E . Scatter plot and bar graph showing that the current of PC1 - CTF / PC2 _ AA is completely abolished when WT PC2 was used , or after introducing R4090W mutation in the putative pore region of PC1 - CTF . Inserted are western blot images showing the expression of the corresponding proteins . Top : anti - PC1 . Bottom : anti - PC2 . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD in bar graph ( * * * P < 0 . 001 , Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Scatter plot and bar graph showing that the current of PC1-CTF/PC2_AA is completely abolished when WT PC2 was used, or after introducing R4090W mutation in the putative pore region of PC1-CTF. Inserted are western blot images showing the expression of the corresponding proteins. Top: anti-PC1. Bottom: anti-PC2. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD in bar graph (***P < 0.001, Student's t-test).


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + and 2 mM Ca2 + ( F ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

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

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ and 2 mM Ca2+ (F), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na + ( G ) , , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

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

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 100 mM Na+ (G), , showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


Representative I - V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2 + ( H ) , showing PC1 - TLD / PC2 _ AA gave rise to current with similar properties as full - length PC1 / PC2 _ AA channel .

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

Representative I-V curves of oocytes injected with the indicated RNAs in a bath solution containing 70 mM Ca2+ (H), showing PC1-TLD/PC2_AA gave rise to current with similar properties as full-length PC1/PC2_AA channel.


I . Co - IP followed by western blot showing the association between PC2 _ AA and the indicated full - length or fragments of PC1 . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-IP followed by western blot showing the association between PC2_AA and the indicated full-length or fragments of PC1. Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.


J . Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2 _ AA and full - length PC1 , PC1 - CTF , or PC1 - TLD at the oocyte surface . Besides the blots shown in the figure , surface samples were also blotted with an anti - PC1 N - terminus antibody , and the cleaved NTF fragment was found associated to the plasma membrane in the full - length sample ( shown in Appendix Fig S4 ) . Bands of full - length ( asterisk ) , GPS - cleaved CTF or expressed CTF fragment ( open circle ) , and TLD ( star ) of PC1 are indicated .

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

J. Surface biotinylation followed by western blot showing the expression of the complexes formed between PC2_AA and full-length PC1, PC1-CTF, or PC1-TLD at the oocyte surface. Besides the blots shown in the figure, surface samples were also blotted with an anti-PC1 N-terminus antibody, and the cleaved NTF fragment was found associated to the plasma membrane in the full-length sample (shown in Appendix Fig S4). Bands of full-length (asterisk), GPS-cleaved CTF or expressed CTF fragment (open circle), and TLD (star) of PC1 are indicated.


A . Representative I - V curves of the PC2 _ AA and PC1 - CTF / PC2 _ AA channels in divalent ion - free bath solutions containing 100 mM of indicated ions , showing the differences in reversal potential .

O O O O O O O O O O O O O O O O O O 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. Representative I-V curves of the PC2_AA and PC1-CTF/PC2_AA channels in divalent ion-free bath solutions containing 100 mM of indicated ions, showing the differences in reversal potential.


B . Bar graphs showing the markedly different reversal potentials of PC2 _ AA channel and the PC1 - CTF / PC2 _ AA channel in bath solutions containing 100 mM of the indicated ions . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * * P < 0 . 001 , Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Bar graphs showing the markedly different reversal potentials of PC2_AA channel and the PC1-CTF/PC2_AA channel in bath solutions containing 100 mM of the indicated ions. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, ***P < 0.001, Student's t-test).


Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( C ) These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars .

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

Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (C) These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.


Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( D ) . These ratios are strikingly different between the two channels . The ratios of the particular ions , which are the values of the bars , are indicted on top of the bars .

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

Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (D). These ratios are strikingly different between the two channels. The ratios of the particular ions, which are the values of the bars, are indicted on top of the bars.


Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions ( B ) , indicating that these amino acids are essential for ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Two PC1 and two PC2 mutations caused dramatic changes in the reversal potential for almost all tested ions (B), indicating that these amino acids are essential for ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Bar graphs showing the reversal potentials of PC1 - CTF / PC2 _ AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions . Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others ( C ) , indicating a less important role of these amino acids in ion permeability . Statistical significance between reversal potentials of all pore mutants and that of the PC1 - CTF / PC2 _ AA channel are indicated . Oocyte numbers are indicated in parentheses . Data are presented as mean ± SD ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Bar graphs showing the reversal potentials of PC1-CTF/PC2_AA and the indicated mutants tested in bath solutions containing 100 mM of the indicated ions. Another two PC1 mutations and one PC2 mutation only led to relatively small changes of reversal potential for some ions but not others (C), indicating a less important role of these amino acids in ion permeability. Statistical significance between reversal potentials of all pore mutants and that of the PC1-CTF/PC2_AA channel are indicated. Oocyte numbers are indicated in parentheses. Data are presented as mean ± SD (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Bar graphs showing the permeability ratios of the indicated ions to that of Na + ( Px : PNa ) ( D ) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases .

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

Bar graphs showing the permeability ratios of the indicated ions to that of Na+ (Px: PNa) (D) Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.


Bar graphs showing the permeability ratios of the indicated ions to that of NMDG + ( Px : PNMDG ) ( E ) . Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases .

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

Bar graphs showing the permeability ratios of the indicated ions to that of NMDG+ (Px: PNMDG) (E). Pore mutations in both PC1 and PC2 lead to significant changes in the permeability ratios in most of the cases.


Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 2 mM Ca2 + ( B ) Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 2 mM Ca2+ (B) Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


Representative currents and a scatter plot and bar graph showing the currents of the full - length WT and mutant PC1s associated with PC2 _ AA in bath solutions containing 100 mM Na + and 70 mM Ca2 + ( C ) . Currents were normalized to the average current of PC1 / PC2 _ AA recorded from the same batch of oocytes . Oocyte numbers in scatter plot and bar graph are indicated in parentheses . Data are presented as mean ± SD in bar graph ( n . s . : not significant , * * P < 0 . 01 , * * * P < 0 . 001 , Student ' s 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative currents and a scatter plot and bar graph showing the currents of the full-length WT and mutant PC1s associated with PC2_AA in bath solutions containing 100 mM Na+ and 70 mM Ca2+ (C). Currents were normalized to the average current of PC1/PC2_AA recorded from the same batch of oocytes. Oocyte numbers in scatter plot and bar graph are indicated in parentheses. Data are presented as mean ± SD in bar graph (n.s.: not significant, **P < 0.01, ***P < 0.001, Student's t-test).


D . Surface biotinylation followed by western blot showing the surface expression of the PC1 _ T3041V / PC2 _ AA complex , but not the PC1 _ L3040H / PC2 _ AA complex , in Xenopus oocytes . PC2 _ AA is completely absent from the surface sample when PC1 _ L3040H was coexpressed , indicating that , in our experimental condition , almost all PC2 _ AA were in the complex with PC1 _ L3040H and trapped in the process of cell surface trafficking . Bands of full - length ( asterisk ) and GPS - cleaved CTF ( open circle ) of PC1 , and 130 kDa PC2 ( star ) are indicated .

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

D. Surface biotinylation followed by western blot showing the surface expression of the PC1_T3041V/PC2_AA complex, but not the PC1_L3040H/PC2_AA complex, in Xenopus oocytes. PC2_AA is completely absent from the surface sample when PC1_L3040H was coexpressed, indicating that, in our experimental condition, almost all PC2_AA were in the complex with PC1_L3040H and trapped in the process of cell surface trafficking. Bands of full-length (asterisk) and GPS-cleaved CTF (open circle) of PC1, and 130 kDa PC2 (star) are indicated.


