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Epithelial cells migrate across wounds to repair injured tissue. Leader cells at the front of migrating sheets often drive this process. However, it is unclear how leaders emerge from an apparently homogeneous epithelial cell population. We characterized leaders emerging from epithelial monolayers in cell culture and found that they activated the stress sensor p53, which was sufficient to initiate leader cell behavior. p53 activated the cell cycle inhibitor p21 WAF1/CIP1 , which in turn induced leader behavior through inhibition of cyclin-dependent kinase activity. p53 also induced crowding hypersensitivity in leader cells such that, upon epithelial closure, they were eliminated by cell competition. Thus, mechanically induced p53 directs emergence of a transient population of leader cells that drive migration and ensures their clearance upon epithelial repair.

In the past 15 years, cell-based microscopy has evolved its focus from observing cell function to aiming to predict it. In particular—powered by breakthroughs in computer vision, large-scale image analysis and machine learning—high-throughput and high-content microscopy imaging have enabled to uniquely harness single-cell information to systematically discover and annotate genes and regulatory pathways, uncover systems-level interactions and causal links between cellular processes, and begin to clarify and predict causal cellular behaviour and decision making. Here we review these developments, discuss emerging trends in the field, and describe how single-cell ‘omics and single-cell microscopy are imminently in an intersecting trajectory. The marriage of these two fields will make possible an unprecedented understanding of cell and tissue behaviour and function.

SUMMARYPredicting how stem cells become patterned and differentiated into target tissues is key for optimising human tissue design. Here, we established DEEP-MAP - for deep learning-enhanced morphological profiling - an approach that integrates single-cell, multi-day, multi-colour microscopy phenomics with deep learning and allows to robustly map and predict cell fate dynamics in real-time without a need for cell state-specific reporters. Using human pluripotent stem cells (hPSCs) engineered to co-express the histone H2B and two-colour FUCCI cell cycle reporters, we used DEEP-MAP to capture hundreds of morphological- and proliferation-associated features for hundreds of thousands of cells and used this information to map and predict spatiotemporally single-cell fate dynamics across germ layer cell fates. We show that DEEP-MAP predicts fate changes as early or earlier than transcription factor-based fate reporters, reveals the timing and existence of intermediate cell fates invisible to fixed-cell technologies, and identifies proliferative properties predictive of cell fate transitions. DEEP-MAP provides a versatile, universal strategy to map tissue evolution and organisation across many developmental and tissue engineering contexts.

Access to primary research data is vital for the advancement of science. To extend the data types supported by community repositories, we built a prototype Image Data Resource (IDR) that collects and integrates imaging data acquired across many different imaging modalities. IDR links data from several imaging modalities, including high-content screening, super-resolution and time-lapse microscopy, digital pathology, public genetic or chemical databases, and cell and tissue phenotypes expressed using controlled ontologies. Using this integration, IDR facilitates the analysis of gene networks and reveals functional interactions that are inaccessible to individual studies. To enable re-analysis, we also established a computational resource based on Jupyter notebooks that allows remote access to the entire IDR. IDR is also an open source platform that others can use to publish their own image data. Thus IDR provides both a novel on-line resource and a software infrastructure that promotes and extends publication and re-analysis of scientific image data.

A key feature of cells is the capacity to activate new functional polarized domains contemporaneously to pre-existing ones. How cells accomplish this is not clear. Here, we show that in fission yeast inhibition of cell polarity at pre-existing domains of polarized cell growth is required to activate new growth. This inhibition is mediated by the ERM-related polarity factor Tea3, which antagonizes the activation of the Rho-GTPase Cdc42 by its co-factor Scd2. We demonstrate that Tea3 acts in a phosphorylation-dependent manner controlled by the PAK kinase Shk1 and that, like Scd2, Tea3 is direct substrate of Shk1. Importantly, we show that Tea3 and Scd2 compete for their binding to Shk1 in vitro, indicating that their biochemical competition for Shk1 underpins their antagonistic roles in controlling polarity. Mathematical modelling of an 9inhibitor-activator9 system mimicking the Tea3-Scd2 antagonism suggests that it can account for several major features of polarity control observed in cells, including the anti-correlated localizations of Tea3 and cell growth areas observed throughout the cell cycle, controlling the period of GTP-Cdc42 oscillations at the cell cortex and modulating the time at which cells trigger new polarized growth. Thus, by preventing pre-existing growth domains from becoming overpowering, polarity inhibition plays a key role in allowing cells to redistribute their polarity-activating machinery to prospective growth sites and control the timing of new growth activation - explaining how a polarity inhibitor can serve as an activator of new polarity zone formation.

