How does kinesin walk?

Chuang WuMolecular Biophysics IIIThursday 19 Jan. 2006

Animation of symmetric hand-over-hand mechanism of kinesin-dependent vesicle transport

along a microtubule

Böhm, Stracke, Unger 2002, 2003 http://www.imb-jena.de/~kboehm/Kinesin.html

Structure of kinesin - homodimer

Fig. 1 of Yildiz, Tomishige, Vale, Selvin 2004 Science

HeadNeck

Stalk

Adapted from Kozielski, Sack, Marx, Thormahlen, Schonbrunn, Biou, Thompson, Mandelkow, Mandelkow 1997 Cell

How does kinesin walk?

• hand–over–hand model

- symmetric mechanism

- asymmetric mechanism

• inchworm model

Hand-over-hand vs inchworm

Yildiz, Tomishige, Vale, Selvin 2004 Science

Method Conclusion

Hua, Chung, Gelles 2002 Science

Immobilize a

kinesin and measure orientations of microtubules

no rotation of stalk

Inconsistent with symmetric hand-over-hand

Yildiz, Tomishige, Vale, Selvin 2004 Science

Fluorescence Imaging One-Nanometer Accuracy

(FIONA),

hand-over-hand mechanism

Kaseda, Higuchi, Hirose 2003 Nature

Generate a single heterodimeric kinesin molecule

hand-over-hand mechanism

Asbury, Fehr, Block 2003 Science

Force-clamp apparatus Asymmetric hand-

over-hand

Work of Hua, Chung, Gelles

• Method: They investigated the kinesin stepping mechanism by immobilizing a Drosophila kinesin derivative through the carboxyl-terminal end of the neck coiled-coil domain and measuring orientations of microtubules moved by single enzyme molecules.

• Conclusion: There’s no rotation of stalk, which is inconsistent with symmetric hand-over-hand movement.

Symmetric hand-over-hand vs inchworm

Hua, Chung, Gelles 2002 Science

Experimental Design

Images from light microscope demonstrate that MT pivots around a single point (cross) on the surface through a restricted range of angles

The microtubule is bound to the heads of a kinesin molecule

Hua, Chung, Gelles 2002 Science

Surface-attached kinesin vs non-attached kinesin

MT orientation over time – a limit range of orientation was observed

Hua, Chung, Gelles 2002 Science

A range of rotation larger than 360 degrees was observed

Movements of microtubules

Hua, Chung, Gelles 2002 Science

The same microtubule in two different time periods.

Displacement and orientation records of two microtubules in 400 and 5 nM ATP.

Conclusions

• Their observations that the kinesin neck coiled coil does not rotate 180 degrees from the beginning of one step to the beginning of the next is inconsistent with symmetric hand-over-hand model.

• The result is consistent with the inchworm type of mechanism.

• They considered a third type of mechanism, named asymmetric hand-over-hand mechanism, in which the three-dimensional structures at the beginning of consecutive 8-nm steps are different.

Work of Yildiz, Tomishige, Vale, Selvin

• Method: Fluorescence Imaging One-Nanometer Accuracy (FIONA), that is capable of tracking the position of a single dye with nanometer accuracy and sub second resolution.

• Conclusion: Kinesin walks hand-over-hand, rather than inchworm

Hand-over-hand vs inchworm

Yildiz, Tomishige, Vale, Selvin 2004 Science

Sites for single fluorescent dye attachments

Yildiz, Tomishige, Vale, Selvin 2004 Science

S43E215 T324

3 residues were mutated to cysteines for fluorescent dye labeling

A single kinesin molecule moving on an immobilized axoneme.

PSF (Point-spread-function)

Yildiz, Tomishige, Vale, Selvin 2004 Science

PSF fit well with Gaussian curve, which confirmed that only a single dye was present on each kinesin analyzed.

Position versus time for kinesin motility

Yildiz, Tomishige, Vale, Selvin 2004 Science

E215C E215C

S43C-T324C heterodimer

Distribution of step sizes

• The step sizes of an individual head of a kinesin dimer and dwell-time analysis support a hand-over-hand mechanism.

Yildiz, Tomishige, Vale, Selvin 2004 Science

Dwell-time histogram

• Dwell time histogram showing the expected exponential decay with a maximum near t=0.

• P(t) = tk2exp(-kt)

Yildiz, Tomishige, Vale, Selvin 2004 Science

• Cy3 fluorophore was attached to E215C and visualized using total internal reflection fluorescence microscopy.

Yildiz, Tomishige, Vale, Selvin 2004 Science

Conclusions

• The results strongly support a hand-over-hand (walking) model for kinesin motility.

Yildiz, Tomishige, Vale, Selvin 2004 Science

Work of Kaseda, Higuchi, Hirose

• Method: Generate a single heterodimeric kinesin molecule by mutating one of the two heads in a nucleotide-binding site

• Conclusion: The heterodimeric kinesin molecule exhibits fast and slow 8-nm steps alternately, providing the first direct evidence for models in which the roles of the two heads alternate every 8-nm step.

Two models of R14A/WT

• Two models explaining the processive movement of kinesin and expected changes in the dwell time when a heterodimeric kinesin is used.

Kaseda, Higuchi, Hirose 2003 Nature

Displacement of R14A/WT in an optical trap

• In most of the cases, the steps seem to be 16 nm

Kaseda, Higuchi, Hirose 2003 Nature

WT/R14A vs

WT/WT

• The observed 16 nm step consists of two successive 8-nm steps.

Kaseda, Higuchi, Hirose 2003 Nature

• A displacement trace showing how step sizes (∆X1, ∆X2) and dwell time (τ) were measured.

Kaseda, Higuchi, Hirose 2003 Nature

Dwell-time differences are greater in WT/R14A

Kaseda, Higuchi, Hirose 2003 Nature

Fast and slow dwell times

• Distribution of the dwell time of the step directly after a step with a long (>100 ms) dwell time (blue), and those following a step with a short (<20 ms) dwell time (orange)

Kaseda, Higuchi, Hirose 2003 Nature

Fast/slow dwell times and force

• Dwell times increase with load.

• The dwell time of the slow step of WT/R14A is at least 10 times longer than that of the fast step at all force levels.

Kaseda, Higuchi, Hirose 2003 Nature

Movement of R14A/R14A homodimer

Kaseda, Higuchi, Hirose 2003 Nature

Conclusions of this work

• A single heterodimeric kinesin showed a step with a long dwell time alternating with one with a short dwell time.

• The results provide the first direct evidence for a model in which the roles of the two heads of a kinesin molecule alternate as it displaces by 8 nm, such as the hand-over-hand model.

Work of Asbury, Fehr, Block

• Method: Force-clamp apparatus to measure the position of kinesin head

• Conclusion: Two head kinesin shows “limp” behavior, which exclude fully symmetric models, such as inchworm and symmetric hand-over-hand mechanism

Asbury, Fehr, Block 2003 Science

“Limp” vs “non-limp”

Asbury, Fehr, Block 2003 Science

Dwell time (s) Dwell time (s)

Conclusion

• The discovery of that kinesin limps implies that it advanced by some form of asymmetric hand-over-hand mechanism

Summary

• Kinesin is a processive motor that takes 8.3-nm center-of-mass steps along microtubules for each ATP hydrolyzed.

• Whether kinesin moves by a “hand-over-hand” or an “inchworm” model has been controversial.

• From these works, we can conclude between these 2 models, kinesin seems to work as a hand-over-hand mechanism.