A , B NR2A protein levels were analyzed in the hippocampus of mice under control conditions ( white bars ; Gadd45a - WT , n = 13 and Gadd45a - KO , n = 14 ) and 1 hour after PA ( black bars ; Gadd45a - WT = 14 , Gadd45a - KO = 14 ) . NR2A signal ( 160 kDa ) was normalized to the loading control ( tubulin ( 55 kDa ) for the total fraction , and synaptotagmin ( 68 kDa ) for the synaptosomal fraction ) and calculated as percentage of control . In Gadd45a - WT samples ( A ) , NR2A protein levels were unchanged in the total fraction during memory formation , while in the synaptosomal fraction , a significant increase was detected ( t20 = 3 . 199 ) . In Gadd45a - KO hippocampi ( B ) , levels of NR2A protein in both fractions remained unaltered during memory consolidation . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni post - hoc test : * * = p < 0 . 01 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O B-TISSUE O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O B-TISSUE O O O O O O B-GENEPROD O O O O O O O O O O O O O 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, B NR2A protein levels were analyzed in the hippocampus of mice under control conditions (white bars; Gadd45a-WT, n=13 and Gadd45a-KO, n=14) and 1 hour after PA (black bars; Gadd45a-WT=14, Gadd45a-KO=14). NR2A signal (160 kDa) was normalized to the loading control (tubulin (55 kDa) for the total fraction, and synaptotagmin (68 kDa) for the synaptosomal fraction) and calculated as percentage of control. In Gadd45a-WT samples (A), NR2A protein levels were unchanged in the total fraction during memory formation, while in the synaptosomal fraction, a significant increase was detected (t20=3.199). In Gadd45a-KO hippocampi (B), levels of NR2A protein in both fractions remained unaltered during memory consolidation. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni post-hoc test: ** = p<0.01.


C Gadd45α pulldown experiments were carried out in wild - type hippocampal lysates ( n = 12 for all groups ) . Percentage of input recovery was unchanged between GFP - FLAG ( green bars ) and Gadd45α - FLAG ( black bars ) for genes that are not regulated by Gadd45α , such as succinate dehydrogenase complex flavoprotein subunit A ( Sdha , no 3 ´ UTR extension ) , and mitogen - activated protein kinase 6 ( Map2K6 , containing 3 ´ UTR extension of 10 kb ) . On the contrary , Gadd45α - FLAG bound fraction revealed a significant increase in Grin2a ( t22 = 4 . 992 ) and Grm5 mRNA ( t22 = 4 . 939 ) as compared to GFP - FLAG fraction . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * * * = p < 0 . 001 .

O B-GENEPROD B-EXP_ASSAY O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD B-EXP_ASSAY O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O 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 Gadd45α pulldown experiments were carried out in wild-type hippocampal lysates (n=12 for all groups). Percentage of input recovery was unchanged between GFP-FLAG (green bars) and Gadd45α-FLAG (black bars) for genes that are not regulated by Gadd45α, such as succinate dehydrogenase complex flavoprotein subunit A (Sdha, no 3´UTR extension), and mitogen-activated protein kinase 6 (Map2K6, containing 3´UTR extension of 10 kb). On the contrary, Gadd45α-FLAG bound fraction revealed a significant increase in Grin2a (t22=4.992) and Grm5 mRNA (t22=4.939) as compared to GFP-FLAG fraction. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: *** = p<0.001.


B Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a - overexpression ( q24 = 4 . 094 ) and Gadd45aK45E - overexpression groups ( q24 = 4 . 726 ) ( n = 9 for all groups ) . Values shown are mean ± SEM ; Tukey ´ s multiple comparison test : * = p < 0 . 05 , * * = p < 0 . 01 .

O B-TISSUE B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Hippocampal Gadd45a mRNA levels were significantly upregulated in both Gadd45a-overexpression (q24 = 4.094) and Gadd45aK45E-overexpression groups (q24 = 4.726) (n=9 for all groups). Values shown are mean ± SEM; Tukey´s multiple comparison test: * = p<0.05, ** = p<0.01.


C Immunohistochemistry for hemagglutinin ( HA , fused to Gadd45α ) in Gadd45a - WT ( left ) , Gadd45a - overexpression ( middle ) and Gadd45aK45E - overexpression ( right ) samples 8 weeks after AAV injection ( n = 3 for all groups ) . Upper row depicts the entire hippocampus , while center and lower rows show magnifications of the CA3 area in which the HA signal ( red ) is not restricted to the nuclei ( blue and white ) . Scale bar : 250 µm .

O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-GENEPROD O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O B-TISSUE O O O O O O O O O O B-TISSUE O O O O B-GENEPROD O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O

C Immunohistochemistry for hemagglutinin (HA, fused to Gadd45α) in Gadd45a-WT (left), Gadd45a-overexpression (middle) and Gadd45aK45E-overexpression (right) samples 8 weeks after AAV injection (n=3 for all groups). Upper row depicts the entire hippocampus, while center and lower rows show magnifications of the CA3 area in which the HA signal (red) is not restricted to the nuclei (blue and white). Scale bar: 250 µm.


D Gadd45α - overexpression was confirmed at the protein level by HA - western blot on hippocampal samples from Gadd45a - WT , Gadd45a - overexpression and Gadd45aK45E - overexpression mice ( n = 3 for all groups ) . Hippocampus scale bars : 250 µm ; CA3 scale bars : 50µm .

O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O B-TISSUE O O O O O O B-TISSUE O O O O O

D Gadd45α-overexpression was confirmed at the protein level by HA-western blot on hippocampal samples from Gadd45a-WT, Gadd45a-overexpression and Gadd45aK45E-overexpression mice (n=3 for all groups). Hippocampus scale bars: 250 µm; CA3 scale bars: 50µm.


