1. Chapter 15
Cytoskeletal Systems

2. Cytoskeletal Systems The interior of a cell is highly structured
The cytoskeleton is a network of interconnected filaments and tubules extending through the cytosol
It plays roles in cell movement and division
It is dynamic and changeable 2

3. Major Structural Elements of the Cytoskeleton
The major structural elements of the cytoskeleton are
Microtubules
Microfilaments
Intermediate filaments

4. Table Microtubules

5. Table 15-1 - Microfilaments

6. Table 15-1 – Intermediate Filaments

7. Eukaryotes Have Three Basic Types of Cytoskeletal Elements
Indirect immunostaining has been used to characterize cytoskeletal elements
Microtubules are composed of tubulin subunits and are about 25 nm in diameter
Micofilaments, 7 nm wide, are composed of actin subunits
Intermediate filaments, 8–12 nm, are variable in composition 7

8. Bacteria Have Cytoskeletal Systems That Are Structurally Similar to Those in Eukaryotes
Recent discoveries show that Bacteria and Archaea have polymer systems that function similarly to eukaryotic cytoskeletal elements
The actin-like MreB protein is involved in DNA segregation
The tubulin-like FtsZ protein is involved in regulating division
Crescentin is a regulator of cell shape 8

9. Figure 15-1a-d

10. Figure 15-1e

11. The Cytoskeleton Is Dynamically Assembled and Disassembled
Microfilaments are essential components of muscle fibrils and microtubules are structural elements of cilia and flagella
These structures are large enough to view by a variety of microscopic techniques
Also, certain drugs can be used to perturb cytoskeletal function

12. Dynamic cytoskeleton
Research has shown that the cytoskeleton is dynamically assembled and disassembled
It includes some remarkably elaborate structures

13. Table 15-2 – first two rows . 13

14. Table 15-2 – remaining two rows. 14

15. Table 15-3

16. Microtubules
Microtubules are the largest of the cytoskeletal components of a cell
There are two types of microtubules
They are involved in a variety of functions in the cell

17. Two Types of Microtubules Are Responsible for Many Functions in the Cell
Cytoplasmic microtubules pervade the cytosol and are responsible for a variety of functions
Maintaining axons
Formation of mitotic and meiotic spindles
Maintaining or altering cell shape
Placement and movement of vesicles

18. Two types of microtubules (MTs)
Axonemal microtubules include the organized and stable microtubules found in structures such as
Cilia
Flagella
Basal bodies to which cilia and flagella attach
The axoneme, the central shaft of a cilium or flagellum, is a highly ordered bundle of MTs

19. Tubulin Heterodimers Are the Protein Building Blocks of Microtubules
MTs are straight, hollow cylinders of varied length that consist of (usually 13) longitudinal arrays of polymers called protofilaments
The basic subunit of a protofilament is a heterodimer of tubulin, one a-tubulin and one b-tubulin
These bind noncovalently to form an ab-heterodimer, which does not normally dissociate

20. Figure 15-2

21. Figure 15-2A

22. Figure 15-2B

23. Figure 15-2C

24. Subunit structure
a and b subunits have very similar 3-D structure, but only 40% amino acid identity
Each has an N-terminal GTP binding domain, a central domain to which colchicine can bind, and a C-terminal domain that interacts with MAPs (microtubule-associated proteins)
All the dimers in the MT are oriented the same way

25. MT polarity and isoforms
Because of dimer orientation, protofilaments have an inherent polarity
The two ends differ both chemically and structurally
Most organisms have several closely related genes for slight variants of a- and b-tubulin, referred to as isoforms

26. Microtubules Can Form as Singlets, Doublets, or Triplets
Cytoplasmic MTs are simple tubes, or singlet MTs, with 13 protofilaments
Some axonemal MTs form doublet or triplet MTs
Doublets and triplets contain one 13-protofilament tubule (the A tubule) and one or two additional incomplete rings (B and C tubules) of 10 or 11 protofilaments

27. Microtubules Form by the Addition of Tubulin Dimers at Their Ends
MTs form by the reversible polymerization of tubulin dimers in the presence of GTP and Mg2+
Dimers aggregate into oligomers, which serve as “nuclei” from which new MTs grow
This process is called nucleation; the addition of more subunits at either end is called elongation

