RESUMO
Constriction kinetics of the cytokinetic ring are expected to depend on dynamic adjustment of contractile ring composition, but the impact of ring component abundance dynamics on ring constriction is understudied. Computational models generally assume that contractile networks maintain constant total amounts of components, which is not always true. To test how compositional dynamics affect constriction kinetics, we first measured F-actin, non-muscle myosin II, septin, and anillin during Caenorhabditis elegans zygotic mitosis. A custom microfluidic device that positioned the cell with the division plane parallel to a light sheet allowed even illumination of the cytokinetic ring. Measured component abundances were implemented in a three-dimensional agent-based model of a membrane-associated contractile ring. With constant network component amounts, constriction completed with biologically unrealistic kinetics. However, imposing the measured changes in component quantities allowed this model to elicit realistic constriction kinetics. Simulated networks were more sensitive to changes in motor and filament amounts than those of crosslinkers and tethers. Our findings highlight the importance of network composition for actomyosin contraction kinetics.
Assuntos
Citoesqueleto de Actina , Citocinese , Animais , Cinética , Citocinese/fisiologia , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Actomiosina/metabolismo , Caenorhabditis elegansRESUMO
Meiotic spindles are positioned perpendicular to the oocyte cortex to facilitate segregation of chromosomes into a large egg and a tiny polar body. In C. elegans, spindles are initially ellipsoid and parallel to the cortex before shortening to a near-spherical shape with flattened poles and then rotating to the perpendicular orientation by dynein-driven cortical pulling. The mechanistic connection between spindle shape and rotation has remained elusive. Here, we have used three different genetic backgrounds to manipulate spindle shape without eliminating dynein-dependent movement or dynein localization. Ellipsoid spindles with flattened or pointed poles became trapped in either a diagonal or a parallel orientation. Mathematical models that recapitulated the shape dependence of rotation indicated that the lower viscous drag experienced by spherical spindles prevented recapture of the cortex by astral microtubules emanating from the pole pivoting away from the cortex. In addition, maximizing contact between pole dynein and cortical dynein stabilizes flattened poles in a perpendicular orientation, and spindle rigidity prevents spindle bending that can lock both poles at the cortex. Spindle shape can thus promote perpendicular orientation by three distinct mechanisms.
Assuntos
Caenorhabditis elegans/metabolismo , Fuso Acromático/metabolismo , Polos do Fuso/metabolismo , Animais , Cromossomos/metabolismo , Dineínas/metabolismo , Feminino , Imunofluorescência , Meiose/fisiologia , Microtúbulos/metabolismoRESUMO
Cytokinesis is the fundamental and ancient cellular process by which one cell physically divides into two. Cytokinesis in animal and fungal cells is achieved by contraction of an actomyosin cytoskeletal ring assembled in the cell cortex, typically at the cell equator. Cytokinesis is essential for the development of fertilized eggs into multicellular organisms and for homeostatic replenishment of cells. Correct execution of cytokinesis is also necessary for genome stability and the evasion of diseases including cancer. Cytokinesis has fascinated scientists for well over a century, but its speed and dynamics make experiments challenging to perform and interpret. The presence of redundant mechanisms is also a challenge to understand cytokinesis, leaving many fundamental questions unresolved. For example, how does a disordered cytoskeletal network transform into a coherent ring? What are the long-distance effects of localized contractility? Here, we provide a general introduction to 'modeling for biologists', and review how agent-based modeling and continuum mechanics modeling have helped to address these questions.