In this paper, we propose a novel application of Generative Adversarial Networks (GAN) to the synthesis of cells imaged by fluorescence microscopy. Compared to natural images, cells tend to have a simpler and more geometric global structure that facilitates image generation. However, the correlation between the spatial pattern of different fluorescent proteins reflects important biological functions, and synthesized images have to capture these relationships to be relevant for biological applications. We adapt GANs to the task at hand and propose new models with casual dependencies between image channels that can generate multi-channel images, which would be impossible to obtain experimentally. We evaluate our approach using two independent techniques and compare it against sensible baselines. Finally, we demonstrate that by interpolating across the latent space we can mimic the known changes in protein localization that occur through time during the cell cycle, allowing us to predict temporal evolution from static images., Comment: The paper appearing at the International Conference on Computer Vision (ICCV) 2017 + its supplementary materials

A key goal of functional genomics is to elucidate how genes and proteins act together in space and time, wired as pathways, to control specific aspects of cell biological function. Here, we develop a method to quantitatively determine proteins’ localization interdependencies at high throughput. We show that this method can be used to systematically obtain weighted, signed and directional pathway relationships, and hence to reconstruct a detailed pathway wiring. As proof-of-principle, we focus on 42 factors that control cell polarity in fission yeast(Schizosaccharomyces pombe)and use high-throughput confocal microscopy and quantitative image analysis to reconstruct their Localization Interdependency Network (LIN). Through this approach we identify 554 pairwise interactions across the factors, including 98% putative new directed links. Validation of an unexpected interaction between two polarity factor subgroups - the polarity landmark proteins and the cell integrity pathway components - by orthogonal phenotyping demonstrates the power of the LIN approach in detecting subtle, systems-level causal connections.

We use micropatterning and strain engineering to encapsulate single living mammalian cells into transparent tubular architectures consisting of three-dimensional (3D) rolled-up nanomembranes. By using optical microscopy, we demonstrate that these structures are suitable for the scrutiny of cellular dynamics within confined 3D-microenvironments. We show that spatial confinement of mitotic mammalian cells inside tubular architectures can perturb metaphase plate formation, delay mitotic progression, and cause chromosomal instability in both a transformed and nontransformed human cell line. These findings could provide important clues into how spatial constraints dictate cellular behavior and function.

Achieving the promises of stem cell science to generate precise disease models and designer cell samples for personalized therapeutics will require harnessing pheno-genotypic cell-level data quantitatively and predictively in the lab and clinic. Those requirements could be met by developing a Big-Data-driven stem cell science strategy and community.

Access to primary research data is vital for the advancement of science. To extend the data types supported by community repositories, we built a prototype Image Data Resource (IDR) that collects and integrates imaging data acquired across many different imaging modalities. IDR links data from several imaging modalities, including high-content screening, super-resolution and time-lapse microscopy, digital pathology, public genetic or chemical databases, and cell and tissue phenotypes expressed using controlled ontologies. Using this integration, IDR facilitates the analysis of gene networks and reveals functional interactions that are inaccessible to individual studies. To enable re-analysis, we also established a computational resource based on Jupyter notebooks that allows remote access to the entire IDR. IDR is also an open source platform that others can use to publish their own image data. Thus IDR provides both a novel on-line resource and a software infrastructure that promotes and extends publication and re-analysis of scientific image data.

The amazing structural variety of cells is matched only by their functional diversity, and reflects the complex interplay between biochemical and mechanical regulation. How both regulatory layers generate specifically shaped cellular domains is not fully understood. Here, we report how cell growth domains are shaped in fission yeast. Based on quantitative analysis of cell wall expansion and elasticity, we develop a model for how mechanics and cell wall assembly interact and use it to look for factors underpinning growth domain morphogenesis. Surprisingly, we find that neither the global cell shape regulators Cdc42-Scd1-Scd2 nor the major cell wall synthesis regulators Bgs1-Bgs4-Rgf1 are reliable predictors of growth domain geometry. Instead, their geometry can be defined by cell wall mechanics and the cortical localization pattern of the exocytic factors Sec6-Syb1-Exo70. Forceful re-directioning of exocytic vesicle fusion to broader cortical areas induces proportional shape changes to growth domains, demonstrating that both features are causally linked., Cell shape is determined by a combination of biochemical regulation and mechanical forces. By imaging the dynamic behaviour of growth regulatory proteins in fission yeast and integrating these data within a mechanical model, Abenza et al. find that exocytosis plays a dominant role in shaping growth domains.

