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How does kinesin walk? Chuang Wu Molecular Biophysics III Thursday 19 Jan. 2006.

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Presentation on theme: "How does kinesin walk? Chuang Wu Molecular Biophysics III Thursday 19 Jan. 2006."— Presentation transcript:

1 How does kinesin walk? Chuang Wu Molecular Biophysics III Thursday 19 Jan. 2006

2 Animation of symmetric hand-over-hand mechanism of kinesin-dependent vesicle transport along a microtubule Böhm, Stracke, Unger 2002, 2003

3 Structure of kinesin - homodimer Fig. 1 of Yildiz, Tomishige, Vale, Selvin 2004 Science Head Neck Stalk Adapted from Kozielski, Sack, Marx, Thormahlen, Schonbrunn, Biou, Thompson, Mandelkow, Mandelkow 1997 Cell

4 How does kinesin walk? hand–over–hand model - symmetric mechanism - asymmetric mechanism inchworm model

5 Hand-over-hand vs inchworm Yildiz, Tomishige, Vale, Selvin 2004 Science

6 MethodConclusion 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

7 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.

8 Symmetric hand-over-hand vs inchworm Hua, Chung, Gelles 2002 Science

9 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

10 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

11 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.

12 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.

13 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

14 Hand-over-hand vs inchworm Yildiz, Tomishige, Vale, Selvin 2004 Science

15 Sites for single fluorescent dye attachments Yildiz, Tomishige, Vale, Selvin 2004 Science S43 E215 T324 3 residues were mutated to cysteines for fluorescent dye labeling A single kinesin molecule moving on an immobilized axoneme.

16 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.

17 Position versus time for kinesin motility Yildiz, Tomishige, Vale, Selvin 2004 Science E215C S43C-T324C heterodimer

18 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

19 Dwell-time histogram Dwell time histogram showing the expected exponential decay with a maximum near t=0. P(t) = tk 2 exp(-kt) Yildiz, Tomishige, Vale, Selvin 2004 Science

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

21 Conclusions The results strongly support a hand-over- hand (walking) model for kinesin motility. Yildiz, Tomishige, Vale, Selvin 2004 Science

22 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.

23 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

24 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

25 WT/R14A vs WT/WT The observed 16 nm step consists of two successive 8-nm steps. Kaseda, Higuchi, Hirose 2003 Nature

26 A displacement trace showing how step sizes (∆X1, ∆X2) and dwell time (τ) were measured. Kaseda, Higuchi, Hirose 2003 Nature

27 Dwell-time differences are greater in WT/R14A Kaseda, Higuchi, Hirose 2003 Nature

28 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

29 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

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

31 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.

32 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

33 Asbury, Fehr, Block 2003 Science

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

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

36 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.


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