) " " " " 21
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
1/10 6 (10-9 ) (10-3 ) 10 80 10 4 1/10 76 (S/N) Approach 1
1 1 cm 1cm 1cm
From Prof. Ishiwata 7nm cm From H.E.Huxley and A.F. Huxley
Yanagida, et. al. Nature 307 (1984), 316 (1985)
Harada, et al. Nature 326 (1987), Kishino et al. Nature 334, (1988) Seeing is Believing 5nm
Cy3- Funatsu, Harada, Saito et al., Nature 374(1995)
nm 1nm cm Vale, Funatsu, Harada, et al. Nature 380 (1996) ATP ATP ADP Pi
3 (2 )-O-Cy3-ATP ATP
ATP hydrolysis cycles Myosin Cy3-ADP Movie Funatsu T., Harada, Y. Tokunaga, M. el al. Nature 374, 95 ATPase ds/dt = -ks
5nm Fa From Askin
DNA 0.61/( /NA) 200nm < 1nm
Heads displacement(nm) (nm) Times(s) N Tanaka H, el al. Boiphys.J.75 98
cm 1cm 1cm ATP 1 ATPase =1:1 Ishijima, Kojima, Tokunaga, et al. Cell 92 (1998)
Laser in Laser out Kitamura, Tokunaga et al. Nature 397(1999) ATP 0.1pN 0.1pN = 1g ( 5.5nm = 5.5 5ms= 5
5.5nm U(x,t) U 0
complex
1) 2) ATP (MyosinV) E = 2 pn 36 nm = 72 10-21 J G ATP -80 10-21 J/mol Efficiency 0% Tanaka, Homma, Iwane, Ikebe et al., Nature 2002
1) 2) ATP 3)
: :
Nature,Dec.14th,2000 DNA ATP
DNA Harada, Funatsu, et al. Biophys. J. (1999)
Harada, Kinoshita, et al. Nature (2001) ATP
ATP F1F0 10nm Sambongi Y, et al. Science 286 00 Noji et al. Nature 386 97
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)
2+ 2+
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
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
Transmitter Membrane Receptor On Signal proteins Expression Off Gene
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
δ
Cy3- AMP)
GTP car1 G- GTP
camp G-protein Signal Miyanaga,Ueda et al.
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.