LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 357

cell contraction, annexin, 35 involved in the interaction of cytoskeleton with
the membrane and gelsolin, which severs and caps actin ilaments. Most

importantly, calcium is involved in the molecular process of actomyosin
contraction. 37 To delineate the contribution of the signalling pathways
activated by a given receptor in the development of mechanical response
in individual cells, one should decouple or isolate their contributions. This
can be achieved using pharmacological inhibitors or activators of molecular
components involved downstream of receptor activation. Alternatively,
silencing RNA can also be used to evaluate the contribution of key structural
and functional proteins. 38 As presented in introduction, force sensing over
individual cells provides a direct readout for the contribution of signalling
pathways in response to the activation of speciic receptors at the cell
surface.

36

17.4 AFM FORCE MEASUREMENT ON INDIVIDUAL CELLS AS
A TOOL TO DELINEATE CELL SIGNALLING EVENTS

Stimulation of cell receptors leads to the activation of several distinct
signalling pathways involving a variety of structural and functional
intracellular proteins and biochemical second messengers. Receptor
activation involves a large variety of biomolecular events, including ligand-
receptor recognition, receptor activation, protein recruitment at speciic
site, production of second messengers, activation or inhibition of kinases
and phosphatases, internalization of receptors, cytoskeleton remodelling,
external adhesion contact remodelling and gene expression. The modulation
of these events is susceptible to produce a speciic signature in the force
signal, mainly in terms of amplitude, kinetics and duration.
AT 1 receptor activation in various cell type, such as endothelial cells,
vascular smooth muscle cells and cardiac myocytes, is well known to
activate several distinct signalling pathways ( Fig. 17.6a ) , including two
pathways signalling through the G-proteins Gq and G 12/13 . 27,28 Activation of
the G-protein Gq leads to phospholipase C (PLC) activation, production of
inositol triphosphate (IP 3 ) from the phosphatidylinositol phosphate lipidic
membrane pool, activation of the IP 3 receptor causing a release of Ca

from
the endoplasmic reticulum into the cytosol and concomitant activation
of numerous kinases. In contrast, G 12/13 activation by the AT 1 receptor
leads to the activation of the small GTP-binding protein RhoA, 39 a process
facilitated by a guanine exchange factor. Once activated, RhoA then activates
its effector RhoA kinase (RhoK). In muscle and nonmuscle cells, both Gq

2+