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Laurence Pelletier - One of the best experts on this subject based on the ideXlab platform.
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Organizational properties of the Pericentriolar Material
The Microtubule Cytoskeleton: Organisation Function and Role in Disease, 2016Co-Authors: David Comartin, Laurence PelletierAbstract:The centrosome is the major microtubule-organizing centre of animal cells. It participates in a number of crucial cellular functions including cell motility, intracellular transport, mitotic spindle assembly/positioning and cilia formation. Centrosome is composed of pair of ninefold symmetric centrioles surrounded by Pericentriolar Material, or PCM. PCM organization undergoes a series of dramatic changes in its organization and function as cells progress through the cell cycle. Indeed, the rather small interphase centrosome increases dramatically in size and microtubule nucleation capacity from interphase to mitosis, a process referred to as centrosome maturation. Until very recently, the PCM was thought to be largely amorphous. However, it has been elegantly demonstrated in several super-resolution studies that the PCM is highly organized and that the higher-order organizational properties are conserved from flies to humans. In this book chapter, we review current knowledge on the organization and composition of PCM in both interphase and mitosis and discuss how the centrosome landscape is altered through post-translational modifications, mainly mitotic phosphorylation, during centrosome maturation.
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Centrosome Biology: The Ins and Outs of Centrosome Assembly
Current Biology, 2015Co-Authors: Suzanna L Prosser, Laurence PelletierAbstract:As a microtubule-organizing center, the centrosome undergoes a dramatic increase in size - via expansion of the Pericentriolar Material - during mitosis. Recent work reveals shared assembly properties of a protein scaffold that facilitates and supports this expansion, a process critical to spindle assembly.
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amorphous no more subdiffraction view of the Pericentriolar Material architecture
Trends in Cell Biology, 2014Co-Authors: Vito Mennella, Laurence Pelletier, Bo Huang, David A AgardAbstract:The centrosome influences the shape, orientation and activity of the microtubule cytoskeleton. The Pericentriolar Material (PCM), determines this functionality by providing a dynamic platform for nucleating microtubules and acts as a nexus for molecular signaling. Although great strides have been made in understanding PCM activity, its diffraction-limited size and amorphous appearance on electron microscopy (EM) have limited analysis of its high-order organization. Here, we outline current knowledge of PCM architecture and assembly, emphasizing recent super-resolution imaging studies that revealed the PCM has a layered structure made of fibers and matrices conserved from flies to humans. Notably, these studies debunk the long-standing view of an amorphous PCM and provide a paradigm to dissect the supramolecular organization of organelles in cells.
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subdiffraction imaging of centrosomes reveals higher order organizational features of Pericentriolar Material
Nature Cell Biology, 2012Co-Authors: Steffen Lawo, Monica Hasegan, Gagan D Gupta, Laurence PelletierAbstract:Centrosomes consist of two centrioles surrounded by Pericentriolar Material (PCM) that nucleates microtubules. The PCM has been considered as amorphous but, using subdiffraction fluorescence imaging, Pelletier and colleagues now reveal the organized structure of human PCM.
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Interaction Proteomics Identify NEURL4 and the HECT E3 Ligase HERC2 as Novel Modulators of Centrosome Architecture
Molecular & Cellular Proteomics, 2012Co-Authors: Abdallah K. Al-hakim, Daniel Durocher, Mikhail Bashkurov, Anne-claude Gingras, Laurence PelletierAbstract:Centrosomes are composed of a centriole pair surrounded by an intricate proteinaceous matrix referred to as Pericentriolar Material. Although the mechanisms underpinning the control of centriole duplication are now well understood, we know relatively little about the control of centrosome size and shape. Here we used interaction proteomics to identify the E3 ligase HERC2 and the neuralized homologue NEURL4 as novel interaction partners of the centrosomal protein CP110. Using high resolution imaging, we find that HERC2 and NEURL4 localize to the centrosome and that interfering with their function alters centrosome morphology through the appearance of aberrant filamentous structures that stain for a subset of Pericentriolar Material proteins including pericentrin and CEP135. Using an RNA interference-resistant transgene approach in combination with structure-function analyses, we show that the association between CP110 and HERC2 depends on nonoverlapping regions of NEURL4. Whereas CP110 binding to NEURL4 is dispensable for the regulation of Pericentriolar Material architecture, its association with HERC2 is required to maintain normal centrosome integrity. NEURL4 is a substrate of HERC2, and together these results indicate that the NEURL4-HERC2 complex participates in the ubiquitin-dependent regulation of centrosome architecture.
Anthony A Hyman - One of the best experts on this subject based on the ideXlab platform.