28. Microtubule assembly
MT formation is slow at first, the lag phase, due to the slow process of nucleation
The elongation phase is much faster
When the mass of MTs reaches a point where the amount of free tubulin is diminished, the assembly is balanced by disassembly; the plateau phase

29. Figure 15-3

30. Critical concentration
Microtubule assembly in vitro depends on concentration of tubulin dimers
The tubulin concentration at which MT assembly is exactly balanced by disassembly is called the critical concentration
MTs grow when the tubulin concentration exceeds the critical concentration and vice versa

31. Addition of Tubulin Dimers Occurs More Quickly at the Plus Ends of Microtubules
The two ends of an MT differ chemically, and one can grow or shrink much faster than the other
This can be visualized by mixing basal bodies (structures found at the base of cilia) with tubulin heterodimers
The rapidly growing MT end is the plus end and the other is the minus end

32. Figure 15-4

33. Microtubule treadmilling
The plus and minus ends of microtubules have different critical concentrations
If the [tubulin subunits] is above the critical concentration for the plus end but below that of the minus end, treadmilling will occur
Treadmilling: addition of subunits at the plus end, and removal from the minus end

34. Figure 15-5

35. Drugs Can Affect the Assembly of Microtubules
Colchicine binds to tubulin monomers, inhibiting their assembly into MTs and promoting MT disassembly
Vinblastin, vincristine are related compounds
Nocodazole inhibits MT assembly, and its effects are more easily reversed than those of colchicine

36. Antimitotic drugs
These drugs are called antimitotic drugs because they interfere with spindle assembly and thus inhibit cell division
They are useful for cancer treatment (vinblastine, vincristine) because cancer cells are rapidly dividing and susceptible to drugs that inhibit mitosis

37. Taxol
Taxol binds tightly to microtubules and stabilizes them, causing a depletion of free tubulin subunits
It causes dividing cells to arrest during mitosis
It is also used in cancer treatment, especially for breast cancer

38. GTP Hydrolysis Contributes to the Dynamic Instability of Microtubules
Each tubulin heterodimer binds two GTP molecules, a-tubulin binds one and b-tubulin binds a second
The GTP bound to the b-subunit is hydrolyzed to GDP after the heterodimer is added to the MT
GTP is needed to promote heterodimer interactions and addition to MTs, but its hydrolysis is not required for MT assembly

39. Dynamic instability
Dynamic instability model: one population of MTs grows by polymerization at the plus ends whereas another population shrinks by depolymerization
Growing MTs have GTP at the plus ends, and shrinking MTs have GDP
The GTP cap at the plus end prevents subunit removal

40. Figure 15-6A

41. GTP-tubulin and dynamic instability
If [GTP-tubulin] is high, it is added to an MT, quickly creating a large GTP-tubulin cap
If the concentration falls, the rate of tubulin addition decreases
At a sufficiently low [GTP-tubulin], the rate of GTP hydrolysis exceeds the rate of subunit addition and the cap shrinks

42. Catastrophe and rescue
If the GTP cap disappears altogether, the MT becomes unstable and loss of GDP-bound subunits is favored
Individual MTs can go through periods of growth and shrinkage; a switch from growth to shrinkage is called microtubule catastrophe
A sudden switch back to growth phase is called microtubule rescue

43. Figure 15-6B

44. Figure 15-7

45. Microtubules Originate from Microtubule- Organizing Centers Within the Cell
MTs originate from a microtubule-organizing center (MTOC)
Many cells have an MTOC called a centrosome near the nucleus
In animal cells the centrosome is associated with two centrioles, surrounded by pericentriolar material

46. Centriole structure
Centriole walls are formed by 9 pairs of triplet microtubules
They are oriented at right angles to each other
They are involved in basal body formation for cilia and flagella
Cells without centrioles have poorly organized mitotic spindles

47. Figure 15-8A,B

48. Figure 15-8C

49. g-tubulin
Centrosomes have large ring-shaped protein complexes in them; these contain g-tubulin (along with gamma tubulin ring proteins: GRiPs)
g-tubulin ring complexes (g-TuRCs) nucleate the assembly of new MTs away from the centrosome
Loss of g-TuRCs prevents a cell from nucleating MTs