Assuntos
Citoesqueleto de Actina/metabolismo , Actomiosina/metabolismo , Citocinese/fisiologia , Hipersensibilidade a Ovo/metabolismo , Animais , Citoesqueleto/metabolismo , Desenvolvimento Embrionário/fisiologia , HumanosRESUMO
The actomyosin cortex is an active material that generates force to drive shape changes via cytoskeletal remodeling. Cytokinesis is the essential cell division event during which a cortical actomyosin ring closes to separate two daughter cells. Our active gel theory predicted that actomyosin systems controlled by a biochemical oscillator and experiencing mechanical strain would exhibit complex spatiotemporal behavior. To test whether active materials in vivo exhibit spatiotemporally complex kinetics, we imaged the C. elegans embryo with unprecedented temporal resolution and discovered that sections of the cytokinetic cortex undergo periodic phases of acceleration and deceleration. Contractile oscillations exhibited a range of periodicities, including those much longer periods than the timescale of RhoA pulses, which was shorter in cytokinesis than in any other biological context. Modifying mechanical feedback in vivo or in silico revealed that the period of contractile oscillation is prolonged as a function of the intensity of mechanical feedback. Fast local ring ingression occurs where speed oscillations have long periods, likely due to increased local stresses and, therefore, mechanical feedback. Fast ingression also occurs where material turnover is high, in vivo and in silico. We propose that downstream of initiation by pulsed RhoA activity, mechanical feedback, including but not limited to material advection, extends the timescale of contractility beyond that of biochemical input and, therefore, makes it robust to fluctuations in activation. Circumferential propagation of contractility likely allows for sustained contractility despite cytoskeletal remodeling necessary to recover from compaction. Thus, like biochemical feedback, mechanical feedback affords active materials responsiveness and robustness.
Assuntos
Actomiosina , Caenorhabditis elegans , Citocinese , Citocinese/fisiologia , Animais , Caenorhabditis elegans/fisiologia , Actomiosina/metabolismo , Fenômenos Biomecânicos , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Retroalimentação Fisiológica , Proteína rhoA de Ligação ao GTP/metabolismo , Embrião não Mamífero/fisiologiaRESUMO
Cytokinesis is the last step of cell division that physically separates the daughter cells. As such, it ensures the proper inheritance of both nuclear and cytoplasmic contents. Accomplishment of cytokinesis in eukaryotes is dictated by several key events: establishment of the division plane, furrow ingression through contraction of an actomyosin ring and abscission via membrane fusion. Most mammalian somatic cells are diploid. Polyploidy can result from cytokinesis failure and may contribute to the development of pathologies such as cancer. However, polyploidy is essential for cellular differentiation and function in some contexts (eg hepatocytes, megakaryocytes and others). Consequently, the degree of ploidy and the achievement of cytokinesis must be tightly regulated throughout an organism and among different cell types. In this review we will highlight several examples of normal and pathological polyploidy, focusing on those caused by a controlled failure or dysregulation of cytokinesis, respectively. Last, we propose therapeutic routes to control cytokinesis to restore or block cell division.
Assuntos
Citocinese/genética , Ploidias , Aneuploidia , Feminino , Células Germinativas/patologia , Doenças Hematológicas/patologia , Hepatócitos/patologia , Humanos , Infertilidade Feminina/patologia , Degeneração Macular/patologia , Megacariócitos/patologia , Músculo Liso Vascular/patologia , Miócitos Cardíacos/patologia , Neoplasias/patologia , Neurofibromatose 2/patologia , Síndrome Oculocerebrorrenal/patologiaRESUMO
Oocytes must be exceptionally large cells in order to support embryonic development. Throughout animal phylogeny, a specialized cell called a syncytium, wherein many nuclei share a continuous cytoplasm, achieves oogenesis. The syncytial nature of germline architecture is key to its function and depends on conserved components of the cortical cytoskeleton. Septins form non-polar cytoskeletal polymers that associate with membranes. In the syncytial germline of the nematode Caenorhabditis elegans, septins are highly enriched on the cortex and generally required for fertility, but the role of septins in the germline is poorly understood. We report that the C. elegans septins, UNC-59 and UNC-61, are important for germline extension during development, the maintenance of its syncytial architecture, and production of oocytes. While much of our findings substantiate the idea that the two C. elegans septins act together, we also found evidence that they have distinct functions. Loss of UNC-61 perturbed germline extension during germline development, while the loss of UNC-59 function severely affected germline architecture in adult hermaphrodites. Consultation of clustering results from a large-scale high-throughput screen suggested that septins are involved in germ cell proliferation and/or differentiation. In sum, our findings implicate a conserved cytoskeletal component in the complex architecture of a germline syncytium.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Septinas/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Oogênese , Células Germinativas/metabolismoRESUMO
During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units in vivo. There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in C. elegans zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA).
