1. Fundamentals of Cell Biology
Chapter 5: The Cytoskeleton and Cellular Architecture

2. Chapter Summary: The Big Picture (1)
Chapter foci:
Cytoskeletal proteins form a skeleton inside the cell
Intermediate filaments provide the cell with mechanical strength
Microtubules are associated with cellular trafficking
Actin is responsible for large-scale movements
Eukaryotic cytoskeletal proteins evolved from early prokaryotes

3. Chapter Summary: The Big Picture (2)
Section topics:
The cytoskeleton is represented by three functional classes of proteins
Intermediate filaments are the strongest, most stable elements of the cytoskeleton
Microtubules organize movement inside a cell
Actin filaments control the movement of cells
Eukaryotic cytoskeletal proteins arose from prokaryotic ancestors

4. The cytoskeleton is represented by three functional classes of proteins
Key Concepts:
The cytoskeleton is a complex mixture of 3 different types of proteins that are responsible for providing mechanical strength to cells and supporting movement of cellular contents.
The most visible form of cytoskeletal proteins are long filaments found in the cytosol, but these proteins also form smaller shapes that are equally important for cellular function.
The structural differences between the 3 protein types underscores their 4 different functions in cells.

5. Cytoskeleton
occupies large portion of cytosol and appears to link organelles to each other and to plasma membrane
3 elements: IFs
MTs
Actin
Elements do not form mixed polymers
Figure 05.01: The cytoskeleton forms an interconnected network of filaments in the cytosol of animal cells.

6. IFs are the strongest, most stable elements of the cytoskeleton
Key Concepts (1):
Intermediate filaments are highly stable polymers that have great mechanical strength.
Intermediate filament polymers are composed of tetramers of individual intermediate filament proteins.
Several different genes encode intermediate filament proteins, and their expression is often cell- and tissue-specific.

7. IFs are the strongest, most stable elements of the cytoskeleton
Key Concepts (2):
Intermediate filament assembly and disassembly are controlled by posttranslational modification of individual intermediate filament proteins.
Specialized intermediate-filament-containing structures protect the nucleus, support strong adhesion by epithelial cells, and provide muscle cells with great mechanical strength.

8. IFs provide mechanical strength to cells
Figure 05.02: Two types of intermediate filaments.
Figure 05.03: Intermediate filaments have the most mechanical strength of the cytoskeletal proteins.

9. IFs are formed from a family of related proteins

10. The primary building block of IFs is a filamentous subunit
α-helices in the central rod domain
Figure 05.04: The central rod domain of intermediate filament proteins forms an alpha helix. The head (and tail) regions form globular shapes.

11. IF subunits form coiled-coil dimers
Figure 05.05: A model for intermediate filament assembly. The coiled coil formed by the dimer formed the structural basis for the strength of intermediate filaments.

12. Heterodimers overlap to form filamentous tetramers
Coiled-coils align to form antiparallel staggered structures

13. Assembly of a mature IF from tetramers occurs in 3 stages
Figure 05.05: A model for intermediate filament assembly. The coiled coil formed by the dimer formed the structural basis for the strength of intermediate filaments.

14. Posttranslational modifications control the shape of intermediate filaments
Chemical modification of IF controls their shape and function
Phosphorylation-dephosphorylation
Glycosylation
Farnesylation
Transglutamination of head and tail domains

15. IFs form specialized structures
Keratins in epithelium
Costameres
Figure 05.06: Keratin expression patterns vary in different epithelial tissues.
Figure 05.07: Costameres link the contractile apparatus of muscle cells to the plasma membrane and extracellular matrix.

16. Microtubules (MT) organize movement inside a cell
Key Concepts (1):
MTs are hollow, tube-shaped polymers comprised of proteins called tubulins.
MTs serve as “roads” or tracks that guide the intracellular movement of cellular contents.
MT formation is initiated at specific sties in the cytosol called MT-organizing centers. The basic building block of a MT is a dimer of two different tubulin proteins.

