;10. Epub 2021 Jan 25.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.

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.
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http://dx.doi.org/10.7554/eLife.62640DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870141PMC
January 2021

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

EMBO J 2020 11 20;39(22):e106249. Epub 2020 Sep 20.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.

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.
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http://dx.doi.org/10.15252/embj.2020106249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667878PMC
November 2020

Homogeneous multifocal excitation for high-throughput super-resolution imaging.

Nat Methods 2020 07 22;17(7):726-733. Epub 2020 Jun 22.

Laboratory for Experimental Biophysics, Institute of Physics, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.

Super-resolution microscopies have become an established tool in biological research. However, imaging throughput remains a main bottleneck in acquiring large datasets required for quantitative biology. Here we describe multifocal flat illumination for field-independent imaging (mfFIFI). By integrating mfFIFI into an instant structured illumination microscope (iSIM), we extend the field of view (FOV) to >100 × 100 µm while maintaining high-speed, multicolor, volumetric imaging at double the diffraction-limited resolution. We further extend the effective FOV by stitching adjacent images for fast live-cell super-resolution imaging of dozens of cells. Finally, we combine our flat-fielded iSIM with ultrastructure expansion microscopy to collect three-dimensional (3D) images of hundreds of centrioles in human cells, or thousands of purified Chlamydomonas reinhardtii centrioles, per hour at an effective resolution of ~35 nm. Classification and particle averaging of these large datasets enables 3D mapping of posttranslational modifications of centriolar microtubules, revealing differences in their coverage and positioning.
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http://dx.doi.org/10.1038/s41592-020-0859-zDOI Listing
July 2020

Centriole foci persist in starfish oocytes despite Polo-like kinase 1 inactivation or loss of microtubule nucleation activity.

Mol Biol Cell 2020 04 19;31(9):873-880. Epub 2020 Feb 19.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

Centrioles must be eliminated or inactivated from the oocyte to ensure that only the two functional centrioles contributed by the sperm are present in the zygote. Such removal can occur during oogenesis, as in , where departure of Polo kinase from centrosomes leads to loss of microtubule nucleating activity and centriole removal. In other species, oocyte-derived centrioles are removed around the time of fertilization through incompletely understood mechanisms. Here, we use confocal imaging of live starfish oocytes and zygotes expressing markers of microtubule nucleating activity and centrioles to investigate this question. We first assay the role of Polo-like kinase 1 (Plk1) in centriole elimination. We find that although Plk1 localizes around oocyte-derived centrioles, kinase impairment with BI-2536 does not protect centrioles from removal in the bat star . Moreover, we uncover that all four oocyte-derived centrioles lose microtubule nucleating activity when retained experimentally in the zygote of the radiate star . Interestingly, two such centrioles nevertheless retain the centriolar markers mEGFP::PACT and pmPoc1::mEGFP. Together, these findings indicate that centrioles can persist when Plk1 activity is impaired, as well as when microtubule nucleating activity is lacking, uncovering further diversity in the mechanisms governing centriole removal.
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http://dx.doi.org/10.1091/mbc.E19-06-0346DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7185973PMC
April 2020

Live imaging screen reveals that TYRO3 and GAK ensure accurate spindle positioning in human cells.

Nat Commun 2019 06 28;10(1):2859. Epub 2019 Jun 28.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015, Lausanne, Switzerland.

