1. Cytoskeleton - Locomotion
Kohidai, Laszlo MD, PhD
Med. habil., Assoc. Professor
Dept. Genetics, Cell & Immunobiology, Semmelweis University
Lecture ED 2014

2. Main functions of cytoskeleton
Determines the shape of the cell
Anchores organelles
Movement of organelles
Tensile strength
Movement of chromosomes
Polarity
Motility

3. Cytoskeleton Microfilaments (actin) Microtubuli (tubulin)
Intermedier filaments
Motor proteins
Actin and microtubule associated proteins

4. Microfilaments
Microtubuli
Intermedier
filaments

5. SLIDING
Globular proteins
Ca2+
ATP
Motor proteins
Fibrillar proteins

6. Microfilaments

7. Polymerization of actin+
ATP
ADP
Depolymerization -
cytochalasin – inh.
phalloidin - stabilizer
ADP
ATP Pi
Polymerization - slow

8. Actin - still in Prokaryots !
((Ent et al. Nature 2001,413, 39)

9. Other actin homologues
((Roeben A et al. J Mol. Biol 2006, 358, 145)

10. Comparison of homologues
Polymerization in both forms
Opposite chirality !!!
((Wickstead and Gull J Cell. Biol 2011, 194, 513)

11. Cyclosis Transitional connections between actin and myosin
Moving
cytoplasm
Stationary
(cortical)
Actin filaments
Chloroplasts
Cell-wall
Plasma membrane
Transitional connections between actin and myosin
Ca2+, temperature- and pH-dependent
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)

12. „Fountain” mechanism Ca2+-dep. requires ATP Mono- Poly- Lobo- podial
Filo-
Reticulo-
Formation of pseudopodium
stress-fibrillums
integrins

13. Cross-linking proteins of actin
contractile bundle
a actinin – in stress fibr.
gel-like network
filamin - cortex
„tight” parallel bundle
fimbrin – in filopodium

14. Migrating keratinocyte
15 mm/sec
Formation of lobopodium
actin-network
microtubuli

15. Regulator proteins of actin polymerisation
gCAP39
Severin
Gelsolin
Villin
CapZ
Tropomodulin - + 
Cofilin
Severin
Gelsolin

16. Actin polymerization – acrosomal-reaction
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)

17. Listeria monocytogenes
local actin polymerization
speed: 10 mm/min
high ability to transmit
in tissues
actin
(Fred Soo & Julie Theriot Laboratory

18. Model of actin nucleation
WASP = Wiscott-Aldrich syndr. prot.

19. Structure of cortical region
(Svitkina, TM, Borisy GG J. Cell Biol. 1999, 145, 1009)

20. Actin – membrane links F-Actin Myosin I. Arp2/3 Profilin - G-actin
Integrin
Filamin

21. Profilin-mechanism Tb4 = thymosin b4
Proline-rich
protein
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)

22. Filamin – Membrane link
actin

23. + Structure of focal contact actin filament a actinin vinculin
paxillin
talin
integrin
fibronectin

24. A plasma membrane – cortex links
Thrombocyte
Muscle
Epithel
Spectrin
tetramer
Glycophorin
Ankyrin
((Lux SE, 1979 Nature 281:426)

25. + + + - - E Electromagnetic field induces the transformation
of cytoskeleton and
formation of pseudopodia
Adhesion plaque + + + - -

26. Myosin head Ca2+-dependent phosphorylation
and its effect on the 3D strcture
ATP - ADP Pi
light chain
heavy chain
a helix
myosin I.
150 kD
monomer
myosin I I.
260 kD
Head: - ATP-ase
- motor
dimer

27. Distribution of myosines in the migrating Dyctiostelium and in dividing cell
myosin I.
(green)
myosin II.
(red)
(Fukui, Y. Mol. Cell Biol 2000, 785))

28. - + Main types of interactions between the globular
and fibrillar components
of cytoskeleton
membrane

29. Non-treated
F-actin blocked
MT-blocked

30. Microtubules

31. Tubulin – still in Prokaryotes ! FtsZ Tubulin
(Margolin Laboratory, University of Texas)

32. Comparison of homologues
Polymerization in both forms
Monomers build helical structure vs. dimers build tubulus
((Wickstead and Gull J Cell. Biol 2011, 194, 513)

