Karsenti Group (Visiting)Publications
Collective behavior of minus-ended motors in mitotic microtubule asters gliding toward DNA.
Athale, C.A., Dinarina, A., Nedelec, F. & Karsenti, E.
Phys Biol. 2014 Jan 29;11(1):016008.
Microtubules (MTs) nucleated by centrosomes form star-shaped structures referred to as asters. Aster motility and dynamics is vital for genome stability, cell division, polarization and differentiation. Asters move either toward the cell center or away from it. Here, we focus on the centering mechanism in a membrane independent system of Xenopus cytoplasmic egg extracts. Using live microscopy and single particle tracking, we find that asters move toward chromatinized DNA structures. The velocity and directionality profiles suggest a random-walk with drift directed toward DNA. We have developed a theoretical model that can explain this movement as a result of a gradient of MT length dynamics and MT gliding on immobilized dynein motors. In simulations, the antagonistic action of the motor species on the radial array of MTs leads to a tug-of-war purely due to geometric considerations and aster motility resembles a directed random-walk. Additionally, our model predicts that aster velocities do not change greatly with varying initial distance from DNA. The movement of asymmetric asters becomes increasingly super-diffusive with increasing motor density, but for symmetric asters it becomes less super-diffusive. The transition of symmetric asters from superdiffusive to diffusive mobility is the result of number fluctuations in bound motors in the tug-of-war. Overall, our model is in good agreement with experimental data in Xenopus cytoplasmic extracts and predicts novel features of the collective effects of motor-MT interactions.
Exploring nucleo-cytoplasmic large DNA viruses in Tara Oceans microbial metagenomes.
Hingamp, P., Grimsley, N., Acinas, S.G., Clerissi, C., Subirana, L., Poulain, J., Ferrera, I., Sarmento, H., Villar, E., Lima-Mendez, G., Faust, K., Sunagawa, S., Claverie, J.M., Moreau, H., Desdevises, Y., Bork, P., Raes, J., de Vargas, C., Karsenti, E., Kandels-Lewis, S., Jaillon, O., Not, F., Pesant, S., Wincker, P. & Ogata, H.
ISME J. 2013 Sep;7(9):1678-95. doi: 10.1038/ismej.2013.59. Epub 2013 Apr 11.
Nucleo-cytoplasmic large DNA viruses (NCLDVs) constitute a group of eukaryotic viruses that can have crucial ecological roles in the sea by accelerating the turnover of their unicellular hosts or by causing diseases in animals. To better characterize the diversity, abundance and biogeography of marine NCLDVs, we analyzed 17 metagenomes derived from microbial samples (0.2-1.6 mum size range) collected during the Tara Oceans Expedition. The sample set includes ecosystems under-represented in previous studies, such as the Arabian Sea oxygen minimum zone (OMZ) and Indian Ocean lagoons. By combining computationally derived relative abundance and direct prokaryote cell counts, the abundance of NCLDVs was found to be in the order of 10(4)-10(5) genomes ml(-1) for the samples from the photic zone and 10(2)-10(3) genomes ml(-1) for the OMZ. The Megaviridae and Phycodnaviridae dominated the NCLDV populations in the metagenomes, although most of the reads classified in these families showed large divergence from known viral genomes. Our taxon co-occurrence analysis revealed a potential association between viruses of the Megaviridae family and eukaryotes related to oomycetes. In support of this predicted association, we identified six cases of lateral gene transfer between Megaviridae and oomycetes. Our results suggest that marine NCLDVs probably outnumber eukaryotic organisms in the photic layer (per given water mass) and that metagenomic sequence analyses promise to shed new light on the biodiversity of marine viruses and their interactions with potential hosts.
Metagenomic 16S rDNA Illumina tags are a powerful alternative to amplicon sequencing to explore diversity and structure of microbial communities.
Logares, R., Sunagawa, S., Salazar, G., Cornejo-Castillo, F.M., Ferrera, I., Sarmento, H., Hingamp, P., Ogata, H., de Vargas, C., Lima-Mendez, G., Raes, J., Poulain, J., Jaillon, O., Wincker, P., Kandels-Lewis, S., Karsenti, E., Bork, P. & Acinas, S.G.
Environ Microbiol. 2013 Aug 18. doi: 10.1111/1462-2920.12250.
Sequencing of 16S rDNA polymerase chain reaction (PCR) amplicons is the most common approach for investigating environmental prokaryotic diversity, despite the known biases introduced during PCR. Here we show that 16S rDNA fragments derived from Illumina-sequenced environmental metagenomes (mi tags) are a powerful alternative to 16S rDNA amplicons for investigating the taxonomic diversity and structure of prokaryotic communities. As part of the Tara Oceans global expedition, marine plankton was sampled in three locations, resulting in 29 subsamples for which metagenomes were produced by shotgun Illumina sequencing (ca. 700 Gb). For comparative analyses, a subset of samples was also selected for Roche-454 sequencing using both shotgun (m454 tags; 13 metagenomes, ca. 2.4 Gb) and 16S rDNA amplicon (454 tags; ca. 0.075 Gb) approaches. Our results indicate that by overcoming PCR biases related to amplification and primer mismatch, mi tags may provide more realistic estimates of community richness and evenness than amplicon 454 tags. In addition, mi tags can capture expected beta diversity patterns. Using mi tags is now economically feasible given the dramatic reduction in high-throughput sequencing costs, having the advantage of retrieving simultaneously both taxonomic (Bacteria, Archaea and Eukarya) and functional information from the same microbial community.
MURF2B, a Novel LC3-Binding Protein, Participates with MURF2A in the Switch between Autophagy and Ubiquitin Proteasome System during Differentiation of C2C12 Muscle Cells.
Pizon, V., Rybina, S., Gerbal, F., Delort, F., Vicart, P., Baldacci, G. & Karsenti, E.
PLoS One. 2013 Oct 4;8(10):e76140. doi: 10.1371/journal.pone.0076140.
The ubiquitin proteasome system and macroautophagy are proteolytic pathways essential in the maintenance of cellular homeostasis during differentiation and remodelling of skeletal muscle. In both pathways, proteins to be degraded are tagged with polyubiquitin. In skeletal muscles, the MURF2 proteins display E3 ubiquitin ligase structure suggesting that they may covalently attach ubiquitin polypeptides to still unknown target proteins. So far only MURF2A isoforms were studied and shown to interact with p62/SQSTM1, a protein implicated in macroautophagic and ubiquitin proteasome system degradations. Here, we analyzed the MURF2B and MURF2A proteins and show that the ratio of the isoforms changes during differentiation of muscle C2C12 cells and that the shift of the isoforms expression follows the sequential activation of autophagic or proteasomal degradation. We also show that MURF2B has a functional domain needed for its interaction with LC3, a protein needed for autophagic vesicles formation. Using specific MURF2 RNAi cells we observed that MURF2A and MURF2B are both needed for the formation of autophagosomes and that in the absence of MURF2B, the cells expressing MURF2A display an activated ubiquitin proteasome system implicated in the degradation of p62/SQSTM1 by UPS. Altogether, our results indicate that MURF2A and MURF2B proteins could participate in the molecular switch between the two ubiquitin degradative pathways.
A holistic approach to marine eco-systems biology.
Karsenti, E., Acinas, S.G., Bork, P., Bowler, C., De Vargas, C., Raes, J., Sullivan, M., Arendt, D., Benzoni, F., Claverie, J.M., Follows, M., Gorsky, G., Hingamp, P., Iudicone, D., Jaillon, O., Kandels-Lewis, S., Krzic, U., Not, F., Ogata, H., Pesant, S., Reynaud, E.G., Sardet, C., Sieracki, M.E., Speich, S., Velayoudon, D., Weissenbach, J. & Wincker, P.
PLoS Biol. 2011 Oct;9(10):e1001177. doi: 10.1371/journal.pbio.1001177. Epub 2011Oct 18.
The structure, robustness, and dynamics of ocean plankton ecosystems remain poorly understood due to sampling, analysis, and computational limitations. The Tara Oceans consortium organizes expeditions to help fill this gap at the global level.
