F Distance between centrosome and cilium in Tg ( actb2 : arl13b - GFP ) embryos fixed and processed for immunohistochemistry against GFP to label the cilium and γ - tubulin to label the centrosome . No difference was found in the distance between the two organelles when close to the apical surface or away from the midline ( n = 50 cells per position from two experiments ; P = 0 . 8279 ; midline mean = 0 . 4956 , s . d = 0 . 1125 ; away from midline mean = 0 . 5004 , s . d = 0 . 1077 ; Student ' s unpaired t - test ) . Bars show mean and standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Distance between centrosome and cilium in Tg(actb2:arl13b-GFP) embryos fixed and processed for immunohistochemistry against GFP to label the cilium and γ-tubulin to label the centrosome. No difference was found in the distance between the two organelles when close to the apical surface or away from the midline (n = 50 cells per position from two experiments; P = 0.8279; midline mean = 0.4956, s.d = 0.1125; away from midline mean = 0.5004, s.d = 0.1077; Student's unpaired t-test). Bars show mean and standard deviation.


A Image sequence from confocal time lapse shows a neuron labelled with membrane and centrosome markers before ( - 16m ) , during ( 0m ) and after axon initiation ( 15m , 31m ) . The centrosome is located on the opposite side of the cell to the nascent axon . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Image sequence from confocal time lapse shows a neuron labelled with membrane and centrosome markers before (-16m), during (0m) and after axon initiation (15m, 31m). The centrosome is located on the opposite side of the cell to the nascent axon. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


B Image sequence from confocal time lapse shows a neuron labelled with membrane and Golgi complex markers before ( - 17m ) , during ( 0m ) and after axon initiation ( 17m , 37m ) . The Golgi complex is located on the opposite side of the cell to the nascent axon . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B Image sequence from confocal time lapse shows a neuron labelled with membrane and Golgi complex markers before (-17m), during (0m) and after axon initiation (17m, 37m). The Golgi complex is located on the opposite side of the cell to the nascent axon. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


C Graph showing distance between centrosome or Golgi complex and base of axon at time of axon initiation and 6 - 8 hours after axon initiation . Bars show mean and standard deviation . At time of axon initiation : centrosome - axon mean = 10 . 1 µm , s . d . = 3 . 3 , n = 26 cells from three experiments ; Golgi complex - axon mean = 9 . 3 µm , s . d . = 3 . 8 , n = 16 cells from two experiments . At 6 - 8 hours after axon initiation : centrosome - axon mean = 4 . 2 µm , s . d . = 2 . 1 , n = 26 cells ; Golgi complex - axon mean = 4 . 3 µm , s . d . = 2 . 1 , n = 27 cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Graph showing distance between centrosome or Golgi complex and base of axon at time of axon initiation and 6-8 hours after axon initiation. Bars show mean and standard deviation. At time of axon initiation: centrosome-axon mean = 10.1 µm, s.d. = 3.3, n = 26 cells from three experiments; Golgi complex-axon mean = 9.3 µm, s.d. = 3.8, n = 16 cells from two experiments. At 6-8 hours after axon initiation: centrosome-axon mean = 4.2 µm, s.d. = 2.1, n = 26 cells; Golgi complex-axon mean = 4.3 µm, s.d. = 2.1, n = 27 cells.


D Transverse reconstruction from confocal time lapse of a neuron labelled with membrane and centrosome markers during axon growth . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D Transverse reconstruction from confocal time lapse of a neuron labelled with membrane and centrosome markers during axon growth. Data information: All scale bars = 10 µm.


E Transverse reconstruction from confocal time lapse of a neuron labelled with membrane and Golgi complex markers during axon growth . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Transverse reconstruction from confocal time lapse of a neuron labelled with membrane and Golgi complex markers during axon growth. Data information: All scale bars = 10 µm.