E Latency to enter the dark compartment for Gadd45a - WT ( white bars , n = 12 ) , Gadd45a - overexpression ( red bars , n = 11 ) and Gadd45aK45E - overexpression mice ( grey bars , n = 11 ) in the PA test . Note that , unlike Gadd45a - overexpression mice , those expressing the mutated form of Gadd45α ( no RNA - binding capacity ) did not show an increase in memory retention . Values shown are mean ± SEM ; 2 - Way ANOVA and Bonferroni Post - hoc test : * = p < 0 . 05 , * * * = p < 0 . 001 .

O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O B-ORGANISM O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O B-ORGANISM O O B-EXP_ASSAY O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Latency to enter the dark compartment for Gadd45a-WT (white bars, n=12), Gadd45a-overexpression (red bars, n=11) and Gadd45aK45E-overexpression mice (grey bars, n=11) in the PA test. Note that, unlike Gadd45a-overexpression mice, those expressing the mutated form of Gadd45α (no RNA-binding capacity) did not show an increase in memory retention. Values shown are mean ± SEM; 2-Way ANOVA and Bonferroni Post-hoc test: * = p<0.05, *** = p<0.001.


( A ) Electron micrographs of MEFs . Scale bar ; 1 μm . High - magnification images depicting representative electron - dense materials are shown . Scale bar ; 0 . 2 μm .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O

(A) Electron micrographs of MEFs . Scale bar; 1 μm. High-magnification images depicting representative electron-dense materials are shown. Scale bar; 0.2 μm.


( B ) Citrate synthase activity ( absorbance / min ) was measured in MEFs . Error bars represent ± SD from eight independent experiments . Data information : For graph , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O B-GENEPROD I-GENEPROD B-EXP_ASSAY O B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Citrate synthase activity (absorbance/min) was measured in MEFs . Error bars represent ±SD from eight independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( C , D ) Oxygen consumption rates ( OCRs ) of MEFs were measured on an XF24 Analyzer . ( C ) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin ( 2 μg / mL ) ( Oligo ) , FCCP ( 2 μM ) , and rotenone ( 1 μM ) plus actinomycin ( 2 μM ) ( R & A ) . ( D ) OCR ( pmol O2 / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-CELL_TYPE O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D) Oxygen consumption rates (OCRs) of MEFs were measured on an XF24 Analyzer. (C) Oxygen consumption profiles for MEFs exposed sequentially to oligomycin (2 μg/mL) (Oligo), FCCP (2 μM), and rotenone (1 μM) plus actinomycin (2 μM) (R & A). (D) OCR (pmol O2/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( E ) ATP production ( pmol / min ) ( n = 8 ) . Error bars represent ± SD from eight independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) ATP production (pmol/min) (n=8). Error bars represent ±SD from eight independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( F ) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype . MEFs were treated with tunicamycin ( 5 μg / ml ) or vehicle control in the presence or absence of bafilomycin A1 ( 400 nM ) , and endogenous LC3 and p62 levels were measured by immunoblotting . Data represent the ratios of LC3 - II to LC3 - I and p62 to actin in the absence of bafilomycin , which were normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative immunoblot of LC3 and p62 in MEFs of indicated genotype. MEFs were treated with tunicamycin (5 μg/ml) or vehicle control in the presence or absence of bafilomycin A1 (400 nM), and endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin in the absence of bafilomycin, which were normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


( G ) Survival rate of MEFs treated with tunicamycin ( 5 μg / ml ) , thapsigargin ( 1 μM ) , or hydrogen peroxide ( 100 μM ) . Error bars represent ± SD from four independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 . 01 .

O O O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Survival rate of MEFs treated with tunicamycin (5 μg/ml), thapsigargin (1 μM), or hydrogen peroxide (100 μM). Error bars represent ±SD from four independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01.


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( A ) Absolute [ Ca2 + ] m changes in MEFs of indicated genotype in response to bradykinin ( 2 . 5 μM ) . ( B ) Basal and peak values of Ca2 + transients ( μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

B-CELL_TYPE O O O B-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (A) Absolute [Ca2+]m changes in MEFs of indicated genotype in response to bradykinin (2.5 μM). (B) Basal and peak values of Ca2+ transients (μM). Error bars represent ±SD from six independent experiments. Data information: For graph P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( C ) Peak values of Ca2 + transients in MEFs transfected with IP3R or shRNA against IP3R , or treated with 2 - AP ( 20 mM ) . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

B-CELL_TYPE O O O B-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (C) Peak values of Ca2+ transients in MEFs transfected with IP3R or shRNA against IP3R, or treated with 2-AP (20 mM). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


MEFs were transfected with mitochondrially targeted Cameleon . Free Ca2 + dynamics in the mitochondrial matrix were visualized using FRET . Mitochondrial [ Ca2 + ] ( [ Ca2 + ] m ) was continuously monitored by FRET imaging data are represented as absolute [ Ca2 + ] in μmol . ( D ) Peak values of Ca2 + transients in MEFs transfected with VDAC1 or shRNA against VDAC1 . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

B-CELL_TYPE O O O B-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-SUBCELLULAR O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