50. Figure 15-9

51. Figure 15-9A

52. Figure 15-9B

53. MTOCs Organize and Polarize the Micotubules Within Cells
MTOCs nucleate and anchor MTs
MTs grow outward from the MTOC with a fixed polarity—the minus ends are anchored in the MTOC
Because of this, dynamic growth and shrinkage of MTs occurs at the plus ends, near the cell periphery

54. Figure 15-10A-C

55. Figure 15-10D

56. Microtubule Stability Is Tightly Regulated in Cells by a Variety of Microtubule-Binding Proteins
Cells regulate MTs with great precision
Some MT-binding proteins use ATP to drive vesicle or organelle transport or to generate sliding forces between MTs
Others regulate MT structure

57. Microtubule-Stabilizing/Bundling Proteins
MAPs, microtubule-associated proteins, bind at regular intervals along a microtubule wall, allowing for interaction with other cellular structures and filaments
A MAP called Tau causes MTs to form tight bundles in axons
MAP2 promotes the formation of looser bundles in dendrites

58. MAPs that promote bundling
MAPs such as Tau and MAP2 have two regions
One region binds to the MT wall and another part of the protein extends at right angles to the MT to allow for interaction with other proteins
The length of the extended “arm” controls the spacing of MTs in the bundle

59. Figure 15-11A

60. +–TIP Proteins
MTs can be stabilized by proteins that “capture” and protect the growing plus ends
These are +–TIP proteins (+-end tubulin interacting proteins)
These decrease the likelihood that MTs will undergo catastrophic subunit loss

61. Figure 15-11B

62. Microtubule-Destabilizing/Severing Proteins
Some proteins promote depolarization of MTs
Stathmin/Op18 binds to tubulin heterodimers and prevents their polymerization
Catastrophins act at the ends of MTs and promote the peeling of subunits from the ends
Proteins such as katanins sever MTs

63. Figure 15-11C

64. Microfilaments
Microfilaments are the smallest of the cytoskeletal filaments
They are best known for their role in muscle contraction
They play a role in cell migration, amoeboid movement, and cytoplasmic streaming 64

65. Additional roles of microfilaments
Development and maintenance of cell shape (via microfilaments just beneath the plasma membrane at the cell cortex)
Structural core of microvilli

66. Actin Is the Protein Building Block of Microfilaments
Actin is a very abundant protein in all eukaryotic cells
Once synthesized, it folds into a globular-shaped molecule that can bind ATP or ADP (G-actin; globular actin)
G-actin molecules polymerize to form microfilaments, F-actin

67. Figure 15-12

68. Figure 15-12A

69. Figure 15-12B

70. Figure 15-12C

71. Different Types of Actin Are Found in Cells
Actin is highly conserved, but there are some variants
Actins can be broadly divided into muscle-specific actins (a-actins) and nonmuscle actins (b- and g-actins)
b- and g-actin localize to different regions of a cell

72. G-Actin Monomers Polymerize into F-Actin Microfilaments
G-actin monomers can polymerize reversibly into filaments with a lag phase, and elongation phase, similar to tubulin assembly
F-actin filaments are composed of two linear strands of polymerized G-actin, wound into a helix
All the actin monomers in the filament have the same orientation

73. Demonstration of microfilament polarity
Myosin subfragment 1 (S1) can be incubated with microfilaments (MFs)
S1 fragments bind and decorate the actin MFs in a distinctive arrowhead pattern
The plus end of an MF is called the barbed end and the minus end is called the pointed end, because of this pattern

74. Figure 15-13A,B

75. Figure 15-13C

76. Polarity of microfilaments
The polarity of MFs is reflected in more rapid addition or loss of G-actin at the plus end than the minus end
After the G-actin monomers assemble onto a microfilament, the ATP bound to them is slowly hydrolysed
So, the growing MF ends have ATP-actin, whereas most of the MF is composed of ADP-actin

77. Specific Drugs Affect Polymerization of Microfilaments
Cytochalasins are fungal metabolites that prevent the addition of new monomers to existing MFs
Latrunculin A is a toxin that sequesters actin monomers and prevents their addition to MFs
Phalloidin stabilizes MFs and prevents their depolymerization