Proper spindle positioning is crucial for spatial cell division control. Spindle positioning in human cells relies on a ternary complex comprising Gαi1-3, LGN and NuMA, which anchors dynein at the cell cortex, thus enabling pulling forces to be exerted on astral microtubules. We develop a live imaging siRNA-based screen using stereotyped fibronectin micropatterns to uncover components modulating spindle positioning in human cells, testing 1280 genes, including all kinases and phosphatases. We thus discover 16 components whose inactivation dramatically perturbs spindle positioning, including tyrosine receptor kinase 3 (TYRO3) and cyclin G associated kinase (GAK). TYRO3 depletion results in excess NuMA and dynein at the cortex during metaphase, similar to the effect of blocking the TYRO3 downstream target phosphatidylinositol 3-kinase (PI3K). Furthermore, depletion of GAK leads to impaired astral microtubules, similar to the effect of downregulating the GAK-interactor Clathrin. Overall, our work uncovers components and mechanisms governing spindle positioning in human cells.
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http://dx.doi.org/10.1038/s41467-019-10446-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6599018PMC
June 2019

Aurora A depletion reveals centrosome-independent polarization mechanism in .

Elife 2019 02 26;8. Epub 2019 Feb 26.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.

How living systems break symmetry in an organized manner is a fundamental question in biology. In wild-type zygotes, symmetry breaking during anterior-posterior axis specification is guided by centrosomes, resulting in anterior-directed cortical flows and a single posterior PAR-2 domain. We uncover that zygotes depleted of the Aurora A kinase AIR-1 or lacking centrosomes entirely usually establish two posterior PAR-2 domains, one at each pole. We demonstrate that AIR-1 prevents symmetry breaking early in the cell cycle, whereas centrosomal AIR-1 instructs polarity initiation thereafter. Using triangular microfabricated chambers, we establish that bipolarity of () embryos occurs effectively in a cell-shape and curvature-dependent manner. Furthermore, we develop an integrated physical description of symmetry breaking, wherein local PAR-2-dependent weakening of the actin cortex, together with mutual inhibition of anterior and posterior PAR proteins, provides a mechanism for spontaneous symmetry breaking without centrosomes.
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http://dx.doi.org/10.7554/eLife.44552DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417861PMC
February 2019

Centriole assembly at a glance.

J Cell Sci 2019 02 20;132(4). Epub 2019 Feb 20.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland.

The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle - from assembly to disappearance - focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form or are eliminated.
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http://dx.doi.org/10.1242/jcs.228833DOI Listing
February 2019

Tissue- and sex-specific small RNAomes reveal sex differences in response to the environment.

PLoS Genet 2019 02 8;15(2):e1007905. Epub 2019 Feb 8.

Department of Ecology and Evolution, University of Lausanne, Lausanne, Switzerland.

RNA interference (RNAi) related pathways are essential for germline development and fertility in metazoa and can contribute to inter- and trans-generational inheritance. In the nematode Caenorhabditis elegans, environmental double-stranded RNA provided by feeding can lead to heritable changes in phenotype and gene expression. Notably, transmission efficiency differs between the male and female germline, yet the underlying mechanisms remain elusive. Here we use high-throughput sequencing of dissected gonads to quantify sex-specific endogenous piRNAs, miRNAs and siRNAs in the C. elegans germline and the somatic gonad. We identify genes with exceptionally high levels of secondary 22G RNAs that are associated with low mRNA expression, a signature compatible with silencing. We further demonstrate that contrary to the hermaphrodite germline, the male germline, but not male soma, is resistant to environmental RNAi triggers provided by feeding, in line with previous work. This sex-difference in silencing efficacy is associated with lower levels of gonadal RNAi amplification products. Moreover, this tissue- and sex-specific RNAi resistance is regulated by the germline, since mutant males with a feminized germline are RNAi sensitive. This study provides important sex- and tissue-specific expression data of miRNA, piRNA and siRNA as well as mechanistic insights into sex-differences of gene regulation in response to environmental cues.
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http://dx.doi.org/10.1371/journal.pgen.1007905DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383947PMC
February 2019

Multicolor single-particle reconstruction of protein complexes.

Nat Methods 2018 10 1;15(10):777-780. Epub 2018 Oct 1.

Laboratory for Experimental Biophysics, Institute of Physics, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.

