( A ) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec ‐ 4 ( d ) or deg ‐ 3 ( d ) alleles in SNT ‐ 1 ‐ deficient mutants . For mec ‐ 4 ( d ) mutant animals , bars denote touch receptor neuron corpses . For deg ‐ 3 ( d ) mutants , bars denote inner labial neuron 1 ( L1 ) sensory neuron and PVC interneuron corpses .

O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE I-CELL_TYPE O O

(A) Number of cell corpses at the L1 stage of development per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles in SNT‐1‐deficient mutants. For mec‐4(d) mutant animals, bars denote touch receptor neuron corpses. For deg‐3(d)mutants, bars denote inner labial neuron 1 (L1) sensory neuron and PVC interneuron corpses.


( B ) Expression of a full ‐ length MEC ‐ 4 : : GFP reporter fusion under the control of the mec ‐ 4 promoter in touch receptor neurons of wild ‐ type , snt ‐ 1 and unc ‐ 57 mutant animals . Representative photos of touch receptor neuron cell bodies are shown . Bar denotes 6 μm . The quantification ( % ) of GFP signal intensity from the animals examined is graphed below ( n = 100 neurons per assay ; P0 . 05 compared with wild type ; t ‐ test ) .

O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE 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 O O O O O O O O O O O O

(B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, snt‐1 and unc‐57 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test).


( C ) Percentage of SNT ‐ 1 ‐ deficient animals that survive near ‐ lethal treatment with sodium azide . This chemical inhibits the activity of the respiratory electron transport complex IV ( cytochrome C oxidase ) and simulates hypoxia .

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

(C) Percentage of SNT‐1‐deficient animals that survive near‐lethal treatment with sodium azide. This chemical inhibits the activity of the respiratory electron transport complex IV (cytochrome C oxidase) and simulates hypoxia.


( D ) Number of touch receptor neuron corpses , at the L1 stage of development , per 100 animals carrying the neurotoxic mec - 4 ( d ) or deg ‐ 3 ( d ) alleles in UNC ‐ 57 ‐ deficient animals .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O

(D) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec-4(d) or deg‐3(d) alleles in UNC‐57‐deficient animals.


( E ) Percentage of UNC ‐ 57 ‐ deficient animals that survive after the hypoxia ‐ inducing treatment with sodium azide .

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

(E) Percentage of UNC‐57‐deficient animals that survive after the hypoxia‐inducing treatment with sodium azide.


( F ) Number of touch receptor neuron corpses , at the L1 stage of development , per 100 animals carrying the neurotoxic mec ‐ 4 ( d ) or deg ‐ 3 ( d ) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin ‐ mediated endocytosis . UNC ‐ 11 and DPY ‐ 23 are adaptor proteins required for the formation of clathrin ‐ coated pits , DYN ‐ 1 is a GTPase necessary for fission of clathrin ‐ coated vesicles and UNC ‐ 26 participates in the uncoating of vesicles . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with wild ‐ type animals , unpaired t ‐ test ) .

O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-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 B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-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

(F) Number of touch receptor neuron corpses, at the L1 stage of development, per 100 animals carrying the neurotoxic mec‐4(d) or deg‐3(d) alleles together with lesions in genes encoding key proteins that participate in all four steps of clathrin‐mediated endocytosis. UNC‐11 and DPY‐23 are adaptor proteins required for the formation of clathrin‐coated pits, DYN‐1 is a GTPase necessary for fission of clathrin‐coated vesicles and UNC‐26 participates in the uncoating of vesicles. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with wild‐type animals, unpaired t‐test).


( A ) Confocal images of touch receptor neurons expressing a pmec ‐ 17APS ‐ 2GFP reporter transgene . During early necrosis , the number of APS ‐ 2 : : GFP puncta , corresponding to clathrin ‐ coated pits and vesicles , in touch receptor neurons ofmec ‐ 4 ( d ) animals does not change significantly , whereas at later stages it decreases . A similar decrease is also observed in unc ‐ 57 ( e1190 ) mutants , defective for clathrin ‐ mediated endocytosis .

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

(A) Confocal images of touch receptor neurons expressing a pmec‐17APS‐2GFP reporter transgene. During early necrosis, the number of APS‐2::GFP puncta, corresponding to clathrin‐coated pits and vesicles, in touch receptor neurons ofmec‐4(d) animals does not change significantly, whereas at later stages it decreases. A similar decrease is also observed in unc‐57(e1190) mutants, defective for clathrin‐mediated endocytosis.


( B ) Confocal images of touch receptor neurons expressing a pmec ‐ 7GFP : : RAB ‐ 5 reporter transgene . The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec ‐ 4 ( d ) animals significantly increases during early necrosis , whereas it declines later .

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

(B) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐5 reporter transgene. The number of fluorescent puncta that correspond to early endosomes in touch receptor neurons of mec‐4(d) animals significantly increases during early necrosis, whereas it declines later.


( C ) Confocal images of touch receptor neurons expressing a pmec ‐ 7GFP : : RAB ‐ 11 reporter transgene . The number of GFP : : RAB ‐ 11 puncta that correspond to recycling endosomes in touch receptor neurons of mec ‐ 4 ( d ) animals increases early during cell death , while it declines as degeneration proceeds . Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus . Bars denote 4 μm . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with control animals , unpaired t ‐ test ) .

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

(C) Confocal images of touch receptor neurons expressing a pmec‐7GFP::RAB‐11 reporter transgene. The number of GFP::RAB‐11 puncta that correspond to recycling endosomes in touch receptor neurons of mec‐4(d) animals increases early during cell death, while it declines as degeneration proceeds. Both early and recycling endosomes tend to accumulate around a swollen structure that is probably the nucleus. Bars denote 4 μm. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).


Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death . ( A ) Depletion of synaptotagmin ( SNT ‐ 1 ) or endophilin ( UNC ‐ 57 ) does not further suppress MEC ‐ 4 ( d ) ‐ induced necrosis in cad ‐ 1 mutants with reduced cathepsin activity or with V ‐ ATPase dysfunction ( B , C ) .

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

Endocytosis functions together with lysosomal proteolysis to facilitate necrotic cell death. (A) Depletion of synaptotagmin (SNT‐1) or endophilin (UNC‐57) does not further suppress MEC‐4(d)‐induced necrosis in cad‐1 mutants with reduced cathepsin activity or with V‐ATPase dysfunction (B, C).


Similarly , no synthetic reduction of necrotic cell death is observed in calpain ‐ deficient animals that also carry lesions in the synaptotagmin ( D ) or endophilin ( E ) genes . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

O O O O O O O O O O O O B-GENEPROD O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Similarly, no synthetic reduction of necrotic cell death is observed in calpain‐deficient animals that also carry lesions in the synaptotagmin (D) or endophilin (E) genes. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


( A ) Synaptotagmin ( SNT ‐ 1 ) deficiency further suppresses MEC ‐ 4 ( d ) ‐ induced necrosis in animals with impaired autophagy .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O

(A) Synaptotagmin (SNT‐1) deficiency further suppresses MEC‐4(d)‐induced necrosis in animals with impaired autophagy.


( B ) Endophilin dysfunction enhances suppression of necrosis in mec ‐ 4 ( d ) ; lgg ‐ 1 ( RNAi ) animals , where autophagy is impaired . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 01 , compared with single mutant control animals , unpaired t ‐ test ) .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-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

(B) Endophilin dysfunction enhances suppression of necrosis in mec‐4(d);lgg‐1(RNAi) animals, where autophagy is impaired. Error bars denote s.e.m. values (n>250 for all populations examined; P0.01, compared with single mutant control animals, unpaired t‐test).


( A ) Depletion of the kinesin 1 heavy chain UNC ‐ 116 suppresses necrosis induced by both mec ‐ 4 ( d ) and deg ‐ 3 ( d ) toxic alleles .

O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O

(A) Depletion of the kinesin 1 heavy chain UNC‐116 suppresses necrosis induced by both mec‐4(d) and deg‐3(d) toxic alleles.


( B ) Expression of a full ‐ length MEC ‐ 4 : : GFP reporter fusion under the control of the mec ‐ 4 promoter in touch receptor neurons of wild ‐ type , unc ‐ 116 and unc ‐ 104 mutant animals . Representative photos of touch receptor neuron cell bodies are shown . Bar denotes 6 μm . The quantification ( % ) of GFP signal intensity from the animals examined is graphed below ( n = 100 neurons per assay ; P0 . 05 compared with wild type ; t ‐ test ) .

(B) Expression of a full‐length MEC‐4::GFP reporter fusion under the control of the mec‐4 promoter in touch receptor neurons of wild‐type, unc‐116 and unc‐104 mutant animals. Representative photos of touch receptor neuron cell bodies are shown. Bar denotes 6 μm. The quantification (%) of GFP signal intensity from the animals examined is graphed below (n=100 neurons per assay; P0.05 compared with wild type; t‐test).


( C ) Percentage of unc ‐ 116 animals that survive after treatment with sodium azide .

