1. B. The Cytoskeletal System and Adhesion Red = Actin Microfilaments Green = Tubulin Microtubules Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings

2. 3 Fibrous Components and Motors Thin Filaments: Actin family of proteins Microtubules: Tubulin family of proteins Intermediate Filaments: Very cell-specific family of proteins The first two have molecular motors

3. 1. Actin Microfilaments Chains of few to many 42kDa monomers Found in all eukaryotic cells (except nematode worm sperm) Highly Conserved: Genes vary <20% from algae to humans; human primary protein sequence <5% Three main isoforms: alpha, beta, gamma Alpha is muscle actin, beta and gamma are found in nearly all cells

4. Figure 16-12 Molecular Biology of the Cell (© Garland Science 2008) ATP Binding ADP Binding

5. Figure 16-74c,d Molecular Biology of the Cell (© Garland Science 2008)

6. Figure 16-47 Molecular Biology of the Cell (© Garland Science 2008)

7. Fig. 6-26 Microvillus Plasma membrane Microfilaments (actin filaments) Intermediate filaments 0.25 µm

8. Figure 16-69a Molecular Biology of the Cell (© Garland Science 2008) Actin tracks allow specific movement between two points

9. Figure 17-49a Molecular Biology of the Cell (© Garland Science 2008) Remember.....

10. The Key Structural Elements 1. 70 Å filaments (f-actin) - polymers of globular monomers (g-actin) formed as double helix 2. Structural polarity –sequences and structures unequally directed towards each of the ends

11. Figure 16-7 Molecular Biology of the Cell (© Garland Science 2008) “Dynamic instability” a. Can form, crosslink, breakdown and reform rapidly b. The cell can build new structures as its needs change

12. 2. Tubulin Microtubules Large hollow tubes of many 110kDa dimers Three main isoforms: alpha, beta, gamma Found in all eukaryotic cells Alpha and beta found in microtubules, gamma found in microtubule organizing center (MTOC) Highly Conserved: Primary sequences vary <10% from algae to humans

13. Figure 16-11 Molecular Biology of the Cell (© Garland Science 2008) GTP BindingGDP Binding

14. Figure 16-30c Molecular Biology of the Cell (© Garland Science 2008) Large-scale, long distance movement of cellular components, including organelles

15. Fig. 6-22 Centrosome Microtubule Centrioles 0.25 µm Longitudinal section of one centriole Microtubules Cross section of the other centriole

16. Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008)

17. Figure 16-85c Molecular Biology of the Cell (© Garland Science 2008) Projections of asters to move the chromosomes in cell division

18. Figure 16-104 Molecular Biology of the Cell (© Garland Science 2008) Directed transport along the neuronal axon

19. Cilia and flagella

21. Give direction to plant cell division and growth and dictate sites of plant wall synthesis

22. Plant microtubules have no obvious MTOC

23. The Key Structural Elements 1. These are also polar – unequally directed towards each end a. most: (–) end toward nucleus and (+) end to cell membrane 2. 250 Å filaments formed as a hollow bundle of 13 protofilaments a. protofilaments are long polymers of tubulin dimers b. dimers are one alpha and one beta globular monomers c. gamma tubulin makes up the MTOC: centriole or basal body

24. Figure 16-16c Molecular Biology of the Cell (© Garland Science 2008) Like actin, microtubules can be changed as the cells needs change

25. 3. Intermediate Filaments Rope-like filament structures formed from 8 protofilaments twisted together Lamins found in nucleus, cytosolic IF’s found in some cells and not others IF’s found only in some animals, including vertebrates, nematodes, molluscs Plants and single-celled organisms have no IF’s

26. Figure 16-19 Molecular Biology of the Cell (© Garland Science 2008)

27. Figure 16-20 Molecular Biology of the Cell (© Garland Science 2008) Keratins in the cytosol

28. Lamins in the Nucleus

29. Sarcomere Support

30. The Key Structural Elements 1. Apolar – very stable, not structurally nor functionally oriented towards one end or the other 2. No motors

31. 4. Bacterial Homologs and Analogs Homologs are genes and proteins that share common sequence Analogs are proteins that share common function

32. Figure 16-26 Molecular Biology of the Cell (© Garland Science 2008) Actin homologs: MreB and Mbl Helical proteins give cell its shape by distributing themselves throughout the cell and directing sites of cell wall synthesis. Mbl+Mbl- Analogous to plant tubulin!