F Plots showing the positions of the centrosome and base of the axon at the time of axon initiation relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 26 cells from three experiments ) . Left - hand plots : centrosome position is not random ( dorsal view P < 0 . 001 , mean = - 100 . 8o ; transverse view P < 0 . 001 , mean = - 33 . 7o ) and axon position is not random ( dorsal view P < 0 . 001 , mean = 101 . 0o ; transverse view P < 0 . 001 , mean = 154 . 8o ; Moore ' s modification of Rayleigh ' s test ) . Centrosome and axon positions are significantly different ( dorsal view 0 . 001 > P ; transverse view 0 . 001 > P ; Moore ' s test for paired data ) . Right - hand plots : vectors linking centrosome and nascent axon of the same cell are not random ( dorsal view P < 0 . 001 , mean = 91 . 0o ; transverse view P < 0 . 001 , mean = 151 . 7o ; Moore ' s modification of Rayleigh ' s test ) . G Plots showing the positions of the Golgi complex ( magenta ) and base of the axon ( grey ) at the time of axon initiation relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 16 cells from two experiments ) . Left - hand plots : Golgi complex position is not random ( dorsal view 0 . 01 < P < 0 . 05 , mean = - 83 . 0o ; transverse view P < 0 . 001 , mean = - 53 . 4o ) and axon position is not random ( dorsal view P < 0 . 001 , mean = 89 . 1o ; transverse view P < 0 . 001 , mean = 147 . 4o ; Moore ' s modification of Rayleigh ' s test ) . Golgi complex and axon positions are significantly different ( dorsal view 0 . 001 > P ; transverse view 0 . 001 > P ; Moore ' s test for paired data ) . Right - hand plots : vectors linking Golgi complex and nascent axon of the same cell are not random ( dorsal view P < 0 . 001 , mean = 98 . 5o ; transverse view P < 0 . 001 , mean = 156 . 8o ; Moore ' s modification of Rayleigh ' s test ) . H Plots showing the positions of the centrosome and base of the axon 6 - 12 hours after axon initiation relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 26 cells ) . Centrosome position is not random ( dorsal view P < 0 . 001 , mean = 67 . 7o ; transverse view P < 0 . 001 , mean = 151 . 6o ) . Axon position is not random ( dorsal view P < 0 . 001 , mean = 74 . 0o ; transverse view P < 0 . 001 , mean = 153 . 7o ; Moore ' s modification of Rayleigh ' s test ) . Centrosome and axon positions are not significantly different ( dorsal view 0 . 5 < P ; transverse view 0 . 1 < P < 0 . 5 ; Moore ' s test for paired data ) . I Plots showing the positions of the Golgi complex and base of the axon 6 - 12 hours after axon initiation relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 27 cells ) . Golgi complex position is not random ( dorsal view P < 0 . 001 , mean = 79 . 9o ; transverse view P < 0 . 001 , mean = 149 . 9o ) . Axon position is not random ( dorsal view P < 0 . 001 , mean = 76 . 0o ; transverse view P < 0 . 001 , mean = 153 . 9o ; Moore ' s modification of Rayleigh ' s test ) . Golgi complex and axon positions are different only in transverse view ( dorsal view 0 . 5 < P ; transverse view 0 . 005 < P < 0 . 01 ; Moore ' s test for paired data ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Plots showing the positions of the centrosome and base of the axon at the time of axon initiation relative to the cell centroid at 0,0 for dorsal and transverse view (n = 26 cells from three experiments). Left-hand plots: centrosome position is not random (dorsal view P < 0.001, mean = -100.8o; transverse view P < 0.001, mean = -33.7o) and axon position is not random (dorsal view P < 0.001, mean = 101.0o; transverse view P < 0.001, mean = 154.8o; Moore's modification of Rayleigh's test). Centrosome and axon positions are significantly different (dorsal view 0.001 > P; transverse view 0.001 > P; Moore's test for paired data). Right-hand plots: vectors linking centrosome and nascent axon of the same cell are not random (dorsal view P < 0.001, mean = 91.0o; transverse view P < 0.001, mean = 151.7o; Moore's modification of Rayleigh's test). G Plots showing the positions of the Golgi complex (magenta) and base of the axon (grey) at the time of axon initiation relative to the cell centroid at 0,0 for dorsal and transverse view (n = 16 cells from two experiments). Left-hand plots: Golgi complex position is not random (dorsal view 0.01 < P < 0.05, mean = -83.0o; transverse view P < 0.001, mean = -53.4o) and axon position is not random (dorsal view P < 0.001, mean = 89.1o; transverse view P < 0.001, mean = 147.4o; Moore's modification of Rayleigh's test). Golgi complex and axon positions are significantly different (dorsal view 0.001 > P; transverse view 0.001 > P; Moore's test for paired data). Right-hand plots: vectors linking Golgi complex and nascent axon of the same cell are not random (dorsal view P < 0.001, mean = 98.5o; transverse view P < 0.001, mean = 156.8o; Moore's modification of Rayleigh's test). H Plots showing the positions of the centrosome and base of the axon 6-12 hours after axon initiation relative to the cell centroid at 0,0 for dorsal and transverse view (n = 26 cells). Centrosome position is not random (dorsal view P < 0.001, mean = 67.7o; transverse view P < 0.001, mean = 151.6o). Axon position is not random (dorsal view P < 0.001, mean = 74.0o; transverse view P < 0.001, mean = 153.7o; Moore's modification of Rayleigh's test). Centrosome and axon positions are not significantly different (dorsal view 0.5 < P; transverse view 0.1 < P < 0.5; Moore's test for paired data). I Plots showing the positions of the Golgi complex and base of the axon 6-12 hours after axon initiation relative to the cell centroid at 0,0 for dorsal and transverse view (n = 27 cells). Golgi complex position is not random (dorsal view P < 0.001, mean = 79.9o; transverse view P < 0.001, mean = 149.9o). Axon position is not random (dorsal view P < 0.001, mean = 76.0o; transverse view P < 0.001, mean = 153.9o; Moore's modification of Rayleigh's test). Golgi complex and axon positions are different only in transverse view (dorsal view 0.5 < P; transverse view 0.005 < P < 0.01; Moore's test for paired data).


