1. CYTOSKELETON (I) Actin filaments
Cell Biology
Lecture 9

2. Readings and Objectives
Russell : Chapter 1 (not complete information)
Cooper: Chapter 12
Objectives
Actin and actin filament dynamism
Actin bundles and network
Actin and myosin: role in contractile assemblies
Role in cellular movement

3. Introduction: Cytoskeleton
A network of protein filaments and tubules, extends from the nucleus to the plasma membrane
Structural framework
cell shape, localize organelles, general organization of the cytoplasm
Movement
Cell movement, internal transport of organelles, muscle contraction
Dynamic structures, continually reorganized
Composed of three main types of protein filaments:
Actin filaments (7 nm)
Intermediate filaments (8-11 nm)
Microtubules ( 25 nm)

4. Structure and Organization of Actin Filaments
Actin filaments (microfilaments): Polymer of Actin, flexible fibers 7 nm in diameter, several µm in length
Actin first isolated from muscle cells in 1942
Abundant in all types of eukaryotic cells
Mammals have 6 actin genes: 4 are expressed in muscle cells and 2 in nonmuscle cells
Highly conserved
Prokaryotic ancestor is MreB

5. Actin and Actin Filament
3-D structure determined in 1990
Actin: globular (G-actin), 375 aa (43 kD), barbed and pointed ends, binds head-tail to nucleate a trimer
Filamentous (F-actin): monomers added to both end
Filament is polar pointed end vs barbed end

6. G-Actin and Actin Filament
Polymerization is reversible
The rate at which monomers are added to filaments is proportional to their concentration
ATP bound actin binds to barbed end with high affinity
ADP-actin has low affinity to the pointed ends
when ATP hydrolyses to ADP
ADP-actin dissociates from filaments more readily than ATP-actin
Therefore, the critical concentration of actin monomers is higher for addition to the pointed end than to the barbed end of actin filaments

7. Treadmilling: polarity of F-actin growth
At cellular actin concentrations
Barbed end of a filament grows 5–10 times faster than the pointed end
ADP-actin dissociates from pointed end
Exchange of ATP for ADP added to barbed-end
Process is called Treadmilling
Dynamic growth
Direction? PointedBarbed

8. Actin Binding Proteins
Actin Binding Proteins (ABP): modulate the Assembly and disassembly of actin filaments
ABD/Actin interaction has diverse functionality
Contribute to the cellular role of actin filaments

9. Actin Remodeling
Some actin-binding proteins bind along the length of actin filaments, stabilizing them or cross-linking them to one another
Others stablize by capping the ends and preventing dissociation
Others promote dissociation, while others regulate the exchange of ATP for ADP.

10. Initiation of Actin Filament polymerization
Nucleation is the rate-limiting step
Formin and the Arp2/3 complex determine where filaments are formed by facilitating nucleation
Formins nucleate long unbranched actin filaments

11. Initiation of Actin Filament polymerization
actin filaments actively turn over and branch extensively
These filaments are nucleated by the Arp2/3 (Actin Related Protein) complex, which binds actin/ATP near the barbed ends

12. Actin Filament Depolymerization
The ADF/cofilin (Actin Depolymerizing Factor) family modifies existing filaments
enhance the rate of dissociation of actin/ADP monomers from the pointed end, and remain bound to the monomers, preventing their reincorporation

13. Actin Filament Severing
ADF/cofilin can also bind to and sever actin filaments
Profilin reverses the ADF/Cofilin effect
Stimulate exchange of bound ADP for ATP and dissociating the actin/ATP monomers from cofilin
Become available for reassembly in two filaments

14. Summary: Assembly of an Actin Filament

15. Actin filament higher order assemblies
Actin bundles—cross-linked into closely packed parallel arrays
Actin networks—cross-linked in arrays that form 3-D meshworks

16. Actin Bundles Two types of actin bundles:
Parallel filaments cross linked by actin-bundling proteins
Have two domains to bind actin and align the filaments
Two types of actin bundles:
Non contractile
filaments (14 nm apart) aligned in parallel, same polarity, barbed ends adjacent to the plasma membrane
Fimbrin: a 68 kD proteins, cross links by its two actin binding domains (ABD)

17. Actin bundles: Intenstinal Microvilli
Actin bundles take part in avariety of cell surface protrusions
cell movement
phagocytosis
absorption of nutrients
Intenstinal microvilli:
Membrane projections, increase absorption surface

