Kinesin: an ATPase that steps along microtubules

Introduction Current Research Meet "Team-Kinesin" Block Lab Publications Back to Block Lab Home

Crystal structure of dimeric kinesin
(Adapted from Kozielski et al., Cell 1997)




Kinesin is one of the world's tiniest motors.

Kinesin molecules use the energy of ATP hydrolysis to move in discrete steps along protein filaments called microtubules in cells. Each molecule of kinesin consists of two catalytically active motor domains, called "heads" that bind both a microtubule and ATP. The two heads are joined by short polypeptide chain, called the "neck linker" to a long coiled-coil stalk that terminates in a distal cargo binding domain.

A single (dimeric) protein molecule -- consisting of only ~800 amino acids, or roughly ten thousand atoms -- is sufficient to move long distances along a microtubule, even against considerable load. By studying the motion of single molecules of kinesin, our aim is to understand how this tiny machine works. Historically, most measurements of kinesins have focused on conventional kinesin (Kinesin-1 or, more commonly, "kinesin"), the founding member of a diverse family of related motor proteins. Kinesin proteins are essential for vesicle transport in neurons, and chromosome movement in dividing cells. By learning how different kinesin proteins work, we take an important step toward comprehending the dynamics of living cells.


We use optical traps to probe the stepping of single molecules of kinesin.

In order to track kinesin motion, we attach the molecules to microscopic beads. Kinesin itself is much too small to see in the optical microscope, so the beads serve as markers that can be tracked with very high precision (to 1 nm or better). The beads also act as "handles", through which we can apply force using an optical trap. Applying tension reduces Brownian motion of the bead, and allows us to observe that kinesin moves in a stepwise fashion, in many sequential increments (thus, kinesin is a processive enzyme). The step size is 8-nm, and corresponds to the spacing of the tubulin dimers that make up the microtubule. For each 8-nm step, kinesin uses a single fuel molecule, hydrolyzing one ATP molecule into ADP and inorganic phosphate. By observing an individual step of kinesin, we observe a single enzymatic turnover of ATP hydrolysis.


Cartoon of a kinesin experiment. The kinesin walks along the microtubule towards its plus-end, while being subjected to a retarding force by the optical trap.



Kinesin steps under constant forward load. The position of a kinesin motor in nanometers versus time at low ATP concentration. In this graph, the 8-nm steps that kinesin takes along the microtubule can readily be seen.
(Figure from Lang et al., Biophysical Journal, 2002 ).



We can apply controlled forces to kinesin with the optical trap.

Our optical traps have become very sophisticated, with automatic feedback control that allows us to apply forces of constant magnitude and direction (any direction in the plane of the coverslip) to the kinesin motors while they move. Studying the effect of load on the speed and regularity of kinesin's stepping motion provides clues about how motion is coupled to the biochemical events of ATP hydrolysis.

For example, our recent data suggests that at least 3 nm of each 8-nm step occurs all at once, during a single biochemical transition (Block et al., PNAS, 2003). This "working stroke" must be well-aligned with the microtubule. The remaining 5 nm may occur simultaneously, or may occur after a slight (< 1 ms) delay. We also find evidence for smaller (< 1 nm) side-to-side motions occurring at other times in the biochemical cycle.




Read more about this work in:
"Optical trap provides new insights into motor molecules -- nature's ultimate nanomachines" in the Stanford Report.

MOVIE: Kinesin-driven bead movement along a microtubule
(Credit: J. Shaevitz)


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In this clip, a micron-scale kinesin-coated bead moves along a microtubule.


MOVIE: Kinesin movement against an optical trap
(Credit: J. Shaevitz)


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In this clip, a freely diffusing kinesin-coated bead is grabbed by the optical trap and positioned above a microtubule. As the bead is moved away from the center of the trap (indicated by the crosshairs), the force on the kinesin increases. Eventually the motor releases microtubule and the bead returns to the trap center. Multiple such "runs" can be observed.




Overview of Current Research Areas:
  1. How exactly do kinesin's two heads move during stepping?


  2. Although the center of mass position of a bead-bound kinesin motor can be measured with exquisite precision using an optical trap, the relative position of each motor head is more difficult to determine. Two broad classes of models have been invoked to explain the movement of kinesin's heads during a step. In hand-over-hand models, the heads exchange leading and trailing roles with every step. By contrast, in inchworm models, one head always leads. By measuring the stepwise motion of individual enzymes, we find that some kinesin molecules exhibit a striking alternation in the dwell times between sequential steps, causing these motors to "limp" along the microtubule. Limping implies that kinesin molecules strictly alternate between two different conformations as they step, indicative of a hand-over-hand mechanism. We are currently investigating the structural origins of limping, and further exploring the details of kinesin's walk along a microtubule.



    Cartoon of kinesin's two heads (colored light gray and dark gray) exchanging positions as kinesin walks "hand-over-hand" along the microtubule.
    A kinesin stepping record showing clear alternation in dwell times between even and odd steps. This quick step, slow step walking mode is called limping. The dotted gray lines are spaced at 8-nm to guide the eye.







