Actin Flashcards
Actin filaments determine the shape of the cell’s … and are necessary for …
surface, whole-cell locomotion
Actin filaments form many types of …. Some of these are dynamic structures, such as the … and … that cells use to explore territory and pull themselves around.
cell-surface projections
lamellipodia, filopodia
Actin filaments are made up of subunits that are … and …: … subunits
Each subunit is in contact with … other subunits.
compact, globular, actin
4
The initial process of nucleus assembly is called …, and it can take quite a long time, depending on ….
filament nucleation, how many subunits must come together to form the nucleus
Nucleation: The instability of smaller aggregates creates a … barrier to nucleation.
kinetic
When polymerization is initiated in a test tube containing a solution of pure individual subunits, there is an initial …, during which … are observed. During this phase, however, …, so that it is followed by a phase of …, during which …
lag phase, no filaments
nuclei are assembling slowly
rapid filament elongation
subunits add quickly onto the ends of the nucleated filaments
After nucleation and elongation, the system approaches a … at which …
steady state
the rate of addition of new subunits to the filament ends is exactly balanced by the rate of subunit dissociation from the ends
The concentration of free subunits left in solution at the steady state is called the …
critical concentration, Cc
The value of the critical concentration is equal to …
the rate constant for subunit loss divided by the rate constant for subunit addition—that is, Cc = k(off)/k(on)
The actin subunit is a … globular polypeptide chain and is thus a … rather than a dimer.
single, monomer
Each actin subunit has a binding site for …
ATP (or ADP)
The actin subunits assemble … to generate filaments with a distinct …
The length of an actin filament is …, allowing for …
head-to-tail, structural polarity
variable, torsional flexibility
The actin filament can be considered to consist of … that …
two parallel protofilaments
twist around each other in a right-handed helix
Actin filaments are … compared with the hollow cylindrical microtubules
relatively flexible
The structural polarity of actin filaments is created by the … of all of their subunits.
regular, parallel orientation
The two ends of the actin filaments are different in ways that have a profound effect on …
filament growth rates
What happens to the rates of dissociation in the absence of ATP hydrolysis?
In the absence of ATP hydrolysis, the free energy difference, and therefore the equilibrium constant (and the critical concentration), must be the same for addition of subunits at either end of the polymer. In this case, the ratio of the forward and backward rate constants, kon/koff, must be identical at the two ends, even though the absolute values of these rate constants may be very different at each end.
The more dynamic of the two ends of a filament, where both … and … are fast, is called the …, and the other end is called the …
growth and shrinkage
plus end
minus end
In actin, a structurally polar filament, the kinetic rate constants for association and dissociation—… and …, respectively—are often much greater at one end than at the other: the … end
k(on) and k(off)
plus
ATP-binding cleft on the monomer points toward the … end, lying …
minus
deep in a cleft near the center of the subunit
The plus ends of actin filaments are usually referred to as “…” ends, and minus ends as “…” ends, because of the … appearance of myosin heads when bound along the filament.
barbed
pointed
arrowhead
Filament elongation proceeds spontaneously when the free energy change (ΔG) for addition of the soluble subunit is … This is the case when the concentration of subunits in solution exceeds the …
less than zero
critical concentration
Actin filament depolymerization proceeds spontaneously when this free energy change is …
greater than zero
A cell can couple an energetically unfavorable process spontaneous …; thus, the free energy released can be used to do mechanical work—in particular, …
polymerisation and depolymerisation
to push or pull an attached load
In addition to their ability to form …, actin subunits are enzymes that can hydrolyse …
noncovalent polymers
a nucleoside triphosphate, ATP
ATP hydrolysis is accelerated when the subunits are …
incorporated into filaments
Shortly after incorporation of an actin subunit into a filament, … occurs, whereby …
ATP hydrolysis
the free phosphate group is released from each subunit, but the nucleoside diphosphate remains trapped in the filament structure
What happens when ATP is hydrolysed? What does this mean for the free energy change upon dissociation?
Much of the free energy released by cleavage of the high-energy phosphate-phosphate bond is stored in the polymer lattice, making the free energy change upon dissociation of the ADP-form polymer higher than the free energy change upon dissociation of the ATP-form polymer.
Cc(…) is greater than Cc(…), which means that for certain concentrations of free subunits, …
ADP, ATP
ADP-form polymers will shrink while ATP-form polymers grow.
