Remote control of myosin and kinesin motors using light-activated gearshifting.

Cytoskeletal motors perform critical force generation and transport functions in eukaryotic cells. Engineered modifications of motor function provide direct tests of protein structure-function relationships and potential tools for controlling cellular processes or for harnessing molecular transport in artificial systems. Here, we report the design and characterization of a panel of cytoskeletal motors that reversibly change gears--speed up, slow down or switch directions--when exposed to blue light. Our genetically encoded structural designs incorporate a photoactive protein domain to enable light-dependent conformational changes in an engineered lever arm. Using in vitro motility assays, we demonstrate robust spatiotemporal control over motor function and characterize the kinetics of the optical gearshifting mechanism. We have used a modular approach to create optical gearshifting motors for both actin-based and microtubule-based transport.

[1]  H. Higuchi,et al.  A mutant of the motor protein kinesin that moves in both directions on microtubules , 2000, Nature.

[2]  David G. Altman,et al.  The power stroke of myosin VI and the basis of reverse directionality , 2007, Proceedings of the National Academy of Sciences.

[3]  J. Sellers,et al.  Myosins: a diverse superfamily. , 2000, Biochimica et biophysica acta.

[4]  D. Manstein,et al.  Recombinant motor domain constructs of Chara corallina myosin display fast motility and high ATPase activity. , 2003, Biochemical and biophysical research communications.

[5]  M. Heidecker,et al.  Light-directed generation of the actin-activated ATPase activity of caged heavy meromyosin. , 1996, Biochemistry.

[6]  Hee-Won Park,et al.  Rotation of the stalk/neck and one head in a new crystal structure of the kinesin motor protein, Ncd , 2003, The EMBO journal.

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

[8]  Kevin H. Gardner,et al.  Structural Basis of a Phototropin Light Switch , 2003, Science.

[9]  M Anson,et al.  Myosin motors with artificial lever arms. , 1996, The EMBO journal.

[10]  B. Imperiali,et al.  Light-triggered myosin activation for probing dynamic cellular processes. , 2011, Angewandte Chemie.

[11]  K. Kohama,et al.  The fastest‐actin‐based motor protein from the green algae, Chara, and its distinct mode of interaction with actin , 1995, FEBS letters.

[12]  M. Alexandre,et al.  A Base-Catalyzed Mechanism for Dark State Recovery in the Avena sativa Phototropin-1 LOV2 Domain† , 2007 .

[13]  F. Kull,et al.  A METAL SWITCH FOR CONTROLLING THE ACTIVITY OF MOLECULAR MOTOR PROTEINS , 2011, Nature Structural &Molecular Biology.

[14]  K. Hellingwerf,et al.  A base-catalyzed mechanism for dark state recovery in the Avena sativa phototropin-1 LOV2 domain. , 2007, Biochemistry.

[15]  J. Christie,et al.  Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin. , 2000, Biochemistry.

[16]  Elisabeth F. Heuston,et al.  A kinesin motor in a force-producing conformation , 2010, BMC Structural Biology.

[17]  W. P. Russ,et al.  Surface Sites for Engineering Allosteric Control in Proteins , 2008, Science.

[18]  H. Sweeney,et al.  The Structural Basis for the Large Powerstroke of Myosin VI , 2007, Cell.

[19]  Z. Bryant,et al.  Engineering myosins for long-range transport on actin filaments , 2013, Nature nanotechnology.

[20]  Samara L. Reck-Peterson,et al.  Engineered, harnessed, and hijacked: synthetic uses for cytoskeletal systems. , 2012, Trends in cell biology.

[21]  Jung-Chi Liao,et al.  Engineered myosin VI motors reveal minimal structural determinants of directionality and processivity. , 2009, Journal of molecular biology.

[22]  Viola Vogel,et al.  Harnessing biological motors to engineer systems for nanoscale transport and assembly. , 2008, Nature nanotechnology.

[23]  B. Kuhlman,et al.  A genetically-encoded photoactivatable Rac controls the motility of living cells , 2009, Nature.

[24]  Muneaki Nakamura,et al.  Engineering controllable bidirectional molecular motors based on myosin , 2012, Nature nanotechnology.

[25]  D. Manstein,et al.  Molecular engineering of a backwards-moving myosin motor , 2004, Nature.

[26]  K. Deisseroth,et al.  Millisecond-timescale, genetically targeted optical control of neural activity , 2005, Nature Neuroscience.

[27]  N. Tamaoki,et al.  A photochromic ATP analogue driving a motor protein with reversible light-controlled motility: controlling velocity and binding manner of a kinesin-microtubule system in an in vitro motility assay. , 2012, Chemical communications.

[28]  Ronald D. Vale,et al.  A lever-arm rotation drives motility of the minus-end-directed kinesin Ncd , 2006, Nature.

[29]  K. Huth.  Transport , 2015, Canadian Medical Association Journal.

[30]  N. Volkmann,et al.  Microscopic evidence for a minus‐end‐directed power stroke in the kinesin motor ncd , 2002, The EMBO journal.

[31]  J. Spudich,et al.  The neck region of the myosin motor domain acts as a lever arm to generate movement. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[32]  A. Hyman,et al.  Preparation of marked microtubules for the assay of the polarity of microtubule-based motors by fluorescence , 1991, Journal of Cell Science.

[33]  M. Yamada,et al.  Photocontrol of kinesin ATPase activity using an azobenzene derivative. , 2007, Journal of biochemistry.

[34]  K. Moffat,et al.  Light-activated DNA binding in a designed allosteric protein , 2008, Proceedings of the National Academy of Sciences.

[35]  B. Hammerich,et al.  Frozen steady states in active systems , 2011, Proceedings of the National Academy of Sciences.

[36]  Josiah P. Zayner,et al.  TULIPs: Tunable, light-controlled interacting protein tags for cell biology , 2012, Nature Methods.

[37]  A. Månsson.  Translational actomyosin research: fundamental insights and applications hand in hand , 2012, Journal of Muscle Research and Cell Motility.

[38]  S. Diez,et al.  Selective control of gliding microtubule populations. , 2012, Nano letters.

[39]  E. Katayama,et al.  Higher plant myosin XI moves processively on actin with 35 nm steps at high velocity , 2003, The EMBO journal.

[40]  Samara L. Reck-Peterson,et al.  Tug-of-War in Motor Protein Ensembles Revealed with a Programmable DNA Origami Scaffold , 2012, Science.

[41]  Christopher A. Voigt,et al.  The promise of optogenetics in cell biology: interrogating molecular circuits in space and time , 2011, Nature Methods.