API delivery is blocked at low temperatures . SEY6210 cells were pulse labeled for 5 min and chased for the times indicated . Identical experiments were performed at 10 ° , 14 ° , and 30 ° C . API was recovered by immunoprecipitation followed by SDS - PAGE and detected by a STORM phosphorimager ( Molecular Dynamics , Sunnyvale , CA ) . The positions of prAPI and mAPI are indicated .

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

API delivery is blocked at low temperatures. SEY6210 cells were pulse labeled for 5 min and chased for the times indicated. Identical experiments were performed at 10°, 14°, and 30°C. API was recovered by immunoprecipitation followed by SDS-PAGE and detected by a STORM phosphorimager (Molecular Dynamics, Sunnyvale, CA). The positions of prAPI and mAPI are indicated.


Precursor API is trapped in a prevacuolar compartment in vps18 cells . ( A ) Yeast strain JSR18Δ1 with plasmid pJSR9 ( vps18 ts ) was grown at the permissive temperature of 26 ° C . Before labeling , the cells were incubated at either 26 ° or 38 ° C for 10 min . Pulse labeling was for 5 min , followed by the indicated chase times .

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

Precursor API is trapped in a prevacuolar compartment in vps18 cells. (A) Yeast strain JSR18Δ1 with plasmid pJSR9 (vps18 ts) was grown at the permissive temperature of 26°C. Before labeling, the cells were incubated at either 26° or 38°C for 10 min. Pulse labeling was for 5 min, followed by the indicated chase times.


( B ) vps18 spheroplasts were shifted to 38 ° C for 5 min , pulse labeled for 5 min , and then chased for either 0 or 60 min . At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer . The Total ( T ) fraction either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 . The supernatant ( S0 ) and pellet ( P0 ) fractions were collected after centrifugation at 10 , 000 g for 5 min .

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

(B) vps18 spheroplasts were shifted to 38°C for 5 min, pulse labeled for 5 min, and then chased for either 0 or 60 min. At each time point the spheroplasts were collected by centrifugation and resuspended in PS0 buffer. The Total (T) fraction either received no treatment, was treated with 50 μg/ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100. The supernatant (S0) and pellet (P0) fractions were collected after centrifugation at 10,000 g for 5 min.


A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment . ( A ) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer . The Total ( T ) fraction ( after differential lysis ) either received no treatment , was treated with 50 μg / ml proteinase K , or received proteinase K in the presence of 0 . 2 % Triton X - 100 .

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

A mutant in the API propeptide causes accumulation of prAPI on a nonvacuolar compartment. (A) Spheroplasts from an ape1Δ strain containing a plasmid bearing P22L API were subjected to differential lysis in PS200 buffer. The Total (T) fraction (after differential lysis) either received no treatment, was treated with 50 μg/ ml proteinase K, or received proteinase K in the presence of 0.2% Triton X-100.


( B ) The supernatant ( S200 ) and pellet ( P200 ) fractions were collected after centrifugation at 5 , 000 g for 5 min . The P200 fraction was resuspended in 10 % Ficoll , and vacuoles were isolated by flotation through 4 % Ficoll . V , vacuole ; I , 4 / 10 % Ficoll interface ; P , gradient pellet . Proteins were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The supernatant (S200) and pellet (P200) fractions were collected after centrifugation at 5,000 g for 5 min. The P200 fraction was resuspended in 10% Ficoll, and vacuoles were isolated by flotation through 4% Ficoll. V, vacuole; I, 4/10% Ficoll interface; P, gradient pellet. Proteins were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


( C ) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min , harvested by centrifugation , and subjected to a nonradioactive chase in either nitrogen - containing ( SMD ) or nitrogen starvation ( SD - N ) medium . Samples were collected at the times indicated and immunoprecipitated with API antiserum . The resulting SDS - PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O 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) ape1Δ cells containing P22L API on a plasmid were pulse labeled for 5 min, harvested by centrifugation, and subjected to a nonradioactive chase in either nitrogen-containing (SMD) or nitrogen starvation (SD-N) medium. Samples were collected at the times indicated and immunoprecipitated with API antiserum. The resulting SDS-PAGE gels were quantified using a Molecular Dynamics STORM phosphorimager.


Precursor API is trapped within a vesicle in cvt17 mutants . ( A ) Precursor API is in a protease - protected compartment in cvt17 mutants . THY32 ( cvt17 ) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig

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

Precursor API is trapped within a vesicle in cvt17 mutants. (A) Precursor API is in a protease-protected compartment in cvt17 mutants. THY32 (cvt17) spheroplasts were subjected to differential lysis and protease treatment in PS200 buffer as in Fig 3


B ) Precursor API is not free in the vacuolar lumen in cvt17 mutants . Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant ( S0 ) and pellet ( P0 ) fractions by centrifugation at 10 , 000 g . The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X - 100 as indicated . All fractions were precipitated with 10 % TCA and resolved by SDS PAGE ; proteins of interest were detected by Western blotting . The positions of prAPI , mAPI , and mPrA are indicated .

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

B) Precursor API is not free in the vacuolar lumen in cvt17 mutants. Differential fractionation was continued by resuspending the P200 fraction in PS0 buffer and separating the supernatant (S0) and pellet (P0) fractions by centrifugation at 10,000 g. The S0 and P0 fractions were treated with proteinase K in the presence and absence of Triton X-100 as indicated. All fractions were precipitated with 10% TCA and resolved by SDS PAGE; proteins of interest were detected by Western blotting. The positions of prAPI, mAPI, and mPrA are indicated.


Precursor API is contained within subvacuolar vesicles in cvt17 mutants . ( A ) Isolated vacuoles ( V ) from cvt17 cells were lysed in PS0 buffer , loaded on top of an Optiprep step gradient , and centrifuged at 170 , 000 g for 60 min as described in Materials and Methods . Fractions VV , 0 / 1 . 06 interface ; LD , 1 . 06 region ; SV , 1 . 06 / 1 . 12 interface ; HD , 1 . 12 region .

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

Precursor API is contained within subvacuolar vesicles in cvt17 mutants. (A) Isolated vacuoles (V) from cvt17 cells were lysed in PS0 buffer, loaded on top of an Optiprep step gradient, and centrifuged at 170,000 g for 60 min as described in Materials and Methods. Fractions VV, 0/1.06 interface; LD, 1.06 region; SV, 1.06/1.12 interface; HD, 1.12 region.


( B ) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X - 100 as indicated . Because of the presence of protease inhibitors in the vesicle isolation procedure , an intermediate - sized API degradation product results from proteinase K treatment . The V fraction represents one - tenth of the load of the Optiprep gradient . Proteins were detected by Western blotting . The positions of prAPI and mALP are indicated .

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

(B) An aliquot of fractions VV and SV was treated with proteinase K either in the presence or absence of Triton X-100 as indicated. Because of the presence of protease inhibitors in the vesicle isolation procedure, an intermediate-sized API degradation product results from proteinase K treatment. The V fraction represents one-tenth of the load of the Optiprep gradient. Proteins were detected by Western blotting. The positions of prAPI and mALP are indicated.


Immuno - EM of cvt17 cells . Yeast strains SEY6210 ( parental wild type ) and THY32 ( cvt17 ) were grown in YPD and prepared for microscopy as described in Materials and Methods . ( A ) Wild - type cells probed with antibody against mature API . ( B ) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion . ( C ) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API . N , nucleus ; V , vacuole ; ( arrows ) API containing vesicles . Bars , 0 . 5 μm .

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

Immuno-EM of cvt17 cells. Yeast strains SEY6210 (parental wild type) and THY32 (cvt17) were grown in YPD and prepared for microscopy as described in Materials and Methods. (A) Wild-type cells probed with antibody against mature API. (B) A cvt17 section showing a cytosolic vesicle containing prAPI probed with antibody against the API proregion. (C) A cvt17 section showing subvacuolar vesicles containing API probed with antibody against mature API. N, nucleus; V, vacuole; (arrows) API containing vesicles. Bars, 0.5 μm.