18. Actin bundles: Intenstinal Microvilli
closely packed parallel bundles of 20 to 30 actin filaments
Relatively permanent
The filaments are cross-linked in part by fimbrin and villlin
The actin bundles are attached to the plasma membrane by the calcium-binding protein calmodulin in association with myosin I
At the base attach to actin cortex

19. Actin bundles: membrane protrusions
Other surface protrusions are transient and form in response to environmental stimuli
Pseudopodia- responsible for phagocytosis and the movement of amoebas
Lamellipodia- broad, sheetlike extensions at the leading edge of fibroblasts
Filopodia- thin projections of the plasma membrane in migrating cells

20. Actin Bundles: Contractile
Contractile bundles: more widely-spaced filaments (40 nm), cross-linked by α-actinin
α-actinin: a 102 kD protein with single ABD and an α-helical spacer
Interacts with actin as a dimer
Increased spacing allows actin interaction with motor protein myosin II
Important in muscle fiber contraction

21. Higher order Actin assembly: Actin Network
Filamin (280 kD) form flexible cross-links
Filamin dimer: flexible V-shaped molecule
actin-binding domains at the end of each arm
dimerization domain
Β-sheet spacer
Binds actin orthogonally, form 3-D network beneath the plasma membrane
network (cell cortex) determines cell shape, and cell movement

22. Actin Network and cell cortex
Red blood cells as mode
lack other cytoskeletal components, so the cortical cytoskeleton is the principal determinant of cell shape
Spectrin, major actin-binding cortex protein
tetramer of two polypeptide chains, α and β
ends of the spectrin tetramers bind actin filaments, resulting in the spectrin-actin network

23. Actin Network and Cell Cortex
Ankyrin links the spectrin-actin network and the plasma membrane
Protein 4.1 is another link that binds spectrin-actin junctions and the transmembrane protein glycophorin

24. Association of Actin filaments with Motor proteins
Brings higher level of functional complexity to cells
Cellular or organismal movement
Intracellular cargo transportation, cell division
Association with motor protein myosin
Myosin is a molecular motor: converts chemical energy (ATP) to mechanical energy  force and movement.
Muscle contraction: model for understanding actin-myosin interactions and the motor activity of myosin molecules

25. Actin-Myosin and Muscle Contraction
muscle fibers, large cells (50 µm in diameter and up to several centimeters in length)
Cytoplasm consists of myofibrilsmyosin filaments and thin actin filaments
sarcomeres, myofibril units of skeletal and cardiac muscle
actin filaments attached at their barbed ends to the Z disc

26. Sacromere: a structural and contractile unit
Titin is extremely large protein; extend from the M line to the Z disc
keep myosin II filaments centered in the sarcomere
maintain the resting tension that allows a muscle to snap back if overextended
Nebulin, associated with actin, regulate assembly of actin filaments

27. Sliding Filament Model
was proposed in 1954
Myosin slides on actin filament
Sarcomere shortens, bringing the Z discs closer
There is no change in the width of the A band, but the I bands and H zone almost disappear

28. Sliding Filament Model
Tropomyosin binds along actin filaments, also bound to troponin
No Ca2+, tropomyosin-troponin block binding of myosin to actin
nerve impulses, stimulate release of Ca2+ from the sarcoplasmic reticulum
Ca2+ binds troponin C, shifts the complex
Allows myosin binding to actin

29. Sliding Filament Model
Myosin II (the type in muscle), large protein with two heavy chains and two pairs of light chains
heavy chains have a globular head region and a long α-helical tail
Tails twist around in a coiled-coil
globular heads bind actin
myosin moves the head groups along the actin filament in the direction of the barbed end

30. Sliding Filament Model
ATP hydrolysis is required
Binding of ATP dissociates myosin from actin
ATP hydrolysis induces a conformational change that displaces the myosin head group

31. Sliding Filament Model
myosin head binds to a new position on the actin filament and Pi is released
The “power stroke”: Myosin head returns to its original conformation, which drives actin filament sliding, and ADP is released

32. Muscle contraction and the role of Actin thin filaments

33. Actin and myosin in cell divison
Cytokinesis—division of a cell following mitosis
A contractile ring of actin and myosin II is assembled underneath the plasma membrane
Contraction of the ring pinches the cell in two

34. Actin and myosin: vesicular transport
Myosin I: much smaller than myosin II,contains a globular head group, acts as a molecular motor
Short tails bind to other structures
Movement of myosin I along actin filament
transport cargo, such as a vesicle