The Experts below are selected from a list of 2190 Experts worldwide ranked by ideXlab platform
Yasushi Hiraoka - One of the best experts on this subject based on the ideXlab platform.
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a cytoplasmic dynein heavy chain is required for oscillatory nuclear movement of meiotic prophase and efficient meiotic recombination in fission yeast
Journal of Cell Biology, 1999Co-Authors: Ayumu Yamamoto, Robert R West, Richard J Mcintosh, Yasushi HiraokaAbstract:Meiotic recombination requires pairing of homologous chromosomes, the mechanisms of which remain largely unknown. When pairing occurs during meiotic prophase in fission yeast, the nucleus oscillates between the cell poles driven by Astral Microtubules. During these oscillations, the telomeres are clustered at the spindle pole body (SPB), located at the leading edge of the moving nucleus and the rest of each chromosome dangles behind. Here, we show that the oscillatory nuclear movement of meiotic prophase is dependent on cytoplasmic dynein. We have cloned the gene encoding a cytoplasmic dynein heavy chain of fission yeast. Most of the cells disrupted for the gene show no gross defect during mitosis and complete meiosis to form four viable spores, but they lack the nuclear movements of meiotic prophase. Thus, the dynein heavy chain is required for these oscillatory movements. Consistent with its essential role in such nuclear movement, dynein heavy chain tagged with green fluorescent protein (GFP) is localized at Astral Microtubules and the SPB during the movements. In dynein-disrupted cells, meiotic recombination is significantly reduced, indicating that the dynein function is also required for efficient meiotic recombination. In accordance with the reduced recombination, which leads to reduced crossing over, chromosome missegregation is increased in the mutant. Moreover, both the formation of a single cluster of centromeres and the colocalization of homologous regions on a pair of homologous chromosomes are significantly inhibited in the mutant. These results strongly suggest that the dynein-driven nuclear movements of meiotic prophase are necessary for efficient pairing of homologous chromosomes in fission yeast, which in turn promotes efficient meiotic recombination.
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oscillatory nuclear movement in fission yeast meiotic prophase is driven by Astral Microtubules as revealed by continuous observation of chromosomes and Microtubules in living cells
Journal of Cell Science, 1998Co-Authors: Daqiao Ding, Yuji Chikashige, Tokuko Haraguchi, Yasushi HiraokaAbstract:Using a computerized fluorescence microscope system to observe fluorescently stained cellular structures in vivo, we have examined the dynamics of chromosomes and Microtubules during the process of meiosis in the fission yeast Schizosaccharomyces pombe. Fission yeast meiotic prophase is characterized by a distinctive type of nuclear movement that is led by telomeres clustered at the spindle-pole body (the centrosome-equivalent structure in fungi): the nucleus oscillates back and forth along the cell axis, moving continuously between the two ends of the cell for some hours prior to the meiotic divisions. To obtain a dynamic view of this oscillatory nuclear movement in meiotic prophase, we visualized Microtubules and chromosomes in living cells using jellyfish green fluorescent protein fused with alpha-tubulin and a DNA-specific fluorescent dye, Hoechst 33342, respectively. Continuous observation of chromosomes and Microtubules in these cells demonstrated that the oscillatory nuclear movement is mediated by dynamic reorganization of Astral Microtubules originating from the spindle-pole body. During each half-oscillatory period, the Microtubules extending rearward from the leading edge of the nucleus elongate to drive the nucleus to one end of the cell. When the nucleus reversed direction, its motion during the second half of the oscillation was not driven by the same Microtubules that drove its motion during the first half, but rather by newly assembled Microtubules. Reversible inhibition of nuclear movement by an inhibitor of microtubule polymerization, thiabendazole, confirmed the involvement of Astral Microtubules in oscillatory nuclear movement. The speed of the movement fluctuated within a range 0 to 15 micron/minute, with an average of about 5 microm/minute. We propose a model in which the oscillatory nuclear movement is mediated by dynamic instability and selective stabilization of Astral Microtubules.
Alexey Khodjakov - One of the best experts on this subject based on the ideXlab platform.