    To learn more about this project, see Asbury et al. Science, 2003 or read "Limping all the way, protein carries stuff of life across cell" in the Stanford Report.



  3. How do kinesin's heads remain synchronized with each other during processive stepping?

    Kinesin is more than the sum of its parts
    When one head of kinesin is removed by using biochemical techniques, the remaining head can still bind and release microtubules and hydrolyze ATP.  Each head can therefore function as an independent enzyme.  However, arbitrarily gluing the two individual heads back together would not form a processive motor.  Doing that would create a molecule where each head randomly cycled through states with different conformations and affinities for the microtubule track independently of what the other head was doing.  It wouldn't take long before both heads happened to let go of the microtubule at the same time.  Whenever both heads let go, kinesin diffuses away into the surrounding solution, ending its processive run.  Since kinesin is known to take ~100 steps on average before detaching from the microtubule, there must be some mechanism to allow the heads to communicate to each other the status of their enzymatic cycling and thereby make sure at least one is holding on to the microtubule at all times.

    Studies with nucleotide analogs
    Evidence of head-head communication is therefore an important goal of our research on kinesin.  We've approached the problem by watching how kinesin steps in the presence of nucleotide analogs, such as AMP-PNP and BeFx.  Nucleotide analogs are small molecules like ATP that the kinesin motor normally uses as its fuel.  Unlike ATP, analogs
    bind to kinesin's rear head and pause its motion since they cannot be used as fuel.  Such pauses are evident in records of kinesin stepping in the figure below.  Furthermore, the pauses are interspersed by short-lived backsteps, shown in red and orange.  To our surprise, the pauses only ended immediately after the motor took one of these backsteps.  Since the pauses were induced by binding of nucleotide analogs, we concluded that the analogs can only unbind from kinesin when it takes a backstep and the analog is bound to the front head.  This result means that there must be some way the heads communicate with each other to determine which is in front and which is in back.  A likely mechanism is the internal strain in the protein thought to build up whenever both heads are attached to the microtubule.
    Kinesin stepping records with BeFx
    Kinesin stepping records in the presence of 2 mM ATP and either 1 mM BeFx (traces 1, 2, and 4) or 1 mM AMP-PNP (trace 3).  Each record shows stepping (black) interrupted by pauses (blue).  Each pause is punctuated by backsteps (red and orange).  The pauses end only after the motor takes a backstep and releases the bound nucleotide analog.

    To learn more about this project, see Guydosh et al. PNAS, 2006.

  4. Besides conventional kinesin, what are the biophysical properties of other kinesin-family proteins?


    (Top) A kinesin dimer binds vesicular cargo and moves along a single microtubule; by contrast tetramers of Eg5 likely crosslink microtubules during cell division and generate sliding forces that help move the two duplicated daughter cells apart. (Bottom) Force vs. velocity curves for kinesin and Eg5. Eg5 walks more slowly, and is less sensitive to force than kinesin.

    The mitotic motor protein, Eg5
    Although much is now known of the stepping mechanism of conventional kinesin, very little is known of the biophysical and biochemical properties of the other members of the diverse family of kinesin-related enzymes. One important subset of this large family of kinesin-related molecular motors generates the forces that drive cell division (or mitosis). Eg5 is one such mitotic kinesin. In its native state, Eg5 consists of four catalytically-active motor heads, each one of which can bind microtubules and use the energy of ATP hydrolysis to perform work.

    To better understand how Eg5 functions in cells, we performed the first single-molecule experiments of Eg5 and demonstrated that individual dimers are mechanically processive, even under significant load. Like kinesin, Eg5 moves in 8-nm steps toward the plus-end of the microtubules, but moves ~10-fold slower, and takes ~10-fold fewer steps, on average, before detaching from the microtubule. Additionally, by measuring Eg5's velocity as a function of ATP concentration and applied force, we found that Eg5 is significantly less sensitive to force. We suspect these differences have evolved to satisfy the distinct physiological demands of cell division and cargo transport. Additional measurements of Eg5 detachment kinetics and force-dependencies are underway.

    To learn more about this work, see Valentine et al. Nature Cell Biology, 2006, and Valentine et al. Cell Division, 2006.

  5. Recent Technical Advances:

    Polymer-based surface treatments that reduce nonspecific surface interactions in single-molecule assays
    Optical trapping assays have stringent requirements for the controlled attachment of microtubules and motors to glass coverslips and polymeric colloidal spheres. Many single-molecule assays have traditionally relied upon fortuitous nonspecific interactions between motors, filaments, and surfaces, but extension to new classes of kinesin family motors has proven difficult, limiting the application of optical trapping techniques to new motor proteins. We have developed protocols using a variety of novel polymer-based surface chemistries that allow for robust, stereospecific attachment of motors and filaments while inhibiting undesirable nonspecific interactions. For more detail, see Fordyce et al., (2007) "Single-Molecule Techniques: A Laboratory Manual" in Cold Spring Harbor Laboratory Press .
    Combined optical tweezers and single-molecule fluorescence
    New tools will be required to determine exactly which of kinesin's biochemical steps correspond to physical motions. Toward this end, we are developing a combined instrument for optical trapping and single molecule fluorescence. By attaching fluorescent reporter molecules to kinesin or ATP, we hope to establish the timing of biochemical events relative to the mechanical steps, and to determine the conformational changes of kinesin during processive stepping. For instrument design and preliminary characterization, see Lang, et al., Nature Methods 2004, and Lang et al., Journal of Biology, 2003.
    Automated two-dimensional force-clamp
    To better study the effects of load on single kinesin proteins moving along a microtubule, we constructed a new instrument that can be operated as a two-dimensional force clamp, allowing the application of loads of fixed magnitude and direction to motor-coated microscopic beads moving in vitro. Flexibility and automation in experimental design are achieved by computer control of both the trap position, using acousto-optic deflectors, and the sample position, using a three-dimensional piezo stage. Sophisticated calibration routines reduce potential sources of error, and improve force and position resolution. For more detail, see Lang et al., Biophysical Journal (2002).