MEFs were transfected with mitochondrially targeted Cameleon. Free Ca2+ dynamics in the mitochondrial matrix were visualized using FRET. Mitochondrial [Ca2+] ([Ca2+]m) was continuously monitored by FRET imaging data are represented as absolute [Ca2+] in μmol. (D) Peak values of Ca2+ transients in MEFs transfected with VDAC1 or shRNA against VDAC1. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( E ) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm ) . Scale bar : 100 nm . Data represent percentage of ERmitochondrial contacts per mitochondria perimeter . Error bars represent ± SD from 40 images MEFs Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O B-EXP_ASSAY O B-CELL_TYPE O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SUBCELLULAR O O O O O O O O O O B-CELL_TYPE O O O O O O O O O O O O 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) Representative electron of MEFs . Full arrowheads designate the limits of the zone of intimate contact between ER and mitochondria 20 nm). Scale bar: 100 nm. Data represent percentage of ERmitochondrial contacts per mitochondria perimeter. Error bars represent ±SD from 40 images MEFs Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( F ) In situ PLA images using anti - IP3R and anti - VDAC1 antibodies . Scale bar : 20 μm . Data represent fluorescent in MEFs normalized LRRK2 + / + MEFs . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O B-EXP_ASSAY O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O O O 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) In situ PLA images using anti-IP3R and anti-VDAC1 antibodies. Scale bar: 20 μm. Data represent fluorescent in MEFs normalized LRRK2+/+ MEFs. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( G ) Peak values of Ca2 + transients in MEFs transfected with synthetic tethering protein ( TOM - mRFP - ER ) to induce artificial tethering of the ER and mitochondria . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O 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) Peak values of Ca2+ transients in MEFs transfected with synthetic tethering protein (TOM-mRFP-ER) to induce artificial tethering of the ER and mitochondria. Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Representative immunoblots of MAM components in MEFs of indicated genotype . MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right .

O O O O B-EXP_ASSAY O B-SUBCELLULAR O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O

(A) Representative immunoblots of MAM components in MEFs of indicated genotype. MAM fraction was extracted from MEFs by the Percoll gradient method and immunoblotted with antibodies indicated at the right.


( C ) Peak values of Ca2 + transients in MEFs transfected with deletion constructs of LRRK2 ( G2019S ) . Error bars represent ± SD from six independent experiments . Data information : For graph , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O 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) Peak values of Ca2+ transients in MEFs transfected with deletion constructs of LRRK2(G2019S). Error bars represent ±SD from six independent experiments. Data information: For graph , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( D ) Binding assays to detect interactions between deletion constructs of LRRK2 ( G2019S ) and E3 ubiquitin ligases . HEK293 cells were transfected with deletion constructs of LRRK2 ( G2019S ) and E3 ubiquitin ligases , and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody against the V5 or HA epitope , as indicated the right of each panel .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O

(D) Binding assays to detect interactions between deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases. HEK293 cells were transfected with deletion constructs of LRRK2(G2019S) and E3 ubiquitin ligases, and cell lysates were immunoprecipitated with antibody against the V5 or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the V5 or HA epitope, as indicated the right of each panel.


Peak values of Ca2 + transients in MEFs transfected with ligase - active MARCH5 ( WT ) , MULAN ( WT ) , and Parkin ( W403A ) [ Parkin ( WA ) ] or dominant - negative forms of MARCH5 ( H43W ) [ MARCH ( HW ) ] , MULAN ( C339A ) [ MULAN ( CA ) ] , Parkin ( C431A ) [ Parkin ( CA ) ] ( E ) Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Peak values of Ca2+ transients in MEFs transfected with ligase-active MARCH5(WT), MULAN(WT), and Parkin(W403A) [Parkin(WA)] or dominant-negative forms of MARCH5(H43W) [MARCH(HW)], MULAN(C339A) [MULAN(CA)], Parkin(C431A) [Parkin(CA)] (E) Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


Peak values of Ca2 + transients in MEFs transfected with active USP30 or inactive USP30 ( C77S ) [ USP ( CS ) ] ( F ) . Error bars represent ± SD from six independent experiments . Data information : For graph the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 .

O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Peak values of Ca2+ transients in MEFs transfected with active USP30 or inactive USP30(C77S) [USP(CS)] (F). Error bars represent ±SD from six independent experiments. Data information: For graph the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05


( A ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2 + / + and LRRK2 ( G2019S ) - expressing MEFs treated with vehicle ( Control ) or tunicamycin ( 1 μg / ml ) . Endogenous PERK , MARCH5 , MULAN and Parkin were immunoprecipitated with antibody , and precipitates were immunoblotted with antibody indicated at the right . Data represent the ratio of phosphorylated to total protein levels . Error bars represent ± SD from independent experiments . Data information : For graph A the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases in LRRK2+/+ and LRRK2(G2019S)-expressing MEFs treated with vehicle (Control) or tunicamycin (1 μg/ml). Endogenous PERK, MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Data represent the ratio of phosphorylated to total protein levels. Error bars represent ±SD from independent experiments. Data information: For graph A the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) Immunoprecipitation / immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin ( 5 μg / ml ) . LRRK2 ( G2019S ) MEFs were also pretreated with LRRK2 - IN - 1 ( 1 μM ) . MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody against the Myc epitope , phosphoserine or mitofin2 as indicated to the right of each panel . Mfn2 : mitofusin 2 , Ub : ubiquitin . Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin . Error bars represent ± SD from four independent experiments . Data information : For graph B , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation/immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in MEFs in the of tunicamycin (5 μg/ml). LRRK2(G2019S) MEFs were also pretreated with LRRK2-IN-1 (1 μM). MEFs were transfected with each E3 ubiquitin ligase and biquitin and lysates were immunoprecipitated with antibody against the Myc or HA epitope. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody against the Myc epitope, phosphoserine or mitofin2 as indicated to the right of each panel. Mfn2: mitofusin 2, Ub: ubiquitin. Data represent ratios of phosphorylated to total E3 ubiquitin ligase and mitofusin2 to actin. Error bars represent ±SD from four independent experiments. Data information: For graph B, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Peak values of Ca2 + transients in MEFs treated with LRRK2 - IN - 1 ( 1 μM ) . Error bars represent ± SD from six independent experiments . Data information : For graph A , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Peak values of Ca2+ transients in MEFs treated with LRRK2-IN-1 (1 μM). Error bars represent ±SD from six independent experiments. Data information: For graph A , the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( B ) ( Upper panel ) Diagram showing full - length PERK Myc at the N - terminus and FLAG at the C - terminus ( M - PERK - F ) . S1P recognition sequence R33SLL is mutated to A33SLL ( M - PERK ( R33A ) - F ) . ( Lower panel ) Immunoblot of PERK and PERK ( R33A ) from transfected MEFs under tunicamycin . MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method . Lysates were immunoprecipitated with antibody against FLAG . Precipitated proteins were subjected to SDS / PAGE , and blots were stained with antibody as indicated to the right .

