2. Cytoskeleton System Xiamixinuer · Yilike Chapter 8

3. Teaching Requirements: 1. Mastering: concepts of the cytoskeleton; structure, chemical composition, and assembly of microtubules and microfilaments. 2. Comprehending: functions of microtubules and microfilaments. 3. Understanding: functions of the cytoskeleton; types and functions of intermediate filaments. 2

4. 3 The cytoskeleton

5. 4 1. Introduction A. Conception of Cytoskeleton (Narrow sense) A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol.

6. 5 The comparison among three types of the cytoskeleton

7. 6 B. Techniques for studying the cytoskeleton  Fluorescent microscopy and Electron microscopy : Immunofluorescence: fluorescently-labeled antibody Fluorescence: microinject into living cells Video microscopy: in vitro motility assays Electron: Triton X-100, Metal replica  Drugs and mutations (about functions)  Biochemical analysis(in vitro)

8. 7 Fluorescence microscopy actin microtubules filamin microfilaments cytoskeleton microtubule s

9. 8 C. The self-assembly and dynamic structure of cytoskeletal filaments  Each type of cytoskeletal filament is constructed from smaller protein subunits.  The cytoskeleton is a network of three filamentous structures.  The cytoskeleton is a dynamic structure with many roles.

10. 9 D. The function of the cytoskeleton Structural support Internal framework maintaining position of the organelles Machinery required for movement of materials and organelles within cells Force generating elements responsible for movement of cells from one place to another

11. 10 2.Microtubule, MT A. Structures: Hollow Tubular structures 25nm in diameter Assembled from protein tubulin The tubulin consists of alpha-beta tubulin heterodimers arranged in rows (protofilaments) Form cytoskeleton, mitotic spindle, centrioles, core of cilia and flagella

12. 11 a and ßTubulin heterodimers are the protein building blocks of MTs

13. 12 Arrangement of protofilaments in singlet, doublet, and triplet MTs SingletDoubletTriplet A B A B C In cilia and flagella In centrioles and basal bodies

14. 13              Assembling process of MT   + -                         PEDAL                                                                                                                                                                                                                                         OUTSIDE OF THE BODY

15. 14                    tubulin  tubulin heterodimer assemble Head tail connection profila ment MT （ 13 ） 1 23 4 5 6 7 89 10 11 12 13 CROSSSECTION      

16. 15 B. MTs assemble from microtubule- organizing centers (MTOCs) Microtubule-organizing centers (MTOCs) :is the region to assemble MT,Where includes  - tubulin. MTOCs:include Centrosome, Mitotic spindle and Basal body.

17. 16 Microtubule-organizing centers (MTOCs) (1) Interphase: Centrosome Dynamic instability (2) Dividing cell: Mitotic spindle Dynamic instability (3) Ciliated cell: Basal body Stability

18. 17 Centrosome is a microtubule organizing center, MTOCs

19. 18 Centrosome containing a pair of centrioles

20. 19 Centrioles Centrioles are short cylinders with a 9 + 0 pattern of microtubule triplets. Centrioles may be involved in microtubule formation and disassembly during cell division and in the organization of cilia and flagella.

21. 20

22. 21  MT are nucleated by a protein complex containing  -tubulin The centrosome is the major MTOC of animal cells

23. 22  Experiments supporting that centrosome is the MTOC Treat cell with colcemid Cytosolic MTs depoly, except those in centrosome Remove colcemid Tublin repoly Expla I: MTOC nucleate poly of tubulins Expla II: MTOC gather MTs in cytosol centrosome + TubulinsMT + TubulinsNo A B  Why the centrosome can act as MTOC?

24. 23 Cilia and flagella Cilia (small and numerous) and flagella (large and single) have a 9 + 2 pattern of microtubules and are involved in cell movement. Cilia and flagella move when the microtubule doublets slide past one another. Each cilium and flagellum has a basal body at its base.

25. 24

26. 25 Basal body structure

27. 26 C. Characteristics of MT assembly Dynamic instability due to the structural differences between a growing and a shrinking microtubule end.  GTP cap;  Catastrophe: accidental loss of GTP cap;  Rescue: regain of GTP cap

28. 27 Microtubules have a plus and minus ends. Typically the minus is for anchoring and the plus is for growing. The transition between MT growth and MT shrinking is controlled in cells by special proteins..