XMAP215-EB1 interaction is required for proper spindle assembly and chromosome segregation in Xenopus egg extract.
Kronja, I., Kruljac-Letunic, A., Caudron-Herger, M., Bieling, P. & Karsenti, E.
Mol Biol Cell. 2009 Jun;20(11):2684-96. Epub 2009 Apr 15.
In metaphase Xenopus egg extracts, global microtubule growth is mainly promoted by two unrelated microtubule stabilizers, end-binding protein 1 (EB1) and XMAP215. Here, we explore their role and potential redundancy in the regulation of spindle assembly and function. We find that at physiological expression levels, both proteins are required for proper spindle architecture: Spindles assembled in the absence of EB1 or at decreased XMAP215 levels are short and frequently multipolar. Moreover, the reduced density of microtubules at the equator of DeltaEB1 or DeltaXMAP215 spindles leads to faulty kinetochore-microtubule attachments. These spindles also display diminished poleward flux rates and, upon anaphase induction, they neither segregate chromosomes nor reorganize into interphasic microtubule arrays. However, EB1 and XMAP215 nonredundantly regulate spindle assembly because an excess of XMAP215 can compensate for the absence of EB1, whereas the overexpression of EB1 cannot substitute for reduced XMAP215 levels. Our data indicate that EB1 could positively regulate XMAP215 by promoting its binding to the microtubules. Finally, we show that disruption of the mitosis-specific XMAP215-EB1 interaction produces a phenotype similar to that of either EB1 or XMAP215 depletion. Therefore, the XMAP215-EB1 interaction is required for proper spindle organization and chromosome segregation in Xenopus egg extracts.
Regulation of microtubule dynamics by reaction cascades around chromosomes.
Athale, C.A., Dinarina, A., Mora-Coral, M., Pugieux, C., Nedelec, F. & Karsenti, E.
Science. 2008 Nov 21;322(5905):1243-1247. Epub 2008 Oct 23.
During spindle assembly, chromosomes generate gradients of microtubule stabilization through a reaction-diffusion process, but how this is achieved is not well understood. We measured the spatial distribution of microtubule aster asymmetry around chromosomes by incubating centrosomes and micropatterned chromatin patches in frog egg extracts. We then screened for microtubule stabilization gradient shapes that would generate such spatial distributions with the use of computer simulations. Only a long-range, sharply decaying microtubule stabilization gradient could generate aster asymmetries fitting the experimental data. We propose a reaction-diffusion model that combines the chromosome generated Ran-guanosine triphosphate-Importin reaction network to a secondary phosphorylation network as a potential mechanism for the generation of such gradients.
Hepatoma up-regulated protein is required for chromatin-induced microtubule assembly independently of TPX2.
Casanova, C.M., Rybina, S., Yokoyama, H., Karsenti, E. & Mattaj, I.W.
Mol Biol Cell. 2008 Nov;19(11):4900-8. Epub 2008 Sep 17.
The production of RanGTP around chromosomes is crucial for spindle microtubule assembly in mitosis. Previous work has shown that hepatoma up-regulated protein (HURP) is a Ran target, required for microtubule stabilization and spindle organization. Here we report a detailed analysis of HURP function in Xenopus laevis mitotic egg extracts. HURP depletion severely impairs bipolar spindle assembly around chromosomes: the few spindles that do form show a significant decrease in microtubule density at the spindle midzone. HURP depletion does not interfere with microtubule growth from purified centrosomes, but completely abolishes microtubule assembly induced by chromatin beads or RanGTP. Simultaneous depletion of the microtubule destabilizer MCAK with HURP does not rescue the phenotype, demonstrating that the effect of HURP is not to antagonize the destabilization activity of MCAK. Although the phenotype of HURP depletion closely resembles that reported for TPX2 depletion, we find no evidence that TPX2 and HURP physically interact or that they influence each other in their effects on spindle microtubules. Our data indicate that HURP and TPX2 have nonredundant functions essential for chromatin-induced microtubule assembly.
Cdk11 is a RanGTP-dependent microtubule stabilization factor that regulates spindle assembly rate.
Yokoyama, H., Gruss, O.J., Rybina, S., Caudron, M., Schelder, M., Wilm, M., Mattaj, I.W. & Karsenti, E.
J Cell Biol. 2008 Mar 10;180(5):867-75. Epub 2008 Mar 3.
Production of Ran-guanosine triphosphate (GTP) around chromosomes induces local nucleation and plus end stabilization of microtubules (MTs). The nuclear protein TPX2 is required for RanGTP-dependent MT nucleation. To find the MT stabilizer, we affinity purify nuclear localization signal (NLS)-containing proteins from Xenopus laevis egg extracts. This NLS protein fraction contains the MT stabilization activity. After further purification, we used mass spectrometry to identify proteins in active fractions, including cyclin-dependent kinase 11 (Cdk11). Cdk11 localizes on spindle poles and MTs in Xenopus culture cells and egg extracts. Recombinant Cdk11 demonstrates RanGTP-dependent MT stabilization activity, whereas a kinase-dead mutant does not. Inactivation of Cdk11 in egg extracts blocks RanGTP-dependent MT stabilization and dramatically decreases the spindle assembly rate. Simultaneous depletion of TPX2 completely inhibits centrosome-dependent spindle assembly. Our results indicate that Cdk11 is responsible for RanGTP-dependent MT stabilization around chromosomes and that this local stabilization is essential for normal rates of spindle assembly and spindle function.
Self-organization in cell biology: a brief history.
Nat Rev Mol Cell Biol. 2008 Mar;9(3):255-62.
Over the past two decades, molecular and cell biologists have made important progress in characterizing the components and compartments of the cell. New visualization methods have also revealed cellular dynamics. This has raised complex issues about the organization principles that underlie the emergence of coherent dynamical cell shapes and functions. Self-organization concepts that were first developed in chemistry and physics and then applied to various morphogenetic problems in biology over the past century are now beginning to be applied to the organization of the living cell.
Tubulin dimers oligomerize before their incorporation into microtubules.
Mozziconacci, J., Sandblad, L., Wachsmuth, M., Brunner, D. & Karsenti, E.
PLoS ONE. 2008;3(11):e3821. Epub 2008 Nov 27.
In the presence of GTP, purified dimers of alpha- and beta-tubulin will interact longitudinally and laterally to self-assemble into microtubules (MTs). This property provides a powerful in vitro experimental system to describe MT dynamic behavior at the micrometer scale and to study effects and functioning of a large variety of microtubule associated proteins (MAPs). Despite the plethora of such data produced, the molecular mechanisms of MT assembly remain disputed. Electron microscopy (EM) studies suggested that tubulin dimers interact longitudinally to form short oligomers which form a tube by lateral interaction and which contribute to MT elongation. This idea is however challenged: Based on estimated association constants it was proposed that single dimers represent the major fraction of free tubulin. This view was recently supported by measurements suggesting that MTs elongate by addition of single tubulin dimers. To solve this discrepancy, we performed a direct measurement of the longitudinal interaction energy for tubulin dimers. We quantified the size distribution of tubulin oligomers using EM and fluorescence correlation spectroscopy (FCS). From the distribution we derived the longitudinal interaction energy in the presence of GDP and the non-hydrolysable GTP analog GMPCPP. Our data suggest that MT elongation and nucleation involves interactions of short tubulin oligomers rather than dimers. Our approach provides a solid experimental framework to better understand the role of MAPs in MT nucleation and growth.
Discrete states of a protein interaction network govern interphase and mitotic microtubule dynamics.
Niethammer, P., Kronja, I., Kandels-Lewis, S., Rybina, S., Bastiaens, P. & Karsenti, E.
PLoS Biol. 2007 Jan;5(2):e29.