A Image sequence from confocal time lapse of a neuron labelled with a membrane marker and lifeact - Ruby before , during ( 0m ) and after axon initiation . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A Image sequence from confocal time lapse of a neuron labelled with a membrane marker and lifeact-Ruby before, during (0m) and after axon initiation. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


B Graph showing time ( minutes ) between actin accumulation and nascent axon initiation ( n = 15 cells from 7 experiments ) . Bars show mean and standard deviation .

B Graph showing time (minutes) between actin accumulation and nascent axon initiation (n = 15 cells from 7 experiments). Bars show mean and standard deviation.


C Image sequence from confocal time lapse of a neuron labelled with lifeact - Ruby and EB3 - GFP before , during ( 0m ) and after axon initiation . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

C Image sequence from confocal time lapse of a neuron labelled with lifeact-Ruby and EB3-GFP before, during (0m) and after axon initiation. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


D Image sequence from confocal time lapse of a neuron labelled with a membrane marker and Kif5c560 - YFP before , during ( 0m ) and after axon initiation . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

D Image sequence from confocal time lapse of a neuron labelled with a membrane marker and Kif5c560-YFP before, during (0m) and after axon initiation. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


E Three time points from confocal time lapse of a triple labelled neuron before , during ( 0m ) and after nascent axon initiation . Images to left show distribution sequence of lifeact - Ruby , kif5c560 - YFP and EB3 - GFP individually . Dual channel merges to the right show relative locations of pairs of fusion proteins . Images to right are maximum projections of transverse reslices of confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E Three time points from confocal time lapse of a triple labelled neuron before, during (0m) and after nascent axon initiation. Images to left show distribution sequence of lifeact-Ruby, kif5c560-YFP and EB3-GFP individually. Dual channel merges to the right show relative locations of pairs of fusion proteins. Images to right are maximum projections of transverse reslices of confocal z-stacks. Data information: All scale bars = 10 µm.


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 ( - 10m ) and during ( 0m to 170m ) nocodazole treatment . Protrusions present before nocodazole addition are retracted upon nocodazole treatment ( 20m ) . Short , transient non - axonal 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 ( - 10m ) and during ( 0h to 170m ) nocodazole treatment . Small protrusions present before nocodazole addition ( - 10m ) are retracted upon nocodazole treatment ( 0m ) . A nascent axon - like protrusion ( long , broad , long - lived ) is extended during nocodazole treatment ( 110m to 170m ) . Transient non - axonal protrusions are also present . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 (-10m) and during (0m to 170m) nocodazole treatment. Protrusions present before nocodazole addition are retracted upon nocodazole treatment (20m). Short, transient non-axonal 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 (-10m) and during (0h to 170m) nocodazole treatment. Small protrusions present before nocodazole addition (-10m) are retracted upon nocodazole treatment (0m). A nascent axon-like protrusion (long, broad, long-lived) is extended during nocodazole treatment (110m to 170m). Transient non-axonal protrusions are also present. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


