Goshima Gohta

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  • Preprint
    Length control of the metaphase spindle
    ( 2005-09-30) Goshima, Gohta ; Wollman, Roy ; Stuurman, Nico ; Scholey, Jonathan M. ; Vale, Ronald D.
    The pole-to-pole distance of the metaphase spindle is reasonably constant in a given cell type; in the case of vertebrate female oocytes, this steady-state length can be maintained for substantial lengths of time, during which time microtubules remain highly dynamic. Although a number of molecular perturbations have been shown to influence spindle length, a global understanding of the factors that determine metaphase spindle length has not been achieved. Using the Drosophila S2 cell line, we depleted or overexpressed proteins that either generate sliding forces between spindle microtubules (Kinesin-5, Kinesin-14, dynein), promote microtubule polymerization (EB1, Mast/Orbit [CLASP], Minispindles [Dis1/XMAP215/TOG]) or depolymerization (Kinesin-8, Kinesin-13), or mediate sister-chromatid cohesion (Rad21) in order to explore how these forces influence spindle length. Using high-throughput automated microscopy and semiautomated image analyses of >4000 spindles, we found a reduction in spindle size after RNAi of microtubule-polymerizing factors or overexpression of Kinesin-8, whereas longer spindles resulted from the knockdown of Rad21, Kinesin-8, or Kinesin-13. In contrast, and differing from previous reports, bipolar spindle length is relatively insensitive to increases in motor-generated sliding forces. However, an ultrasensitive monopolar-to-bipolar transition in spindle architecture was observed at a critical concentration of the Kinesin-5 sliding motor. These observations could be explained by a quantitative model that proposes a coupling between microtubule depolymerization rates and microtubule sliding forces. By integrating extensive RNAi with high-throughput image-processing methodology and mathematical modeling, we reach to a conclusion that metaphase spindle length is sensitive to alterations in microtubule dynamics and sister-chromatid cohesion, but robust against alterations of microtubule sliding force.
  • Article
    Augmin-dependent microtubule nucleation at microtubule walls in the spindle
    (Rockefeller University Press, 2013-07-01) Kamasaki, Tomoko ; O’Toole, Eileen ; Kita, Shigeo ; Osumi, Masako ; Usukura, Jiro ; McIntosh, J. Richard ; Goshima, Gohta
    The formation of a functional spindle requires microtubule (MT) nucleation from within the spindle, which depends on augmin. How augmin contributes to MT formation and organization is not known because augmin-dependent MTs have never been specifically visualized. In this paper, we identify augmin-dependent MTs and their connections to other MTs by electron tomography and 3D modeling. In metaphase spindles of human cells, the minus ends of MTs were located both around the centriole and in the body of the spindle. When augmin was knocked down, the latter population of MTs was significantly reduced. In control cells, we identified connections between the wall of one MT and the minus end of a neighboring MT. Interestingly, the connected MTs were nearly parallel, unlike other examples of end–wall connections between cytoskeletal polymers. Our observations support the concept of augmin-dependent MT nucleation at the walls of existing spindle MTs. Furthermore, they suggest a mechanism for maintaining polarized MT organization, even when noncentrosomal MT initiation is widespread.
  • Preprint
    Shortening of microtubule overlap regions defines membrane delivery sites during plant cytokinesis
    ( 2016-12) de Keijzer, Jeroen ; Kieft, Henk ; Ketelaar, Tijs ; Goshima, Gohta ; Janson, Marcel E.
    Different from animal cells that divide by constriction of the cortex inwards, cells of land plants divide by initiating a new cell wall segment from their centre. For this, a disk-shaped, membrane-enclosed precursor termed the cell plate is formed that radially expands towards the parental cell wall. The synthesis of the plate starts with the fusion of vesicles into a tubulo-vesicular network. Vesicles are putatively delivered to the division plane by transport along microtubules of the bipolar phragmoplast network that guides plate assembly. How vesicle immobilisation and fusion are then locally triggered is unclear. In general, a framework for how the cytoskeleton spatially defines cell plate formation is lacking. Here we show that membranous material for cell plate formation initially accumulates along regions of microtubule overlap in the phragmoplast of the moss Physcomitrella patens. Kinesin-4 mediated shortening of these overlaps at the onset of cytokinesis proved to be required to spatially confine membrane accumulation. Without shortening, the wider cell plate membrane depositions evolved into cell walls that were thick and irregularly shaped. Phragmoplast assembly thus provides a regular lattice of short overlaps on which a new cell wall segment can be scaffolded. Since similar patterns of overlaps form in central spindles of animal cells, involving the activity of orthologous proteins, we anticipate that our results will help uncover universal features underlying membrane-cytoskeleton coordination during cytokinesis.