33. Polymerization of tubulin
GTP
GTP
GTP
GTP
Polymerization - fast
Protofilament (strait)
GDP
GDP
GDP
GDP
Protofilament (curved)
Depolymerization

34. Dynamics of microtubule-assembly
Nucleation Elongation - +
incorporation
balanced
release

35. Role of g-tubulin in nucleation
(Wiease et al. Curr.Opin.Struct.Biol. 1999, 9, 250)

36. Microtubular systems in the cells - Centrosome - Cilia / flagellum
Interphase cell
Dividing cell
Neuron
centrosome
Basal body
Cilla
spindle
axon
Microtubular
systems
in the cells
- Centrosome
- Cilia / flagellum
- Mitotic system
- Vesicular transport

37. MTOC = Microtubulus organizing center
g-tubulin
specific
region of
the cortex
((Brinkley, B.R. Encyclop. Neurosci. 1987, 665)

38. Network of microtubuli
Protofilaments
a-b dimer
alpha tubulin
beta tubulin
24 nm
Fibroblast

39. Cilia
cilia
flagellum
Paramecium

40. A B
dynein-arms
nexin
tubulin
(13 ill. 11 protofilaments)

41. Composition of dynein-arms
ATP-independent binding
ATP-dependent hydrolysis
The arm moves toward the - pole

42. The role of dynein arms
in beating of cilia
Bending
„Telescoping”
Proteolysis

43. Molecules composing the cilia
more than 250 types of molecules
70% a and b tubulin
dynein arms
outer - 9 polypeptides - ATP-ase
inner – composition varies
radial spokes polypeptides

44. Microtubules of mitotic spindle and kinetochore

45. Arrangement of actin during cell-division

46. Intermedier filaments

47. Crescentin

48. Mechanical characterization of cytoskeleton components
intermedier filament
i.e. vimentin
microtubule
= rupture
deformation
actin filament
force

49. Role of intermedier filaments
Buffer against external mechanical stress
Tissue specificity
Epithel – keratin
Connective tissue
Muscles
Neuroglia
Neurones - neurofilaments
Nucleus – lamines
(lamina fibrosa)
} vimentin

50. Structure of intermedier filamentums
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)

51. Domain structures of intermedier filamentums
H2N-
a helical domain
-COOH
keratins
vimentin
neurofilam. prot.
nuclear prot

52. Intermedier filaments
Keratin filaments
Vimentin-like filaments
! They DO NOT co-polymerize !

53. Microvilli myosin I. actin villin „terminal web”
a rigid bundle composed by actin mol.s
actin + on the apical part
villin is the linker molecule of actins
„terminal web” = intermedier fil. + spectrin
anchoreb by myosin I. and calmodulin to the surface membrane

54. SEM structure of microvilli
actin bundle
linker molecules
„terminal web”

55. Intermedier filaments
Neuro-filaments
– many cross-linkers
Glial filaments
– few cross-linkers
The number of protein cross-links between the
intermedier filaments varies in different tissues

56. Microtubuli associated proteins
(MAP-s)

57. Groups of MAP-s Structural MAP-s - MT-assembly - links to MF and to IF
Motor proteins
- sliding on MT
Enzymes, signal molecules
- glycolytic enzymek
- kinases
Shape and polarity
of the cell
Membrane transports
Assembly of molecules

58. Motor-proteins

59. Structure of motor-proteins
motor domain
motor
domain
assoc.
polypeptides
„stalk”
Kinesin
Myosin
Dynein

60. Motor proteins - + kinesin dynein microtubule kinesin dynein heavy
chain
light chain
kinesin
dynein

61. kinesin - +
dynein
cAMP
cAMP
pigment cells

62. Kinesin
ADP
ATP
ADP
ATP
ADP
ATP
ADP
ADP-Pi

63. MT-motor proteins and the transported elements
(Hirokawa, N. Science 1998, 279:519

64. Dynein – membrane relations
(Hirokawa, N. Science 1998, 279:519)

65. There are other mechanisms
over sliding …

66. Locomotion – with spasmoneme of Vorticella

67. Spasmoneme spring Contracts 40% in few msecs Velocity: 8 cm˛/sec
Negative
charges
Neutralization with Ca2+

68. Actin spring in sperm of horse-shoe crab Limulus polyphemus!
acrosome
actin bundle
The extension does not involve a myosin
motor or actin polymerization
The bundle is crystalline in its coiled and
uncoiled states