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Microtubules assemble near most kinetochores during early prometaphase in human cells
Journal of Cell Biology, 2018Co-Authors: Vitali Sikirzhytski, Alexey Khodjakov, Fioranna Renda, Valentin Magidson, Irina Tikhonenko, Bruce F McewenAbstract:For proper segregation during cell division, each chromosome must connect to the poles of the spindle via microtubule bundles termed kinetochore fibers (K-fibers). K-fibers form by two distinct mechanisms: (1) capture of Astral Microtubules nucleated at the centrosome by the chromosomes’ kinetochores or (2) attachment of kinetochores to noncentrosomal Microtubules with subsequent transport of the minus ends of these Microtubules toward the spindle poles. The relative contributions of these alternative mechanisms to normal spindle assembly remain unknown. In this study, we report that most kinetochores in human cells develop K-fibers via the second mechanism. Correlative light electron microscopy demonstrates that from the onset of spindle assembly, short randomly oriented noncentrosomal Microtubules appear in the immediate vicinity of the kinetochores. Initially, these Microtubules interact with the kinetochores laterally, but end-on attachments form rapidly in the first 3 min of prometaphase. Conversion from lateral to end-on interactions is impeded upon inhibition of the plus end–directed kinetochore-associated kinesin CenpE.
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relative contributions of chromatin and kinetochores to mitotic spindle assembly
Journal of Cell Biology, 2009Co-Authors: Christopher B Oconnell, Jadranka Loncarek, Petr Kalab, Alexey KhodjakovAbstract:During mitosis and meiosis in animal cells, chromosomes actively participate in spindle assembly by generating a gradient of Ran guanosine triphosphate (RanGTP). A high concentration of RanGTP promotes microtubule nucleation and stabilization in the vicinity of chromatin. However, the relative contributions of chromosome arms and centromeres/kinetochores in this process are not known. In this study, we address this issue using cells undergoing mitosis with unreplicated genomes (MUG). During MUG, chromatin is rapidly separated from the forming spindle, and both centrosomal and noncentrosomal spindle assembly pathways are active. MUG chromatin is coated with RCC1 and establishes a RanGTP gradient. However, a robust spindle forms around kinetochores/centromeres outside of the gradient peak. When stable kinetochore microtubule attachment is prevented by Nuf2 depletion in both MUG and normal mitosis, chromatin attracts Astral Microtubules but cannot induce spindle assembly. These results support a model in which kinetochores play the dominant role in the chromosome-mediated pathway of mitotic spindle assembly.
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kinetochore driven formation of kinetochore fibers contributes to spindle assembly during animal mitosis
Journal of Cell Biology, 2004Co-Authors: Helde Maiato, Alexey Khodjakov, Conly L RiedeAbstract:It is now clear that a centrosome-independent pathway for mitotic spindle assembly exists even in cells that normally possess centrosomes. The question remains, however, whether this pathway only activates when centrosome activity is compromised, or whether it contributes to spindle morphogenesis during a normal mitosis. Here, we show that many of the kinetochore fibers (K-fibers) in centrosomal Drosophila S2 cells are formed by the kinetochores. Initially, kinetochore-formed K-fibers are not oriented toward a spindle pole but, as they grow, their minus ends are captured by Astral Microtubules (MTs) and transported poleward through a dynein-dependent mechanism. This poleward transport results in chromosome bi-orientation and congression. Furthermore, when individual K-fibers are severed by laser microsurgery, they regrow from the kinetochore outward via MT plus-end polymerization at the kinetochore. Thus, even in the presence of centrosomes, the formation of some K-fibers is initiated by the kinetochores. However, centrosomes facilitate the proper orientation of K-fibers toward spindle poles by integrating them into a common spindle.
Timothy J Mitchison - One of the best experts on this subject based on the ideXlab platform.
