INVESTIGATING MAMMALIAN CELL NANOMECHANICS WITH SIMULTANEOUS OPTICAL AND ATOMIC FORCE MICROSCOPY - Life at the Nanoscale: Atomic Force Microscopy of Live Cells - page 382

A simultaneous AFM and LSCM was used to acquire confocal stacks before
and after the stress-relaxation experiments allowing us to examine the 3D
deformation and relaxation of the cytoarchitecture. 3 The two stacks were
then subtracted to produce an image in which contrast is generated from
the movement of speciic structures relative to their initial positions. Several
general phenomena were observed to occur during the viscous relaxation
and deformation of the architecture in this cell type. F-actin ilaments were
not observed to signiicantly deform or remodel under 2 nN and up to 10 nN
of force. This is in contrast to mouse NIH3T3 ibroblasts ( Fig. 18.7 ) in which
F-actin ilaments were observed to deform readily.
The MT and IF networks clearly deform in response to force applied
above the nucleus ( Fig. 18.9 ), as evidenced by the formation of ilamentous
structures in three dimensions after subtraction. MT deformation notably
occurs throughout the cell, including at the cell edge often >30 μ m away
from the point of force. Furthermore, ilaments do not appear to move in a

(a) (b)

(c)

Figure 18.9. (a) A subtraction image of GFP-actin before and after the stress-
relaxation experiment reveals no signiicant F-actin deformation in human ibroblast
cells (scale bars = 10 μ m). 3 However, the microtubule cytoskeleton (b) reveals
signiicant deformation and as evidenced by ilamentous contrast in the subtraction
image. (c) A zoom of the area in (b) presented as a green-red overlay demonstrates
how ilaments move both towards and away from the contact point (white cross).