Single-particle reconstruction (SPR) from electron microscopy (EM) images is widely used in structural biology, but it lacks direct information on protein identity. To address this limitation, we developed a computational and analytical framework that reconstructs and coaligns multiple proteins from 2D super-resolution fluorescence images. To demonstrate our method, we generated multicolor 3D reconstructions of several proteins within the human centriole, which revealed their relative locations, dimensions and orientations.
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http://dx.doi.org/10.1038/s41592-018-0140-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6173288PMC
October 2018

Integrated Microfluidic Device for Drug Studies of Early Embryogenesis.

Adv Sci (Weinh) 2018 May 8;5(5):1700751. Epub 2018 Mar 8.

Laboratory of Microsystems Ecole Polytechnique Fédérale de Lausanne (EPFL) Lausanne 1015 Switzerland.

Small molecules inhibitors are powerful tools for studying multiple aspects of cell biology and stand at the forefront of drug discovery pipelines. However, in the early () embryo, which is a powerful model system for cell and developmental biology, the use of small molecule inhibitors has been limited by the impermeability of the embryonic eggshell, the low-throughput manual embryo isolation methods, and the lack of well-controlled drug delivery protocols. This work reports a fully integrated microfluidic approach for studies of early embryogenesis, including the possibility of testing small molecule inhibitors with increased throughput and versatility. The setup enables robust on-chip extraction of embryos from gravid adult worms in a dedicated pillar array chamber by mechanical compression, followed by rapid fluidic transfer of embryos into an adjacent microtrap array. Parallel analysis of ≈100 embryos by high-resolution time-lapse imaging from the one-cell stage zygote until hatching can be performed with this device. The implementation of versatile microfluidic protocols, in particular time-controlled and reversible drug delivery to on-chip immobilized embryos, demonstrates the potential of the device for biochemical and pharmacological assays.
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http://dx.doi.org/10.1002/advs.201700751DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980161PMC
May 2018

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

Nat Nanotechnol 2018 08 21;13(8):696-701. Epub 2018 May 21.

Laboratory for Bio- and Nano-Instrumentation, Swiss Federal Institute of Technology Lausanne (EPFL), Lausanne, Switzerland.

The self-assembly of protein complexes is at the core of many fundamental biological processes, ranging from the polymerization of cytoskeletal elements, such as microtubules, to viral capsid formation and organelle assembly. 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.
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http://dx.doi.org/10.1038/s41565-018-0149-4DOI Listing
August 2018

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

IEEE Trans Med Imaging 2018 05;37(5):1235-1246

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.
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http://dx.doi.org/10.1109/TMI.2018.2795464DOI Listing
May 2018

PI(4,5)P forms dynamic cortical structures and directs actin distribution as well as polarity in embryos.

Development 2018 05 30;145(11). Epub 2018 May 30.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland

Asymmetric division is crucial for embryonic development and stem cell lineages. In the one-cell embryo, a contractile cortical actomyosin network contributes to asymmetric division by segregating partitioning-defective (PAR) proteins to discrete cortical domains. In the current study, we found that the plasma membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP) localizes to polarized dynamic structures in zygotes, distributing in a PAR-dependent manner along the anterior-posterior (A-P) embryonic axis. PIP cortical structures overlap with F-actin, and coincide with the actin regulators RHO-1 and CDC-42, as well as ECT-2. Particle image velocimetry analysis revealed that PIP and F-actin cortical movements are coupled, with PIP structures moving slightly ahead of F-actin. Importantly, we established that PIP cortical structure formation and movement is actin dependent. Moreover, we found that decreasing or increasing the level of PIP resulted in severe F-actin disorganization, revealing interdependence between these components. Furthermore, we determined that PIP and F-actin regulate the sizing of PAR cortical domains, including during the maintenance phase of polarization. Overall, our work establishes that a lipid membrane component, PIP, modulates actin organization and cell polarity in embryos.
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http://dx.doi.org/10.1242/dev.164988DOI Listing
May 2018

The Rise of the Cartwheel: Seeding the Centriole Organelle.