Microtubule organization is key to eukaryotic cell structure and function. In most animal cells, interphase microtubules organize around the centrosome, the major microtubule organizing centre (MTOC). Interphase microtubules can also become organized independently of a centrosome, but how acentrosomal microtubules arrays form and whether they are functionally equivalent to centrosomal arrays remains poorly understood. Here, we show that the interphase microtubule arrays of fission yeast cells can persist independently of nuclear-associated MTOCs, including the spindle pole body (SPB)--the centrosomal equivalent. By artificially enucleating cells, we show that arrays can form de novo (self-organize) without nuclear-associated MTOCs, but require the microtubule nucleator mod20-mbo1-mto1 (refs 3-5), the bundling factor ase1 (refs 6,7), and the kinesin klp2 (refs 8,9). Microtubule arrays in enucleated and nucleated cells are morphologically indistinguishable and similarly locate to the cellular axis and centre. By simultaneously tracking nuclear-independent and SPB-associated microtubule arrays within individual nucleated cells, we show that both define the cell centre with comparable precision. We propose that in fission yeast, nuclear-independent, self-organized, acentrosomal microtubule arrays are structurally and functionally equivalent to centrosomal arrays.

Hereditary spastic paraplegias (HSPs) are neurodegenerative diseases caused by mutations in more than 20 genes, which lead to progressive spasticity and weakness of the lower limbs. The most frequently mutated gene causing autosomal dominant HSP is SPG4, which encodes spastin, a protein that belongs to the family of ATPases associated with various cellular activities (AAAs). A number of studies have suggested that spastin regulates microtubule dynamics. We have studied the ATPase activity of recombinant human spastin and examined the effect of taxol-stabilized microtubules on this activity. We used spastin translated from the second ATG and provide evidence that this is the physiologically relevant form. We showed that microtubules enhance the ATPase activity of the protein, a property also described for katanin, an AAA of the same spastin subgroup. Furthermore, we demonstrated that human spastin has a microtubule-destabilizing activity and can bundle microtubules in vitro, providing new insights into the molecular pathogenesis of HSP.

Natural variation within species reveals aspects of genome evolution and function. The fission yeast Schizosaccharomyces pombe is an important model for eukaryotic biology, but researchers typically use one standard laboratory strain. To extend the usefulness of this model, we surveyed the genomic and phenotypic variation in 161 natural isolates. We sequenced the genomes of all strains, finding moderate genetic diversity (π = 3 × 10(-3) substitutions/site) and weak global population structure. We estimate that dispersal of S. pombe began during human antiquity (∼340 BCE), and ancestors of these strains reached the Americas at ∼1623 CE. We quantified 74 traits, finding substantial heritable phenotypic diversity. We conducted 223 genome-wide association studies, with 89 traits showing at least one association. The most significant variant for each trait explained 22% of the phenotypic variance on average, with indels having larger effects than SNPs. This analysis represents a rich resource to examine genotype-phenotype relationships in a tractable model.

Super-resolution imaging techniques represent the latest wave of imaging technology and as such are bound to advance future fungal cell microscopy. A conventional light microscope is diffraction limited to a resolution of ~ 250 nm, restricting its capability to allow to accurately measure and distinguish structures in biological samples. Super-resolution techniques, mainly developed over the past decade, circumvent the resolution barrier achieving resolutions that can be well below the conventional limit. Three super-resolution platforms are outlined in this chapter—structured illumination microscopy, stimulated emission depletion microscopy and localization microscopy. Each method differs in terms of maximum achievable resolution, ease of use, phototoxicity and applicability to 3D and live-cell imaging. The value and potential of these techniques to fungal cell biology is demonstrated through review of specific applications, with an emphasis on accurately estimating object size and resolving the finer organization of larger structures.