  • Article
    Physical properties of the cytoplasm modulate the rates of microtubule polymerization and depolymerization
    (Elsevier, 2022-02-28) Molines, Arthur T. ; Lemière, Joë ; Gazzola, Morgan ; Steinmark, Ida Emilie ; Edrington, Claire H. ; Hsu, Chieh-Ting ; Real-Calderon, Paula ; Suhling, Klaus ; Goshima, Gohta ; Holt, Liam J. ; Thery, Manuel ; Brouhard, Gary J. ; Chang, Fred
    The cytoplasm is a crowded, visco-elastic environment whose physical properties change according to physiological or developmental states. How the physical properties of the cytoplasm impact cellular functions in vivo remains poorly understood. Here, we probe the effects of cytoplasmic concentration on microtubules by applying osmotic shifts to fission yeast, moss, and mammalian cells. We show that the rates of both microtubule polymerization and depolymerization scale linearly and inversely with cytoplasmic concentration; an increase in cytoplasmic concentration decreases the rates of microtubule polymerization and depolymerization proportionally, whereas a decrease in cytoplasmic concentration leads to the opposite. Numerous lines of evidence indicate that these effects are due to changes in cytoplasmic viscosity rather than cellular stress responses or macromolecular crowding per se. We reconstituted these effects on microtubules in vitro by tuning viscosity. Our findings indicate that, even in normal conditions, the viscosity of the cytoplasm modulates the reactions that underlie microtubule dynamic behaviors.
  • Article
    Augmin : a protein complex required for centrosome-independent microtubule generation within the spindle
    (Rockefeller University Press, 2008-04-28) Goshima, Gohta ; Mayer, Mirjam ; Zhang, Nan ; Stuurman, Nico ; Vale, Ronald D.
    Since the discovery of γ-tubulin, attention has focused on its involvement as a microtubule nucleator at the centrosome. However, mislocalization of {gamma}-tubulin away from the centrosome does not inhibit mitotic spindle formation in Drosophila melanogaster, suggesting that a critical function for γ-tubulin might reside elsewhere. A previous RNA interference (RNAi) screen identified five genes (Dgt2–6) required for localizing γ-tubulin to spindle microtubules. We show that the Dgt proteins interact, forming a stable complex. We find that spindle microtubule generation is substantially reduced after knockdown of each Dgt protein by RNAi. Thus, the Dgt complex that we name "augmin" functions to increase microtubule number. Reduced spindle microtubule generation after augmin RNAi, particularly in the absence of functional centrosomes, has dramatic consequences on mitotic spindle formation and function, leading to reduced kinetochore fiber formation, chromosome misalignment, and spindle bipolarity defects. We also identify a functional human homologue of Dgt6. Our results suggest that an important mitotic function for γ-tubulin may lie within the spindle, where augmin and γ-tubulin function cooperatively to amplify the number of microtubules.
  • Preprint
    RNAi screening identifies the armadillo repeat-containing kinesins responsible for microtubule-dependent nuclear positioning in Physcomitrella patens
    ( 2015-01) Miki, Tomohiro ; Nishina, Momoko ; Goshima, Gohta
    Proper positioning of the nucleus is critical for the functioning of various cells. Actin and myosin have been shown to be crucial for the localisation of the nucleus in plant cells, whereas microtubule (MT)-based mechanisms are commonly utilised in animal and fungal cells. In this study, we combined live cell microscopy with RNA interference (RNAi) screening or drug treatment and showed that MTs and a plant-specific motor protein, armadillo repeat-containing kinesin (kinesin-ARK), are required for nuclear positioning in the moss Physcomitrella patens. In tip-growing protonemal apical cells, the nucleus was translocated to the centre of the cell after cell division in an MT-dependent manner. When kinesin-ARKs were knocked down using RNAi, the initial movement of the nucleus towards the centre took place normally; however, before reaching the centre, the nucleus was moved back to the basal edge of the cell. In intact (control) cells, MT bundles that are associated with kinesin-ARKs were frequently observed around the moving nucleus. In contrast, such MT bundles were not identified after kinesin-ARK downregulation. An in vitro MT-gliding assay showed that kinesin-ARK is a plus-end-directed motor protein. These results indicate that MTs and the MT-based motor drive nuclear migration in the moss cells, thus showing a conservation of the mechanism underlying nuclear localisation among plant, animal, and fungal cells.
