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Cited by 121Microtubule and mitosis dynamicsAdvanced Fluorescence Microscopy TechniquesForce Microscopy Techniques and Applications

Direct observation of the binding state of the kinesin head to the microtubule

Open access
Guydosh, Nicholas R., Block, Steven M.·2009·Nature
DOI 10.1038/nature08259PMC PMC2859689
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Abstract

The dimeric motor protein kinesin-1 converts chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules. Cargo attached to the kinesin stalk moves processively in 8-nm increments as its twin motor domains (heads) carry out an asymmetric, 'hand-over-hand' walk. The extent of individual head interactions with the microtubule during stepping, however, remains controversial. A major experimental limitation has been the lack of a means to monitor the attachment of an individual head to the microtubule during movement, necessitating indirect approaches. Here we report the development of a single-molecule assay that can directly report head binding in a walking kinesin molecule, and show that only a single head is bound to the microtubule between steps at low ATP concentrations. A bead was linked to one of the two kinesin heads by means of a short DNA tether and used to apply rapidly alternating hindering and assisting loads with an optical trap. The time-dependent difference between forwards and backwards displacements of the bead alternated between two discrete values during stepping, corresponding to those intervals when the linked head adopted a bound or an unbound state. The linked head could only rebind the microtubule once ATP had become bound to its partner head.

Integrity signals

Registry

Crossref · 10.1038/nature08259

Cites

30 of 30 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

13 of 34 cited sources checked · 5 supported · 0 flagged · accuracy not yet measured

  • Findingcitation

    The stall force fell by half when load was applied to a single head instead of the stalk, from about 6 pN to about 3 pN.

    “The stall force was halved when loads were applied to a single head instead of the stalk (falling from ∼6 pN to ∼3 pN)15”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Block, S. M., Asbury, C. L., Shaevitz, J. W. & Lang, M. J. Probing the kinesin reaction cycle with a 2D optical force clamp. Proc. Natl Acad. Sci. USA 100, 2351–2356 (2003) source

      The Results and Discussion (pages unavailable) report that backward loads stalled kinesin at roughly 4–6 pN, consistent with the claim’s approximate 6 pN baseline. Our copy does not report applying load to a single head instead of the stalk, a stall force of about 3 pN under that condition, or a halving of stall force. (LLM, conf=0.85)

      Experimental — accuracy not yet measured
  • Premisecitation

    Cryo-EM structures of microtubule-bound kinesin show that the neck linker conformation is tightly coupled to the nucleotide state of the head.

    “Cryo-EM structures of MT-bound kinesin17,18 (see also C.V. Sindelar & K.H. Downing, manuscript in preparation) show that the conformation of the kinesin neck linker is tightly coupled to the state of the nucleotide bound to the head.”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [17] Sindelar, C. V. & Downing, K. H. The beginning of kinesin’s force-generating cycle visualized at 9-Å resolution. J. Cell Biol. 177, 377–385 (2007) source

      In “The Switch II Helix is ‘ADP-like’” (page not supplied), the cryo-EM map of nucleotide-free, microtubule-bound kinesin suggests that its neck linker is disordered. The Abstract and Summary propose a mechanism by which the bound nucleotide could affect the neck linker, but this paper examines only the nucleotide-free state; it does not demonstrate tight coupling across nucleotide states. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [18] Kikkawa, M. & Hirokawa, N. High-resolution cryo-EM maps show the nucleotide binding pocket of KIF1A in open and closed conformations. EMBO J. 25, 4187–4194 (2006) source
  • Interpretationreasoning

    Strain from an unfavourable neck-linker conformation also explains why ATP does not bind prematurely to the front, nucleotide-free head of a two-heads-bound kinesin molecule.

    “Strain produced by an unfavourable neck-linker conformation also explains the observation23-25 that ATP does not bind prematurely to the front, nucleotide-free head of 2-HB kinesin molecule (Fig. 4, state 3).”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [23] Klumpp, L. M., Hoenger, A. & Gilbert, S. P. Kinesin’s second step. Proc. Natl Acad. Sci. USA 101, 3444–3449 (2004) source
    • cites [24] 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) source
    • cites [25] Guydosh, N. R. & Block, S. M. Backsteps induced by nucleotide analogs suggest the front head of kinesin is gated by strain. Proc. Natl Acad. Sci. USA 103, 8054–8059 (2006) source

      The paper’s discussion of front-head gating (page unavailable) says that strain between the two bound heads reduces the leading head’s ATP affinity, preventing premature binding until the rear head detaches. It does not specifically identify an unfavourable neck-linker conformation as the source of that strain. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
  • Interpretationreasoning

    The inability of one head to bind the microtubule naturally explains why microtubule-stimulated ADP release is inhibited until the microtubule-attached head binds ATP and docks its neck linker.