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.

Delegates at scientific meetings can come from diverse backgrounds and use very different methods in their research. Promoting interactions between these ‘distant’ delegates is challenging but such interactions could lead to novel interdisciplinary collaborations and unexpected breakthroughs. We have developed a network-based ‘speed dating’ approach that allows us to initiate such distant interactions by pairing every delegate with another delegate who might be of interest to them, but whom they might never have encountered otherwise. Here we describe our approach and its algorithmic implementation.

Every cell has a characteristic shape key to its fate and function. That shape is not only the product of genetic design and of the physical and biochemical environment, but it is also subject to inheritance. However, the nature and contribution of cell shape inheritance to morphogenetic control is mostly ignored. Here, we investigate morphogenetic inheritance in the cylindrically-shaped fission yeast Schizosaccharomyces pombe. Focusing on sixteen different ‘curved’ mutants - a class of mutants which often fail to grow axially straight – we quantitatively characterize their dynamics of cell shape inheritance throughout generations. We show that mutants of similar machineries display similar dynamics of cell shape inheritance, and exploit this feature to show that persistent axial cell growth in S. pombe is secured by multiple, separable molecular pathways. Finally, we find that one of those pathways corresponds to the swc2-swr1-vps71 SWR1/SRCAP chromatin remodelling complex, which acts additively to the known mal3-tip1-mto1-mto2 microtubule and tea1-tea2-tea4-pom1 polarity machineries.

The post-genomic era has produced a variety of new investigation technologies, techniques and approaches that may offer exciting insights into many long-standing questions of scientific research. The microtubule cytoskeleton is a highly conserved system that shows a high degree of internal complexity, is known to be integral to many cell systems and functions on a fundamental level. After decades of study, much is still unknown about microtubules in vivo from the control of dynamics in living cells to their responses to environmental changes and responses to other cellular processes. In the present article, we examine some outstanding questions in the microtubule field and propose a combination of emerging interdisciplinary approaches, i.e. high-throughput functional genomics techniques, quantitative and super-resolution microscopy, and in silico modelling, that could shed light on the systemic regulation of microtubules in cells by networks of regulatory factors. We propose that such an integrative approach is key to elucidate the function of the microtubule cytoskeleton as a complete responsive integral biological system.

SummaryUnderstanding cells as integrated systems requires that we systematically decipher how single genes affect multiple biological processes and how processes are functionally linked. Here, we used multiprocess phenotypic profiling, combining high-resolution 3D confocal microscopy and multiparametric image analysis, to simultaneously survey the fission yeast genome with respect to three key cellular processes: cell shape, microtubule organization, and cell-cycle progression. We identify, validate, and functionally annotate 262 genes controlling specific aspects of those processes. Of these, 62% had not been linked to these processes before and 35% are implicated in multiple processes. Importantly, we identify a conserved role for DNA-damage responses in controlling microtubule stability. In addition, we investigate how the processes are functionally linked. We show unexpectedly that disruption of cell-cycle progression does not necessarily affect cell size control and that distinct aspects of cell shape regulate microtubules and vice versa, identifying important systems-level links across these processes.

Cell polarity is regulated by evolutionarily conserved polarity factors whose precise higher-order organization at the cell cortex is largely unknown. Here we image frontally the cortex of live fission yeast cells using time-lapse and super-resolution microscopy. Interestingly, we find that polarity factors are organized in discrete cortical clusters resolvable to ~50–100 nm in size, which can form and become cortically enriched by oligomerization. We show that forced co-localization of the polarity factors Tea1 and Tea3 results in polarity defects, suggesting that the maintenance of both factors in distinct clusters is required for polarity. However, during mitosis, their co-localization increases, and Tea3 helps to retain the cortical localization of the Tea1 growth landmark in preparation for growth reactivation following mitosis. Thus, regulated spatial segregation of polarity factor clusters provides a means to spatio-temporally control cell polarity at the cell cortex. We observe similar clusters in Saccharomyces cerevisiae and Caenorhabditis elegans cells, indicating this could be a universal regulatory feature., Cell polarity is generated and maintained by the spatial accumulation of polarity factors. By imaging fission yeast cells ‘end-on’, the authors show that the polarity factors Tea1 and Tea3 segregate into distinct clusters, and that surprisingly, their segregation is critical for cell polarization.

