Biomedical Engineering Reference
In-Depth Information
similarly demonstrates that actin network growth and polymerization are suf-
ficient for generating protrusive forces.
The maximal force that can be generated by the polymerization motor
can be estimated from the differences in the free energy of an actin subunit
in solution and bound to a filament. Taking the intracellular actin monomer
concentration to be
50
μ
M [45], this amounts to a few pico newtons per
filament (reviewed in [48]), comparable to other molecular motors. The elastic
Brownian ratchet model [53] provides a mechanism for how this free energy
can be translated into mechanical force that can push the cell membrane
forward. In this model, thermal fluctuations cause an actin filament to bend
away from the membrane, introducing transient gaps that occasionally allow
an additional monomer to be incorporated into the filament. The elastic force
of the lengthened filament then exerts a pushing force on the membrane.
The intrinsic polar behavior of a large number of individual actin filaments
and their ability to generate protrusive forces must be coordinated to enable
large-scale protrusion of the cell as a whole. The dendritic nucleation model
describes how this is achieved at the leading edge of a moving cell. Actin
filaments are oriented with their barbed ends towards the leading edge, so
that the addition of new subunits to the growing barbed ends provides the
protrusive force that drives the lamellipodium forward [38, 53]. New filaments
are nucleated mainly in the vicinity of the leading edge by branching off the
sides of existing actin filaments through the action of the Arp2/3 branching
protein complex [20, 54]. This results in the formation of a dense dendritic
actin meshwork. As shown in Figure 2.3, this meshwork is highly ordered in
keratocytes with filaments oriented with their barbed ends at approximately
±
∼
10
−
35
◦
with respect to the leading edge [55]. This organization allows the in-
trinsically polar behavior of individual actin filaments to be translated into
large-scale treadmilling of the cell as a whole, with assembly of the dendritic
meshwork at the front and disassembly toward the rear (reviewed in [1, 7]).
The actin in the lamellipodium is highly dynamic, constantly being assem-
bled and disassembled from the meshwork with a half-life of tens of seconds
[34, 56, 57]. With such a rapid turnover rate, it is obviously essential to inves-
tigate the dynamics of the actin meshwork and not just its static structure.
Initially, the pattern of filamentous-actin flow was studied by photoactivation
of fluorescent actin that enabled tracking of the movement of a spatially de-
fined subset of actin filaments within a rapidly moving keratocyte. The actin
network in the front of the lamellipodium was found to remain nearly station-
ary with respect to the substrate (i.e., in the laboratory frame of reference),
and therefore moved rearward with respect to the cell's leading edge (i.e., in
the frame of reference that moves with the cell) at the same rate as net cell
translocation [34, 39]. More detailed mapping of the actin meshwork flow has
been facilitated by fluorescent speckle microscopy (FSM) [56, 57, 58, 59, 60].
FSM allows simultaneous mapping of actin meshwork flow across the entire
lamellipodium with high temporal and spatial resolution. As shown in Figure
2.4 the introduction of low levels (estimated to be
<
0
.
1
μ
M final concentration