RESUMO
Septins, a conserved family of filament-forming proteins, contribute to eukaryotic cell division, polarity, and membrane trafficking. Septins are thought to act in these processes by scaffolding other proteins to the plasma membrane. The mechanisms by which septins associate with the plasma membrane are not well understood but can involve two polybasic domains and/or an amphipathic helix. We discovered that the genomes of organisms throughout phylogeny, but not most commonly used model organisms, encode one or more septins predicted to have transmembrane domains. The nematode Caenorhabditis elegans, which was thought to express only two septin proteins, UNC-59 and UNC-61, translates multiple isoforms of UNC-61, and one isoform, UNC-61a, is predicted to contain a transmembrane domain. UNC-61a localizes specifically to the apical membrane of the C. elegans vulva and is important for maintaining vulval morphology. UNC-61a partially compensates for the loss of the other two UNC-61 isoforms, UNC-61b and UNC-61c. The UNC-61a transmembrane domain is sufficient to localize a fluorophore to membranes in mammalian cells, and its deletion from UNC-61a recapitulates the phenotypes of unc-61a null animals. The localization and loss-of-function phenotypes of UNC-61a and its transmembrane domain suggest roles in cell polarity and secretion and help explain the cellular and tissue biological underpinnings of C. elegans septin null alleles' enigmatically hypomorphic phenotypes. Together, our findings reveal a novel mechanism of septin-membrane association with profound implications for the dynamics and regulation of this association.
RESUMO
Contractile force generation by the cortical actomyosin cytoskeleton is essential for a multitude of biological processes. The actomyosin cortex behaves as an active material that drives local and large-scale shape changes via cytoskeletal remodeling in response to biochemical cues and feedback loops. Cytokinesis is the essential cell division event during which a cortical actomyosin ring generates contractile force to change cell shape and separate two daughter cells. Our recent work with active gel theory predicts that actomyosin systems under the control of a biochemical oscillator and experiencing mechanical strain will exhibit complex spatiotemporal behavior, but cytokinetic contractility was thought to be kinetically simple. To test whether active materials in vivo exhibit spatiotemporally complex kinetics, we used 4-dimensional imaging with unprecedented temporal resolution and discovered sections of the cytokinetic cortex undergo periodic phases of acceleration and deceleration. Quantification of ingression speed oscillations revealed wide ranges of oscillation period and amplitude. In the cytokinetic ring, activity of the master regulator RhoA pulsed with a timescale of approximately 20 seconds, shorter than that reported for any other biological context. Contractility oscillated with 20-second periodicity and with much longer periods. A combination of in vivo and in silico approaches to modify mechanical feedback revealed that the period of contractile oscillation is prolonged as a function of the intensity of mechanical feedback. Effective local ring ingression is characterized by slower speed oscillations, likely due to increased local stresses and therefore mechanical feedback. Fast ingression also occurs where material turnover is high, in vivo and in silico . We propose that downstream of initiation by pulsed RhoA activity, mechanical positive feedback, including but not limited to material advection, extends the timescale of contractility beyond that of biochemical input and therefore makes it robust to fluctuations in activation. Circumferential propagation of contractility likely allows sustained contractility despite cytoskeletal remodeling necessary to recover from compaction. Our work demonstrates that while biochemical feedback loops afford systems responsiveness and robustness, mechanical feedback must also be considered to describe and understand the behaviors of active materials in vivo .
