How does actin polymerization drive protrusion?

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How does actin polymerization drive protrusion?
Hypothesis 1
Hypothesis 2
Hypothesis 3
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Evidence for 1 The Acrosome reaction
Stages during fertilization of a sea-urchin egg
Elongation of the acrosomal process results from
a burst of actin polymerization at the tip. This
allows the sperm to penetrate the jelly coat
surounding the egg
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Evidence for 2 Gel Swelling mechanism of
protrusion

• 1. Protrusion in Dictyostelium starts as a bleb
  and actin fills in behind.
• 2. During the acrosome reaction
• Increased osmolarity, decreases rate of acrosomal
  actin filament elongation.
• Decreases in osmolarity, increase rate of
  acrosomal actin polymerization.

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Evidence for 3Myosin I driven protrusion
Actin filament sliding mechanism of protrusion
Myosin I at leading edge

• Myosin I walks toward end while associated
  with the plasma membrane
• Actin filaments slide rearwards, relative to
  membrane
• This may provide space for actin monomers to add
  to ends

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To understand how actin polymerization drives
protrusion we need to know

• 1. Where the nucleation of actin filaments occurs
  
• 2. How high rates of actin polymerization are
  maintained at the protruding edge
• 3. How polymerization generates a protrusive
  force
• To be covered later in this course

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APBs involved in regulating actin dynamics
Lodish 5th Ed. Chapter 19, p786-791

• 1. Dynamics
• Thymosin ?-4 (G-actin sequesterer)
• Profilin (Increases rate of polymerization)
• Gelsolin (Increases rate of actin filament
  turnover)
• Capping proteins (Increases rate of
  polymerization)
• Arp2/3 (Nucleation )

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Thymosin ?-4 and Profilin are monomer
sequestering proteins

• Fact The Cc for actin filament polymerization is
  0.1uM, the total actin concentration in a cell is
  200uM. 40 of actin in cells is unpolymerized.
  Why ?

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Microinjection of excess TB4 into cells causes
loss of stress fibers

• Although actin stress fibers are relatively
  stable turnover of actin monomers is occurring.
• Monomers leaving a stress fiber will be rapidly
  sequestered by TB4. Gradually the stress fiber
  will disappear.
• The equilibrium is shifted toward increasing
  monomer concentration at the expense of f-actin.

Before
After
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Profilin increases the rate of actin
polymerization

• Profilin binds to actin opposite the ATP binding
  cleft
• allows exchange of ADP for ATP, contrasts with
  T?-4
• Profilin-actin complex to binds readily to the
  end of the actin filament (affinity of complex gt
  than single actin monomer
• A conformational change in the complex occurs
  after binding to end actin filament, causing
  profilin to fall off

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Profilin competes with T?-4, for actin monomers

• When a small amount of profilin is activated it
  completes with thymosin for G-actin and rapidly
  adds it to the end of F-actin
• The activity of profilin is regulated by
• phosphorylation, binding to inositol
  phospho-lipids
• The activity of profilin is increased close to
  the plasma membrane by binding to
• acidic membrane phospholipids, certain proline
  rich proteins that localize at the plasma membrane

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Role of profilin during the Acrosome reaction
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Functions of the actin cytoskeleton dependent on
polymerization

• The acrosome reaction.
• The rapid formation of an acrosomal process
  penetrates the thick jelly coat of the sea urchin
  egg allowing nuclear fusion between sperm and
  egg.
• Before fertilization short actin filaments lie in
  a pocket at the head of the sperm together with
  many profilin-actin complexes
• Upon contact with the egg, the acrosomal vesicle
  is exocytosed, uncovering ends of actin
  filaments.
• At the same time, profilin (of the profilin-actin
  complex) is activated resulting in the rapid
  addition of G-actin to the exposed ends of the
  pre-existing actin filaments
• This results in an explosive elongation of the
  acrosomal process
• The acrosomal process contacts the egg plasma
  membrane and fuses with it.
• The sperm and egg nuclei fuse.

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Experiment to demonstrate the location of newly
polymerized actin

• 1. A fibroblast was microinjected with rhodamine
  (red) labeled actin monomers
• 2. Cell was fixed shortly after microinjection.
• 3. The cytoskeleton was stained with fluorescein
  phalloidin (green).

• New actin polymerization occurs within the actin
  cortex that lies just beneath the plasma membrane
• Actin polymerization in this location can form a
  variety of surface structures
• Microvilli, filopodia, lamellipodia
• Nucleation of actin filament growth is regulated
  by external signals
• Nucleation is initiated by a comlex of 7 proteins
  called the ARP2/3 complex

All actin is labeled in a lamellipodium
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The role of Arp2/3 in protrusion

• Arp2/3 is a highly conserved complex of 7
  proteins, including 2 actin related proteins
  (Arp2 and Arp3)
• Identified first in the cortical (submembranous)
  actin of amoebae
• Found in highly dynamic actin structures in many
  cell types
• e.g. Listeria (actin tails), edge of
  lamellipodia, cortical actin patches (yeast)

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Arp2/3 nucleates actin filament assembly

• Arp2/3 is present at high ( 10uM) concentrations
  in motile cells e.g. leukocytes
• Arp2 and Arp3 are 45 similar to actin monomers

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Arp2/3 provides a template for actin filament
growth

• Arp2/3 nucleates actin filament by binding to the
  - end of the actin filament
• Arp2/3 can bind to the sides of pre-existing
  actin filaments, resulting in the development of
  a branching mesh of actin filaments
• Nucleation is more efficient when ARP2/3 is bound
  to the side of an actin filament

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Distribution of Arp2/3 in a moving cell
Svitkina and Borisy 1999
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How is Arp2/3 activated

• WASp, Wiskott-Aldrich Syndrome protein, mutated
  protein leads to bleeding, immunodeficiency - is
  rich in proline
• WASp is activated when it binds PIP2 and active
  Cdc42 (small GTPase)
• VCA domain of WASp is necessary for Arp2/3
  activation - binds actin and ARP2/3, --increases
  affinity of ARP2/3 to side of filament
• Other (proline rich) activators of ARP2/3 include
  VASP (Vasodilator-stimulated phosphoprotein),
  Scar/WAVE family proteins

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A conformational change occurs when Arp2/3
activated

• VCA domain of WASp becomes more compact when
  bound to G-actin
• A conformational change occurs so that ARP2 and
  ARP3 move closer together, to form a template for
  actin filament growth
• In budding yeast and Dictyostelium Myosin I may
  bind (via SH3 domains) ARP2/3 possibly
  transporting it to the protruding edge.