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(Svitkina et al., 1997). This means that a cell extending its leading edge at
0.2 mm per second must remodel the branched network in 5 s or less. Given
this brief lifetime, the branched zone will be seen only in lamellae that are
actively expanding at the time of preparation for microscopy. Choosing
keratocytes, cells that move rapidly at a constant rate, may have contributed
to the success of Svitkina and Borisy (Svitkina et al., 1997) in preserving
branches that were missing in earlier studies.
Remodelling must involve two steps: dissociation of branches; and the
conversion of short filaments into long filaments. How does this happen? ATP
hydrolysis and phosphate dissociation destabilize branches and are also likely
to be the timer for disassembly. The rate constant for ATP hydrolysis is
0.3 s 71 (Blanchoin and Pollard, 2002) and nothing has yet been found that
influences this reaction rate. Thus newly assembled ATP-actin subunits
hydrolyse their bound ATP with a half-time of about 2 s. Phosphate
dissociation is much slower, with a half-time of 350 s (Carlier, 1987; Blanchoin
and Pollard, 1999), far too slow to account for debranching in cells. However,
ADF/cofilin strongly accelerates phosphate dissociation from ADP-P i actin
filaments (Blanchoin and Pollard, 1999) to rates that keep pace with
hydrolysis. Rate of phosphate dissociation depends on the concentration of
active ADF/cofilin and involves a very low-a nity transient interaction of
ADP/cofilin with the filament. Phosphorylation of ADF/cofilin by LIM
kinase downstream of PAK (p21-activated kinase) (Edwards et al., 1999),
blocks this and other interactions of ADF/cofilin with actin (Blanchoin et al.,
2000c) and is expected to slow phosphate dissociation and to stabilize
Short filaments might be converted to long filaments by subunit addition to
the filament ends or by end-to-end annealing, a very favourable reaction
(Andrianantroandro et al., 2001). Capping barbed ends is expected to prevent
both of these reactions, so the cell must have a mechanism to avoid it. VASP
appears to inhibit capping near the leading edge (Bear et al., 2002), so it may
also promote annealing in the presence of capping protein.
Actin filaments in the lamella turn over on a time scale of tens of seconds.
Both the pioneering photoactivation observations (Theriot and Mitchison,
1991) and recent speckle microscopy experiments (Watanabe and Mitchison,
2002) show that depolymerization occurs broadly behind the leading edge.
Pure actin filaments are stable for days, treadmilling at less than 0.1 subunits
per second at steady state and capping will stabilize their dynamic barbed
ends. So, how do filaments turn over rapidly in cells? ADF/cofilin is the prime
candidate to drive filament disassembly in cells, since ADF/cofilin and profilin
increase the turnover of filaments in vitro (Carlier et al., 1997; Rosenblatt et
al., 1997). The higher a nity of ADF/cofilins for ADP-actin monomers than
ADP-actin filaments provides the thermodynamic basis for their ability to
depolymerize filaments (Blanchoin and Pollard, 1999) but does not reveal the
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