C Graph showing duration ( minutes ) of nascent axon - like protrusions in nocodazole treated cells and non - axonal 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 minutes , s . d . = 20 . 33 . Control non - axons : n = 40 protrusions from 10 cells from three experiments ; mean = 17 . 15 minutes , s . d . = 8 . 804 . Nocodazole non - axons : n = 41 protrusions from 16 cells from three experiments ; mean = 16 . 182 minutes , s . d . = 7 . 147 . One - way ANOVA with multiple comparisons : P < 0 . 0001 ; nocodazole nascent axon vs nocodazole non - axon P < 0 . 0001 ; control non - axon vs nocodazole non - axon P = 0 . 9132 . D Graph showing length ( µm ) of nascent axon - like protrusions and non - axonal protrusions in control and nocodazole treated cells . Bars show mean and standard deviation . Control nascent axons were analysed after 60 minutes : n = 18 protrusions from 18 cells ; mean = 14 . 092 µm , s . d . = 5 . 149 . Nocodazole nascent axons : n = 14 protrusions from 14 cells ; mean = 9 . 596 µm , s . d . = 2 . 371 . Control non - axons : n = 40 protrusions from 10 cells ; mean = 5 . 802 µm , s . d . = 2 . 553 . Nocodazole non - axons : n = 41 protrusion from 16 cells ; mean = 4 . 407 µm , 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 non - axon P < 0 . 0001 ; nocodazole nascent axon vs nocodazole non - axon P < 0 . 0001 ; control non - axon vs nocodazole non - axon P = 0 . 1485 . E Graph showing width ( µm ) at the base of nascent axon - like protrusions and non - axonal 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 µm , s . d . = 3 . 163 . Nocodazole nascent axons : n = 14 protrusions from 14 cells from three experiments ; mean = 4 . 188 µm , s . d . = 1 . 637 . Control non - axons from three experiments : n = 40 protrusions from 10 cells ; mean = 1 . 061 µm , s . d . = 0 . 652 . Nocodazole non - axons from three experiments : n = 41 protrusions from 16 cells ; mean = 0 . 984 µm , 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 non - axon P < 0 . 0001 ; nocodazole nascent axon vs nocodazole non - axon P < 0 . 0001 ; control non - axon vs nocodazole non - axon P = 0 . 9958 . Data information : * * * P < 0 . 001 . * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Graph showing duration (minutes) of nascent axon-like protrusions in nocodazole treated cells and non-axonal 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 minutes, s.d. = 20.33. Control non-axons: n = 40 protrusions from 10 cells from three experiments; mean = 17.15 minutes, s.d. = 8.804. Nocodazole non-axons: n = 41 protrusions from 16 cells from three experiments; mean = 16.182 minutes, s.d. = 7.147. One-way ANOVA with multiple comparisons: P < 0.0001; nocodazole nascent axon vs nocodazole non-axon P < 0.0001; control non-axon vs nocodazole non-axon P = 0.9132. D Graph showing length (µm) of nascent axon-like protrusions and non-axonal protrusions in control and nocodazole treated cells. Bars show mean and standard deviation. Control nascent axons were analysed after 60 minutes: n = 18 protrusions from 18 cells; mean = 14.092 µm, s.d. = 5.149. Nocodazole nascent axons: n = 14 protrusions from 14 cells; mean = 9.596 µm, s.d. = 2.371. Control non-axons: n = 40 protrusions from 10 cells; mean = 5.802 µm, s.d. = 2.553. Nocodazole non-axons: n = 41 protrusion from 16 cells; mean = 4.407 µm, 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 non-axon P < 0.0001; nocodazole nascent axon vs nocodazole non-axon P < 0.0001; control non-axon vs nocodazole non-axon P = 0.1485. E Graph showing width (µm) at the base of nascent axon-like protrusions and non-axonal 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 µm, s.d. = 3.163. Nocodazole nascent axons: n = 14 protrusions from 14 cells from three experiments; mean = 4.188 µm, s.d. = 1.637. Control non-axons from three experiments: n = 40 protrusions from 10 cells; mean = 1.061 µm, s.d. = 0.652. Nocodazole non-axons from three experiments: n = 41 protrusions from 16 cells; mean = 0.984 µm, 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 non-axon P < 0.0001; nocodazole nascent axon vs nocodazole non-axon P < 0.0001; control non-axon vs nocodazole non-axon P = 0.9958. Data information: *** P < 0.001. **** P < 0.0001.


F Plots showing nascent axon position on the soma of control cells relative to the cell centroid at 0 , 0 for dorsal and transverse view ( 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 view ( n = 14 cells from three experiments ) . Numbers show total count of axons in each quadrant . H Plots showing the position of non - axonal protrusions on the soma of control cells relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 40 protrusions from 10 cells from three experiments ) . Numbers show total count of protrusions in each quadrant . I Plots showing the position of non - axonal protrusions on the soma of nocodazole treated cells relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 41 protrusions from 16 cells from three experiments ) . Numbers show total count of protrusions originating in each quadrant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Plots showing nascent axon position on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse view (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 view (n = 14 cells from three experiments). Numbers show total count of axons in each quadrant. H Plots showing the position of non-axonal protrusions on the soma of control cells relative to the cell centroid at 0,0 for dorsal and transverse view (n = 40 protrusions from 10 cells from three experiments). Numbers show total count of protrusions in each quadrant. I Plots showing the position of non-axonal protrusions on the soma of nocodazole treated cells relative to the cell centroid at 0,0 for dorsal and transverse view (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 . Data information : * * * P < 0 . 001 . * * * * P < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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. Data information: *** P < 0.001. **** P < 0.0001.