The cytoplasm of eukaryotic cells is thought to adopt discrete "states" corresponding to different steady states of protein networks that govern changes in subcellular organization. For example, in Xenopus eggs, the interphase to mitosis transition is induced solely by activation of cyclin-dependent kinase 1 (CDK1) that phosphorylates many proteins leading to a reorganization of the nucleus and assembly of the mitotic spindle. Among these changes, the large array of stable microtubules that exists in interphase is replaced by short, highly dynamic microtubules in metaphase. Using a new visual immunoprecipitation assay that quantifies pairwise protein interactions in a non-perturbing manner in Xenopus egg extracts, we reveal the existence of a network of interactions between a series of microtubule-associated proteins (MAPs). In interphase, tubulin interacts with XMAP215, which is itself interacting with XKCM1, which connects to APC, EB1, and CLIP170. In mitosis, tubulin interacts with XMAP215, which is connected to EB1. We show that in interphase, microtubules are stable because the catastrophe-promoting activity of XKCM1 is inhibited by its interactions with the other MAPs. In mitosis, microtubules are short and dynamic because XKCM1 is free and has a strong destabilizing activity. In this case, the interaction of XMAP215 with EB1 is required to counteract the strong activity of XKCM1. This provides the beginning of a biochemical description of the notion of "cytoplasmic states" regarding the microtubule system.
Self-organisation processes in living matter.
Interdisciplinary Science Reviews June 2007 32(2) 163-175
Apart from the theory of evolution, there is no general theory of life. In addition to its special chemistry involving molecules carrying information, living matter has the property of being built of self-organising systems far from thermodynamic equilibrium. Although this may seem obvious, the implications of this fact for our understanding of life are far from familiar. In this review, I attempt to explain how the concept of self-organisation can change our view of living matter. I discuss more specifically some recent progress made in the understanding of how reaction diffusion and collective molecular behaviours are, in large part, responsible for the generation of cellular and subcellular dynamic patterns characteristic of living matter.
Modelling microtubule patterns.
Karsenti, E., Nedelec, F. & Surrey, T.
Nat Cell Biol. 2006 Nov;8(11):1204-11.
The cellular cytoskeleton is well studied in terms of its biological and physical properties, making it an attractive subject for systems approaches. Here, we describe the experimental and theoretical strategies used to study the collective behaviour of microtubules and motors. We illustrate how this led to the beginning of an understanding of dynamic cellular patterns that have precise functions.
Gradients in the self-organization of the mitotic spindle.
Bastiaens, P., Caudron, M., Niethammer, P. & Karsenti, E.
Trends Cell Biol. 2006 Mar;16(3):125-34. Epub 2006 Feb 14.
Recent evidence points at a role of protein interaction gradients around chromatin in mitotic spindle morphogenesis in large eukaryotic cells. Here, we explain how gradients can arise over distances of tens of microns around supramolecular structures from mixtures of soluble molecules. We discuss how coupled sets of such reaction diffusion processes generate the spatial information that determines the local dynamics of microtubules required to form a bipolar spindle. We argue that such reaction diffusion processes are involved in the self-organization of supramolecular structures in the cell.
Microtubule-dependent transport and organization of sarcomeric myosin during skeletal muscle differentiation.
Pizon, V., Gerbal, F., Diaz, C.C. & Karsenti, E.
EMBO J 2005 Nov 2;24(21):3781-92. Epub 2005 Oct 20.
It has been proposed that microtubules (MTs) participate in skeletal muscle cell differentiation. However, it is still unclear how this happens. To examine whether MTs could participate directly in the organization of thick and thin filaments into sarcomeres, we observed the concomitant reorganization and dynamics of MTs with the behavior of sarcomeric actin and myosin by time-lapse confocal microscopy. Using green fluorescent protein (GFP)-EB1 protein to label MT plus ends, we determined that MTs become organized into antiparallel arrays along fusing myotubes. Their dynamics and orientation was found to be different across the thickness of the myotubes. We observed fast movements of Dsred-myosin along GFP-MTs. Comparison of GFP-EB1 and Dsred-myosin dynamics revealed that myosin moved toward MT plus ends. Immuno-electron microscopy experiments confirmed that myosin was actually associated with MTs in myotubes. Finally, we confirmed that MTs were required for the stabilization of myosin-containing elements prior to incorporation into mature sarcomeres. Collectively, our results strongly suggest that MTs become organized into a scaffold that provides directional cues for the positioning and organization of myosin filaments during sarcomere formation.
Spatial coordination of spindle assembly by chromosome-mediated signaling gradients.
Caudron, M., Bunt, G., Bastiaens, P. & Karsenti, E.
Science 2005 Aug 26;309(5739):1373-6.
During cell division, chromosomes are distributed to daughter cells by the mitotic spindle. This system requires spatial cues to reproducibly self-organize. We report that such cues are provided by chromosome-mediated interaction gradients between the small guanosine triphosphatase (GTPase) Ran and importin-beta. This produces activity gradients that determine the spatial distribution of microtubule nucleation and stabilization around chromosomes and that are essential for the self-organization of microtubules into a bipolar spindle.
TPX or not TPX?
Mol Cell 2005 Aug 19;19(4):431-2.
An Aurora A regulatory module has been identified in two different proteins: TPX2 in Xenopus laevis and TPXL-1 in C. elegans. The diverse roles of these two proteins in spindle assembly leave us to beckon the true C. elegans TPX2 ortholog to center stage.
Cytoskeleton: spindle saga.
Nature 2004 Dec 2;432(7017):563-4. Europe PMC
Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts.
Brunet, S., Sardon, T., Zimmerman, T., Wittmann, T., Pepperkok, R., Karsenti, E. & Vernos, I.
Mol Biol Cell 2004 Dec;15(12):5318-28. Epub .
TPX2 has multiple functions during mitosis, including microtubule nucleation around the chromosomes and the targeting of Xklp2 and Aurora A to the spindle. We have performed a detailed domain functional analysis of TPX2 and found that a large N-terminal domain containing the Aurora A binding peptide interacts directly with and nucleates microtubules in pure tubulin solutions. However, it cannot substitute the endogenous TPX2 to support microtubule nucleation in response to Ran guanosine triphosphate (GTP) and spindle assembly in egg extracts. By contrast, a large C-terminal domain of TPX2 that does not bind directly to pure microtubules and does not bind Aurora A kinase rescues microtubule nucleation in response to RanGTP and spindle assembly in TPX2-depleted extract. These and previous results suggest that under physiological conditions, TPX2 is essential for microtubule nucleation around chromatin and functions in a network of other molecules, some of which also are regulated by RanGTP.
Eg5 causes elongation of meiotic spindles when flux-associated microtubule depolymerization is blocked.
Shirasu-Hiza, M., Perlman, Z.E., Wittmann, T., Karsenti, E. & Mitchison, T.J.
Curr Biol 2004 Nov 9;14(21):1941-5.
In higher eukaryotes, microtubules (MT) in both halves of the mitotic spindle translocate continuously away from the midzone in a phenomenon called poleward microtubule flux. Because the spindle maintains constant length and microtubule density, this microtubule translocation must somehow be coupled to net MT depolymerization at spindle poles. The molecular mechanisms underlying both flux-associated translocation and flux-associated depolymerization are not well understood, but it can be predicted that blocking pole-based destabilization will increase spindle length, an idea that has not been tested in meiotic spindles. Here, we show that simultaneous addition of two pole-disrupting reagents p50/dynamitin and a truncated version of Xklp2 results in continuous spindle elongation in Xenopus egg extracts, and we quantitatively correlate this elongation rate with the poleward translocation of stabilized microtubules. We further use this system to demonstrate that this poleward translocation requires the activity of the kinesin-related protein Eg5. These results suggest that Eg5 is responsible for flux-associated MT translocation and that dynein and Xklp2 regulate flux-associated microtubule depolymerization at spindle poles.
Homology-based functional proteomics by mass spectrometry: application to the Xenopus microtubule-associated proteome.
Liska, A.J., Popov, A.V., Sunyaev, S., Coughlin, P., Habermann, B., Shevchenko, A., Bork, P., Karsenti, E. & Shevchenko, A.
Proteomics 2004 Sep;4(9):2707-21.
