Tug-of-War in Motor Protein Ensembles Revealed with a Programmable DNA Origami Scaffold

Push Me, Release, Pull You In eukaryotic cells, nearly all long-distance transport of cargos is carried out by the microtubule-based motors kinesin and dynein. These opposite-polarity motors move cargos bidirectionally so that they reach their cellular destinations with spatial and temporal specificity. To understand transport by motor ensembles, Derr et al. (p. 662, published online 11 October; see the Persective by Diehl) used a DNA scaffold for building an artificial cargo that could be programmed to bind different numbers and types of molecular motors with defined geometry. A cargo with multiple copies of the same motor was transported with minimal interference, suggesting that similar-polarity motors can coordinate without the need for additional cellular factors. However, ensembles of opposite-polarity motors frequently engaged in a sort of “tug of war,” which could only be resolved by releasing one motor from the microtubule track. Thus, within the cell, it is likely that regulation is required for bidirectional transport. Two microtubule motors attached to the same cargo cooperate or compete, depending on relative directionality. Cytoplasmic dynein and kinesin-1 are microtubule-based motors with opposite polarity that transport a wide variety of cargo in eukaryotic cells. Many cellular cargos demonstrate bidirectional movement due to the presence of ensembles of dynein and kinesin, but are ultimately sorted with spatial and temporal precision. To investigate the mechanisms that coordinate motor ensemble behavior, we built a programmable synthetic cargo using three-dimensional DNA origami to which varying numbers of DNA oligonucleotide-linked motors could be attached, allowing for control of motor type, number, spacing, and orientation in vitro. In ensembles of one to seven identical-polarity motors, motor number had minimal affect on directional velocity, whereas ensembles of opposite-polarity motors engaged in a tug-of-war resolvable by disengaging one motor species.

[1]  R. Vale,et al.  Directional instability of microtubule transport in the presence of kinesin and dynein, two opposite polarity motor proteins , 1992, The Journal of cell biology.

[2]  T. Mitchison,et al.  Microtubule polymerization dynamics. , 1997, Annual review of cell and developmental biology.

[3]  Ronald D Vale,et al.  The Directional Preference of Kinesin Motors Is Specified by an Element outside of the Motor Catalytic Domain , 1997, Cell.

[4]  J. Ashby.  References and Notes , 1999 .

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

[6]  Ronald D Vale,et al.  The Molecular Motor Toolbox for Intracellular Transport , 2003, Cell.

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

[8]  M. Welte,et al.  Bidirectional Transport along Microtubules , 2004, Current Biology.

[9]  R. Lipowsky,et al.  Cooperative cargo transport by several molecular motors. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[10]  P. Rothemund.  Folding DNA to create nanoscale shapes and patterns , 2006, Nature.

[11]  Enrico Gratton,et al.  Organelle transport along microtubules in Xenopus melanophores: evidence for cooperation between multiple motors. , 2006, Biophysical journal.

[12]  R. Vallee,et al.  The cellular roles of the lissencephaly gene LIS1, and what they tell us about brain development. , 2006, Genes & development.

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

[14]  John M. Walker,et al.  Molecular Motors , 2007, Methods in Molecular Biology™.

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

[16]  S. Gross,et al.  Stepping, Strain Gating, and an Unexpected Force-Velocity Curve for Multiple-Motor-Based Transport , 2008, Current Biology.

[17]  E. Fisher,et al.  Cytoplasmic dynein could be key to understanding neurodegeneration , 2008, Genome Biology.

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

[19]  Shawn M. Douglas,et al.  Self-assembly of DNA into nanoscale three-dimensional shapes , 2009, Nature.

[20]  Roop Mallik,et al.  Tug-of-war between dissimilar teams of microtubule motors regulates transport and fission of endosomes , 2009, Proceedings of the National Academy of Sciences.

[21]  Adam H. Marblestone,et al.  Rapid prototyping of 3D DNA-origami shapes with caDNAno , 2009, Nucleic acids research.

[22]  Pamela E. Constantinou,et al.  Negative interference dominates collective transport of kinesin motors in the absence of load. , 2009, Physical chemistry chemical physics : PCCP.

[23]  Yosuke Tanaka,et al.  Molecular Motors in Neurons: Transport Mechanisms and Roles in Brain Function, Development, and Disease , 2010, Neuron.

[24]  Collective Behavior of Antagonistically Acting Kinesin-1 Motors , 2010 .

[25]  Cécile Leduc,et al.  Collective behavior of antagonistically acting kinesin-1 motors. , 2010, Physical review letters.

[26]  F. Simmel,et al.  Single-molecule kinetics and super-resolution microscopy by fluorescence imaging of transient binding on DNA origami. , 2010, Nano letters.

[27]  Pamela E. Constantinou,et al.  The mechanochemistry of integrated motor protein complexes. , 2010, Journal of biomechanics.

[28]  Pamela E. Constantinou,et al.  Two kinesins transport cargo primarily via the action of one motor: implications for intracellular transport. , 2010, Biophysical journal.

[29]  L. Goldstein,et al.  Stable Kinesin and Dynein Assemblies Drive the Axonal Transport of Mammalian Prion Protein Vesicles , 2011, Cell.

[30]  Michael R Diehl,et al.  Cooperative Responses of Multiple Kinesins to Variable and Constant Loads* , 2011, The Journal of Biological Chemistry.

[31]  R. Lipowsky,et al.  Transient binding of dynein controls bidirectional long-range motility of early endosomes , 2011, Proceedings of the National Academy of Sciences.

[32]  Clare C. Yu,et al.  Mechanical stochastic tug-of-war models cannot explain bidirectional lipid-droplet transport , 2011, Proceedings of the National Academy of Sciences.

[33]  H. Sweeney,et al.  Myosin Va and myosin VI coordinate their steps while engaged in an in vitro tug of war during cargo transport , 2011, Proceedings of the National Academy of Sciences.

[34]  Samara L. Reck-Peterson,et al.  Lis1 is an initiation factor for dynein-driven organelle transport , 2012, The Journal of cell biology.

[35]  Tuning Multiple Motor Travel via Single Motor Velocity , 2012, Traffic.

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

[37]  Samara L. Reck-Peterson,et al.  Structural Basis for Microtubule Binding and Release by Dynein , 2012, Science.

[38]  O. Zhapparova,et al.  Bidirectional transport of organelles: unity and struggle of opposing motors , 2012, Cell biology international.

[39]  Peng Yin,et al.  Submicrometre geometrically encoded fluorescent barcodes self-assembled from DNA. , 2012, Nature chemistry.

[40]  Simon L. Bullock,et al.  Single-molecule assays reveal that RNA localization signals regulate dynein-dynactin copy number on individual transcript cargoes , 2012, Nature Cell Biology.

[41]  Hailong Lu,et al.  Collective Dynamics of Elastically Coupled Myosin V Motors* , 2012, The Journal of Biological Chemistry.

[42]  K. Gothelf,et al.  Multilayer DNA origami packed on hexagonal and hybrid lattices. , 2012, Journal of the American Chemical Society.

[43]  Gregg M. Gallatin,et al.  Nanomanufacturing with DNA Origami: Factors Affecting the Kinetics and Yield of Quantum Dot Binding , 2012 .