TENSION ON THE LINKER GATES THE ATP-DEPENDENT RELEASE OF DYNEIN FROM MICROTUBULES

Cytoplasmic dynein is a dimeric motor that transports intracellular cargoes towards the minus-end of microtubules (MTs). In contrast to other processive motors, stepping of the dynein motor domains (heads) is not precisely coordinated. Therefore, the mechanism of dynein processivity remains unclear. Here, by engineering the mechanical and catalytic properties of the motor, we show that dynein processivity minimally requires a single active head and a second inert MT binding domain. Processivity arises from a high ratio of MT-bound to unbound time, and not from interhead communication. Additionally, nucleotide-dependent microtubule release is gated by tension on the linker domain. Intramolecular tension sensing is observed in dynein’s stepping motion at high interhead separations. We developed a quantitative model for the stepping characteristics of dynein and its response to chemical and mechanical perturbation.

[1]  Shin'ichi Ishiwata,et al.  Kinesin–microtubule binding depends on both nucleotide state and loading direction , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  S. Block,et al.  Versatile optical traps with feedback control. , 1998, Methods in enzymology.

[3]  Samara L. Reck-Peterson,et al.  Single-Molecule Analysis of Dynein Processivity and Stepping Behavior , 2006, Cell.

[4]  R. Vale,et al.  Crystal Structure of the Dynein Motor Domain , 2011, Science.

[5]  Steven M. Block,et al.  Kinesin Moves by an Asymmetric Hand-OverHand Mechanism , 2003 .

[6]  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.

[7]  Peter A. Combs,et al.  Cytoplasmic Dynein Moves Through Uncoordinated Stepping of the AAA+ Ring Domains , 2012, Science.

[8]  S. Burgess,et al.  Helix sliding in the stalk coiled coil of dynein couples ATPase and microtubule binding , 2009, Nature Structural &Molecular Biology.

[9]  Tomohiro Shima,et al.  Two modes of microtubule sliding driven by cytoplasmic dynein , 2006, Proceedings of the National Academy of Sciences.

[10]  Zev Bryant,et al.  Contribution of the myosin VI tail domain to processive stepping and intramolecular tension sensing , 2010, Proceedings of the National Academy of Sciences.

[11]  K. Sutoh,et al.  The 2.8-Å Crystal Structure of the Dynein Motor Domain , 2012 .

[12]  K. Sutoh,et al.  Head-head coordination is required for the processive motion of cytoplasmic dynein, an AAA+ molecular motor. , 2006, Journal of structural biology.

[13]  Samara L. Reck-Peterson,et al.  Lis1 Acts as a “Clutch” between the ATPase and Microtubule-Binding Domains of the Dynein Motor , 2012, Cell.

[14]  Hideo Higuchi,et al.  Alternate fast and slow stepping of a heterodimeric kinesin molecule , 2003, Nature Cell Biology.

[15]  Samara L. Reck-Peterson,et al.  Regulatory ATPase Sites of Cytoplasmic Dynein Affect Processivity and Force Generation*S⃞ , 2008, Journal of Biological Chemistry.

[16]  S. McKinney,et al.  Nonblinking and long-lasting single-molecule fluorescence imaging , 2006, Nature Methods.

[17]  Toshio Ando,et al.  Video imaging of walking myosin V by high-speed atomic force microscopy , 2010, Nature.

[18]  R. Vallee,et al.  Dynein: An ancient motor protein involved in multiple modes of transport. , 2004, Journal of neurobiology.

[19]  S. Lindquist,et al.  Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein. , 2002, Molecular cell.

[20]  R. Vale,et al.  Kinesin Walks Hand-Over-Hand , 2004, Science.

[21]  Steven M Block,et al.  Kinesin motor mechanics: binding, stepping, tracking, gating, and limping. , 2007, Biophysical journal.

[22]  Andreas Hoenger,et al.  Kinesin's second step. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

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

[24]  Koen Visscher,et al.  An objective, model-independent method for detection of non-uniform steps in noisy signals , 2008, Comput. Phys. Commun..

