1. Actin By Enrique M. De La Cruz & E. Michael Ostap
Chapter 8
Actin By
Enrique M. De La Cruz & E. Michael Ostap

2. 8.1 Introduction
Cell motility is a fundamental and essential process for all eukaryotic cells.
Actin filaments form many different cellular structures.
Proteins associated with the actin cytoskeleton produce forces required for cell motility.

3. The polymerization of actin can provide forces that drive the:
8.1 Introduction
The actin cytoskeleton is dynamic and reorganizes in response to intracellular and extracellular signals.
The polymerization of actin can provide forces that drive the:
extension of cellular processes
movement of some organelles

4. 8.2 Actin is a ubiquitously expressed cytoskeletal protein
Actin is a ubiquitous and essential protein found in all eukaryotic cells.
Actin exists as:
a monomer called G-actin
a filamentous polymer called F-actin

5. 8.3 Actin monomers bind ATP and ADP
The actin monomer is a 43 kDa molecule that has four subdomains.
A nucleotide and a divalent cation bind reversibly in the cleft of the actin monomer.

6. 8.4 Actin filaments are structurally polarized polymers
In the presence of physiological concentrations of monovalent and divalent cations, actin monomers polymerize into filaments.
The actin filament is structurally polarized and the two ends are not identical.

7. 8.5 Actin polymerization is a multistep and dynamic process
De novo actin polymerization is a multistep process that includes nucleation and elongation steps.
The rates of monomer incorporation at the two ends of an actin filament are not equal.
The barbed end of an actin filament is the fast growing end.

8. 8.6 Actin subunits hydrolyze ATP after polymerization
ATP hydrolysis by subunits in an actin filament is essentially irreversible.
This makes actin polymerization a nonequilibrium process.
The critical concentration for actin assembly depends on whether actin has bound ATP or ADP.

9. 8.6 Actin subunits hydrolyze ATP after polymerization
The critical concentration of ATP-actin is lower than that of ADP-actin.
In the presence of ATP, the two ends of the actin filament have different critical concentrations.

10. 8.7 Actin-binding proteins regulate actin polymerization and organization
For the actin cytoskeleton to drive motility, the cell must be able to regulate actin polymerization and depolymerization.
Actin-binding proteins:
associate with monomers or filaments
influence the organization of actin filaments in cells

11. 8.8 Actin monomer-binding proteins influence polymerization
The two major actin monomer-binding proteins in many eukaryotic cells are:
thymosin β4
profilin

12. In metazoan cells, thymosin β4:
8.8 Actin monomer-binding proteins influence polymerization
In metazoan cells, thymosin β4:
sequesters actin monomers
maintains a cytosolic pool of ATP-actin that can be utilized for rapid filament elongation
Profilin-actin monomer complexes contribute to filament elongation at barbed ends but not at pointed ends.

13. 8.9 Nucleating proteins control cellular actin polymerization
Nucleating proteins allow the cell to control the time and place of de novo filament formation.
The Arp2/3 complex and formins nucleate filaments in vivo.

14. Arp2/3 nucleation generates a branched filament network.
8.9 Nucleating proteins control cellular actin polymerization
Arp2/3 nucleation generates a branched filament network.
Formin proteins nucleate unbranched filaments.
Arp2/3 is activated at cell membranes by proteins:
Scar
WASP
WAVE

15. 8.10 Capping proteins regulate the length of actin filaments
Capping proteins inhibit actin filament elongation.
Capping proteins function at either the barbed or pointed ends of actin filaments.

16. Capping protein and gelsolin:
8.10 Capping proteins regulate the length of actin filaments
Capping protein and gelsolin:
inhibit elongation at barbed ends
inhibited by phospholipids of the plasma membrane
Tropomodulin is a protein that caps the pointed end of actin filaments.

17. 8.11 Severing and depolymerizing proteins regulate actin filament dynamics
Actin filaments must disassemble to maintain a soluble pool of monomers.
Members of the cofilin/ADF family of proteins sever and accelerate the depolymerization of actin filaments.

18. Actin filaments with bound ADP are targets for cofilin/ADF proteins.
8.11 Severing and depolymerizing proteins regulate actin filament dynamics
Severing increases the number of filament ends available for assembly and disassembly.
Cofilin/ADF binds cooperatively and changes the twist of actin filaments.
Actin filaments with bound ADP are targets for cofilin/ADF proteins.

19. 8.12 Crosslinking proteins organize actin filaments into bundles and orthogonal networks
Crosslinking proteins connect actin filaments to form:
bundles
orthogonal networks
Actin bundles and networks are mechanically very strong.