    “The inability of one head to bind the MT offers a natural explanation for the observation7,20-22 that the MT-stimulated release of ADP is inhibited until the MT-attached head binds ATP and docks its neck linker (Fig. 4, state 2).”
    Quote in the paperSubstantial claim1 of 4 sources checked
    • cites [7] Hackney, D. D. Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. Proc. Natl Acad. Sci. USA 91, 6865–6869 (1994) source
    • cites [20] Ma, Y.-Z. & Taylor, E. W. Interacting head mechanism of microtubule-kinesin ATPase. J. Biol. Chem. 272, 724–730 (1997) source

      The “affinity site” section (page unavailable) says the ADP-containing head is held in a weakly bound or detached state and that ATP binding to the other head promotes its ADP release. It does not identify neck-linker docking as the required step, and it describes the second head as weakly bound or detached rather than unable to bind the microtubule at all. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [21] Gilbert, S. P., Moyer, M. L. & Johnson, K. A. Alternating site mechanism of the kinesin ATPase. Biochemistry 37, 792–799 (1998) source
    • cites [22] Crevel, I., Carter, N., Schliwa, M. & Cross, R. Coupled chemical and mechanical reaction steps in a processive Neurospora kinesin. EMBO J. 18, 5863–5872 (1999) source
  • Findingown data

    At low ATP concentrations, only one head is attached to the microtubule between steps.

    “and show that only a single head is bound to the microtubule between steps at low ATP concentrations.”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 17
  • May combine two claims. 31

  • Classified as probably not a substantial claim.

    Premisecitation

    The two motor domains of kinesin perform an asymmetric hand-over-hand walk.

    “as its twin motor domains (heads) carry out an asymmetric, hand-over-hand walk4-7.”
    Quote in the paperSubstantial claim1 of 4 sources checked
    • cites [4] Yildiz, A., Tomishige, M., Vale, R. D. & Selvin, P. R. Kinesin walks hand-over-hand. Science 303, 676–678 (2004) source
    • cites [5] Kaseda, K., Higuchi, H. & Hirose, K. Alternate fast and slow stepping of a heterodimeric kinesin molecule. Nature Cell Biol. 5, 1079–1082 (2003) source
    • cites [6] Asbury, C. L., Fehr, A. N. & Block, S. M. Kinesin moves by an asymmetric hand-over-hand mechanism. Science 302, 2130–2134 (2003) source
    • cites [7] Hackney, D. D. Evidence for alternating head catalysis by kinesin during microtubule-stimulated ATP hydrolysis. Proc. Natl Acad. Sci. USA 91, 6865–6869 (1994) source

  • May combine two claims.

    Premisecitation

    Single-molecule fluorescence methods resolved head motions by tagging heads with fluorophores, but had limited spatiotemporal resolution and could not apply controlled loads.

    “Single-molecule fluorescence techniques have successfully resolved motions of the heads by tagging these individually with fluorophores, but such approaches have suffered from limited spatiotemporal resolution and lack the ability to apply controlled loads”
    Quote in the paperSubstantial claim2 of 4 sources checked
    • cites [4] Yildiz, A., Tomishige, M., Vale, R. D. & Selvin, P. R. Kinesin walks hand-over-hand. Science 303, 676–678 (2004) source
    • cites [8] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R. D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008) source

      The Single Molecule Fluorescence Motility Assays section describes labeling a kinesin head with a quantum dot to track its movement and reports a 70 ms temporal resolution that required slowing the motors to avoid merging steps. The Optical Trapping Assays section applies controlled loads using a separate method. Our copy does not establish a limitation in spatial resolution or explicitly say that fluorescence methods cannot apply controlled loads; no page numbers are available in the supplied copy. (LLM, conf=0.82)