Characterization of API - containing vesicles . ( A ) Fractionations were performed as in Fig . 5 . The total ( T ) , vacuole ( V ) , vacuolar vesicle ( VV ) , and subvacuolar vesicle ( SV ) fractions are shown . Proteins were detected by immunoblotting as indicated in the figure .

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

Characterization of API-containing vesicles. (A) Fractionations were performed as in Fig. 5. The total (T), vacuole (V), vacuolar vesicle (VV), and subvacuolar vesicle (SV) fractions are shown. Proteins were detected by immunoblotting as indicated in the figure.


( B ) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager . The relative mobility of the molecular mass standards are indicated on the left . Polypeptides enriched in the subvacuolar vesicles are indicated on the right .

O O O O O O O O O O O O O O O O O O O O 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) SYPRO orange staining detected by fluorescent scanning mode on a Molecular Dynamics STORM phosphorimager. The relative mobility of the molecular mass standards are indicated on the left. Polypeptides enriched in the subvacuolar vesicles are indicated on the right.


C . Proteins from whole cell extracts and culture supernatants of PAKΔretS , the derivative tssA1 mutant ( ΔA1 ) ( both carrying the pBBR plasmid ) and the complemented ΔA1 mutant carrying pBBR - tssA1 ( ΔA1 + A1 ) were analysed by Western blot . Polyclonal antibodies directed against TssA1 ( A1 ) , Hcp1 , VgrG1a and Tse3 were used . The anti - VgrG1a antibody detects VgrG1a , VgrG1b and VgrG1c , as indicated on the left . Cytoplasmic RNA polymerase ( RNAP ) was monitored using monoclonal antibody directed against its β - subunit .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Proteins from whole cell extracts and culture supernatants of PAKΔretS, the derivative tssA1 mutant (ΔA1) (both carrying the pBBR plasmid) and the complemented ΔA1 mutant carrying pBBR-tssA1 (ΔA1+A1) were analysed by Western blot. Polyclonal antibodies directed against TssA1 (A1), Hcp1, VgrG1a and Tse3 were used. The anti-VgrG1a antibody detects VgrG1a, VgrG1b and VgrG1c, as indicated on the left. Cytoplasmic RNA polymerase (RNAP) was monitored using monoclonal antibody directed against its β-subunit.


A . BTH experiments showing that TssA1 is able to self - interact in vivo . A graphical representation of β - galactosidase activity from co - transformants of E . coli DHM1 cells producing TssA1 ( A1 ) fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in quadruplicate .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. BTH experiments showing that TssA1 is able to self-interact in vivo. A graphical representation of β-galactosidase activity from co-transformants of E. coli DHM1 cells producing TssA1 (A1) fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in quadruplicate.


B . In vitro cross - linking experiments of purified His6 - TssA1 . About 30 μg of purified His6 - TssA1 was cross - linked ( 30 min , 1h and 2h ) at room temperature using increasing amount of ethylene glycol - bis ( succinimidylsuccinate ) EGS ( 2 mM and 5 mM ) where indicated . Western blot analysis of cross - linked products using an anti - His6 monoclonal antibody is shown . The cross - linked species are highlighted with stars with the corresponding oligomeric state indicated on the right ( 1x = monomer ; 2x = dimer ; 6x = hexamer ) . Molecular weight markers ( kDa ) are indicated on the left .

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

B. In vitro cross-linking experiments of purified His6-TssA1. About 30 μg of purified His6-TssA1 was cross-linked (30 min, 1h and 2h) at room temperature using increasing amount of ethylene glycol-bis(succinimidylsuccinate) EGS (2 mM and 5 mM) where indicated. Western blot analysis of cross-linked products using an anti-His6 monoclonal antibody is shown. The cross-linked species are highlighted with stars with the corresponding oligomeric state indicated on the right (1x = monomer; 2x = dimer; 6x = hexamer). Molecular weight markers (kDa) are indicated on the left.


C . Analysis of the oligomeric state of TssA1 by AUC . Sedimentation data of His6 - TssA1 ( 0 . 56 mg / ml ) recorded at a rotor speed of 20 , 000 rpm is shown . The left panel shows the sedimentation boundary fits and for clarity , only every third scan is shown in the fitted data plots . The experimental absorbance data are shown as black circles , whereas the boundary fits are shown as grey lines . The right panel shows the size - distribution analysis c ( s ) , obtained from fitting the scan boundaries using SEDFIT , revealing a dodecameric peak ( Mr ~ 479 kDa ) at S20 , w value of 16 . 6 S with a minor peak ( Mr ~ 1 MDa ) at S20 , w value of 29 . 01 S .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the oligomeric state of TssA1 by AUC. Sedimentation data of His6-TssA1 (0.56 mg/ml) recorded at a rotor speed of 20,000 rpm is shown. The left panel shows the sedimentation boundary fits and for clarity, only every third scan is shown in the fitted data plots. The experimental absorbance data are shown as black circles, whereas the boundary fits are shown as grey lines. The right panel shows the size-distribution analysis c(s), obtained from fitting the scan boundaries using SEDFIT, revealing a dodecameric peak (Mr ~ 479 kDa) at S20,w value of 16.6 S with a minor peak (Mr ~1 MDa) at S20,w value of 29.01 S.


A . A representative micrograph of the TssA1 complex ( left panel ) showing discrete ring - shaped particles with a diameter of ~ 260 Å . The rotationally symmetrised average of the data set ( inset panel ) presents two distinct rings ( numbered ) , disclosing the layered nature of the TssA1 complex . Representative class averages of the TssA1 complex ( right panel ) demonstrating the wide range of conformations taken by the ring - shaped structures . Scale bars are 500 Å in the left panel , 130 Å in the inset and 100 Å in the right panels , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A representative micrograph of the TssA1 complex (left panel) showing discrete ring-shaped particles with a diameter of ~260 Å. The rotationally symmetrised average of the data set (inset panel) presents two distinct rings (numbered), disclosing the layered nature of the TssA1 complex. Representative class averages of the TssA1 complex (right panel) demonstrating the wide range of conformations taken by the ring-shaped structures. Scale bars are 500 Å in the left panel, 130 Å in the inset and 100 Å in the right panels, respectively.


B . Double MSA classification : ( i ) representative orientation class averages show TssA1 rings arranged into four - fold ( 4x ) , five - fold ( 5x ) and six - fold ( 6x ) symmetry - like complexes ( indicated in red , blue and green , respectively ) ; ( ii ) sub - class averages obtained from sub - classification of the corresponding orientation class ; and ( iii ) calculated difference images showing some white or black peaks ( coloured circles ) at the outer ring region . Scale bar is 100 Å .

B. Double MSA classification: (i) representative orientation class averages show TssA1 rings arranged into four-fold (4x), five-fold (5x) and six-fold (6x) symmetry-like complexes (indicated in red, blue and green, respectively); (ii) sub-class averages obtained from sub-classification of the corresponding orientation class; and (iii) calculated difference images showing some white or black peaks (coloured circles) at the outer ring region. Scale bar is 100 Å.


D . SEC - MALS analysis of the purified His6 - TssA11 - 245 . The TssA11 - 245 elution peak is shown and the corresponding mass is indicated . The molar mass and the differential refractive index ( RI ) are plotted versus the elution volume . Data were analysed using the ASTRA method ( Slotboom , Duurkens et al . , 2008 ) .

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

D. SEC-MALS analysis of the purified His6-TssA11-245. The TssA11-245 elution peak is shown and the corresponding mass is indicated. The molar mass and the differential refractive index (RI) are plotted versus the elution volume. Data were analysed using the ASTRA method (Slotboom, Duurkens et al., 2008).


