graduate school of medicine, the University of Tokyo ...
Transcript of graduate school of medicine, the University of Tokyo ...
![Page 1: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/1.jpg)
How molecular motor acts
2006.11.27. graduate school of medicine, the University of Tokyo Nobutaka Hirokawa
Life Science Seen from Molecular Motor
![Page 2: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/2.jpg)
Low-angle, rotary shadowing electronmicrograph
Kinesin
KIF1A
![Page 3: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/3.jpg)
![Page 4: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/4.jpg)
![Page 5: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/5.jpg)
Okada et al.Science 283:1152-, 1999
![Page 6: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/6.jpg)
![Page 7: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/7.jpg)
![Page 8: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/8.jpg)
![Page 9: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/9.jpg)
![Page 10: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/10.jpg)
![Page 11: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/11.jpg)
Optical Trapping Nanometry
Spatial resolution < 0.2 nmTemporal resolution < 1 ms
0.2μm beads⇒Small viscous drag
⇒ High temporal resolution
(Response time ≦ 0.25 ms)
Okada,Higuchi, and Hirokawa Nature 424:574-2003
![Page 12: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/12.jpg)
Immobilization of KIF1A to Bead
![Page 13: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/13.jpg)
Bidirectional and Step-wise Movement of a Single KIF1A Monomer
![Page 14: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/14.jpg)
One ATP Hydrolysis Triggers One Stepping Movement.
Time (s)8 9 10
4080
120
-20 0 20
(nm)
(ms)-400
4080
120160
Cycle Time (ms)
0
200
400
600
800
0 200 400 600 800
![Page 15: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/15.jpg)
Step size of KIF1A is 8×n nm, and varies stochastically.
02468
10
-32 32160-16step (nm)
12
![Page 16: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/16.jpg)
Plus-end biased binding of KIF1A to the microtubule: ensemble average
Time (s)
on
0
2
4
-0.2 0 0.2
// MT⊥ MTControl
+-end
//^
![Page 17: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/17.jpg)
Flush Ratchet Model
α β α β α βα β
- +
KIF1AATP
Phosphate
drelease ~0 nm
Dwell Detach
Load F Drag -gv
1D-diffusion under load
dbind ~3 nm
ADPBind
① ②
③④
![Page 18: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/18.jpg)
![Page 19: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/19.jpg)
Microtubule Structure Kikkawa et al.
JCB, 1994: Nature, 1995
Microtubule Microtubule-Kinesin complex
![Page 20: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/20.jpg)
KIF1A-Microtubule Complex: cryo EM(15Å resolution)
Kikkawa et al. Cell 100: 241-, 2000
![Page 21: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/21.jpg)
KIF1A-Microtubule Complex: cryo EM(15Å resolution)
![Page 22: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/22.jpg)
![Page 23: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/23.jpg)
![Page 24: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/24.jpg)
![Page 25: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/25.jpg)
![Page 26: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/26.jpg)
ADP ATP(AMPPNP)
View from outside
![Page 27: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/27.jpg)
ADP ATPView from outside
![Page 28: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/28.jpg)
![Page 29: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/29.jpg)
Tarzan Model
“Drag force” = diffusion might reflect the fluctuation of theflexible tether between KIF1A and tubulin (K-loop & E-hook).
TarzanTarzan
![Page 30: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/30.jpg)
k i
Kikkawa et al. Nature 411: 439- ,2001
![Page 31: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/31.jpg)
![Page 32: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/32.jpg)
Structural Biological Analysis of kinesin motors
KIF1A alternately uses two loops to bind microtubules
Nitta,R. et al. Science 305:678- , 2004
![Page 33: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/33.jpg)
Construction of KIF1A Motor Domain
![Page 34: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/34.jpg)
Chemo-mechanical cycle of kinesin motors
![Page 35: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/35.jpg)
Overall architecture of KIF1A
ATP(AMPPNP)
ADP-Pi (1)(ADP-AlF3)
ADP-Pi (2)(ADP-VO4)
ADP
![Page 36: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/36.jpg)
Conformational changes of two switch regions
![Page 37: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/37.jpg)
KIF1A*=ATP- Microtubule
![Page 38: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/38.jpg)
KIF1A=ADP-Pi -Microtubule
![Page 39: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/39.jpg)
KIF1A*=ADP-Pi -Microtubule
![Page 40: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/40.jpg)
KIF1A=ADP -Microtubule
![Page 41: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/41.jpg)
Nucleotide construct
wild type L12 L11 L8
AMPPNP 4.2±1.3 6.0±1.4 20.2±4.0 25.0±6.0
ADP 6.8±2.5 23.5±8.4 12.3±4.0 26.5±5.0
ATP 10.8±1.8 40.5±11.8 nd nd
ADP-AlFx 5.9±1.5 7.1±1.7 nd nd
ADP-Vi 21.4±4.3 167±66 nd nd
Equilibrium Binding Constants of KIF1A mutant
![Page 42: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/42.jpg)
Design principle
of Walker-
type NTPase
s
![Page 43: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/43.jpg)
Design principle of Walker-type NTPases
![Page 44: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/44.jpg)
Model for the processive movement of the monomeric kinesin KIF1A
![Page 45: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/45.jpg)
![Page 46: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/46.jpg)
![Page 47: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/47.jpg)
Univ. Tokyo
Takao NAKATAYoshimitsu KANAIYasuko NODASumio TERADAYosuke TAKEISen TAKEDAYasushi OKADAYosuke TANAKAMasahiko KAWAGISHIMitsutoshi SETOU
Laurent GUILLAUDRichard WONGJunlin TENGHarukata MIKIDae-Hyung SEOGChunjie ZHAOTerunaga NAKAGAWAMasahide KIKKAWAHiroaki YAJIMARyo NITTATadayuki OGAWA
UCSFRobert J. FletterickElena P. Sabrin
Univ. Hospital TokyoJunko TAKITAYasuhide HAYASHI
Niigata Univ. Brain Res. Inst.
Masaaki SAITOShoji TSUJI
![Page 48: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/48.jpg)
![Page 49: graduate school of medicine, the University of Tokyo ...](https://reader030.fdocument.pub/reader030/viewer/2022012412/616c30bbdb8cd53496479064/html5/thumbnails/49.jpg)