Actin filaments

Monomer

ATP-actin (G-actin)

*assembles into filaments

Associated motor protein

myosin

Broad function

motility, contractility (muscle)

placement of actin w/in a cell → form outline

Actin monomer

• disassembly, ADP-G-actin = (-) end

• assembly, ATP-G-actin = (+) end

How does the interconversion of the nucleotide state affect assembly/disassembly of actin subunits?

• ATP-G-actin binds to the plus end (growing nearly x10 faster than the minus end)

• ATP hydrolysis can’t keep up with the plus end/lags behind, but catches up with additions of ADP-F-actin on the minus end (bound less tightly and eventually released)

movement of actin → treadmills backward (from + to - end)

*subunits add predominately to filament + ends

What would happen if a mutation prevented actin’s ability to bind ATP?

there would be no assembly and disassembly would occur until it reaches the critical concentration

What would happen if a mutation prevented actin’s ability to hydrolyze ATP?

the filament would assemble (G-actin → F-actin), there would be no disassembly

*filaments will continue to grow

Profilin

binds to free subunits to assist w/ association to growing filaments

*promotes actin assembly and controls assembly @ the plasma membrane

Thymyosin

binds to free subunits to prevent their association to filaments

Cap Z

blocks gain/loss of actin @ + end

*allows elongation/dissociation @ - end

Arp 2/3

nucleates assembly to connect filaments together in a branching formation

Tropomodulin

blocks gain/loss of actin @ the - end

*allows elongation/dissociation @ the + end

Cofilin

promotes depolymerization in established filaments

*cleaves actin by twisting adjacent F-actin monomers in the filament

What properties do all myosin types share?

*associated motor protein

• 20 different types

• structure → common head and specific tail domains

• 2 heavy chains (w/ ATP binding sites)

• 2 copies of each 2 light chains

What makes the types of myosin different?

some move towards the (+) end → I, II, IV

some move towards the (-) end → (only VI)

Myosin II

only myosin capable of producing contractile force

• forms bipolar filaments → can pull actin in 2 directions @ once (others only move in 1 direction towards a pole)

Cross bridge cycle

1. a myosin head w/o a bound nucleotide is locked tightly onto an actin filament (rigor configuration)

2. ATP binds to the cleft on the back of the head → conformation change of actin-binding site

3. reduces the affinity of the head for actin allowing it to move along the filament

4. the cleft closes around the ATP → causing the head to cock back

5. ATP hydrolysis occurs, but the ADP and Pi remain bound to the actin

6. weak binding of the myosin head to a new site on the actin causes release of Pi and tight binding of the head to actin

7. release triggers power stroke → generates force, the head loses its bound ADP (starts new cycle)

8. @ the end, the myosin head again locks tightly to actin in rigor conformation

How does ATP hydrolysis lead to mechanical movement w/in a sarcomere?

1. the ADP and Pi remain bound to actin

2. weak binding of the myosin head to a new site on actin → release of Pi and tight binding of head to actin

3. release triggers power stroke/force-generating change

4. head loses bound ADP and locks tightly to actin in rigor conformation (starts over)

Skeletal muscle

Ca2+ binds to troponin, moving tropomyosin and uncovering actin → allows myosin to bind → mechanical action

Smooth muscle

Ca2+ binds to calmodulin → activates/binds to myosin light chain kinase → phosphorylation of MLC → contraction

How are smooth and skeletal muscles similar?

contractions are triggered by an increase in cytosolic Ca2+

What happens during asthma attacks?

phosphorylation of MLC kinase by PKA inhibits MLC kinase activation by Ca2+/calmodulin

Why is albuterol useful for treating asthma attacks?

PKA adds inhibitory phosphate onto MLC kinase → prevents association w/ Ca2+/calmodulin

• no association = no activation of MLC kinase

• no phosphorylation of MLC = no filaments and no association w/ actin to cause contraction of smooth muscle cells

What does a migrating cell need to move in specific directions?

must use extracellular cues to establish which portion of the cell acts as the front/back ends

GTPase proteins (cellular migration)

cdc42, Rac, Rho

Cdc42

establishes filopodia and polarity/directionality in the cell @ the leading edge

Rac

changes extend and establish the front/leading edge of the cell

Rho

changes contract the rear end of the cell towards the middle

*keeps polarity in the front

How are Rac and Rho related?

they mutually inhibit one another

• are either highly expressed/constitutively active in invasive cancers

Scratch assays

detect the rate of migration of cells growth in a dish

Compared to healthy cells, what would you expect to see in a scratch assay of cancerous cells?

increased wound closure/migration

Process of cellular migration

1) Arp2/3-dependent mechanism extends one or more lamellipodia @ leading edge

2) lamellipodia adhere to substratum by formation of focal adhesions → integrin connect actin and ECM proteins (fibronectin, collagen)

3) actin-myosin II-dependent contraction @ rear end propels bulk of cytoplasm forward

4) deadhesion and endocytic recycling @ back of cell → trailing edge stays attached to substratum until tail detaches; membrane/integrins @ rear of cell and transports to front for reuse in making new adhesions

Actin assembly

can produce force for movement

When does the power stroke occur?

during the release of Pi

Microtubules

polymers of tubulin, basis of mitotic spindle

Microfilaments

composed of actin, contribute to shape/organization of plasma membrane (eukaryotic cells)

IFs

only type of cytoskeletal filament not used as tracks by motor proteins