A . Co - purification experiments using His6 - TssA1 and untagged TssB1C1 . Western blot analysis of the Ni - NTA outputs using antibodies directed against the His6 - tag ( upper panel ) and TssB1 ( lower panel ) . I , Input ; FT , Flow through ; W , Wash and E , Elution .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Co-purification experiments using His6-TssA1 and untagged TssB1C1. Western blot analysis of the Ni-NTA outputs using antibodies directed against the His6-tag (upper panel) and TssB1 (lower panel). I, Input; FT, Flow through; W, Wash and E, Elution.


B . SEC elution fraction of the co - purified His6 - TssA1 and TssB1C1 . SDS - PAGE showing the presence of the three proteins His6 - TssA1 ( A1 ) , TssB1 ( B1 ) and TssC1 ( C1 ) . Molecular weight markers ( kDa ) are indicated on the left .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. SEC elution fraction of the co-purified His6-TssA1 and TssB1C1. SDS-PAGE showing the presence of the three proteins His6-TssA1 (A1), TssB1 (B1) and TssC1 (C1). Molecular weight markers (kDa) are indicated on the left.


C . Representative micrographs of the immunogold - labelled complex His6 - TssA1 / TssB1C1 . The presence of gold particles is indicated with white arrows . Two close - up views of TssB1C1 sheath displaying gold particles at one extremity are shown ( inset panels ) . Scale bars are 2 , 000 Å and 500 Å for images 1 and 2 - 6 , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Representative micrographs of the immunogold-labelled complex His6-TssA1/TssB1C1. The presence of gold particles is indicated with white arrows. Two close-up views of TssB1C1 sheath displaying gold particles at one extremity are shown (inset panels). Scale bars are 2,000 Å and 500 Å for images 1 and 2-6, respectively.


D . BTH experiment showing interaction between TssA1 ( A1 ) and Hcp1 . A graphical representation of β - galactosidase activity from E . coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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

D. BTH experiment showing interaction between TssA1 (A1) and Hcp1. A graphical representation of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits is shown. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


B . Cartoon representations of the dodecameric gp6 _ 334C structure ( PDB code 3H3W ) . Chains A and B of gp6 dimers are shown in magenta and green respectively , and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange . A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly , is shown on the right .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Cartoon representations of the dodecameric gp6_334C structure (PDB code 3H3W). Chains A and B of gp6 dimers are shown in magenta and green respectively, and the conserved blocks mapped onto the assembled dodecameric gp6 structure are shown in orange. A close up view showing the conserved blocks present in regions of gp6 involved in ring assembly, is shown on the right.


A Fluorescence microscopy images showing the presence / absence of TssBC sheaths ( seen as fluorescent foci ) in the ∆ B1 : : B1 - sfGFP ( top panel ) , ∆ B1 ∆ A1 : : B1 - sfGFP , ∆ B1 ∆ E1 : : B1 - sfGFP cells ( middle panels ) and in the complemented strains ∆ B1 ∆ A1 : : B1 - sfGFP + A1 and ∆ B1 ∆ E1 : : B1 - sfGFP + E1 ( bottom panels ) . Scale bars are 2 μm . B . Quantification of TssB1 - sfGFP foci and of diffuse fluorescence in various P . aeruginosa backgrounds imaged in panel A . Values are normalized to the total number of bacteria ( % ) . Data were obtained from three independent experiments , with images and values from representative replicates being shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence microscopy images showing the presence/absence of TssBC sheaths (seen as fluorescent foci) in the ∆B1::B1-sfGFP (top panel), ∆B1∆A1::B1-sfGFP, ∆B1∆E1::B1-sfGFP cells (middle panels) and in the complemented strains ∆B1∆A1::B1-sfGFP + A1 and ∆B1∆E1::B1-sfGFP + E1 (bottom panels). Scale bars are 2 μm. B. Quantification of TssB1-sfGFP foci and of diffuse fluorescence in various P. aeruginosa backgrounds imaged in panel A. Values are normalized to the total number of bacteria (%). Data were obtained from three independent experiments, with images and values from representative replicates being shown.


BTH screen for interactions between TssA1 and T6SS components of interest . A graphical representation ( bottom ) of β - galactosidase activity from E . coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits and images of corresponding spots on X - gal LB agar plates ( top ) are shown . The combination of T25 / T18 fusion proteins are indicated as 25 or 18 followed by the name of the fused T6SS protein . T6SS proteins are abbreviated as follows : A1 = TssA1 , E1 = TssE1 , F1 = TssF1 , G1 = TssG1 and K1 = TssK1 . The values shown on the y axis correspond to the activity in Miller units . In each case , average activity from two independent experiments is shown and error bars indicate the standard deviation ( S . D . ) . Experiments were carried out in triplicate .

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

BTH screen for interactions between TssA1 and T6SS components of interest. A graphical representation (bottom) of β-galactosidase activity from E. coli DHM1 cells producing the indicated proteins fused to the adenylate cyclase T25 or adenylate cyclase T18 subunits and images of corresponding spots on X-gal LB agar plates (top) are shown. The combination of T25/T18 fusion proteins are indicated as 25 or 18 followed by the name of the fused T6SS protein. T6SS proteins are abbreviated as follows: A1=TssA1, E1=TssE1, F1=TssF1, G1=TssG1 and K1=TssK1. The values shown on the y axis correspond to the activity in Miller units. In each case, average activity from two independent experiments is shown and error bars indicate the standard deviation (S.D.). Experiments were carried out in triplicate.


B . Cartoon representation of the gp6 / gp8 baseplate subcomplex of the T4 phage . The gp6 ring structure ( PDB code 3H3W ) is shown using the same colour code described in figure 5 . The chains A and B of the gp8 dimers ( PDB code 1PDM ) are shown in blue and yellow , respectively . The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange . A close up view of the gp8 structure ( bottom ) shows that the conserved blocks are involved in dimerization and interaction with gp6 .

B. Cartoon representation of the gp6/gp8 baseplate subcomplex of the T4 phage. The gp6 ring structure (PDB code 3H3W) is shown using the same colour code described in figure 5. The chains A and B of the gp8 dimers (PDB code 1PDM) are shown in blue and yellow, respectively. The conserved blocks mapped onto the dimeric structure of gp8 are shown in orange. A close up view of the gp8 structure (bottom) shows that the conserved blocks are involved in dimerization and interaction with gp6.


A . Structure of the sheath / baseplate complex of the bacteriophage T4 , adapted from Kostyuchenko et al . ( Kostyuchenko , Leiman et al . , 2003 ) . A colour code with corresponding gp protein is provided . The proposed corresponding homolog in the T6SS is indicated with parentheses .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Structure of the sheath/baseplate complex of the bacteriophage T4, adapted from Kostyuchenko et al. (Kostyuchenko, Leiman et al., 2003). A colour code with corresponding gp protein is provided. The proposed corresponding homolog in the T6SS is indicated with parentheses.


The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects . The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2 , or by mCherry - tagged PI4Kβ1 expressed from a genomic fragment . Plants shown are one month old . Scale bar , 10 cm .

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

The Arabidopsis pi4kβ1 pi4kβ2 double mutant displays a growth defect and cytokinetic defects. The growth phenotype of the Arabidopsis pi4kβ1 pi4kβ2 double mutant can be complemented by ectopic expression of PI4Kβ1 or PI4Kβ2, or by mCherry-tagged PI4Kβ1 expressed from a genomic fragment. Plants shown are one month old. Scale bar, 10 cm.


The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants , as shown by propidium iodide ( PI ) staining of five - day - old roots . Arrowhead , oblique cell wall . Insets , magnifications of areas showing cell wall stubs . Scale bars , 50 µm .