The application of functional proteomics to important model organisms with unsequenced genomes is restricted because of the limited ability to identify proteins by conventional mass spectrometry (MS) methods. Here we applied MS and sequence-similarity database searching strategies to characterize the Xenopus laevis microtubule-associated proteome. We identified over 40 unique, and many novel, microtubule-bound proteins, as well as two macromolecular protein complexes involved in protein translation. This finding was corroborated by electron microscopy showing the presence of ribosomes on spindles assembled from frog egg extracts. Taken together, these results suggest that protein translation occurs on the spindle during meiosis in the Xenopus oocyte. These findings were made possible due to the application of sequence-similarity methods, which extended mass spectrometric protein identification capabilities by 2-fold compared to conventional methods.
The mitotic spindle and actin tails.
Karsenti, E. & Nedelec, F.
Biol Cell 2004 Apr;96(3):237-40.
To segregate their chromosomes, eukaryotic cells rely on a dynamic structure made of microtubules: the mitotic spindle. This structure can form in cells lacking centrosomes, because their chromosomes also nucleate microtubules. This second assembly pathway is observed even in some cells that naturally have centrosomes, for example when the centrosomes are ablated by laser surgery. Recent results have started to address the complementary question of whether centrosome-nucleated microtubules alone could sustain the formation of a functional mitotic spindle. We wonder in this respect whether lower eukaryotes such as yeasts are different from higher eukaryotes such as vertebrates.
Stathmin-tubulin interaction gradients in motile and mitotic cells.
Niethammer, P., Bastiaens, P. & Karsenti, E.
Science 2004 Mar 19;303(5665):1862-6.
The spatial organization of the microtubule cytoskeleton is thought to be directed by steady-state activity gradients of diffusible regulatory molecules. We visualized such intracellular gradients by monitoring the interaction between tubulin and a regulator of microtubule dynamics, stathmin, using a fluorescence resonance energy transfer (FRET) biosensor. These gradients were observed both during interphase in motile membrane protrusions and during mitosis around chromosomes, which suggests that a similar mechanism may contribute to the creation of polarized microtubule structures. These interaction patterns are likely to reflect phosphorylation of stathmin in these areas.
Stu2p and XMAP215: turncoat microtubule-associated proteins?
Popov, A.V. & Karsenti, E.
Trends Cell Biol 2003 Nov;13(11):547-50. Europe PMC
Long-range communication between chromatin and microtubules in Xenopus egg extracts.
Carazo-Salas, R.E. & Karsenti, E.
Curr Biol 2003 Sep 30;13(19):1728-33.
The mitotic spindle of animal cells is a bipolar array of microtubules that guides chromosome segregation during cell division. It has been proposed that during spindle assembly chromatin can positively influence microtubule stability at a distance from its surface throughout its neighboring cytoplasm. However, such an "a distance" effect has never been visualized directly. Here, we have used centrosomal microtubules and chromatin beads to probe the regulation of microtubule behavior around chromatin in Xenopus egg extracts. We show that, in this system, chromatin does affect microtubule formation at a distance, inducing preferential orientation of centrosomal microtubules in its direction. Moreover, this asymmetric distribution of microtubules is translated into a directional migration of centrosomal asters toward chromatin and their steady-state repositioning within 10 microm of chromatin. To our knowledge, this is the first direct evidence of a long-range guidance effect at the sub-cellular level.
Importin alpha-regulated nucleation of microtubules by TPX2.
Schatz, C.A., Santarella, R., Hoenger, A., Karsenti, E., Mattaj, I.W., Gruss, O.J. & Carazo-Salas, R.E.
EMBO J 2003 May 1;22(9):2060-70.
The importin alpha-regulated microtubule-associated protein TPX2 is known to be critical for meiotic and mitotic spindle formation in vertebrates, but its detailed mechanism of action and regulation is not understood. Here, the site of interaction on TPX2 for importin alpha is mapped. A TPX2 mutant that cannot bind importin alpha is constitutively active in the induction of microtubule-containing aster-like structures in Xenopus egg extract, demonstrating that no other importin alpha or RanGTPase target is required to mediate microtubule assembly in this system. Further, recombinant TPX2 is shown to induce the formation and bundling of microtubules in dilute solutions of pure tubulin. In this purified system, importin alpha prevents TPX2-induced microtubule formation, but not TPX2-tubulin interaction or microtubule bundling. This demonstrates that TPX2 has more than one mode of interaction with tubulin and that only one of these types of interaction is abolished by importin alpha. The data suggest that the critical early function in spindle formation regulated by importin alpha is TPX2-mediated microtubule nucleation.
Self-organisation and forces in the microtubule cytoskeleton.
Nedelec, F., Surrey, T. & Karsenti, E.
Curr Opin Cell Biol. 2003 Feb;15(1):118-24.
Modern microscopy techniques allow us to observe specifically tagged proteins in live cells. We can now see directly that many cellular structures, for example mitotic spindles, are in fact dynamic assemblies. Their apparent stability results from out-of-equilibrium stochastic interactions at the molecular level. Recent studies have shown that the spindles can form even after centrosomes are destroyed, and that they can even form around DNA-coated beads devoid of kinetochores. Moreover, conditions have been produced in which microtubule asters interact even in the absence of chromatin. Together, these observations suggest that the spindle can be experimentally deconstructed, and that its defining characteristics can be studied in a simplified context, in the absence of the full division machinery.
Transient association of titin and myosin with microtubules in nascent myofibrils directed by the MURF2 RING-finger protein.
Pizon, V., Iakovenko, A., Van Der Ven, P.F., Kelly, R., Fatu, C., Furst, D.O., Karsenti, E. & Gautel, M.
J Cell Sci 2002 Dec 1;115(Pt 23):4469-82.
Assembly of muscle sarcomeres is a complex dynamic process and involves a large number of proteins. A growing number of these have regulatory functions and are transiently present in the myofibril. We show here that the novel tubulin-associated RING/B-box protein MURF2 associates transiently with microtubules, myosin and titin during sarcomere assembly. During sarcomere assembly, MURF2 first associates with microtubules at the exclusion of tyrosinated tubulin. Then, MURF2-labelled microtubules associate transiently with sarcomeric myosin and later with A-band titin when non-striated myofibrils differentiate into mature sarcomeres. Finally, MURF2 labelled microtubules disappear from the sarcomere after the incorporation of myosin filaments and the elongation of titin. This suggests that the incorporation of myosin into nascent sarcomeres and the elongation of titin require an active, microtubule-dependent transport process and that MURF2-associated microtubules play a role in the alignment and extension of nascent sarcomeres. MURF2 is expressed in at least four isoforms, of which a 27 kDa isoform is cardiac specific. A C-terminal isoform is generated by alternative reading frame use, a novelty in muscle proteins. In mature cardiac sarcomeres, endogenous MURF2 can associate with the M-band, and is translocated to the nucleus. MURF2 can therefore act as a transient adaptor between microtubules, titin and nascent myosin filaments, as well as being involved in signalling from the sarcomere to the nucleus.
Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells.
Gruss, O.J., Wittmann, M., Yokoyama, H., Pepperkok, R., Kufer, T., Sillje, H., Karsenti, E., Mattaj, I.W. & Vernos, I.
Nat Cell Biol 2002 Nov;4(11):871-9.
In Xenopus laevis egg extracts, TPX2 is required for the Ran-GTP-dependent assembly of microtubules around chromosomes. Here we show that interfering with the function of the human homologue of TPX2 in HeLa cells causes defects in microtubule organization during mitosis. Suppressing the expression of human TPX2 by RNA interference leads to the formation of two microtubule asters that do not interact and do not form a spindle. Our results suggest that in vivo, even in the presence of duplicated centrosomes, spindle formation requires the function of TPX2 to generate a stable bipolar spindle with overlapping antiparallel microtubule arrays. This indicates that chromosome-induced microtubule production is a general requirement for the formation of functional spindles in animal cells.
XMAP215 is required for the microtubule-nucleating activity of centrosomes.
Popov, A.V., Severin, F. & Karsenti, E.
Curr Biol 2002 Aug 6;12(15):1326-30.
