バイオトポロジー 7月2日

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

構造が生み出す機能

生体組織

問い
生体組織はどのように形造られるのか？

アクチン（actin）

チューブリン（tubulin)アクチンの様々な機能
red : actin
green : bacteria

筋収縮
http://www.ludwig.ucl.ac.uk/buzz_
html/assays2_content.htm

食作用

J. Cell Biol. Svitkina et al. 160 (3): 409.

G-actin
red : actin

細胞運動blue : actinorange : microtubules

http://www5.mfour.med.kyoto-u.ac.jp/rho.html

構造体の支持細胞質分裂

http://raven.zoology.washington.edu/
celldynamics/research/cytokinesis/

Myoblast cell

E (ATP, GTP)Journal of Muscle Research and Cell Motility (2005) 26:343–354

MyofibrilE (ATP, GTP)

Energy dissipative assembly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
・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
２ 生体分子モーターの能動的自己組織化
・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

Salt
5nm

2mm

KCl＝10nm

Actin

E.D. 4e/nm
Lp 10um

Polycation？

F-Actin

Poly (L-lysine)
F-actinNH CH CO n
CH 2 4
NH 3+ Cl

10μm

10μm

Poly x, y-ionene bromide
Br[(CH2)x10mm

CH3N+CH3(CH2)yBr-CH3N+]nCH3Bioconjugate Chem.; 14(6); 1185-1190 (2003)

50nm
F-actinF-actin+PDMAPAA-Q

50nm

Ave. Length (mm)304030108206Ave. thickness (nm)

7060
50

4-810-710-61010

CA: Actin (M)Biomacromolecules 6 (6), 3005 (2005)

Thickness (nm)

N=310080

N=5N = 26 N = 186

60200nm

40NH CH CO n
CH 2 4
NH 3+ Cl

2010010110210

3

Degree of Polymerization (n)F-actins + polycation

Nucleus

Actin bundle?
D is determined by D*

D*  4 / 2g (G / D  0)

Di DLL is determined by CA / CN(CN : nucleus concentration)

 LiΔg

G  G  G  gV  Sbulksurf

π D2L
π D 2  2DLg 
42

 : Surface energy per unit areag : Energy gain per unit volum e / g : Nucleus size

 (gD2 D)L   (gL) D2  (L)DBiochemistry, 45(34), 10313 (2006)

Anisotropic growth of actin bundle
LActual pathD  D*D*

DInverse relationship between g and D
D* = g -1gGg increases

D*D

Inverse relationship between binding energy and D受動的自己組織化の例
Fig. 1. Examples of static
self-assembly. (A) Crystal
structure of a ribosome. (B)Self-assembled peptideamphiphilenanoÞbers. (C)An array of millimetersizedpolymeric plates assembled
at a water/perßuorodecalininterface bycapillary interactions. (D)Thin Þlm of a nematic liquid
crystal on an isotropic
substrate. (E) Micrometersized
metallic polyhedra
folded from planar substrates.
(F) A three-dimensional
aggregate of micrometer
plates assembled bycapillary forces. [Imagecredits: (A) from (24); (B)from (25); (C) from (26);(D) from (27); (E) from(28); (F) from (29)]

Science, 295, 2418 (2002)

(mm/s)1.5

0.5

6,10-ionene

6,4-ionene

3,3-ionene

PDMAPAA-Q

poly-Lysine

1native

3.3s

Velocity

20
Biomacromolecules, 6, 845-849 (2005).

nR  nL
Polarity( P) nR  nL
P=1.0

P=0
L

Polymer

PDMAPAA-Q
p-Lysine
3,3-ionene
6,4-ionene
6,10-ionene

Polarity

0.89 (n=23)*
0.76 (n=21)
0.50 (n=22)
0.42 (n=17)0.49 (n=22)*n is the number of samples for the average.

R1.4(mm/s)

PDMAPAA-Q
poly-lysine

Velocity

3.3s

16,103,3-0.6

Native6,4-ionene

0.200.2 0.4P

0.60.81

Biomacromolecules 6, 3005-3009 (2005).