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 ( - 10m ) and during ( 0h to 144m ) nocodazole treatment . A nascent axon - like protrusion is extended during nocodazole treatment ( 92m to 144m ) but kif5c560 - YFP does not accumulate there . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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 (-10m) and during (0h to 144m) nocodazole treatment. A nascent axon-like protrusion is extended during nocodazole treatment (92m to 144m) but kif5c560-YFP does not accumulate there. Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


A Transverse section from a confocal z - stack showing the whole neural tube of a utr - mCherry embryo , showing localisation to the basal surface . B Transverse sections from a confocal z - stack of a non - apical progenitor labelled with a membrane marker in a utr - mCherry embryo to identify the basal surface of the spinal cord . Image sequence from confocal time - lapse shows the non - apical progenitor ( - 44m ) undergoing mitosis to produce two neurons ( - 22m ) , of which one is not in contact with the basal surface . Both neurons extend nascent axons ( 0m ) . Data information : All scale bars = 5 µm .

A Transverse section from a confocal z-stack showing the whole neural tube of a utr-mCherry embryo, showing localisation to the basal surface. B Transverse sections from a confocal z-stack of a non-apical progenitor labelled with a membrane marker in a utr-mCherry embryo to identify the basal surface of the spinal cord. Image sequence from confocal time-lapse shows the non-apical progenitor (-44m) undergoing mitosis to produce two neurons (-22m), of which one is not in contact with the basal surface. Both neurons extend nascent axons (0m). Data information: All scale bars = 5 µm.


C Transverse sections from a confocal z - stack of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr - mCherry embryo to identify the basal surface of the spinal cord . Three different sections show the middle of the soma , the axon initiation site , and the axon tip . Green and magenta peaks in graphs show relative positions of cell membrane and basal surface , respectively . D Transverse sections from confocal z - stacks of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr - mCherry embryo injected with lamMO . Three different sections show the middle of the soma , the axon initiation site , and the axon tip . Green and magenta peaks in graphs show relative positions of cell membrane and basal surface , respectively . E Graphs showing distance ( µm ) between the basal edge of the soma , the axon initiation site or the axon tip and the basal surface of the spinal cord . Measurements were performed by measuring between basal - most green and magenta peaks in graphs of relative grey values ( C , D ) . Bars show mean and standard deviation . WT : n = 5 cells from one experiment ; lamMO : n = 11 cells from two experiments . Soma : WT mean = 2 . 007 µm , s . d . = 1 . 055 ; lamMO mean = 1 . 871 µm , s . d . = 1 . 830 . Student ' s two - tailed test , P = 0 . 880 . Axon initiation site : WT mean = 1 . 366 µm , s . d . = 3 . 13 ; lamMO mean = 2 . 353 µm , s . d . = 2 . 125 . Student ' s two - tailed test , P = 0 . 468 . Axon tip : WT mean = 0 . 637 µm , s . d . = 1 . 078 ; lamMO mean = 2 . 903 µm , s . d . = 2 . 0433 . Student ' s two - tailed test , P = 0 . 037 . Data information : All scale bars = 5 µm . * P < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C Transverse sections from a confocal z-stack of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr-mCherry embryo to identify the basal surface of the spinal cord. Three different sections show the middle of the soma, the axon initiation site, and the axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. D Transverse sections from confocal z-stacks of a neuron at the time of nascent axon initiation labelled with a membrane marker in a utr-mCherry embryo injected with lamMO. Three different sections show the middle of the soma, the axon initiation site, and the axon tip. Green and magenta peaks in graphs show relative positions of cell membrane and basal surface, respectively. E Graphs showing distance (µm) between the basal edge of the soma, the axon initiation site or the axon tip and the basal surface of the spinal cord. Measurements were performed by measuring between basal-most green and magenta peaks in graphs of relative grey values (C, D). Bars show mean and standard deviation. WT: n = 5 cells from one experiment; lamMO: n = 11 cells from two experiments. Soma: WT mean = 2.007 µm, s.d. = 1.055; lamMO mean = 1.871 µm, s.d. = 1.830. Student's two-tailed test, P = 0.880. Axon initiation site: WT mean = 1.366 µm, s.d. = 3.13; lamMO mean = 2.353 µm, s.d. = 2.125. Student's two-tailed test, P = 0.468. Axon tip: WT mean = 0.637 µm, s.d. = 1.078; lamMO mean = 2.903 µm, s.d. = 2.0433. Student's two-tailed test, P = 0.037. Data information: All scale bars = 5 µm. * P < 0.05.


F Image sequence from confocal time lapse shows a neuron in a Sly - / - embryo labelled with membrane and centrosome markers before ( - 40m ) , during ( 0 ) and after axon initiation ( 20m ) . Images are transverse reconstructions from confocal z - stacks .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F Image sequence from confocal time lapse shows a neuron in a Sly-/- embryo labelled with membrane and centrosome markers before (-40m), during (0) and after axon initiation (20m). Images are transverse reconstructions from confocal z-stacks.