O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O B-SMALL_MOLECULE O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-CELL_TYPE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O

(B)(Upper panel) Diagram showing full-length PERK Myc at the N-terminus and FLAG at the C-terminus (M-PERK-F). S1P recognition sequence R33SLL is mutated to A33SLL (M-PERK(R33A)-F). (Lower panel) Immunoblot of PERK and PERK(R33A) from transfected MEFs under tunicamycin. MAM fraction and cytosol were extracted from transfected MEFs by the Percoll gradient method. Lysates were immunoprecipitated with antibody against FLAG. Precipitated proteins were subjected to SDS/PAGE, and blots were stained with antibody as indicated to the right.


( C ) In vitro kinase assay using isolated PERK , isolated E3 ubiquitin ligase and [ γ - 32P ] ATP . Reaction mixture was subjected to SDS / PAGE . Blots were with antibody against Myc , FLAG or phosphoserine . [ γ - 32P ] ATP - incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ) .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY O O O O

(C) In vitro kinase assay using isolated PERK, isolated E3 ubiquitin ligase and [γ-32P]ATP. Reaction mixture was subjected to SDS/PAGE. Blots were with antibody against Myc, FLAG or phosphoserine. [γ-32P]ATP-incorporated E3 ubiquitin ligases were visualized by autoradiography ( blot was exposed for 24 , and right blot for 36 ).


( D ) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases , HA - tagged Ubl , and His - tagged mitofusin2 in the presence of E1 enzyme , UbcH7 and ATP . E3 ubiquitin ligases were initially phosphorylated PERK or PERK ( K618R ) in vitro . Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination . itofusin 2 with Ni - NTA subjected to SDS / PAGE . Blots were with antibody against HA or mitofusin2 . Mfn2 : mitofusin 2 , Ub : ubiquitin .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-SMALL_MOLECULE O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O

(D) In vitro ubiquitination assay using phosphorylated E3 ubiquitin ligases, HA-tagged Ubl, and His-tagged mitofusin2 in the presence of E1 enzyme, UbcH7 and ATP. E3 ubiquitin ligases were initially phosphorylated PERK or PERK(K618R) in vitro. Phosphorylated E3 ubiquitin ligases were in vitro ubiquitination. itofusin 2 with Ni-NTA subjected to SDS/PAGE. Blots were with antibody against HA or mitofusin2. Mfn2: mitofusin 2, Ub: ubiquitin.


( E ) Immunoprecipitation / Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2 ( G2019S ) - expressing MEFs transfected with the increasing amounts of LRRK2 - d1 - V5 ( 1 , 5 , 25 μg / 106 cells ) treated with tunicamycin ( 1 μg / ml ) . Endogenous MARCH5 , MULAN and Parkin were immunoprecipitated with antibody , and precipitates were immunoblotted with antibody indicated at the right . Endogenous mitofusin 2 was immunoblotted with anti - mitofusin 2 antibody . Data represent the ratio of phosphorylated to total protein levels of MARCH5 , MULAN and Parkin , and the ratio of mitofusin 2 to actin . Error bars represent ± SD from independent experiments . Data information : For graph E , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 , * * P < 0 .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-EXP_ASSAY B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-CELL_TYPE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-EXP_ASSAY O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD I-GENEPROD O B-EXP_ASSAY O O O B-GENEPROD I-GENEPROD O O O O O O O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O 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) Immunoprecipitation/Immunoblot of phosphorylated E3 ubiquitin ligases and mitofusin 2 in LRRK2(G2019S)-expressing MEFs transfected with the increasing amounts of LRRK2-d1-V5 (1, 5, 25 μg/106 cells) treated with tunicamycin (1 μg/ml). Endogenous MARCH5, MULAN and Parkin were immunoprecipitated with antibody, and precipitates were immunoblotted with antibody indicated at the right. Endogenous mitofusin 2 was immunoblotted with anti-mitofusin 2 antibody. Data represent the ratio of phosphorylated to total protein levels of MARCH5, MULAN and Parkin, and the ratio of mitofusin 2 to actin. Error bars represent ±SD from independent experiments. Data information: For graph E, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05, **P < 0.01


( A ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2 . HEK293 cells were transfected with PERK and either MARCH5 , MULAN or Parkin , LRRK2 , LRRK2 ( D1994A ) or LRRK2 ( S1292D ) . Cell lysates were immunoprecipitated with antibody against Myc or V5 , and precipitates were immunoblotted with antibody indicated the right .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_LINE O O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O

(A) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK and the binding to mutant LRRK2. HEK293 cells were transfected with PERK and either MARCH5, MULAN or Parkin, LRRK2, LRRK2(D1994A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc or V5, and precipitates were immunoblotted with antibody indicated the right.


( B ) In vitro kinase assay for auto - phosphorylated LRRK2 using isolated LRRK2 mutants and [ γ - 32P ] ATP with or without LRRK2 - IN - 1 ( 1 μM ) . Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS / PAGE . Blots were with antibody against V5 or phosphoserine . [ γ - 32P ] ATP - incorporated LRRK2 visualized by autoradiography .