Bioessays 2018 04 6;40(4):e1700241. Epub 2018 Mar 6.

School of Life Sciences, Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology (EPFL) Lausanne, Switzerland.

The cartwheel is a striking structure critical for building the centriole, a microtubule-based organelle fundamental for organizing centrosomes, cilia, and flagella. Over the last 50 years, the cartwheel has been described in many systems using electron microscopy, but the molecular nature of its constituent building blocks and their assembly mechanisms have long remained mysterious. Here, we review discoveries that led to the current understanding of cartwheel structure, assembly, and function. We focus on the key role of SAS-6 protein self-organization, both for building the signature ring-like structure with hub and spokes, as well as for their vertical stacking. The resemblance of assembly intermediates in vitro and in vivo leads us to propose a novel registration step in cartwheel biogenesis, whereby stacked SAS-6-containing rings are put in register through interactions with peripheral elements anchored to microtubules. We conclude by evoking some avenues for further uncovering cartwheel and centriole assembly mechanisms.
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http://dx.doi.org/10.1002/bies.201700241DOI Listing
April 2018

Interaction between the centriolar protein SAS-5 and microtubules facilitates organelle assembly.

Mol Biol Cell 2018 03 24;29(6):722-735. Epub 2018 Jan 24.

Department of Biochemistry, University of Oxford, Oxford OX1 3QU, United Kingdom

Centrioles are microtubule-based organelles that organize the microtubule network and seed the formation of cilia and flagella. New centrioles assemble through a stepwise process dependent notably on the centriolar protein SAS-5 in SAS-5 and its functional homologues in other species form oligomers that bind the centriolar proteins SAS-6 and SAS-4, thereby forming an evolutionarily conserved structural core at the onset of organelle assembly. Here, we report a novel interaction of SAS-5 with microtubules. Microtubule binding requires SAS-5 oligomerization and a disordered protein segment that overlaps with the SAS-4 binding site. Combined in vitro and in vivo analysis of select mutants reveals that the SAS-5-microtubule interaction facilitates centriole assembly in embryos. Our findings lead us to propose that the interdependence of SAS-5 oligomerization and microtubule binding reflects an avidity mechanism, which also strengthens SAS-5 associations with other centriole components and, thus, promotes organelle assembly.
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http://dx.doi.org/10.1091/mbc.E17-06-0412DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6003225PMC
March 2018

ZYG-1 promotes limited centriole amplification in the C. elegans seam lineage.

Dev Biol 2018 02 4;434(2):221-230. Epub 2018 Jan 4.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland. Electronic address:

Genome stability relies notably on the integrity of centrosomes and on the mitotic spindle they organize. Structural and numerical centrosome aberrations are frequently observed in human cancer, and there is increasing evidence that centrosome amplification can promote tumorigenesis. Here, we use C. elegans seam cells as a model system to analyze centrosome homeostasis in the context of a stereotyped stem like lineage. We found that overexpression of the Plk4-related kinase ZYG-1 leads to the formation of one supernumerary centriolar focus per parental centriole during the cell cycle that leads to the sole symmetric division in the seam lineage. In the following cell cycle, such supernumerary foci function as microtubule organizing centers, but do not cluster during mitosis, resulting in the formation of a multipolar spindle and then aneuploid daughter cells. Intriguingly, we found also that supernumerary centriolar foci do not assemble in the asymmetric cell divisions that precedes or that follows the symmetric seam cell division, despite the similar presence of GFP::ZYG-1. Furthermore, we established that supernumerary centrioles form earlier during development in animals depleted of the heterochronic gene lin-14, in which the symmetric division is precocious. Conversely, supernumerary centrioles are essentially not observed in animals depleted of lin-28, in which the symmetric division is lacking. These findings lead us to conclude that ZYG-1 promotes limited centriole amplification solely during the symmetric division in the C. elegans seam lineage.
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http://dx.doi.org/10.1016/j.ydbio.2018.01.001DOI Listing
February 2018

Centriole Biogenesis: From Identifying the Characters to Understanding the Plot.

