Regulation of actin filament dynamics in vivo
Fig. 16-52, Alberts
G-actin
•
Drugs that destabilize actin
filaments e.g. cytochalasins
– Cytochalasin D (binds +end of
f-actin)
– Latrunculin (binds G-actin)
F-actin
•
Drugs that stabilize actin
filaments, e.g. phalloidins
– (bind to sides of f-actin)
The Roles of Actin Binding Proteins
Dynamics/structure
Structure: Filamin,
ABP-120, 240 etc.,
Structure:Fimbrin,
Villin, -actinin
Actin-Membrane Binding:
Vinculin, Ponticulin, -actinin
Dynamics:Cofilin,
ADF
Dynamics:Gelsolin
Actin binding proteins that regulate dynamics
Lodish 5th Ed. Chapter 19, p786-791
• Actin binding proteins (APB’s) modulate the function of the actin
cytoskeleton
• 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 )
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
?
•
Proteins in the cytoplasm sequester or bind to actin monomers - preventing
them from polymerizing. Factors which influence the binding of these proteins
to actin monomers will affect the rate of actin polymerization.
Active sequesterers
Inactive sequesterers
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.
After
Before
The 3D structure of the actin-profilin complex
Fig. 18-14
•
Profilin is a weak actin sequesterer
•
It binds opposite the ATP binding cleft
(in contrast to T-4)
•
This allows exchange of ADP for ATP
to occur (recharging the monomer)
•
Profilin facilitates addition of actin
monomers to the growing + end of Factin
– Because affinity of actin-profilin
complex > than single actin monomer
– it speeds up rate of polymerization at
+end
Regulation of actin polymerization by profilin
Fig. 18-15
Regulation of actin polymerization by profilin
(explanation of previous slide)
•
A)Before a signaling event, profilin is inactive because it is bound to PIP2 in the
plasma membrane.
•
B1) Following a signaling event PIP2 is cleaved by phospholipase C, releasing
profilin – which is now active.
B2) When a small amount of profilin is activated it completes with T-4 for G-actin
and rapidly adds it to the +end of F-actin.
•
•
C) In addition to increasing the rate of actin polymerization at the + end, profilin
sequesters ADP-G-actin, allowing exchange of ADP for ATP – thus increasing the
pool of ATP-G actin (which indirectly increases the rate of actin polymerization).
•
The activity of profilin is maintained close to the plasma membrane because the
activated profilin binds to acidic membrane phospholipids and certain proline rich
proteins (will talk about these later) that localize at the plasma membrane.
Actin binding proteins modulate the structure of the actin
cytoskeleton
Fig. 16-65 and 66, Alberts
•
•
Actin forms a wide variety of structures in the cell, some are more permanent
than others. Many different structures can coexist in the same cell.
There are 3 basic types of actin structure
– contractile bundles-containing -actinin,
– gel-like networks containing filamin
– tight parallel bundles containing fimbrin.
filamin
 -actinin
fimbrin
Actin filaments and actin-binding proteins in a
microvillus
•
•
•
•
Microvilli are permanent finger-like projections (0.08um wide, 0.5-10um in length)
Each structure contains a core of actin filaments cross-linked by proteins such as fimbrin,
villin and myosin I (crosslinks F-actin to the plasma membrane)
They are found were the cell membrane faces a fluid environment.
They increase surface area up to 20 times in gut epithelial cells
Actin structures formed
by ABP’s
Filopodia
(fimbrin)
Actin meshworks form ruffles
on cell surface (filamin)
( -actinin)
ABP’s can link actin filaments to the plasma membrane
• Function: They attach to actin cortex to integral membrane proteins
• Provide stiffness to cell membranes and immobilizes some membrane
proteins.
Filamin provides rigidity to lamellipodia and support for
the plasma membrane
•
•
•
•
•
Cells that lack filamin, cannot move because cells cannot
form rigid protrusions
Cells form blebs because the actin cortex is not stiff enough
to prevent the cell’s cytoplasm from “bulging - out”
When the filamin gene is transfected into the same cell
type, cells can move and do not form blebs
Filamin crosslinks actin
filaments into 3-dimensional
gels and flat sheets
The hinge region of filamin
allows actin filaments to
change orientation with respect
to each other
Actin binding proteins that link the plasma membrane to
the actin cytoskeleton
• The actin filaments within cellular
surface features, must be linked
to the membrane since the actin
cytoskeleton exerts protrusive
and contractile forces on it.
– e.g. endocytosis, phagocytosis
– protrusion and retraction of the cell
edge during cell movement
ERM proteins attach actin filaments to the plasma membrane Ch.
16, p.936
•
•
•
•
ABPs that crosslink the actin cytoskeleton to the plasma membrane are important for cell
shape and strength of the plasma membrane
Ezrin, radixin, moesin were the first three ERM proteins discovered
C-term. domain binds actin, N-term. domain binds transmembrane proteins such as
CD44
The activity of these proteins is regulated by extra and intracellular signal
– Folded state - inactive, unfolded state, active