Dynein is regulated by the stability of its microtubule track

How dynein motors accurately move cargoes is an important question. In budding yeast, dynein moves the mitotic spindle to the predetermined site of cytokinesis by pulling on astral microtubules. In this study, using high-resolution imaging in living cells, we discover that spindle movement is regulated by changes in microtubule plus-end dynamics that occur when dynein generates force. Mutants that increase plus-end stability increase the frequency and duration of spindle movements, causing positioning errors. We find that dynein plays a primary role in regulating microtubule dynamics by destabilizing microtubules. In contrast, the dynactin complex counteracts dynein and stabilizes microtubules through a mechanism involving the shoulder subcomplex and the cytoskeletal-associated protein glycine-rich domain of Nip100/p150glued. Our results support a model in which dynein destabilizes its microtubule substrate by using its motility to deplete dynactin from the plus end. We propose that interplay among dynein, dynactin, and the stability of the microtubule substrate creates a mechanism that regulates accurate spindle positioning.

[1]  P. Philippsen,et al.  Mechanism of nuclear movements in a multinucleated cell , 2017, bioRxiv.

[2]  S. M. Markus,et al.  The dynein cortical anchor Num1 activates dynein motility by relieving Pac1/LIS1-mediated inhibition , 2015, The Journal of cell biology.

[3]  Wei-Lih Lee,et al.  Improved Plasmids for Fluorescent Protein Tagging of Microtubules in Saccharomyces cerevisiae , 2015, Traffic.

[4]  Kai Zhang,et al.  The structure of the dynactin complex and its interaction with dynein , 2015, Science.

[5]  T. Schroer,et al.  Structural organization of the dynein-dynactin complex bound to microtubules , 2015, Nature Structural &Molecular Biology.

[6]  A. Yildiz,et al.  THE AAA3 DOMAIN OF CYTOPLASMIC DYNEIN ACTS AS A SWITCH TO FACILITATE MICROTUBULE RELEASE , 2014, Nature Structural &Molecular Biology.

[7]  E. Holzbaur,et al.  Dynactin functions as both a dynamic tether and brake during dynein-driven motility , 2014, Nature Communications.

[8]  Gira Bhabha,et al.  Activation of cytoplasmic dynein motility by dynactin-cargo adapter complexes , 2014, Science.

[9]  Ha Thanh Thi Hoang,et al.  In vitro reconstitution of a highly processive recombinant human dynein complex , 2014, The EMBO journal.

[10]  D. Sept,et al.  Genome-wide Analysis Reveals Novel and Discrete Functions for Tubulin Carboxy-Terminal Tails , 2014, Current Biology.

[11]  T. Stearns,et al.  Methods in yeast genetics , 2013 .

[12]  E. Holzbaur,et al.  Dynactin Subunit p150Glued Is a Neuron-Specific Anti-Catastrophe Factor , 2013, PLoS biology.

[13]  M. Steinmetz,et al.  Molecular Mechanism of Action of Microtubule-Stabilizing Anticancer Agents , 2013, Science.

[14]  I. Cheeseman,et al.  Cortical Dynein and Asymmetric Membrane Elongation Coordinately Position the Spindle in Anaphase , 2013, Cell.

[15]  Frank Jülicher,et al.  Cortical Dynein Controls Microtubule Dynamics to Generate Pulling Forces that Position Microtubule Asters , 2012, Cell.

[16]  M. Srayko,et al.  Visualization of dynein-dependent microtubule gliding at the cell cortex: implications for spindle positioning , 2011, The Journal of cell biology.

[17]  J. Cooper,et al.  Functional interaction between dynein light chain and intermediate chain is required for mitotic spindle positioning , 2011, Molecular biology of the cell.

[18]  Wei-Lih Lee,et al.  Quantitative analysis of Pac1/LIS1‐mediated dynein targeting: Implications for regulation of dynein activity in budding yeast , 2011, Cytoskeleton.

[19]  S. O’Rourke,et al.  Caenorhabditis elegans EFA-6 limits microtubule growth at the cell cortex , 2010, Nature Cell Biology.

[20]  P. Philippsen,et al.  Dynamics of Multiple Nuclei in Ashbya gossypii Hyphae Depend on the Control of Cytoplasmic Microtubules Length by Bik1, Kip2, Kip3, and Not on a Capture/Shrinkage Mechanism , 2010, Molecular biology of the cell.

[21]  A. Hyman,et al.  Membrane Invaginations Reveal Cortical Sites that Pull on Mitotic Spindles in One-Cell C. elegans Embryos , 2010, PloS one.

[22]  D. Sept,et al.  Neurodegeneration mutations in dynactin impair dynein-dependent nuclear migration , 2009, Proceedings of the National Academy of Sciences.

[23]  J. Yates,et al.  The human kinetochore Ska1 complex facilitates microtubule depolymerization-coupled motility. , 2009, Developmental cell.

[24]  Wei-Lih Lee,et al.  Motor- and Tail-Dependent Targeting of Dynein to Microtubule Plus Ends and the Cell Cortex , 2009, Current Biology.