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Method: In vitro analysis of Pericentriolar Material assembly
Methods in Cell Biology, 2015Co-Authors: Jeffrey B Woodruff, Anthony A HymanAbstract:Centrosomes are major microtubule-organizing centers in eukaryotic cells and play a critical role in embryonic development and asymmetric cell division. Centrosomes comprise a pair of centrioles surrounded by an amorphous proteinaceous meshwork called the Pericentriolar Material (PCM). Robust deposition of PCM around the centrioles is essential for a centrosome to achieve full microtubule nucleating potential. Despite the wealth of information on PCM composition and function, the mechanism and regulation of PCM assembly have been difficult to ascertain, due in part to the lack of an in vitro system. Here, we describe methods to establish an in vitro system to study PCM assembly in Caenorhabditis elegans. Specifically, we describe (1) how to express and purify the C. elegans PCM proteins SPD-5, SPD-2, and PLK-1 from baculovirus-infected insect cells, (2) how to assemble these proteins into PCM-like structures in vitro, and (3) how to quantify this assembly process in a semiautomated fashion.
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Pericentriolar Material structure and dynamics.
Philosophical Transactions of the Royal Society B, 2014Co-Authors: Jeffrey B Woodruff, Oliver Wueseke, Anthony A HymanAbstract:A centrosome consists of two barrel-shaped centrioles embedded in a matrix of proteins known as the Pericentriolar Material (PCM). The PCM serves as a platform for protein complexes that regulate o...
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The C. elegans Pericentriolar Material components SPD-2 and SPD-5 are monomeric in the cytoplasm prior to incorporation into the PCM matrix
Molecular Biology of the Cell, 2014Co-Authors: Oliver Wueseke, Jakob Bunkenborg, Marco Y Hein, Jeffrey B Woodruff, Andrea Zinke, Jens S. Andersen, Valeria Viscardi, Karen Oegema, Anthony A HymanAbstract:Centrosomes are the main microtubule-organizing centers in animal cells. Centrosomes consist of a pair of centrioles surrounded by a matrix of Pericentriolar Material (PCM) that assembles from cytoplasmic components. In Caenorhabditis elegans embryos, interactions between the coiled-coil proteins SPD-5 and SPD-2 and the kinase PLK-1 are critical for PCM assembly. However, it is not known whether these interactions promote the formation of cytoplasmic complexes that are added to the PCM or whether the components interact only during incorporation into the PCM matrix. Here we address this problem by using a combination of live-cell fluorescence correlation spectroscopy, mass spectrometry, and hydrodynamic techniques to investigate the native state of PCM components in the cytoplasm. We show that SPD-2 is monomeric, and neither SPD-2 nor SPD-5 exists in complex with PLK-1. SPD-5 exists mostly as a monomer but also forms complexes with the PP2A-regulatory proteins RSA-1 and RSA-2, which are required for microtubule organization at centrosomes. These results suggest that the interactions between SPD-2, SPD-5, and PLK-1 do not result in formation of cytoplasmic complexes, but instead occur in the context of PCM assembly.
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centrosomes are autocatalytic droplets of Pericentriolar Material organized by centrioles
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: David Zwicker, Steffen Jaensch, Markus Decker, Anthony A Hyman, Frank JülicherAbstract:Centrosomes are highly dynamic, spherical organelles without a membrane. Their physical nature and their assembly are not understood. Using the concept of phase separation, we propose a theoretical description of centrosomes as liquid droplets. In our model, centrosome Material occurs in a form soluble in the cytosol and a form that tends to undergo phase separation from the cytosol. We show that an autocatalytic chemical transition between these forms accounts for the temporal evolution observed in experiments. Interestingly, the nucleation of centrosomes can be controlled by an enzymatic activity of the centrioles, which are present at the core of all centrosomes. This nonequilibrium feature also allows for multiple stable centrosomes, a situation that is unstable in equilibrium phase separation. Our theory explains the growth dynamics of centrosomes for all cell sizes down to the eight-cell stage of the Caenorhabditis elegans embryo, and it also accounts for data acquired in experiments with aberrant numbers of centrosomes and altered cell volumes. Furthermore, the model can describe unequal centrosome sizes observed in cells with perturbed centrioles. We also propose an interpretation of the molecular details of the involved proteins in the case of C. elegans. Our example suggests a general picture of the organization of membraneless organelles.