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

(C) Percentage of unc‐116 animals that survive after treatment with sodium azide.


( D ) Depletion of the monomeric kinesin UNC ‐ 104 protects neurons against MEC ‐ 4 ( d ) ‐ induced degeneration .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O

(D) Depletion of the monomeric kinesin UNC‐104 protects neurons against MEC‐4(d)‐induced degeneration.


( E ) unc ‐ 104 mutant animals are more resistant to hypoxic death induced by sodium azide .

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

(E) unc‐104 mutant animals are more resistant to hypoxic death induced by sodium azide.


( F ) Combined inactivation of endocytosis and kinesin ‐ mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC ‐ 4 ( d ) ‐ induced degeneration . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P0 . 001 , compared with control animals , unpaired t ‐ test ) .

O O O O O O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE I-CELL_TYPE O B-GENEPROD I-GENEPROD I-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

(F) Combined inactivation of endocytosis and kinesin‐mediated intracellular trafficking does not enhance protection of touch receptor neurons against MEC‐4(d)‐induced degeneration. Error bars denote s.e.m. values (n>250 for all populations examined; P0.001, compared with control animals, unpaired t‐test).


Genetic interaction between kinesin ‐ mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death . Dysfunction of either kinesin 1 heavy chain ( UNC ‐ 116 ) or the monomeric kinesin UNC ‐ 104 does not significantly enhance suppression of neurodegeneration in aspartyl protease ‐ deficient mutant animals ( A , B ) , in animals with compromised lysosomal acidification ( C , D ) , or in calpain protease ‐ deficient mutants animals ( E , F ) . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

O 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 I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Genetic interaction between kinesin‐mediated intracellular trafficking and cellular proteolytic pathways mediating necrotic cell death. Dysfunction of either kinesin 1 heavy chain (UNC‐116) or the monomeric kinesin UNC‐104 does not significantly enhance suppression of neurodegeneration in aspartyl protease‐deficient mutant animals (A, B), in animals with compromised lysosomal acidification (C, D), or in calpain protease‐deficient mutants animals (E, F). Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


( A ) Kinesin 1 heavy chain ( UNC ‐ 116 ) deficiency .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O

(A) Kinesin 1 heavy chain (UNC‐116) deficiency.


( B ) Monomeric kinesin UNC ‐ 104 deficiency . In both cases , no significant synthetic protection of neurons is observed in LGG ‐ 1 / LC3 ‐ depleted animals with impaired autophagy . Error bars denote s . e . m . values ( n > 250 for all populations examined ; P > 0 . 5 , compared with single mutant control animals , unpaired t ‐ test ) .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD I-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

(B) Monomeric kinesin UNC‐104 deficiency. In both cases, no significant synthetic protection of neurons is observed in LGG‐1/LC3‐depleted animals with impaired autophagy. Error bars denote s.e.m. values (n>250 for all populations examined; P>0.5, compared with single mutant control animals, unpaired t‐test).


A Normalized firefly luciferase luminescence ( compared to renilla luciferase ) as a function of external IAA concentration . Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP ( in the case of the control ) both under the control of a constitutive CaMV 35S promoter . Where indicated , 10 µM NPA was added . B Normalized firefly luciferase luminescence ( compared to renilla luciferase ) as a function of external NPA concentration in the presence of 100nM IAA in control GFP - ( blue or red ) or PIN1 - ( green or purple ) transformed protoplasts .

O O B-ORGANISM B-GENEPROD B-EXP_ASSAY O O O B-ORGANISM B-GENEPROD O O O O O O B-SMALL_MOLECULE O O B-ORGANISM B-CELL_TYPE 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 B-ORGANISM B-GENEPROD O O O O O O O B-SMALL_MOLECULE O O O O O B-ORGANISM B-GENEPROD B-EXP_ASSAY O O O B-ORGANISM B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O B-CELL_TYPE O

A Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external IAA concentration. Arabidopsis protoplast cells were transiently transformed with either AtPIN1 or GFP (in the case of the control) both under the control of a constitutive CaMV 35S promoter. Where indicated, 10 µM NPA was added. B Normalized firefly luciferase luminescence (compared to renilla luciferase) as a function of external NPA concentration in the presence of 100nM IAA in control GFP- (blue or red) or PIN1- (green or purple) transformed protoplasts.


A , B Native PAGE separation reveals distinct detergent - sensitive PIN1 - complexes . Plasma membrane preparations from dark grown Arabidopsis cell suspension cultures were solubilized with ( a ) Complexiolyte 27 ( a mixture of ionic and non - ionic detergents ) , and ( b ) Complexiolyte 47 ( lower stringency , non - ionic detergent ) , ( both Logopharm ) ; first dimension BN - PAGE , second dimension SDS - PAGE , blots stained with anti - PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions . Values are given in KDa .

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

A, B Native PAGE separation reveals distinct detergent-sensitive PIN1-complexes. Plasma membrane preparations from dark grown Arabidopsis cell suspension cultures were solubilized with (a) Complexiolyte 27 (a mixture of ionic and non-ionic detergents), and (b) Complexiolyte 47 (lower stringency, non-ionic detergent), (both Logopharm); first dimension BN-PAGE, second dimension SDS-PAGE, blots stained with anti-PIN1 antibody revealing distinct PIN1 complex populations at the indicated positions. Values are given in KDa.


c ) Molecular abundance ( abundancenormspec values calculated as summarized peptide PVs divided by the number of MS - accessible protein specific amino acids ) of PIN subunits and ABCB19 in anti - GFP affinity purification of CL47 - solubilized PIN1 - GFP - expressing roots with a GFP - specific DARPin . Note the significant heteromerization of several endogenous PINs with PIN1 - GFP and the absence of other abundant interaction partners ( ABC19B shown as an example ) .

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

c) Molecular abundance (abundancenormspec values calculated as summarized peptide PVs divided by the number of MS-accessible protein specific amino acids) of PIN subunits and ABCB19 in anti-GFP affinity purification of CL47-solubilized PIN1-GFP-expressing roots with a GFP-specific DARPin. Note the significant heteromerization of several endogenous PINs with PIN1-GFP and the absence of other abundant interaction partners (ABC19B shown as an example).


a ) BN - PAGE was performed with PIN1 - containing microsomes prepared from either PIN1 - expressing HEK293T cells or a dark - grown Arabidopsis cell suspension culture . Prior to solubilization with 1 % dodecyl maltoside , microsomes were incubated with either 10 µM NPA or 10 µM quercetin .

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

a) BN-PAGE was performed with PIN1-containing microsomes prepared from either PIN1-expressing HEK293T cells or a dark-grown Arabidopsis cell suspension culture. Prior to solubilization with 1% dodecyl maltoside, microsomes were incubated with either 10 µM NPA or 10 µM quercetin.


b ) PIN1 dimer stability induced by NPA after expression in HEK cells . Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50 % ( v / v ) CL27 . Each measurement given ( three for each concentration ) represents the mean of three gel lanes for wild type ( circles ) or triple S2523 , S253E , S261E phosphomimetic sequences ( crosses ) ( example images are given in the figure inset ) .

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

b) PIN1 dimer stability induced by NPA after expression in HEK cells. Relative distribution between monomer and dimer after NPA treatment is given relative to distribution of untreated samples after solubilization with 50% (v/v) CL27. Each measurement given (three for each concentration) represents the mean of three gel lanes for wild type (circles) or triple S2523, S253E, S261E phosphomimetic sequences (crosses) (example images are given in the figure inset).


a ) Summed 2D projections of PLA interactions ( left ) and co - localisation ( right ) ( n = 3 ) between PIN1 and PIN4 laterally bisected 2D - projected heat map . Treatments as indicated . b ) Quantification of positive PLA interactions . n . t ( no treatment ) and 10µM NPA , n = 4 ; 10µM IAA and 10µM IAA + 10µM NPA ; n = 3 . Error bars indicate standard deviation .

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

a) Summed 2D projections of PLA interactions (left) and co-localisation (right) (n=3) between PIN1 and PIN4 laterally bisected 2D-projected heat map. Treatments as indicated. b) Quantification of positive PLA interactions. n.t (no treatment) and 10µM NPA, n=4; 10µM IAA and 10µM IAA+ 10µM NPA; n=3. Error bars indicate standard deviation.


a ) Microsomes from HEK293 cells expressing PIN1 - RFP were separated under native conditions in the presence of NPA or an anti - PIN1 Fab fragment . 1 ) PIN1 - RFP , 2 ) PIN1 - RFP + Fab , 3 ) PIN1 - RFP + 10 µM NPA , 4 ) PIN1 - RFP + Fab + 10 µM NPA , 5 ) PIN1 - RFP + 10 µM Quercetin ( Q ) , 6 ) PIN1 - RFP + Fab + 10 µM Quercetin ( Q ) . Protein complexes were solubilized and separated under native conditions as described before . Western blots were performed with an anti - RFP monoclonal antibody .

