1. Cytoskeleton System Chapter 9 1. Introduction
A. Conception of Cytoskeleton (Narrow sense)
A complex network of interconnected microfilaments, microtubules and intermediate filaments that extends throughout the cytosol.
Microbubules
Microfilamemts
Intermediate filaments

2. 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
Quick freezing-deep etching EM
Biochemical analysis (in vitro)
Difference centrifugation; SDS-PAGE
Drugs and mutations (about functions)
SSDS

3. 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 strucrure with many roles.

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

5. 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.

6. (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----

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

8. 2.2 Assembly
◆ Mechanism of actin polymerization: 3 phases of G-actin polymerization.
◆ Critical concentration (Cc). In steady state, G-actin monomers only exchange with subunits at the filament ends but there is no net change in the total mass of filaments.
◆ During the elongation state, one end of the filament, the (+) end, elongates five to ten times faster than does the opposite (-) end. This is because Cc value is much lower for G-actin addition at the (+) end than for addition at the (-) end.

9. Figure 6-17 The three phases of G-actin polymerization in vitro.

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

11. (5)The nucleation of actin filaments at the PM is frequently regulated by external signals, allowing the cell to change its shape and stiffness rapidly in response to changes in its external environment.
This nucleation is catalyzed by a complex of proteins that includes two actin-related proteins, or ARPs(Arp2 and Arp3).

12. Actin arrays in a cell.

13. 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

14. D. Actin-binding proteins
The structures and functions of cytoskeleton are mainly controlled by its binding proteins

15. 2.4 microfilament-binding proteins
Actin binding proteins control the structure and behavior of actin filament.
◆ actin binding proteins
e.g. proflin (promote acting assembly), thymosin beta4 (inhibits actin assembly). Some cytosolic proteins control actin polymerization.
◆ microfilament-binding proteins
◆ 3 different types of stalbe actin filament structures：
◆ Parallel bundle: MFs isotactic parallel arrange，mainly found in microvillus and filopodium (丝状伪足).
◆ Contractile bundle: MFs anti-parallel arrange, mainly found in stress fibers (应力纤维) and contractive ring of mitosis(有丝分裂收缩环 )。
◆ Gel-like network: MFs cross-linked arrange, most be found in cell cortex (cytosol, 细胞皮层).

16. (2) MF-binding proteins

17. 成核蛋白（nucleating proteins）: actin-related proteins, ARPs
单体–隔离蛋白（monomer-sequestering protein）: thymosin
封端（加帽）蛋白（End-blocking(capping) proteins）:capZ
单体–聚合蛋白（monomer-polymerizing proteins）:抑制蛋白（profilin）是一种与ATP-肌动蛋白单体结合的蛋白质
肌动蛋白纤维解聚蛋白（actin filament-depolymerizing proteins）: cofilin、ADF以及蚕食蛋白与肌动蛋白纤维的减端结合，大大促进肌动蛋白纤维解聚成单体。
交联蛋白（cross-linking proteins）: ABP280和细丝蛋白,促进形成近于正交相互联系的纤维松散网络
纤维–切割蛋白（filament-severing proteins）: gelsolin
膜结合蛋白（membrane-binding proteins）:连接膜与肌动蛋白的蛋白质包括联结蛋白（vinculin），ERM家族的成员包括埃兹蛋白（ezrin）、根蛋白（radixin）和膜突蛋白（moesin）

18. Model of the complementary roles of profilin and thymosin β4 in regulating polymerization of G-actin.

19. Actin filaments are likewise strongly affected by the binding of accessory proteins along their sides.
Actin filaments in most cells are stabilized by the binding of tropomyosin, an elongated protein. Which can prevent the filament from interacting with other proteins.
Another important actin filament binding protein, cofilin, present in all eucaryotic cells, which destabilized actin filaments(also called actin depolymerizing factor).
Cofilin binds along the length of the actin filament, forcing the filament to twist a little more tightly.
In addition, cofilin binding cause a large increase in the rate of actin filament treadmilling.

20. The modular structures of four actin-cross-linking proteins
The formation of two types of actin filament bundles:
Contractile bundle mediated by α-actinin
parallel bundle mediated by fimbrin.

21. Actin filaments are often nucleated at the plasma membrane
Actin filaments are often nucleated at the plasma membrane. The highest density of actin filaments is at the cell periphery forming cell cortex.
Gel-like network
Filamin cross-links actin filaments into a three-dimensional network with the physical properties of a gel.
Loss of filamin causes abnormal cell motility

22. E. Functions of MFs
(1) Maintain cell ’ s shape and enforce PM

23. (2) Cell migration (Fibroblast et al)
Platelet activation is a controlled sequence of actin filament severing,uncapping, elongation,recapping, and cross-linking.

24. (3) Microvillus: Support the projecting membrane of intestinal epithelial cells

26. (4) Stress fibers Composed of actin filaments and myosin-II
Focal contacts MFs
Focal contacts
Stress Fibers
Response to tension
Response to tension

27. (5) Contractile ring: For cytokinesis

28. (6) Muscle contraction
Organization of skeletal muscle tissue

29. Sarcomere

30. 皮肤 ?蛋白
平原皮肤球 ??蛋白
同伴皮肤 ?蛋白

31. Proteins play important roles in muscle contraction
Myosin: The actin motor portein
Myosin II--Dimer
ATPase
Mainly in muscle cells
Thick filamemts

32. Light-chain phosphorylation and the regulation of the assembly of myosin II into thick filaments

33. Tropomyosin, Tm and Tropnin, Tn
Ropelike molecule
Complex, Ca2+-subunit
Regulate MF to bind to the head of myosin
Control the position of Tm on the surface of MF