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The Caenorhabditis elegans centrosomal protein SPD-2 is required for both Pericentriolar Material recruitment and centriole duplication
Current Biology, 2004Co-Authors: Laurence Pelletier, Eva Hannak, Carrie Cowan, Nurhan Ozlu, Thomas Müller-reichert, Bianca Habermann, Martine Ruer, Anthony A HymanAbstract:Background: The centrosome is composed of a centriole pair and Pericentriolar Material (PCM). By marking the site of PCM assembly, the centrioles define the number of centrosomes present in the cell. The PCM, in turn, is responsible for the microtubule (MT) nucleation activity of centrosomes. Therefore, in order to assemble a functional bipolar mitotic spindle, a cell needs to control both centriole duplication and PCM recruitment. To date, however, the molecular mechanisms that govern these two processes still remain poorly understood. Results: Here we show that SPD-2 is a novel component of the C. elegans centrosome. SPD-2 localizes to the centriole throughout the cell cycle and accumulates on the PCM during mitosis. We show that SPD-2 requires SPD-5 for its accumulation on the PCM and that in the absence of SPD-2, centrosome assembly fails. We further show that centriole duplication is also defective in spd-2(RNAi) embryos, but not in spd-5(RNAi) embryos, where PCM recruitment is efficiently blocked. Conclusions: Taken together, our results suggest that SPD-2 may link PCM recruitment and centriole duplication in C. elegans. SPD-2 shares homology with a human centrosome protein, suggesting that this key component of the C. elegans centrosome is evolutionarily conserved.
Tomer Avidorreiss - One of the best experts on this subject based on the ideXlab platform.
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plk1 polo phosphorylates sas 4 at the onset of mitosis for an efficient recruitment of Pericentriolar Material to centrosomes
Cell Reports, 2018Co-Authors: Anand Ramani, Aruljothi Mariappan, Marco Gottardo, Sunit Mandad, Henning Urlaub, Tomer AvidorreissAbstract:Summary Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a Pericentriolar Material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4’s localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.
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sas 4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome
Nature Communications, 2011Co-Authors: Jayachandran Gopalakrishnan, Vito Mennella, David A Agard, Stephanie Blachon, Bo Zhai, Andrew H Smith, Timothy L Megraw, Daniela Nicastro, Steven P Gygi, Tomer AvidorreissAbstract:Cell division and cilium formation are dependent on centrosomes that consist of two centrioles and Pericentriolar Material (PCM). In this study, the Sas-4 protein is shown to be important in mediating the formation of cytoplasmic PCM complexes and the incorporation of this Material into centrosomes.
Tomer Avidor-reiss - One of the best experts on this subject based on the ideXlab platform.
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Plk1/Polo phosphorylates Sas-4 at the onset of mitosis for an efficient recruitment of Pericentriolar Material to centrosomes.
Cell Reports, 2018Co-Authors: Anand Ramani, Tomer Avidor-reiss, Aruljothi Mariappan, Marco Gottardo, Sunit Mandad, Henning Urlaub, Maria Riparbelli, Giuliano Callaini, Alain Debec, Regina FeederleAbstract:Summary Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a Pericentriolar Material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4’s localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion.
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Plk1/Polo Phosphorylates Sas-4 at the Onset of Mitosis for an Efficient Recruitment of Pericentriolar Material to Centrosomes
Elsevier, 2018Co-Authors: Anand Ramani, Tomer Avidor-reiss, Aruljothi Mariappan, Marco Gottardo, Sunit Mandad, Henning Urlaub, Maria Riparbelli, Giuliano Callaini, Alain Debec, Regina FeederleAbstract:Summary: Centrosomes are the major microtubule-organizing centers, consisting of centrioles surrounded by a Pericentriolar Material (PCM). Centrosomal PCM is spatiotemporally regulated to be minimal during interphase and expands as cells enter mitosis. It is unclear how PCM expansion is initiated at the onset of mitosis. Here, we identify that, in Drosophila, Plk1/Polo kinase phosphorylates the conserved centrosomal protein Sas-4 in vitro. This phosphorylation appears to occur at the onset of mitosis, enabling Sas-4’s localization to expand outward from meiotic and mitotic centrosomes. The Plk1/Polo kinase site of Sas-4 is then required for an efficient recruitment of Cnn and γ-tubulin, bona fide PCM proteins that are essential for PCM expansion and centrosome maturation. Point mutations at Plk1/Polo sites of Sas-4 affect neither centrosome structure nor centriole duplication but specifically reduce the affinity to bind Cnn and γ-tubulin. These observations identify Plk1/Polo kinase regulation of Sas-4 as essential for efficient PCM expansion. : Ramani et al. show that Plk1/Polo phosphorylates Drosophila Sas-4 at the onset of mitosis. Cell-cycle-specific modification of Sas-4 determines the spatiotemporal localization of Sas-4 in centrosomes, which is required for an efficient recruitment of PCM proteins in mitosis. Keywords: Pericentriolar Material, centrosomes, Sas-4, Drosophila melanogaster, Plk1, centrosome maturatio
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Conserved TCP domain of Sas-4/CPAP is essential for Pericentriolar Material tethering during centrosome biogenesis.