In most living cells, the cytoplasmic G-actin concentration (…) is extraordinarily … than the critical concentration (Cc) of actin filaments (…)
∼100 µM
∼0.1 µM
In living cells, most of the free actin subunits are in the … form, as …
ATP
the free concentration of ATP is much higher than that of ADP
If the rate of subunit addition is high (rapid filament growth), then it is likely that … before …, so that the tip of the polymer remains in …, forming an “…”
a new subunit will add on to the polymer,
the nucleotide in the previously added subunit has been hydrolyzed
the T form, ATP cap
At an intermediate concentration of free subunits, it is possible for the rate of subunit addition to be … than nucleotide hydrolysis at the plus end, but … than nucleotide hydrolysis at the minus end. In this case, the plus end of the filament is in the …, the minus end in the …
faster, slower
ATP conformation
ADP conformation
During …, subunits are recruited at the plus end of the polymer in the T form and shed from the minus end in the D form, maintaining the actin filament at a ….. This occurs at … subunit concentration
treadmilling
constant length
an intermediate
The … that occurs gives rise to …, making treadmilling possible.
ATP hydrolysis
the difference in the free energy of the association/dissociation reactions at the plus and minus ends of the actin filament
The high G-actin concentration is maintained by F-actin disassembly by …, and via the prevention of spontaneous G-actin nucleation by G-actin–sequestering proteins, … and …, and the blockage of F-actin polymerization by …
ADF/cofilin
profilin and thymosin-β4
F-actin–capping proteins
Actin monomers bound to … are in a locked state, where they cannot associate with either the plus or minus ends of actin filament and cannot…. This results in …
thymosin-β4
hydrolyze or exchange their bound nucleotide
depolymerisation
Thymosin-β4 complexes with G-actin on a … ratio, and has a … concentration in the cell of …
1:1
high, 0.55mm
Thymosin-β4 is a … protein: …kD
small, 5
Thymosin β4 functions like a … for … as represented in the following reaction: …
buffer, monomeric actin
F-actin ↔ G-actin + Thymosin β4 ↔ G-actin/Thymosin β4
Profilin binds to … & sequesters it from the pool of polymerizable actin monomers. However, profilin also catalyzes the exchange of actin-bound … thereby converting poorly polymerizing … into …
monomeric actin, polymerizable actin monomers
ADP to ATP
ADP-actin monomers
readily polymerizing ATP-actin monomers
Profilin has a higher affinity for … than for … actin monomers
ATP-, ADP-
Explain the mechanism through which profilin regulates lipid metabolism
Profilin negatively regulates PIP2 by inhibiting the binding of PIP2 to PLC (a membrane bound enzyme), thereby inhibiting the dissociation of PIP2 into DAG and IP3.
Under normal circumstances PLC would hydrolyse PIP2 into DAG and IP3. DAG would then go on to activate PKC, which acts as a kinase, activating proteins through phosphorylation, and IP3 acts as a second messenger, increasing the cytosolic Ca2+. Both of these lead to a cellular response
Explain how the inhibition of PIP2 by profilin can be overcome.
When the EGF receptor is activated by binding EGF, it phosphorylates PLC. Phosphorylated PLC can overcome the inhibition by profilin. PLC then hydrolysis PIP2, which releases IP3 and DAG, and simultaneously releasing profilin from the membrane, allowing it to interact with actin subunits in the membrane.
What are the dual roles of profilin?
Profilin is a negative regulator of the phosphoinositide signaling pathway in addition to its established function as an inhibitor of actin polymerization.
What are some properties of the myosin superfamily?
All myosins share similar motor domains, but their C-terminal tails (tail region) and N-terminal extensions (neck region) are very diverse. Many myosins form dimers, with two motor domains per molecule, but a few (such as I, IX, and XIV) seem to function as monomers, with just one motor domain. Myosin VI, despite its overall structural similarity to other family members, is unique in moving toward the minus end (instead of the plus end) of an actin filament.
First identified in skeletal muscle.
They were isolated on their ability to hydrolyse ATP on binding actin.
Myosin II
A myosin II molecule is composed of two heavy chains (each about 2000 amino acids long) and four light chains.
The heavy chains are involved in Ca2+ binding.
The light chains are of two distinct types, and one copy of each type is present on each myosin head: an essential and a regulatory light chain.
Dimerization occurs when the two α helices of the heavy chains wrap around each other to form a coiled-coil, driven by the association of regularly spaced hydrophobic amino acids (see Figure 3-11). The coiled-coil arrangement makes an extended rod in solution, and this part of the molecule is called the tail.
Myosin II activation
Each myosin head binds and hydrolyses ATP, using the energy of ATP hydrolysis to walk toward the plus end of an actin filament.
Myosin II is always associated with contractile activity in muscle and nonmuscle cells.
Myosin II regulation
The myosin II can exist in two different conformational states in such cells, an extended state that is capable of forming bipolar filaments, and a bent state in which the tail domain apparently interacts with the motor head. Phosphorylation of the regulatory light chain by the calcium-dependent myosin light-chain kinase (MLCK) causes the myosin II to preferentially assume the extended state, which promotes its assembly into a bipolar filament and leads to cell contraction
Myosin I
Only has one head, doesn’t form filaments.
The myosin I proteins contain a second actin-binding site or a membrane-binding site in their tails, and they are generally involved in intracellular organization and the protrusion of actin-rich structures at the cell surface.
