Biomolecular Motors: Topology in Biology, Structural Integration. and Emergence of Functions

Transcription

1 Biotopology: 4 th July 2014 To understand the basic concepts of Topology from the viewpoint of Biology and To focus on natural topological phenomena Biomolecular Motors: Topology in Biology, Structural Integration and Emergence of Functions Arif Md. Rashedul KABIR Faculty of Science, Hokkaido University

2 What is Topology? Space The study of geometric properties and spatial relations unaffected by the continuous change of shape or size of objects.

3 Topology and Nature Topology in Biology Structure Function Integrated Structure Emergent Function opology in biology Offer divers functions

4 Levels of organization in nature

5 Topology inside cell Biomolecular Motor Protein System Actin- Myosin Microtubule- Kinesin Cytoskeleton Molecular Biology of The Cell, 5 th Edition

6 Motor protein systems in cellular activities red : actin green : bacteria Phagocytosis Muscle contraction Cell movement red : actin blue : actin orange : microtubules Structural support Cytokinesis

7 Actin-Myosin: Cell locomotion Cell shape regulation

8 Microtubule-Kinesin: Intracellular transport Organelles regulation

9 Topological Variation of Actin Polymerization

10 Actin Polymerization: Cell Movement

11 Actin web: Cell Movement

12 Actin web: Cell Movement Treelike web of polymerized Actins Molecular Biology of The Cell, 5 th Edition

13 Actin web: Cell Movement Actin polymerization enables cell motility.

14 Movement of Listeria monocytogenes: use of polymerization force kt F [ A1 ] ln K C F : Force generated by polymerization. : Increase in length due to incorporation of one molecule. K C : Critical concentration in the absence of an external force. [A I ]: Concentration of free molecules.

15 Linear motor proteins Myosin: Actin associated motor Molecular Biology of The Cell, 5 th Edition

16 Linear motor proteins Kinesin and Dynein: Microtubule s motor Kinesin Dynein

17 time (s) Diffusion: How fast is it? Diffusion speed depends on the size of body t=x 2 2D D=k B T/6phr h k B length ( m) Diffusion time as a function of the length for a typical value of the diffusion coefficient (D=100 m 2 /s) of a protein in water.

18 How to communicate inside cell? Active transport Biomolecular motor driven transportation

19 Linear motor driven bending motion Slipper animalcule Cilium Dynein MT ref: Nikon ref: Heuser et al. JCB,2009

20 Cilia and Flagella Motile structures from Microtubules Motion in sperm, protozoan etc. Molecular Biology of The Cell, 5 th Edition

21 Organelle transportation by Kinesin and Dynein

22 Melanosome movement regulation Change in skin color of fish Transport of pigment granules in African cichlid fish.

23 Myosin mediated muscle contraction

24 Molecular communication system Broadcast of Information Natural Computing -by Yasuhiro Suzuki, Masami Hagiya, Hiroshi Umeo, Andrew Adamatzky

25 Stress, deformation and topology Physical Impact Microtubule fracture Cargo Traumatic Brain Injury (TBI) Impaired cellular transport Memory loss Mechanical stress Microtubule Breakage Interrupted cellular transport Min. D., et al., Exp. Neurol 2012, 233, 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 Important to know What is mechanism of microtubule breakage on molecular level? What fracture mode induces severe damage of microtubule? 1 Faul M, Xu L, Wald MM, Coronado VG. Traumatic Brain Injury in the United States: Emergency Department Visits, Hospitalizations and Deaths Atlanta (GA): Centers for Disease Control and Prevention, National Center for Injury Prevention and Control; Finkelstein E, Corso P, Miller T and associates.

26 Different fracture modes Mode Ⅰ: Opening mode Mode Ⅱ: Shear mode Mode Ⅲ: Torsional mode? Optical trap MT MT breakage by optical tweezers Young s modulus of MT: 0.29 MPa Broken Guo et al, Biophysical journal, 2006, 90, µm

27 What and how to do? Microtubule Substrate 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.

28 Stretch chamber DC motor Vertical stepping motor DC motor OMEC-2BG (Sigma Koki) Output velocity: µm/s PC N 2 PDMS PC Lens Kinesin anti-gfp antibody Casein MT Stress direction PDMS Young s modulus: E=1.86 MPa

29 Stretch chamber How it works? Stretcher 3 cm Δ L Strain, (%) 100 L L= Length change of PDMS L= Initial length of PDMS

30 Microtubule fragmentation Elongation Microtubule Kinesin: 900 nm Strain: 14% Strain rate: 1.4 %/s Before applying stress Fragmentation 10 µm After applying stress Shear stress causes fragmentation of MTs.