[25]  G. Neale,et al.  Enhancing Drug Accumulation in Saccharomyces cerevisiae by Repression of Pleiotropic Drug Resistance Genes with Chimeric Transcription Repressors , 2008, Molecular Pharmacology.

[26]  C. Doe,et al.  Lis1/dynactin regulates metaphase spindle orientation in Drosophila neuroblasts. , 2008, Developmental biology.

[27]  J. Cooper,et al.  Dynactin Function in Mitotic Spindle Positioning , 2008, Traffic.

[28]  J. Labbé,et al.  Heterotrimeric G protein signaling functions with dynein to promote spindle positioning in C. elegans , 2007, The Journal of cell biology.

[29]  Cleopatra Kozlowski,et al.  Cortical Microtubule Contacts Position the Spindle in C. elegans Embryos , 2007, Cell.

[30]  Anthony A. Hyman,et al.  Spindle Oscillations during Asymmetric Cell Division Require a Threshold Number of Active Cortical Force Generators , 2006, Current Biology.

[31]  M. Steinmetz,et al.  Key interaction modes of dynamic +TIP networks. , 2006, Molecular cell.

[32]  Mohan L Gupta,et al.  Plus end-specific depolymerase activity of Kip3, a kinesin-8 protein, explains its role in positioning the yeast mitotic spindle , 2006, Nature Cell Biology.

[33]  Trisha N Davis,et al.  The Dam1 kinetochore complex harnesses microtubule dynamics to produce force and movement. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Andrew D. Stephens,et al.  A microtubule-binding domain in dynactin increases dynein processivity by skating along microtubules , 2006, Nature Cell Biology.

[35]  J. Cooper,et al.  The offloading model for dynein function , 2005, The Journal of cell biology.

[36]  Kazuo Sutoh,et al.  Distinct functions of nucleotide-binding/hydrolysis sites in the four AAA modules of cytoplasmic dynein. , 2004, Biochemistry.

[37]  K. Thorn,et al.  Optimized cassettes for fluorescent protein tagging in Saccharomyces cerevisiae , 2004, Yeast.

[38]  Marileen Dogterom,et al.  Dynamic instability of microtubules is regulated by force , 2003, The Journal of cell biology.

[39]  J. Labbé,et al.  PAR Proteins Regulate Microtubule Dynamics at the Cell Cortex in C. elegans , 2003, Current Biology.

[40]  J. Cooper,et al.  The role of the lissencephaly protein Pac1 during nuclear migration in budding yeast , 2003, The Journal of cell biology.

[41]  P. Gönczy.  Mechanisms of spindle positioning: focus on flies and worms. , 2002, Trends in cell biology.

[42]  Claudia J. Bode,et al.  Epothilone and paclitaxel: unexpected differences in promoting the assembly and stabilization of yeast microtubules. , 2002, Biochemistry.

[43]  M. Longtine,et al.  PCR-based engineering of yeast genome. , 2002, Methods in enzymology.

[44]  M. Han,et al.  Cytoplasmic dynein light intermediate chain is required for discrete aspects of mitosis in Caenorhabditis elegans. , 2001, Molecular biology of the cell.

[45]  Kyung S. Lee,et al.  A Novel Function of Saccharomyces cerevisiae CDC5 in Cytokinesis , 2001, The Journal of cell biology.

[46]  Anthony A. Hyman,et al.  Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo , 2001, Nature.

[47]  J. Cooper,et al.  Microtubule Interactions with the Cell Cortex Causing Nuclear Movements in Saccharomyces cerevisiae , 2000, The Journal of cell biology.

[48]  E. Salmon,et al.  The polarity and dynamics of microtubule assembly in the budding yeast Saccharomyces cerevisiae , 1999, Nature Cell Biology.

[49]  John G. White,et al.  The dynactin complex is required for cleavage plane specification in early Caenorhabditis elegans embryos , 1998, Current Biology.

[50]  Tim Stearns,et al.  Microtubules Orient the Mitotic Spindle in Yeast through Dynein-dependent Interactions with the Cell Cortex , 1997, The Journal of cell biology.

[51]  C. Waterman-Storer,et al.  The p150Glued component of the dynactin complex binds to both microtubules and the actin-related protein centractin (Arp-1). , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[52]  J. Cooper,et al.  Ultrastructural analysis of the dynactin complex: an actin-related protein is a component of a filament that resembles F-actin , 1994, The Journal of cell biology.

[53]  B. Matsumoto,et al.  Kinetic stabilization of microtubule dynamic instability in vitro by vinblastine. , 1993, Biochemistry.

[54]  B. Byers,et al.  Behavior of spindles and spindle plaques in the cell cycle and conjugation of Saccharomyces cerevisiae , 1975, Journal of bacteriology.

[55]  R. Ebstein,et al.  DURATION OF STIMULUS AND LATENT PERIODS PRECEDING FURROW FORMATION IN SAND DOLLAR EGGS. , 1965, The Journal of experimental zoology.