O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY B-GENEPROD O O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O B-GENEPROD O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-GENEPROD O O B-EXP_ASSAY O

(B) In vitro kinase assay for auto-phosphorylated LRRK2 using isolated LRRK2 mutants and [γ-32P]ATP with or without LRRK2-IN-1 (1 μM). Mutant LRRK2 precipitated with antibody against V5 was subjected to SDS/PAGE. Blots were with antibody against V5 or phosphoserine. [γ-32P]ATP-incorporated LRRK2 visualized by autoradiography.


( C ) Binding assays to detect interactions between phosphomimetic LRRK2 ( S1292D ) or phosphorylation - defective LRRK2 ( S1292A ) and E3 ubiquitin ligases . HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope . Precipitated proteins were subjected to SDS / PAGE , for immunoblotwith antibody against V5 or Myc .

O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-CELL_LINE O O O O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O B-EXP_ASSAY O O O B-GENEPROD O B-GENEPROD O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O B-GENEPROD O B-GENEPROD O

(C) Binding assays to detect interactions between phosphomimetic LRRK2(S1292D) or phosphorylation-defective LRRK2(S1292A) and E3 ubiquitin ligases. HEK293 cells were transfected with LRRK2 mutants and E3 ubiquitin ligases lysates were immunoprecipitated with antibody against Myc or V5 epitope. Precipitated proteins were subjected to SDS/PAGE, for immunoblotwith antibody against V5 or Myc.


( D ) Immunoprecipitation / mmunoblot of E3 ubiquitin ligases by PERK - ΔN in the presence of LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . HEK293 cells were transfected with MARCH5 , MULAN , Parkin , and PERK - ΔN LRRK2 ( S1292A ) or LRRK2 ( S1292D ) . Cell lysates were immunoprecipitated with antibody against Myc , and precipitates were immunoblotted with antibody indicated the right .

O O O B-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O

(D) Immunoprecipitation/mmunoblot of E3 ubiquitin ligases by PERK-ΔN in the presence of LRRK2(S1292A) or LRRK2(S1292D). HEK293 cells were transfected with MARCH5, MULAN, Parkin, and PERK-ΔN LRRK2(S1292A) or LRRK2(S1292D). Cell lysates were immunoprecipitated with antibody against Myc, and precipitates were immunoblotted with antibody indicated the right.


( A ) Immunoblot of LC3 and p62 . MEFs were transfected with PERK - ΔN or PERK shRNA . Endogenous LC3 and p62 levels were measured by immunoblotting . Data represent the ratios of LC3 - II to LC3 - I and p62 to actin normalized LRRK + / + MEFs . Error bars represent ± SD from four independent experiments . Data information : For graph A , the P values was determined by a Mann - Whitney U test . ns = not significant , * P < 0 . 05 .

O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-CELL_TYPE O O O O O O O O O O O O O O O O O O O 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) Immunoblot of LC3 and p62. MEFs were transfected with PERK-ΔN or PERK shRNA. Endogenous LC3 and p62 levels were measured by immunoblotting. Data represent the ratios of LC3-II to LC3-I and p62 to actin normalized LRRK+/+ MEFs. Error bars represent ±SD from four independent experiments. Data information: For graph A, the P values was determined by a Mann-Whitney U test. ns = not significant, *P < 0.05.


( B ) Peak values of Ca2 + transients in MEFs transfected with PERK - ΔN or PERK shRNA . Error bars represent ± SD from six independent experiments .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE B-EXP_ASSAY O B-CELL_TYPE O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O

(B) Peak values of Ca2+ transients in MEFs transfected with PERK-ΔN or PERK shRNA. Error bars represent ±SD from six independent experiments.


D ) In the 6 months old p32cKO heart , p62 and multi - ubiquitin staining patterns were co - localized . Scale bar , 5 µm . E ) In the 6 months old p32cKO heart , the staining patterns of p62 and the mitochondrial marker protein , pyruvate dehydrogenase ( PDH ) , were co - localized . Scale bar , 5 µm .

O O O O O O O B-GENEPROD B-TISSUE O B-GENEPROD O O O B-SMALL_MOLECULE B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD B-TISSUE O O B-EXP_ASSAY O O B-GENEPROD O O B-SUBCELLULAR O O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O

D) In the 6 months old p32cKO heart, p62 and multi-ubiquitin staining patterns were co-localized. Scale bar, 5 µm. E) In the 6 months old p32cKO heart, the staining patterns of p62 and the mitochondrial marker protein, pyruvate dehydrogenase (PDH), were co-localized. Scale bar, 5 µm.


( F ) Western blot analysis of ubiquitinated proteins using an anti - multi - ubiquitin antibody in the 6 months old WT and p32cKO heart ( n = 4 mice per group ) . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * * p < 0 . 005 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD B-TISSUE O O O O B-ORGANISM 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-GENEPROD O O O O O O O O O O

(F) Western blot analysis of ubiquitinated proteins using an anti-multi-ubiquitin antibody in the 6 months old WT and p32cKO heart (n = 4 mice per group). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( B ) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart . Tissues were excited at a wavelength of 540 ( upper panel ) or 470 ( lower panel ) and emission spectra were collected with a confocal microscope at wavelengths ( band path ) of 580 - 630 nm ( upper panel ) or 510 - 560 nm ( lower panel ) . Enlarged view of the white squares is shown right . Scale bars , 20 µm . The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM ( three months old mice , n = 11 ; 6 months old mice , n = 9 - 14 , 9 months old mice , n = 9 - 15 , 12 months old mice , n = 11 - 21 ) . Statistical significance was assessed by Student ' s t - test , * * * p < 0 . 005 .

