Molecular Biology of the Cell by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morgan, Martin Raff, Keith Roberts, Peter Walter by by Bruce Alberts, Alexander Johnson, Julian Lewis, David Morg

900 Chapter 16: The Cytoskeleton
100
% actin subunits in filaments
nucleation
(lag phase)
actin
subunits
elongation
(growth phase)
concentration of
monomers at
steady state = C c
steady state
(equilibrium phase)
actin filament
with subunits
coming on and off
growing
actin filament
100
% actin subunits in filaments
elongation
(growth phase)
C c unchanged by
addition of nuclei
growing
actin filament
steady state
(equilibrium phase)
actin filament
with subunits
coming on and off
0
oligomers
0
preformed filament seeds added here
(A)
time after salt addition
(B)
time after salt addition
Figure 16–13 The time course of actin polymerization in a test tube. (A) Polymerization of pure actin subunits into filaments
occurs after a lag phase. (B) Polymerization occurs more rapidly in the presence of preformed fragments of actin filaments,
which act as nuclei for filament growth. As indicated, the % free subunits after polymerization reflects the critical concentration
(C c ), at which there is no net change in polymer. Actin polymerization is often studied by observing the change in the light
emission from a fluorescent probe, called pyrene, that has been covalently attached to the actin. Pyrene-actin fluoresces more
brightly when it is incorporated into actin filaments.
phase of rapid filament elongation during which subunits are added quickly to
the ends of the nucleated filaments (Figure 16–13A). Finally, as the concentration
of actin monomers declines, the system approaches a steady state at which the
rate of addition of new subunits to the filament ends exactly balances the rate
of subunit dissociation. The concentration of free subunits left in solution at this
MBoC6 m16.10/16.13
point is called the critical concentration, C c . As explained in Panel 16–2, the value
of the critical concentration is equal to the rate constant for subunit loss divided
by the rate constant for subunit addition—that is, C c = k off /k on , which is equal
to the dissociation constant, K d , and the inverse of the equilibrium constant, K
(see Figure 3–44). In a test tube, the C c for actin polymerization—that is, the free
actin monomer concentration at which the fraction of actin in the polymer stops
increasing—is about 0.2 μM. Inside the cell, the concentration of unpolymerized
actin is much higher than this, and the cell has evolved mechanisms to prevent
most of its monomeric actin from assembling into filaments, as we discuss later.
The lag phase in filament growth is eliminated if preexisting seeds (such as
fragments of actin filaments that have been chemically cross-linked) are added to
the solution at the beginning of the polymerization reaction (Figure 16–13B). The
cell takes great advantage of this nucleation requirement: it uses special proteins
to catalyze filament nucleation at specific sites, thereby determining the location
at which new actin filaments are assembled.
Actin Filaments Have Two Distinct Ends That Grow at Different
Rates
Due to the uniform orientation of asymmetric actin subunits in the filament, the
structures at its two ends are different. This orientation makes the two ends of
each polymer different in ways that have a profound effect on filament growth
rates. The kinetic rate constants for actin subunit association and dissociation—
k on and k off , respectively—are much greater at the plus end than the minus end.
This can be seen when an excess of purified actin monomers is allowed to assemble
onto polarity-marked filaments—the plus end of the filament elongates up to
ten times faster (see Figure 16–12). If filaments are rapidly diluted so that the free
subunit concentration drops below the critical concentration, the plus end also
depolymerizes faster.
It is important to note, however, that the two ends of an actin filament have the
same net affinity for actin subunits, if all of the subunits are in the same nucleotide