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

a) Microsomes from HEK293 cells expressing PIN1-RFP were separated under native conditions in the presence of NPA or an anti-PIN1 Fab fragment. 1) PIN1-RFP, 2) PIN1-RFP+Fab, 3) PIN1-RFP+10 µM NPA, 4) PIN1-RFP+Fab+10 µM NPA, 5) PIN1-RFP + 10 µM Quercetin (Q), 6) PIN1-RFP+Fab+10 µM Quercetin (Q). Protein complexes were solubilized and separated under native conditions as described before. Western blots were performed with an anti-RFP monoclonal antibody.


b ) Fourteen - day - old Arabidopsis seedlings ( wild type , brown ; pPIN1 : : Mab9B2 ; blue ) grown on AM containing 0 . 2 µM NPA . Scale bar = 1 cm .

O O O O O O O B-ORGANISM O O O O O O O O O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O

b) Fourteen-day-old Arabidopsis seedlings (wild type, brown; pPIN1::Mab9B2; blue) grown on AM containing 0.2 µM NPA. Scale bar = 1 cm.


c ) NPA - affected lateral root density of nine - day - old Arabidopsis seedlings expressing Mab9B2 scFv fragments ( wild type sample sizes were between 16 and 21 plants ; Mab9B2 sample sizes were between 12 and 31 plants ) . Bars indicate standard error .

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

c) NPA-affected lateral root density of nine-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments (wild type sample sizes were between 16 and 21 plants; Mab9B2 sample sizes were between 12 and 31 plants). Bars indicate standard error.


d ) NPA - affected apical hook angle in dark - grown three - day - old Arabidopsis seedlings expressing Mab9B2 scFv fragments . ( wild type sample sizes were between 38 and 50 plants ; Mab9B2 sample sizes were between 33 and 45 plants ) . e ) Gravitropic curvature in four - day old Arabidopsis seedlings expressing Mab9B2 scFv fragments . ( wild type sample sizes were between 15 and 21 plants ; Mab9B2 sample sizes were between 14 and 20 plants ) .

O O B-SMALL_MOLECULE O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-ORGANISM O O B-GENEPROD 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 B-EXP_ASSAY I-EXP_ASSAY O O O O O B-ORGANISM O O B-GENEPROD 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) NPA-affected apical hook angle in dark-grown three-day-old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 38 and 50 plants; Mab9B2 sample sizes were between 33 and 45 plants). e) Gravitropic curvature in four-day old Arabidopsis seedlings expressing Mab9B2 scFv fragments. (wild type sample sizes were between 15 and 21 plants; Mab9B2 sample sizes were between 14 and 20 plants).


A ) Auto - Ub of recombinant CHN - 1 and CHN - 1 - UFD - 2 was carried out using the E2 UBE2D1 . Time - dependent ( 0 , 10 , 20 , 40 , 80 , 120 min ) CHN - 1 auto - Ub was performed as indicated using wild - type ubiquitin . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Below , a graph representing the nmol of ubiquitylated CHN - 1 vs . time for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2 ( cyan ) . Plotted data are the mean of three technical replicates . Error bars represent the standard error of measurement ( SEM ) ; statistical and significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [ CHN - 1 vs Time , CHN - 1 + UFD - 2 vs Time , and CHN - 1 vs CHN - 1 + UFD - 2 with increasing time ] .

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

A) Auto-Ub of recombinant CHN-1 and CHN-1-UFD-2 was carried out using the E2 UBE2D1. Time-dependent (0, 10, 20, 40, 80, 120 min) CHN-1 auto-Ub was performed as indicated using wild-type ubiquitin. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Below, a graph representing the nmol of ubiquitylated CHN-1 vs. time for CHN-1 alone (black) or CHN-1+UFD-2 (cyan). Plotted data are the mean of three technical replicates. Error bars represent the standard error of measurement (SEM); statistical and significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [CHN-1 vs Time, CHN-1+UFD-2 vs Time, and CHN-1 vs CHN-1+UFD-2 with increasing time].


B ) CHN - 1 auto - Ub was performed in the presence of UFD - 2 with the increasing molar concentration as indicated . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , signal quantification of the unmodified CHN - 1 ( yellow ) , ubiquitylated fraction < 70 kDa ( cyan ) and > 70 kDa ( magenta ) , plotted as a percentage of different CHN - 1 species present in the indicated condition . Plotted data are the mean from the three technical replicates . Error bars represent the SEM ; statistical significance was determined using a two - way ANOVA test . ( * * P < 0 . 01 )

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

B) CHN-1 auto-Ub was performed in the presence of UFD-2 with the increasing molar concentration as indicated. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, signal quantification of the unmodified CHN-1 (yellow), ubiquitylated fraction <70 kDa (cyan) and >70 kDa (magenta), plotted as a percentage of different CHN-1 species present in the indicated condition. Plotted data are the mean from the three technical replicates. Error bars represent the SEM; statistical significance was determined using a two-way ANOVA test. (**P < 0.01)


C ) Auto - Ub was performed as indicated using recombinant CHN - 1 and UFD - 2P951A , UBE2D1 E2 , UbWT , UbNoK , or Ub with substitutions of lysines 29 , 48 , and 63 to arginines ( Ub3KTR ) . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

C) Auto-Ub was performed as indicated using recombinant CHN-1 and UFD-2P951A, UBE2D1 E2, UbWT, UbNoK, or Ub with substitutions of lysines 29, 48, and 63 to arginines (Ub3KTR). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


E ) CHN - 1 auto - Ub was performed in the presence of recombinant C . elegans UFD - 2 and S . cerevisiae Ufd - 2p and UBE2D1 . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

E) CHN-1 auto-Ub was performed in the presence of recombinant C. elegans UFD-2 and S. cerevisiae Ufd-2p and UBE2D1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


B ) CHN - 1 auto - Ub was performed as indicated using increasing molar concentrations ( 0 . 5 , 1 , 2 , 3 , 4 µM ) of UBE2D1 without and with a complexing equimolar concentration ( 1 : 1 ) of recombinant CHN - 1 and UFD - 2P951A ( 1 . 5 µM CHN - 1 with 1 . 5 µM of recombinant UFD - 2P951A ) . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , a graph representing the nmol of ubiquitylated CHN - 1 vs . UBE2D1 ( µM ) for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2P951A ( cyan ) . Plotted data are the mean from the three technical replicates . Error bars represent the SEM ; statistical significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [ nmol of ubiquitylated CHN - 1 vs UBE2D1 ( µM ) in the presence of CHN - 1 or CHN - 1 + UFD - 2P951A and CHN - 1 vs CHN - 1 + UFD - 2P951A with increasing UBE2D1 ( µM ) ] .

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

B) CHN-1 auto-Ub was performed as indicated using increasing molar concentrations (0.5, 1, 2, 3, 4 µM) of UBE2D1 without and with a complexing equimolar concentration (1:1) of recombinant CHN-1 and UFD-2P951A (1.5 µM CHN-1 with 1.5 µM of recombinant UFD-2P951A). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, a graph representing the nmol of ubiquitylated CHN-1 vs. UBE2D1 (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan). Plotted data are the mean from the three technical replicates. Error bars represent the SEM; statistical significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [nmol of ubiquitylated CHN-1 vs UBE2D1(µM) in the presence of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1(µM)].


C ) Discharging assay of Ub - charged UBE2D1 was carried out in the presence of CHN - 1 or CHN - 1 - UFD - 2P951A . The experimental sample was run together with the control with and without a reducing agent ( 50 mM DTT ) . The reaction was stopped after 30 min by boiling the samples in 1x SDS sample buffer . Proteins were resolved via SDS - PAGE and immunoblotted with anti - UBE2D1 antibodies . Below , quantification of uncharged UBE2D1 plotted as the uncharged UBE2D1 fraction vs . UBE2D1 - Ub ( µM ) . The graph representing the UBE2D1 fraction vs . UBE2D1 - Ub ( µM ) for CHN - 1 alone ( black ) or CHN - 1 + UFD - 2P951A ( cyan ) and Plotted data are the mean of three technical replicates . Significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [ UBE2D1 vs UBE2D1 - Ub ( µM ) in the presene of CHN - 1 or CHN - 1 + UFD - 2P951A and CHN - 1 vs CHN - 1 + UFD - 2P951A with increasing UBE2D1 - Ub ( µM ) ] .

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

C) Discharging assay of Ub-charged UBE2D1 was carried out in the presence of CHN-1 or CHN-1-UFD-2P951A. The experimental sample was run together with the control with and without a reducing agent (50 mM DTT). The reaction was stopped after 30 min by boiling the samples in 1x SDS sample buffer. Proteins were resolved via SDS-PAGE and immunoblotted with anti-UBE2D1 antibodies. Below, quantification of uncharged UBE2D1 plotted as the uncharged UBE2D1 fraction vs. UBE2D1-Ub (µM). The graph representing the UBE2D1 fraction vs. UBE2D1-Ub (µM) for CHN-1 alone (black) or CHN-1+UFD-2P951A (cyan) and Plotted data are the mean of three technical replicates. Significance was determined using Pearson correlation coefficients which define the statistical relation between two continuous variables [UBE2D1 vs UBE2D1-Ub(µM) in the presene of CHN-1 or CHN-1+UFD-2P951A and CHN-1 vs CHN-1+UFD-2P951A with increasing UBE2D1-Ub(µM)].