O O O B-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O O O O B-GENEPROD B-TISSUE O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-SMALL_MOLECULE B-EXP_ASSAY O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O O O O O O B-ORGANISM O O O O O 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) Autofluorescence showing lipofuscin localization around the nucleus in the 6 months old p32cKO heart. Tissues were excited at a wavelength of 540 (upper panel) or 470 (lower panel) and emission spectra were collected with a confocal microscope at wavelengths (band path) of 580-630 nm (upper panel) or 510-560 nm (lower panel). Enlarged view of the white squares is shown right. Scale bars, 20 µm. The quantification of the ratio of autofluorescence of DAPI staining is presented in the plot in the right panel as mean ± SEM (three months old mice, n = 11; 6 months old mice, n = 9-14, 9 months old mice, n = 9-15, 12 months old mice, n = 11-21). Statistical significance was assessed by Student's t-test, ***p < 0.005.


( D ) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts . Quantification is shown in the lower panel ( n = 4 mice per group ) . GAPDH was used as an internal control . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * * p < 0 . 005 .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O B-ORGANISM O O O O B-GENEPROD O O O 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-GENEPROD O O O O O O O O O O

(D) Western blot analysis of Lamp2 in the 6 months old WT and p32cKO hearts. Quantification is shown in the lower panel (n = 4 mice per group). GAPDH was used as an internal control. Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, ***p < 0.005.


( A ) LC - MS / MS metabolomic analysis of NAD + , NADH , NADP + , nicotinamide and NAAD in the 6 months old WT and p32cKO hearts . NAD + and NADP + showed significantly different levels between WT and p32cKO hearts ( n = 12 ) . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT vs p32cKO , * * p < 0 . 001 , * * * p < 0 . 0002 .

O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-GENEPROD B-TISSUE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O B-GENEPROD B-TISSUE O O 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-GENEPROD O O O O O O O O O O O O O O O O O O

(A) LC-MS/MS metabolomic analysis of NAD+, NADH, NADP+, nicotinamide and NAAD in the 6 months old WT and p32cKO hearts. NAD+ and NADP+ showed significantly different levels between WT and p32cKO hearts (n = 12). Error bars are presented as mean ± SD. Student's t-test was performed on WT vs p32cKO, **p < 0.001, ***p < 0.0002.


( B ) Real - time PCR analysis of RNA expression of NAD - synthesizing enzymes in the 6 months old WT and p32cKO hearts . Error bars are presented as mean ± SD . Student ' s t - test was performed on WT mice vs p32cKO mice ( n = 6 ) , * * * p < 0 . 005 , * p < 0 . 05 . The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows . NAM : nicotinamide , NA : nicotinic acid , QA : quinolic acid , NMN : nicotinamide mononucleotide .

O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD B-TISSUE O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD B-ORGANISM O O O 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-SMALL_MOLECULE O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O

(B) Real-time PCR analysis of RNA expression of NAD-synthesizing enzymes in the 6 months old WT and p32cKO hearts. Error bars are presented as mean ± SD. Student's t-test was performed on WT mice vs p32cKO mice (n = 6), ***p < 0.005, *p < 0.05. The right panel shows the NAD synthesis pathway and mRNA expression levels are indicated by red arrows. NAM: nicotinamide, NA: nicotinic acid, QA: quinolic acid, NMN: nicotinamide mononucleotide.


( G ) Nmnat3 mRNA expression in 3T3 - L1 cells after 1 mM CAP treatment for 72 h . The HIF1α inhibitor ( 20 or 50 µM ) was added 2 h before the CAP treatment . Error bars are presented as mean ± SD of three independent experiments . Student ' s t - test was performed on WT cells vs WT cells treated CAP and ( or ) HIF1α inhibitor , * * p < 0 . 01 , * p < 0 . 05 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O B-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O 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-SMALL_MOLECULE O O O O B-GENEPROD O O O O O O O O O O O O O O O O O

(G) Nmnat3 mRNA expression in 3T3-L1 cells after 1 mM CAP treatment for 72 h. The HIF1α inhibitor (20 or 50 µM) was added 2 h before the CAP treatment. Error bars are presented as mean ± SD of three independent experiments. Student's t-test was performed on WT cells vs WT cells treated CAP and (or) HIF1α inhibitor,**p < 0.01, *p < 0.05.


( I ) Nmnat3 mRNA expression in 3T3 - L1 cells after 150 µM CoCl2 treatment for 72 h ( n = 3 ) . Error bars are presented as mean ± SD of three independent experiments . Statistical significance was assessed by Student ' s t - test , * * p < 0 . 01 .

O O O B-GENEPROD B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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) Nmnat3 mRNA expression in 3T3-L1 cells after 150 µM CoCl2 treatment for 72 h (n = 3). Error bars are presented as mean ± SD of three independent experiments. Statistical significance was assessed by Student's t-test, **p < 0.01.


( A ) Lysosomal acidification is impaired in p32KO MEFs . Representative images of WT and p32KO MEFs , stained with dextran - Oregon Green ( 488 ) and dextran - TMRM . Scale bar , 5 µm . Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification , and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification . Dextran - Oregon Green is quenched under acidic pH , and thus an increased green : red ratio denotes impaired lysosomal acidification . The average ± SEM of the green : red ratio in at least 30 cells in two independent experiments is presented in the plot ( normalized to WT green / red = 1 ) . Statistical significance was assessed by Student ' s t - test , * p < 0 . 05 , * * * p < 0 . 001 .