The irregularities in the pattern of root epidermal cell division orientation of the pi4kβ1 pi4kβ2 double mutant is also complemented by ectopic expression of PI4Kβ variants, as shown by propidium iodide (PI) staining of five-day-old roots. Arrowhead, oblique cell wall. Insets, magnifications of areas showing cell wall stubs. Scale bars, 50 µm.


Quantifications of cell wall stubs , oblique cell walls and root meristem size ( wild type , n = 15 roots ; pi4kβ1 , n = 15 roots ; pi4kβ2 , n = 14 roots ; pi4kβ1 pi4kβ2 , n = 18 roots ; pPI4Kβ1 : PI4Kβ1 , n = 14 roots , pPI4Kβ2 : PI4Kβ2 , n = 15 roots ) . n . d . , not detected . Data are mean ± SD . Lowercase letters indicate a significant increase of oblique cell walls ( Welch ANOVA with Games - Howell post - hoc test ; p < 0 . 0001 ) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant ( one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 001 ) .

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

Quantifications of cell wall stubs, oblique cell walls and root meristem size (wild type, n = 15 roots; pi4kβ1, n = 15 roots; pi4kβ2, n = 14 roots; pi4kβ1 pi4kβ2, n = 18 roots; pPI4Kβ1:PI4Kβ1, n = 14 roots, pPI4Kβ2:PI4Kβ2, n = 15 roots). n.d., not detected. Data are mean ± SD. Lowercase letters indicate a significant increase of oblique cell walls (Welch ANOVA with Games-Howell post-hoc test; p < 0.0001) or a significant reduction of root meristem size in the pi4kβ1 pi4kβ2 double mutant (one-way ANOVA with post-hoc Tukey HSD; p < 0.001).


Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG - PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters , as indicated , shown for one - month - old plants . Scale bar , 10 cm . Complementation with the pKNOLLE - driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2 / M phase .

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

Complementation of the pi4kβ1 pi4kβ2 phenotype by functional FLAG-PI4Kβ1 expressed from the intrinsic pPI4Kβ1 or by the pKNOLLE promoters, as indicated, shown for one-month-old plants. Scale bar, 10 cm. Complementation with the pKNOLLE-driven construct indicates functionality of PI4Kβ1 during cell plate formation in G2/M phase.


PI staining of pKNOLLE : FLAG - PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects . Arrowhead , cell wall stub in the pi4kβ1 pi4kβ2 double mutant . Scale bars , 50 µm .

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

PI staining of pKNOLLE:FLAG-PI4Kβ1 expressed in the pi4kβ1 pi4kβ2 double mutant background indicates rescue of cytokinetic defects. Arrowhead, cell wall stub in the pi4kβ1 pi4kβ2 double mutant. Scale bars, 50 µm.


In vivo localization of mCherry - PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five - day - old complemented pi4kβ1 pi4kβ2 plants . The mCherry - PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole ( VP ) mode with the pinhole set to 2 . Arrowheads , nascent cell plates decorated by mCherry - PI4Kβ1 . Scale bar , 20 µm . Whole - mount immunostaining of five - day - old seedlings expressing mCherry - PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti - tubulin ( red ) and anti - mCherry ( green ) antibodies , and DAPI ( blue ) . ( І ) , ( ІІ ) , magnifications of regions marked in b , representing early and late cytokinetic stages . Scale bars , 20 µm .

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

In vivo localization of mCherry-PI4Kβ1 expressed from the pPI4Kβ1 promoter in root tips of five-day-old complemented pi4kβ1 pi4kβ2 plants. The mCherry-PI4Kβ1 distribution was imaged using a Zeiss LSM880 in Airyscan Virtual Pinhole (VP) mode with the pinhole set to 2. Arrowheads, nascent cell plates decorated by mCherry-PI4Kβ1. Scale bar, 20 µm. Whole-mount immunostaining of five-day-old seedlings expressing mCherry-PI4kβ1 in the pi4kβ1 pi4kβ2 double mutant background using anti-tubulin (red) and anti-mCherry (green) antibodies, and DAPI (blue). (І), (ІІ), magnifications of regions marked in b, representing early and late cytokinetic stages. Scale bars, 20 µm.


Relative localization of mCherry - PI4Kβ1 and the TGN marker , ARF1 , during cytokinesis . Four - day - old seedlings were immunostainned with anti - mCherry ( green ) and anti - ARF1 ( red ) antibodies . Scale bars are 10 µm .

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

Relative localization of mCherry-PI4Kβ1 and the TGN marker, ARF1, during cytokinesis. Four-day-old seedlings were immunostainned with anti-mCherry (green) and anti-ARF1 (red) antibodies. Scale bars are 10 µm.


The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five - day - old root tip meristems . Images shown are from 3 roots ( wild type ) and 7 roots ( pi4kβ1 pi4kβ2 double mutant ) . In wild type controls ( A ) , vesicles fused at the cell division plane to give rise to tubular network ( TN ) of a nascent cell plate . White arrowheads , fused vesicles at the division plane . n = 8 cells . In the pi4kβ1 pi4kβ2 double mutant ( B ) , vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse . The nuclei of both wild type and double mutant cells were at telophase or interphase , as judged by the presence of decondensed chromatin and a nuclear envelope . At this stage , failed cell plate formation was never seen in wild type ( A ) . Black arrowheads , unfused vesicles . n = 6 cells . Unfused vesicles clustered around a cell wall stub ( arrow ) . White arrowheads , light vesicles ; black arrowheads , dark vesicles ; black arrow , cell wall stub . n = 8 cells . No cross wall was seen in this cell with two nuclei . n = 6 cells . n , nucleus ; ne , nuclear envelope . Scale bars in magnified figures , 2 μm ; in insets , 1 μm .

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

The ultrastructure of cytokinetic defects was analyzed by transmission electron microscopy of five-day-old root tip meristems. Images shown are from 3 roots (wild type) and 7 roots (pi4kβ1 pi4kβ2 double mutant). In wild type controls (A), vesicles fused at the cell division plane to give rise to tubular network (TN) of a nascent cell plate. White arrowheads, fused vesicles at the division plane. n = 8 cells. In the pi4kβ1 pi4kβ2 double mutant (B), vesicles were delivered to the cell division plane at a similar stage as wild type but did not fuse. The nuclei of both wild type and double mutant cells were at telophase or interphase, as judged by the presence of decondensed chromatin and a nuclear envelope. At this stage, failed cell plate formation was never seen in wild type (A). Black arrowheads, unfused vesicles. n = 6 cells. Unfused vesicles clustered around a cell wall stub (arrow). White arrowheads, light vesicles; black arrowheads, dark vesicles; black arrow, cell wall stub. n = 8 cells. No cross wall was seen in this cell with two nuclei. n = 6 cells. n, nucleus; ne, nuclear envelope. Scale bars in magnified figures, 2 μm; in insets, 1 μm.


The effects of reduced association of PtdIns ( 4 ) P with the cell plate on the recruitment of CLC2 - GFP were analyzed in root meristem cells of five - day - old plants . Top , Cell plate - associated fluorescence of the PtdIns ( 4 ) P reporter , 2 × mCherryFAPP1 - PH , was monitored at the end of cytokinesis in five - day - old wild type controls or pi4kβ1 pi4kβ2 double mutants , as indicated . Scale bars , 10 µm . Bottom , The intensity of 2 × mCherryFAPP1 - PH signals was quantified at the end of cytokinesis at the cell plate ( left plot ) and at the TGN ( right plot ) . Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane ( wild type , n = 37 cells , 24 roots ; pi4kβ1 pi4kβ2 , n = 55 cells , 41 roots ) . TGN intensities were recorded using the Airyscan VP mode , and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity ( wild type , n = 30 cells , 22 roots ; pi4kβ1 pi4kβ2 , n = 37 cells , 31 roots ) . * * * , significant differences ( p < 0 . 0001 ) according to two - tailed Mann - Whitney U - tests .