D ) CHN - 1 auto - Ub was performed as indicated in the presence of Ube2W - Ub or Ube2W - UbFLAG without and with equimolar concentration of recombinant CHN - 1 and UFD - 2P951A . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . For each sample , the quantified relative signal after probing the blot using anti - CHN - 1 antibodies is shown as a ratio above the respective signal . Below , a schematic of the CHN - 1 - UbFLAG ( cyan ) , CHN - 1 - Ub ( magenta ) , and unmodified CHN - 1 signal ( black ) presented as the ratio and the signal fold change between CHN - 1 and CHN - 1 - UFD - 2P951A .

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

D) CHN-1 auto-Ub was performed as indicated in the presence of Ube2W-Ub or Ube2W-UbFLAG without and with equimolar concentration of recombinant CHN-1 and UFD-2P951A. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. For each sample, the quantified relative signal after probing the blot using anti-CHN-1 antibodies is shown as a ratio above the respective signal. Below, a schematic of the CHN-1-UbFLAG (cyan), CHN-1-Ub (magenta), and unmodified CHN-1 signal (black) presented as the ratio and the signal fold change between CHN-1 and CHN-1-UFD-2P951A.


A ) Auto - Ub of recombinant CHN - 1 using UBE2D1 was performed alone or complexed with UFD - 2 in the presence of recombinant DAF - 21 , DAF - 21ΔEEVD , HSP - 1 , or HSP - 1ΔEEVD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

A) Auto-Ub of recombinant CHN-1 using UBE2D1 was performed alone or complexed with UFD-2 in the presence of recombinant DAF-21, DAF-21ΔEEVD, HSP-1, or HSP-1ΔEEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


B ) Top , schematics of the HSP - 1 peptide sequence ( cyan ) aligned with the C - terminal sequence of full - length HSP - 1 ( amino acids 600 - 640 ) used in the ubiquitylation reaction . Auto - Ub of recombinant CHN - 1 was performed as indicated HSP - 1 ‑ derived peptide . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , quantification of the CHN - 1 modifications ( unmodified , multi - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) or CHN - 1 + HSP - 1 peptide ( cyan ) . Plotted data are the mean of three technical replicates .

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

B) Top, schematics of the HSP-1 peptide sequence (cyan) aligned with the C-terminal sequence of full-length HSP-1 (amino acids 600-640) used in the ubiquitylation reaction. Auto-Ub of recombinant CHN-1 was performed as indicated HSP-1‑derived peptide. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black) or CHN-1+HSP-1 peptide (cyan). Plotted data are the mean of three technical replicates.


C ) Top , schematics of the UFD - 2 peptide sequences aligned with the C - terminal sequence of full - length UFD - 2 ( UFD - 2 amino acids 864 - 914 ) for PEPTIDE 1 ( yellow ) and PEPTIDE 2 ( cyan ) and ( UFD - 2 amino acids 648 - 678 ) for PEPTIDE 3 ( magenta ) used in the ubiquitylation reaction . Below , auto - Ub of recombinant CHN - 1 was performed as indicated in the presence of UFD - 2 ‑ derived peptides . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies . Right , quantification of the CHN - 1 modifications ( unmodified , multi - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) , CHN - 1 + PEPTIDE 1 ( yellow ) , CHN - 1 + PEPTIDE 2 ( cyan ) , or CHN - 1 + PEPTIDE 3 ( magenta ) . Plotted data are the mean of three technical replicates .

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

C) Top, schematics of the UFD-2 peptide sequences aligned with the C-terminal sequence of full-length UFD-2 (UFD-2 amino acids 864-914) for PEPTIDE 1 (yellow) and PEPTIDE 2 (cyan) and (UFD-2 amino acids 648-678) for PEPTIDE 3 (magenta) used in the ubiquitylation reaction. Below, auto-Ub of recombinant CHN-1 was performed as indicated in the presence of UFD-2‑derived peptides. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies. Right, quantification of the CHN-1 modifications (unmodified, multi-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black), CHN-1+PEPTIDE 1 (yellow), CHN-1+PEPTIDE 2 (cyan), or CHN-1+PEPTIDE 3 (magenta). Plotted data are the mean of three technical replicates.


D ) Auto - Ub of recombinant CHN - 1 was performed as indicated in the presence of recombinant HSP - 1 or HSP - 1EEYD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

D) Auto-Ub of recombinant CHN-1 was performed as indicated in the presence of recombinant HSP-1 or HSP-1EEYD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


E ) Auto - Ub of recombinant CHN - 1 was performed as indicated alone or with UFD - 2 or UFD - 2EEVD . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

E) Auto-Ub of recombinant CHN-1 was performed as indicated alone or with UFD-2 or UFD-2EEVD. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


F ) CHN - 1R230A auto - Ub was performed as indicated in the presence of recombinant UFD - 2 and HSP - 1 . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - CHN - 1 antibodies .

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

F) CHN-1R230A auto-Ub was performed as indicated in the presence of recombinant UFD-2 and HSP-1. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-CHN-1 antibodies.


A ) To detect CHN - 1 and UFD - 2 , indicated lysates of young adult worms were subjected to immunoblotting with anti - CHN - 1 and anti - UFD - 2 antibodies . Tubulin served as a loading control . Right , quantification of the CHN - 1 signals normalized to tubulin levels and plotted as N2 ( wild - type ; black ) , ufd - 2 ( tm1380 ) ( cyan ) , and UFD - 2P951A ( magenta ) . Plotted data are the mean of three biological replicates .

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

A) To detect CHN-1 and UFD-2, indicated lysates of young adult worms were subjected to immunoblotting with anti-CHN-1 and anti-UFD-2 antibodies. Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels and plotted as N2 (wild-type; black), ufd-2(tm1380) (cyan), and UFD-2P951A (magenta). Plotted data are the mean of three biological replicates.


B ) CHN - 1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor ( MG - 132 , 10µM ) and DUB inhibitor ( NEM , 100mM ) . Tubulin served as a loading control . Right , quantification of the CHN - 1 signals normalized to tubulin levels plotted as control ( black ) , or MG - 132 treated ( magenta ) in N2 ( wild - type ) and ufd - 2 ( tm1380 ) . Plotted data are the mean of three biological replicates .

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

B) CHN-1 protein levels were determined in the indicated lysates of young adult worms treated with a proteasome inhibitor (MG-132, 10µM) and DUB inhibitor (NEM, 100mM). Tubulin served as a loading control. Right, quantification of the CHN-1 signals normalized to tubulin levels plotted as control (black), or MG-132 treated (magenta) in N2 (wild-type) and ufd-2(tm1380). Plotted data are the mean of three biological replicates.


B ) Ubiquitylation of recombinant AHCY - 1 was performed as indicated . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - AHCY - 1 antibodies . Right , quantification of the AHCY - 1 modifications ( unmodified , oligo - monoubiquitylated , poly - ubiquitylated ) when CHN - 1 alone ( black ) , CHN - 1 - UFD - 2 ( magenta ) , or CHN - 1 - UFD - 2EEVD ( cyan ) . Plotted data are the mean of three technical replicates .

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

B) Ubiquitylation of recombinant AHCY-1 was performed as indicated. Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Right, quantification of the AHCY-1 modifications (unmodified, oligo-monoubiquitylated, poly-ubiquitylated) when CHN-1 alone (black), CHN-1-UFD-2 (magenta), or CHN-1-UFD-2EEVD (cyan). Plotted data are the mean of three technical replicates.


C ) Protein level of endogenous AHCY - 1 in N2 ( wild - type ) , chn - 1 ( by155 ) , CHN - 1 OE , and ufd - 2 ( tm1380 ) young adult worms treated with the proteasome inhibitor ( MG - 132 , 10 µM ) and DUB inhibitor ( NEM , 100 mM ) . Protein samples were resolved via SDS - PAGE and immunoblotted with anti - AHCY - 1 antibodies . Tubulin served as a loading control . Right , quantification of the modified AHCY - 1 signals plotted as Ub - modified AHCY - 1 species normalized to unmodified endogenous AHCY - 1 signal and plotted for N2 ( wild - type ; black ) , chn - 1 ( by155 ) ( magenta ) , CHN - 1 OE ( yellow ) , and ufd - 2 ( tm1380 ) ( cyan ) . Plotted data are the mean of three biological replicates .

