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; two-tailed; 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), and a significant reduction to 53.46 ± 1.37% following 7 days of 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; two-tailed; 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; two-tailed; 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).