Annu Rev Cell Dev Biol 2017 10 16;33:23-49. Epub 2017 Aug 16.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), CH-1015, Lausanne, Switzerland; email:

The centriole is a beautiful microtubule-based organelle that is critical for the proper execution of many fundamental cellular processes, including polarity, motility, and division. Centriole biogenesis, the making of this miniature architectural wonder, has emerged as an exemplary model to dissect the mechanisms governing the assembly of a eukaryotic organelle. Centriole biogenesis relies on a set of core proteins whose contributions to the assembly process have begun to be elucidated. Here, we review current knowledge regarding the mechanisms by which these core characters function in an orderly fashion to assemble the centriole. In particular, we discuss how having the correct proteins at the right place and at the right time is critical to first scaffold, then initiate, and finally execute the centriole assembly process, thus underscoring fundamental principles governing organelle biogenesis.
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http://dx.doi.org/10.1146/annurev-cellbio-100616-060454DOI Listing
October 2017

Identification of Chlamydomonas Central Core Centriolar Proteins Reveals a Role for Human WDR90 in Ciliogenesis.

Curr Biol 2017 Aug 3;27(16):2486-2498.e6. Epub 2017 Aug 3.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne 1015, Switzerland. Electronic address:

Centrioles are evolutionarily conserved macromolecular structures that are fundamental to form cilia, flagella, and centrosomes. Centrioles are 9-fold symmetrical microtubule-based cylindrical barrels comprising three regions that can be clearly distinguished in the Chlamydomonas reinhardtii organelle: an ∼100-nm-long proximal region harboring a cartwheel; an ∼250-nm-long central core region containing a Y-shaped linker; and an ∼150-nm-long distal region ending at the transitional plate. Despite the discovery of many centriolar components, no protein has been localized specifically to the central core region in Chlamydomonas thus far. Here, combining relative quantitative mass spectrometry and super-resolution microscopy on purified Chlamydomonas centrioles, we identified POB15 and POC16 as two proteins of the central core region, the distribution of which correlates with that of tubulin glutamylation. We demonstrated that POB15 is an inner barrel protein within this region. Moreover, we developed an assay to uncover temporal relationships between centriolar proteins during organelle assembly and thus established that POB15 is recruited after the cartwheel protein CrSAS-6 and before tubulin glutamylation takes place. Furthermore, we discovered that two poc16 mutants exhibit flagellar defects, indicating that POC16 is important for flagellum biogenesis. In addition, we discovered that WDR90, the human homolog of POC16, localizes to a region of human centrioles that we propose is analogous to the central core of Chlamydomonas centrioles. Moreover, we demonstrate that WDR90 is required for ciliogenesis, echoing the findings in Chlamydomonas. Overall, our work provides novel insights into the identity and function of centriolar central core components.
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http://dx.doi.org/10.1016/j.cub.2017.07.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399476PMC
August 2017

TRACMIT: An effective pipeline for tracking and analyzing cells on micropatterns through mitosis.

PLoS One 2017 26;12(7):e0179752. Epub 2017 Jul 26.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.

