Biomolecular Motors: Topology in

Biology, Structural Integration

and Emergence of Functions

Arif Md. Rashedul KABIR

Faculty of Science, Hokkaido University

Biotopology: 4th July 2014 To understand the basic concepts of Topology from the viewpoint of Biology and

To focus on natural topological phenomena

What is Topology?

Space

The study of geometric properties and spatial relations unaffected by the continuous change of shape or size of objects.

Topology and Nature

Topology in Biology

Structure Function

Integrated Structure

Emergent Function

Topology in biology Offer divers functions

Levels of organization in nature

Biomolecular Motor Protein System

Actin- Myosin

Microtubule- Kinesin

Cytoskeleton

Topology inside cell

Molecular Biology of The Cell, 5th Edition

Phagocytosis Cell movement

Cytokinesis

Muscle contraction

red : actin

green : bacteria

blue : actin

orange : microtubules

red : actin

Structural support

Motor protein systems in cellular activities

Actin-Myosin: Cell locomotion

Cell shape regulation………

Microtubule-Kinesin:

Intracellular transport

Organelles regulation………

Topological Variation of Actin

Polymerization

Actin Polymerization: Cell Movement

Actin web: Cell Movement

Treelike web of polymerized Actins

Actin web: Cell Movement

Molecular Biology of The Cell, 5th Edition

Actin polymerization enables

cell motility.

Actin web: Cell Movement

Movement of Listeria monocytogenes: use of polymerization force

CK

AkTF

][ln 1

F : Force generated by polymerization. : Increase in length due to incorporation of one molecule. KC: Critical concentration in the absence of an external force. [AI]: Concentration of free molecules.

Linear motor proteins

Myosin: Actin associated motor

Molecular Biology of The Cell, 5th Edition

Linear motor proteins Kinesin and Dynein: Microtubule’s motor

Kinesin

Dynein

10-2

10-1

100

101

102

103

104

100

101

102

103

tim

e (

s)

length (m)

Diffusion time as a function of the length for a typical value of the diffusion coefficient (D=100m2/s) of a protein in water.

t=𝑥2 2𝐷

Diffusion: How fast is it?

D=kBT/6phr

h

kB

Diffusion speed depends on the size of body.

Biomolecular motor driven transportation

How to communicate inside cell?

…………Active transport

Linear motor driven

bending motion

MT

Cilium

Dynein

ref: Heuser et al. JCB,2009 ref: Nikon

Slipper animalcule

Cilia and Flagella Motile structures from Microtubules

Motion in sperm, protozoan etc. Molecular Biology of The Cell, 5th Edition

Organelle transportation

by Kinesin and Dynein

Transport of pigment granules in African cichlid fish.

Melanosome movement regulation Change in skin color of fish

Myosin mediated muscle contraction

Molecular communication system Broadcast of Information

‘Natural Computing’ -by Yasuhiro Suzuki, Masami Hagiya, Hiroshi Umeo, Andrew Adamatzky

Physical Impact Cargo

Microtubule fracture

Important to know…… What is mechanism of microtubule breakage on molecular level? What fracture mode induces severe damage of microtubule?

Stress, deformation and topology

1Faul M, Xu L, Wald MM, Coronado VG. Traumatic Brain Injury in the United States: Emergency Department Visits, Hospitalizations and Deaths 2002–2006. Atlanta (GA): Centers for Disease Control and Prevention, National Center for Injury Prevention and Control; 2010. Finkelstein E, Corso P, Miller T and associates.

Impaired cellular transport Memory loss

Traumatic Brain Injury (TBI)

10 million TBI’s per year world-wide 1.7 million TBI’s per year in the U.S.1

-52,000 Deaths -275,000 Hospitalizations

Mechanical stress ↓

Microtubule Breakage ↓

Interrupted cellular transport

Min. D., et al., Exp. Neurol 2012, 233, 364

Different fracture modes Mode Ⅰ: Opening mode

Mode Ⅱ: Shear mode

Mode Ⅲ: Torsional mode

5 µm Guo et al, Biophysical journal, 2006, 90, 2093

Broken

MT

Optical trap MT breakage by optical tweezers

Young’s modulus of MT: 0.29 MPa

?

What and how to do?

I. Establish an experimental setup for studying effect of shear stress on MTs.

II. Perform quantitative investigation on the behavior of MTs under shear stress.

Substrate

Microtubule

DC motor OMEC-2BG (Sigma Koki) Output velocity:1.5-100µm/s

PC PC N2

MT

PDMS

Stress direction

Lens

Vertical stepping motor

DC motor

Stretch chamber

Kinesin

anti-GFP antibody

Casein

5 cm Stretch chamber

PDMS Young’s modulus:

E=1.86 MPa

3 cm

Stretcher

Stretch chamber How it works?

100, L

Δ L (%) Strain

∆L= Length change of PDMS L= Initial length of PDMS

Microtubule fragmentation Microtubule

Before applying stress

Elongation

Kinesin: 900 nM

Strain: 14%

Strain rate: 1.4 %/s

Fragmentation 10 µm

Shear stress causes fragmentation of MTs.

