1. Key Points in Constitution of Cytoskeleton Network 1.Polymerization of monomer 1.Regulation of assembly and disassembly 1.Formation of network by associated protein 1.Perform function by motor proteins

2. Cytoskeleton functions are difficult to assay due to 1.Formation of large protein complex 2.Force generation without chemical change

3. Components of cytoskeleton

4. Microtubule is made up of 13 protofilaments

5. Tubulin dimers constitute protofilament

6. Nucleation of microtubule is GTP and Mg++- dependent

7. A threshold concentration of tubulin (critical concentration, Cc) Is indispensibe for microtubule elongation

8. Flourescence– labeled microtubule demonstrates that microtubue is a dynamic structure

9. Dynamic instability of microtubule: consequence of delay hydrolysis of GTP after assembly

10. MTOC (microtubule organization center, centrosome)

11. Centrosome is the place within a cell where microtubule minus end is protected

12. Centrosome is located at peri-nuclear region

13. Centrosome is the center of a cell

14. Centrosome comprises centriole and pericentriole material

15. Structure of a centriole

16. Cilia: derivative of centrioles

17.  -tubulin is the major component of pericentriole material

18. Microtubule dynamic can be manipulated experimentally

20. Microtubule associated proteins (MAP) enhance microtubule stability

21. Microtubules interact with other cytoskeleton via MAPs

22. Pigment granules move along the microtubule tracks

23. Dynein and kinesin are microtubule motors

24. Identification and characterization of microtubule motors

25. Dynein and kinesin move cargos along opposite direction

26. Molecular structure of kinesin

28. A model for moving microtubule cargo through motor proteins

29. Intracellular organelles distribute along microtubule networks Golgi ER MT

31. Intermediate filaments occur only in multicellular organisms

32. Intermediate filaments are the bona fide cellular skeleton

34. Intermdiate filament proteins are made up of helical rod domain and non-helical domain

35. Intermediate filament is a non-polar structure and its basic structural unit is the staggered tetramer of two coiled coil dimers

36. Assembly of Intermediate filaments (also called 10 nm filaments) is dependent on rod domain aggregation

37. Nuclear lamin disassembly is dependent on phosphorylation of non-helical domain

38. Intermediate filaments function as intercellular bridge to link neighboring cells via desmosomes

39. Keratins are divided into 2 groups based on their pI

40. Expression of keratin pairs are tissue specific

41. Keratins are more amenable to deforming force than other cytoskeletal proteins

42. Keratins are the first cytoskeletal protein found to be related to human diseases

43. Numerous hot spots are found in human keratin genes

44. Keratin mutation affects its assembly and mechanical property

45. Methods to generate transgenic mice

46. Transgenic mice overexpressing k14 mutant display Epidermolysis bullosa Simplex (EBS) phenotype

47. Ultrastructure of epidermal cells displaying EBS

49. Actins occupy up to 5% total cellular protein

50. F actin is made up of G (globular) actins

51. Polymerization of actin is ATP and K +, Mg 2+ - dependent

52. Amount of actin within a cell is kept dynamically equilibrium

53. S1 myosin fragment can be used to detect actin threadmilling

54. Barbed end (plus end) of actin contains newly decorated actin

55. Profilin and thymosin compete with each other for actin monomer binding

57. Cytochalasin inhibits growth of actin fiber

58. Cellular structures made up of actin fibers

59. Cell cortex contains actin fibers arranged in different array and density

60. Stress fibers are not culture artifact

61. Actin binding proteins

62.  -actinin and fimbrin are bundling proteins

63. Filamin is an actin-binding gel forming protein

64. Myosins are actin motor proteins Figure 16 – 51 Myosin II. (A) A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long (green) and four light chains (blue). The light chains are of two distinct types, and one copy of each type is present on each myosin head. Dimerization occurs when the two a helices of the heavy chains wrap around each other to form a coiled- coil, driven by the association of regularly spaced hydrophobic amino acids (see Figure 3 – 11). The coiled-coil arrangement makes an extended rod in solution, and this part of the molecule is called the tail. (B) The two globular heads and the tail can be clearly seen in electron micrographs of myosin molecules shadowed with platinum. (B, courtesy of David Shotton.)

65. Mysoin derivatives

66. Actin moves along myosin

67. Myosin mutant mice displays defective heart Figure 16 – 75 Effect on the heart of a subtle mutation in cardiac myosin. Left, normal heart from a 6-day old mouse pup. Right, heart from a pup with a point mutation in both copies of its cardiac myosin gene, changing Arg 403 to Gln. Both atria are greatly enlarged (hypertrophic), and the mice die within a few weeks of birth. (From D. Fatkin et al., J. Clin. Invest. 103:147, 1999.)

68. Summary of actin binding proteins

72. Figure 16 – 50 The dramatic effects of Rac, Rho, and Cdc42 on actin organization in fibroblasts. In each case, the actin filaments have been labeled with fluorescent phalloidin, and focal contacts have been located with an antibody against vinculin. (A) Serum-starved fibroblasts have actin filaments primarily in the cortex, and relatively few focal contacts. (B) Microinjection of a constitutively activated form of Rho causes the rapid assembly of many prominent stress fibers and focal contacts. (C) Microinjection of a constitutively activated form of Rac, a closely related monomeric GTPase, causes the formation of an enormous lamellipodium that extends from the entire circumference of the cell. (D) Microinjection of a constitutively activated form of Cdc42, another Rho family member, causes the protrusion of many long filopodia at the cell periphery that form adhesive contacts with the substratum. The distinct global effects of these three GTPases on the organization of the actin cytoskeleton are mediated by the actions of dozens of other protein molecules that are regulated by the GTPases. Many of these target proteins resemble the various actin-associated proteins that we have discussed in this chapter. (From A. Hall, Science 279:509 – 514, 1998. © AAAS.)

75. Figure 16 – 90 A model for protrusion of the actin meshwork at the leading edge. Two time points during advance of the lamellipodium are illustrated, with newly assembled structures at the later time point shown in a lighter color. Nucleation is mediated by the ARP complex at the front. Newly nucleated actin filaments are attached to the sides of preexisting filaments, primarily at a 70° angle. Filaments elongate, pushing the plasma membrane forward because of some sort of anchorage of the array behind. At a steady rate, actin filament plus ends become capped. After newly polymerized actin subunits hydrolyze their bound ATP in the filament lattice, the filaments become susceptible to depolymerization by cofilin. This cycle causes a spatial separation between net filament assembly at the front and net filament disassembly at the rear, so that the actin filament network as a whole can move forward, even though the individual filaments within it remain stationary with respect to the substratum.