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

C) Protein level of endogenous AHCY-1 in N2 (wild-type), chn-1(by155), CHN-1 OE, and ufd-2(tm1380) young adult worms treated with the proteasome inhibitor (MG-132, 10 µM) and DUB inhibitor (NEM, 100 mM). Protein samples were resolved via SDS-PAGE and immunoblotted with anti-AHCY-1 antibodies. Tubulin served as a loading control. Right, quantification of the modified AHCY-1 signals plotted as Ub-modified AHCY-1 species normalized to unmodified endogenous AHCY-1 signal and plotted for N2 (wild-type; black), chn-1(by155) (magenta), CHN-1 OE (yellow), and ufd-2(tm1380) (cyan). Plotted data are the mean of three biological replicates.


D ) Representative images of GFP : : AHCY - 1 fluorescence in chn - 1 ( by155 ) , ufd - 2 ( tm1380 ) , and CHN - 1 OE background . Scale bar = 200µm . Below , quantification of the AHCY - 1 GFP signal plotted as fluorescence intensity for GFP : : AHCY - 1 expressing worms ( control ; black ) , chn - 1 ( by155 ) ( magenta ) , ufd - 2 ( tm1380 ) ( cyan ) or CHN - 1 OE ( yellow ) . Plotted data are the mean of three biological replicates .

O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O B-ORGANISM O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O

D) Representative images of GFP::AHCY-1 fluorescence in chn-1(by155), ufd-2(tm1380), and CHN-1 OE background. Scale bar = 200µm. Below, quantification of the AHCY-1 GFP signal plotted as fluorescence intensity for GFP::AHCY-1 expressing worms (control; black), chn-1(by155) (magenta), ufd-2(tm1380) (cyan) or CHN-1 OE (yellow). Plotted data are the mean of three biological replicates.


a , Protein extracts from drpr null ( w ; drpr ∆ 5 / drpr ∆ 5 ) pupae at puparium formation ( 0 h ) and wild - type ( Canton - S ) salivary glands6 h , 12 h and 14 h after puparium formation , analysed by western blotting with anti - Drpr antibody .

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

a, Protein extracts from drpr null (w; drpr∆5/drpr∆5) pupae at puparium formation (0 h) and wild-type (Canton-S) salivary glands6 h, 12 h and 14 h after puparium formation, analysed by western blotting with anti-Drpr antibody.


b , Samples from control animals ( + / w ; + / drpr ∆ 5 ) , n = 12 , and drpr null mutants ( w ; drpr ∆ 5 / drpr ∆ 5 ) , n = 47 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . c , Quantification of data from b and d . d , Samples from control animals ( + / w ; + / UAS - drprIR ) , n = 11 , and those with salivary gland - specific knockdown of drpr ( fkh - Gal4 / w ; UAS - drprIR / + ) , n = 19 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation .

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

b, Samples from control animals (+/w; +/drpr∆5), n = 12, and drpr null mutants (w; drpr∆5/drpr∆5), n = 47, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. c, Quantification of data from b and d. d, Samples from control animals (+/w; +/UAS-drprIR), n = 11, and those with salivary gland-specific knockdown of drpr (fkh-Gal4/w; UAS-drprIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation.


e , Control animals ( + / w ; + / UAS - drpr - IIR ) , n = 9 , and those with salivary gland - specific knockdown of drpr - I ( fkh - Gal4 / w ; UAS - drpr - IIR / + ) , n = 20 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . f , Quantification of data from e .

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

e, Control animals (+/w; +/UAS-drpr-IIR), n = 9, and those with salivary gland-specific knockdown of drpr-I (fkh-Gal4/w; UAS-drpr-IIR/+), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. f, Quantification of data from e.


g , Samples from drpr null animals ( + / w ; + / UAS - Drpr - I ; drpr ∆ 5 / drpr ∆ 5 ) , n = 9 , and those with salivary gland - specific expression of Drpr - I ( fkh - Gal4 / w ; UAS - Drpr - I / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 20 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . h , Quantification of data from g .

O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O 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 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 O

g, Samples from drpr null animals (+/w; +/UAS-Drpr-I; drpr∆5/drpr∆5), n = 9, and those with salivary gland-specific expression of Drpr-I (fkh-Gal4/w; UAS-Drpr-I/+; drpr∆5/drpr∆5), n = 20, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.


a , Samples from animals with salivary gland - specific expression of p35 ( fkh - Gal4 / + ; UAS - p35 / + ) , n = 18 ( left ) , drpr null animals ( + / w ; + / UAS - p35 ; drpr ∆ 5 / drpr ∆ 5 ) , n = 10 ( middle ) , and drpr null animals with salivary gland - specific expression of p35 ( fkh - Gal4 / w ; UAS - p35 / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 16 ( right ) , analysed by histology for the presence of salivary gland material 24 h after puparium formation . Red circles , cell fragments ; yellow circle , gland fragments .

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

a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments.


a , Samples from animals with salivary gland - specific expression of p35 ( fkh - Gal4 / + ; UAS - p35 / + ) , n = 18 ( left ) , drpr null animals ( + / w ; + / UAS - p35 ; drpr ∆ 5 / drpr ∆ 5 ) , n = 10 ( middle ) , and drpr null animals with salivary gland - specific expression of p35 ( fkh - Gal4 / w ; UAS - p35 / + ; drpr ∆ 5 / drpr ∆ 5 ) , n = 16 ( right ) , analysed by histology for the presence of salivary gland material 24 h after puparium formation . Red circles , cell fragments ; yellow circle , gland fragments . b , Quantification of data from a .

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

a, Samples from animals with salivary gland-specific expression of p35 (fkh-Gal4/+; UAS-p35/+), n = 18 (left), drpr null animals (+/w; +/UAS-p35; drpr∆5/drpr∆5), n = 10 (middle), and drpr null animals with salivary gland-specific expression of p35 (fkh-Gal4/w; UAS-p35/+; drpr∆5/drpr∆5), n = 16 (right), analysed by histology for the presence of salivary gland material 24 h after puparium formation. Red circles, cell fragments; yellow circle, gland fragments. b, Quantification of data from a.


c , Salivary glands dissected from animals expressing drprIR specifically in GFP - marked clone cells ( heat shock ( hs ) flp / w ; UAS - drprIR / + ; act , cd2 , FRT > Gal4 , UAS - GFP / + ) 6 h and 14 h after puparium formation . Stained with GFP antibody ( green ) , to label cells expressing drpr IR , and Lamin antibody ( red ) .

O O B-TISSUE I-TISSUE O O O O B-GENEPROD 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 B-GENEPROD O O O B-GENEPROD 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 B-GENEPROD O O O B-GENEPROD O O O O O

c, Salivary glands dissected from animals expressing drprIR specifically in GFP-marked clone cells (heat shock (hs)flp/w; UAS-drprIR/+; act,cd2, FRT > Gal4, UAS-GFP/+) 6 h and 14 h after puparium formation. Stained with GFP antibody (green), to label cells expressing drpr IR, and Lamin antibody (red).


a , Samples from animals with salivary gland - specific knockdown of Atg12 ( fkh - Gal4 / w ; UAS - Atg12IR / + ) , n = 21 , and those with salivary gland - specific knockdown of both Atg12 and drpr - I ( fkh - Gal4 / w ; UAS - Atg12IR / + ; UAS - drpr - IIR / + ) , n = 19 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . b , Quantification of data from a .

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

a, Samples from animals with salivary gland-specific knockdown of Atg12 (fkh-Gal4/w; UAS-Atg12IR/+), n = 21, and those with salivary gland-specific knockdown of both Atg12 and drpr-I (fkh-Gal4/w; UAS-Atg12IR/+; UAS-drpr-IIR/+), n = 19, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. b, Quantification of data from a.


c , GFP - LC3 was expressed in salivary glands of control animals ( + / w ; UAS - GFP - LC3 / + ; fkh - Gal4 / + ) and those with salivary gland - specific knockdown of drpr ( w ; UAS - drprIR / UAS - GFP - LC3 ; fkh - Gal4 / + ) . Salivary glands were dissected 6 h and 14 h after puparium formation , imaged for GFP - LC3 , and LC3 puncta were quantified using Zeiss Automeasure software . d , Quantification of data from c . Error bars represent s . e . m . ; n ≥ 10 ; p 0 . 0000001 .

O O B-GENEPROD O B-GENEPROD O O O B-TISSUE I-TISSUE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O O O O O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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

c, GFP-LC3 was expressed in salivary glands of control animals (+/w; UAS-GFP-LC3/+; fkh-Gal4/+) and those with salivary gland-specific knockdown of drpr (w; UAS-drprIR/UAS-GFP-LC3; fkh-Gal4/+). Salivary glands were dissected 6 h and 14 h after puparium formation, imaged for GFP-LC3, and LC3 puncta were quantified using Zeiss Automeasure software. d, Quantification of data from c. Error bars represent s.e.m.; n ≥ 10; p  0.0000001.


e , LAMP1 - GFP was expressed in control animals ( tub - LAMP - GFP / w ; + / fkh - Gal4 ) and those with salivary gland - specific knockdown of drpr ( tub - LAMP - GFP / w ; UAS - drprIR / + ; fkh - Gal4 / + ) . Salivary glands were dissected 14 h after puparium formation , imaged for LAMP - GFP , and LAMP puncta were quantified using Zeiss Automeasure software . f , Quantification of data from e . Error bars represent s . e . m . ; n ≥ 10 ; p 0 . 05 .

