1. Cytoskeleton and locomotion
Láng, Orsolya MD, PhD
Dept. Genetics, Cell & Immunobiology, Semmelweis University
Lecture EPh 2016

2. Main components of the eukaryotic cytoskeleton
Microfilaments:
actin
7nm
Microtubules:
tubulins (a, b)
25 nm
Intermediate filaments:
lamin
cell specific prot. (e.g. vimentin)
8-12 nm
+ Associated proteins (e.g. motor proteins)

3. Filamentous structures in the cytoplasm of walled bacteria
weak sequence homology but
crystallography appreciate their striking structural properties and similarity to eukaryotic cytoskeletal elements
Microfilament like:
MreB
Microtubule like:
FtsZ, TubZ
Intermediate filament like:
Crescentin, Parm
Nature Cell Biology12, 731–733 (2010)

4. Function of the cytoskeleton
Tissue level:
muscle movement
Cellular level:
determines shape of the cell
motility of the cells
cell adhesion
mitosis, meiosis
Subcellular level:
anchors organelles
organization of organelles
provides tensile strength
movement of chromosomes
organizing cell polarity
Intracellular movement of vesicles
- Endocytosis – clathrin-mediated endocytosis and phagocytosis
Dynamic
Adaptable
Stable
Strong

5. Cytoskeletal filaments are dynamic and adaptable

6. Stability of cytoskeletal filaments

7. Strong cytoskeletal filaments
Intermediate filaments- resistant to stretching forces

8. Microfilaments

9. Molecular structure of actin
G-actin
( Garland Science Molecular Biology of the Cell 2008)

10. Polymerazition of actin+
ATP
ADP
Depolymerization -
ADP
ATP Pi
Polymerization - slow
Drugs: cytochalasin – inhibition (cap at + end)
phalloidin - stabilizer

11. Treadmilling of actin filament
Video:

12. Actin and accessory proteins
Molecular Biology of the Cell (© Garland Science 2008)

13. Actin binding proteins (ABPs)
3 groups:
banding and cross linking proteins
regulatory proteins: polymerization/depolymerization, severing proteins,capping proteins
Motor proteins
- sliding on MF (myosin)
Organisation of
actin filaments
Sliding

14. Organisation of actin filaments
Molecular Biology of the Cell (© Garland Science 2008)

15. Cross-linking proteins I.
Contractile bundle
Parallel bundle
Molecular Biology of the Cell (© Garland Science 2008)

16. Cross-linking proteins II.
plasma membrane
Gel-like network
Molecular Biology of the Cell (© Garland Science 2008)

17.  Regulatory proteins - + gCAP39 Tropomodulin Severin Gelsolin Villin
CapZ
Tropomodulin - + 
Cofilin
Severin
Gelsolin
Profilin: G actin pool
Thymosin: actin sequestring
Formin: actin polymerizing protein

18. Actin polymerisation – moition I.
Acrosomal reaction
(Lodish, H. et al. Mol. Cell Biol. 2000, 767)

19. Actin polymerisation – motion II. Listreia Monocytogenes infection

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

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

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

23. Filamin – Membrane link
actin

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

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

26. Microvilli At bottom: (spectrin, myosin, intermediate filaments)
It is called: terminal web

27. Motor proteins: myosins
General structure:
Globular head and fibrillar tail
Heavy chains and light chains
Head: motor domain with ATP-ase activity
ADP- straight
Direction: + end motors

28. Myosin II molecule
Molecular Biology of the Cell (© Garland Science 2008)

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

30. Microtubules

31. Molecular structure of tubulin dimers and microtubules
GTP
Molecular Biology of the Cell (© Garland Science 2008)

32. Polymerisation of microtubules
Drugs:
Colchicine and colcemid– inhibition (binds subunit)
Taxol - stabilizer
Molecular Biology of the Cell (© Garland Science 2008)

33. Polymerisation of microtubules
Dynamic instability
and treadmilling
Video:
Molecular Biology of the Cell (© Garland Science 2008)

34. Role of g-tubulin in nucleation of microtubules at - end
Molecular Biology of the Cell (© Garland Science 2008)

35. Microtubule associated proteins (MAPs)
Structural MAP-s
- MT-assembly
links to MF and to IF
(eg. tau, MAPs1 and MAPs2)
Motor proteins
- sliding on MT
(e.g. kinesin and dynein family)
Enzymes, signal molecules
- glycolytic enzymes
- kinases
Shape and polarity
of the cell
Vesicular transports
Assembly of molecules

36. Structure of motor-proteins
motor domain
motor
domain
assoc.
polypeptides
„stalk”
Kinesin
Myozin
Dynein

37. - + ? Motor proteins + kinesin dynein ?

