Signal Receptor Ion Channel Transmitter Energy transducion and transport Receptor A signal Energy transduction Cross talk Nuclear Cascades B signal As

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2 Signal Receptor Ion Channel Transmitter Energy transducion and transport Receptor A signal Energy transduction Cross talk Nuclear Cascades B signal Assembly ATP Transport Genetic information DNA RNA polymerase

3 1/10 6 (10-9 ) (10-3 ) /10 76 (S/N) Approach 1

4 1 1 cm 1cm 1cm

5 From Prof. Ishiwata 7nm cm From H.E.Huxley and A.F. Huxley

6 Yanagida, et. al. Nature 307 (1984), 316 (1985)

7 Harada, et al. Nature 326 (1987), Kishino et al. Nature 334, (1988) Seeing is Believing 5nm

9 Cy3- Funatsu, Harada, Saito et al., Nature 374(1995)

10 nm 1nm cm Vale, Funatsu, Harada, et al. Nature 380 (1996) ATP ATP ADP Pi

11 3 (2 )-O-Cy3-ATP ATP

12 ATP hydrolysis cycles Myosin Cy3-ADP Movie Funatsu T., Harada, Y. Tokunaga, M. el al. Nature 374, 95 ATPase ds/dt = -ks

13 5nm Fa From Askin

14 DNA 0.61/( /NA) 200nm < 1nm

15 Heads displacement(nm) (nm) Times(s) N Tanaka H, el al. Boiphys.J.75 98

16 cm 1cm 1cm ATP 1 ATPase =1:1 Ishijima, Kojima, Tokunaga, et al. Cell 92 (1998)

17 Laser in Laser out Kitamura, Tokunaga et al. Nature 397(1999) ATP 0.1pN 0.1pN = 1g ( 5.5nm = 5.5 5ms= 5

18 5.5nm U(x,t) U 0

19 complex

1) 2) ATP (MyosinV) E = 2 pn 36 nm = 72 10-21 J G ATP -80 10-21

20 1) 2) ATP (MyosinV) E = 2 pn 36 nm = J G ATP J/mol Efficiency 0% Tanaka, Homma, Iwane, Ikebe et al., Nature 2002

21 1) 2) ATP 3)

22 : :

23 Nature,Dec.14th,2000 DNA ATP

24 DNA Harada, Funatsu, et al. Biophys. J. (1999)

25 Harada, Kinoshita, et al. Nature (2001) ATP

26 ATP F1F0 10nm Sambongi Y, et al. Science Noji et al. Nature

nm Ryanodine receptor Ryanodine 10 µm 633 nm

putative model of ryanodine-ryr interaction

27 nm Ryanodine receptor Ryanodine 10 µm 633 nm 532 nm Cy5/anti-RYR/RYR Bodipy FL-X A putative model of ryanodine-ryr interaction Ide,Takeuchi At nanomolar concentration.ryanodine, a plant alkaloid, locks the channel into a long-lived open subconductance state. RYR is predicted to have one high affinity ryanodine binding sites. SRW Chen et al., Biophysical J. (2002)

28 2+ 2+

29 Single -molecule imaging in tip-dip lipid bilayer Searching new method for singlemolecule imaging more convenient more popular Application of patch electrode Simultaneous measurement Diminishing the bilayer area Observation of all bilayer membrane Decrease of capacitive noise

30 Imaging and electrical records of nachr using tip-dip method Electrical records +Ach 0.6M KCl 2.5mM HEPES ph7.4 1mM CaCl 2 50µM DIDS 5mM DTT 100nM Ach (bath) V: 100mV 10pA 2sec +antagonist 10µM tubocurarine 1kHz low-pass filtered Imaging 5µm 3sec average Single molecule of nachr might be observed nachr was labeled with cy5-maleimide (labeled cystein residue was not specified) Molar dye/protein ratio was 0.3 Em: 633 nm Epi fluorescence observation

31 Transmitter Membrane Receptor On Signal proteins Expression Off Gene

32 DAG IP 3 PIP 2 PLCγ P GTP P P Grb2 msos Ras Ras GDP GTP P Shc GDP P Raf MEK P EGF Ras Raf1 EGF Ras Raf

33 δ

35 Cy3- AMP)

37 GTP car1 G- GTP

38 camp G-protein Signal Miyanaga,Ueda et al.

Neurite Growth NGF signal Fast lateral diffusion

40 NGF Nerve cell Growth cone 70x time lapse Single-molecule Imaging of NGF/NGFR Complexes Inducing Neurite Growth NGF signal Fast lateral diffusion Retrograde transport signal Endocytosis Inactivation of NGFR Tani, et al.