LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 355

GFP-actin distribution in the late phase of the stimulation demonstrates
an extensive rearrangement of the actin structures, most notably the loss
of f-actin component ( Fig. 17.4b at 10 minutes) in the apical region and
the increase in actin density at the basal region of the cell ( Fig. 17.4c , see
arrowhead at 10 minutes). This reorganization of the actin cytoskeleton
is concomitant with the spreading of the cell body and is most certainly
responsible for the luctuation in the AFM signal in the late phase of the
stimulation. Essentially, these observations point towards the implication
of the actin cytoskeleton in the luctuating AFM signal observed in the late
phase of the stimulation as well as the cell spreading observed in the phase
contrast micrographs. In this kind of experiment, the contribution of the
actin cytoskeleton is often discriminated by pretreating the cells with the
actin depolymerizing drug latrunculin A before their stimulation by AngII.
In this case, the mechanical response is largely abolished, 23 which conirms
the essential contribution of the actin cytoskeleton in the development of
the mechanical response.
17.3.2 Fluorescence Quanficaon of the Intracellular Calcium
Level in Relaon to the AFM Force Signal
Stimulation of AT 1 receptor is well known to be linked to the activation
of several distinct signalling pathways, most notably the Gq pathway ( Fig.
17.5a ) , which is a key regulator of the cytosolic calcium concentration. 27,28
Temporal relationship between the receptor activation and the mechanical
response can also be established by measuring intracellular Ca 2+ level
as an independent biochemical indicator. Changes in intracellular Ca 2+
concentration are easily detected using the luorescent Ca 2+ probe FURA-
2/AM. 29,30 In this procedure, the cells are loaded with FURA-2/AM and
the calcium quantiied in individual cells by calculating the ratio of the
luorescence emission at 510 nm between the calcium-bound probe
(excitation at 340 nm) and the calcium-free probe (excitation at 380 nm).
Using an inverted luorescence microscope, 24,31 cytosolic calcium release can
be recorded simultaneously to force monitoring with a time resolution on
the order of 1 second ( Fig. 17.5b,c ) . Following the stimulation with AngII,
an increase in cytosolic Ca 2+ is observed and reaches a maximum within
few minutes and typically returns to baseline level. A strong correlation
exists between the mechanical and the Ca 2+ signal, thus conirming that the
mechanical response occurs as a direct consequence of AT 1 -R activation. It
can also be noted that the Ca 2+ signal occurs prior to the onset in the force
signal by approximately 30 seconds, which is consistent with the possibility
that the mechanical signal occurs as a consequence of an increased
intracellular Ca 2+ level.