O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-TISSUE I-TISSUE O O O O O O O O O O O B-GENEPROD O B-GENEPROD 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

e, LAMP1-GFP was expressed in control animals (tub-LAMP-GFP/w; +/fkh-Gal4) and those with salivary gland-specific knockdown of drpr (tub-LAMP-GFP/w; UAS-drprIR/+; fkh-Gal4/+). Salivary glands were dissected 14 h after puparium formation, imaged for LAMP-GFP, and LAMP puncta were quantified using Zeiss Automeasure software. f, Quantification of data from e. Error bars represent s.e.m.; n ≥ 10; p  0.05.


g , Samples from drpr mutant animals ( + / w ; + / UAS - Atg16A ; drprDgr ; 5 / drprDgr ; 5 ) , n = 10 , and those with salivary gland - specific expression of Atg16A ( fkh - Gal4 / w ; UAS - Atg16A / + ; drprDgr ; 5 / drprDgr ; 5 ) , n = 16 , analysed by histology for the presence of salivary gland material ( red circles ) 24 h after puparium formation . h , Quantification of data from g .

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

g, Samples from drpr mutant animals (+/w; +/UAS-Atg16A; drprDgr;5/drprDgr;5), n = 10, and those with salivary gland-specific expression of Atg16A (fkh-Gal4/w; UAS-Atg16A/+; drprDgr;5/drprDgr;5), n = 16, analysed by histology for the presence of salivary gland material (red circles) 24 h after puparium formation. h, Quantification of data from g.


a , Protein extracts from drpr null ( w ; drprDgr ; 5 / drprDgr ; 5 ) pupae at puparium formation ( 0 h ) and from the fat bodies of wild - type ( Canton - S ) third - instar larvae were analysed by western blotting with anti - Drpr antibody . Third - instar larvae were either fed or starved for 4 h .

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

a, Protein extracts from drpr null (w; drprDgr;5/drprDgr;5) pupae at puparium formation (0 h) and from the fat bodies of wild-type (Canton-S) third-instar larvae were analysed by western blotting with anti-Drpr antibody. Third-instar larvae were either fed or starved for 4 h.


b , We either fed Wild - type ( Canton - S ) third - instar larvae or starved them for 4 h , and dissected fat bodies , stained them with anti - Drpr antibody , and imaged them for Drpr ( green ) . We stained nuclei with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) ( blue ) .

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

b, We either fed Wild-type (Canton-S) third-instar larvae or starved them for 4 h, and dissected fat bodies, stained them with anti-Drpr antibody, and imaged them for Drpr (green). We stained nuclei with 4′,6-diamidino-2-phenylindole (DAPI) (blue).


c , We starved third - instar larvae expressing GFP - Atg8a in all cells , and drprIR specifically in dsRed - marked clone cells ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , for 4 h . We dissected larval fat bodies and imaged them for GFP - Atg8a ( green ) and dsRed ( red ) .

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

c, We starved third-instar larvae expressing GFP-Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), for 4 h. We dissected larval fat bodies and imaged them for GFP-Atg8a (green) and dsRed (red).


d , Salivary glands of animals expressing GFP - Atg8a in all cells , and drprIR specifically in dsRed - marked clone cells ( hsflp / w ; UAS - drprIR / + ; hsGFPAtg8a , act , cd2 , FRT > Gal4 , UAS - dsRed / + ) , dissected 14 h after puparium formation . Imaged for GFP - Atg8a ( green ) and dsRed ( red ) . We stained nuclei with Hoechst ( blue ) .

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

d, Salivary glands of animals expressing GFP-Atg8a in all cells, and drprIR specifically in dsRed-marked clone cells (hsflp/w; UAS-drprIR/+; hsGFPAtg8a, act,cd2, FRT > Gal4, UAS-dsRed/+), dissected 14 h after puparium formation. Imaged for GFP-Atg8a (green) and dsRed (red). We stained nuclei with Hoechst (blue).


( a ) Effect of steroids on MERS - CoV replication . Vero cells were infected with MERS - CoV at MOI = 0 . 01 in the presence of steroids for 24 h . The viral titer in the cell supernatant was quantified by standard plaque assay using Vero / TMPRSS2 cells . Cell viability in the presence of steroids was quantified by WST assay at 24 hours post - infection ( hpi ) .

O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM O O B-CELL_LINE O O O O B-ORGANISM I-ORGANISM I-ORGANISM 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 B-EXP_ASSAY I-EXP_ASSAY O B-CELL_LINE O B-GENEPROD O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O

(a) Effect of steroids on MERS-CoV replication. Vero cells were infected with MERS-CoV at MOI = 0.01 in the presence of steroids for 24 h. The viral titer in the cell supernatant was quantified by standard plaque assay using Vero/TMPRSS2 cells. Cell viability in the presence of steroids was quantified by WST assay at 24 hours post-infection (hpi).


( b ) Ciclesonide escape mutant . Vero cells treated with ciclesonide were infected with parental MERS - CoV or ciclesonide escape mutant . Viral titer was measured as described in panel a .

O O O B-SMALL_MOLECULE O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O B-ORGANISM I-ORGANISM I-ORGANISM O B-ORGANISM I-ORGANISM I-ORGANISM O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O

(b) Ciclesonide escape mutant. Vero cells treated with ciclesonide were infected with parental MERS-CoV or ciclesonide escape mutant. Viral titer was measured as described in panel a.


( c ) Vero cells were infected with the parental MERS - CoV / EMC strain ( Re - EMC / MERS ) or the recombinant mutant strain ( Re - Nsp15 - A25V ) with an amino acid substitution at A25V in NSP15 . Viral titer was measured as described in panel a .

O O O B-CELL_LINE O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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

(c) Vero cells were infected with the parental MERS-CoV/EMC strain (Re-EMC/MERS) or the recombinant mutant strain (Re-Nsp15-A25V) with an amino acid substitution at A25V in NSP15. Viral titer was measured as described in panel a.


( a ) Intracellular SARS - CoV - 2 RNA ( 6 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 1 in the presence of 10 □ μM compounds for 6 h . Cellular viral RNA was quantified by real - time PCR using the E gene primer / probe set .

O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM O O O O O O B-CELL_LINE O B-GENEPROD O O O O B-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM I-ORGANISM 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 I-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O

(a) Intracellular SARS-CoV-2 RNA (6 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 1 in the presence of 10□μM compounds for 6 h. Cellular viral RNA was quantified by real-time PCR using the E gene primer/probe set.


( b ) Culture Medium SARS - CoV - 2 RNA ( 27 hpi ) . VeroE6 / TMPRSS2 cells were infected with SARS - CoV - 2 at MOI = 0 . 01 in the presence of ciclesonide for 27 h . Viral RNA in culture medium was quantified by real - time PCR using the E gene primer / probe set . Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay .

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

(b) Culture Medium SARS-CoV-2 RNA (27 hpi). VeroE6/TMPRSS2 cells were infected with SARS-CoV-2 at MOI = 0.01 in the presence of ciclesonide for 27 h. Viral RNA in culture medium was quantified by real-time PCR using the E gene primer/probe set. Cell viability in the presence of ciclesonide was quantified at 27 hpi by WST assay.


( B ) CRMP2 expression in adult cortex ( layers VI - IV ) , corpus callosum ( CC ) and hippocampal area CA1 . CRMP2 ( green ) is expressed throughout the cortex and hippocampus , CRMP2A ( red ) is particularly strongly expressed in a subset of callosal axons . Nuclei are counterstained with Hoechst 33342 . Scale bars : 50 μm .

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

(B) CRMP2 expression in adult cortex (layers VI - IV), corpus callosum (CC) and hippocampal area CA1. CRMP2 (green) is expressed throughout the cortex and hippocampus, CRMP2A (red) is particularly strongly expressed in a subset of callosal axons. Nuclei are counterstained with Hoechst 33342. Scale bars: 50 μm.


( C ) Whole - mount immunohistochemistry with anti - neurofilaments antibody for visualization of peripheral nerves . In crmp2 - / - embryos , the growth of ophthalmic branch of the trigeminal nerve is increased and axons are defasciculated ( first and second row , arrowheads ) . Similarly , we detected increased growth and branching of lateral branches of spinal nerves in crmp2 - / - embryos ( third row , arrowheads ) . Trigeminal nerve ( V ) and its branches ( Op - ophthalmic , Mx - maxillar , Md - mandibular ) are indicated . Scale bars : 500 μm .

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

(C) Whole-mount immunohistochemistry with anti-neurofilaments antibody for visualization of peripheral nerves. In crmp2-/- embryos, the growth of ophthalmic branch of the trigeminal nerve is increased and axons are defasciculated (first and second row, arrowheads). Similarly, we detected increased growth and branching of lateral branches of spinal nerves in crmp2-/- embryos (third row, arrowheads). Trigeminal nerve (V) and its branches (Op - ophthalmic, Mx - maxillar, Md - mandibular) are indicated. Scale bars: 500 μm.


