Biology Reference
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Mitochondria are dynamic structures, which display basal movements
driven by the cytoskeleton. Thus, to measure and distinguish baseline
displacements from displacements caused by the AFM tip, we designed the
following experiment that included a built-in control for each cell measured. 18
NIH3T3 cells were cultured in 60 mm culture dishes. Dishes were mounted
on the temperature-controlled stage of a simultaneous AFM-luorescence
microscope
that was used to deliver precise forces to living cells. Prior to
image capture, the AFM tip was irst optically positioned ~2 μ m above the
cell and the time to contact was approximately 250 ms ( Fig. 18.2 ).
Then image capture was started at 1 frame/sec, and after collecting
several images of basal movement of mitochondria, the tip was brought into
contact with the cell at an applied force of 10 nN. The contact time of the
tip was ~3 seconds, and the total imaging time was typically 10 seconds.
By creating two luorescence image overlays (images 1 + 2, prior to the
perturbation and images 2 + 3, after perturbation) we are able to qualitatively
observe that the AFM tip does indeed produce increased displacements
of the mitochondria ( Fig. 18.2 ). Besides the obvious displacement around
the centre of the cell, displacements further away towards the cell edge are
also visible. To produce a quantitative displacement analysis, we used the
Particle Tracker plug-in for ImageJ. For each cell measured, displacements
were calculated for the average basal displacements in addition to the
average perturbed displacements of individual mitochondrial structures.
The results reveal that ~80% of cells displayed an increase in
mitochondrial displacement over the basal movements within each cell.
We found that the average basal displacement of mitochondria was 114 ±
6 nm. However, after pushing with the AFM tip, the average displacement
increased to 160 ± 10 nm (
< 8E-7) ( Fig. 18.3 ). Therefore, locally applied
forces over the nucleus induced a statistically signiicant rearrangement
of mitochondria at the cell edges, increasing ~40% following indentation
at an average distance of ~26 μ m from the point of contact. Moreover,
mitochondria are often observed to move both towards and away from the
point of contact (Fig. 18.4). In our analysis, it is clear that the mitochondria
around the nucleus also moves in response to the tip; however, it is dificult
to separate the 2D and 3D components of the motion using standard
luorescence microscopy, and we leave that analysis for a future study with
confocal microscopy (see section 18.3.2).
To investigate the role of the cytoskeleton in transmitting force, we
used the anti-cytoskeletal drugs cytochalasin D (CytD) and nocodazole
to selectively disrupt both the actin and MT networks, respectively. 54 Cells
were incubated for 30 minutes with each of the drugs (10 μ M nocodazole,
P
 
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