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Fig. 5 A. Image sequence from confocal time lapse of a neuron that does not extend a nascent axon during nocodazole treatment labelled with a membrane marker and lifeact-Ruby before (?10 m) and during (0 m to 170 m) nocodazole treatment. Protrusions present before nocodazole addition are retracted upon nocodazole treatment (20 m). Short, transient nonaxonal protrusions are extended during nocodazole treatment. Images are transverse reconstructions from confocal z-stacks.
B. Image sequence from confocal time lapse of a neuron that extends a nascent axon during nocodazole treatment labelled with a membrane marker and lifeact-Ruby before (?10 m) and during (0 h to 170 min) nocodazole treatment. Small protrusions present before nocodazole addition (?10 m) are retracted upon nocodazole treatment (0 m). A nascent axon-like protrusion (long, broad, long-lived) is extended during nocodazole treatment (110 m to 170 m). Transient nonaxonal protrusions are also present. Images are transverse reconstructions from confocal z-stacks.
B. Graph showing duration (minutes) of nascent axon-like protrusions in nocodazole-treated cells and nonaxonal protrusions in control and nocodazole-treated cells. Control nascent axons were not analysed as they do not retract. Bars show mean and standard deviation. Nocodazole nascent axons: n = 14 protrusions from 14 cells from three experiments; mean = 65.17 min, s.d. = 20.33. Control nonaxons: n = 40 protrusions from 10 cells from three experiments; mean = 17.15 min, s.d. = 8.804. Nocodazole nonaxons: n = 41 protrusions from 16 cells from three experiments; mean = 16.182 min, s.d. = 7.147. One-way ANOVA with multiple comparisons: P < 0.0001; nocodazole nascent axon vs nocodazole nonaxon P < 0.0001; control nonaxon vs nocodazole nonaxon P = 0.9132.
C. Graph showing length (?m) of nascent axon-like protrusions and nonaxonal protrusions in control and nocodazole-treated cells. Bars show mean and standard deviation. Control nascent axons were analysed after 60 min: n = 18 protrusions from 18 cells; mean = 14.092 ?m, s.d. = 5.149. Nocodazole nascent axons: n = 14 protrusions from 14 cells; mean = 9.596 ?m, s.d. = 2.371. Control nonaxons: n = 40 protrusions from 10 cells; mean = 5.802 ?m, s.d. = 2.553. Nocodazole nonaxons: n = 41 protrusion from 16 cells; mean = 4.407 ?m, s.d. = 2.024. One-way ANOVA with multiple comparisons: P < 0.0001; control nascent axon vs nocodazole nascent axon P = 0.0002; control nascent axon vs control nonaxon P < 0.0001; nocodazole nascent axon vs nocodazole nonaxon P < 0.0001; control nonaxon vs. nocodazole nonaxon P = 0.1485.
D. Graph showing width (?m) at the base of nascent axon-like protrusions and nonaxonal protrusions in control and nocodazole-treated cells. Bars show mean and standard deviation. Control nascent axons: n = 19 protrusions from 19 cells from six experiments; mean = 7.806 ?m, s.d. = 3.163. Nocodazole nascent axons: n = 14 protrusions from 14 cells from three experiments; mean = 4.188 ?m, s.d. = 1.637. Control nonaxons from three experiments: n = 40 protrusions from 10 cells; mean = 1.061 ?m, s.d. = 0.652. Nocodazole nonaxons from three experiments: n = 41 protrusions from 16 cells; mean = 0.984 ?m, s.d. = 0.757. One-way ANOVA with multiple comparisons: P < 0.0001; control nascent axon vs nocodazole nascent axon P < 0.0001; control nascent axon vs control nonaxon P < 0.0001; nocodazole nascent axon vs nocodazole nonaxon P < 0.0001; control nonaxon vs nocodazole nonaxon P = 0.9958.
F. Plots showing nascent axon position on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse views (n = 19 cells from six experiments). Numbers show total count of axons in each quadrant.
G. Plots showing nascent axon position on the soma of nocodazole-treated cells relative to the cell centroid at 0,0 for dorsal and transverse views (n = 14 cells from three experiments). Numbers show total count of axons in each quadrant.
H. Plots showing the position of nonaxonal protrusions on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse views (n = 40 protrusions from 10 cells from three experiments). Numbers show total count of protrusions in each quadrant.
I. Plots showing the position of nonaxonal protrusions on the soma of nocodazole-treated cells relative to the cell centroid at 0,0 for dorsal and transverse views (n = 41 protrusions from 16 cells from three experiments). Numbers show total count of protrusions originating in each quadrant.
J. Graph showing the ratio of kif5c560-YFP fluorescence intensity in the soma compared to the axon with or without nocodazole. Bars show mean and standard deviation. Control: n = 6 cells from four experiments; mean = 1.81, s.d. = 1.59. Nocodazole: n = 7 cells from four experiments; mean = 0.75, s.d. = 0.19.
K. Image sequence from confocal time lapse of a neuron that extends a nascent axon during nocodazole treatment labelled with a membrane marker and kif5c560-YFP before (?10 m) and during (0 h to 144 min) nocodazole treatment. A nascent axon-like protrusion is extended during nocodazole treatment (92 m to 144 m), but kif5c560-YFP does not accumulate there. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 ?m. ***P < 0.001. ****P < 0.0001.
Source data are available online for this figure.