( D , E ) Quantification of areas innervated by ophthalmic branches of trigeminal ganglion and spinal nerve , normalized to WT littermates ( each dot depicts one embryo ) . Area of ophthalmic branch in crmp2 - / - was increased by 66 % ( p < 0 . 05 , n = 10 , 3 litters ) . For spinal nerves , total area in knockouts was increased by 45 % ( p < 0 . 05 , WT n = 8 , crmp2 - / - n = 7 , 3 litters ) . Mean ± SD , * p < 0 . 05 , t - test .

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

(D, E) Quantification of areas innervated by ophthalmic branches of trigeminal ganglion and spinal nerve, normalized to WT littermates (each dot depicts one embryo). Area of ophthalmic branch in crmp2-/- was increased by 66% (p<0.05, n=10, 3 litters). For spinal nerves, total area in knockouts was increased by 45% (p<0.05, WT n=8, crmp2-/- n=7, 3 litters). Mean ± SD, * p<0.05, t-test.


( F ) Growth of motor neurons from E11 . 5 - E12 . 5 WT and crmp2 - / - spinal cord explants in microfluidic chambers . Excerpts from 14 hour time - lapse imaging are shown . Upon application of Sema3A into distal compartment , WT axons tend to stop or slow down their growth unlike crmp2 - / - axons . Purple lines highlight the growth path of individual axons in a distal chamber . Scale bars : 100 μm .

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

(F) Growth of motor neurons from E11.5 - E12.5 WT and crmp2-/- spinal cord explants in microfluidic chambers. Excerpts from 14 hour time-lapse imaging are shown. Upon application of Sema3A into distal compartment, WT axons tend to stop or slow down their growth unlike crmp2-/- axons. Purple lines highlight the growth path of individual axons in a distal chamber. Scale bars: 100 μm.


( G ) Quantification of the fraction of growing ( > 50 μm ) axons in one imaging field ( WT : control 76 . 8 ± 5 . 7 % , Sema3A 57 . 7 ± 10 . 6 % , p < 0 . 001 , crmp2 - / - : control 75 . 4 ± 10 . 4 % , Sema3A 75 . 4 ± 6 . 7 % , p = 0 . 99 ) , n = 3 experiments per genotype , 7 - 8 explants per condition . Mean ± SD . * * * p < 0 . 001 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test .

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

(G) Quantification of the fraction of growing (>50 μm) axons in one imaging field (WT: control 76.8±5.7%, Sema3A 57.7±10.6%, p<0.001, crmp2-/-: control 75.4±10.4%, Sema3A 75.4±6.7%, p=0.99), n=3 experiments per genotype, 7-8 explants per condition. Mean ± SD. *** p<0.001, 2-way ANOVA with Bonferroni's multiple comparisons test.


( A ) CRMP2 deficiency leads to callosal hypoplasia . Shortening of corpus callosum ( arrowheads ) is apparent in both sagittal ( first row ) and coronal sections ( second row ) of adult brains . Scale bars : 1 mm .

O O O B-GENEPROD O O O B-TISSUE B-DISEASE O B-EXP_ASSAY O B-TISSUE I-TISSUE O O O O O O O B-TISSUE O O O O O B-TISSUE I-TISSUE O O O O O O B-TISSUE O O O O O O O

(A) CRMP2 deficiency leads to callosal hypoplasia. Shortening of corpus callosum (arrowheads) is apparent in both sagittal (first row) and coronal sections (second row) of adult brains. Scale bars: 1 mm.


( B ) Quantification of callosal length in 30 - days old mice ( P30 , n = 3 , crmp2 - / - is 80 . 8 ± 5 % of WT , p = 0 . 003 ) and adult mice ( n = 3 , crmp2 - / - is 80 . 5 ± 3 % of WT , p = 0 . 002 ) , mean ± SD , * p < 0 . 05 , t - test .

O O O O O B-TISSUE O O O O O O B-ORGANISM 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 B-ORGANISM 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

(B) Quantification of callosal length in 30-days old mice (P30, n=3, crmp2-/- is 80.8±5% of WT, p=0.003) and adult mice (n=3, crmp2-/- is 80.5±3% of WT, p=0.002), mean ± SD, * p<0.05, t-test.


( C ) Labeling of adult corpus callosum with anti - neurofilaments antibody in sagittal sections . Outline depicts missing posterior part of the tract in crmp2 - / - mice . Caudal part of the corpus callosum in WTs is located dorsally above the habenular commissure ( hbc ) , while in crmp2 - / - mice callosum terminates rostrally before reaching the hbc ( arrows ) . PC indicates posterior commissure . Scale bars : 500 µm .

O O O O O O B-TISSUE I-TISSUE O O O B-SUBCELLULAR O O B-TISSUE O O O O O O O O O O O B-GENEPROD O O O B-ORGANISM O O O O O B-TISSUE I-TISSUE O O O O O O O B-TISSUE I-TISSUE O B-TISSUE O O O O B-GENEPROD O O O B-ORGANISM B-TISSUE O O O O O B-TISSUE O O O O B-TISSUE O B-TISSUE I-TISSUE O O O O O O O

(C) Labeling of adult corpus callosum with anti-neurofilaments antibody in sagittal sections. Outline depicts missing posterior part of the tract in crmp2-/- mice. Caudal part of the corpus callosum in WTs is located dorsally above the habenular commissure (hbc), while in crmp2-/- mice callosum terminates rostrally before reaching the hbc (arrows). PC indicates posterior commissure. Scale bars: 500 µm.


( D ) The growth of GFP - labeled callosal axons in the contralateral cortex at P6 ( embryos were electroporated at E15 . 5 ) . Note the disorganized paths of crmp2 - / - axons . Scale bars : 200 μm .

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

(D) The growth of GFP-labeled callosal axons in the contralateral cortex at P6 (embryos were electroporated at E15.5). Note the disorganized paths of crmp2-/- axons. Scale bars: 200 μm.


( F ) Quantification of tortuosity of axons ( WT n = 6 pups , crmp2 - / - n = 3 pups ) upon their exit from the callosal tract ( WT 1 . 016 ± 0 . 003 vs . crmp2 - / - 1 . 044 ± 0 . 012 , p < 0 . 001 ) , mean ± SD , * * * p < 0 . 001 , t - test .

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

(F) Quantification of tortuosity of axons (WT n=6 pups, crmp2-/- n=3 pups) upon their exit from the callosal tract (WT 1.016±0.003 vs. crmp2-/- 1.044±0.012, p<0.001), mean ± SD, ***p<0.001, t-test.


( G ) DiI - labeled callosal axons from P9 oblique brain sections and their reconstruction in Neurolucida 360 ( WT n = 6 , crmp2 - / - n = 9 ) . Scale bars : 200 μm .

O O O B-SMALL_MOLECULE O O B-TISSUE B-SUBCELLULAR O O O B-TISSUE O O O B-EXP_ASSAY 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

(G) DiI-labeled callosal axons from P9 oblique brain sections and their reconstruction in Neurolucida 360 (WT n=6, crmp2-/- n=9). Scale bars: 200 μm.


( H ) Polar histograms of callosal axons reconstructed Note the broader range of axon growth angles in crmp2 - / - mice .

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

(H) Polar histograms of callosal axons reconstructed Note the broader range of axon growth angles in crmp2-/- mice.


( I ) Left : schematic representation of polar histogram analysis by clustering the traced axons ( WT n = 6 pups , crmp2 - / - n = 9 pups ) into 3 groups based on the growth angles . Right : proportion of axons growing in selected clusters ( WT , 0 ° - ± 20 ° : 67 . 3 ± 10 . 3 % ; ± 20 ° - ± 40 ° : 21 . 2 ± 5 . 3 % ; ± 40 ° - ± 60 ° : 6 . 22 ± 3 . 2 % ; crmp2 - / - , 58 . 5 ± 5 % , p = 0 . 047 , 27 . 7 ± 4 . 3 % , p = 0 . 022 , 8 . 64 ± 2 . 25 % , p = 0 . 11 ) , mean ± SD , * p < 0 . 05 , t - test .

O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O B-ORGANISM O B-GENEPROD O O O O O O B-ORGANISM O O O O O O O B-EXP_ASSAY O O O O B-EXP_ASSAY 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 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 O O O O O O O O O O O

(I) Left: schematic representation of polar histogram analysis by clustering the traced axons (WT n=6 pups, crmp2-/- n=9 pups) into 3 groups based on the growth angles. Right: proportion of axons growing in selected clusters (WT, 0°-±20°: 67.3±10.3%; ±20°-±40°: 21.2±5.3%; ±40°-±60°: 6.22±3.2%; crmp2-/-, 58.5±5%, p=0.047, 27.7±4.3%, p=0.022, 8.64±2.25%, p=0.11), mean ± SD, *p<0.05, t-test.