29. 28  Drugs affect the assembly of MTs (1) Colchicine Binding to tubulin dimers, prevent MTs polymerization (2) Taxol Binding to MTs, stabilize MTs These compounds are called antimitotic drugs, and have application in medical practice as anticancer drugs

30. 29 D. Microtuble-associated proteins (MAPs) MAPs modulate MT structure, assembly, and function Katanin like proteinsMAPs Tau: In axon, cause MTs to form tight bundles MAP2: In dendrites, cause MTs to form looser bundles MAP1B: In both axons and dendrites to form crossbridge between microtubules Control organization

31. 30 MAP2 associated with brain MTs

32. 5. Functions of MTs A. Maintenance of cell shape(constitute the centriols and cilia or flagella). B. Cell motility (see in cilia or flagella). C. Chromosome movements in cell division D. Organelle movement (MT associated motor proteins: kinesins: towards + end (anterograde transport) Golgi to ER or PM traffic;dyneins: towards - end (retrograde transport) ER to Golgi traffic.) 31

33. 32 5. Functions of MTs A. Maintenance of cell shape(constitute the centriols and cilia or flagella).

34. 33 No centrioles in Plant and fungi A pair of centrioles are surrounded by electron dense pericentriolar material. Centrioles contain nine evenly spaced fibrils, each containing three microtubules, A, B and C tubules. A tubule is connected to the center of the centriole by a radial spoke. Centrioles are in pairs and at right angles to each other. Structure Constitute the centriols and cilia or flagella

35. 34 Constitute the centriols and cilia or flagella

36. 5. Functions of MTs B. Cell motility (see in cilia or flagella). 35

37. 36 A comparison of the beating of flagella and cilia

38. 37 Ultrastructure of a eukaryotic flagellum or cilium

39. 38 SPERM MOVEMENT CILIA MOVEMENT

40. 39 Motor molecules and the cytoskeleton

41. 40 Motor molecules and the organelle

42. 41 Dyenin arms responsible for sliding Crosslinks and spokes responsible for bending

43. 42 B. Transport in the cytoplasm MT associated motor proteins: kinesins: towards + end (anterograde transport) Golgi to ER or PM traffic dyneins: towards - end (retrograde transport) ER to Golgi traffic

44. 43 C. Movement of chromosomes

45. 44 3. Microfilament, MF A. MFs are made of actin and involved in cell motility.  Using ATP, G-actin polymerizes to form MF(F-actin)

46. 45 G-actin F-actin PLUS END MINUS END

47. 46 G-actinDimerTrimer F-actin +end -end Assembly of MF

48. 47 B. MF assembly and disassembly  Characteristics: (1) Within a MF, all the actin monomers are oriented in the same direction, so MF has a polarity Myosin is molecular motor for actins.

49. 48 (2) In vitro, (Polymerization) both ends of the MF grow, but the plus end faster than the minus. Because actin monomers tend to add to a filament’s plus end and leave from its minus end---- “Tread-milling”

50. 49 (3) Dynamic equilibrium between the G-actin and polymeric forms, which is regulated by ATP hydrolysis and G-actin concentration.

51. 50 (4) Dynamic equilibrium is required for the cell functions. Some MFs are temporary and others permanent.

52. 51 C. Specific drugs affect polymer dynamics Cytochalasins: Prevent the addition of new monomers to existing MFs, which eventually depolymerize. Phalloidin: A cyclic peptide from the death cap fungus, blocks the depolymerization of MF Those drugs disrupt the monomer-polymer equilibrium, so are poisonous to cells

53. 52 D. Actin-binding proteins The structures and functions of cytoskeleton are mainly controlled by its binding proteins (1) Monomer-sequestering proteins Bind with actin monomers and prevent them from polymerizing. thymosin and ( profilin) Promoting the assembly of MF

54. 53 (2) MF-binding proteins

55. Functions of MF (1) Maintenance of cell shape and enforce PM to change cell shape( i.e.Microvillus: Support the projecting membrane of intestinal epithelial cells) (2) Cell migration or motility (as in pseudopodia) (3) To form contractile ring in cell division: At cytokinesis (4) Muscle contraction ： Sarcomere is the unit of the muscle cells. (5)Cytoplasm streaming 54

