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生物分子モーターの集積と
その創発的機能
角五 彰
北海道大学大学院先端生命科学研究院
バイオトポロジー 7月2日
構造が生み出す機能
生体組織
生体組織はどのように形造られるのか?
問い
アクチン(actin)
チューブリン(tubulin)
G-actin食作用
細胞運動
細胞質分裂
筋収縮http://www.ludwig.ucl.ac.uk/buzz_
html/assays2_content.htm
red : actin
green : bacteria
http://raven.zoology.washington.edu/celldynamics/research/cytokinesis/
blue : actin
orange : microtubules
J. Cell Biol. Svitkina et al. 160 (3): 409.
http://www5.mfour.med.kyoto-u.ac.jp/rho.html
red : actin
構造体の支持
アクチンの様々な機能
Myoblast cell
Myofibril
E (ATP, GTP)
Energy dissipative assembly
E (ATP, GTP)
Journal of Muscle Research and Cell Motility (2005) 26:343–354
アクチン関連タンパクのな相互作用が重要?
内容
生体分子モーターの受動的自己組織化
2 生体分子モーターの能動的自己組織化
・静電的な相互作用を利用した自己組織化・構造形成メカニズム
・分子モーター集合体の機能発現・運動機能 構造相関に関する考察
・化学エネルギーで駆動する自己組織化・集合体の多様な構造と運動機能・運動機能 構造相関に関する考察
・集合体の構造制御
自己組織化の時空間制御
10μm
F-actin
CH3 CH3
[(CH2)x N+ (CH2)y N+]n
CH3 CH3
Poly x, y-ionene bromide
Br- Br-
10mm
Poly (L-lysine)
10μm
NH CH CO
CH2
NH3+4
n
Cl
Actin
Salt
KCl
F-Actin
E.D. 4e/nm Lp 10um
10nm
2mm
5nm
Polycation
=
Bioconjugate Chem.; 14(6); 1185-1190 (2003)
?
50nm
50nm
+PDMAPAA-Q
F-actin
F-actin
10
20
30
40
50
6070
4
6
8
10
30
10-8
10-7
10-6
Ave
. L
en
gth
(m
m)
CA: Actin (M)
Ave
. thic
kn
ess (n
m)
Biomacromolecules 6 (6), 3005 (2005)
20
40
60
80
100
100
101
102
103
Thic
kness
(nm
)
Degree of Polymerization (n)
N = 3 N = 5 N = 26 N = 186
200nm
NH CH CO
CH2
NH3+4
n
Cl
L is determined by CA / CN
(CN : nucleus concentration)D is determined by D*
L
D
eunit volumper gain Energy :g
areaunit per energy Surface :
size Nucleus:/ gSgVGGG surfbulk
ii LΔg
D
2
22
4
2 DLDπg
LDπ
gD* 2/4 )0/( DG
F-actins + polycation Nucleus Actin bundle
?
DLDgLLDgD )()()2( 2
Biochemistry, 45(34), 10313 (2006)
D
L
D*
Actual path
Anisotropic growth of actin bundle
D D*
D*
G
g increases
D
D* = g -1
Inverse relationship between binding energy and D
g
Inverse relationship between g and D
Fig. 1. Examples of staticself-assembly. (A) Crystalstructure of a ribosome. (B)Self-assembled peptideamphiphilenanoÞbers. (C)An array of millimetersizedpolymeric plates assembledat a water/perßuorodecalininterface bycapillary interactions. (D)Thin Þlm of a nematic liquidcrystal on an isotropicsubstrate. (E) Micrometersizedmetallic polyhedrafolded from planar substrates.(F) A three-dimensionalaggregate of micrometerplates assembled bycapillary forces. [Imagecredits: (A) from (24); (B)from (25); (C) from (26);(D) from (27); (E) from(28); (F) from (29)]
受動的自己組織化の例
Science, 295, 2418 (2002)
0
0.5
1
1.5
2
Velo
cit
y3
.3s(m
m/s
)
na
tive
3,3
-io
ne
ne
po
ly-L
ysin
e
6,4
-io
ne
ne
6,1
0-io
ne
ne
PD
MA
PA
A-Q
Biomacromolecules, 6, 845-849 (2005).
P=1.0 P=0LR
LR
nn
nnPPolarity
)(
p-Lysine
3,3-ionene
Polymer
6,4-ionene
6,10-ionene
Polarity
0.76 (n=21)
0.50 (n=22)
0.42 (n=17)
0.49 (n=22)
PDMAPAA-Q 0.89 (n=23)*
*n is the number of samples for the average.
LR
P
Ve
loc
ity
3.3
s (
mm
/s)
6,4-ionene
3,3-
PDMAPAA-Q
poly-lysine
6,10-
10.80
1.4
1
0.6
0.4
Native
0.2
0.2 0.6
Biomacromolecules 6, 3005-3009 (2005).
□
バンドルの極性は核形成段階で決まるのではないか
?
F-actin + polycationNucleus Actin bundle
バンドルの太さと極性の関係は?
