Actin and Cell Motility

What are the three main classes of cytoskeletal filaments?

actin, tubulin, intermediate filaments

What are the common properties of cytoskeletal filaments?

composed of polymers, highly regulated by other factors, can be either dynamic or stable

What do actin filaments aid in?

cell migration, muscle cell contraction, cell shape

What do microtubules aid in?

position organelles, function as roads to transport vesicles, function in mitosis to separate chromosomes

What are intermediate filaments made up of?

various fibrous proteins such as keratins, vimentins, and neurofilaments

What do intermediate filaments aid in?

mechanical strength for tissues, support for nuclear envelope

What is diameter of actin?

7-9 nm

What is diameter of microtubules?

25 nm

What is the diameter of intermediate filaments?

10nm

What is the structure of actin?

stiff but brittle, resist deformation well but break under moderate force

What does a shallow slope for actin mean?

they don’t deform much before failing

What is actin ideal for?

maintaining rigid cell shape and forming contractile structures

What does a steep slope for microtubules mean?

accumulate deformation under low force but break abruptly without much resistance to that deformation

What are microtubules ideal for?

long-rage intracellular transport and resisting compression forces

Which cytoskeletal filament is the most flexible and durable?

intermediate filaments

What are intermediate filaments ideal for?

mechanical strength, especially in cells under stress

What is a common theme in nature?

nothing is static

What are different structures that actin can form?

lamellipodium, filopodium, stress fibers

What is the structure of lamellipodium?

branched and crosslinked filaments

What is the structure of filopodium?

bundle of parallel filaments

What is the structure of stress fibers?

antiparallel contractile structures

What is the molecular structure of actin?

globular protein of 42 kDa, two subdomains connected by a cleft

What can the two subdomains of actin bind?

ATP, ADP, and Mg2+

What is the polarity of actin filaments determined by?

orientation of monomers within filaments

What do actin monomers organize into?

a helical filament

What type of bonds are between actin subunits?

non-covalent bonds

Why is it important that actin subunits are bound non-covalently?

so they can be easily separated

What is F-actin?

a helical polymer of globular actin monoers

What is G-actin?

globular actin monomers

What is the structure of F-actin?

right-handed, double-helical structure

What happens every 13 actin subunits or 6 turns?

the helical pattern repeats, corresponds to a length of about 36-37 nm

How does actin form?

G-actin assembles into dimers, then trimers, then F-actin

What is nucleation?

the creating of the first trimer

What part of actin binds ATP?

monomers (G-actin)

What controls actin filament dynamics?

ATP hydrolysis

When does G-actin occur?

in low ionic concentrations

When does F-actin occur?

high ionic concentrations

What is actin filament polarity decoded by?

myosin

What is the plus end of actin also known as?

the barbed end

What is the minus end of actin also known as?

the pointed end

What end of actin grows faster?

the plus end

What type of actin polymerizes more efficiently?

ATP-bound

What happens after actin polymerization?

the actin ATPase activity hydrolyzes ATP to ADP

What does ATP hydrolysis act as for actin polymerization?

a molecular clock since older actin filaments with ADP are more unstable and tend to disassemble

Where does the actin filament predominantly grow?

the plus end

Which part of the filament is old?

the minus end

Where is most of the ATP on an actin filament found?

the plus end

Where is most of the ADP on an actin filament found?

the minus end

What would happens if a molecular of ATP that couldn’t be hydrolyzed attached to actin?

stabilize F-action, stop it from disassembling, becomes static

Can actin monomers be added or removed from either end of the filament?

yes

What is Cc?

critical concentration

Why is there preferential addition of monomers to the plus end of actin?

because the Cc is lower for the plus end than the minus end

What happens when the [G-actin] > Cc?

filaments will grow

What happens when the [G-actin] < Cc?

filaments will disassemble

What happens when [G-actin] = Cc?

no net change in filament length

What are the 3 phases of actin polymerization?

nucleation, elongation, steady state

What happens in nucleation?

G-actin/ATP aggregate to form an unstable dimer and not until a third actin joins is the nucleus stable enough to promote more polymerization

What happens in elongation?

addition of G-actin/ATP monomers occurs at both ends, continues until G-actin falls below Cc

What happens in steady-state?

G-actin monomers exchange with subunits in the filament but no change in filament size, internal subunits slowly hydrolyze ATP

What is treadmilling?

how we describe the nature of actin polymerization and depolymerization

At what concentrations does actin treadmilling occur?

above the Cc for the plus end and below the Cc for the minus end

What is treadmilling the result of?

different rates of polymerization at both ends

How many actin binding proteins (ABP) have been found?

more than 100

What are three activities of ABPs?

sequestration, polymerization, depolymerization

What is the first problem with actin?

there is too much of it

How much of the actin pool is polymerized?

50%

Why is actin polymerization occurring all the time?

sequestration

What is thymosin?

sequesters G-actin, prevents assembly into filaments

What is profilin?

promotes actin polymerization, binds opposite to ATP binding region allowing exchange of ADP for ATP

What are lamellipodia characterized by?

a highly branching actin network at the leading edge of a cell

What are filopodia often drawn as?

a bundle of actin filaments resembling a spike like structure at the plasma membrane

What does the Arp2/3 complex do?

makes lamellipodia

What Arp2/3 proteins do?

promotes formation of branches, nucleates microfilaments upon activation

Where are Arp2/3 enriched?

at the leading edge

What do formin proteins do?

make filopodia

What are formin proteins?

potent filament nucleator and processive plus end polymerase

What is the structure of formin?

homodimer that bind to each other via the FH2 domain

What doe formin have an affinity for?

the barbed end

What is cofilin?

14kDa, actin depolymerizing factor, filament depolyermizing and fragmenting protein

What does cofilin do?

works to disassemble old filaments

Where is cofilin kept?

behind the leading edge of polymerizing actin in order to break it down so that the cell can move forward

What controls the availability of free actin?

thymosin and profilin

What sets the speed/structure of polymerization?

Arp2/3 and formins

What alters the longevity of filaments?

cofilin

What does bacterial ActA do?

binds and activates Arp2/3 at its pole

What do thermal fluctuations allow for?

insertion of additional actin subunits at the plus end near the membrane, resulting in a net forward pushing force

What are chemical modifiers of actin used in the lab?

cytochalasin D, phalloidin

What does cytochalasin D do?

binds to the plus end of actin filaments and prevents addition of actin subunits, induces F-actin depolymerization

What does phalloidin do?

prevents depolymerization of existing filaments

What are the molecular motors of microfilaments?

myosin

What are the molecular motors of microtubules?

kinesin, dynein

What is actin decorated in?

myosin motor domain

What is a fundamental property of actin?

it can form contractile structures with myosin motor proteins

What is mused to move myosin motor domain?

ATP

What do all myosins (except for myosin VI) move towards?

the plus end

What is myosin II (conventional) used for?

muscle contractions, ameboid movement and cytokinesis

What is mysoin-I used for?

endocytosis, ameboid movement

What is myosin V used for?

transport of membrane-bounded vesicles

What is myosin VI used for?

minus end directed transport of vesicles from golgi to the cell surface, endocytosis, organization of the plasma membrane and stereocilia

What is myosin II composed of?

a heavy chain that has a globular head domain and a long sequence that dimerizes, 2 light chains per heavy chain