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are incubated for
1 h under constant, gentle rotation to prevent the magnetic beads
(and latex beads, if included) from settling to the tube surface. This ensures that beads
are well dispersed through the depth of the sample. For all mechanical measurements,
we select beads that are located in the center of the tube to minimize wall effects. For
cross-linked networks, the tubulin solution is first incubated at 35
C for 3 min in a
small microcentrifuge tube, and then streptavidin is added to the desired molar ratio
of streptavidin:biotin-labeled tubulin. The solution is well mixed by gently pipetting
using a cutoff P20 pipette tip and then immediately loaded into the capillary tube,
which is sealed with vacuum grease and incubated at 35
Cfor
1 h under constant
rotation, as described earlier. In all cases, after 1 h, the networks are immediately used
for imaging and mechanical testing. We have found that the structures are robust for at
least several hours, and in some cases as long as 12-18 h, but at longer times, the
network structures degrade. Using confocal microscopy, we observe an increase in
background fluorescence and fewer and fainter MT filaments, indicating that depoly-
merization can occur at long times even in the presence of taxol.
6.1.2
Structure determination
6.1.2.1
Steady-state network morphology determined by confocal
microscopy
In order to ensure quality control, we observe the steady-state structure of every MT
network we generate, and we discard any samples that show network collapse, bead
aggregation, or other structural irregularities (
Yang et al., 2012
). We have found this
to be particularly important when testing new concentrations or cross-linking condi-
tions; however, given the protocol described above, our sample failure rate is typi-
cally
10% for fresh tubulin samples. This failure rate increases when tubulin
aliquots are subjected to multiple freeze-thaw cycles, suggesting that the quality
of the tubulin protein is an important determinant of network quality. Additionally,
we can determine the network mesh size and the extent of spatial heterogeneity with
this technique (see
Section 1.2.3
). It is possible to image the networks directly through
the thin rectangular tubes, which have a wall thickness of 90
m
m. We have found that
the use of this nonstandard thickness introduces minor spherical aberrations, but these
have not caused any significant difficulties in our analysis of the images, and the ben-
efits of using small (
L) volumes greatly outweigh the disadvantages of using
nonstandard coverslips. For applications in which such aberrations are unacceptable,
use of an objective lens with coverslip thickness correction is recommended.
Confocal microscopy images are obtained using an inverted Fluoview 500 laser
scanning system (Olympus) (
Fig. 6.1
). Steady-state structures are determined by col-
lecting two-dimensional scans (typically 1024
5
m
1024 pixels) of rhodamine-labeled
MT networks using 561 nm laser excitation and a 60
NA 1.4 oil-immersion objec-
tive, with a scan rate of
200 nm/pixel. In order to
achieve the largest dynamic range within the image, PMT gain and offset are man-
ually increased until a small number of pixels are saturated and a small number are
10 s/scan and magnification of
