Statistical Appendix
1. Analysis of comet tail directionality in control neurons
1a. sector analysis
Quantitive analysis of the comet tails directional propagation in 5 different cells was
performed relative to the long-axis of the axon. We analyzed 419 comets and found
that 24h after micro-injection of EB3-GFP encoding mRNA, 69.53% ±1.97% of the
MT's plus ends labeled by EB3-GFP polymerize anterogradely towards the tip of the
axons (within a sector confined between +60º and -60º; n>500 comet tails). It should
be noted that the trajectory of the EB3-GFP comet tails is very close to the long axis
(0º) of the axon (Figure 3B). A fraction of 14.23% ± 1.76% of the EB3-GFP comet
tails translocated retrogradely (defined as comet tails propagation within the sector
confined by +120º and -120º relative to the long axis of the axon). The rest of the
EB3-GFP comets (16.24 ± 0.77%) pointed perpendicular to the longitudinal axis of
the axon (towards the sectors bordered between 60º and 120º).
48h after the initial observation we analyzed the trajectories 401 comets in the same
AOIs and found them to be unaltered (74.44 ± 2.98%, 12.12 ± 1.71% and 13.43 ±
2.08% for anterograde, retrograde and perpendicular sectors respectively. Chi square
test; χ2=4.772; df=2, P=0.0920). We used paired t-test to assess changes in each
sector and found no significant alterations in these individual sectors (paired t-test;
anterograde sector; n=5 AOIs; two-tailed; alpha =0.05; p= 0.2176, retrograde sector;
n=5 AOIs; two-tailed; alpha =0.05; p= 0.4234; lateral sector; n=5 AOIs; two-tailed;
alpha =0.05; p= 0.2526) Sector division of EB3-GFP following 96h was 69.96 ±
3.13%, 14.29 ± 1.93% and 15.75 ± 1.43% for anterograde, retrograde and
perpendicular sectors respectively (n=392 comets), and following 7 days 68.30 ±
1.79%, 15.70 ± 1.71% and 16.00 ± 2.55% for anterograde, retrograde and
perpendicular sectors respectively (n=416 comets). No significant difference was
found between initial observation 96h or 7 days (Chi square test; 96h; χ2=0.020; df=2,
P=0.9903; 7 days; χ2=0.711; df=2, P=0.7007). No significant alterations were found
in individual sectors (paired t-test; 96h; anterograde sector; n=5 AOIs; two-tailed;
alpha =0.05; p= 0.9452, retrograde sector; n=5 AOIs; two-tailed; alpha =0.05; p=
0.9779; lateral sector; n=5 AOIs; two-tailed; alpha =0.05; p=0.8137; 7 days;
anterograde sector; n=5 AOIs; two-tailed; alpha =0.05; p= 0.6501, retrograde sector;
n=5 AOIs; two-tailed; alpha =0.05; p= 0.5377; lateral sector; n=5 AOIs; two-tailed;
alpha =0.05; p= 0.9142).
1b. equivalent vector
To simplify the presentation of the polar orientation of the entire MTs population we
developed a weighted vector whose size represents the overall polarity of the MTs
within the axon. Each EB3-GFP within the AOI was assigned an angle according to
our analysis. For each given angle, we arbitrarily chose a vector with a set size of 1
arbitrary units (AU) and found the x and y components of the movement vectors.
Thereafter, we summed up 50 x and y components, and received a weighed vector
whose size represents the overall polarity of MTs in the neuron. If EB3-GFP comets
translocated in different directions, the size of weighed vector should have been
smaller than in the case of uniform MTs polarity. We created weighed vectors for 5
control neurons and found that the average size of equivalent vector was 33.23 AU
during initial observation and 32.04 AU 48h later. This -3.58% change was not found
significant (paired t-test; n=4; two-tailed; alpha =0.05; p=0.6979). Average vectors
were 30.71 and 30.15 at 96h and 7 days respectively. These -7.58% and -9.72%
changes in vector length relative to initial observation were not significant (96h, n=4;
two-tailed; alpha =0.05; p=0. 0.3697; 7 days, n=4; two-tailed; alpha =0.05; p=
0.2569).