      Experimental — accuracy not yet measured
    • cites [9] Mori, T., Vale, R. D. & Tomishige, M. How kinesin waits between steps. Nature 450, 750–754 (2007) source
    • cites [14] Asenjo, A. B. & Sosa, H. A mobile kinesin-head intermediate during the ATP-waiting state. Proc. Natl Acad. Sci. USA 106, 5657–5662 (2009) source

      The Results describe a probe on one kinesin head reporting alternating mobility during walking, and Materials and Methods describes labeling individual heads with a fluorescent probe. The Results also identify missed events caused by the system’s 200-ms time resolution. Our copy does not establish a spatial-resolution limitation or state that fluorescence methods cannot apply controlled loads. (LLM, conf=0.86)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationown data

    The data do not support models in which both kinesin heads bind the microtubule until ATP binding triggers release of the rear head, nor models in which one head is parked against the forward face of its microtubule-bound partner in the waiting state.

    “Our data do not support models where both kinesin heads bind to the MT until ATP binding triggers release of the rear head4,8, nor where one head is parked against the forward face of its MT-bound partner in the waiting state10.”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [4] Yildiz, A., Tomishige, M., Vale, R. D. & Selvin, P. R. Kinesin walks hand-over-hand. Science 303, 676–678 (2004) source
    • cites [8] Yildiz, A., Tomishige, M., Gennerich, A. & Vale, R. D. Intramolecular strain coordinates kinesin stepping behavior along microtubules. Cell 134, 1030–1041 (2008) source

      Citation 10 is attached to the waiting-state model, not the preceding two-head-bound model. In “A Model for Communication between the Two Kinesin Heads” (page unavailable), the paper describes a weak-binding head positioned behind the bound head, consistent with rejecting a forward-parked position. It does not say that its own data rule out a head parked against the forward face of its partner. (LLM, conf=0.75)

      Experimental — accuracy not yet measured
    • cites [10] Alonso, M. C. et al. An ATP gate controls tubulin binding by the tethered head of kinesin-1. Science 316, 120–123 (2007) source

  • May combine two claims.

    Findingown data

    With the bead linked to one head but unloaded, motors moved an average of 980 ± 80 nm before detaching, a processivity equivalent to unlabelled kinesin.

    “With the bead linked to one of the two heads, but unloaded, motors travelled an average distance of 980 ± 80 nm (mean ± s.e.m.) before detaching (Supplementary Fig. 2), displaying a processivity equivalent to that of unlabelled kinesin15.”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [15] Block, S. M., Asbury, C. L., Shaevitz, J. W. & Lang, M. J. Probing the kinesin reaction cycle with a 2D optical force clamp. Proc. Natl Acad. Sci. USA 100, 2351–2356 (2003) source

      The citation is attached to the claim that processivity was equivalent to that of unlabelled kinesin. The Results discuss how sideways loads affect run length, and Materials and Methods describe measuring mean run lengths, but our copy gives no comparison with head-linked beads or unlabelled kinesin. Searches for 980 ± 80 nm and head-linked bead processivity found no supporting passage; page numbers were unavailable. (LLM, conf=0.76)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Findingown data

    The authors overcame these limitations by attaching an optically trapped bead directly to one head rather than the stalk, without disabling motor function.

    “We overcame these limitations by linking an optically-trapped bead directly to one of the two heads, rather than to the stalk (Fig. 1a), without disabling the motor function of the molecule.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Under hindering loads, head-linked beads stepped 16.3 ± 0.1 nm toward the microtubule plus end, as expected for individual heads walking hand-over-hand along protofilaments with 8.2-nm tubulin-dimer spacing.

    “When head-linked beads were subjected to hindering loads under force-clamped conditions, we observed steps of 16.3 ± 0.1 nm toward the plus end of the MT (Fig. 1b and Supplementary Fig. 4a), exactly as expected for individual heads executing a hand-overhand walk along protofilaments with an 8.2-nm tubulin-dimer spacing.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The tubulin-dimer spacing along microtubule protofilaments is 8.2 nm, so two successive advances correspond to 16.4 nm.

  • May combine two claims.

    Findingown data

    Unloaded bead-linked motors had average speeds of 581 ± 9 nm/sec, comparable to wild-type motors.