After applying stress

Microtubule fragmentation

Kinesin changes rigidity of Microtubule

Kinesin works as a softening agent for Microtubule.

Kinesin (nM)

50

100

200

600

900

1300

Buckling of MT by compression

Compression

Kinesin: 30 nM

Compression rate: 1.4 %/s

10 µm

Compression causes buckling of MTs.

Before compression

After 14% compression

Role of Kinesin in compression induced deformation of MTS

10 µm

Kinesin (nM)

10

30

50

100

200

Polycation

Actin

Salt KCl

F-Actin

E.D. 4e/nm Lp 10um

10nm

m

5nm

Actin bundle

＝

Bioconjugate Chem 14(6); 1185-1190 (2003)

pI=4.7

Poly x, y-ionene

Poly (L-lysine)

NH CH CO

CH2

NH3+4

n

Cl

CH2 CH

CO

NH

CH2

N+

3

CH3

CH3H3C

n

Cl

PDMAPAA-Q

10μm

F-actin

Biomacromol., 9, 537-542 (2008)

Structural integration of Actin

50nm

L

D

Anisotropic growth of bundles

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

p

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)

Anisotropic nucleation growth model

Kinesin

Microtubule(MT）

streptavidin(St)

＋ATP

Biotin(Bt)

Biomacromolecules 9, 2277–2282 (2008)

Ref. Nano let.,2005

Active self-organization

Structural Integration of Microtubules

Active Self-Organization

・Cross-linker conc.（St/Bt) ・MT conc.

Parameters

Polymorphism

Single

St/Bt: 1/100

Tub: 672nM

Bundles

+ATP 5mM ~4h

St/Bt: 1/16

Tub: 672nM

Network

+ATP 5mM ~4h

Tub: 3360 nM

St/Bt: 1/8

20μm（X100 speed）

+ATP 5mM

~4h

St/Bt: 1/16

Tub: 672nM

20μm（X100 speed）

Network

Bundle

Single

Ring

Phase diagram for MT assembly

Soft matter 2011

20μm）

32±2 x10-24 Nm2

62±9 x10-24 Nm2

Langmuir 2010

Major Parameters

C.C.W : C.W. =93:7(n=238)

Handedness of the rotation

Microtubule: 240nM Kinesin: 63nM

Biomacromolecules 9, 2277–2282 (2008)

Left handed CCW CW Right handed

Scenario for preferential rotation

Supertwist in PFs arrangement

0

10

20

30

40

50

11 12 13 14 15 16 17

30min24h

Perc

enta

ge o

f to

tal M

Ts

Number of PFs

Increase in PFs of MT

TEM Image

Right

Left

Ref. J.mol.Biol.,2000

,M

EIR

M : bending moment [Nm]

R : radius of curvature [m]

EI : flexural rigidity [Nm2]

I : Second moments[m4]

43

2 4)

2( rnnI

p

p

2r

22.114

15 R

R

R

28.1min30

24 R

R h

Experimental

n 1 2

Cross-section

Effect of PFs(n) on bending moment

Theoretical

Ave. 4.5μm、Mean 3.7μm Ave. 5.8μm、Mean 4.8μm

0

5

10

15

20

25

0 2 4 6 8 10 12 14 16

30 min

Even

ts (

nu

mb

er)

Ring diameter (m)

0

5

10

15

0 2 4 6 8 10 12 14 16

24 h

Events

(num

ber)

Ring diameter (m)

→

Increase in Ring diameter

with Incubation time

30min 24h

28.1min30

24 R

R h

T. Hashimoto et. al: PNAS 2007 R. Kuroda et. al: Current Biology 2004

Chirality shift in nature

10m

Size distribution

Important parameter for

ring formation

1.Bulk and Surface E

2.Bending E

3.Confrmational E

10m

Size variation of MT rings

4)(s

bsb

D

D

Bending rigidity rigidity bundle :b

rigidy filament :s

diameter bundle :bD

diameter filament :sD

Thermodynamics of MT ring formation

ΔG~Bulk＋Surface＋bending＋Conformation

＋Coulomb E＋Counter ion S

ln3

2

3

3/23/2

2

3/23/23/2

3/2

Tk

l

e

TkLG

B

s

Bcoil

: effective binding energy : effective surface energy : density of the polymer chain

k : rigidity of the polymer chain L : contour length : dielectric constant ls: Bjerrum length Soft-matter, 2012

ln3

2

3

3/23/2

2

3/23/23/2

3/2

Tk

l

e

TkLG

B

s

Bcoil

Soft-matter

Thermodynamics of MT ring formation

MT ring formation at Air/buffer interface

Yield >90% (previsou~0.4%)

Soft matter (2012) Front cover

Conclusions:

1. Topological variance is a very common

phenomenon observed in nature.

2. Diverse structural variation is the key

to highly complex and versatile activities

of natural bodies.