G Plots showing axon position on the soma relative to the cell centroid at 0 , 0 for dorsal and transverse view in Sly - / - embryos ( n = 18 cells from three experiments ) . Axon position is not random ( dorsal view P < 0 . 001 , mean = 98 . 8o ; transverse view P < 0 . 001 , mean = 161 . 1o ; Moore ' s modification of Rayleigh ' s test ) . H Plots showing merge of WT and Sly - / - axon positions on the cell body relative to cell centroid at 0 , 0 for dorsal and transverse views . Axon positions in WT and Sly - / - are not significantly different in dorsal view ( 0 . 2 < P < 0 . 5 ) but are in transverse view ( P < 0 . 001 ; Batschelet ' s alternative to Hotelling test ) . WT : n = 86 cells from eight experiments ; Sly - / - : n = 18 cells from three experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G Plots showing axon position on the soma relative to the cell centroid at 0,0 for dorsal and transverse view in Sly-/- embryos (n = 18 cells from three experiments). Axon position is not random (dorsal view P < 0.001, mean = 98.8o; transverse view P < 0.001, mean = 161.1o; Moore's modification of Rayleigh's test). H Plots showing merge of WT and Sly-/- axon positions on the cell body relative to cell centroid at 0,0 for dorsal and transverse views. Axon positions in WT and Sly-/- are not significantly different in dorsal view (0.2 < P < 0.5) but are in transverse view (P < 0.001; Batschelet's alternative to Hotelling test). WT: n = 86 cells from eight experiments; Sly-/-: n = 18 cells from three experiments.


I Graph showing distance between centrosome and base of axon at time of axon initiation in WT and Sly - / - embryos . Bars show mean and standard deviation . WT : n = 26 cells from three experiments , mean = 10 . 13 µm , s . d . = 3 . 35 . Sly - / - : n = 15 cells from two experiments , mean = 12 . 41 µm , s . d . = 3 . 281 . One - way ANOVA , P = 0 . 123 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

I Graph showing distance between centrosome and base of axon at time of axon initiation in WT and Sly-/- embryos. Bars show mean and standard deviation. WT: n = 26 cells from three experiments, mean = 10.13 µm, s.d. = 3.35. Sly-/-: n = 15 cells from two experiments, mean = 12.41 µm, s.d. = 3.281. One-way ANOVA, P = 0.123.


J Plots showing centrosome position relative to the cell centroid at 0 , 0 for dorsal and transverse view in Sly - / - embryos ( n = 15 cells from two experiments ) . Centrosome position is not random ( dorsal view P < 0 . 001 , mean = - 129 . 0o ; transverse view 0 . 001 < P < 0 . 005 , mean = - 57 . 0o ; Moore ' s modification of Rayleigh ' s test ) . K Plots showing merge of WT and Sly - / - centrosome positions on the cell body relative to cell centroid at 0 , 0 for dorsal and transverse views . Centrosome positions are not significantly different between WT and Sly - / - ( dorsal view 0 . 2 < P < 0 . 2 , transverse view 0 . 1 < P < 0 . 2 ; Batschelet ' s alternative to Hotelling test ) . WT : n = 26 cells from three experiments ; Sly - / - : n = 15 cells from two experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

J Plots showing centrosome position relative to the cell centroid at 0,0 for dorsal and transverse view in Sly-/- embryos (n = 15 cells from two experiments). Centrosome position is not random (dorsal view P < 0.001, mean = -129.0o; transverse view 0.001 < P < 0.005, mean = -57.0o; Moore's modification of Rayleigh's test). K Plots showing merge of WT and Sly-/- centrosome positions on the cell body relative to cell centroid at 0,0 for dorsal and transverse views. Centrosome positions are not significantly different between WT and Sly-/- (dorsal view 0.2 < P < 0.2, transverse view 0.1 < P < 0.2; Batschelet's alternative to Hotelling test). WT: n = 26 cells from three experiments; Sly-/-: n = 15 cells from two experiments.


L Plots showing the positions of the centrosome and base of the axon in Sly - / - embryos at the time of axon initiation relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 15 cells from two experiments ) . Left - hand plots : centrosome position is not random ( dorsal view P < 0 . 001 , mean = - 129 . 0o ; transverse view P < 0 . 001 , mean = - 57 . 0 ) and axon position is not random ( dorsal view P < 0 . 001 , mean = 95 . 1o ; transverse view P < 0 . 001 , mean = 168 . 8o , Moore ' s modification of Rayleigh ' s test ) . Centrosome and axon positions are significantly different ( dorsal view 0 . 001 > P ; transverse view 0 . 001 > P ; Moore ' s test for paired data ) . Right - hand plots : vectors connecting centrosome and nascent axon from the same cell are not random ( dorsal view P < 0 . 001 , mean = 66 . 1o ; transverse view P < 0 . 001 , mean = 150 . 9o ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

