INVESTIGATING MAMMALIAN CELL NANOMECHANICS WITH SIMULTANEOUS OPTICAL AND ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 386

differential TFM analysis 103 in which we measured the change in traction
forces as a function of time. This is in comparison with the absolute traction
forces that are typically determined by removing the cells with trypsin after
an experiment to measure the unstressed bead positions. 94,99 Foregoing the
trypsin step allowed us to measure more cells per dish and quickly obtain
a reliable statistical sample. Traction analysis was carried out using the
LIBTRC-2.0 analysis libraries developed and kindly provided by Professor
M. Dembo (Boston University).
Cells on the GXG gels described earlier did not display any signiicant
traction force dynamics when left unperturbed. However, the cells
demonstrated a signiicant increase in cellular traction force over time in
response to applied loads. What is particularly important to notice is that
applied forces to the cell nucleus are not merely transmitted through the cell
and to the substrate in a circular deformation proile. In reality, the applied
force is converted into biochemical signalling which results in localized
“hot spots” randomly distributed over the cell contact area as seen in Fig.
18.11 . These areas of large magnitude traction forces are discontinuous,
heterogeneous and increase over time in response to a constant applied force
to the nucleus. Consistent with our imaging of cytoskeletal deformation,
force appears to be rapidly transduced throughout the cell (increase in
cellular traction observed within 30 seconds) and applied forces are not
simply transmitted through the cell as if it behaves as an isotropic and
continuous medium.
To directly probe the origin of the cellular traction forces, we transiently
transfected the cells with zyxin-RFP which is a protein found in stable focal
adhesions and known to be mechanically regulated. Simultaneous imaging

(a)

(c)

(b)

Figure 18.11. Traction force maps of a single cell over 2 minutes in the absence of
any applied forces (a) and with a constant 10 nN force applied to the nucleus (b)
(scale bar = 15 μ m). From visual inspection, it is clear that the cell generates transient
changes in traction forces in the absence of mechanical stimuli. However, a mechanical
stimulus results in the generation of distinct “hot spots” in which traction forces
increase rapidly. The average traction force per cell is plotted as a function of time in
(c). Traction forces in control cells (red) do not vary signiicantly over time but rapidly
increase in cells that are mechanically stimulated (black).