( A , B ) ( A ) Coronal sections of P14 brains stained for calbindin and VgluT1 . Infrapyramidal bundle ( IPB ) progresses into hippocampal CA3 region in both WT and crmp2 - / - mice . Nuclei are counterstained with Hoechst 33342 . ( B ) Details from depicted regions in ( A ) , arrowheads show the course of IPB . Scale bars : 100 μm .

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

(A, B) (A) Coronal sections of P14 brains stained for calbindin and VgluT1. Infrapyramidal bundle (IPB) progresses into hippocampal CA3 region in both WT and crmp2-/- mice. Nuclei are counterstained with Hoechst 33342. (B) Details from depicted regions in (A), arrowheads show the course of IPB. Scale bars: 100 μm.


( C , D ) ( C ) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2 - / - mice . No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2 - / - suggesting mature synapses . Scale bars : 500 μm . ( D ) Details from depicted regions in ( C ) , arrowheads show the course of IPB . Scale bars : 100 μm .

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

(C, D) (C) Staining for calbindin shows unpruned infrapyramidal bundle in adult crmp2-/- mice. No VGluT2 signal in hippocampal CA3 region is present in WT and crmp2-/- suggesting mature synapses. Scale bars: 500 μm. (D) Details from depicted regions in (C), arrowheads show the course of IPB. Scale bars: 100 μm.


( E , F ) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2 - / - mice ( arrowheads ) . Scale bars : 100 μm .

O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O B-GENEPROD O O O B-ORGANISM O O O O O O O O O O

(E, F) Staining for calbindin and VgluT1 shows mature synapses in the main bundle in both genotypes and in the IPB in crmp2-/- mice (arrowheads). Scale bars: 100 μm.


( G ) Quantification of IPB index ( length of the IPB vs . main bundle , WT 0 . 43 ± 0 . 05 , crmp2 - / - 0 . 78 ± 0 . 05 , p < 0 . 001 ) counted from adult coronal sections ( WT n = 4 mice , crmp2 - / - n = 5 mice ) . Mean ± SD , * * * p < 0 . 001 , t - test .

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

(G) Quantification of IPB index (length of the IPB vs. main bundle, WT 0.43±0.05, crmp2-/- 0.78±0.05, p<0.001) counted from adult coronal sections (WT n=4 mice, crmp2-/- n=5 mice). Mean ± SD, ***p<0.001, t-test.


( A ) Upper row : DiI tracing of the visual cortex axons at P9 ( before pruning ) , sagittal sections . Branching point of the tract is shown . Lower row : visual cortex axons in adult mice after pruning period . Note significantly reduced number of axons continuing into pyramidal tract in WT ( dashed arrow ) . ( arrows - 2 branches of corticospinal visual axons ) . Maximum projections are shown . Scale bars : 200 μm .

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

(A) Upper row: DiI tracing of the visual cortex axons at P9 (before pruning), sagittal sections. Branching point of the tract is shown. Lower row: visual cortex axons in adult mice after pruning period. Note significantly reduced number of axons continuing into pyramidal tract in WT (dashed arrow). (arrows - 2 branches of corticospinal visual axons). Maximum projections are shown. Scale bars: 200 μm.


( C ) Quantification of VP ( visual pruning ) index ( fluorescence intensity of pyramidal axons after vs . before the branch point ) . In WT animals after the pruning period , only a minor part of axons descends towards the pyramidal tract ( WT : P9 0 . 65 ± 0 . 09 , adults 0 . 17 ± 0 . 04 , p = 0 . 006 ) . However , in crmp2 - / - mice , corticospinal axons are still largely present , and their VP indexes are not significantly different between adult and P9 stages ( crmp2 - / - : P9 0 . 54 ± 0 . 18 , adults 0 . 62 ± 0 . 27 , p = 0 . 24 ) . Mean ± SD , * * p < 0 . 01 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test . P9 : n = 3 pups / genotype , adults : n = 5 mice / genotype )

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

(C) Quantification of VP (visual pruning) index (fluorescence intensity of pyramidal axons after vs. before the branch point). In WT animals after the pruning period, only a minor part of axons descends towards the pyramidal tract (WT: P9 0.65±0.09, adults 0.17±0.04, p=0.006). However, in crmp2-/- mice, corticospinal axons are still largely present, and their VP indexes are not significantly different between adult and P9 stages (crmp2-/-: P9 0.54±0.18, adults 0.62±0.27, p=0.24). Mean ± SD, **p<0.01, 2-way ANOVA with Bonferroni's multiple comparisons test. P9: n=3 pups/genotype, adults: n=5 mice/genotype)


( A ) Time - lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation . Upper panel : Axon growth without Semaphorin stimulation . Middle panel : stimulation with Sema3A ( 0 hours ) ( 1nM , n = 669 axons for WT , 761 axons for knockout ) causes retraction of both WT and crmp2 - / - neurons . Lower panel : stimulation with Sema3F ( 5 nM , n = 602 axons for WT , 955 axons for knockout ) causes axon retraction in WT , but not in crmp2 - / - neurons . Red triangles depict growing axons , yellow retracting axons and blue asterisks indicate steady non - growing axons . Scale bars : 100 μm ( whole image field ) and 50 μm ( magnified ) .

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

(A) Time-lapse imaging of DIV7 cultured hippocampal neurons before and after Semaphorin stimulation. Upper panel: Axon growth without Semaphorin stimulation. Middle panel: stimulation with Sema3A (0 hours) (1nM, n=669 axons for WT, 761 axons for knockout) causes retraction of both WT and crmp2-/- neurons. Lower panel: stimulation with Sema3F (5 nM, n=602 axons for WT, 955 axons for knockout) causes axon retraction in WT, but not in crmp2-/- neurons. Red triangles depict growing axons, yellow retracting axons and blue asterisks indicate steady non-growing axons. Scale bars: 100 μm (whole image field) and 50 μm (magnified).


( C ) Quantification of retracting axons ( number of retracting vs . steady axons , 3 experiments ) . WT : control 13 . 4 ± 5 % , Sema3A 31 . 2 ± 6 % ( p < 0 . 001 ) , Sema3F 36 . 9 ± 8 . 7 % ( p < 0 . 001 ) ; crmp2 - / - : control 19 . 8 ± 1 . 8 % , Sema3A 28 . 5 ± 4 . 4 % ( p < 0 . 05 ) , Sema3F 22 . 4 ± 4 . 1 % ( p > 0 . 99 ) , mean ± SD are shown . * * * p < 0 . 001 , * p < 0 . 05 , 2 - way ANOVA with Bonferroni ' s multiple comparisons test .

O O O O O B-EXP_ASSAY B-SUBCELLULAR O B-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY B-SUBCELLULAR 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 B-GENEPROD 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 B-GENEPROD 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 O O O O O O O O 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) Quantification of retracting axons (number of retracting vs. steady axons, 3 experiments). WT: control 13.4±5%, Sema3A 31.2±6% (p<0.001), Sema3F 36.9±8.7% (p<0.001); crmp2-/-: control 19.8±1.8%, Sema3A 28.5±4.4% (p<0.05), Sema3F 22.4±4.1% (p>0.99), mean ± SD are shown. ***p<0.001, *p<0.05, 2-way ANOVA with Bonferroni's multiple comparisons test.


( A , B ) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2 - / - mice at P30 . In adults , however , spine density in crmp2 - / - granule cells is increased comparing to WT . Scale bars : 5 μm . ( C ) Quantification of dendritic spine density in DG granule cells in the inner molecular layer ( 50 - 100 μm away from the soma ) . WT , P30 : 12 . 5 ± 2 . 2 spines / 10 μm , adults : 9 . 95 ± 2 . 3 ; crmp2 - / - , P30 : 12 . 86 ± 2 . 1 ( p > 0 . 99 ) , adults : 12 . 88 ± 2 . 9 ( p < 0 . 001 ) , mean ± SD , * * * p < 0 . 001 , 2 - way ANOVA with Bonferroni correction . P30 : 3 animals / genotype , WT = 61 dendrites , knockout = 64 dendrites ; adults : 3 animals / genotype , WT = 41 dendrites , knockout = 37 dendrites .

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

(A, B) Dendritic spine density in DiOlistically labeled DG granule cells is similar in WT and crmp2-/- mice at P30. In adults, however, spine density in crmp2-/- granule cells is increased comparing to WT. Scale bars: 5 μm. (C) Quantification of dendritic spine density in DG granule cells in the inner molecular layer (50 - 100 μm away from the soma). WT, P30: 12.5±2.2 spines/10 μm, adults: 9.95±2.3; crmp2-/-, P30: 12.86±2.1 (p>0.99), adults: 12.88±2.9 (p<0.001), mean ± SD, ***p<0.001, 2-way ANOVA with Bonferroni correction. P30: 3 animals/genotype, WT=61 dendrites, knockout=64 dendrites; adults: 3 animals/genotype, WT=41 dendrites, knockout=37 dendrites.