L Plots showing the positions of the centrosome and base of the axon in Sly-/- embryos at the time of axon initiation relative to the cell centroid at 0,0 for dorsal and transverse view (n = 15 cells from two experiments). Left-hand plots: centrosome position is not random (dorsal view P < 0.001, mean = -129.0o; transverse view P < 0.001, mean = -57.0) and axon position is not random (dorsal view P < 0.001, mean = 95.1o; transverse view P < 0.001, mean = 168.8o, Moore's modification of Rayleigh's test). Centrosome and axon positions are significantly different (dorsal view 0.001 > P; transverse view 0.001 > P; Moore's test for paired data). Right-hand plots: vectors connecting centrosome and nascent axon from the same cell are not random (dorsal view P < 0.001, mean = 66.1o; transverse view P < 0.001, mean = 150.9o).


D . Global measurement of the severing of filaments exposed to 20 nM mCherry - cofilin - 1 from time t = 0 onwards . N = 20 ( from 1 representative experiment ) and 50 ( 1 experiment ) for non - oxidized and oxidized actin filaments , respectively . P - value = 8 . 10 - 8 ( log - rank test ) . Data information : Thick solid and dashed lines are survival fractions calculated from the experimental data . Thin grey lines are single exponential fits . 95 % confidence intervals are shown as shaded surfaces .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D. Global measurement of the severing of filaments exposed to 20 nM mCherry-cofilin-1 from time t=0 onwards. N = 20 (from 1 representative experiment) and 50 (1 experiment) for non-oxidized and oxidized actin filaments, respectively. P-value = 8.10-8 (log-rank test). Data information: Thick solid and dashed lines are survival fractions calculated from the experimental data. Thin grey lines are single exponential fits. 95% confidence intervals are shown as shaded surfaces.


B . Global quantification of the severing events on oxidized and non - oxidized filaments exposed to unlabelled S3D - cofilin1 from time t = 0 onwards . From top to bottom , N = 50 ( from 1 experiment ) , 50 ( 1 experiment ) , 100 ( 2 experiments ) filaments . Data information All lines are survival fractions calculated from the experimental data 95 % confidence intervals are shown as shaded surfaces .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B. Global quantification of the severing events on oxidized and non-oxidized filaments exposed to unlabelled S3D-cofilin1 from time t=0 onwards. From top to bottom, N = 50 (from 1 experiment), 50 (1 experiment), 100 (2 experiments) filaments. Data information All lines are survival fractions calculated from the experimental data 95% confidence intervals are shown as shaded surfaces.


A . Time - lapse images of Tpm - saturated actin filament depolymerizing from their barbed end ( BE ) , with or without MICAL1 . Depolymerization is initiated at time t = 0 from a non - oxidized actin filament . The increase in the depolymerization rate reflects the oxidation of actin with time . Tpm is labeled with neonGreen ( nGr ) , and actin is unlabeled .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Time-lapse images of Tpm-saturated actin filament depolymerizing from their barbed end (BE), with or without MICAL1. Depolymerization is initiated at time t=0 from a non-oxidized actin filament. The increase in the depolymerization rate reflects the oxidation of actin with time. Tpm is labeled with neonGreen (nGr), and actin is unlabeled.


C . Time - lapse images showing the disassembly of Tpm domains . Filaments were saturated with 100 nM nGr - Tpm1 . 8 before the experiment and Tpm was removed from solution at time t = 0 ( the solution contains 0 . 6 µM unlabeled G - actin to prevent filament depolymerization ) . PE and BE indicate F - actin pointed and barbed end , respectively .

C. Time-lapse images showing the disassembly of Tpm domains. Filaments were saturated with 100 nM nGr-Tpm1.8 before the experiment and Tpm was removed from solution at time t=0 (the solution contains 0.6 µM unlabeled G-actin to prevent filament depolymerization). PE and BE indicate F-actin pointed and barbed end, respectively.


A . Time - lapse images of filaments , oxidized or not , bare or Tpm - saturated , exposed to 1 µM mCherry - cofilin - 1 , from time t = 0 onwards .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Time-lapse images of filaments, oxidized or not, bare or Tpm-saturated, exposed to 1 µM mCherry-cofilin-1, from time t=0 onwards.


A . Time - lapse of actin filaments constantly exposed to nGr - Tpm1 . 8 ( yellow ) , WT mCh - cofilin - 1 ( blue ) and S3D - cofilin - 1 , and to MICAL1 from time t = 100s onwards . Here we mixed WT with an excess of S3D - cofilin - 1 to mimic cellular conditions in which most of cofilin - 1 is inhibited by phosphorylation . While Tpm prevents cofilin from binding to non - oxidized filaments , MICAL1 rapidly oxidizes actin , allowing cofilin to bind and sever filaments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Time-lapse of actin filaments constantly exposed to nGr-Tpm1.8 (yellow), WT mCh-cofilin-1 (blue) and S3D-cofilin-1, and to MICAL1 from time t=100s onwards. Here we mixed WT with an excess of S3D-cofilin-1 to mimic cellular conditions in which most of cofilin-1 is inhibited by phosphorylation. While Tpm prevents cofilin from binding to non-oxidized filaments, MICAL1 rapidly oxidizes actin, allowing cofilin to bind and sever filaments.