    “Remarkably, the unloaded speeds of bead-linked motors under these conditions averaged 581 ± 9 nm/sec (mean ± s.e.m.), comparable to unloaded speeds recorded for wild-type motors (Supplementary Fig. 3).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    The small rearward motion under hindering load averaged −1.7 ± 0.2 nm; adding it to the forward motion of 18.2 ± 0.2 nm gave a net displacement of 16.5 ± 0.3 nm, statistically indistinguishable from the expected 16.4 nm.

    “The small rearward motion under hindering load averaged −1.7 ± 0.2 nm. When added to the forward motion, 18.2 ± 0.2 nm, this yields a net displacement of 16.5 ± 0.3 nm, statistically indistinguishable from the expected advance of 16.4 nm (twice the tubulin-dimer spacing).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The tubulin-dimer spacing along microtubule protofilaments is 8.2 nm, so two successive advances correspond to 16.4 nm.

  • May combine two claims.

    Findingown data

    The dwell intervals after overshoot and recovery motions depended on ATP concentration, and both lengthened when ATP was reduced.

    “The durations of dwell intervals following the overshoot and recovery motions were sensitive to the ATP concentration, and both increased when the ATP level was reduced (Fig. 1d and Supplementary Fig. 5).”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    Two additional constructs, A128C and S181C, successfully produced 16-nm steps when a bead was linked to a single head (data not shown).

    “Two additional constructs were generated that successfully generated 16-nm steps when bead were linked to a single head, A128C and S181C (data not shown).”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Findingown data

    Assisting loads greater than 1.7 pN often made DNA-linked kinesin abruptly and irreversibly lose activity and stick to the coverglass; such loads may trigger motor unfolding and were avoided.

    “Assisting loads greater than 1.7 pN often caused the DNA-linked kinesin irreversibly and abruptly to lose activity and then stick to the coverglass surface. Such loads may trigger unfolding of the motor and were therefore avoided.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Cargo connected to the kinesin stalk advances processively in 8-nm steps.

    “Cargo attached to the kinesin stalk moves processively in 8-nm increments3”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [3] Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993) source

  • May combine two claims.

    Premisecitation

    Optical-trapping assays usually track and load a bead attached to the C-terminus of the common stalk, so the displacements of the two heads are not resolved.

    “Optical-trapping assays for kinesin typically involve tracking and loading a bead attached to the C-terminus of the common stalk of the protein3 in place of its natural cargo. Because beads attached to the stalk tend to report the position of the molecule as a whole, the displacements generated by the two separate heads remain unresolved.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [3] Svoboda, K., Schmidt, C. F., Schnapp, B. J. & Block, S. M. Direct observation of kinesin stepping by optical trapping interferometry. Nature 365, 721–727 (1993) source

  • Classified as probably not a substantial claim.

    Premisecitation

    Kinesin-1 is a dimeric motor protein that turns the chemical energy of ATP hydrolysis into mechanical work for moving cargo along microtubules.

    “The dimeric motor kinesin-1 couples chemical energy from ATP hydrolysis into mechanical work used to transport cargo along microtubules1,2.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [1] Block, S. M. Kinesin motor mechanics: binding, stepping, tracking, gating, and limping. Biophys. J. 92, 2986–2995 (2007) source
    • cites [2] Vale, R. D. The molecular motor toolbox for intracellular transport. Cell 112, 467–480 (2003) source

  • May combine two claims.

    Interpretationreasoning

    Because the 70-bp dsDNA oligomer is shorter than half a persistence length, the tether is stiff and transmits head motions directly to the bead.

    “Because this short oligomer subtends less than half a persistence length, the tether is stiff and transmits motions of the head directly to the bead.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Recent work suggests that kinesin does not uniformly adopt a two-heads-bound configuration in the presence of apyrase.

    “In support of this, recent work suggests that kinesin does not uniformly adopt a 2-HB configuration in the presence of apyrase9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Mori, T., Vale, R. D. & Tomishige, M. How kinesin waits between steps. Nature 450, 750–754 (2007) source

  • May combine two claims.

    Interpretationreasoning

    The halved stall force is consistent with the motor's energetic efficiency being unaffected by head linkage, because half the load is moved through twice the distance per step.

    “which is consistent with the energetic efficiency of the motor being unaffected by the head linkage, because half the load is moved through twice the distance per step (Supplementary Fig. 3).”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    The initial and final dwells of all records were excluded from analysis.