20. Actin bundles help form:
8.12 Crosslinking proteins organize actin filaments into bundles and orthogonal networks
Actin crosslinking proteins have two binding sites for actin filaments.
Actin bundles help form:
Stereocilia
Filopodia
Orthogonal actin networks form:
sheets (lamellae)
gels

21. 8.13 Actin and actin-binding proteins work together to drive cell migration
Interactions among actin and proteins that bind actin monomers and filaments regulate the growth and organization of protrusive structures in cells.
The addition of actin monomers to the barbed ends of actin filaments located at the cell’s plasma membrane pushes the membrane outward.

22. 8.14 Small G proteins regulate actin polymerization
Members of the Rho family of small G proteins regulate actin polymerization and dynamics.
Activation of Rho, Rac, and Cdc42 proteins induces formation of, respectively:
Lamellipodia
Filopodia
Contractile filaments

23. 8.15 Myosins are actin-based molecular motors with essential roles in many cellular processes
Myosin proteins are energy transducing machines that use ATP to:
power motility
generate force along actin filaments
The myosin superfamily of actin-based molecular motors consists of at least eighteen classes
Many classes have multiple isoforms.

24. Some myosins power muscle and cellular contractions.
8.15 Myosins are actin-based molecular motors with essential roles in many cellular processes
Some myosins power muscle and cellular contractions.
Others power membrane and vesicle transport.
Myosins play key roles in regulating cell shape and polarity.
Myosins participate in signal transduction and sensory perception pathways.

25. 8.16 Myosins have three structural domains
Myosin family members have three structural domains termed the:
head (or motor) domain
regulatory domain
tail domain
The motor domain:
contains the ATP- and actin-binding sites
is responsible for converting the energy from ATP hydrolysis into mechanical work.

26. The tail domain of myosin:
8.16 Myosins have three structural domains
In most myosins, the regulatory domain acts as a force transducing lever arm.
The tail domain of myosin:
interacts with cargo proteins or lipid
determines its biological function

27. 8.17 ATP hydrolysis by myosin is a multistep reaction
Members of the myosin superfamily share a conserved reaction pathway for the hydrolysis of ATP.
Myosin’s affinity for actin depends on whether ATP, ADP-Pi, or ADP is bound to the nucleotide-binding site of myosin.

28. Myosins with bound ATP or ADP-Pi are in weak binding states.
8.17 ATP hydrolysis by myosin is a multistep reaction
Myosins with bound ATP or ADP-Pi are in weak binding states.
In its weak binding states, myosin rapidly associates and dissociates from actin.
ATP hydrolysis:
“activates” myosin
occurs while myosin is detached from actin

29. 8.17 ATP hydrolysis by myosin is a multistep reaction
Myosin’s force-generating powerstroke accompanies phosphate release after myosin-ADPPi rebinds actin.
Myosins with either bound ADP or with no nucleotide bound are in strong binding states.

30. Myosins in the weak binding states do not bear force.
8.17 ATP hydrolysis by myosin is a multistep reaction
Myosin in its strong binding states remains attached to actin for longer times.
Myosins in the weak binding states do not bear force.
Myosins in the strong binding states resist movement if external forces are applied.

31. 8.18 Myosin motors have kinetic properties suited for their cellular roles
The ATPase cycle mechanism is conserved among all myosins.
The ATPase cycle kinetics of different myosins are tuned for specific biological functions.

32. Low duty ratio myosins spend most of their time detached from actin.
8.18 Myosin motors have kinetic properties suited for their cellular roles
Myosins with high duty ratios spend a large fraction of their cycle time attached to actin.
Low duty ratio myosins spend most of their time detached from actin.
Some high-duty ratio myosins are processive and “walk” along actin filaments for long distances.

33. 8.19 Myosins take nanometer steps and generate piconewton forces
A single myosin motor generates enough force (several piconewtons) to transport biological molecules and vesicles.
The stroke size of a myosin is proportional to the length of its “lever arm.”

34. 8.20 Myosins are regulated by multiple mechanisms
The force-generating activity and cellular localization of myosins are regulated.
Myosin function is regulated:
by phosphorylation
by interactions with actin- and myosinbinding proteins

35. 8.21 Myosin-II functions in muscle contraction
Myosin-II is the motor that powers muscle contraction.
Actin and myosin-II are the major components of the sarcomere.
The sarcomere is the fundamental contractile unit of striated muscle.