34. Thick and thin filaments sliding model

35. Excitation-contraction coupling process
Action potential
Ca2+ rise in cytosol Tn Tm
Sliding

36. Schematic diagram showing how a Ca2+-release channel in the sarcoplasmic reticulum membrane is thought to be opened by a voltage-sensitive transmembrane protein in the adjacent T-tubule membrane

37. F. Smooth muscle cell(平滑肌细胞 ) contraction
Smooth and nonmuscle cell contraction are regulated in a manner distinct from that of skeletal muscle cells
Bind to MLCK
Ca2+ rise
Ca2+ -calmodulin
Regulate light chain Phosphorylate
Myosin interact with actin
Contraction
SLOW

38. 3.Microtubule, MT A. Structures: Tubulin heterodimers
are the protein building
blocks of MTs
A. Structures:

39. Arrangement of protofilaments in singlet, double, and triplet MTsC
In cilia and flagella
In centrioles and basal bodies

40. B. MTs assemble from microtubule-organizing centers (MTOCs)
(1) Interphase: Centrosome
Dynamic instability
(2) Dividing cell:
Mitotic spindle
Dynamic instability
(3) Ciliated cell: Basal body
Stability

41. Basal body structure

42. 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

43. Why the centrosome can act as MTOC
Structure
No centrioles in Plant and fungi

44. MT are nucleated by a protein complex containing ?-tubulin
The centrosome is the major MTOC of animal cells

45. 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

46. Microtuble-associated proteins (MAPs)
MAPs modulate MT structure, assembly, and function
Control organization
Motor MAPs
Nonmotor MAPs
Tau: In axon, cause MTs to form tight bundles
MAP2: In dendrites, cause MTs to form looser bundles

47. The importance of MAPs for neurite formation
Like dendrite
Like axon

48. Organization of MT bundles by MAPs . Spacing of MTs depends on MAPs
Insect cell expressing MAP2
Insect cell expressing tau
From J. Chen et al Nature 360: 674

49. The effects of proteins that bind to MT ends
(A)The transition between Mt growth and Mt shrinking is controlled in cells by special proteins.
??蛋白
(B)Capping proteins help to localize Mt in budding yeast cell.

50. 5. Functions of MTs
1. Maintain cell shape

51. 2. Motor proteins and intracellular transport
（1）Motor proteins: 3 superfamily
Kinesin dependent-MT (2KHC+2KLC),
N-/M-Kinesin向正极运动；C-kinesin向负极运动
头部具MT 和ATP结合位点；迄今已报道600余种
Cytoplasmic Dynein dependent-MT (2/3HC+ more LC)
14um/s ; C端为马达结构域（结合ATP）;N端为易变尾部
Axonemal arm dyneins: 与纤毛鞭毛运动有关
Myosin dependent-MF

52. (2) Kinesin is a plus-end directed MT motor protein
Intracellular transport of membrane-bounded vesicles, proteins: Directionality
(2) Kinesin is a plus-end directed MT motor protein
1985年发现的驱动蛋白分子--“常规驱动蛋白”（conventional kinesin），只是相关蛋白超家族中的一个成员，驱动蛋白-相关蛋白（kinesin-related proteins）超家族简称为KRPs或KLPs（kinesin-like proteins）。根据基因组序列分析，估计哺乳动物能产生50种以上不同的KLPs。KLPs的头部都含有相关的氨基酸序列，反映出它们具有共同的进化祖先，并且在沿微管运动方面具有相似的作用。比较而言，KLPs的尾部有较大变异，具有各种不同的序列，反映出不同的驱动蛋白成员运送不同的货物。
Hand-over-hand model

53. Comparison of the mechanochemical cycles of kinesin and myosin II.
??星期三解释的 1 分子 ATP, 先前 ?8 nm, 相当于 ?秒 1 um.
Motor proteins generate force by coupling ATP hydrolysis to conformational changes.

54. (3) Dynein is a minus-end directed motor protein
Axonemal and cytoplasimic dyneins
MW=1500KDa
胞质动力蛋白至少有两种作用：
1. 在有丝分裂期间作为纺锤体定位和染色体移动的产力装置 。
2. 作为在胞质中定位高尔基复合体和将小泡和细胞器移向减端的微管马达蛋白。
Mediate: 动力蛋白激活蛋白复合体 （dynactin complex）

55. Intracellular transport in nerve cells
Mt organization in fibroblasts and neurons.

56. Movement of pigment granules: color adjustment

57. The placement of organelles

58. 3. Movement of mitotic spindle and chromosomes

59. 4. Cilia and flagella: Structure and movement
Size and length: The same diameter, flagella
are often much longer
Movement: Cilia: Beating;
Flagella: Bending motion
Ciliary dynein

60. Structure:

61. Microtubule sliding causes cilia/flagella to bend
Dyneins
Crosslinks and spokes

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

63. 1. IFs assemble from fibrous subunits

64. Assembly Characteristics of IFs
Monomers: Fibrous proteins
Antiparallel tetramer: No polarity
Almost no IF monomers within cell
But IFs are still dynamic polymers in the cell
IF typing serves as a diagnostic tool in medicine

65. 2. IF proteins are tissue-specific

66. 3. Function of IFs: Confer mechanical strength on tissues
Disruption of keratin networks causes blistering

67. IFs are cross-linked and bundled into strong arrays;
IFs are ropelike fibers with a diameter of around 10nm;
IFs are made of IF proteins, which constitute a large and heterogeneous family.
Less is understood about the mechanism of assembly and disassembly of IFs than of actin filaments and microtubules, but they are clearly highly dynamic structures in most cell types.

68. Summary: Cytoskeletal functions

69. Summary of cytoskeleton
1. 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)
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.