□アクチンバンドルの成長メカニズム
F-actinActin Bundle

Polycation~100nm

1~10mmF-actin + polycation
Nucleus

Actin bundle

太さ成長

長さ成長?
バンドルの極性は核形成段階で決まるのではないか
バンドルの太さと極性の関係は？

F-actin

Arrow headMyosin
Head

200nm

bundle Myosin

1 n nR  nL
Ppolarity  n i 1 nR  nL60
50401300.8200.6

100.4

000.05 0.1 0.15 0.2
KCl concentration CS⇒

0.2

Polarity

Head

Poly-Lys(n=26, 10-5M)

70

Average Thickness D (nm)Polarity assay

Poly-Lys(n=26)

60-6Thickness D (nm)C =10 M, C =0.1M
P

50

S-4

C =10 M, C =0.1M
PS

40
30
2020min

105min00

5 10 15 20 25 30 35 40 0Time (min)5 10 15 20 25 30 35 40

Time (min)vNucleusformation

Dif P ~ R 1 ~ N  v(CP , C A , Dif P , Dif A )=0.5~0.6
R : Gyration radiusN : Polymerization degree

DifP：Diffusion coefficient

N73

Nucleation
10min.
Time1338

N52620min.

Polarity

0.350.59

CP =10-6 M⇒

CP =10-4 M

直径

核形成速度

極性Parameter
塩濃度⇒

ポリマー濃度⇒
ポリマー重合度⇒D~ /gP  VD

Langmuir,25(3),1554-1557 (2009)

能動的自己組織化の例
Fig. 2. Examples of dynamic
self-assembly. (A) An optical
micrograph of a cell with ßuorescently
labeled cytoskeleton
and nucleus; microtubules(;24 nm in diameter) are colored
red. (B) Reaction-diffusion
waves in a Belousov-Zabatinski
reaction in a 3.5-inch
Petri dish. (C) A simple aggregateof three millimeter-sized,rotating, magnetized disks interactingwith one another via
vortex-vortex interactions. (D)A school of Þsh. (E) Concentricrings formed by charged metallic
beads 1 mm in diameter
rolling in circular paths on adielectric support. (F) Convection
cells formed above a micropatterned
metallic support.
The distance between the centersof the cells is ;2 mm.
[Image credits: (A) from (30);(B) from (26); (C) from (31)]

Science, 295, 2418 (2002)

Microtubule: 9.6nM
Streptavidin: 2nM+ATP 5mM~0.5h後
MTs bundle formation

20μm（X100 speed）

Rotary motors

10mm

Rotate for ~2days
Biomacromolecules 9, 2277–2282 (2008)

C.C.W : C.W. =93:7(n=238)
Microtubule: 240nM
Kinesin: 63nM

Microtubule (GTP, +Taxol)

32±2 x10-24 Nm2

Microtubule (GTP→GMPCPP)

62±9 x10-24 Nm2

Mickey and Howard. (1995). J Cell Biol 130(4), 909-917.

GMPCPP-MT

Microtubule: 240nM
Streptavidin: 240nM+ATP 5mM~4h後
Linear shaped MT bundles
Length: 100μm~
GTP-MT

20μm（X100 speed）
Langmuir in pressラチェットモーター機構kT[ A1 ]Fln
KCA force generated by polymerization F in the case of G- or F-actin is defined by thefollowing 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 Carlier
Dynamique du Cytosquelette, Laboratoire d’Enzymologie et Biochimie structurales,
C.N.R.S., Avenue de la Terrasse, 91198 Gif-sur-Yvette, Francebis(4-(N,N-dimethylamino)phenyl)(4-vinylphenyl)methyl leucohydroxide（Leucohydroxide)

HCCH2CH2HC

λ ＞270nm
H3C

CH3NN

H3COH

CH3Δ

H3CNC

H3C+CH3N

OH

CH3Masahiro Irie and Dawan Kuntchankun,Macromol.1986,19,2476-2480

*HC C
H2xO

H
C C
H2y*

NH
(CH2)3N(CH3)3+

N(CH3)2(CH3)2N
OH

poly(Leucohydroxide-co-DMAPAA-Q)

Fluorescence image

0 min

10 min20 min30 min10μm

UV-irradiation time
λ ＞270nm

J. Biomedical Materials Research Prat A, (2008).

10μm

光（X100 speed)

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
Actin+Monocation

Bright field

Radicalpolymerization

Photo-induced
Growth of actin

10mm
Fluorescenceimage

UV irradiation

10mm

10mm

Liposome encapsulated F-actin

10mm

f ~ 5mm

10mm

f 5mm ~

Liposome encapsulated G-actin

10mm

10mm

f 2 ~ 3mm

f 3 ~ 5mm

Langmuir, 24(20), 11975 (2008)

課題
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