The study of gene and protein interaction networks has improved our understanding of the multiple, systemic levels of regulation found in eukaryotic and prokaryotic organisms. Here we carry out a large-scale analysis of the protein-protein interaction (PPI) network of fission yeast (Schizosaccharomyces pombe) and establish a method to identify ‘linker’ proteins that bridge diverse cellular processes - integrating Gene Ontology and PPI data with network theory measures. We test the method on a highly characterized subset of the genome consisting of proteins controlling the cell cycle, cell polarity and cytokinesis and identify proteins likely to play a key role in controlling the temporal changes in the localization of the polarity machinery. Experimental inspection of one such factor, the polarity-regulating RNB protein Sts5, confirms the prediction that it has a cell cycle dependent regulation. Detailed bibliographic inspection of other predicted ‘linkers’ also confirms the predictive power of the method. As the method is robust to network perturbations and can successfully predict linker proteins, it provides a powerful tool to study the interplay between different cellular processes., Author Summary Analysis of protein interaction networks has been of use as a means to grapple with the complexity of the interactome of biological organisms. So far, network based approaches have only been used in a limited number of organisms due to the lack of high-throughput experiments. In this study, we investigate by graph theoretical network analysis approaches the protein-protein interaction network of fission yeast, and present a new network measure, linkerity, that predicts the ability of certain proteins to function as bridges between diverse cellular processes. We apply this linkerity measure to a highly conserved and coupled subset of the fission yeast network, consisting of the proteins that regulate cell cycle, polarized cell growth, and cell division. In depth literature analysis confirms that several proteins identified as linkers of cell polarity regulation are indeed also associated with cell cycle and/or cell division control. Similarly, experimental testing confirms that a mostly uncharacterized polarity regulator identified by the method as an important linker is regulated by the cell cycle, as predicted.

SPG4, the gene encoding for spastin, a member of the ATPases associated with various cellular activities (AAA) family, is mutated in around 40% of cases of autosomal dominant hereditary spastic paraplegia (AD-HSP). This group of neurodegenerative diseases is characterized by a progressive spasticity and lower limb weakness with degeneration of terminal axons in cortico-spinal tracts and dorsal columns. Spastin has two main domains, a microtubule interacting and endosomal trafficking (MIT) domain at the N-terminus and the C-terminus AAA domain. Early studies suggested that spastin interacts with microtubules similarly to katanin, a member of the same subgroup of AAA. Recent evidence confirmed that spastin possesses microtubule-severing activity but can also bundle microtubules in vitro. Understanding the physiologic and pathologic involvement of these activities and their regulation is critical in the study of HSP.

Mol Syst Biol. 3: 95 We want to see how, in some cases at least, the forms of living things, and of the parts of living things, can be explained by physical considerations.…D'Arcy Thompson (1917) Proper organization of microtubule polymers is crucial to the form and function of all eukaryotic cells. Whether growing, dividing or polarizing, different cell types—be they neurons, plant cells or fungal cells—organize specialized microtubule patterns appropriate for their needs. Is it possible to identify the set of factors sufficient to organize microtubules in a specific cell, and to understand how the cell regulates those factors spatially and temporally such that they collectively sustain that particular cellular pattern of microtubules? In a recent issue of Cell , Marcel Janson and collaborators (Janson et al , 2007) use an elegant combination of quantitative microscopy, in vitro assays and computer simulations to try to elucidate the minimal set of components required to stably organize microtubule patterns in a unicellular eukaryote, the fission yeast Schizosaccharomyces pombe . Microtubule organization in a given cell is …

Hereditary spastic paraplegias (HSPs) are neurodegenerative diseases caused by mutations in more than 20 genes, which lead to progressive spasticity and weakness of the lower limbs. The most frequently mutated gene causing autosomal dominant HSP is SPG4, which encodes spastin, a protein that belongs to the family of ATPases associated with various cellular activities (AAAs). A number of studies have suggested that spastin regulates microtubule dynamics. We have studied the ATPase activity of recombinant human spastin and examined the effect of taxol-stabilized microtubules on this activity. We used spastin translated from the second ATG and provide evidence that this is the physiologically relevant form. We showed that microtubules enhance the ATPase activity of the protein, a property also described for katanin, an AAA of the same spastin subgroup. Furthermore, we demonstrated that human spastin has a microtubule-destabilizing activity and can bundle microtubules in vitro, providing new insights into the molecular pathogenesis of HSP.