33. Figure 16-27a Molecular Biology of the Cell (© Garland Science 2008) Actin homolog: ParM Analogous to tubulin!

34. Figure 16-25a Molecular Biology of the Cell (© Garland Science 2008) Tubulin homolog: FtsZ As a bacterium divides: after the two chromosome copies have been segregated, the Z-ring forms and then contracts to split the daughter cells. Two rings reform in the daughters. This is analogous to actin!

35. Figure 16-28 Molecular Biology of the Cell (© Garland Science 2008) Intermediate filament homolog: Crescentin Creates the curved shape by forming a cortical band

36. b. Molecular motors allow filament- based intracellular transport The three basic types 1. Myosins: move along microfilaments towards the positive end (mostly), Some myosin gene products move toward negative end 2. Kinesins: move along microtubules towards the positive end (away from the MTOC) 3. Dyneins: move along microtubules towards the negative end (toward the MTOC)

37. Figure 16-54a Molecular Biology of the Cell (© Garland Science 2008) Globular heads on fibrous tails: a. Heads have ATPase activity, dictate filament track, direction and speed b. Tails have “cargo” binding and myosin thick filament organizing activity Molecular Motors are ATPases that Couple Hydrolysis with Movement

38. Kinesins and Myosins are Evolutionarily Related 1. Both have single head (type I ) and double head (type II) designs 2. Little sequence similarity but remarkable similarities in tertiary structures Dyneins are Something Else 1. They are an ancient family of proteins found in all eukaryotes a. Cytosolic dyneins move most structures, ciliar dyneins move cilia and flagella 2. Generally similar structure to the others – can have trimer heads

39. Figure 16-67 Molecular Biology of the Cell (© Garland Science 2008) The motor cargo complex grabs onto meshwork and transmembrane proteins.

40. “Power Stroke” mechanism to couple movement to ATP metabolism 1. At the end of one cycle the motor is bound to actin at 45 o 2. ATP bindingFilament Release 3. ATP HydrolysisConformational Relaxation to 90 o 4. Release of P i Filament Binding 5. Release of ADP Conformational Stroke to 45 o 6. The shift in the motor back to 45 o moves the cargo forward

41. 2. Regulation of Cell Function via Control of the Cytoskeleton

42. a. Actin filaments are assembled and disassembled rapidly and thus are able to help regulate long-term activities, change and movement Assembly of specific actin structures and cellular functions is dependent on the expression and activity of microfilament associated proteins (MFAP’s) in specific cellular locations

43. Figure 16-10 Molecular Biology of the Cell (© Garland Science 2008) Actin fiber assembly is spontaneous if only actin and ATP are present and the bonds between subunits are non-covalent Nucleation is the rate-limiting step in assembly and the first control

44. Figure 16-34c Molecular Biology of the Cell (© Garland Science 2008) To make a dense actin network, or gel, the cell must express and activate ARP complexes at that site Activating factor can follow internal or external signals

45. Figure 16-36 Molecular Biology of the Cell (© Garland Science 2008) Formin facilitates nucleation and causes rapid extension Expressed with ARP complexes, leads to very rapid assemblage and branching

46. Thymosin and Profilin Regulate Speed

47. Figure 16-43 Molecular Biology of the Cell (© Garland Science 2008) Caps mean slow growth and stabilized fibers

48. Figure 16-49a Molecular Biology of the Cell (© Garland Science 2008) Lateral association of double helical fibers by bundling proteins

49. Figure 16-51 Molecular Biology of the Cell (© Garland Science 2008) Stabilization of gel structures

50. Figure 16-50a Molecular Biology of the Cell (© Garland Science 2008) Microvilli assemblage

51. Many changing cell functions are regulated by actin fiber reorganization, which requires coordinated disassembly of existing fibers followed by nucleation and polymerization at a new location