Proceedings of the National Academy of Sciences of the United States of America, 2014Co-Authors: Xiangdong Zheng, Marcus L. Basiri, Alain Debec, Li Ming Gooi, Arpit Wason, Elke Gabriel, Narges Zare Mehrjardi, Qian Yang, Xingrun Zhang, Tomer Avidor-reissAbstract:Pericentriolar Material (PCM) recruitment to centrioles forms a key step in centrosome biogenesis. Deregulation of this process leads to centrosome aberrations causing disorders, one of which is autosomal recessive primary microcephaly (MCPH), a neurodevelopmental disorder where brain size is reduced. During PCM recruitment, the conserved centrosomal protein Sas-4/CPAP/MCPH6, known to play a role in centriole formation, acts as a scaffold for cytoplasmic PCM complexes to bind and then tethers them to centrioles to form functional centrosomes. To understand Sas-4's tethering role, we determined the crystal structure of its T complex protein 10 (TCP) domain displaying a solvent-exposed single-layer of β-sheets fold. This unique feature of the TCP domain suggests that it could provide an "extended surface-like" platform to tether the Sas-4-PCM scaffold to a centriole. Functional studies in Drosophila, human cells, and human induced pluripotent stem cell-derived neural progenitor cells were used to test this hypothesis, where point mutations within the 9-10th β-strands (β9-10 mutants including a MCPH-associated mutation) perturbed PCM tethering while allowing Sas-4/CPAP to scaffold cytoplasmic PCM complexes. Specifically, the Sas-4 β9-10 mutants displayed perturbed interactions with Ana2, a centrosome duplication factor, and Bld-10, a centriole microtubule-binding protein, suggesting a role for the β9-10 surface in mediating protein-protein interactions for efficient Sas-4-PCM scaffold centriole tethering. Hence, we provide possible insights into how centrosomal protein defects result in human MCPH and how Sas-4 proteins act as a vehicle to tether PCM complexes to centrioles independent of its well-known role in centriole duplication.
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Tubulin nucleotide status controls Sas-4-dependent Pericentriolar Material recruitment
Nature Cell Biology, 2012Co-Authors: Jayachandran Gopalakrishnan, Yiu-cheung Frederick Chim, Marcus L. Basiri, Dorothy A. Lerit, Nasser M. Rusan, Tomer Avidor-reissAbstract:Avidor-Reiss and colleagues show that the nucleotide status of tubulin regulates recruitment of Pericentriolar Material. Binding of GTP-bound tubulin to the Sas-4 centrosomal protein prevents the Sas-4-dependent formation of centrosomal protein complexes, whereas the Sas-4-stimulated hydrolysis of tubulin–GTP into tubulin–GDP has the opposite effect.
David A Agard - One of the best experts on this subject based on the ideXlab platform.
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amorphous no more subdiffraction view of the Pericentriolar Material architecture
Trends in Cell Biology, 2014Co-Authors: Vito Mennella, Laurence Pelletier, Bo Huang, David A AgardAbstract:The centrosome influences the shape, orientation and activity of the microtubule cytoskeleton. The Pericentriolar Material (PCM), determines this functionality by providing a dynamic platform for nucleating microtubules and acts as a nexus for molecular signaling. Although great strides have been made in understanding PCM activity, its diffraction-limited size and amorphous appearance on electron microscopy (EM) have limited analysis of its high-order organization. Here, we outline current knowledge of PCM architecture and assembly, emphasizing recent super-resolution imaging studies that revealed the PCM has a layered structure made of fibers and matrices conserved from flies to humans. Notably, these studies debunk the long-standing view of an amorphous PCM and provide a paradigm to dissect the supramolecular organization of organelles in cells.
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subdiffraction resolution fluorescence microscopy reveals a domain of the centrosome critical for Pericentriolar Material organization
Nature Cell Biology, 2012Co-Authors: Vito Mennella, Bettina Keszthelyi, Kent L Mcdonald, B Chhun, Gregory C Rogers, Bo Huang, David A AgardAbstract:Centrosomes, the microtubule nucleation centre of most cells, consist of two centrioles surrounded by Pericentriolar Material (PCM). The PCM has been considered as amorphous but, using subdiffraction fluorescence microscopy approaches, Agard and colleagues now reveal the organized structure of the PCM of Drosophila centrosomes.
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sas 4 provides a scaffold for cytoplasmic complexes and tethers them in a centrosome
Nature Communications, 2011Co-Authors: Jayachandran Gopalakrishnan, Vito Mennella, David A Agard, Stephanie Blachon, Bo Zhai, Andrew H Smith, Timothy L Megraw, Daniela Nicastro, Steven P Gygi, Tomer AvidorreissAbstract:Cell division and cilium formation are dependent on centrosomes that consist of two centrioles and Pericentriolar Material (PCM). In this study, the Sas-4 protein is shown to be important in mediating the formation of cytoplasmic PCM complexes and the incorporation of this Material into centrosomes.