Biology Reference
In-Depth Information
different actin polymerization transients has not been studied. However, it is
clear that no one mechanism can account for all of the barbed ends and cell
behaviour resulting from chemotactic stimulation. In neutrophils, both
uncapping of barbed ends and the nucleation activity of the Arp2/3 complex
generate free barbed ends in response to FMLP. In this system, interaction
between the uncapping and Arp2/3-complex pathways through phospho-
inositides appears to be essential for optimum nucleation (Glogauer et al.,
2000). In macrophages the stimulation of the Arp2/3 complex by WASp is
essential for chemotaxis but a role for uncapping has not yet been studied. In
carcinoma cells, both cofilin and the Arp2/3 complex are required for
lamellipodium extension: cofilin is needed to form barbed ends and the Arp2/3
complex for the growth of branched filaments (Condeelis, 2001). In addition,
there may be a strong cooperation in vivo between the cofilin and Arp2/3
complex-dependent pathways, as inhibition of cofilin by microinjection of
function-blocking antibodies is su
cient to inhibit the EGF-stimulated
generation of all barbed ends (Bailly et al., 2001). Cooperation between
cofilin and the Arp2/3 complex has been studied in vitro and involves
stimulation of the nucleation activity of the Arp2/3 complex by cofilin-
severing activity: the Arp2/3 complex prefers to bind to the sides of filaments
newly polymerized from the barbed ends generated by cofilin severing
(Ichetovkin et al., 2002).
Concerning which of the three mechanisms for barbed end generation is
responsible for the early actin polymerization transient, one would predict a
mechanism with very rapid kinetics since the early transient peaks by 5 s in
Dictyostelium and by 35-50 s in carcinoma cells (Bailly et al., 1999).
Uncapping by capping protein requires competition between PIP
2
and the
barbed end for binding to capping protein. However, PIP
2
is believed to be
hydrolysed by the activation of PLC as an early event after stimulation with
EGF. This would slow the uncapping of capping protein from barbed ends
until after re-synthesis of PIP
2
. On the other hand, cofilin-mediated severing
would be activated by PIP
2
hydrolysis during the initial PLC activation and
cofilin severing is a very fast event (Chan et al., 2000). At least 50% of
cofilin in resting carcinoma cells is in the dephosphorylated form (Zebda et
al., 2000) and that fraction of cofilin bound to PIP
2
could be activated
directly by PIP
2
hydrolysis. Additionally, activation of cofilin at the plasma
membrane does not require a recruitment step since the PIP
2
-associated
cofilin would be pre-localized to PIP
2
-rich regions. Finally, cofilin severs
crosslinked actin filaments at concentrations 50 to 100-fold lower than
previously measured for actin filaments in solution (Figure 11.5) which
would make cofilin-mediated severing of tethered filaments at the plasma
membrane extremely e
cient. In comparison, sequential recruitment of
WASp family members to the plasma membrane and then the Arp2/3
complex is a slow process in carcinoma cells taking at least 60 s (Condeelis,