52. Rapid disassembly of fibers SeveringDepolymerization

53. Combine disassembly with localized assembly Profilin promotes ATP exchange and ARP nucleation

54. Figure 16-90 Molecular Biology of the Cell (© Garland Science 2008)

55. Lamellipodia

56. Assembly of the contractile ring followed by severing is required in cytokinesis

57. Figure 13-46 Molecular Biology of the Cell (© Garland Science 2008) Pseudopod-Driven Phagocytosis in Neutrophils

58. Figure 13-47a Molecular Biology of the Cell (© Garland Science 2008) Actin polymerization is required to extend pseudopods Actin depolymerization is required to seal off the phagosome

59. Regulation of the active actin structure is associated with known Rho-family signaling pathways stress fibers microspikes or filopodia lamellipodia

60. Figure 16-98b Molecular Biology of the Cell (© Garland Science 2008)

61. Figure 16-98a Molecular Biology of the Cell (© Garland Science 2008)

62. b. Microtubules are also assembled and disassembled rapidly and are used to regulate movement and positioning of materials, including the cell

63. Figure 16-30a Molecular Biology of the Cell (© Garland Science 2008)  -Tubulin ring complex (  -TuRC) nucleates assembly from the MTOC and remains associated with the minus end

64. Figure 16-30b Molecular Biology of the Cell (© Garland Science 2008) A high concentration of free tubulin subunits is required for assembly and elongation

65. Figure 16-41 Molecular Biology of the Cell (© Garland Science 2008) MAP2, Tau and Plus-end tracking proteins (TIPs) bind and stabilize microtubules

66. Figure 16-45a Molecular Biology of the Cell (© Garland Science 2008) Green = MT Red = +Tip protein Long-term stable structures

67. Expression of stathmin protein will bind free tubulin subunits and inhibit assembly and elongation Each stathmin protein binds two dimers Phosphorylation of stathmin will inactivate it

68. Microtubule Severing

69. Figure 16-44 Molecular Biology of the Cell (© Garland Science 2008)

70. Disassembly and Reassembly in Mitosis

71. Figure 16-19 Molecular Biology of the Cell (© Garland Science 2008) c. Intermediate filaments are the most stable element in the cytoskeleton but their structure can be regulated to facilitate specific cell activities Remember....

72. The aminoterminal head and carboxyterminal tail appear to be key points of regulatory control

73. Glycosylation and phosphorylation of intermediate filaments. Hyder C L et al. J Cell Sci 2011;124:1363-1372 ©2011 by The Company of Biologists Ltd

74. Disassembly of nucleus in mitosis

75. Nuclear Envelope Fragmentation

76. CELLULAR ADHESION

77. Figure 19-46 Molecular Biology of the Cell (© Garland Science 2008) Hemidesmosomes bind intermediate filaments to anchor proteins to integrins to ECM fibrous proteins Very stable

78. Figure 19-45 Molecular Biology of the Cell (© Garland Science 2008) Focal adhesions bind microfilaments to anchor proteins to integrins to ECM fibrous proteins Can be stable or used for migration

79. Figure 19-48b Molecular Biology of the Cell (© Garland Science 2008) Internal binding and external binding are linked

80. Figure 19-17a Molecular Biology of the Cell (© Garland Science 2008) Very stable Desmosomes bind intermediate filaments to anchor proteins to their own cadherins which bind to the adjacent cell’s cadherins

81. Adherens junctions bind microfilaments to anchor proteins to their own cadherins which bind to the adjacent cell’s cadherins

82. Figure 19-7 Molecular Biology of the Cell (© Garland Science 2008) A fairly large protein family, all have homophillic binding and are Ca ++ -dependent

83. Figure 19-24 Molecular Biology of the Cell (© Garland Science 2008) Tight Junctions can block molecules as small as ions from passing between cells

84. Figure 19-26a Molecular Biology of the Cell (© Garland Science 2008)

85. When there is an infection or inflammation, endothelial cells will express selectins on their surface that specifically bind WBC. WBC also express integrins that bind strongly, stop their rolling and aid in escape

86. Figure 19-20 Molecular Biology of the Cell (© Garland Science 2008) NCAM is found on a variety of cell types and mediates homo- philic binding. Ig superfamily cell adhesion molecules ICAM is found on endothelial cells and binds heterophilically to integrins on white blood cells.

87. This is a large gene family