    “The initial and final dwells of all records were not included in the analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    The linked head then detaches from the microtubule and moves ahead of its bound partner.

    “The linked head then releases the MT and moves ahead of its bound partner (state 3).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Therefore, for both heads to bind the microtubule simultaneously, the rear head must bind ATP or ADP-Pi while the front head holds ADP or no nucleotide.

    “Therefore, if both heads are to bind the MT simultaneously, the rear head must bind ATP (or ADP-Pi) while the front head holds ADP or no nucleotide.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    Only data recorded after the final force-clamp update and before load reversal were included, to allow the feedback to equilibrate.

    “Only data recorded after the final force-clamp update and before load reversal were included in analysis to allow time for the feedback to equilibrate.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Labelled and unlabelled kinesin were readily distinguished by a gel shift in native PAGE.

    “Labelled and unlabelled kinesin was readily distinguished by a gel shift in native PAGE.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Interpretationreasoning

    The actual dependence of dwell times on ATP concentration may be stronger than reported, but this does not change any conclusions.

    “The actual dependence of dwell times on the ATP concentration may therefore be even stronger than that reported here (but does not change any of our conclusions).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    The motor core is tilted when bound to ATP or ADP-Pi analogues, allowing the neck linker to dock.

    “By contrast, the motor core is tilted when bound to ATP or to analogues of ADP-Pi, allowing the neck linker to dock.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Hwang, W., Lang, M. J. & Karplus, M. Force generation in kinesin hinges on cover-neck bundle formation. Structure 16, 62–71 (2008) source

  • May combine two claims.

    Interpretationreasoning

    The rear head hydrolyzes ATP, releases Pi, and detaches from the microtubule, regenerating the starting configuration with the molecule advanced by 16.4 nm over two steps.

    “Finally, the rear head hydrolyzes ATP, releases Pi, and unbinds the MT to regenerate the starting configuration (state 1), but with the molecule advanced by 16.4 nm (2 steps).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The tubulin-dimer spacing along microtubule protofilaments is 8.2 nm, so two successive advances correspond to 16.4 nm.

  • May combine two claims.

    Interpretationreasoning

    ATP binding to the microtubule-bound head then lets the free head rebind the microtubule in its proper orientation and release ADP.

    “Subsequent binding of ATP to the MT-bound head allows the free head to rebind the MT in its proper orientation and release ADP (state 4).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The data support a model in which kinesin binds ATP and takes its step from a one-head-bound state.

    “Our data support a model for the reaction cycle (Fig. 4) where kinesin binds ATP and takes its step from a 1-HB state.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Data were recorded from samples in which only 10-35% of beads moved.

    “Data were recorded from samples where just 10-35 % of beads moved.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    Labelling efficiency was below 25%, ensuring that more than 93% of labelled kinesins carried a single DNA oligomer.

    “The labelling efficiency was <25 %, ensuring >93 % of labelled kinesins were attached to a single DNA oligomer.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Abrupt jumps in the running difference record indicate that the linked head adopts both bound and unbound states during the kinetic cycle, consistent with a one-head-bound waiting state.

    “Accordingly, abrupt jumps in the running difference record signify that the linked head adopts both bound and unbound states during the kinetic cycle, consistent with a 1-HB waiting state.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    The authors attributed these 8-nm-stepping beads to double motor linkages or conjugation between DNA and stalk residues and excluded them from analysis.

    “We attributed these to beads carrying double motor linkages or to conjugation between the DNA and stalk residues, and excluded them from the analysis.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Study design and conductown data

    Because recovery motions were small, some were missed by the step-finding procedure, so some overshoot-recovery pairs appeared as a single 16-nm transition.

    “Because of their small amplitudes, a fraction of recovery motions were missed by our step-finding procedure, causing some pairs of overshoot and recovery motions to appear as a single, 16-nm transition.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationreasoning

    The mechanical work available to detach a bound head under 1.7 pN over 2 nm would be less than 3.4 pN nm; because this is below thermal energy, any increase in detachment rate would be limited to a factor of 2-3, too small to explain the observations.