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co movement of Astral Microtubules organelles and f actin by dynein and actomyosin forces in frog egg cytoplasm
eLife, 2020Co-Authors: James F Pelletier, Christine M Field, Sebastian Furthauer, Matthew Sonnett, Timothy J MitchisonAbstract:How bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed that dynein-based, inward organelle transport generates length-dependent pulling forces that move centrosomes and MTs outwards, while other components of cytoplasm are static. We imaged aster movement by dynein and actomyosin forces in Xenopus egg extracts and observed outward co-movement of MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and soluble fluorescein. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster periphery, then mostly halted inside the aster, while dynein-coated beads moved to the aster center at a constant rate, suggesting organelle movement is limited by brake proteins or other sources of drag. These observations call for new models in which all components of the cytoplasm comprise a mechanically integrated aster gel that moves collectively in response to dynein and actomyosin forces.
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co movement of Astral Microtubules organelles and f actin suggests aster positioning by surface forces in frog eggs
bioRxiv, 2020Co-Authors: James F Pelletier, Christine M Field, Sebastian Furthauer, Matthew Sonnett, Timothy J MitchisonAbstract:How bulk cytoplasm generates forces to separate post-anaphase microtubule (MT) asters in Xenopus laevis and other large eggs remains unclear. Previous models proposed dynein-based organelle transport generates length-dependent forces on Astral MTs that pull centrosomes through the cytoplasm, away from the midplane. In Xenopus egg extracts, we co-imaged MTs, endoplasmic reticulum (ER), mitochondria, acidic organelles, F-actin, keratin, and fluorescein in moving and stationary asters. In asters that were moving in response to dynein and actomyosin forces, we observed that all cytoplasmic components moved together, i.e., as a continuum. Dynein-mediated organelle transport was restricted by interior MTs and F-actin. Organelles exhibited a burst of dynein-dependent inward movement at the growing aster surface, then mostly halted inside the aster. Dynein-coated beads were slowed by F-actin, but in contrast to organelles, beads did not halt inside asters. These observations call for new models of aster positioning based on surface forces and internal stresses.
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increased lateral microtubule contact at the cell cortex is sufficient to drive mammalian spindle elongation
Molecular Biology of the Cell, 2017Co-Authors: Joshua Guild, Timothy J Mitchison, Miriam Bracha Ginzberg, Christina L Hueschen, Sophie DumontAbstract:The spindle is a dynamic structure that changes its architecture and size in response to biochemical and physical cues. For example, a simple physical change, cell confinement, can trigger centrosome separation and increase spindle steady-state length at metaphase. How this occurs is not understood, and is the question we pose here. We find that metaphase and anaphase spindles elongate at the same rate when confined, suggesting that similar elongation forces can be generated independent of biochemical and spindle structural differences. Furthermore, this elongation does not require bipolar spindle architecture or dynamic Microtubules. Rather, confinement increases numbers of Astral Microtubules laterally contacting the cortex, shifting contact geometry from "end-on" to "side-on." Astral Microtubules engage cortically anchored motors along their length, as demonstrated by outward sliding and buckling after ablation-mediated release from the centrosome. We show that dynein is required for confinement-induced spindle elongation, and both chemical and physical centrosome removal demonstrate that Astral Microtubules are required for such spindle elongation and its maintenance. Together the data suggest that promoting lateral cortex-microtubule contacts increases dynein-mediated force generation and is sufficient to drive spindle elongation. More broadly, changes in microtubule-to-cortex contact geometry could offer a mechanism for translating changes in cell shape into dramatic intracellular remodeling.
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a model for cleavage plane determination in early amphibian and fish embryos
Current Biology, 2010Co-Authors: Timothy J Mitchison, Martin Wuhr, Edwin S Tan, Sandra K Parker, William H DetrichAbstract:Current models for cleavage plane determination propose that metaphase spindles are positioned and oriented by interactions of their Astral Microtubules with the cellular cortex, followed by cleavage in the plane of the metaphase plate [1, 2]. We show that in early frog and fish embryos, where cells are unusually large, Astral Microtubules in metaphase are too short to position and orient the spindle. Rather, the preceding interphase aster centers and orients a pair of centrosomes prior to nuclear envelope breakdown, and the spindle assembles between these prepositioned centrosomes. Interphase asters center and orient centrosomes with dynein-mediated pulling forces. These forces act before Astral Microtubules contact the cortex; thus, dynein must pull from sites in the cytoplasm, not the cell cortex as is usually proposed for smaller cells. Aster shape is determined by interactions of the expanding periphery with the cell cortex or with an interaction zone that forms between sister-asters in telophase. We propose a model to explain cleavage plane geometry in which the length of Astral Microtubules is limited by interaction with these boundaries, causing length asymmetries. Dynein anchored in the cytoplasm then generates length-dependent pulling forces, which move and orient centrosomes.
