LABEL-FREE MONITORING OF CELL SIGNALLING PROCESSES THROUGH AFM-BASED FORCE MEASUREMENTS - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 348

for a preselected biochemical signalling event does not necessarily mean
a lack of receptor activation. Thus, because of the ability of ligands to
stabilize or stimulate subsets of receptor activities, a receptor activation
screen should not rely on a single speciic assay, but rather on an integrated
approach to measure multiple signalling events simultaneously. Unlike
label-dependent cell assays that measure speciic cellular events, cell-based
biosensing technologies such as those based on impedance mesurement 1-3
or evanescent ield at surface 4,5 have the potential to provide an integrated
cellular response. The recognition of this need for more in-depth analysis
of biological activity beyond simple speciicity, selectivity and afinity is
clear. Hence, what is required is a high content information on the action
of molecules on drug targets and, especially, a more precise proile of the
molecular pathways induced by a particular candidate drug molecule.
In this chapter, we present an experimental strategy based on atomic
force microscope (AFM) force sensing on individual cells to report on the
effects of external pharmacological stimuli on cellular functions. This kind
of cell-based biosensing allows for label-free, multimodal and real-time
monitoring of cellular responses and signalling pathways in recombinant
cell models as well as primary cell cultures related to physiologically and
pathophysiological models. Exposition of receptors present at the external
surface of cells to external chemical or biochemical messenger entities
(hormones, neurotransmitters, ions, light, scent, taste, etc) leads to the
activation of a variety of intracellular molecular signalling pathways which
are often associated with change in cell morphology, cell motility or speciic
proteins expression. Here we show that AFM-based force measurements
in conjunction with luorescence imaging of intracellular component can
ingerprint the contribution of signalling pathways subsequent to the
activation of speciic receptor at the cell membrane.

17.2 AFMBASED MONITORING OF CELLULAR RESPONSES

Because of its great sensitivity, AFM-based force measurement can detect
small magnitude morphological changes, which are not readily detectable
with conventional optical imaging techniques. Nevertheless, microscopic
observations in conjunction with mechanical assays based on lexible
substrates have provided a wealth of information pertaining to cell
contraction and locomotion.

6-8

Nanoscaled approaches such as optical and

magnetic traps, were established

as valuable tools for studying the mechanical response in individual cells
in various physiologically relevant contexts. Force measurement with the

6,7,9-11

micropipette aspiration

8

and AFM

12-18,23