  • Article
    Reconstitution of dynamic microtubules with Drosophila XMAP215, EB1, and Sentin
    (Rockefeller University Press, 2012-11-26) Li, Wenjing ; Moriwaki, Takashi ; Tani, Tomomi ; Watanabe, Takashi ; Kaibuchi, Kozo ; Goshima, Gohta
    Dynamic microtubules (MTs) are essential for various intracellular events, such as mitosis. In Drosophila melanogaster S2 cells, three MT tip-localizing proteins, Msps/XMAP215, EB1, and Sentin (an EB1 cargo protein), have been identified as being critical for accelerating MT growth and promoting catastrophe events, thus resulting in the formation of dynamic MTs. However, the molecular activity of each protein and the basis of the modulation of MT dynamics by these three factors are unknown. In this paper, we showed in vitro that XMAP215msps had a potent growth-promoting activity at a wide range of tubulin concentrations, whereas Sentin, when recruited by EB1 to the growing MT tip, accelerated growth and also increased catastrophe frequency. When all three factors were combined, the growth rate was synergistically enhanced, and rescue events were observed most frequently, but frequent catastrophes restrained the lengthening of the MTs. We propose that MT dynamics are promoted by the independent as well as the cooperative action of XMAP215msps polymerase and the EB1–Sentin duo.
  • Article
    Drosophila kinesin-8 stabilizes the kinetochore–microtubule interaction
    (Rockefeller University Press, 2018-12-11) Edzuka, Tomoya ; Goshima, Gohta
    Kinesin-8 is required for proper chromosome alignment in a variety of animal and yeast cell types. However, it is unclear how this motor protein family controls chromosome alignment, as multiple biochemical activities, including inconsistent ones between studies, have been identified. Here, we find that Drosophila kinesin-8 (Klp67A) possesses both microtubule (MT) plus end–stabilizing and –destabilizing activity, in addition to kinesin-8's commonly observed MT plus end–directed motility and tubulin-binding activity in vitro. We further show that Klp67A is required for stable kinetochore–MT attachment during prometaphase in S2 cells. In the absence of Klp67A, abnormally long MTs interact in an “end-on” fashion with kinetochores at normal frequency. However, the interaction is unstable, and MTs frequently become detached. This phenotype is rescued by ectopic expression of the MT plus end–stabilizing factor CLASP, but not by artificial shortening of MTs. We show that human kinesin-8 (KIF18A) is also important to ensure proper MT attachment. Overall, these results suggest that the MT-stabilizing activity of kinesin-8 is critical for stable kinetochore–MT attachment.
  • Preprint
    Clustering of a kinesin-14 motor enables processive retrograde microtubule-based transport in plants
    ( 2015-05) Jonsson, Erik ; Yamada, Moe ; Vale, Ronald D. ; Goshima, Gohta
    The molecular motors kinesin and dynein drive bidirectional motility along microtubules (MTs) in most eukaryotic cells1,2. Land plants, however, are a notable exception, since they contain a large number of kinesins but lack cytoplasmic dynein, the foremost processive retrograde transporter3,4. It remains unclear how plants achieve retrograde cargo transport without dynein. Here, we have analyzed the motility of the six members of minus-end-directed kinesin-14 motors in the moss Physcomitrella patens and found that none are processive as native dimers. However, when artificially clustered into as little as dimer of dimers, the type-VI kinesin-14 (a homologue of Arabidopsis KCBP [kinesin-like calmodulin binding protein]) exhibited highly processive and fast motility (up to 0.6 μm/s). Multiple kin14-VI dimers attached to liposomes also induced transport of this membrane cargo over several microns. Consistent with these results, in vivo observations of GFP-tagged kin14-VI in moss cells revealed fluorescent punctae that moved processively towards the minus ends of the cytoplasmic MTs. These data suggest that clustering of a kinesin-14 motor serves as a dynein-independent mechanism for retrograde transport in plants.
  • Article
    Five factors can reconstitute all three phases of microtubule polymerization dynamics
    (Rockefeller University Press, 2016-10-31) Moriwaki, Takashi ; Goshima, Gohta
    Cytoplasmic microtubules (MTs) undergo growth, shrinkage, and pausing. However, how MT polymerization cycles are produced and spatiotemporally regulated at a molecular level is unclear, as the entire cycle has not been recapitulated in vitro with defined components. In this study, we reconstituted dynamic MT plus end behavior involving all three phases by mixing tubulin with five Drosophila melanogaster proteins (EB1, XMAP215Msps, Sentin, kinesin-13Klp10A, and CLASPMast/Orbit). When singly mixed with tubulin, CLASPMast/Orbit strongly inhibited MT catastrophe and reduced the growth rate. However, in the presence of the other four factors, CLASPMast/Orbit acted as an inducer of pausing. The mitotic kinase Plk1Polo modulated the activity of CLASPMast/Orbit and kinesin-13Klp10A and increased the dynamic instability of MTs, reminiscent of mitotic cells. These results suggest that five conserved proteins constitute the core factors for creating dynamic MTs in cells and that Plk1-dependent phosphorylation is a crucial event for switching from the interphase to mitotic mode.