2. Analysis of comet tail directionality in paclitaxel incubated neurons
2a. sector analysis
A representative analysis from one neuron is given on Figure 4. We analyzed 1037
comet tails before paclitaxel incubation, and 390, 370 and 165 comets 96h, 48h and 7
days later respectively. We examined the first 48h following 100nM paclitaxel
incubation and found a significant reduction in the anterograde sector from 69.00
±1.5% to 53.69. ± 2.81% (paired t-test; n=5 AOIs; two-tailed; alpha =0.05; p=
0.0011) significant change in the retrograde sector from 16.39 ± 1.34% to 27.68 ±
2.53% (paired t-test; n=5 AOIs; two-tailed; alpha =0.05; p= 0.0028) and a non-
significant increase in the perpendicular sectors from 14.61% ± 1% to 18.63 ± 3.34%
(paired t-test; n=5 AOIs; two-tailed; alpha =0.05; p= 0.3455). However, the overall
sector distribution significantly changed (Chi square test; χ2=48.124; df=2,
P<0.0001). Following 96h of 100nM paclitaxel incubation we found a significant
reduction in the anterograde sector to 47.93 ± 1.81% (paired t-test; n=5 AOIs; twotailed; alpha =0.05; p= 7.5014e-005), significant growth in the retrograde sector to
34.07% ±1.97% (paired t-test; n=5 AOIs; two-tailed; alpha =0.05; p= 1.3224e-004)
and a non-significant increase in the perpendicular sector to 17.94 ± 2.78% (n=5
AOIs; two-tailed; alpha =0.05; p= 0.3952). In addition, the overall sector distribution
was significantly changed from initial observation (Chi square test; χ2=96.310; df=2,
P<0.0001). Following 7 days of 100nM paclitaxel incubation we analyzed EB3-GFP
comets at the same AOIs as before the exposure, and found a significant reduction in
the anterograde sector to 45.64% ± 1.79 (paired t-test; n=3 AOIs; two-tailed; alpha
=0.05; p= 0.0041), significant change in the retrograde sector to 32.03% ± 4.06%
(paired t-test; n=3 AOIs; two-tailed; alpha =0.05; p= 0.0310) and a non-significant
increase in the perpendicular sectors to 22.33 ± 1.67% (n=3 AOIs; two-tailed; alpha
=0.05; p= 0.0640). In addition, the overall sector distribution significantly changed
(Chi square test; χ2=98.406; df=2, P<0.0001). These results point out a reduction in
dominant anterograde sector of MTs polymerization trajectories, in favor of
retrogradely and perpendicularily aligned polymerizing MTs in 100nM paclitaxel.
We repeated the procedure in neurons incubated with 10nM paclitaxel by analyzing
1019 comet tails before paclitaxel incubation, and 902, 573 and 171 comets 96h, 48h
and 7 days later respectively. The sectorial change was milder than in 100nM
paclitaxel. We found a significant reduction in the antetrograde sector from 68.20 ±
1.09% before paclitaxel incubation to 57.56 ± 0.73% (n=7 AOIs; two-tailed; alpha
=0.05; p= 0.0097), a significant reduction to 53.16 ± 1.61% following 96h 10nM
paclitaxel incubation (n=7 AOIs; two-tailed; alpha =0.05; p= 0.0127),
significant reduction to 53.46 ± 1.37% following 7 days of
and a
10nM paclitaxel
incubation (n=3 AOIs; two-tailed; alpha =0.05; p= 0.0427). There was a significant
change in the retrograde sector from 19.28 ± 4.89% to 28.37% ± 0.63% (n=7 AOIs;
two-tailed; alpha =0.01; p= 0.0034), to 28.99 ± 0.99% (n=7 AOIs; two-tailed; alpha
=0.05; p= 0.0106) and to 37.29 ± 1.62% (n=3 AOIs; two-tailed; alpha =0.01; p=
0.0052) following paclitaxel incubation of 48h , 96h and 7 days respectively. We
found non-significant changes in the perpendicular sectors from 12.52% ± 0.68%
before incubation to 14.06 ± 0.44% in the first 48h of paclitaxel incubation (n=7
AOIs; two-tailed; alpha =0.05; p=0.4843), 17.85± 0.84% 96h from paclitaxel
incubation (n=7 AOIs; two-tailed; alpha =0.05; p= 0.0865) and 9.25 ± 1.74% 7 days
from paclitaxel incubation (n=3 AOIs; two-tailed; alpha =0.05; p=0.6805) . The
overall sector distribution significantly changed during the first 48h after 10nM
paclitaxel incubation (Chi square test; χ2= 87.869; df=2, p<0.0001) with a further
significant change in sector distribution up to 96h (Chi square test; χ2= 113.443; df=2,
p<0.0001), and up to 7 days (Chi square test; χ2= 41.429; df=2, p<0.0001).