The use of micropatterns has transformed investigations of dynamic biological processes by enabling the reproducible analysis of live cells using time-lapse fluorescence microscopy. With micropatterns, thousands of individual cells can be efficiently imaged in parallel, rendering the approach well suited for screening projects. Despite being powerful, such screens remain challenging in terms of data handling and analysis. Typically, only a fraction of micropatterns is occupied in a manner suitable to monitor a given phenotypic output. Moreover, the presence of dying or otherwise compromised cells complicates the analysis. Therefore, focusing strictly on relevant cells in such large time-lapse microscopy dataset poses interesting analysis challenges that are not readily met by existing software packages. This motivated us to develop an image analysis pipeline that handles all necessary image processing steps within one open-source platform to detect and analyze individual cells seeded on micropatterns through mitosis. We introduce a comprehensive image analysis pipeline running on Fiji termed TRACMIT (pipeline for TRACking and analyzing cells on micropatterns through MITosis). TRACMIT was developed to rapidly and accurately assess the orientation of the mitotic spindle during metaphase in time-lapse fluorescence microscopy of human cells expressing mCherry::histone 2B and plated on L-shaped micropatterns. This solution enables one to perform the entire analysis from the raw data, avoiding the need to save intermediate images, thereby decreasing data volume and thus reducing the data that needs to be processed. We first select micropatterns containing a single cell and then identify anaphase figures in the time-lapse recording. Next, TRACMIT tracks back in time until metaphase, when the angle of the mitotic spindle with respect to the micropattern is assessed. We designed the pipeline to allow for manual validation of selected cells with a simple user interface, and to enable analysis of cells plated on micropatterns of different shapes. For ease of use, the entire pipeline is provided as a series of Fiji/ImageJ macros, grouped into an ActionBar. In conclusion, the open source TRACMIT pipeline enables high-throughput analysis of single mitotic cells on micropatterns, thus accurately and efficiently allowing automatic determination of spindle positioning from time-lapse recordings.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0179752PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5528263PMC
October 2017

Computer simulations reveal mechanisms that organize nuclear dynein forces to separate centrosomes.

Mol Biol Cell 2017 Nov 12;28(23):3165-3170. Epub 2017 Jul 12.

Swiss Institute for Experimental Cancer Research, School of Life Sciences, Swiss Federal Institute of Technology, 1015 Lausanne, Switzerland

Centrosome separation along the surface of the nucleus at the onset of mitosis is critical for bipolar spindle assembly. Dynein anchored on the nuclear envelope is known to be important for centrosome separation, but it is unclear how nuclear dynein forces are organized in an anisotropic manner to promote the movement of centrosomes away from each other. Here we use computational simulations of embryos to address this fundamental question, testing three potential mechanisms by which nuclear dynein may act. First, our analysis shows that expansion of the nuclear volume per se does not generate nuclear dynein-driven separation forces. Second, we find that steric interactions between microtubules and centrosomes contribute to robust onset of nuclear dynein-mediated centrosome separation. Third, we find that the initial position of centrosomes, between the male pronucleus and cell cortex at the embryo posterior, is a key determinant in organizing microtubule aster asymmetry to power nuclear dynein-dependent separation. Overall our work reveals that accurate initial centrosome position, together with steric interactions, ensures proper anisotropic organization of nuclear dynein forces to separate centrosomes, thus ensuring robust bipolar spindle assembly.
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http://dx.doi.org/10.1091/mbc.E16-12-0823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5687019PMC
November 2017

Zika virus causes supernumerary foci with centriolar proteins and impaired spindle positioning.

Open Biol 2017 01;7(1)

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology Lausanne (EPFL), 1015, Lausanne, Switzerland

Zika virus (ZIKV) causes congenital microcephaly. Although ZIKV can impair cell cycle progression and provoke apoptosis, which probably contributes to disease aetiology through depletion of neural progenitor cells, additional cellular mechanisms may be important. Here, we investigated whether ZIKV infection alters centrosome number and spindle positioning, because such defects are thought to be at the root of inherited primary autosomal recessive microcephaly (MCPH). In addition to HeLa cells, in which centrosome number and spindle positioning can be well monitored, we analysed retinal epithelial cells (RPE-1), as well as brain-derived microglial (CHME-5) and neural progenitor (ReN) cells, using immunofluorescence. We established that ZIKV infection leads to supernumerary foci containing centriolar proteins that in some cases drive multipolar spindle assembly, as well as spindle positioning defects in HeLa, RPE-1 and CHME-5 cells, but not in ReN cells. We uncovered similar phenotypes in HeLa cells upon infection with dengue virus (DENV-2), another flavivirus that does not target brain cells and does not cause microcephaly. We conclude that infection with Flaviviridae can increase centrosome numbers and impair spindle positioning, thus potentially contributing to microcephaly in the case of Zika.
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http://dx.doi.org/10.1098/rsob.160231DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5303270PMC
January 2017

Chemical Genetic Screen Identifies Natural Products that Modulate Centriole Number.