31 Microtubule fragmentation

32 Kinesin changes rigidity of Kinesin (nm) Microtubule Kinesin works as a softening agent for Microtubule.

33 Buckling of MT by compression Compression Kinesin: 30 nm Compression rate: 1.4 %/s Before compression After 14% compression 10 µm Compression causes buckling of MTs.

34 Role of Kinesin in compression induced deformation of MTS Kinesin (nm) µm

35 Structural integration of Actin Actin 5nm Salt KCl pi=4.7 10nm m = F-Actin E.D. 4e/nm Lp 10um PDMAPAA-Q Polycation Actin bundle F-actin Poly (L-lysine) CH 2 CH CO n 10μm NH CH CO CH 2 NH Cl n Poly x, y-ionene H 3 C NH CH 2 3 N+ CH 3 Cl CH 3 50nm Biomacromol., 9, (2008) Bioconjugate Chem 14(6); (2003)

36 Anisotropic growth of bundles L D

37 Anisotropic nucleation growth model F-actins + polycation Nucleus Actin bundle D is determined by D* D* 4 / 2 g ( G / D 0) L is determined by C A / C N (C N : nucleus concentration) L G G bulk π D 2 L g 4 D G surf D i Li gv π D 2 2pDL 2 Δg S :Surface energy per unit area g :Energy gain per unit volume / g:nucleus size ( gd 2 D) L ( gl) D2 ( L) D Biochemistry, 45(34), (2006)

38 Active self-organization Structural Integration of Microtubules Kinesin ATP Microtubule(MT streptavidin(st) Biotin(Bt) Ref. Nano let.,2005 Biomacromolecules 9, (2008)

39 Active Self-Organization Parameters Cross-linker conc. (St/Bt) MT conc. Polymorphism

40 Single Tub: 672nM St/Bt: 1/100

41 Bundles +ATP 5mM ~4h Tub: 672nM St/Bt: 1/16

42 Network +ATP 5mM ~4h Tub: 3360 nm St/Bt: 1/8

43

44 +ATP 5mM ~4h Tub: 672nM St/Bt: 1/16 20μm(X100 speed)

45 20μm(X100 speed)

46 Phase diagram for MT assembly Network Ring Bundle Single Soft matter 2011

47 Major Parameters 20μm) Langmuir ±2 x10-24 Nm 2 62±9 x10-24 Nm 2

48 Handedness of the rotation C.C.W : C.W. =93:7(n=238) Microtubule: 240nM Kinesin: 63nM Biomacromolecules 9, (2008)

49 Supertwist in PFs arrangement Scenario for preferential rotation Right handed CW CCW Left handed

50

51 Percentage of total MTs Increase in PFs of MT 30min 24h TEM Image Ref. J.mol.Biol.,2000 Right Number of PFs Left

52 Effect of PFs(n) on bending moment n 1 2 2r R Cross-section R EI M, I 2 p ( n 3 n) r p M : bending moment [Nm] R : radius of curvature [m] EI : flexural rigidity [Nm 2 ] I : Second moments[m 4 ] R R Theoretical R24 h R 30min 1.28 Experimental

53 Events (number) Events (number) Increase in Ring diameter with Incubation time Ave. 4.5μm Mean 3.7μm Ave. 5.8μm Mean 4.8μm min 30min R24 h R 30min h 24 h Ring diameter ( m) Ring diameter ( m)

54 Chirality shift in nature T. Hashimoto et. al: PNAS 2007 R. Kuroda et. al: Current Biology 2004

55 Size distribution 10 m

56 Important parameter for ring formation 1.Bulk and Surface E 2.Bending E 3.Confrmational E

57 Size variation of MT rings 10 m Bending rigidity D b s ( D b s 4 ) b s D b D s :bundle rigidity :filament rigidy :bundle diameter :filament diameter

58 hermodynamics of MT ring formation ΔG~Bulk+Surface+bending+Conformation +Coulomb E+Counter ion S G coil 2/3 L 2/3 3 2 k B T 2/3 2/3 2 e l s 2/3 kbt 3 2/3 ln : 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

59 hermodynamics of MT ring formation 3 2/3 2/3 2/3 Gcoil L k T 2/3 B 2 2 e l s 2/3 kbt 3 2/3 ln Soft-matter

60 MT ring formation at Air/buffer interface Yield >90% (previsou~0.4%) Soft matter (2012) Front cover

61 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.