    “Taking 2 nm as a (generous) estimate of the distance between the bound and transition states along the reaction coordinate for head unbinding16, the mechanical work available to detach a bound head would be less than (1.7 pN × 2 nm) = 3.4 pN nm. Because this value is less than thermal energy (kBT ≈ 4 pN nm), any increase in detachment rate would be limited to a factor of 2-3, and too small to a”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [16] Taniguchi, Y., Nishiyama, M., Ishii, Y. & Yanagida, T. Entropy rectifies the Brownian steps of kinesin. Nature Chem. Biol. 1, 342–347 (2005) source

  • May combine two claims.

    Interpretationreasoning

    Assisting load helps pull the linked head forward and also creates a small torque between the neck linker and the DNA attachment; to relieve this torque, the linked head rotates to a rearward-facing orientation free of the microtubule during overshoot dwells.

    “In this case, the application of assisting load not only helps to pull this head forward, but also supplies a small torque, generated between the neck linker domain (joining the head to the stalk) and the point of attachment of the DNA. To relieve torque, the linked head rotates to adopt a rearward-facing orientation, free of the MT, during overshoot dwells.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The kinesin neck linker can act as a free swivel.

  • May combine two claims.

    Interpretationreasoning

    Because low ATP or high load makes the transition from this state rate-limiting, it is the longest-lived state of the cycle under the conditions studied.

    “Because low ATP levels or high loads cause the transition from this state to become rate-limiting, it is the longest-lived state of the cycle (therefore the ‘waiting state’) under the conditions explored here.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationcitation

    In that study, distances between kinesin heads were estimated from FRET at 100 Hz and filtered with a 100-ms window, so the proposed structure likely represents an average over positions.

    “In that study, estimates of distance between the kinesin heads were obtained from FRET measurements recorded at 100 Hz and filtered by a 100-ms window, so the proposed structure likely corresponds to an average over positions.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Mori, T., Vale, R. D. & Tomishige, M. How kinesin waits between steps. Nature 450, 750–754 (2007) source

  • May combine two claims.

    Interpretationown data

    The unbound head is mobile and can be pulled around its bound partner or rotated with an optical trap.

    “The unbound head is mobile, and can be pulled about its bound partner or even rotated with an optical trap.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Interpretationreasoning

    N62C was chosen as the attachment point because it lies in a solvent-exposed loop on the back of the motor domain, away from motility-critical regions such as the neck linker and nucleotide binding pocket.

    “This residue was selected as an attachment point because it is situated in a solvent-exposed loop on the back side of the motor domain, distal from regions known to be critical for motility, such as the neck linker and nucleotide binding pocket.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Kinesin fractions were confirmed by mass spectrometry and SDS-PAGE.

    “Kinesin fractions were confirmed by mass spectrometry and SDS-PAGE.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Overshoot and recovery dwell times were computed only when the intervening recovery step was smaller than −6 nm.

    “Times of overshoot and recovery dwell intervals (Fig. 1d) were computed only when the intervening recovery step was smaller than −6 nm.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductnone given

    In the absence of load, video at 30 Hz and image processing tracked the centroid of a moving bead, and bead release was determined from an increase in positional variance.

    “In the absence of load, video frame capture (at 30 Hz) followed by image processing was used to track the centroid of a moving bead. Bead release from the MT was determined from the increase in its positional variance.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The final dwell interval and step transition in each stepping record were excluded from analysis.

    “The final dwell interval and step transition in any given stepping record were excluded from the analysis.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Under alternating load, step positions under hindering and assisting load were merged, and steps separated by a single load reversal were treated as one transition at the midpoint.

    “Under alternating load, step positions detected under hindering and assisting load were merged. Any steps separated by a single load reversal (14 ms) were taken as a single step transition at the midpoint between these.”
    Quote in the paperNot a substantial claim
43 of 43

  • The paper relies on this without support.

    The tubulin-dimer spacing along microtubule protofilaments is 8.2 nm, so two successive advances correspond to 16.4 nm.

    “along protofilaments with an 8.2-nm tubulin-dimer spacing”
    “statistically indistinguishable from the expected advance of 16.4 nm (twice the tubulin-dimer spacing)”

  • The paper relies on this without support.

    The kinesin neck linker can act as a free swivel.

    “The neck linker region consists of a single polypeptide chain (∼13 residues) and can act as a free swivel.”

  • The paper relies on this without support.

    The established biochemical reaction cycle for kinesin is as used in the proposed model.