38. kinesin - +
dynein
cAMP
cAMP
pigment cells

39. Kinesin
ADP
ATP
ADP
ATP
ADP
ATP
ADP
ADP-Pi

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

41. In cells: MTOC = Microtubule organizing center
g-tubulin
9x3 microtubules A,B,C
2 centrioles at a right angle
Organisator:
Gamma-tubulin in pericentriolar matrix
- end of forming microtubule
(Brinkley, B.R. Encyclop. Neurosci. 1987, 665)

42. Centrosome
Molecular Biology of the Cell (© Garland Science 2008)

43. Cilia
cilia
flagellum
Paramecium

44. Structure of cilia B A tubulin (13 ill. 11 protofilaments) dynein-arms
nexin
tubulin
(13 ill. 11 protofilaments)

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

46. Role of the dynein arms in beating cilia
Telescopic effect
Beating

47. Microtubules of mitotic spindle and kinetochore

48. How motor proteins can organise the position of cell organelles (ER, Golgi) ?
(Hirokawa, N. Science 1998, 279:519

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

50. Intermediate filaments

51. Structure of an intermediate filaments Antiparallel tetramer
Monomer
Parallel dimer
Antiparallel tetramer
Protofilaments
Intermedaite
filaments
Molecular Biology of the Cell (© Garland Science 2008)

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

53. Role of intermedier filaments
Buffer against external mechanical stress
Tissue specificity !!!
Epithel – keratin
Connective tissue
Muscles
Neuroglia
Neurones(axon) - neurofilaments
} vimentin-like
Vimentin
Desmin
Glial protein
Exception:
Nucleus – lamines (A,B,C) →(lamina fibrosa)

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

55. Axon of a neuron Glial cell Axon cross-section
Neurofilament (NF-H)
Glial cell
Glial filaments
Axon cross-section
Cross bridges are formed by non-helical C terminus
few cross bridges
Molecular Biology of the Cell (© Garland Science 2008)

56. Intermediate filaments and the cytoskeleton
In the hypothetical epithelial cell depicted, the three key filament systems of the cytoskeleton, microfilaments (MFs), microtubules (MTs) and intermediate filaments (IFs), are connected to each other by dimeric complexes of plakin-type molecules such as plectin and BPAG1. In addition, a multitude of MT-associated proteins and actin-binding proteins, including motor proteins, are thought to increase the complexity of these interactions. IFs are coupled to IF-anchoring plaques of cell–cell junctions (desmosomes) by desmoplakin, a prototype plaque molecule (plakin), and to those of cell–matrix junctions (hemidesmosomes) by plectin and BPAG1. The transmembrane proteins that mediate the contact with the neighbouring cells and with the extracellular matrix (ECM) are desmosomal cadherins and integrins, respectively. IFs are furthermore coupled to the outer nuclear membrane (ONM) by plectin and nesprin-3, whereas nesprin-2 anchors the MF system to the nucleus. On the inner side of the nuclear envelope, a layer of nuclear IF proteins (lamins) is attached to pores and inner nuclear membrane (INM) proteins as well as to chromatin. The membrane proteins of the INM might be linked to those of the ONM and thereby provide a mechanical continuum reaching from the ECM to chromatin. The number of newly identified INM and ONM proteins is increasing steadily and is represented here only in a schematic manner. ER, endoplasmic reticulum; MTOC, microtubule-organizing centre; NPC, nuclear pore complex.
outer nuclear membrane protein
inner nuclear membrane protein

57. Cell locomotion/ movement
Cellular level:
Ciliar movement
Amoeboid mocevent
Mesenchymal migration
Collective migration
Tissue level:
muscle movement – skeletal muscle

58. Cilia - flagellum

59. Amoeboid movement
chemoattractant

60. Composition of thick filament in a sarcomer

61. Composition of sarcomer
Working unit bordered by two Z-lines/disc
Z line – a-actinin + desmin
Thin filament – a-helical actin
molecules + tropomyosins + troponin
Thick filament – myosin II molecules
+ MBP (myosin binding proteins)
Other proteins: titin, distrophin
Troponin:
Tn-T binds tropomyosin
Tn-C binds Ca2+(4 Ca2+/mol = calmodulin)
Tn-I inhibitor

63. Skeletal muscle - Sarcomere

64. Skeletal muscle - Sarcoplasmic reticulum

65. X-linked recessive inheritance
Duschenne muscular distrophy

66. Useful videos and linkes: