How%20does%20kinesin%20walk? - PowerPoint PPT Presentation

Title: How%20does%20kinesin%20walk?


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How does kinesin walk?

• Chuang Wu
• Molecular Biophysics III
• Thursday 19 Jan. 2006

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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
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Structure of kinesin - homodimer
Fig. 1 of Yildiz, Tomishige, Vale, Selvin 2004
Science
Adapted from Kozielski, Sack, Marx, Thormahlen,
Schonbrunn, Biou, Thompson, Mandelkow, Mandelkow
1997 Cell
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How does kinesin walk?

• handoverhand model
• - symmetric mechanism
• - asymmetric mechanism
• inchworm model

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Hand-over-hand vs inchworm
Yildiz, Tomishige, Vale, Selvin 2004 Science
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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
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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 Theres no rotation of stalk, which
  is inconsistent with symmetric hand-over-hand
  movement.

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Symmetric hand-over-hand vs inchworm
Hua, Chung, Gelles 2002 Science
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Experimental Design
The microtubule is bound to the heads of a
kinesin molecule

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

Hua, Chung, Gelles 2002 Science
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Surface-attached kinesin vs non-attached kinesin
MT orientation over time a limit range of
orientation was observed
A range of rotation larger than 360 degrees was
observed
Hua, Chung, Gelles 2002 Science
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Movements of microtubules
The same microtubule in two different time
periods.
Displacement and orientation records of two
microtubules in 400 and 5 nM ATP.
Hua, Chung, Gelles 2002 Science
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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.

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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

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Hand-over-hand vs inchworm
Yildiz, Tomishige, Vale, Selvin 2004 Science
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Sites for single fluorescent dye attachments
3 residues were mutated to cysteines for
fluorescent dye labeling
A single kinesin molecule moving on an
immobilized axoneme.
Yildiz, Tomishige, Vale, Selvin 2004 Science
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PSF (Point-spread-function)
PSF fit well with Gaussian curve, which confirmed
that only a single dye was present on each
kinesin analyzed.
Yildiz, Tomishige, Vale, Selvin 2004 Science
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Position versus time for kinesin motility
E215C
E215C
S43C-T324C heterodimer
Yildiz, Tomishige, Vale, Selvin 2004 Science
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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
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Dwell-time histogram

• Dwell time histogram showing the expected
  exponential decay with a maximum near t0.
• P(t) tk2exp(-kt)

Yildiz, Tomishige, Vale, Selvin 2004 Science
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• Cy3 fluorophore was attached to E215C and
  visualized using total internal reflection
  fluorescence microscopy.

Yildiz, Tomishige, Vale, Selvin 2004 Science
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Conclusions

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

Yildiz, Tomishige, Vale, Selvin 2004 Science
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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.

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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
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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
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WT/R14A vs WT/WT

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

Kaseda, Higuchi, Hirose 2003 Nature
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• A displacement trace showing how step sizes (?X1,
  ?X2) and dwell time (t) were measured.

Kaseda, Higuchi, Hirose 2003 Nature
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Dwell-time differences are greater in WT/R14A
Kaseda, Higuchi, Hirose 2003 Nature
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Fast and slow dwell times

• Distribution of the dwell time of the step
  directly after a step with a long (gt100 ms) dwell
  time (blue), and those following a step with a
  short (lt20 ms) dwell time (orange)

Kaseda, Higuchi, Hirose 2003 Nature
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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
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Movement of R14A/R14A homodimer
Kaseda, Higuchi, Hirose 2003 Nature
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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.

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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

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Asbury, Fehr, Block 2003 Science
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Limp vs non-limp
Dwell time (s)
Dwell time (s)
Asbury, Fehr, Block 2003 Science
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Conclusion

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

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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.