Vale Ronald D.

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Vale
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Ronald D.
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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 : 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.