17. MTs organize movement inside a cell
Key Concepts (2):
MTs have structural polarity, which determines the direction of the molecular transport they support. This polarity is caused by the binding orientation of the proteins in the tubulin dimer.
The stability of MTs is determined, at least in part, by the type of guanine nucleotides bound by the tubulin dimers within it.
Dynamic instability is caused by the rapid growth and shrinkage of MTs at one end, which permits cells to rapidly reorganize their MTs.

18. MTs organize movement inside a cell
Key Concepts (3):
MT-binding proteins play numerous roles in controlling the location, stability, and function of microtubules.
Dyneins and kinesins are the motor proteins that use ATP energy to transport molecular “cargo” along MTs.
Cilia and flagella are specialized MT-based structures responsible for motility in some cells.

19. MT cytoskeleton is a network of "roads" for molecules "pass to and fro"

20. MT assembly begins at a MT-organizing center (MTOC)
Figure 05.08: The distribution of microtubules in a human epithelial cell. The microtubules are stained green and the DNA is stained red.

21. Figure 05.09: The structure and location of the centrosome.
The MTOC contains the gamma tubulin ring complex (γTuRC) that nucleates MT formation
Centrioles
Pericentriolar material
gamma (γ ) tubulin  
Figure 05.09: The structure and location of the centrosome.

22. The primary building block of MTs is an alpha-beta tubulin dimer
α - and β -tubulin bind together to form stable dimer
If purified α-β  tubulin dimers bound to GTP are concentrated enough (critical concentration), they spontaneously form MTs  
Figure 05.10: A three dimensional model of the dimer formed by α- and β-tubulin.
Figure 05.11: In vitro assembly of microtubules is spontaneous and GTP-dependent. The graph represents the turbidity of a solution of α-β tubulin dimers over time.

23. MTs are hollow "tubes" composed of 13 protofilaments
Polymers of dimers  sheet composed of 13 protofilaments  folds into a tube
GTP binding and hydrolysis regulate MT polymerization and disassembly
Figure 05.12: A simple model of microtubule assembly.

24. The growth and shrinkage of MTs is called dynamic instability
Some microtubules rapidly grow and shrink in cells = dynamic instability
Elongation is at the
+ end by GTP-bound dimers
Figure 05.13: Growth and shrinkage of microtubules in a living cell. The microtubules have been tagged with a fluorescent molecule, and recorded by video over time.
Figure 05.14: The growth of microtubules begins at the gamma tubulin ring and continues as long as the plus end contains GTP-bound tubulin dimers.

25. Figure 05.15: Two fates of the plus ends of microtubules.
Catastrophe?
What happens when the supply of GTP-bound tubulin dimers runs out?
1) MT depolymerizes at the + end OR
2) Capping proteins prevent depolymerization
Figure 05.15: Two fates of the plus ends of microtubules.

26. Some MTs exhibit treadmilling
In cases where neither end of MT is stabilized, tubulin dimers are added to the + end and lost from the - end
Overall length of these MTs remains fairly constant, but the dimers are always in flux
Figure 05.16: Treadmilling in microtubules.

27. Benefits of dynamic instability
Allows cells to have
flexibility with trafficking during cell movement
ability to exert force by bonding with cargo molecules
Figure 05.17: Microtubules exert enough force to move cargo by dynamic instability.
Figure 05.18: Longitudinal and lateral bonds make microtubules strong.

28. MT-associated proteins regulate the stability and function of MTs
“MAPs” = capping proteins, rescue-associated proteins, and proteins that govern the motion
motor protein = special type of MAP that transports organelles/vesicles
Dyneins and kinesins

29. Motors
Figure 05.19: The structure of dynein and kinesis, the two most common motor proteins that bind to microtubules.
Figure 05.20: How a microtubule motor protein moves along a microtubule.

30. Cilia and Flagella Axoneme Sliding dynein = whip movement
Figure 05.24: The coordinated motion of a cilium and a flagellum.
Figure 05.23: The structure of an axoneme.