Microtubules are essential structures that organize the cytoplasm and form the mitotic spindle. Their number and orientation depend on the rate of nucleation events and their dynamics. Microtubules are often, but not always, nucleated off a single cytoplasmic element, the centrosome. One microtubule-associated protein, XMAP215, is also a resident centrosomal protein. In this study, we have found that XMAP215 is a key component for the microtubule-nucleating activity of centrosomes. We show that depletion of XMAP215 from Xenopus egg extracts impairs their ability to reconstitute the microtubule nucleation potential of salt-stripped centrosomes. We also show that XMAP215 immobilized on polymer beads induces the formation of microtubule asters in egg extracts as well as in solutions of pure tubulin. Formation of asters by XMAP215 beads indicates that this protein is able to anchor nascent microtubules via their minus ends. The aster-forming activity of XMAP215 does not require gamma-tubulin in pure tubulin solutions, but it is gamma-tubulin-dependent in egg extracts. Our results indicate that XMAP215, a resident centrosomal protein, contributes to the microtubule-nucleating activity of centrosomes, suggesting that, in vivo, the formation of asters by centrosomes requires factors additional to gamma-tubulin.
L'auto-organisation au coeur de la division cellulaire.
La Recherche 2002 (9) 52-55
Des Molécules à la cellule: L'origine de la stabilité dynamique dans les systèmes vivants.
Médecine Sciences 2002 18(12) 1173-1174
The mitotic spindle: a self-made machine.
Karsenti, E. & Vernos, I.
Science 2001 Oct 19;294(5542):543-7.
The mitotic spindle is a highly dynamic molecular machine composed of tubulin, motors, and other molecules. It assembles around the chromosomes and distributes the duplicated genome to the daughter cells during mitosis. The biochemical and physical principles that govern the assembly of this machine are still unclear. However, accumulated discoveries indicate that chromosomes play a key role. Apparently, they generate a local cytoplasmic state that supports the nucleation and growth of microtubules. Then soluble and chromosome-associated molecular motors sort them into a bipolar array. The emerging picture is that spindle assembly is governed by a combination of modular principles and that their relative contribution may vary in different cell types and in various organisms.
Physical properties determining self-organization of motors and microtubules.
Surrey, T., Nédélec, F., Leibler, S. & Karsenti, E.
Science 2001 May 11;292(5519):1167-71.
In eukaryotic cells, microtubules and their associated motor proteins can be organized into various large-scale patterns. Using a simplified experimental system combined with computer simulations, we examined how the concentrations and kinetic parameters of the motors contribute to their collective behavior. We observed self-organization of generic steady-state structures such as asters, vortices, and a network of interconnected poles. We identified parameter combinations that determine the generation of each of these structures. In general, this approach may become useful for correlating the morphogenetic phenomena taking place in a biological system with the biophysical characteristics of its constituents.
Ran-GTP coordinates regulation of microtubule nucleation and dynamics during mitotic-spindle assembly.
Carazo-Salas, R.E., Gruss, O.J., Mattaj, I.W. & Karsenti, E.
Nat Cell Biol 2001 Mar;3(3):228-34.
It was recently reported that GTP-bound Ran induces microtubule and pseudo-spindle assembly in mitotic egg extracts in the absence of chromosomes and centrosomes, and that chromosomes induce the assembly of spindle microtubules in these extracts through generation of Ran-GTP. Here we examine the effects of Ran-GTP on microtubule nucleation and dynamics and show that Ran-GTP has independent effects on both the nucleation activity of centrosomes and the stability of centrosomal microtubules. We also show that inhibition of Ran-GTP production, even in the presence of duplicated centrosomes and kinetochores, prevents assembly of a bipolar spindle in M-phase extracts.
XMAP215 regulates microtubule dynamics through two distinct domains.
Popov, A.V., Pozniakovsky, A., Arnal, I., Antony, C., Ashford, A.J., Kinoshita, K., Tournebize, R., Hyman, A.A. & Karsenti, E.
EMBO J 2001 Feb 1;20(3):397-410.
XMAP215 belongs to a family of proteins involved in the regulation of microtubule dynamics. In this study we analyze the function of different parts of XMAP215 in vivo and in Xenopus egg extracts. XMAP215 has been divided into three fragments, FrN, FrM and FrC (for N-terminal, middle and C-terminal, respectively). FrN co-localizes with microtubules in egg extracts but not in cells, FrC co- localizes with microtubules and centrosomes both in egg extracts and in cells, while FrM does not co- localize with either centrosomes or microtubules. In Xenopus egg extracts, FrN stimulates microtubule growth at plus-ends by inhibiting catastrophes, while FrM has no effect, and FrC suppresses microtubule growth by promoting catastrophes. Our results suggest that XMAP215 is targeted to centrosomes and microtubules mainly through its C-terminal domain, while the evolutionarily conserved N-terminal domain contains its microtubule-stabilizing activity.
Ran induces spindle assembly by reversing the inhibitory effect of importin alpha on TPX2 activity.
Gruss, O.J., Carazo-Salas, R.E., Schatz, C.A., Guarguaglini, G., Kast, J., Wilm, M., Le Bot, N., Vernos, I., Karsenti, E. & Mattaj, I.W.
Cell 2001 Jan 12;104(1):83-93.
The small GTPase Ran, bound to GTP, is required for the induction of spindle formation by chromosomes in M phase. High concentrations of Ran.GTP are proposed to surround M phase chromatin. We show that the action of Ran.GTP in spindle formation requires TPX2, a microtubule-associated protein previously known to target a motor protein, Xklp2, to microtubules. TPX2 is normally inactivated by binding to the nuclear import factor, importin alpha, and is displaced from importin alpha by the action of Ran.GTP. TPX2 is required for Ran.GTP and chromatin-induced microtubule assembly in M phase extracts and mediates spontaneous microtubule assembly when present in excess over free importin alpha. Thus, components of the nuclear transport machinery serve to regulate spindle formation in M phase.
Cell form as an essential determinant of cell function.
Academic Press, San Diego
The use of dominant negative mutants to study the function of mitotic motors in the in vitro spindle assembly assay in Xenopus egg extracts.
Boleti, H., Karsenti, E. & Vernos, I.
Methods Mol Biol 2001;164:173-89. Europe PMC
Xkid, a chromokinesin required for chromosome alignment on the metaphase plate.
Antonio, C., Ferby, I., Wilhelm, H., Jones, M., Karsenti, E., Nebreda, A.R. & Vernos, I.
Cell 2000 Aug 18;102(4):425-35
Metaphase chromosome alignment is a key step of animal cell mitosis. The molecular mechanism leading to this equatorial positioning is still not fully understood. Forces exerted at kinetochores and on chromosome arms drive chromosome movements that culminate in their alignment on the metaphase plate. In this paper, we show that Xkid, a kinesin-like protein localized on chromosome arms, plays an essential role in metaphase chromosome alignment and in its maintenance. We propose that Xkid is responsible for the polar ejection forces acting on chromosome arms. Our results show that these forces are essential to ensure that kinetochores and chromosome arms align on a narrow equatorial plate during metaphase, a prerequisite for proper chromosome segregation.
TPX2, A novel xenopus MAP involved in spindle pole organization
Wittmann, T., Wilm, M., Karsenti, E. & Vernos, I.
J Cell Biol 2000 Jun 26;149(7):1405-18
TPX2, the targeting protein for Xenopus kinesin-like protein 2 (Xklp2), was identified as a microtubule-associated protein that mediates the binding of the COOH-terminal domain of Xklp2 to microtubules (Wittmann, T., H. Boleti, C. Antony, E. Karsenti, and I. Vernos. 1998. J. Cell Biol. 143:673-685). Here, we report the cloning and functional characterization of Xenopus TPX2. TPX2 is a novel, basic 82.4-kD protein that is phosphorylated during mitosis in a microtubule-dependent way. TPX2 is nuclear during interphase and becomes localized to spindle poles in mitosis. Spindle pole localization of TPX2 requires the activity of the dynein-dynactin complex. In late anaphase TPX2 becomes relocalized from the spindle poles to the midbody. TPX2 is highly homologous to a human protein of unknown function and thus defines a new family of vertebrate spindle pole components. We investigated the function of TPX2 using spindle assembly in Xenopus egg extracts. Immunodepletion of TPX2 from mitotic egg extracts resulted in bipolar structures with disintegrating poles and a decreased microtubule density. Addition of an excess of TPX2 to spindle assembly reactions gave rise to monopolar structures with abnormally enlarged poles. We conclude that, in addition to its function in targeting Xklp2 to microtubule minus ends during mitosis, TPX2 also participates in the organization of spindle poles.