O O O B-SUBCELLULAR B-EXP_ASSAY O O O B-GENEPROD B-CELL_TYPE O O O O O O B-GENEPROD B-CELL_TYPE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD B-CELL_TYPE O B-SUBCELLULAR B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O O B-CELL_TYPE O O O O B-SUBCELLULAR B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lysosomal acidification is impaired in p32KO MEFs. Representative images of WT and p32KO MEFs, stained with dextran-Oregon Green (488) and dextran-TMRM. Scale bar, 5 µm. Addition of 1 mM NMN for 48 h to p32KO MEFs rescued lysosomal acidification, and 1 mM CAP treatment of WT MEFs for 48 h decreased lysosomal acidification. Dextran-Oregon Green is quenched under acidic pH, and thus an increased green:red ratio denotes impaired lysosomal acidification. The average ± SEM of the green:red ratio in at least 30 cells in two independent experiments is presented in the plot (normalized to WT green/red = 1). Statistical significance was assessed by Student's t-test, *p < 0.05, ***p < 0.001.


( E ) Nmnat3 ( v1 ) overexpression in p32KO MEFs rescued lysosomal function . LysoTracker Red - stained p32KO MEFs were quantified by flow cytometry . Error bars are presented as mean ± SEM of 4 independent experiments . Statistical significance was assessed by Student ' s t - test , * * * p < 0 . 001 .

O O O B-GENEPROD O O O O O B-GENEPROD B-CELL_TYPE O B-SUBCELLULAR O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY B-GENEPROD B-CELL_TYPE O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O 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) Nmnat3(v1) overexpression in p32KO MEFs rescued lysosomal function. LysoTracker Red-stained p32KO MEFs were quantified by flow cytometry. Error bars are presented as mean ± SEM of 4 independent experiments. Statistical significance was assessed by Student's t-test, ***p < 0.001.


( A ) WT MEFs were stained with dextran - Oregon Green and dextran - TMRM to examine lysosomal acidification after 10 nM FK866 treatment . FK866 decreased lysosomal acidification , which was rescued by 1 mM NMN pretreatment . The stained MEFs were quantified using a microscope ( BZ - X800 , KEYENCE ) . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

O O O O B-CELL_TYPE O B-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O B-SUBCELLULAR B-EXP_ASSAY O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE O B-SUBCELLULAR B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY B-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O 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) WT MEFs were stained with dextran-Oregon Green and dextran-TMRM to examine lysosomal acidification after 10 nM FK866 treatment. FK866 decreased lysosomal acidification, which was rescued by 1 mM NMN pretreatment. The stained MEFs were quantified using a microscope (BZ-X800, KEYENCE). Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( F ) To examine autophagosome and autolysosome formation , we used DAPRed , which indicates autophagosomes , and DALGreen , which indicates autolysosomes . After starvation , co - localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells . Scale bars , 10 µm . Quantification is shown on the right . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * * p < 0 . 001 .

O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O O B-SUBCELLULAR O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O B-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O 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) To examine autophagosome and autolysosome formation, we used DAPRed, which indicates autophagosomes, and DALGreen, which indicates autolysosomes. After starvation, co-localization of both staining agents was observed upon FK866 or bafilomycin A treatment in WT MEF cells. Scale bars, 10 µm. Quantification is shown on the right. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, ***p < 0.001.


( D ) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs , the membrane pellet ( bound ) and supernatant ( free ) were subjected to immunoblotting with GAPDH and PGK1 antibodies .

O O O O O O O O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD O O

(D) After adding various concentrations of NaCl to the lysosomal fraction of WT MEFs, the membrane pellet (bound) and supernatant (free) were subjected to immunoblotting with GAPDH and PGK1 antibodies.


( I ) After addition of GAP , NAD and Pi , ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin / luciferase kit . Iodoacetate ( IA ; 4 µM ) was used to inhibit GAPDH activity . Three independent experiments were performed . Error bars are presented as mean ± SEM of three independent experiments . Statistical significance was assessed by one - way ANOVA , * * p < 0 . 002 .

O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE O B-SMALL_MOLECULE B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O 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) After addition of GAP, NAD and Pi, ATP production in the lysosomal fraction isolated by MAG10 was measured using a luciferin/luciferase kit. Iodoacetate (IA; 4 µM) was used to inhibit GAPDH activity. Three independent experiments were performed. Error bars are presented as mean ± SEM of three independent experiments. Statistical significance was assessed by one-way ANOVA, **p < 0.002.


B Image sequence from confocal time lapse shows the early steps in neuronal differentiation . Two transient basal protrusions are extended along the basal surface of the neural tube ( 6h 26 to 14h 01 ) and then retracted ( 15h 31 to 17h 31 ) . The apical attachment is also retracted ( 17h 31 to 19h 53 ; e . g . - 1h 30 , ) before the axon is extended ( 20h 38 to 21h 50 ) . Main images are maximum projections and insets are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O B-EXP_ASSAY O O O O O O O B-CELL_TYPE B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

B Image sequence from confocal time lapse shows the early steps in neuronal differentiation. Two transient basal protrusions are extended along the basal surface of the neural tube (6h 26 to 14h 01) and then retracted (15h 31 to 17h 31). The apical attachment is also retracted (17h 31 to 19h 53; e.g. -1h 30,) before the axon is extended (20h 38 to 21h 50). Main images are maximum projections and insets are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


C Image sequence from confocal time lapse shows a neuron before , during and after axon initiation . Prior to axon initiation , the neuron extends multiple small , transient pre - axonal protrusions ( - 2h to - 20m ) . The nascent axon is extended ( 0h ) and maintained for a short period ( 0h to 30m ) before axon growth begins ( 45m to 2h 20 ) . Images are transverse reconstructions from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

C Image sequence from confocal time lapse shows a neuron before, during and after axon initiation. Prior to axon initiation, the neuron extends multiple small, transient pre-axonal protrusions (-2h to -20m). The nascent axon is extended (0h) and maintained for a short period (0h to 30m) before axon growth begins (45m to 2h 20). Images are transverse reconstructions from confocal z-stacks. Data information: All scale bars = 10 µm.


