LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 358

and G 12/13 are largely involved in the regulation of contraction, motility and
actin-based cytoskeleton dynamics. To test their implication in force

generation observed following the stimulation of the AT 1 receptor, cells are
pretreated with the inhibitor blebbistatin. This agent is known to inhibit
myosin-II ATPase activity and lower actin-myosin afinity, 44 thus interfering
directly with the actomyosin contraction mechanism. Figure 17.6b shows
a representative mechanical response recorded by AFM where the initial
contractile response is totally abolished by blebbistatin, which indicates
that the actomyosin contractile machinery plays a prominent role in the
development of the mechanical response following the stimulation of the
AT 1 receptor.
Actomyosin-dependent contractile response requires the phosphoryl-
ation of the regulatory myosin light chain (MLC). As illustrated in Fig. 17.6a ,
phosphorylation-dependent activation of MLC was previously shown to
involve two alternative signalling pathways. The irst one involves MLCK,
a kinase regulated through the G-protein Gq and the second is due to the
direct phosphorylation of MLC by RhoK, which is regulated by the G 12/13
signalling pathway. The Rho-kinase also has the capability to inactivate the
MLC phosphatase. To test whether MLCK contributes to the mechanical
response detected by AFM, cells are pretreated with an MLCK inhibitor (ML-
9). The AngII-induced mechanical response in Fig. 17.6c is similar to the
one shown in Fig. 17.2 and therefore indicates that the inhibition of MLCK
has virtually no effect on the mechanical response of the cell to AngII. This
is conirmed by phase contrast images, which shows that the spreading
behaviour of the cell is not affected by ML-9. In contrast, pretreatment of the
cells with an inhibitor of the kinase RhoK (Y-27632) largely diminished the
initial mechanical response ( Fig. 17.6d ) . This result points towards a main
contribution of RhoK in the regulation of the mechanical response induced
by AngII. Additionally, the apparent decrease in the signal luctuation
normally observed after the stimulation (6.30 ± 0.46 nm for AngII vs. 2.23
± 0.28 nm for Y-27632/AngII) as well as the decrease in the spreading of
the cell observed in the phase contrast micrograph is consistent with the
role of RhoK in cytoskeleton remodelling. Naturally, the contribution of
several other signalling elements in the mechanical response remains to be
evaluated since AT 1 receptor signalling is not limited to classical G-proteins
but also to G-protein independent pathways involving a variety of protein
effectors such as Cdc42, Jak, β-arrestin and Src.

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Given the fact that AFM-
based force measurement requires no labelling, the contribution of these
components could be tested using appropriate modulators (inhibitor,
activator, silencing RNA) for their activities.

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