Structural transitions at microtubule ends correlate with their dynamic properties in Xenopus egg extracts.
Arnal, I., Karsenti, E. & Hyman, A.A.
J Cell Biol 2000 May 15;149(4):767-74
Microtubules are dynamically unstable polymers that interconvert stochastically between growing and shrinking states by the addition and loss of subunits from their ends. However, there is little experimental data on the relationship between microtubule end structure and the regulation of dynamic instability. To investigate this relationship, we have modulated dynamic instability in Xenopus egg extracts by adding a catastrophe-promoting factor, Op18/stathmin. Using electron cryomicroscopy, we find that microtubules in cytoplasmic extracts grow by the extension of a two- dimensional sheet of protofilaments, which later closes into a tube. Increasing the catastrophe frequency by the addition of Op18/stathmin decreases both the length and frequency of the occurrence of sheets and increases the number of frayed ends. Interestingly, we also find that more dynamic populations contain more blunt ends, suggesting that these are a metastable intermediate between shrinking and growing microtubules. Our results demonstrate for the first time that microtubule assembly in physiological conditions is a two-dimensional process, and they suggest that the two-dimensional sheets stabilize microtubules against catastrophes. We present a model in which the frequency of catastrophes is directly correlated with the structural state of microtubule ends.
Control of microtubule dynamics by the antagonistic activities of XMAP215 and XKCM1 in Xenopus egg extracts.
Tournebize, R., Popov, A., Kinoshita, K., Ashford, A.J., Rybina, S., Pozniakovsky, A., Mayer, T.U., Walczak, C.E., Karsenti, E. & Hyman, A.A.
Nat Cell Biol 2000 Jan;2(1):13-19
Microtubules are dynamic polymers that move stochastically between periods of growth and shrinkage, a property known as dynamic instability. Here, to investigate the mechanisms regulating microtubule dynamics in Xenopus egg extracts, we have cloned the complementary DNA encoding the microtubule-associated protein XMAP215 and investigated the function of the XMAP215 protein. Immunodepletion of XMAP215 indicated that it is a major microtubule-stabilizing factor in Xenopus egg extracts. During interphase, XMAP215 stabilizes microtubules primarily by opposing the activity of the destabilizing factor XKCM1, a member of the kinesin superfamily. These results indicate that microtubule dynamics in Xenopus egg extracts are regulated by a balance between a stabilizing factor, XMAP215, and a destabilizing factor, XKCM1.
Le mouvement: de l'aleatoire au determinisme.
Medecine Sciences 2000 16 719-721
Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation.
Carazo-Salas, R.E., Guarguaglini, G., Gruss, O.J., Segref, A., Karsenti, E. & Mattaj, I.W.
Nature 1999 Jul 8;400(6740):178-81
Chromosomes are segregated by two antiparallel arrays of microtubules arranged to form the spindle apparatus. During cell division, the nucleation of cytosolic microtubules is prevented and spindle microtubules nucleate from centrosomes (in mitotic animal cells) or around chromosomes (in plants and some meiotic cells). The molecular mechanism by which chromosomes induce local microtubule nucleation in the absence of centrosomes is unknown, but it can be studied by adding chromatin beads to Xenopus egg extracts. The beads nucleate microtubules that eventually reorganize into a bipolar spindle. RCC1, the guanine-nucleotide-exchange factor for the GTPase protein Ran, is a component of chromatin. Using the chromatin bead assay, we show here that the activity of chromosome-associated RCC1 protein is required for spindle formation. Ran itself, when in the GTP-bound state (Ran-GTP), induces microtubule nucleation and spindle-like structures in M-phase extract. We propose that RCC1 generates a high local concentration of Ran-GTP around chromatin which in turn induces the local nucleation of microtubules.
Tobacco BY-2 cell-free extracts induce the recovery of microtubule nucleating activity of inactivated mammalian centrosomes.
Stoppin-Mellet, V., Peter, C., Buendia, B., Karsenti, E. & Lambert, A.M.
Biochim Biophys Acta 1999 Feb 4;1449(1):101-6
The structure and the molecular composition of the microtubule- organizing centers in acentriolar higher plant cells remain unknown. We developed an in vitro complementation assay where tobacco BY-2 extracts can restore the microtubule-nucleating activity of urea-inactivated mammalian centrosomes. Our results provide first evidence that soluble microtubule-nucleating factors are present in the plant cytosolic fraction. The implication for microtubule nucleation in higher plants is discussed.
La forma come fattore determinante della funzione cellulare.
In "Enciclopedia Italiana ed. Frontiere Della Vita" E. Treccani (ed.). Vol. II. 1999, Roma, pp. 77-98
Role of xklp3, a subunit of the Xenopus kinesin II heterotrimeric complex, in membrane transport between the endoplasmic reticulum and the Golgi apparatus.
Le Bot, N., Antony, C., White, J., Karsenti, E. & Vernos, I.
J Cell Biol 1998 Dec 14;143(6):1559-73.
The function of the Golgi apparatus is to modify proteins and lipids synthesized in the ER and sort them to their final destination. The steady-state size and function of the Golgi apparatus is maintained through the recycling of some components back to the ER. Several lines of evidence indicate that the spatial segregation between the ER and the Golgi apparatus as well as trafficking between these two compartments require both microtubules and motors. We have cloned and characterized a new Xenopus kinesin like protein, Xklp3, a subunit of the heterotrimeric Kinesin II. By immunofluorescence it is found in the Golgi region. A more detailed analysis by EM shows that it is associated with a subset of membranes that contain the KDEL receptor and are localized between the ER and Golgi apparatus. An association of Xklp3 with the recycling compartment is further supported by a biochemical analysis and the behavior of Xklp3 in BFA-treated cells. The function of Xklp3 was analyzed by transfecting cells with a dominant-negative form lacking the motor domain. In these cells, the normal delivery of newly synthesized proteins to the Golgi apparatus is blocked. Taken together, these results indicate that Xklp3 is involved in the transport of tubular-vesicular elements between the ER and the Golgi apparatus.
Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubule-associated protein, and dynein.
Wittmann, T., Boleti, H., Antony, C., Karsenti, E. & Vernos, I.
J Cell Biol 1998 Nov 2;143(3):673-85.
Xklp2 is a plus end-directed Xenopus kinesin-like protein localized at spindle poles and required for centrosome separation during spindle assembly in Xenopus egg extracts. A glutathione-S-transferase fusion protein containing the COOH-terminal domain of Xklp2 (GST-Xklp2-Tail) was previously found to localize to spindle poles (Boleti, H., E. Karsenti, and I. Vernos. 1996. Cell. 84:49-59). Now, we have examined the mechanism of localization of GST-Xklp2-Tail. Immunofluorescence and electron microscopy showed that Xklp2 and GST-Xklp2-Tail localize specifically to the minus ends of spindle pole and aster microtubules in mitotic, but not in interphase, Xenopus egg extracts. We found that dimerization and a COOH-terminal leucine zipper are required for this localization: a single point mutation in the leucine zipper prevented targeting. The mechanism of localization is complex and two additional factors in mitotic egg extracts are required for the targeting of GST-Xklp2-Tail to microtubule minus ends: (a) a novel 100-kD microtubule-associated protein that we named TPX2 (Targeting protein for Xklp2) that mediates the binding of GST-Xklp2-Tail to microtubules and (b) the dynein-dynactin complex that is required for the accumulation of GST-Xklp2-Tail at microtubule minus ends. We propose two molecular mechanisms that could account for the localization of Xklp2 to microtubule minus ends.