RESUMO
Anillin is a highly conserved multidomain protein that interacts with cytoskeletal components as well as their regulators. Throughout phylogeny, Anillins contribute to cytokinesis, the cell shape change that occurs at the end of meiosis and mitosis to separate a cell into daughter cells. Failed cytokinesis results in binucleation, which can lead to genomic instability. Study of Anillin in several model organisms has provided us with insight into how the cytoskeleton is coordinated to ensure that cytokinesis occurs with high fidelity. Here we review Anillin's interacting partners and the relevance of these interactions in vivo. We also discuss questions of how these interactions are coordinated, and finally provide some perspective regarding Anillin's role in cancer.
Assuntos
Proteínas Contráteis/metabolismo , Citocinese , Animais , Ciclo Celular , Proteínas Contráteis/química , Humanos , Proteínas/metabolismoRESUMO
During cytokinesis, constriction of a cortical contractile ring generates a furrow that partitions one cell into two. The contractile ring contains three filament systems: actin, bipolar myosin II filaments, and septins, GTP-binding hetero-oligomers that polymerize to form a membrane-associated lattice. The contractile ring also contains a potential filament crosslinker, Anillin, that binds all three filament types. Here, we show that the contractile ring possesses an intrinsic symmetry-breaking mechanism that promotes asymmetric furrowing. Asymmetric ingression requires Anillin and the septins, which promote the coalescence of components on one side of the contractile ring, but is insensitive to a 10-fold reduction in myosin II levels. When asymmetry is disrupted, cytokinesis becomes sensitive to partial inhibition of contractility. Thus, asymmetric furrow ingression, a prevalent but previously unexplored feature of cell division in metazoans, is generated by the action of two conserved furrow components and serves a mechanical function that makes cytokinesis robust.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas Contráteis/metabolismo , Citocinese , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/embriologia , Drosophila melanogaster , Embrião não Mamífero/citologia , Miosina Tipo II/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Many cells and tissues exhibit chirality that stems from the chirality of proteins and polymers. In the Caenorhabditis elegans zygote, actomyosin contractility drives chiral rotation of the entire cortex circumferentially around the division plane during anaphase. How contractility is translated to cell-scale chirality, and what dictates handedness, are unknown. Septins are candidate contributors to cell-scale chirality because they anchor and cross-link the actomyosin cytoskeleton. We report that septins are required for anaphase cortical rotation. In contrast, the formin CYK-1, which we found to be enriched in the posterior in early anaphase, is not required for cortical rotation but contributes to its chirality. Simultaneous loss of septin and CYK-1 function led to abnormal and often reversed cortical rotation. Our results suggest that anaphase contractility leads to chiral rotation by releasing torsional stress generated during formin-based polymerization, which is polarized along the cell anterior-posterior axis and which accumulates due to actomyosin network connectivity. Our findings shed light on the molecular and physical bases for cellular chirality in the C. elegans zygote. We also identify conditions in which chiral rotation fails but animals are developmentally viable, opening avenues for future work on the relationship between early embryonic cellular chirality and animal body plan.
Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Septinas/metabolismo , Zigoto/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Actomiosina/metabolismo , Anáfase , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/fisiologia , Polaridade Celular , Forminas/metabolismo , Forminas/fisiologia , Lateralidade Funcional/fisiologia , Microtúbulos/metabolismo , Rotação , Septinas/fisiologia , Zigoto/fisiologiaRESUMO
Actomyosin cortical contractility drives many cell shape changes including cytokinetic furrowing. While positive regulation of contractility is well characterized, counterbalancing negative regulation and mechanical brakes are less well understood. The small GTPase RhoA is a central regulator, activating cortical actomyosin contractility during cytokinesis and other events. Here we report how two novel cytokinetic ring components, GCK-1 (germinal center kinase-1) and CCM-3 (cerebral cavernous malformations-3), participate in a negative feedback loop among RhoA and its cytoskeletal effectors to inhibit contractility. GCK-1 and CCM-3 are recruited by active RhoA and anillin to the cytokinetic ring, where they in turn limit RhoA activity and contractility. This is evidenced by increased RhoA activity, anillin and nonmuscle myosin II in the cytokinetic ring, and faster cytokinetic furrowing, following depletion of GCK-1 or CCM-3. GCK-1 or CCM-3 depletion also reduced RGA-3 levels in pulses and increased baseline RhoA activity and pulsed contractility during zygote polarization. Together, our results suggest that GCK-1 and CCM-3 regulate cortical actomyosin contractility via negative feedback. These findings have implications for the molecular and cellular mechanisms of cerebral cavernous malformation pathologies.
Assuntos
Caenorhabditis elegans/citologia , Citocinese , Retroalimentação Fisiológica , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Polaridade Celular , Estabilidade Proteica , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
Mitotic cell rounding is the process of cell shape change in which a flat interphase cell becomes spherical at the onset of mitosis. Rearrangement of the actin cytoskeleton, de-adhesion, and an increase in cortical rigidity accompany mitotic cell rounding. The molecular mechanisms that contribute to this process have not been defined. We show that RhoA is required for cortical retraction but not de-adhesion during mitotic cell rounding. The mitotic increase in cortical rigidity also requires RhoA, suggesting that increases in cortical rigidity and cortical retraction are linked processes. Rho-kinase is also required for mitotic cortical retraction and rigidity, indicating that the effects of RhoA on cell rounding are mediated through this effector. Consistent with a role for RhoA during mitotic entry, RhoA activity is elevated in rounded, preanaphase mitotic cells. The activity of the RhoA inhibitor p190RhoGAP is decreased due to its serine/threonine phosphorylation at this time. Cumulatively, these results suggest that the mitotic increase in RhoA activity leads to rearrangements of the cortical actin cytoskeleton that promote cortical rigidity, resulting in mitotic cell rounding.
Assuntos
Citoesqueleto de Actina/metabolismo , Adesão Celular/fisiologia , Citoesqueleto/metabolismo , Células Eucarióticas/metabolismo , Mitose/fisiologia , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Anáfase/efeitos dos fármacos , Anáfase/fisiologia , Adesão Celular/efeitos dos fármacos , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Inibidores Enzimáticos/farmacologia , Células Eucarióticas/citologia , Células Eucarióticas/efeitos dos fármacos , Células HeLa , Humanos , Interfase/efeitos dos fármacos , Interfase/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Mitose/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Quinases Associadas a rhoRESUMO
New methods to directly visualize Rho GTPases reveal how a protein called RhoGDI regulates the activity of these 'molecular switches' at the plasma membrane.
Assuntos
Inibidores de Dissociação do Nucleotídeo Guanina , Proteínas rho de Ligação ao GTP , Membrana Celular , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Cytokinesis is the subject of intense study, but mechanisms underlying contractility and cell shape change in cytokinesis are still being defined. Furthermore, it is unknown how contractile mechanisms vary among cell types and throughout development. Recent findings uncover differential molecular requirements for cytokinesis depending on cell fate and embryonic context.