2b. equivalent vector
Based on the angle histogram the equivalent vectors were drawn and averaged in 5
neurons incubated with 100nM paclitaxel. The average vector exhibited -53.16%
decrease from initial observation to 48h later. The change was found significant using
paired t-test (n=5; two-tailed; alpha =0.05; p=6.3773e-005). Equivalent vectors
derived from 96h and 7days following 100nM incubation were shortened by -46.15%
and -66.19% respectively relative to initial observation (96h, n=5; two-tailed; alpha
=0.01; p= 0.0048; 7 days, n=3; two-tailed; alpha =0.001; p= 2.8083e-004), meaning
the major shortening of equivalent vector occurred within first 48h.
Based on the angle histogram of 7 neurons incubated with 10nM, the equivalent
vectors were measured. The average vector decreased significantly by -35.24%
during first 48h of observation (n=7; two-tailed; alpha =0.05; p=0.0107). 96h and 7
days following incubation, the vector was shortened by -45.91% and –62.47%
respectively relative to initial observation. These changes were found significant
using paired t-test (96h; n=7; two-tailed; alpha =0.01; p=0.0079; 7 days; n=3; twotailed; alpha =0.01; p=0.0017). These results imply that paclitaxel effect over MTs
polarity is time dependent.
3. Analysis of SR101 labeled organelle transport in control neurons and
paclitaxel incubated neurons
To image retrograde transport, pinocytotic vesicles were labeled by the fluid phase
pinocytotic marker SR101. We defined a motile organelle as a vesicle, which moves
faster than 0.015µm/s. According to this cutoff, the percentage of dynamic vesicles
was 92.71 ± 3.61%, 54.6 ±10.21% and 40.44 ± 7.46% of vesicles in control neurons,
10nM and 100nM paclitaxel incubated neurons respectively. The percentage of
motile organelles significantly differs between control and 10nM (t-test, n=5; twotailed; alpha =0.05; p=0.016) or between control and 100nM paclitaxel (t-test, n=5 for
control and 6 for 100nM; two-tailed; alpha =0.01; p=0.00037) but not between 10nM
and 100nM paclitaxel (t-test, n=5 for 10nM and 6 for 100nM; two-tailed; alpha
=0.05; p=0.296).
Among vesicles defined as motile, the percentage of organelles translocating
retrogradely was calculated. The percentage of motile retrograde organelles was
96.71 ± 1.35%, 71.03% ± 14.67 and 72.96% ± 5.76% for control neurons, 10nM and
100nM paclitaxel incubated neurons respectively. The percentage of retrograde
organelles significantly differs between control and 100nM paclitaxel (t-test, n=5 for
control and 6 for 100nM; two-tailed; alpha =0.01; p=0.0082) but not between control
and 10nM paclitaxel (t-test, n=5; two-tailed; alpha =0.05; p=0.55) and between 10nM
and 100nM paclitaxel (t-test, n=5 for control and 6 for 100nM; two-tailed; alpha
=0.01; p=0.907).