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.
  • Preprint
    Phase separation of signaling molecules promotes T cell receptor signal transduction
    ( 2016-03) Su, Xiaolei ; Ditlev, Jonathon ; Hui, Enfu ; Xing, Wenmin ; Banjade, Sudeep ; Okrut, Julia ; King, David S. ; Taunton, Jack ; Rosen, Michael K. ; Vale, Ronald D.
    Activation of various cell surface receptors triggers the reorganization of downstream signaling molecules into micron- or submicron-sized clusters. However, the functional consequences of such clustering has been unclear. We biochemically reconstituted a 12-component signaling pathway on model membranes, beginning with T cell receptor (TCR) activation and ending with actin assembly. When TCR phoshophorylation was triggered, downstream signaling proteins spontaneously separated into liquid-like clusters that promoted signaling outputs both in vitro and in human Jurkat T cells. Reconstituted clusters were enriched in kinases but excluded phosphatases, and enhanced actin filament assembly by recruiting and organizing actin regulators. These results demonstrate that protein phase separation can create a distinct physical and biochemical compartment that facilitates signaling.
  • 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.
  • Article
    Dynamics of myosin, microtubules, and Kinesin-6 at the cortex during cytokinesis in Drosophila S2 cells
    (Rockefeller University Press, 2009-08) Vale, Ronald D. ; Spudich, James A. ; Griffis, Eric R.
    Signals from the mitotic spindle during anaphase specify the location of the actomyosin contractile ring during cytokinesis, but the detailed mechanism remains unresolved. Here, we have imaged the dynamics of green fluorescent protein–tagged myosin filaments, microtubules, and Kinesin-6 (which carries activators of Rho guanosine triphosphatase) at the cell cortex using total internal reflection fluorescence microscopy in flattened Drosophila S2 cells. At anaphase onset, Kinesin-6 relocalizes to microtubule plus ends that grow toward the cortex, but refines its localization over time so that it concentrates on a subset of stable microtubules and along a diffuse cortical band at the equator. The pattern of Kinesin-6 localization closely resembles where new myosin filaments appear at the cortex by de novo assembly. While accumulating at the equator, myosin filaments disappear from the poles of the cell, a process that also requires Kinesin-6 as well as possibly other signals that emanate from the elongating spindle. These results suggest models for how Kinesin-6 might define the position of cortical myosin during cytokinesis.
  • 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
    A composition-dependent molecular clutch between T cell signaling condensates and actin
    (eLife Sciences Publications, 2019-07-03) Ditlev, Jonathon ; Vega, Anthony R ; Köster, Darius Vasco ; Su, Xiaolei ; Tani, Tomomi ; Lakoduk, Ashley M ; Vale, Ronald D. ; Mayor, Satyajit ; Jaqaman, Khuloud ; Rosen, Michael K.
    During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments. We found that basic regions of Nck and N-WASP/WASP promote association and co-movement of LAT condensates with actin, indicating conversion of weak individual affinities to high collective affinity upon phase separation. Condensates lacking these components were propelled differently, without strong actin adhesion. In cells, LAT condensates lost Nck as radial actin transitioned to the concentric network, and engineered condensates constitutively binding actin moved aberrantly. Our data show that Nck and WASP form a clutch between LAT condensates and actin in vitro and suggest that compositional changes may enable condensate movement by distinct actin networks in different regions of the IS. https://doi.org/10.7554/eLife.42695.001