Chapter 8
Actin
By
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.
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
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.
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.
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
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.
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.
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
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.
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.
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.
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.
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.
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
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.
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.
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.
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
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.
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
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.
8.18 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.
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.
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.