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

The effects of reduced association of PtdIns(4)P with the cell plate on the recruitment of CLC2-GFP were analyzed in root meristem cells of five-day-old plants. Top, Cell plate-associated fluorescence of the PtdIns(4)P reporter, 2×mCherryFAPP1-PH, was monitored at the end of cytokinesis in five-day-old wild type controls or pi4kβ1 pi4kβ2 double mutants, as indicated. Scale bars, 10 µm. Bottom, The intensity of 2×mCherryFAPP1-PH signals was quantified at the end of cytokinesis at the cell plate (left plot) and at the TGN (right plot). Signal intensities at the cell plate were recorded along the dashed lines indicated in the images and normalized to intensities at the apical plasma membrane (wild type, n = 37 cells, 24 roots; pi4kβ1 pi4kβ2, n = 55 cells, 41 roots). TGN intensities were recorded using the Airyscan VP mode, and the mean TGN intensity was normalized to mean apical and basal plasma membrane intensity (wild type, n=30 cells, 22 roots; pi4kβ1 pi4kβ2, n=37 cells, 31 roots). ***, significant differences (p<0.0001) according to two-tailed Mann-Whitney U-tests.


Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of wild type seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks .

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

Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of wild type seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.


Image series from live cell time lapse analysis of CLC2 - GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4 - 64 , as indicated . Arrowheads , appearance of CLC2 - GFP at the cell plate . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Scale bars , 10 μm . Images are from median focal planes chosen from time lapse series recorded as 3D stacks .

Image series from live cell time lapse analysis of CLC2-GFP at the cell plate in roots of pi4kβ1 pi4kβ2 seedlings costained with FM4-64, as indicated. Arrowheads, appearance of CLC2-GFP at the cell plate. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Scale bars, 10 μm. Images are from median focal planes chosen from time lapse series recorded as 3D stacks.


Left , quantification of the time of CLC2 - GFP appearance at the cell plate in wild type ( B ) or the pi4kβ1 pi4kβ2 double mutant ( C ) ( wild type , n = 10 cells , 7 roots ; pi4kβ1 pi4kβ2 , n = 24 cells , 19 roots ) . Right , quantification of CLC2 - GFP at the TGN in interphase cells . Five - day - old roots expressing CLC2 - GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity ( wild type , n = 14 cells , 2 roots ; pi4kβ1 pi4kβ2 , n = 15 cells , 2 roots ) .

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

Left, quantification of the time of CLC2-GFP appearance at the cell plate in wild type (B) or the pi4kβ1 pi4kβ2 double mutant (C) (wild type, n = 10 cells, 7 roots; pi4kβ1 pi4kβ2, n = 24 cells, 19 roots). Right, quantification of CLC2-GFP at the TGN in interphase cells. Five-day-old roots expressing CLC2-GFP were subjected to immunostaining and the mean TGN intensity was normalized to plasma membrane intensity (wild type, n = 14 cells, 2 roots; pi4kβ1 pi4kβ2, n = 15 cells, 2 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Five - day - old seedlings were immunostained with anti - KNOLLE ( green ) and counterstained with DAPI ( blue ) . In the pi4kβ1 pi4kβ2 double mutant , diffusion of KNOLLE occurred at the plasma membrane ( arrowheads ) . Scale bars , 10 μm . Quantitative analysis of lateral diffusion of KNOLLE at the plasma membrane ( PM ) from ( A ) . Data are mean ± SD from four independent experiments . * * , a significant difference ( p < 0 . 01 ) according to a two - tailed Student ' s t test ( untreated wild type , n = 97 cells , 25 roots ; untreated pi4kβ1 pi4kβ2 , n = 142 cells , 44 roots ; BFA treated wild type , n = 126 cells , 28 roots ; BFA treated pi4kβ1 pi4kβ2 , n = 183 cells , 43 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Five-day-old seedlings were immunostained with anti-KNOLLE (green) and counterstained with DAPI (blue). In the pi4kβ1 pi4kβ2 double mutant, diffusion of KNOLLE occurred at the plasma membrane (arrowheads). Scale bars, 10 μm. Quantitative analysis of lateral diffusion of KNOLLE at the plasma membrane (PM) from (A). Data are mean ± SD from four independent experiments. **, a significant difference (p<0.01) according to a two-tailed Student's t test (untreated wild type, n= 97 cells, 25 roots; untreated pi4kβ1 pi4kβ2, n = 142 cells, 44 roots; BFA treated wild type, n = 126 cells, 28 roots; BFA treated pi4kβ1 pi4kβ2, n = 183 cells, 43 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Enhanced accumulation of KNOLLE at the cell plate at the end of cytokinesis in the pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were immunostained with anti - KNOLLE antibodies ( green ) and counterstained with DAPI ( blue ) . Scale bars , 10 μm . Quantification of KNOLLE intensity at the cell plate from ( C ) , normalized to the signal intensity of intracellular compartments . * * * , a significant difference ( p < 0 . 0001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 26 cells , 20 roots ; pi4kβ1 pi4kβ2 , n = 41 cells , 37 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of KNOLLE at the cell plate at the end of cytokinesis in the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were immunostained with anti-KNOLLE antibodies (green) and counterstained with DAPI (blue). Scale bars, 10 μm. Quantification of KNOLLE intensity at the cell plate from (C), normalized to the signal intensity of intracellular compartments. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 26 cells, 20 roots; pi4kβ1 pi4kβ2, n = 41 cells, 37 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . Panels a - f display data obtained using cytokinetic cells Enhanced accumulation of PIN2 - GFP at the cell plate at the end of cytokinesis in the the pi4kβ1 pi4kβ2 double mutant . Five - day - old seedlings were stained with 2 μM FM 4 - 64 for 3 minutes at room temperature . Scale bars , 10 μm . Quantification of PIN2 - GFP signal at the cell plate from ( E ) , normalized to the intensity at the apical plasma membrane intensity , as indicated by the dashed lines . * * * , a significant difference ( p < 0 . 001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 28 cells , 16 roots ; pi4kβ1 pi4kβ2 , n = 45 cells , 26 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. Panels a-f display data obtained using cytokinetic cells Enhanced accumulation of PIN2-GFP at the cell plate at the end of cytokinesis in the the pi4kβ1 pi4kβ2 double mutant. Five-day-old seedlings were stained with 2 μM FM 4-64 for 3 minutes at room temperature. Scale bars, 10 μm. Quantification of PIN2-GFP signal at the cell plate from (E), normalized to the intensity at the apical plasma membrane intensity, as indicated by the dashed lines. ***, a significant difference (p<0.001) according to a two-tailed Mann-Whitney U-test (wild type, n = 28 cells, 16 roots; pi4kβ1 pi4kβ2, n = 45 cells, 26 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . panels g - j display data from the analysis of interphase cells . Internalization of PIN2 - GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min , then washed and incubated with 50 μM CHX and 25 μM BFA . Scale bars , 10 μm . Quantification of punctate signals from ( G ) induced by BFA in wild type and pi4kβ1 pi4kβ2 double mutants . Data are mean ± SD . * , a significant difference ( p < 0 . 05 ) according to a two - tailed Student ' s t test ; ( wild type , n = 116 cells , 6 roots ; pi4kβ1 pi4kβ2 , n = 110 cells , 7 roots ) .