Chembiochem 2016 11 23;17(21):2063-2074. Epub 2016 Sep 23.

Swiss Institute for Experimental Cancer Research (ISREC), Swiss Federal Institute of Technology Lausanne (EPFL), 1015, Lausanne, Switzerland.

Centrioles are microtubule-based organelles found in most eukaryotic cells and that are critical for the formation of cilia and flagella, as well as of centrosomes in animal cells. The number of centrioles must be strictly regulated in proliferating cells in order to ensure genome integrity upon cell division. Despite their importance, however, the mechanisms governing centriole assembly and number control remain incompletely understood, owing in part to a paucity of available small-molecule compounds for dissection and alteration of the underlying processes. Here we have developed a chemical genetic approach to identify small-molecule compounds capable of modulating centriole numbers in human cells. High-throughput screening of ≈2600 natural compounds identified 14 candidate molecules that either diminish (ten compounds) or augment (four compounds) the number of centrioles per cell. We investigated the mechanisms of action of four of these compounds and discovered that two of them potentially reduce centriole number through effects on NF-κB signalling. Moreover, we established that one further compound blocks cell cycle progression and probably indirectly causes an augmentation of centriole number. The last compound analysed induces, in addition to excess centrioles, exceptionally long primary cilia-like structures. Overall, our analysis demonstrates that natural products constitute a rich source of tool compounds useful for unravelling and manipulating the mechanisms governing centriole assembly and number control.
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http://dx.doi.org/10.1002/cbic.201600327DOI Listing
November 2016

KAT2A/KAT2B-targeted acetylome reveals a role for PLK4 acetylation in preventing centrosome amplification.

Nat Commun 2016 10 31;7:13227. Epub 2016 Oct 31.

Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), 67404 Illkirch, France.

Lysine acetylation is a widespread post-translational modification regulating various biological processes. To characterize cellular functions of the human lysine acetyltransferases KAT2A (GCN5) and KAT2B (PCAF), we determined their acetylome by shotgun proteomics. One of the newly identified KAT2A/2B substrate is polo-like kinase 4 (PLK4), a key regulator of centrosome duplication. We demonstrate that KAT2A/2B acetylate the PLK4 kinase domain on residues K45 and K46. Molecular dynamics modelling suggests that K45/K46 acetylation impairs kinase activity by shifting the kinase to an inactive conformation. Accordingly, PLK4 activity is reduced upon in vitro acetylation of its kinase domain. Moreover, the overexpression of the PLK4 K45R/K46R mutant in cells does not lead to centrosome overamplification, as observed with wild-type PLK4. We also find that impairing KAT2A/2B-acetyltransferase activity results in diminished phosphorylation of PLK4 and in excess centrosome numbers in cells. Overall, our study identifies the global human KAT2A/2B acetylome and uncovers that KAT2A/2B acetylation of PLK4 prevents centrosome amplification.
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http://dx.doi.org/10.1038/ncomms13227DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5095585PMC
October 2016

The Human Centriolar Protein CEP135 Contains a Two-Stranded Coiled-Coil Domain Critical for Microtubule Binding.

Structure 2016 08 28;24(8):1358-1371. Epub 2016 Jul 28.