[25]  Francesco S. Pavone,et al.  Ultrafast force-clamp spectroscopy of single molecules reveals load dependence of myosin working stroke , 2012, Nature Methods.

[26]  A. Carter,et al.  Insights into dynein motor domain function from a 3.3 Å crystal structure , 2012, Nature Structural &Molecular Biology.

[27]  J. Gelles,et al.  Processive Movement by a Kinesin Heterodimer with an Inactivating Mutation in One Head , 2008, Biochemistry.

[28]  Arne Gennerich,et al.  Walking the walk: how kinesin and dynein coordinate their steps. , 2009, Current opinion in cell biology.

[29]  William O. Hancock,et al.  Neck Linker Length Determines the Degree of Processivity in Kinesin-1 and Kinesin-2 Motors , 2010, Current Biology.

[30]  Samara L. Reck-Peterson,et al.  The Affinity of the Dynein Microtubule-binding Domain Is Modulated by the Conformation of Its Coiled-coil Stalk*[boxs] , 2005, Journal of Biological Chemistry.

[31]  J. Scholey,et al.  Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A. , 2005, Molecular biology of the cell.

[32]  R. Vale,et al.  Spindly, a novel protein essential for silencing the spindle assembly checkpoint, recruits dynein to the kinetochore , 2007, The Journal of cell biology.

[33]  Samara L. Reck-Peterson,et al.  Force-Induced Bidirectional Stepping of Cytoplasmic Dynein , 2007, Cell.

[34]  Paul R. Selvin,et al.  Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization , 2003, Science.

[35]  J. McIntosh,et al.  The Molecular Architecture of Axonemes Revealed by Cryoelectron Tomography , 2006, Science.

[36]  Samara L. Reck-Peterson,et al.  Dynein achieves processive motion using both stochastic and coordinated stepping , 2011, Nature Structural &Molecular Biology.

[37]  Kazuo Sutoh,et al.  The coordination of cyclic microtubule association/dissociation and tail swing of cytoplasmic dynein , 2007, Proceedings of the National Academy of Sciences.

[38]  N. Katsanis,et al.  Microtubule transport defects in neurological and ciliary disease , 2005, Cellular and Molecular Life Sciences CMLS.

[39]  K. Sutoh,et al.  The 2.8 Å crystal structure of the dynein motor domain , 2012, Nature.

[40]  Richard B. Vallee,et al.  An extended microtubule-binding structure within the dynein motor domain , 1997, Nature.

[41]  S. Varambally,et al.  Structure and Functional Role of Dynein's Microtubule-Binding Domain , 2008, Science.

[42]  S. Burgess,et al.  Dynein structure and power stroke , 2003, Nature.

[43]  Corey W. Liu,et al.  Characterization of the FKBP.rapamycin.FRB ternary complex. , 2005, Journal of the American Chemical Society.

[44]  J. Spudich.  Molecular Motors Take Tension in Stride , 2006, Cell.

[45]  K. Sutoh,et al.  ATP hydrolysis cycle–dependent tail motions in cytoplasmic dynein , 2005, Nature Structural &Molecular Biology.

[46]  Yasushi Hiraoka,et al.  Mutations in Dynein Link Motor Neuron Degeneration to Defects in Retrograde Transport , 2003, Science.

[47]  Richard B. Vallee,et al.  Multiple modes of cytoplasmic dynein regulation , 2012, Nature Cell Biology.

[48]  Genji Kurisu,et al.  X-ray structure of a functional full-length dynein motor domain , 2011, Nature Structural &Molecular Biology.

[49]  J. Howard,et al.  Kinesin's processivity results from mechanical and chemical coordination between the ATP hydrolysis cycles of the two motor domains. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[50]  Ronald D. Vale,et al.  Intramolecular Strain Coordinates Kinesin Stepping Behavior along Microtubules , 2008, Cell.

[51]  R. Vallee,et al.  LIS1 and NudE Induce a Persistent Dynein Force-Producing State , 2010, Cell.

[52]  S. Burgess,et al.  AAA+ Ring and Linker Swing Mechanism in the Dynein Motor , 2009, Cell.