4 - Actin (micro)filaments

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what properties do actin and microtubules share?
are polymers that are highly dynamic (can grow from one end of the other end) and they are polarized (must know which end motor proteins are moving towards)
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what fuel do actin microfilaments use for formation?
ATp binding proteins
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List all the different structure made out of actin?
microvill, cell cortex, adherens belt, filopodia, lamellipoidum/leading edge, stress fibres and conttractile ring
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Describe the structure of actin?
globular protein (G-actin), divided by a central cleft that binds ATP. filaments appear as 2 strands of subunits. One repeating unit consists of 28 subunits (14 in each strand)-72nm length. filament has a clockwise helical twist ->symmetry every 36nm
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what happens to ATP-G-actin monomers during nucleation/elongation?
form stable oligomers of actin (filament nuclei) slowly by bumping into each other -> actin filament grows by addition of subunits to both ends until steady state is reached
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What happens during steady state to actin filament?
the ends of F-actin filaments are in a steady state with monomeric G-actin. This is achieved at the critical concentration Cc. Below the Cc the filament will disassemble, above the Cc the filament will get longer
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What happens if preformed filaments are added?
added there is no lag (nucleation) phase but the Cc remains constant.
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How does addition/dissociation of ATP-G-actin filaments vary at the plus and minus end?
addition is is much (10X) faster at the plus (+) end than the minus (-) end, whereas the rate of dissociation is similar. . ATP-actin is added preferentially at the plus end while ADP-actin disassembles at the minus end, giving rise to treadmilling
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How can treadmilling of actin filaments be demonstrated?
by laser photobleaching in a stationary HeLa cell. GFP labelles actin -> near the leading edge a bundle of actin filaments was zapped with a laser. The bleached zone is seen to move backwards - away from the leading edge of the cell.
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Role of Cytochalasin B, D
Binds monomers and barbed ends, inhibits polymerization
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Role of Tolytoxin
Binds monomers and barbed ends, inhibits polymerization 1000 X more effective than cytochalasin B
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Role of Latrunculin A
Binds monomers and inhibits polymerization
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Role of Phalloidin
Binds and stabilizes filaments, can be labeled with a fluorescent dye, making it very useful for staining actin filaments in cells
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Role of Botulinum toxin
This ADP ribosylates the monomer, stabilising G actin.
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What are actin binding/ accessory proteins needed for?
Monomer binding proteins, Filament nucleators, Filament capping proteins, Filament severing proteins, Filament side binding stabilisers, Bundling proteins, Cross-linking proteins, Membrane linkers, motors
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How concentrated is G-actin in cells?
is 1000 times more concentrated than the critical concentration for actin filament formation. However most G-actin is bound to thymosin-b4 and this cannot be incorporated into filaments
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Role of profilin?
binds G-actin more weakly than thymosin-b4, but stronger than actin + ends. Uniquely it allows ADP/ATP exchange and can promote ATP replacement and promotes actin incorporation into filaments. Profilin is mostly at the plasma membrane bound to PIP2.
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What does cofilin do?
) binds to the sides of ADP-actin in the filament, inducing them to fragment. In this manner cofilin replenishes the pool of free ADP-actin which can be recharged by profilin to be used again
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How does dissociation of ADP-actin filaments occur?
the first requiring cofilin which chops off 18-20mers and a secondary step which requires Aip1 (actin-interacting protein 1) which chops the 18-20mers to monomers
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What do capping proteins do?
bind to the ends of filaments to regulate the dynamics and treadmilling of actin filaments so actin filaments don't grow and shrink in an unregulated manner
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Examples of capping proteins?
CapZ (regulated by lipids at the plasmamembrane - so filaments are less likely to be capped near the cell cortex and thus can grow) and tropomodulin (binds the minus ends of filaments and in doing so promotes filament growth)
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What is formin?
multi-domain protein containing Rho GTPase binding domain (RBD), a profilin-ATP-actin binding domain (FH1) ) and an filament nucleating domain (FH2)
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What happens to formin when RBD is not bound to Rho?
binds and inhibits the FH2 domain. When Rho GTPase becomes activated formin is recruited to the plasmamembrane. This causes a conformational shift which releases the FH1 and FH2 domains to trigger filament formation.
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What does the Arp2/Arp3 complex do?
induces branching of actin filaments. The angle at which branch filaments are nucleated is fixed at 70o. The Arp2/Arp3 complex is frequently located near the cell membrane and can be activated by proteins such as WASp
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What is WASp?
is a multi-domain protein containing Rho GTPase binding domain (RBD), an actin binding domainand an Arp2/Arp3 complex binding domain
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What happens to WASp when RBD is not bound to Rho?
RBD prevents binding to the Arp2/Arp3 complex. When Cdc42 GTPase becomes activated WASp is recruited to the plasmamembrane. This causes a conformational shift which releases the Arp2/Arp3 binding domain to trigger branch formation.
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What are microvilli?
stable extensions of the plasma membrane produced by the close packing of actin filaments by villin and fimbrin which are short cross-linking proteins - if villin or fibrin are over-expressed in a fibroblast they will make microvilli
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Difference between alpha actin and fimbrin?
alpha-actin form dimers which bundle either parallel or anti-parallel filaments in contractile bundles.contrast fimbrin only bundles filaments of the same polarity and packs them more tightly
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Role of filamin?
dimers with itself in such a way that the actin binding sites are at an angle to one another. Filamin-actin networks form gels that resist rapid deformation, but adjust to tension slowly. So cells resist abrupt forces, but slowly change shape.
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What is dystropin?
a very large skeletal muscle protein which makes up 5% of membrane-associated cytoskeleton in voluntary muscle cells and links cortical cytoskeleton actin filaments to the extracellular matrix via a transmembrane glycoprotein - Duchenne muscular dys.
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what fuel do actin microfilaments use for formation?

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Card 3

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List all the different structure made out of actin?

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Card 4

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Describe the structure of actin?

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Card 5

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what happens to ATP-G-actin monomers during nucleation/elongation?

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