The role of nucleation in patterning microtubule networks.
Hyman, A.A. & Karsenti, E.
This is a review article.
J Cell Sci 1998 Aug 111 ( Pt 15) 2077-2083
Control of microtubule nucleation is important for many microtubule dependent processes in cells. Traditionally, research has focused on nucleation of microtubules from centrosomes. However, it is clear that microtubules can nucleate from non-centrosome dependent sites. In this review we discuss the consequences of non-centrosome dependent microtubule nucleation for formation of microtubule patterns, concentrating on the assembly of mitotic spindles.
A model for the proposed roles of different microtubule-based motor proteins in establishing spindle bipolarity.
Walczak, C.E., Vernos, I., Mitchison, T.J., Karsenti, E. & Heald, R.
Curr Biol 1998 Jul 30-Aug 13;8(16):903-13.
BACKGROUND: In eukaryotes, assembly of the mitotic spindle requires the interaction of chromosomes with microtubules. During this process, several motor proteins that move along microtubules promote formation of a bipolar microtubule array, but the precise mechanism is unclear. In order to examine the roles of different motor proteins in building a bipolar spindle, we have used a simplified system in which spindles assemble around beads coated with plasmid DNA and incubated in extracts from Xenopus eggs. Using this system, we can study spindle assembly in the absence of paired cues, such as centrosomes and kinetochores, whose microtubule-organizing properties might mask the action of motor proteins. RESULTS: We blocked the function of individual motor proteins in the Xenopus extracts using specific antibodies. Inhibition of Xenopus kinesin-like protein 1 (Xklp1) led either to the dissociation of chromatin beads from microtubule arrays, or to collapsed microtubule bundles on beads. Inhibition of Eg5 resulted in monopolar microtubule arrays emanating from chromatin beads. Addition of antibodies against dynein inhibited the focusing of microtubule ends into spindle poles in a dose-dependent manner. Inhibition of Xenopus carboxy-terminal kinesin 2 (XCTK2) affected both pole formation and spindle stability. Co-inhibition of XCTK2 and dynein dramatically increased the severity of spindle pole defects. Inhibition of Xklp2 caused only minor spindle pole defects. CONCLUSIONS: Multiple microtubule-based motor activities are required for the bipolar organization of microtubules around chromatin beads, and we propose a model for the roles of the individual motor proteins in this process.
Microtubule dynamics, molecular motors and chromosome behavior.
Vernos, I. & Karsenti, E.
In "Dynamics of Cell Division", Endow, S. & Glover, D. (eds.), 1998, Oxford University Press, New York, pp. 97-123
In vitro assays for mitotic spindle assembly and function.
Heald, R., Tournebize, R., Vernos, I., Murray, A., Hyman, A. & Karsenti, E.
In "Cell Biology: A laboratory handbook", 2nd Ed., Vol. 2., Celis, J. (ed.), 1998, Academic Press, pp. 326-335
Three-dimensional reconstruction of the mammalian centriole from cryoelectron micrographs: the use of common lines for orientation and alignment.
Kenney, J., Karsenti, E., Gowen, B. & Fuller, S.D.
J Struct Biol 1997 Dec;120(3):320-8
The microtubule organizing center of the animal cell (S. D. Fuller et al., 1992, Curr. Opin. Struct. Biol. 2, 264-274; D. M. Glover et al., 1993, Sci. Am. 268, 62-68; E. B. Wilson, 1925), (The Cell in Development and Heredity) comprises two centrioles and the pericentriolar material. We have completed several three-dimensional reconstructions of individual centrioles from tilt series of cryoelectron micrographs. The reconstruction procedure uses minimization of the common lines residual to define the orientation of the centriolar minefold symmetry axis and then uses this symmetry to generate a structure by weighted backprojection to 28-nm resolution. Many of the features of these reconstructions agree with previous, conventional transmission electron microscopy studies (M. Paintrand et al., 1992, J. Struct. Biol. 108, 107-128). The microtubule barrel of the centriole is roughly 500 nm long and 300 nm in diameter and the microtubule bundles appear to taper toward the distal end. In addition, we see a handedness to the pericentriolar material at the base (distal end) of the centriole which is opposite to the skew of the microtubule triplets. The region at which the microtubule barrel joins this base is intriguingly complex and includes an internal cylindrical feature which is a site of gamma tubulin localization.
Reconstruction of the centrosome cycle from cryoelectron micrographs.
Chretien, D., Buendia, B., Fuller, S.D. & Karsenti, E.
J Struct Biol 1997 Nov;120(2):117-33
The absence of detailed in vitro studies leaves the molecular events involved in the centrosome cycle poorly characterized. Most earlier studies have employed electron microscopy of thin or thick sections of cells. Here we have analyzed the structure of centrosomes isolated from nonsynchronized human lymphoblastic KE37 cells using cryoelectron microscopy of vitrified specimens. The centrosomes were classified into five categories depending on the number of centrioles (one or two), the respective orientation of the two centrioles in a pair (orthogonal or disoriented), and the presence or absence of appendages at the distal extremity of the centrioles (referred to as mature and immature, respectively). A detailed analysis of the centriole dimensions in these categories allowed us to reconstruct the centrosome cycle in KE37 cells. Our results suggest that centriole assembly is completed only when the mother centriole of an immature orthogonal pair separates from its daughter in preparation to centrosome duplication. Our study shows that an in vitro approach based on cryoelectron microscopy of vitrified specimens can be used to obtain detailed structural information on the centrosome cycle. Copyright 1997 Academic Press.
Mitotic chromatin regulates phosphorylation of Stathmin/Op18.
Andersen, S.S., Ashford, A.J., Tournebize, R., Gavet, O., Sobel, A., Hyman, A.A. & Karsenti, E.
Nature 1997 Oct 9 389(6651) 640-643
Meiotic and mitotic spindles are required for the even segregation of duplicated chromosomes to the two daughter cells. The mechanism of spindle assembly is not fully understood, but two have been proposed that are not mutually exclusive. The 'search and capture' model suggests that dynamic microtubules become progressively captured and stabilized by the kinetochores on chromosomes, leading to spindle assembly. The 'local stabilization' model proposes that chromosomes change the state of the cytoplasm around them, making it more favourable to microtubule polymerization. It has been shown that Stathmin/Op18 inhibits microtubule polymerization in vitro by interaction with tubulin, and that overexpression in tissue culture cells of non-phosphorylatable mutants of Stathmin/Op18 prevents the assembly of mitotic spindles. We have used Xenopus egg extracts and magnetic chromatin beads to show that mitotic chromatin induces phosphorylation of Stathmin/Op18. We have also shown that Stathmin/Op18 is one of the factors regulated by mitotic chromatin that governs preferential microtubule growth around chromosomes during spindle assembly.
XMAP310: a Xenopus rescue-promoting factor localized to the mitotic spindle.
Andersen, S.S. & Karsenti, E.
J Cell Biol. 1997 Nov 17;139(4):975-83.
To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Dominguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289-1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.
Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts
Heald, R., Tournebize, R., Blank, T., Sandaltzopoulos, R., Becker, P., Hyman, A.A. & Karsenti, E.
Nature 1996 Aug 1 382(6590) 420-425
Functional nuclei and mitotic spindles are shown to assemble around DNA- coated beads incubated in Xenopus egg extracts. Bipolar spindles assemble in the absence of centrosomes and kinetochores, indicating that bipolarity is an intrinsic property of microtubules assembling around chromatin in a mitotic cytoplasm. Microtubules nucleated at dispersed sites with random polarity rearrange into two arrays of uniform polarity. Spindle-pole formation requires cytoplasmic dynein- dependent translocation of microtubules across one another. It is proposed that spindles form in the absence of centrosomes by motor- dependent sorting of microtubules according to their polarity.
Morphogenetic properties of microtubules and mitotic spindle assembly.
Hyman, A.A. & Karsenti, E.
This is a review article.
Cell 1996 Feb 9 84(3) 401-410 Europe PMC
Xklp2, a novel Xenopus centrosomal kinesin-like protein required for centrosome separation during mitosis.