    Meet "Team Kinesin":

    Braulio Gutierrez-Medina

    Bason Clancy

    Nick Guydosh

    Johan Andreasson




    Former Members: Present Position:
    Megan Valentine Assistant Professor, UC Santa Barbara
    Polly Fordyce Postdoctoral Fellow, UC San Francisco
    Joshua Shaevitz Assistant Professor of Physics, Princeton University
    Chip Asbury Assistant Professor of Physiology and Biophysics, University of Washington
    Matt Lang Assistant Professor of Mechanical and Biological Engineering, M.I.T.
    Mark Schnitzer Assistant Professor of Biological Sciences and Applied Physics, Stanford University
    Koen Visscher Associate Professor of Physics, University of Arizona
    Steve Gross Associate Professor, Developmental and Cell Biology and Biomedical Engineering, U.C. Irvine
    Christoph Schmidt Professor of Physics, University of Göttingen
    Karel Svoboda Group Leader, HHMI/Janelia Farm



    Selected Block Lab Publications on Kinesin:

    • Block, S.M. Kinesin motor mechanics: Binding, stepping, tracking, gating, and limping. Biophysical Journal 92: 2986-2995 (2007) (Full Text PDF).

    • Guydosh, N.R., Block, S.M. Not so lame after all: Kinesin still walks with a hobbled head. The Journal of General Physiology 130(5): 441-444 (2007) ( Full Text PDF).

    • Valentine, M.T., Fordyce, P.F., Block, S.M. Eg5 steps it up! Cell Division 1 31 (2006) (Full Text PDF).

    • Valentine, M.T., Fordyce, P.M., Krzysiak, T.C., Gilbert, S.P., Block, S.M. Individual dimers of the mitotic kinesin motor Eg5 step processively and support substantial loads in vitro. Nature Cell Biology 8(5):470-477 (2006) (Full Text PDF).

    • Guydosh, N.R., Block, S.M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain. PNAS 103(21):8054-8059 (2006) (Full Text PDF; Click here for Supplemental Materials).

    • Asbury, C.L., Fehr, A.N., Block, S.M. Kinesin Moves by an Asymmetric Hand-Over-Hand Mechanism. Science 302:2130. (2003) (Full Text PDF).

    • Rosenfeld S.S., Fordyce P.M., Jefferson G.M., King P.H., Block S.M. Stepping and stretching: How kinesin uses internal strain to walk processively. J. Biol. Chem. 278:18550-18556 (2003) (Full Text PDF).

    • Block, S.M., Asbury, C.L., Shaevitz, J.W., Lang, M.J. Probing the kinesin reaction cycle with a 2D optical force clamp. PNAS 100:2351-2356 (2003) (Full Text PDF).

    • Schnitzer, M.J., Visscher, K. and Block, S.M. Mechanism of force production by single kinesin motors. Nature Cell Biology 2 (2000) (Full Text PDF).

    • Visscher, K., Schnitzer, M.J. and Block, S.M. Single kinesin molecules studied with a molecular force clamp. Nature 400: 184-189 (1999) (Full Text PDF).

    • Block, S.M. Kinesin: What gives? Cell 93: 5-8 (1998) (Full Text PDF).

    • Block, S.M. Leading the procession: new insights into kinesin motors. J. Cell Biol. 140: 1281-1284 (1998) (Full Text PDF).

    • Schnitzer, M.J. and Block, S.M. Kinesin hydrolyses one ATP per 8-nm step. Nature 388: 386-390 (1997) (Full Text PDF).

    • Block, S.M. Nanometers and picoNewtons: the macromolecular mechanics of kinesin. Trends Cell Biol. 5: 169-175 (1995) (Full Text PDF).

    • Svoboda, K., Mitra, P. and Block, S.M. Fluctuation analysis of kinesin movement. Biophysical Journal 68: 69s (1995).

    • Svoboda, K. and Block, S.M. Force and velocity measured for single kinesin molecules. Cell 77: 773-784 (1994).

    • Svoboda, K., Schmidt, C.F., Schnapp, B.J., and Block, S.M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365: 721-727 (1993).

    • Block, S.M., Goldstein, L.S.B., and Schnapp, B.J. Bead movement by single kinesin molecules studied with optical tweezers. Nature 348: 348-352 (1990).