D Plots showing axon position on the soma ( e . g . asterisk in Figure 1C 0h ) relative to the cell centroid at 0 , 0 for dorsal and transverse view ( n = 86 cells from 8 experiments ) . Axon position is not random ( dorsal view P < 0 . 001 , mean = 95 . 3o ; transverse view P < 0 . 001 , mean = 148 . 9o ) . Data analysed using Moore ' s modification of the Rayleigh ' s test .

O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O 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 Plots showing axon position on the soma (e.g. asterisk in Figure 1C 0h) relative to the cell centroid at 0,0 for dorsal and transverse view (n = 86 cells from 8 experiments). Axon position is not random (dorsal view P < 0.001, mean = 95.3o; transverse view P < 0.001, mean = 148.9o). Data analysed using Moore's modification of the Rayleigh's test.


F Lateral and transverse reconstructions of DoLA neuron at the time of axon initiation ( 0h ) and during axon growth ( 4h ) . Data information : All scale bars = 10 µm .

O O O O O O O B-CELL_TYPE O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O

F Lateral and transverse reconstructions of DoLA neuron at the time of axon initiation (0h) and during axon growth (4h). Data information: All scale bars = 10 µm.


G - M Plots showing axon position on the soma relative to cell centroid at 0 , 0 in dorsal and transverse view for DoLA ( G ; n = 7 cells ) , CiA ( H ; n = 10 cells ) , CiD ( I ; n = 3 cells ) , VeLD ( J ; n = 5 cells ) , CoSA ( K ; n = 11 cells ) , CoB ( L ; n = 6 cells ) and UCoD ( M ; 15 cells ) neuronal subtypes .

O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CELL_TYPE O O

G-M Plots showing axon position on the soma relative to cell centroid at 0,0 in dorsal and transverse view for DoLA (G; n = 7 cells), CiA (H; n = 10 cells), CiD (I; n = 3 cells), VeLD (J; n = 5 cells), CoSA (K; n = 11 cells), CoB (L; n = 6 cells) and UCoD (M; 15 cells) neuronal subtypes.


A Image sequence showing centrosome position of a neuron labelled with membrane and centrosome labels during apical process retraction . The centrosome is retracted close to the tip of the apical process . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O B-SUBCELLULAR O O O B-CELL_TYPE O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

A Image sequence showing centrosome position of a neuron labelled with membrane and centrosome labels during apical process retraction. The centrosome is retracted close to the tip of the apical process. Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


B ( i ) Image sequence showing a neuron labelled with a membrane marker during apical retraction without any observable abscission event ( n = 64 / 72 cells from 6 experiments ) . ( ii ) High resolution images of boxed section in ( i ) . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O B-CELL_TYPE O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O

B (i) Image sequence showing a neuron labelled with a membrane marker during apical retraction without any observable abscission event (n = 64/72 cells from 6 experiments). (ii) High resolution images of boxed section in (i). Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


C ( i ) Low magnification overview of the spinal cord of a cilium reporter line , Tg ( actb2 : arl13b - GFP ) from a single confocal slice . ( ii ) High resolution image sequence of boxed section in ( i ) . One GFP - labelled cilium moves from apical surface towards the basal surface of the spinal cord ( n = 13 cilia from two experiments ) . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O B-TISSUE I-TISSUE O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

C (i) Low magnification overview of the spinal cord of a cilium reporter line, Tg(actb2:arl13b-GFP) from a single confocal slice. (ii) High resolution image sequence of boxed section in (i). One GFP-labelled cilium moves from apical surface towards the basal surface of the spinal cord (n = 13 cilia from two experiments). Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


D ( i ) Low magnification overview of the spinal cord of a Tg ( actb2 : arl13b - GFP ) embryo labelled with centrin - RFP from a single confocal slice . ( ii ) Image sequence of boxed section in ( i ) . A cilium and centrosome move together from apical surface towards the basal surface of the spinal cord ( n = 2 cells from one experiment ) . Insets show high magnification of cilium - centrosome pair . Images are maximum projections from confocal z - stacks . Data information : All scale bars = 10 µm .

O O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

D (i) Low magnification overview of the spinal cord of a Tg(actb2:arl13b-GFP) embryo labelled with centrin-RFP from a single confocal slice. (ii) Image sequence of boxed section in (i). A cilium and centrosome move together from apical surface towards the basal surface of the spinal cord (n = 2 cells from one experiment). Insets show high magnification of cilium-centrosome pair. Images are maximum projections from confocal z-stacks. Data information: All scale bars = 10 µm.


E Graph showing length of cilium as it moves from apical surface ( 0 % ) to close to basal surface ( 100 % . Cilium length did not change ( P > 0 . 05 for n = 6 / 7 cilia from two experiments ; non - linear regression ) .

O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

E Graph showing length of cilium as it moves from apical surface (0%) to close to basal surface (100%. Cilium length did not change (P > 0.05 for n = 6/7 cilia from two experiments; non-linear regression).


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 B-SUBCELLULAR O B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-SUBCELLULAR O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O O B-CELL_TYPE O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-EXP_ASSAY 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 B-EXP_ASSAY O O O O B-CELL_TYPE O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-EXP_ASSAY 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 B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR 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-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR 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 B-EXP_ASSAY O O O O B-CELL_TYPE O O B-SUBCELLULAR O B-SUBCELLULAR O O B-SUBCELLULAR B-EXP_ASSAY 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 B-EXP_ASSAY O O O O B-CELL_TYPE O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR B-EXP_ASSAY 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 B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O 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 B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY 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 .

O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O

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 .

O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

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 .

O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-SUBCELLULAR O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O

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 B-EXP_ASSAY O O O O O O B-CELL_TYPE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O 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-EXP_ASSAY 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 B-EXP_ASSAY O O O O B-CELL_TYPE O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O B-CELL_TYPE O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O B-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-EXP_ASSAY 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 B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O 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-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O 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-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O B-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O 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.