Assuntos
Citocinese , Desenvolvimento Embrionário , Divisão Celular , Forma Celular , Contração MuscularRESUMO
Cell shape changes such as cytokinesis are driven by the actomyosin contractile cytoskeleton. The molecular rearrangements that bring about contractility in nonmuscle cells are currently debated. Specifically, both filament sliding by myosin motors, as well as cytoskeletal cross-linking by myosins and nonmotor cross-linkers, are thought to promote contractility. Here we examined how the abundance of motor and nonmotor cross-linkers affects the speed of cytokinetic furrowing. We built a minimal model to simulate contractile dynamics in the Caenorhabditis elegans zygote cytokinetic ring. This model predicted that intermediate levels of nonmotor cross-linkers are ideal for contractility; in vivo, intermediate levels of the scaffold protein anillin allowed maximal contraction speed. Our model also demonstrated a nonlinear relationship between the abundance of motor ensembles and contraction speed. In vivo, thorough depletion of nonmuscle myosin II delayed furrow initiation, slowed F-actin alignment, and reduced maximum contraction speed, but partial depletion allowed faster-than-expected kinetics. Thus, cytokinetic ring closure is promoted by moderate levels of both motor and nonmotor cross-linkers but attenuated by an over-abundance of motor and nonmotor cross-linkers. Together, our findings extend the growing appreciation for the roles of cross-linkers in cytokinesis and reveal that they not only drive but also brake cytoskeletal remodeling.
Assuntos
Actomiosina/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Citocinese , Citoesqueleto/metabolismo , Actinas/metabolismo , Animais , Caenorhabditis elegans/citologia , Microtúbulos/metabolismo , Miosina Tipo II/metabolismoRESUMO
Germ cells in most animals are connected by intercellular bridges, actin-based rings that form stable cytoplasmic connections between cells promoting communication and coordination [1]. Moreover, these connections are required for fertility [1, 2]. Intercellular bridges are proposed to arise from stabilization of the cytokinetic ring during incomplete cytokinesis [1]. Paradoxically, proteins that promote closure of cytokinetic rings are enriched on stably open intercellular bridges [1, 3, 4]. Given this inconsistency, the mechanism of intercellular bridge stabilization is unclear. Here, we used the C. elegans germline as a model for identifying molecular mechanisms regulating intercellular bridges. We report that bridges are actually highly dynamic, changing size at precise times during germ cell development. We focused on the regulation of bridge stability by anillins, key regulators of cytokinetic rings and cytoplasmic bridges [1, 4-7]. We identified GCK-1, a conserved serine/threonine kinase [8], as a putative novel anillin interactor. GCK-1 works together with CCM-3, a known binding partner [9], to promote intercellular bridge stability and limit localization of both canonical anillin and non-muscle myosin II (NMM-II) to intercellular bridges. Additionally, we found that a shorter anillin, known to stabilize bridges [4, 7], also regulates NMM-II levels at bridges. Consistent with these results, negative regulators of NMM-II stabilize intercellular bridges in the Drosophila egg chamber [10, 11]. Together with our findings, this suggests that tuning of myosin levels is a conserved mechanism for the stabilization of intercellular bridges that can occur by diverse molecular mechanisms.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Proteínas de Membrana/genética , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Diferenciação Celular , Proteínas Contráteis/genética , Proteínas Contráteis/metabolismo , Citocinese , Células Germinativas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismoRESUMO
The size of mitotic chromosomes is coordinated with cell size in a manner dependent on nuclear trafficking. In this study, we conducted an RNA interference screen of the Caenorhabditis elegans nucleome in a strain carrying an exceptionally long chromosome and identified the centromere-specific histone H3 variant CENP-A and the DNA decatenizing enzyme topoisomerase-II (topo-II) as candidate modulators of chromosome size. In the holocentric organism C. elegans, CENP-A is positioned periodically along the entire length of chromosomes, and in mitosis, these genomic regions come together linearly to form the base of kinetochores. We show that CENP-A protein levels decreased through development coinciding with chromosome-size scaling. Partial loss of CENP-A protein resulted in shorter mitotic chromosomes, consistent with a role in setting chromosome length. Conversely, topo-II levels were unchanged through early development, and partial topo-II depletion led to longer chromosomes. Topo-II localized to the perimeter of mitotic chromosomes, excluded from the centromere regions, and depletion of topo-II did not change CENP-A levels. We propose that self-assembly of centromeric chromatin into an extended linear array promotes elongation of the chromosome, whereas topo-II promotes chromosome-length shortening.