Boleti, H., Karsenti, E. & Vernos, I.
Cell 1996 Jan 12 84(1) 49-59
We describe a novel Xenopus plus end-directed kinesin-like protein (KLP), Xklp2, localized on centrosomes throughout the cell cycle and on spindle pole microtubules during metaphase. Using mitotic spindles assembled in Xenopus egg extracts and different recombinant GST-Xklp2 mutants, we show that this motor is targeted to spindle poles through its C-terminal domain. Xklp2-truncated polypeptides lacking the motor domain block centrosome separation and disrupt preassembled metaphase spindles. Antibodies directed against the tail of Xklp2 have a similar effect. These results show that Xklp2 protein is required for centrosome separation and maintenance of spindle bipolarity. This study is an example of the application of the dominant negative mutant effect on spindle assembly in Xenopus egg extracts, demonstrating the usefulness of this approach in probing the function of proteins in this system.
Motors involved in spindle assembly and chromosome segregation.
Vernos, I. & Karsenti, E.
Curr Opin Cell Biol. 1996 Feb;8(1):4-9.
During the past two years, major advances have been made in our understanding of the role of motor proteins in chromosome-microtubule interactions in the spindle. The discovery of kinesin-like proteins (KLPs) associated with chromosome arms has shed some light on the mechanism of chromosome congression and the establishment of spindle bipolarity. Recent results also indicate that kinetochore KLPs may tether the ends of growing and shrinking microtubules to kinetochores during chromosome movements. Finally, new data indicate that phosphorylation of KLPs may be one of the mechanisms by which they are targeted to specific spindle domains.
The role of microtubule dependent motors in centrosome movements and spindle pole organization during mitosis.
Karsenti, E., Boleti, H. & Vernos, I.
Sem. Cell Biol. 1996 7 367-378
Chromosomes take the lead in spindle assembly.
Vernos, I. & Karsenti, E.
Trends Cell Biol 1995 Aug;5(8):297-301.
Several kinesin-like motor proteins have recently been found associated with chromosome arms. They seem to be involved in the so-called 'polar ejection forces' that contribute to the congression of chromosomes on the metaphase plate, and at least one of them is essential for the maintenance of spindle bipolarity. The discovery of these molecules changes our view of the mechanism of spindle assembly and chromosome movement.
Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates.
Chretien, D., Fuller, S.D. & Karsenti, E.
J Cell Biol 1995 Jun;129(5):1311-28
Observation of microtubule growth at different rates by cryo-electron microscopy reveals that the ends range from blunt to long, gently curved sheets. The mean sheet length increases with the growth rate while the width of the distributions increases with the extent of assembly. The combination of a concentration dependent growth rate of the tubulin sheet with a variable closure rate of the microtubule cylinder, results in a model in which stochastic fluctuations in sheet length and tubulin conformation confine GTP-tubulins to microtubule ends. We propose that the variability of microtubule growth rate observed by video microscopy (Gildersleeve, R. F., A. R. Cross, K. E. Cullen, A. P. Fagen, and R. C. Williams. 1992. J. Biol. Chem. 267: 7995- 8006, and this study) is due to the variation in the rate of cylinder closure. The curvature of the sheets at the end of growing microtubules and the small oligomeric structures observed at the end of disassembling microtubules, indicate that tubulin molecules undergo conformational changes both during assembly and disassembly.
Xklp1, a chromosomal Xenopus kinesin-like protein essential for spindle organization and chromosome positioning.
Vernos, I., Raats, J., Hirano, T., Heasman, J., Karsenti, E. & Wylie, C.
Cell 1995 Apr 7 81(1) 117-127
Xklp1 is a novel Xenopus kinesin-like protein with a motor domain at the amino terminus, nuclear localization sequences in the stalk, and a putative zinc finger-like sequence in the tail. It is nuclear during interphase and chromosomal during mitosis. During late anaphase, a fraction of the protein relocalizes to the spindle interzone and accumulates in the midbody during telophase. Depletion of Xklp1 protein by antisense oligo knockout in oocytes leads to defective mitosis during the first cell cycles following fertilization. The bipolarity of spindles assembled in vitro in the presence of anti-Xklp1 antibodies is unstable, and the chromosomes fail to congress on the metaphase plate.
Approaching microtubule structure with the scanning tunneling microscope (STM).
Maaloum, M., Chretien, D., Karsenti, E. & Horber, J.K.H.
J Cell Sci 1994 Nov 107 ( Pt 11) 3127-3131
We demonstrate that the scanning tunneling microscope can be used to obtain information about arrangement of tubulin subunits in the microtubule wall. Long rows of subunits with a periodicity of 3.8 +/- 0.4 nm were clearly visible in the images of microtubules. The separation between the rows of subunits was 4.8 +/- 0.4 nm. Close inspection of two images revealed another periodicity of 7.8 +/- 0.4 nm in the contour levels of the protofilaments. This indicates that alpha and beta tubulin monomers can be resolved. In these areas the monomers were arranged according to a 'B-type' lattice. Scanning tunneling microscope images confirm that the lateral contacts between tubulin monomers in adjacent protofilaments are compatible with a three-start, left-handed helix model. This study demonstrates that scanning tunneling microscopy can give direct information on the structure and organization of macromolecular assemblies and can complement the classical methods of electron microscopy and X-ray scattering.
Effect on microtubule dynamics of XMAP230, a microtubule-associated protein present in Xenopus laevis eggs and dividing cells.
Andersen, S.S., Buendia, B., Dominguez, J.E., Sawyer, A. & Karsenti, E.
J Cell Biol. 1994 Dec;127(5):1289-99.
The reorganization from a radial [corrected] interphase microtubule (MT) network into a bipolar spindle at the onset of mitosis involves a dramatic change in MT dynamics. Microtubule-associated proteins (MAPs) and other factors are thought to regulate MT dynamics both in interphase and in mitosis. In this study we report the purification and functional in vitro characterization of a 230-KD MAP from Xenopus egg extract (XMAP230). This protein is present in eggs, oocytes, testis and a Xenopus tissue culture cell line. It is apparently absent from non-dividing cells in which an immunologically related 200-kD protein is found. XMAP230 is composed of two isoforms with slightly different molecular masses and pIs. It is localized to interphase MTs, dissociates from MTs at the onset of prophase and specifically binds to spindle MTs during metaphase and anaphase. The dissociation constant of XMAP230 is 500 nM, the stoichiometry of binding to MTs is between 1:8 and 1:4, and the in vivo concentration is approximately 200 nM. Both isoforms are phosphorylated and have reduced affinity for microtubules in mitotic extracts. Analysis of the effect of XMAP230 on MT dynamics by video microscopy shows that it increases the growth rate, decreases the shrinking rate of MTs and strongly suppresses catastrophes. These results suggest that in vivo, XMAP230 participates in the control of the MT elongation rate, stabilizes MTs and locally modulates MT dynamics during mitosis.
Lattice defects in microtubules: protofilament numbers vary within individual microtubules.
Chretien, D., Metoz, F., Verde, F., Karsenti, E. & Wade, R.H.
J Cell Biol 1992 Jun;117(5):1031-40
We have used cryo-electron microscopy of vitrified specimens to study microtubules assembled both from three cycle purified tubulin (3x- tubulin) and in cell free extracts of Xenopus eggs. In vitro assembled 3x-tubulin samples have a majority of microtubules with 14 protofilaments whereas in cell extracts most microtubules have 13 protofilaments. Microtubule polymorphism was observed in both cases. The number of protofilaments can change abruptly along individual microtubules usually by single increments but double increments also occur. For 3x-tubulin, increasing the magnesium concentration decreases the proportion of 14 protofilament microtubules and decreases the average separation between transitions in these microtubules. Protofilament discontinuities may correspond to dislocation-like defects in the microtubule surface lattice.
Role of type 1 and type 2A protein phosphatases in the cell cycle.
Karsenti, E., Verde, F. & Felix, M.A.
Adv. Prot. Phosph. 1991 6 453-482