LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 362

of intracellular lipases or kinases will most likely inluence the mechanical
and morphological state of a given cell and should thus be detectable by
AFM. This is demonstrated by AFM experiments in various receptor and
cellular contexts ( Fig. 17.8 ). First the stimulation of the toll-like receptor in
endothelial cells using the lipopolysaccharide (LPS, an endotoxin found at
the surface of Gram-negative bacteria involved in inlammatory response of
the endothelium) produces a very strong contractile-like response followed
by an important reorganization of the cell body as previously observed in
various physiological contexts. 61 Stimulation of ligand-gated ion channels
such as the ryanodine receptor found in a variety of muscle cells is well
known to trigger large amplitude contractile responses because of cytosolic
calcium mobilization. Ryanodine receptors are also expressed in other
marginally mechanically competent cells such as the HEK-293. 23,62 In Fig.
17. 8b , ryanodine receptors are stimulated with caffeine, which increases
the sensitivity of the receptor for intracellular calcium. Here again the AFM
response is consistent with the contraction of the cell body occurring as a
result of the activation of this receptor. Considering the central role played
by the second messenger calcium, one can assume that any factor inluencing
its cytosolic level will be susceptible to generate mechanical activity at the
cellular level. Figure 17.8c shows AFM-based monitoring of the highly motile
glioblastoma cell line U251. Because of their motile behaviour, the baseline
recorded on these cells usually exhibit sustained mechanical activity, which
can nevertheless be stimulated by affecting cytosolic calcium level with the
membrane ionophore ionomycin. 63 Hence, the results presented earlier
clearly illustrate that AFM-based force experiment performed on individual
cells represents a powerful tool to probe cellular processes involving
morphological or mechanical activity, as controlled by a very large variety
of ligands, receptors, enzymes or second messenger at the cellular level.
The examples presented in this chapter show that AFM-based force
measurements allow for the detection of a large variety of cellular events
originating from G-protein-coupled receptors, receptor tyrosine kinase and
ligand-gated ion channels which are activated by a variety of extracellular
signals. As mentioned earlier, the modulation of these events is susceptible
to produce speciic signatures in the force signal, mainly in terms of
amplitude, kinetic and duration, that could be use to evaluate the mechanism
of action and the potential of new drugs. In conclusion, future studies
should aim at using a variety of cell models, receptor/ligands systems and
pharmacological modulators to mechanically “ingerprint” the principal
signalling pathways and deconvolute them from each other through AFM-
based force experiments in conjunction with luorescence imaging and
monitoring of cell components.