78. Cells Can Dynamically Assemble Actin into a Variety of Structures
Cells can regulate where and how MFs are assembled
Cells that crawl have lamellipodia and filopodia at their leading edge, allowing them to move along a surface
Cells that adhere tightly to the underlying substratum have organized bundles called stress fibers

79. Figure 15-14A

80. Rapidly moving cells
Rapidly moving cells don’t have such striking actin bundles
The cell cortex, just beneath the plasma membrane, has actin crosslinked into a gel or loose lattice

81. Filopodia and lamellipodia
In filopodia, at the leading edge, microfilaments form highly oriented, polarized cables with the plus ends toward the tip of the protrusion
The actin in lamellipodia is less well organized than in filopodia

82. Figure 15-14B

83. Figure 15-15

84. Actin-Binding Proteins Regulate the Polymerization, Length, and Organization of Actin
Cells can precisely control where actin assembles and the structure of the resulting network
They use a variety of actin-binding proteins to do so
Control occurs at the nucleation, elongation, and severing of MFs, and the association of MFs into networks 84

85. Figure 15-16

86. Proteins That Regulate Polymerization
If the concentration of ATP-bound G-actin is high, microfilaments will assemble until the G-actin is limiting
In the cell, a large amount of free G-actin is not available because it is bound by thymosin 4
Profilin competes with thymosin 4 for G-actin binding 86

87. Proteins that regulate polymerization
ADF/cofilin is known to bind ADP-G-actin and F-actin and is thought to increase turnover of ADP-actin at the minus end of MFs
ADF/cofilin also severs filaments, creating new plus ends in the process 87

88. Proteins That Cap Actin Filaments
Whether MFs can grow depends on whether their filament ends are capped
Capping proteins bind the ends of a filament to prevent further loss or addition of subunits
CapZ binds to plus ends to prevent addition of subunits there; tropomodulins bind to minus ends, preventing loss of subunits there 88

89. Proteins That Crosslink Actin Filaments
Often, actin networks form as loose networks of crosslinked filaments
One of the proteins important in the formation of these networks is filamin
Filamin act as splices, joining two MFs together where they intersect 89

90. Proteins That Sever Actin Filaments
MFs are broken up by proteins that sever and/or cap them
Gelsolin breaks actin MFs and caps the newly exposed plus ends, preventing further polymerization 90

91. Proteins That Bundle Actin Filaments
Some actin-containing structures can be highly ordered
Actin may be bundled into tightly organized arrays, called focal contacts or focal adhesions
-actinin is a protein that is prominent in such structures
Fascin in filopodia keeps the actin tightly bundled 91

92. Microvilli
Actin bundles in microvilli are the best-studied examples of ordered actin structures
Microvilli are prominent features of intestinal mucosal cells; they increase the surface area of the cells
The core of a microvillus consists of a tight bundle of microfilaments with the ends pointed toward the tip 92

93. Crosslinks
The MFs are connected to the plasma membrane by crosslinks made of myosin I and calmodulin
The MFs in the bundle are tightly bound together by crosslinking proteins fimbrin and villin 93

94. Figure 15-17

95. Figure 15-17A

96. Figure 15-17B

97. The terminal web
At the base of the microvillus, the MF bundle extends into a network of filaments called the terminal web
The filaments of the terminal web are composed mainly of myosin and spectrin, which connect the MFs to each other, to proteins in the membrane, and perhaps also to intermediate filaments

98. Figure 15-18

99. Proteins That Link Actin to Membranes
MFs are connected to the plasma membrane and exert force on it during cell movement or cytokinesis
This (indirect) connection to the membrane requires one or more linking proteins
One group of such proteins is the band 4.1, ezrin, radixin, and moesin family; another is spectrin and ankyrin

100. Figure 15-19

101. Figure 15-19A

102. Figure 15-19B

103. Proteins That Promote Actin Branching and Growth
Besides loose networks and bundles, actin can form a dendritic (treelike) network
A complex of actin-related proteins, the Arp2/3 complex, nucleates new branches on the sides of filaments
Arp2/3 branching is activated by a family of proteins that includes WASP (Wiskott-Aldrich syndrome protein) and WAVE/Scar

104. Figure 15-20A

105. Figure 15-20B

106. Long actin filaments
For some cell functions long actin filaments are needed
In this case, actin polymerization is regulated independently of the Arp2/3 complex, through proteins called formins
Formins move along the end of the growing filament as they promote polymerization