Dilute Myosin
Motor proteins also have a significant role in organelle transport along actin filaments. The first myosin shown to mediate organelle motility was myosin V, a two-headed myosin. In mice, mutations in the myosin V gene result in a “dilute” phenotype, in which fur color looks faded. Myosin V is associated with the surface of melanosomes, and it is able to mediate their actin-based movement. In dilute mutant mice, the melanosomes are not delivered to the keratinocytes efficiently, and pigmentation is defective.
Severing proteins
- An important actin-filament binding protein present in all eucaryotic cells is cofilin, which destabilizes actin filaments. Also called actin depolymerizing factor, cofilin is unusual in that it binds to actin in both the filament and free subunit forms (1:1)
- Most actin-severing proteins are members of the gelsolin superfamily, whose severing activity is activated by high levels of cytosolic Ca2+ (above 0.1 microM). The Ca2+ activates gelsolin, which cleaves the capped filaments into tiny fragments, each now capped by gelsolin.
PIP2 regulation of severing proteins
The regulation of plus end capping proteins by the inositol phospholipid PIP2 is especially important: an increase in PIP2 in the cytosolic leaflet of the plasma membrane uncaps plus ends. The uncapping makes the plus ends available for elongation, thereby promoting actin filament polymerization at the cell surface.
PIP2 signal transduction
Actin filaments are capped by CapZ, surrounded by a large pool of actin monomers bound to profilin → activation of an intracellular signal transduction cascade → massive increase of ca2+ in cytosol → activates gelsolin → gelsolin cleaves the capped filaments into tiny fragments, each capped by gelsolin → the same signal pathway causes a rise in PIP2 levels → inactivates gelsolin and CapZ and cofilin
Capping proteins
In muscle cells, where actin filaments are exceptionally long-lived, the filaments are known to be specially capped at both ends—by CapZ at the plus end and by tropomodulin at the minus end.
Tropomodulin
Tropomodulin binds only to the minus end of actin filaments that have been coated by tropomyosin and have thereby already been somewhat stabilized.
Actin Binding Protein Characteristics
- Many actin binding proteins regulated by Ca2+.
2. Most have EF hands (2 helical loops in parvalbumin with a Ca2+ binding loop)
Gelsolin regulation
- PIP2: actin/gelsolin binding inhibited by PIP2
Cofilin regulation by PIP2
EGF induces a rapid loss of PIP2 through PLC activity, resulting in a release and activation of a membrane-bound pool of cofilin. Upon release, cofilin binds to and severs F-actin, which is coincident with actin polymerization and lamellipod formation.
Actin at the Cell Cortex
- Tight parallel bundles: these form filopodia. Same polarity, 10-20nm apart, exclude myosin.
- Gel-like network: loosely spaced, found in the cell cortex,.
- Contractile bundles: F-actin filaments have an opposite polarity and lie 30-60nm apart. They form stress fibers. They bind myosin II / tropomyosin.
Parallel bundles formation
Fimbrin: 2 EF Hands, 2F-actin binding domains
Fimbrin excludes myosin because of the packing.
Fimbrin excludes alpha-actinin and vice versa.
Gel-like network formation
Filamin: forms a dimer, with self-association in the tail region. The head (for actin binding) is homologous to spectrin.
Contractile bundles formation
alpha-Actinin: forms a dimer
F-actin binding domains of alpha-actinin and fimbrin are homologous.
Tropomyosin
Selected actin filaments in most cells are stabilized by the binding of tropomyosin, an elongated protein that binds simultaneously to seven adjacent actin subunits in one protofilament. The binding of tropomyosin along an actin filament can prevent the filament from interacting with filamin; for this reason, the regulation of tropomyosin binding is an important step in muscle contraction.
How is filament structure determined?
- If fimbrin binds → myosin excluded because of packing; alpha-actinin excluded → parallel bundles
- If tropomyosin binds → stabilises F-actin filaments → allows for binding of myosin II & inhibits filamin binding → contractile bundles
- If filamin binds → excludes both tropomyosin and alpha-actinin, as well as inhibiting myosin binding → gel-like networks
Actin at cell perimeter
Spectrin is concentrated just beneath the plasma membrane, where it forms a two-dimensional web held together by short actin filaments; spectrin links this web to the plasma membrane because it has separate binding sites for peripheral membrane proteins, which are themselves positioned near the lipid bilayer by integral membrane proteins. The resulting network creates a stiff cell cortex that provides mechanical support for the overlying plasma membrane, allowing the red blood cell to spring back to its original shape after squeezing through a capillary.
Spectrin
Spectrin is a long, flexible protein made out of four elongated polypeptide chains (two α subunits and two β subunits - antiparallel), arranged so that the two actin-filament-binding sites are about 200 nm apart.
Spectrin connection to plasma membran
Via band 3 and ankyrin.