James W Nelson - One of the best experts on this subject based on the ideXlab platform.
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cell division orientation is coupled to cell cell adhesion by the e cadherin lgn complex
Nature Communications, 2017Co-Authors: Martijn Gloerich, Julie M Bianchini, Kathleen A Siemers, Daniel Cohen, James W NelsonAbstract:Both cell-cell adhesion and oriented cell division play prominent roles in establishing tissue architecture, but it is unclear how they might be coordinated. Here, we demonstrate that the cell-cell adhesion protein E-cadherin functions as an instructive cue for cell division orientation. This is mediated by the evolutionarily conserved LGN/NuMA complex, which regulates cortical attachments of Astral spindle Microtubules. We show that LGN, which adopts a three-dimensional structure similar to cadherin-bound catenins, binds directly to the E-cadherin cytosolic tail and thereby localizes at cell-cell adhesions. On mitotic entry, NuMA is released from the nucleus and competes LGN from E-cadherin to locally form the LGN/NuMA complex. This mediates the stabilization of cortical associations of Astral Microtubules at cell-cell adhesions to orient the mitotic spindle. Our results show how E-cadherin instructs the assembly of the LGN/NuMA complex at cell-cell contacts, and define a mechanism that couples cell division orientation to intercellular adhesion.
Wieland B Huttner - One of the best experts on this subject based on the ideXlab platform.
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specific polar subpopulations of Astral Microtubules control spindle orientation and symmetric neural stem cell division
eLife, 2014Co-Authors: Felipe Morabermudez, Fumio Matsuzaki, Wieland B HuttnerAbstract:A stem cell can divide in two ways. Either it can split symmetrically into two identical daughter stem cells, or it can split asymmetrically into a stem cell and a specialist cell. The structure that forms inside the dividing cell to separate pairs of chromosomes—called the mitotic spindle—also partitions the molecules that determine what kind of cell each daughter cell will become. The mitotic spindle is made up of protein Microtubules. Astral Microtubules connect the spindle to a structure found at the inner face of the cell membrane called the cell cortex. This helps the spindle to orient itself correctly and control the plane of cell division. This is particularly important in cells that are different at their top and bottom, like polarized neural stem cells. To divide symmetrically, these cells need to split vertically from top to bottom. Then, to divide asymmetrically they tilt the cell division plane off-vertical. Classical studies on neuroblasts from the fruit fly Drosophila have shown that a big, 90° reorientation, from vertical to horizontal underlies this change. However, in the primary stem cells of the mammalian brain, subtle off-vertical tilting suffices for asymmetric divisions to occur. This tilting must be finely regulated: if not, neurodevelopmental disorders, such as microcephaly and lissencephaly, may arise. Mora-Bermudez et al. investigated how mammalian cortical stem cells control such subtle spindle orientation changes by taking images of developing brain tissue from genetically modified mice. These show that not all Astral Microtubules affect whether the spindle reorients, as was previously thought. Instead, only those connecting the spindle to the cell cortex at the top and bottom of the cell—the apical/basal Astrals—are involved. A decrease in the number of apical/basal Astrals enables the spindle to undergo small reorientations. Mora-Bermudez et al. therefore propose a model in which the spindle becomes less strongly anchored when the number of apical/basal Astrals is reduced. This makes the spindle easier to tilt, allowing neural stem cells to undergo asymmetric divisions to produce neurons. The decrease in the number of apical/basal Astrals appears to be caused by a reduction in the amount of a molecule that is known to help link the Microtubules to the cell cortex. This reduction occurs only in the cortex at the top of the cell. Mora-Bermudez et al. were also able to manipulate this process by adding very low doses of a microtubule inhibitor called nocodazole, which reduced the number of only the apical/basal Astrals, increasing the ability of the spindle to reorient.
