W. Richard Bowen and Nidal Hilal 4

198 7. MICRO/NANOENgINEERINg ANd AFM FOR CELLULAR SENSINg
mechanical stress across the plasma membrane and convey the traction
force that develops in the cytoskeleton to the ECM. Unbound integrins
are mobile within the cell membrane and readily form clusters and focal
adhesions in a tension-dependent manner [11]. Integrins also participate
in other signalling transductions that regulate cell growth [12].
During the initial phase of cell adhesion, interactions between the
integrins and the ECM ligands are independent of force, but rapidly the
resultant adhesion induces the activation of Rac and Cdc42 protein pathways,
leading to the formation of filopodia and lamellipodia (Figure 7.1).
These structures create a small adhesion site, called a focal complex (in
the order of ~1 �m). From this point, cells start to exert traction forces on
the ECM, with about 0.8–0.9 nN �m �2 being exerted by lamellipodia [10].
Filopodia are effectively the “antennae” of the cell, formed at the leading
edge. Focal adhesions and focal complexes recruit the same core proteins
[13]; however, focal adhesions are much larger in size and integrins packing
density, and produce larger forces of a few nanonewton per square
micrometre [14]. Focal complexes can mature into focal adhesions if there
is an increase in force at the adhesion site [10, 15, 16], or by the activation
of the Rho pathway [13]. Thus, physical tension between a cell and the
ECM appears to be essential for focal adhesion formation and any subsequent
firm adhesion.
Finally, it should be noted that cell adhesion, spreading and migration
require assembly and disassembly of multiple focal adhesions. This is
regulated by integrin–ligand binding events and can be stimulated by
properties associated with the ECM (e.g. ligand densities and stiffness
of the matrix) as well as by intracellular signals [9, 17, 18]. To date, the
detailed mechanisms that regulate the organisation of these adhesive
complexes are yet to be elucidated. However, abundant evidence suggests
a highly dynamic feedback loop between a cell and its microenvironment,
which is constantly modulated by delicate changes in a vast range of
(bio)chemical and physical parameters.
7.2 EngInEErIng tHE ECM For ProbIng
CEll SEnSIng
A typical animal cell is �10–100 �m in size. The major components of
focal adhesion sites, such as integrins, talin and vinculin, are proteins with
dimensions in the range of several nanometres. Both of these size scales
require sophisticated tools for visualisation and manipulation of these
functional components. However, the local microenvironments of cells are
inherently heterogeneous and dynamically remodelled at different stages
in the life cycle of a cell. All of these factors can lead to great uncertainty
and variability in the interpretation of observations. Nevertheless, it is