Biomedical Engineering Reference
In-Depth Information
1. PIP
2
, in conjunction with GTP-bound Cdc42, is a strong activa-
tor of N-WASP, which in turn activates Arp2/3 (15).
2. Activated Arp2/3 mediates actin polymerization by nucleating
the sides of preexisting actin filaments. This promotes the formation of
the branched filament network found in lamellipods (16).
3. Actin polymerization by Arp2/3 can drive lamellipod protrusion
(17).
4. Ruffles form at the very same time and locations as PIP
2
local-
ization (18).
Taken together, these facts suggest that the localization of phosphoinositi-
des at the leading edge plays a crucial role in lamellipod extension.
1.2. Dynamics
The principle of the experiments used to study the spatiotemporal dynamics
in response to various chemoattractant gradients is illustrated in Figure 2b. Mo-
tile cells are transfected with chimeric proteins made by fusing a fluorescent
protein either to the molecule of interest, or to a "marker" molecule that binds
specifically to the molecule of interest and thus "reports" on it. The marker
molecules commonly used for reporting on PIP
2
and PIP
3
are the pleckstrin ho-
mology (PH) domains derived from various proteins (19). The transfected cells
are then exposed to various chemoattractant concentration profiles and the
movement of the fluorescent chimeric proteins is visualized using confocal mi-
croscopy. The chemoattractant profiles imposed include
steady or time-varying
gradients
, obtained by appropriate manipulation of the chemoattractant flow rate
through a micropipette, and
steady uniform profiles
, obtained by immersing the
cell in chemoattractant. Each chemoattractant profile reveals particular aspects
of the dynamics associated with gradient sensing.
Steady and time-varying chemoattractant gradients
show the existence
of
amplification
and help identify the first amplified component. In response to
such chemoattractant profiles, it has been observed that
When G-proteins are absent (20) or inactive (14), there is no po-
larization. Evidently, the chemoattractant profile is transmitted to
the cell through the receptors and G-proteins.
The receptors remain uniformly distributed in both neutrophils (21)
and
Dictyostelium
(22). Furthermore, receptor occupancy (23) and
G-protein activity (24,25) are not significantly polarized. It follows
that receptors and G-proteins are required for transmitting the ex-
tracellular signal, but they are not the source of the amplification.