Actin BundleF-actin Polycation
1~10mm
~100nm
太さ成長 長さ成長
アクチンバンドルの成長メカニズム
0
10
20
30
40
50
60
70
0.2
0.4
0.6
0.8
1
0 0.05 0.1 0.15 0.2
Avera
ge T
hic
kn
ess
D (
nm
)
Po
larity
KCl concentration CS
Arrow head
Myosin Head
200nm
Polarity assay
Myosin Head
bundle
Poly-Lys(n=26, 10-5M)
⇒
n
i LR
LR
polaritynn
nn
nP
1
1
F-actin
0 5 10 15 20 25 30 35 40
0
10
20
30
40
50
60
Time (min)
CP=10
-6M, C
S=0.1M
Th
ick
ne
ss
D (
nm
)
0 5 10 15 20 25 30 35 40
Time (min)
CP=10
-4M, C
S=0.1M
Poly-Lys(n=26)
5min20min
),,,(
formation Nucleus
AifPifAP DDCCv
v
R : Gyration radiusN : Polymerization degree
NRDPif ~~ 1
=0.5~0.6
DifP:Diffusion coefficient
5 26N
0.59
CP =10-4 M
0.35Polarity
CP =10-6 M
NucleationTime
73 1338
20min.10min.
N
⇒
⇒
⇒
⇒
Parameter
直径 核形成速度 極性
塩濃度
ポリマー濃度
ポリマー重合度
gD /~
DVPLangmuir,25(3),1554-1557 (2009)
Fig. 2. Examples of dynamicself-assembly. (A) An opticalmicrograph of a cell with ßuorescentlylabeled cytoskeletonand nucleus; microtubules(;24 nm in diameter) are coloredred. (B) Reaction-diffusionwaves in a Belousov-Zabatinskireaction in a 3.5-inchPetri dish. (C) A simple aggregateof three millimeter-sized,rotating, magnetized disks interactingwith one another viavortex-vortex interactions. (D)A school of Þsh. (E) Concentricrings formed by charged metallicbeads 1 mm in diameterrolling in circular paths on adielectric support. (F) Convectioncells formed above a micropatternedmetallic support.The distance between the centersof the cells is ;2 mm.[Image credits: (A) from (30);(B) from (26); (C) from (31)]
能動的自己組織化の例
Science, 295, 2418 (2002)
20μm(X100 speed)
Microtubule: 9.6nMStreptavidin: 2nM
MTs bundle formation
+ATP 5mM~0.5h後
10mm
Rotary motors
Rotate for ~2days
Biomacromolecules 9, 2277–2282 (2008)
C.C.W : C.W. =93:7(n=238)
Microtubule: 240nMKinesin: 63nM
Microtubule (GTP, +Taxol)
Microtubule (GTP→GMPCPP)
32±2 x10-24 Nm2
62±9 x10-24 Nm2
Mickey and Howard. (1995). J Cell Biol 130(4), 909-917.
Microtubule: 240nMStreptavidin: 240nM
Linear shaped MT bundlesLength: 100μm~
+ATP 5mM~4h後
20μm(X100 speed)
Langmuir in press
GMPCPP-MT
GTP-MT
ラチェットモーター機構
CK
AkTF
][ln 1
A force generated by polymerization F in the case of G- or F-actin is defined by the
following equation , where is the increase in length due to incorporation of one actin,
KC is the critical concentration in the absence of an external force, and [AI] is the actin
concentration.
リステリア運動
Dominique Pantaloni, Christophe Le Clainche and Marie-France CarlierDynamique du Cytosquelette, Laboratoire d’Enzymologie et Biochimie structurales,
C.N.R.S., Avenue de la Terrasse, 91198 Gif-sur-Yvette, France
poly(Leucohydroxide-co-DMAPAA-Q)
N(CH3)2
CH
O
CH
2
NH
(CH2)3
N(CH3)3 (CH
3)2N
CH
OH
CH
2
** x
y
+
NCH3
CH3
NCH3
CH3OH
CHCH2
C+
NCH3
CH3
NCH3
CH3
CHCH2
HO
λ >270nm
Δ
Masahiro Irie and Dawan Kuntchankun,Macromol.1986,19,2476-2480
bis(4-(N,N-dimethylamino)phenyl)(4-vinylphenyl)methyl leucohydroxide
(Leucohydroxide)
0 min 20 min10 min 30 min
UV-irradiation time
10μm
λ >270nm
Fluorescence image
J. Biomedical Materials Research Prat A, (2008).
(X100 speed)
光
10μm
Fluorescenceimage
Bright field
10mm
10mm
Actin+Monocation
Photo-inducedGrowth of actin
Radical-polymerization
UV irradiation
UVをトリガーとした脂質二重膜内アクチンバンドル形成
f ~ 5mm
Liposome encapsulated G-actinLiposome encapsulated F-actin
f 5mm ~ f 2 ~ 3mm f 3 ~ 5mm
10mm
10mm 10mm 10mm 10mm
Langmuir, 24(20), 11975 (2008)
課題
講義中に出てきたトピックス(複数可)をトポロジーという観点から自由に批評せよ。(A4枚)