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）