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cells. The average natural displacement was ~50% lower in cells treated with
either one of the two drugs, in comparison with the natural displacement
in untreated cells. These data suggest that the cytoskeletal network has an
important role to play in governing natural motions of mitochondria within
living cells. It shows that natural mitochondrial motion is strongly dependent
on both intact actin ilaments and the MT network, conirming indings on
the cytoskeleton's role in mitochondrial transport.
78,82,83
To examine the role focal adhesions play in governing force transduction
through the cytoplasm, we treated the cells with retinoic acid (RA).
Retinoids are naturally occurring derivatives of retinol (vitamin A) and
have an important role in gene regulation and control in a variety of cellular
and tissue processes, including proliferation, cell differentiation and
apoptosis.
84,85
These compounds also have wider functions relected in their
diverse effects on the regulation of speciic genes,
86
including impacting on
cell adhesion mediated by integrin cell adhesion receptors.
87
RA has been
shown to stimulate keratinocyte growth in culture and also to inhibit the
ECM molecules ibronectin (FN) and thrombospondin.
87
Similar results on FN
inhibition were observed on 3T3 ibroblasts. Adhesion to the substrate was
also reduced after treatments with RA, together with a decrease in attachment
and spreading.
87,88
Treatment with 20
μ
M RA led to a distinct decrease
in the number of focal adhesions by ~50% while leaving the cytoskeleton
intact
(
Fig. 18.5b,c
). Concomitant with the decrease in FAs was a decrease
in the basal movement of mitochondria and no effect of applied forces on
mitochondrial displacements in a fashion similar to CytD and nocodazole-
treated cells (
Fig. 18.5a
).
In each case of drug treatment, the cellular Young's moduli were
also observed to decrease signiicantly
74
(
Fig. 18.6
). Moreover, force
curves measured with pyramidal tips and cantilevers modiied with 19
μ
m microspheres demonstrate that although the absolute value of the
Young's modulus was dependent on tip geometry, the relative decrease in
Young's modulus remains approximately constant (
Fig. 18.6
). These data
demonstrate that the local and global mechanical properties of the cell
are signiicantly impaired after treatment with the drugs. Importantly, it is
clear that the cell requires an intact actin and MT cytoskeleton in addition
to strong connections to the microenvironment via focal adhesions to
maintain and regulate its stiffness. Moreover, all three of these elements of
the cytoarchitecture are required for the transmission of force throughout
the cell. The data presented thus far have revealed that the mechanical
properties of the cell are regulated through the complex interplay of
several architectural elements. By tracking the displacement of intracellular
74
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