Laboratory of Biomolecular Research, Department of Biology and Chemistry, Paul Scherrer Institut, 5232 Villigen, Switzerland. Electronic address:

Centrioles are microtubule-based structures that play important roles notably in cell division and cilium biogenesis. CEP135/Bld10p family members are evolutionarily conserved microtubule-binding proteins important for centriole formation. Here, we analyzed in detail the microtubule-binding activity of human CEP135 (HsCEP135). X-ray crystallography and small-angle X-ray scattering in combination with molecular modeling revealed that the 158 N-terminal residues of HsCEP135 (HsCEP135-N) form a parallel two-stranded coiled-coil structure. Biochemical, cryo-electron, and fluorescence microscopy analyses revealed that in vitro HsCEP135-N interacts with tubulin, protofilaments, and microtubules and induces the formation of microtubule bundles. We further identified a 13 amino acid segment spanning residues 96-108, which represents a major microtubule-binding site in HsCEP135-N. Within this segment, we identified a cluster of three lysine residues that contribute to the microtubule bundling activity of HsCEP135-N. Our results provide the first structural information on CEP135/Bld10p proteins and offer insights into their microtubule-binding mechanism.
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http://dx.doi.org/10.1016/j.str.2016.06.011DOI Listing
August 2016

Discovery of a Selective Aurora A Kinase Inhibitor by Virtual Screening.

J Med Chem 2016 Aug 20;59(15):7188-211. Epub 2016 Jul 20.

Department of Chemistry and Biochemistry, National Center of Competence in Research NCCR Chemical Biology and NCCR TransCure, University of Berne , Freiestrasse 3, 3012 Berne, Switzerland.

Here we report the discovery of a selective inhibitor of Aurora A, a key regulator of cell division and potential anticancer target. We used the atom category extended ligand overlap score (xLOS), a 3D ligand-based virtual screening method recently developed in our group, to select 437 shape and pharmacophore analogs of reference kinase inhibitors. Biochemical screening uncovered two inhibitor series with scaffolds unprecedented among kinase inhibitors. One of them was successfully optimized by structure-based design to a potent Aurora A inhibitor (IC50 = 2 nM) with very high kinome selectivity for Aurora kinases. This inhibitor locks Aurora A in an inactive conformation and disrupts binding to its activator protein TPX2, which impairs Aurora A localization at the mitotic spindle and induces cell division defects. This phenotype can be rescued by inhibitor-resistant Aurora A mutants. The inhibitor furthermore does not induce Aurora B specific effects in cells.
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http://dx.doi.org/10.1021/acs.jmedchem.6b00709DOI Listing
August 2016

Aurora A kinase regulates proper spindle positioning in C. elegans and in human cells.

J Cell Sci 2016 08 22;129(15):3015-25. Epub 2016 Jun 22.

Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne CH-1015, Switzerland

Accurate spindle positioning is essential for error-free cell division. The one-cell Caenorhabditis elegans embryo has proven instrumental for dissecting mechanisms governing spindle positioning. Despite important progress, how the cortical forces that act on astral microtubules to properly position the spindle are modulated is incompletely understood. Here, we report that the PP6 phosphatase PPH-6 and its associated subunit SAPS-1, which positively regulate pulling forces acting on spindle poles, associate with the Aurora A kinase AIR-1 in C. elegans embryos. We show that acute inactivation of AIR-1 during mitosis results in excess pulling forces on astral microtubules. Furthermore, we uncover that AIR-1 acts downstream of PPH-6-SAPS-1 in modulating spindle positioning, and that PPH-6-SAPS-1 negatively regulates AIR-1 localization at the cell cortex. Moreover, we show that Aurora A and the PP6 phosphatase subunit PPP6C are also necessary for spindle positioning in human cells. There, Aurora A is needed for the cortical localization of NuMA and dynein during mitosis. Overall, our work demonstrates that Aurora A kinases and PP6 phosphatases have an ancient function in modulating spindle positioning, thus contributing to faithful cell division.
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http://dx.doi.org/10.1242/jcs.184416DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6203311PMC
August 2016