Fission yeast ( Schizosaccharomyces pombe ) cells grow longitudinally in a manner dependent on a polarized distribution of their interphase microtubules. We found that this distribution required sliding of microtubules toward the cell center along preexisting microtubules. This sliding was mediated by the minus end–directed kinesin motor Klp2, which helped microtubules to become properly organized with plus ends predominantly oriented toward the cell ends and minus ends toward the cell center. Thus, interphase microtubules in the fission yeast require motor activities for their proper organization.

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.

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.

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.

Cell polarity—the intrinsic property of cells to take different shape, migration or growth patterns suited to their function—is one of those things intuitively we are all aware of. It is part of our collective imagination, and indeed if prompted all of us immediately think of cells as

La segregation correcte du materiel genetique au moment de la mitose depend de la formation du fuseau mitotique, assemblage de microtubules qui se reorientent autour des chromosomes. Les mecanismes qui controlent l'assemblage et la reorientation de cette structure dynamique sont tres complexes et peu connus. Une GTPase nucleaire de la famille de Ras, Ran, impliquee jusqu'a maintenant dans le transport nucleo-cytoplasmique, s'avere un acteur essentiel de ce processus. Pendant l'interphase, la forme active, Ran-GTP, est nucleaire, et la forme inactive, Ran-GDP, cytoplasmique. Lors de la mitose, Ran-GTP induit la polymerisation des microtubules et leur organisation en un fuseau bipolaire, probablement sous l'effet inducteur de la chromatine elle-meme. Ran-GTP, agirait durant la mitose comme au cours du transport nucleocytoplasmique lors de l'interphase, d'echange, agirait en liberant des proteines nucleaires directement responsables de l'assemblage des polymeres de tubuline. Ces donnees, en suggerant l'implication directe de la chromatine dans l'assemblage des microtubules par l'intermediaire de Ran, confirment que la formation du fuseau est un processus en perpetuelle activite.

The mitotic spindle of animal cells is a bipolar array of microtubules that guides chromosome segregation during cell division [1]. It has been proposed that during spindle assembly chromatin can positively influence microtubule stability at a distance from its surface throughout its neighboring cytoplasm [2]. 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 μm of chromatin. To our knowledge, this is the first direct evidence of a long-range guidance effect at the sub-cellular level.

Les microtubules sont, à chaque division cellulaire, organisés en fuseau bipolaire pour assurer la transmission des chromosomes aux cellules filles. Dès la sortie d’interphase, les microtubules deviennent extrêmement labiles, ce qui facilite leur réorganisation. Parallèlement, des mécanismes de restabilisation locale des microtubules doivent être mis en place pour l’assemblage du fuseau. Tout d’abord, la chromatine favorise la polymérisation et la stabilisation des microtubules autour d’elle. Cet « effet chromatine » est principalement relayé par une enzyme, la GTPase Ran. Il est renforcé par l’activité de moteurs moléculaires liés à la chromatine, qui ancrent des microtubules aux bras des chromosomes. Enfin, les kinétochores, structures protéiques accolées à une région limitée de la chromatine, stabilisent d’autres microtubules qui s’organisent en « fibres kinétochoriennes » robustes et contribuent au maintien du fuseau bipolaire dans la cellule., The partition of the genetic material of cells in two identical lots during cell division relies on the assembly of a structure composed of microtubules, the bipolar spindle. As a cell prepares its division, changes in the state of its cytoplasm lead to the disassembly and to the global instability of its microtubule network. However, in order for the spindle to form, its microtubules must persist amid this destabilizing cytoplasm. A large body of experimental data now shows that the chromosomes themselves are the source of this persistence. They carry out this task through a chromatin-induced reversal of the dominating microtubule depolymerization regime, thereby resulting in a net microtubule polymerization locally around chromosomes, and through the capture, by kinetochores, of microtubules associated to each spindle pole and the assembly of kinetochore-associated microtubule bundles (K-fibres). In this review, we discuss the mechanisms and molecules that allow chromosomes to locally induce microtubule polymerization and stabilization during spindle assembly.