Biology Reference
In-Depth Information
A
B
10 4
8
6
10 2
4
2
10 0
0
20
60
100
140
0
10
20
30
r (pixels)
Concentration ( m M)
FIGURE 6.2
Determination of mesh size. (A) Distribution of distances between nearest-neighbor MT pixels
measured from confocal images of entangled MT networks of 5
m
M (squares), 10
m
M
P 0 e -r/x ,
where r is the nearest-neighbor distance and x is the characteristic mesh size. (B) Mesh size
of entangled MT networks as a function of tubulin concentration. Each measurement is
averaged over 28-57 images.
Adapted from Yang et al. (2012) . Reproduced by permission of The Royal Society of Chemistry.
(diamonds), and 15
m
M (circles). The distribution is fitted to an exponential, P(x)
¼
distances between filament intersections but is implemented in Cartesian coordinates
to take advantage of the natural axes of the microscope images. The distribution of
distances is plotted and fitted to an exponential P ( x )
P 0 e r / x , where r is the nearest-
neighbor distance and x is the characteristic mesh size ( Fig. 6.2 ). We find that using
an ensemble of 30-60 2D images is sufficient to obtain a robust average value of x .
Some care is required in the interpretation of x . Although x varies to some extent with
the threshold cutoff, previous studies have demonstrated that x provides a good ap-
proximation of the average 3D mesh size, but underestimates the maximum pore di-
ameter ( Lin et al., 2007 ).
¼
6.1.2.4 Analysis of average filament length
It is difficult to assess the distribution of MT contour lengths from confocal micros-
copy images of dense 3D networks. Instead, we determine MT lengths by polymer-
izing a small volume of MTs (typically at concentrations of 10-50
M) in a
microcentrifuge tube using the same chemical conditions, incubation times, and tem-
peratures that are used in generating networks for mechanical analysis. We find that a
molar ratio of 1:6 rhodamine tubulin to total tubulin is sufficiently bright, and we
include biotinylated tubulin at a molar ratio of 1:14 biotinylated to total tubulin di-
mers to enable specific attachment of the MTs to the surface via biotin-streptavidin
bonds. After polymerization, we dilute the MTs by a factor of 100 to a final con-
centration of
m
250 nM into G-PEM80 buffer supplemented with 100
m
M taxol to
Search WWH ::




Custom Search