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

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Internalization of PIN2-GFP was tracked overtime in live roots pretreated with 50 μM CHX for 30 min, then washed and incubated with 50 μM CHX and 25 μM BFA. Scale bars, 10 μm. Quantification of punctate signals from (G) induced by BFA in wild type and pi4kβ1 pi4kβ2 double mutants. Data are mean ± SD. *, a significant difference (p<0.05) according to a two-tailed Student's t test; (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 110 cells, 7 roots).


Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation . panels g - j display data from the analysis of interphase cells . Delayed internalization of FM 4 - 64 from the plasma membrane in the pi4kβ1 pi4kβ2 double mutant . Four - day - old seedlings were pulsed with 2 μM FM 4 - 64 for 3 minutes on ice and then fluorescence was recorded for 20 min . Scale bars , 10 μm . Quantification of intracellular FM 4 - 64 signal from ( i ) , normalized to apical and basal plasma membrane intensities . * * * , a significant difference ( p < 0 . 0001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 116 cells , 6 roots ; pi4kβ1 pi4kβ2 , n = 66 cells , 9 roots ) .

Experiments were performed to address possible effects of the pi4kβ1 pi4kβ2 double mutant on endocytosis of markers relevant for cell plate formation. panels g-j display data from the analysis of interphase cells. Delayed internalization of FM 4-64 from the plasma membrane in the pi4kβ1 pi4kβ2 double mutant. Four-day-old seedlings were pulsed with 2 μM FM 4-64 for 3 minutes on ice and then fluorescence was recorded for 20 min. Scale bars, 10 μm. Quantification of intracellular FM 4-64 signal from (i), normalized to apical and basal plasma membrane intensities. ***, a significant difference (p<0.0001) according to a two-tailed Mann-Whitney U-test (wild type, n = 116 cells, 6 roots; pi4kβ1 pi4kβ2, n = 66 cells, 9 roots).


pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Immunostaining of meristem cells of five - day - old plants using anti - tubulin ( green ) counterstained with DAPI ( false colour red ) . Asterisks , multiple nuclei in one cell ; arrowheads , multiple phragmoplasts in one cell . Dashed lines indicate the outlines of exemplary multinucleated cells . Scale bars , 10 µm .

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

pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Immunostaining of meristem cells of five-day-old plants using anti-tubulin (green) counterstained with DAPI (false colour red). Asterisks, multiple nuclei in one cell; arrowheads, multiple phragmoplasts in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.


pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Whole - mount immunostaining of root tips of five - day - old seedlings . Green , anti - KNOLLE ; red , anti - tubulin ; blue , DAPI . ( І ) , ( ІІ ) , magnifications of areas highlighted in dashed boxes . Arrowheads , solid phragmoplasts with thicker cell plates . Scale bars , 20 µm .

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

pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Whole-mount immunostaining of root tips of five-day-old seedlings. Green, anti-KNOLLE; red, anti-tubulin; blue, DAPI. (І), (ІІ), magnifications of areas highlighted in dashed boxes. Arrowheads, solid phragmoplasts with thicker cell plates. Scale bars, 20 µm.


pi4kβ1 pi4kβ2 double mutant phenotype were recorded . Quantification of the incidences of multinucleated cells , multiple phragmoplasts and solid phragmoplasts during late cytokinesis ( multinucleated cells : wild type , n = 316 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 673 cells , 44 roots ; multiple phragmoplasts : wild type , n = 64 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 188 cells , 44 roots ; solid phragmoplast in late cytokinesis : wild type , n = 41 cells , 29 roots ; pi4kβ1 pi4kβ2 , n = 89 cells , 44 roots ) . Data are mean ± SD from three independent experiments . n . d . not detected .

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

pi4kβ1 pi4kβ2 double mutant phenotype were recorded. Quantification of the incidences of multinucleated cells, multiple phragmoplasts and solid phragmoplasts during late cytokinesis (multinucleated cells: wild type, n = 316 cells, 29 roots; pi4kβ1 pi4kβ2, n = 673 cells, 44 roots; multiple phragmoplasts: wild type, n = 64 cells, 29 roots; pi4kβ1 pi4kβ2, n = 188 cells, 44 roots; solid phragmoplast in late cytokinesis: wild type, n = 41 cells, 29 roots; pi4kβ1 pi4kβ2, n = 89 cells, 44 roots). Data are mean ± SD from three independent experiments. n.d. not detected.


The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging . Five - day - old seedlings expressing mCherry - TUA5 were imaged at 1 frame per 2 min . Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 . Cells are outlined by dashed lines in the first and last frames of each series . White arrowhead , ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant . Scale bars , 10 µm . Plot profiles obtained from dashed lines marked in ( A ) . Black arrowhead , ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant .

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

The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging. Five-day-old seedlings expressing mCherry-TUA5 were imaged at 1 frame per 2 min. Times are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0. Cells are outlined by dashed lines in the first and last frames of each series. White arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant. Scale bars, 10 µm. Plot profiles obtained from dashed lines marked in (A). Black arrowhead, ectopic stabilization of microtubules in central phragmoplasts of the pi4kβ1 pi4kβ2 double mutant.


The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging . 3D projections from time points selected from ( A ) , when the transition to a ring phragmoplast has occurred in wild type , but solid phragmoplast persisted in the double mutant . Scale bars , 10 µm . Duration of phragmoplast persistence during cytokinesis , determined from initiation to disbanding . * * * , a significant difference ( p < 0 . 001 ) according to a two - tailed Mann - Whitney U - test ( wild type , n = 12 cells , 9 roots ; pi4kβ1 pi4kβ2 , n = 21 cells , 16 roots ) .

The dynamic transitions of phragmoplast microtubules were recorded by live cell time lapse imaging. 3D projections from time points selected from (A), when the transition to a ring phragmoplast has occurred in wild type, but solid phragmoplast persisted in the double mutant. Scale bars, 10 µm. Duration of phragmoplast persistence during cytokinesis, determined from initiation to disbanding. ***, a significant difference (p<0.001) according to a two-tailed Mann-Whitney U-test (wild type, n = 12 cells, 9 roots; pi4kβ1 pi4kβ2, n = 21 cells, 16 roots).


The dynamics of the microtubule bundling protein , MAP65 - 3 , were recorded in live cell time lapse analyses of cytokinetic cells from root tips of wild type controls and pi4kβ1 pi4kβ2 double mutants . Image series of 3D projections of GFP - MAP65 - 3 costained with FM 4 - 64 in root tips of four - day - old wild type seedlings . The best angle for visualization was obtained by rotation of the x - axis . Images are representative for 8 cells from 5 roots . Scale bar , 10 µm . Image series of 3D projections of GFP - MAP65 - 3 costained with FM 4 - 64 in root tips of four - day - old pi4kβ1 pi4kβ2 seedlings . The best angle for visualization was obtained by rotation of the x - axis . Images are representative for 11 cells from 10 roots . Arrowheads , persisting signal of GFP - MAP65 - 3 in the center of the cell division plane . Scale bars , 10 µm . Plot profiles were obtained from medial confocal planes at time points displaying the transition from disc to ring phragmoplasts , marked by dashed lines in ( A ) and ( B ) . Arrowhead , persisting signal of GFP - MAP65 - 3 in the center of the cell division plane . Times in ( A - C ) are given relative to the instance when the cell plate contacted the peripheral plasma membrane , defined as t0 .

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

The dynamics of the microtubule bundling protein, MAP65-3, were recorded in live cell time lapse analyses of cytokinetic cells from root tips of wild type controls and pi4kβ1 pi4kβ2 double mutants. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old wild type seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 8 cells from 5 roots. Scale bar, 10 µm. Image series of 3D projections of GFP-MAP65-3 costained with FM 4-64 in root tips of four-day-old pi4kβ1 pi4kβ2 seedlings. The best angle for visualization was obtained by rotation of the x-axis. Images are representative for 11 cells from 10 roots. Arrowheads, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Scale bars, 10 µm. Plot profiles were obtained from medial confocal planes at time points displaying the transition from disc to ring phragmoplasts, marked by dashed lines in (A) and (B). Arrowhead, persisting signal of GFP-MAP65-3 in the center of the cell division plane. Times in (A-C) are given relative to the instance when the cell plate contacted the peripheral plasma membrane, defined as t0.


