Chapter 8

ActinBy

Enrique M. De La Cruz & E. Michael Ostap

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.

• 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

8.1 Introduction

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

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.

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.

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.

8.6 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.

• 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.

8.6 Actin subunits hydrolyze ATP after polymerization

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

8.8 Actin monomer-binding proteins influence polymerization

• The two major actin monomer-binding proteins in many eukaryotic cells are:– thymosin β4 – profilin

• 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.

8.8 Actin monomer-binding proteins influence polymerization

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.

• 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

8.9 Nucleating proteins control cellular actin polymerization

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.

• 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.

8.10 Capping proteins regulate the length of actin filaments

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.

• 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.

8.11 Severing and depolymerizing proteins regulate actin filament dynamics

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.

• Actin crosslinking proteins have two binding sites for actin filaments.

• Actin bundles help form:– Stereocilia– Filopodia

• Orthogonal actin networks form:– sheets (lamellae) – gels

8.12 Crosslinking proteins organize actin filaments into bundles and orthogonal networks

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.

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

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.

• 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.

8.15 Myosins are actin-based molecular motors with essential roles in many cellular processes

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.

• 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

8.16 Myosins have three structural domains

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.

• 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

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.

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.

8.17 ATP hydrolysis by myosin is a multistep reaction

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.

• 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.

8.18 Myosin motors have kinetic properties suited for their cellular roles

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.”

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

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.