31. Actin filaments control the movement of cells
Key Concepts (1):
Actin filaments are thin polymers of actin proteins.
Actin filaments are responsible for large-scale changes in cell shape, including most cell movement.
Actin filament polymerization is initiated at numerous sites in the cytosol by actin-nucleating proteins.
Actin filaments have structural polarity, which determines the direction that force is exerted on them by myosin motor proteins.

32. Actin filaments control the movement of cells
Key Concepts (2):
The stability of actin filaments is deteremined by the type of adenine nucleotides bound by the actin proteins within them.
Actin-binding proteins play numerous roles in controlling the location, stability, and function of actin filaments.
Cell migration is a complex process, requiring assembly and disassembly of different types of actin filament networks.

33. The building block of actin filaments is the actin monomer
Smallest diameter of cytoskeletal filaments – 7nm “microfilament”
Great tensile strength
Structural polarity
+ end – barbed end
- end – pointed end
Often bound to myosin
Figure 05.25: The general structure of an actin filament. The lateral and longitudinal bonds holding actin monomers together are indicated at right.
Figure 05.26: An electron micrograph of an actin filament partially coated with mysoin proteins.

34. Actin found in wide variety of locations and configurations
Figure 05.27: A number of different actin filament-based structures in cells.

35. ATP binding/hydrolysis regulate actin filament polymerization and disassembly
+ ATP = polymerization
ATPADP = depolymerization
Figure 05.28: The structure of an actin monomer. A ribbon model, derived from a crystalized form of the protein.

36. Actin polymerization occurs in 3 stages
Figure 05.29: The three stages of actin filament assembly in vitro.

37. Actin filaments have structural polarity
Actin filaments undergo treadmilling
Figure 05.30: Treadmilling in actin filaments. Note the similarity of this treadmilling with that shown for microtubules in Figure 5-16.

38. 6 classes of proteins bind to actin to control its polymerization/organization
Monomer-binding proteins regulate actin polymerization
Nucleating proteins regulate actin polymerization
Figure 05.31: The structure and function of profilin, an actin monomer-binding protein.
Figure 05.32: ARP2/3 nucleates the formation of a new actin filament off the side of an existing filament.

39. 6 classes of proteins bind to actin to control its polymerization/organization
3. Capping proteins affect the length and stability of actin filaments 4&5. Severing and depolymerizing proteins control actin filament disassembly 6. Cross-linking proteins organize actin filaments into bundles and networks
Figure 05.34: Three forms of crosslinked actin filaments created by different crosslinking proteins.

40. Cell Migration
Actin-binding motor proteins exert force on actin filaments to induce cell movement
Cell migration is a complex, dynamic reorganization of an entire cell
Migrating cells produce three characteristic forms of actin filaments: filopodia, lamellopodia, and contractile filaments

41. Filopodia
Figure 05.35: Different forms of actin in stationary and migrating cells.

42. Myosins are a family of actin-binding motor proteins
myosins = multisubunit proteins organized into 3 structural domains
Motor
Regulatory
Tail
Figure 05.36: Myosin proteins contain three funtional domains

43. Figure 05.37: The contractile cycle of myosin.
Myosins move towards one end of the actin filaments
myosin V crawls towards the - end, all other myosins crawl towards the + end
Allows for movement of cell
Figure 05.37: The contractile cycle of myosin.

44. Striated muscle contraction is a well-studied example of cell movement
Figure 05.38: The anatomy of a skeletal muscle. The sarcomere contains actin and myosin arranged in parallel bundles.

45. Eukkaryotic cytoskeletal proteins arose from prokaryotic ancestors
Modern prokaryotic cells express a number of cytoskeletal proteins that are homologous to eukaryotic cytoskeletal proteins and behave similarly
Vimentin (IF)
FtsZ (MT)
MreB and ParM (actin)
Shared properties seem to include protection of DNA, compartmentalization and motility.