56. Functions of MF (1) Maintenance of cell shape and enforce PM to change cell shape( i.e.Microvillus: Support the projecting membrane of intestinal epithelial cells) 55

57. 56 Microvillus: Support the projecting membrane of intestinal epithelial cells

58. 57 A structure role of microfilaments Microfilaments (Actin filaments) Microvilli Intermediate filaments

59. 58 E. Functions of MFs (2) Cell migration (Fibroblast et al)  Platelet activation is a controlled sequence of actin filament severing,uncapping, elongation,recapping, and cross-linking.

60. 59 (3) To form contractile ring in cell division : At cytokinesis E. Functions of MFs

61. 60 E.Functions of MFs (4) Muscle contraction  Organization of skeletal muscle tissue

62. 61  Sarcomere

63. 5)Cytoplasm streaming:The streaming of cytoplasm in a circular motion around the cell observed in some plants, particularly young sieve tube elements. 62

64. 63  Proteins play important roles in muscle contraction Myosin: The actin motor protein ATPase Binding sites Myosin II--Dimer Mainly in muscle cells Thick filamemts

65. 64 Tropomyosin, Tm and Tropnin, Tn Ropelike molecule Regulate MF to bind to the head of myosin Complex, Ca 2+ -subunit Control the position of Tm on the surface of MF

66. 65  Excitation- contraction coupling process

67. 66 Intermediate filaments, IFs IFs are the most abundant and stable components of the cytoskeleton

68. 67 Class I. + II. (MW 40 - 70 000) Cytokeratins epithelial cells ( > 20 isoforms, skin, hair, nails) Class III. (MW ~53 000) Vimentin cells of mesenchymal origin Desmin muscle GFAPs astroglial cells (= Glial fibrillary acidic proteins) Class IV. (MW 130, 100 and 60 000) Neurofilament proteins in neural cells Class V. (MW = 65-75 000) Nuclear lamins inside surface of the inner nuclear membrane most dynamic INTERMEDIATE FILAMENT PROTEIN MONOMERS: (cell-type-specific) (~ 310 amino acids)

69. 68 monomer coiled-coil dimer staggered anti-parallel tetramer two tetramers helical array of tetramers made of 8 protofilaments

70. Functions of IF 1.Maintenance of cell shape, 2.Anchore of nucleus and certain other organelles, 3.Formation of nuclear lamina 69

71. 70 The comparison among three types of the cytoskeleton

72. 71 Summary of cytoskeleton Three types of cytoskeletal filaments are common to many eucaryotic cells and are fundamental to the spatial organization of these cells. Three types of cytoskeletal filaments are common to many eucaryotic cells and are fundamental to the spatial organization of these cells. The set of accessory proteins is essential for the controlled assembly of the cytoskeletal filaments(includes the motor proteins: myosins, dynein and kinesin) The set of accessory proteins is essential for the controlled assembly of the cytoskeletal filaments(includes the motor proteins: myosins, dynein and kinesin) Cytoskeletal systems are dynamic and adaptable. Cytoskeletal systems are dynamic and adaptable. Nucleation is rate-limiting step in the formation of a cytoskeletal polymer. Nucleation is rate-limiting step in the formation of a cytoskeletal polymer. Regulation of the dynamic behavior and assembly of the cytoskeletal filaments allows eucaryotic cells to build an enormous range of structures from the three basic filaments systems. Regulation of the dynamic behavior and assembly of the cytoskeletal filaments allows eucaryotic cells to build an enormous range of structures from the three basic filaments systems.

73. Homework for cytoskeleton system 1.Conception types and the functions of the cytoskeleton 2.Structures of MT 3.building blocks of MTs and MFs 4.Arrangement of protofilaments 5.MTOC and its elements 6.Specific drugs stabilize MTs or MF 7.Functions of MTs and MFs 8.Cytoskeletal systems are dynamic and adaptable. Nucleation is rate-limiting step in the formation of a cytoskeletal polymer.Regulation of the dynamic behavior and assembly of the cytoskeletal filaments allows eucaryotic cells to build an enormous range of structures from the three basic filaments systems. 72