107. Figure 15-20C

108. Cell Signaling Regulates Where and When Actin-Based Structures Assemble
Both plasma membrane lipids and several small G proteins related to Ras regulate the formation, stability, and breakdown of MFs

109. Inositol Phospholipids
Phosphatidylinositol-4,5-bisphosphate (PIP2) can bind to profilin, CapZ, and proteins such a ezrin
PIP2 recruits these proteins to the membrane and regulates their interactions with actin
CapZ binds tightly to PIP2 resulting in its removal from the end of a MF, promoting disassembly

110. Rho Family GTPases
The cytoskeleton of cells exposed to certain growth factors can undergo a dramatic change
Many signals that result in these changes act via a family of monomeric G proteins called Rho GTPases
Three key family members are Rho, Rac, and Cdc42

111. Rho family GTPases, effect on cytoskeleton
Activation of the Rho pathway results in formation of stress fibers
Rac activation results in extension of lamellipodia
Cdc42 activation results in the formation of filopodia

112. Figure 15-21A-D

113. Figure 15-21E

114. Regulation of Rho GTPases
Rho GTPases are stimulated by guanine-nucleotide exchange factors (GEFs) through the exchange of bound GDP for GTP
GTPase activating proteins (GAPs) inactivate Rho GTPases by causing them to hydrolyze their bound GTPs to GDP
Guanine-nucleotide dissociation inhibitors (GDIs) sequester inactive Rho GTPases in the cytosol

115. Intermediate Filaments
Intermediate filaments are the most stable and least soluble cytoskeletal components and are not polarized
An abundant intermediate filament (IF) is keratin, an important component of structures that grow from skin in animals
IFs may support the entire cytoskeleton

116. Figure 15-22

117. Intermediate Filament Proteins Are Tissue Specific
IFs differ greatly in amino acid composition from tissue to tissue
They are grouped into six classes

118. Classes of intermediate filament proteins
Class I: acidic keratins
Class II: basic or neutral keratins
Proteins of classes I and II make up the tonofilaments found in epithelial surfaces covering the body and lining its cavities

119. Classes of intermediate filament proteins (continued)
Class III: includes vimentin (connective tissue), desmin (muscle cells), and glial fibrillary acidic (GFA) protein (glial cells)
Class IV: These are the neurofilament (NF) proteins found in neurofilaments of nerve cells

120. Classes of intermediate filament proteins (continued)
Class V: includes the nuclear lamins A, B, and C that form a network along the inner surface of the nuclear membrane
Class VI: Neurofilaments in the nerve cells of embryos are made of nestin
Animal cells can be distinguished based on the types of IF proteins they contain—intermediate filament typing

121. Table 15-4

122. Intermediate Filaments Assemble from Fibrous Subunits
IF proteins are fibrous rather than globular
All have a homologous central rodlike domain conserved in size, secondary structure, and to some extent, in sequence
Flanking the central helical domain are N- and C-terminal domains that differ greatly among IF proteins

123. Intermediate filament assembly
The basic structural unit consists of two IF polypeptides intertwined into a coiled-coil
The two polypeptides are aligned in parallel
Two such dimers align laterally to form a tetrameric protofilament
Protofilaments overlap to build up a filamentous structure about 8 protofilaments thick

124. Figure 15-23

125. Figure 15-23A

126. Figure 15-23B

127. Figure 15-23C

128. Figure 15-23D

129. Intermediate Filaments Confer Mechanical Strength on Tissues
Cellular architecture depends on the unique properties of the cytoskeletal elements working together
MTs resist bending when a cell is compressed whereas MFs serve as contractile elements that generate tension
IFs are elastic and can withstand tensile forces

130. The Cytoskeleton Is a Mechanically Integrated Structure
IFs are important structural determinants in many cells and tissues; they are thought to have a tension-bearing role
IFs are not static structures; they are dynamically transported and remodeled
The nuclear lamina, on the inner surface of the nuclear envelope, disassemble at the onset of mitosis and reassemble afterward

131. Integration of cytoskeletal elements
Plakins are linker proteins that connect intermediate filaments, microfilaments, and microtubules
One plakin, called plectin, is found at sites where intermediate filaments connect to MFs and MTs

132. Figure 15-24