    “which is consistent with the established biochemical reaction cycle for kinesin”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Preventing the rear head from binding supports efficient forward motion by lowering backstepping, futile hydrolysis, and entry into states where both heads detach and processivity is lost.

    “The inhibition of the rear head from binding leads to efficient, unidirectional forward motion by reducing backstepping, futile hydrolysis, and access to states where both heads are released, leading to loss of processivity34.”

    From A universal pathway for kinesin stepping (2011) · cites this paper as [34]

    The paper’s discussion (indexed under Abstract; page unavailable) proposes gating that prevents the unbound head from rebinding the microtubule prematurely, and describes forward stepping from a one-head-bound state. It does not say that inhibiting the rear head’s binding reduces backstepping, futile hydrolysis, or loss of processivity through both-head detachment; those proposed benefits are not supported by the passages found. (LLM, conf=0.84)

    Experimental — accuracy not yet measured

  • At low ATP, WT kinesin is predominantly in State {1}, with leading head nucleotide-free and strongly bound, trailing head carrying ADP detached or transiently weakly associated.

    “At low ATP concentrations, WT kinesin is found predominantly in this state, where the leading head is without nucleotide and strongly bound to the microtubule, while the trailing head, carrying ADP, remains detached from the microtubule34, or is perhaps transiently (weakly) associated36,37.”

    From A universal pathway for kinesin stepping (2011) · cites this paper as [34]

  • In the ATP-waiting state, the trailing ADP-bearing head is mobile and detached from the microtubule, so it cannot put substantial tension on the leading head.

    “It is also supported by results showing that in the ATP-waiting state, the trailing head carrying ADP is mobile and free of the microtubule34, and therefore unable to exert substantial tension on the leading head.”

    From A universal pathway for kinesin stepping (2011) · cites this paper as [34]

  • WT kinesin usually adopts a configuration with one head strongly bound to the microtubule and the other ADP-bearing head largely detached, especially at low ATP.

    “This conformational state is partially suppressed in WT kinesin, which tends to adopt a configuration where just one head is strongly attached to the microtubule39,40, while the partner head, carrying ADP, is more-or-less free of the microtubule34–36, particularly at low ATP concentrations.”

    From A universal pathway for kinesin stepping (2011) · cites this paper as [34]

  • The proposed mechanism agrees with a previously conjectured model for WT kinesin gating at low ATP, where generally only one head is bound.

    “Such a mechanism is fully consistent with a model conjectured previously to explain gating in WT kinesin at low ATP concentrations, where only one head is generally bound34.”

    From A universal pathway for kinesin stepping (2011) · cites this paper as [34]

Cites

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Authors
Guydosh, N. R., & Block, S. M.
Affiliations
Stanford University (US)
Corresponding author
Block, Steven M.
ORCID
Guydosh, Nicholas R.
Venue
Nature
Publisher
Springer Science and Business Media LLC
Published
2009-08-19
Volume
461
Issue
7260
Pages
125-128
ISSN
  • 0028-0836 (ISSN-L)
  • 1476-4687
DOI
10.1038/nature08259
PMC
PMC2859689
OpenAlex
W1998198313
Cited by
121
References
30
Citations by year
2026: 2 · 2025: 5 · 2024: 2 · 2023: 4 · 2022: 3 · 2021: 4 · 2020: 4 · 2019: 4 · 2018: 3 · 2017: 6 · 2016: 9 · 2015: 12 · 2014: 16 · 2013: 8 · 2012: 13
FWCI
5.78
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature (publishedVersion)
  • PubMed (publishedVersion)
  • PubMed Central (acceptedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
Funders
National Institute of General Medical Sciences (R01GM051453, GM51453)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Biophysics › Advanced Fluorescence Microscopy Techniques
  • Physical Sciences › Physics and Astronomy › Atomic and Molecular Physics, and Optics › Force Microscopy Techniques and Applications
Keywords
kinesin-1, microtubule binding, single-molecule force spectroscopy, ATP hydrolysis, optical trapping, processive motor
MeSH terms
Adenosine Triphosphate, Animals, DNA, Drosophila melanogaster, Microspheres, Microtubules, Movement, Protein Binding, Time Factors, Kinesins, Optical Tweezers

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read under definition version 3 (extract-v3) on 2026-09-28.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-09-28.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

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