E and F . TLC analysis of cGAMP , which is indicative of cGAS activation , upon incubation with E ) tRNA or F ) total RNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . The FL - hcGAS concentration was 0 . 265 mg / mL . The tRNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 85 mg / mL . The extracted total RNA concentrations from low to high were 0 . 025 , 0 . 05 , 0 . 1 , 0 . 25 , and 0 . 5 mg / mL . NC indicates the negative control that has only the FL - hcGAS . dsDNA ISD at a concentration of 0 . 05 mg / mL was used as a positive control . All the samples were prepared in 20 mM HEPES at pH 7 . 5 and 150 mM NaCl .

O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E and F. TLC analysis of cGAMP, which is indicative of cGAS activation, upon incubation with E) tRNA or F) total RNA (n=2, biological replicates, data from one representative independent biological replicate are shown). The FL-hcGAS concentration was 0.265 mg/mL. The tRNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.85 mg/mL. The extracted total RNA concentrations from low to high were 0.025, 0.05, 0.1, 0.25, and 0.5 mg/mL. NC indicates the negative control that has only the FL-hcGAS. dsDNA ISD at a concentration of 0.05 mg/mL was used as a positive control. All the samples were prepared in 20 mM HEPES at pH 7.5 and 150 mM NaCl.


F . Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments .

F. Western blot analysis of the OptiPrep gradient fractions with or without DNase and RNase treatments.


G . The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells ( n = 13 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . H . The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells ( n = 9 , biological replicates , data from one representative independent biological replicate are shown ) . The scale bar represents 10 µm . The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area ( yellow circle ) . I . Pearson ' s correlation coefficient ( CC ) analysis of the fluorescence signals of cGAS vs . RNA ( n = 13 , biological replicates ) , and mCherry vs . RNA ( n = 9 , biological replicates ) . The median values were calculated and used for evaluation of significance . * * * P < 0 . 001 ( two - tailed Mann - Whitney test ) .

O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

G. The three images on the left showing fluorescence signals of endogenous cGAS and RNA in HeLa cells (n=13, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots the signal intensities of cGAS versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). H. The three images on the left showing the fluorescence signals of mCherry and RNA in HEK293T cells (n=9, biological replicates, data from one representative independent biological replicate are shown). The scale bar represents 10 µm. The scatterplot on the right plots signal intensities of mCherry versus RNA at each pixel in the indicated cytoplasmic area (yellow circle). I. Pearson's correlation coefficient (CC) analysis of the fluorescence signals of cGAS vs. RNA (n=13, biological replicates), and mCherry vs. RNA (n=9, biological replicates). The median values were calculated and used for evaluation of significance. ***P < 0.001 (two-tailed Mann-Whitney test).


D . Fluorescence microscopy images showing the replacement of FTSC - tRNA by Cy5 - ISD at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . E . Fluorescence microscopy images showing the replacement of FTSC - tRNA by TAMRA - 380 bp - dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC - tRNA ( n = 2 , biological replicates , data from one representative independent biological replicate are shown ) . In each square , a representative area that is 1 / 16 of the raw image is displayed . The scale bars represent 20 µm . F . Quantification of the replacement in " D " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of Cy5 - ISD over FTSC - tRNA in the four parts were calculated , averaged and plotted . G . Quantification of the replacement in " E " . For each square , the raw image used was evenly divided into four parts . Fluorescence signal ratios of TAMRA - 380 bp - dsDNA over FTSC - tRNA in the four parts were calculated , averaged and plotted .

O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O

D. Fluorescence microscopy images showing the replacement of FTSC-tRNA by Cy5-ISD at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. E. Fluorescence microscopy images showing the replacement of FTSC-tRNA by TAMRA-380 bp-dsDNA at indicated concentrations in preformed granules of hcGAS and FTSC-tRNA (n=2, biological replicates, data from one representative independent biological replicate are shown). In each square, a representative area that is 1/16 of the raw image is displayed. The scale bars represent 20 µm. F. Quantification of the replacement in "D". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of Cy5-ISD over FTSC-tRNA in the four parts were calculated, averaged and plotted. G. Quantification of the replacement in "E". For each square, the raw image used was evenly divided into four parts. Fluorescence signal ratios of TAMRA-380 bp-dsDNA over FTSC-tRNA in the four parts were calculated, averaged and plotted.