Medial confocal planes from z - stacks from ( a ) and ( b ) demonstrating attachment of the FM 4 - 64 - stained cell plate with the peripheral plasma membrane , defining t0 . Scale bars , 10 µm .

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

Medial confocal planes from z-stacks from (a) and (b) demonstrating attachment of the FM 4-64-stained cell plate with the peripheral plasma membrane, defining t0. Scale bars, 10 µm.


3D reconstruction of ring and disc - shaped immunofluorescence patterns for GFP - MAP65 - 3 and microtubules , based on super resolution structured illumination microscopy ( SIM ) , costained with DAPI . Top panels , wild type ; bottom panels , pi4kβ1 pi4kβ2 double mutants . Scale bars , 10 µm .

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3D reconstruction of ring and disc-shaped immunofluorescence patterns for GFP-MAP65-3 and microtubules, based on super resolution structured illumination microscopy (SIM), costained with DAPI. Top panels, wild type; bottom panels, pi4kβ1 pi4kβ2 double mutants. Scale bars, 10 µm.


The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants . Crosses of pi4kβ1 pi4kβ2 and mpk4 - 2 mutants resulted in combined genotypes with increased growth defects , as indicated . 14 - day - old seedlings are shown . Scale bars , 1 cm . Top , The severe growth defects of the combined genotypes were accompanied by increased tissue distortion , as shown by 3D projections after propidium iodide staining of root tips of 14 - day - old seedlings . Scale bars , 100 µm . Bottom , Multinucleation of cells was increased in the combined genotypes , as evident from immunofluorescence of microtubules ( red ) counterstained with DAPI ( blue ) . Asterisks , multiple nuclei in one cell . Dashed lines indicate the outlines of exemplary multinucleated cells . Scale bars , 10 µm .

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The functional relations of PI4Kβ isoforms with MPK4 were tested by assessing phenotypes of crosses of the corresponding mutants. Crosses of pi4kβ1 pi4kβ2 and mpk4-2 mutants resulted in combined genotypes with increased growth defects, as indicated. 14-day-old seedlings are shown. Scale bars, 1 cm. Top, The severe growth defects of the combined genotypes were accompanied by increased tissue distortion, as shown by 3D projections after propidium iodide staining of root tips of 14-day-old seedlings. Scale bars, 100 µm. Bottom, Multinucleation of cells was increased in the combined genotypes, as evident from immunofluorescence of microtubules (red) counterstained with DAPI (blue). Asterisks, multiple nuclei in one cell. Dashed lines indicate the outlines of exemplary multinucleated cells. Scale bars, 10 µm.


Quantification of cytokinetic defects , as shownin the bottom panels of ( B ) . Cytokinetic defects were enhanced in pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) ( for multinucleated cells , including the percentage of multinucleated cells and the average number of nuclei in multinucleated cells , both according to a Welch ANOVA with Games - Howell post - hoc test ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ( wild type , n = 1650 cells , 13 roots ; pi4kβ1 pi4kβ2 , n = 1441 cells , 19 roots ; mpk4 - 2 , n = 1429 cells , 16 roots ; pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) , n = 574 cells , 16 roots ) ; and the incidence of cells displaying KNOLLE fluorescence at the cell plate according to a one - way ANOVA with post - hoc Tukey HSD ; p < 0 . 05 , lowercase letters ( wild type , n = 1981 cells , 13 roots ; pi4kβ1 pi4kβ2 , n = 1758 cells , 19 roots ; mpk4 - 2 , n = 1721 cells , 16 roots , pi4kβ1 ( - / - ) pi4kβ2 ( + / - ) mpk4 - 2 ( - / - ) , n = 660 cells , 16 roots ) .

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Quantification of cytokinetic defects, as shownin the bottom panels of (B). Cytokinetic defects were enhanced in pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-) (for multinucleated cells, including the percentage of multinucleated cells and the average number of nuclei in multinucleated cells, both according to a Welch ANOVA with Games-Howell post-hoc test; ***, p < 0.001; **,p < 0.01 (wild type, n = 1650 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1441 cells, 19 roots; mpk4-2, n = 1429 cells, 16 roots; pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 574 cells, 16 roots); and the incidence of cells displaying KNOLLE fluorescence at the cell plate according to a one-way ANOVA with post-hoc Tukey HSD; p < 0.05, lowercase letters (wild type, n = 1981 cells, 13 roots; pi4kβ1 pi4kβ2, n = 1758 cells, 19 roots; mpk4-2, n = 1721 cells, 16 roots, pi4kβ1(-/-) pi4kβ2(+/-) mpk4-2(-/-), n = 660 cells, 16 roots).


Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . PI4Kβ1 interacts with MPK4 in yeast two hybrid tests . MPK4 was used as a bait , while PI4Kβ1 was divided into two fragments to be used as prey . The results are representative for three independent experiments .

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Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. PI4Kβ1 interacts with MPK4 in yeast two hybrid tests. MPK4 was used as a bait, while PI4Kβ1 was divided into two fragments to be used as prey. The results are representative for three independent experiments.


Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . In vivo co - immunoprecipitation of MPK4 and PI4Kβ1 from 14 - day - old plants . PI4Kβ1 was specifically co - precipitated with MPK4 - myc using anti - myc antibodies . Arrowhead , Migration of PI4Kβ1 . The results are representative for three independent experiments .

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Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. In vivo co-immunoprecipitation of MPK4 and PI4Kβ1 from 14-day-old plants. PI4Kβ1 was specifically co-precipitated with MPK4-myc using anti-myc antibodies. Arrowhead, Migration of PI4Kβ1. The results are representative for three independent experiments.


Experiments were performed to assess possible physical interaction , colocalization at the cell plate , and functional interplay of PI4Kβ1 and MPK4 . Coordination of MPK4 - YFP and mCherry - PI4Kβ1 during cytokinesis in five - day - old root meristem cells . MPK4 co - localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates . Images were obtained with an LSM880 in Airyscan super - resolution ( SR ) mode . Scale bars , 10 µm .

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Experiments were performed to assess possible physical interaction, colocalization at the cell plate, and functional interplay of PI4Kβ1 and MPK4. Coordination of MPK4-YFP and mCherry-PI4Kβ1 during cytokinesis in five-day-old root meristem cells. MPK4 co-localized with PI4Kβ1 from early to late cytokinesis and gradually concentrated at the leading edges of cell plates. Images were obtained with an LSM880 in Airyscan super-resolution (SR) mode. Scale bars, 10 µm.


Live cell time lapse of MPK4 - YFP and mRFPFAPP1 - PH during cytokinesis , obtained with an LSM880 in Airyscan resolution versus sensitivity ( R - S ) mode . The time series is representative for 6 cells from 5 roots . Arrowheads indicate the initiation and leading edges of the growing cell plate . Scale bar , 10 µm .

Live cell time lapse of MPK4-YFP and mRFPFAPP1-PH during cytokinesis, obtained with an LSM880 in Airyscan resolution versus sensitivity (R-S) mode. The time series is representative for 6 cells from 5 roots. Arrowheads indicate the initiation and leading edges of the growing cell plate. Scale bar, 10 µm.


Left panels , The distribution of mRFPFAPP1 - PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls ( top ) and mpk4 - 2 mutants ( bottom ) . Intensity profiles were recorded as indicated by the dashed lines . Scale bars , 10 µm . Right top , intensity of mRFPFAPP1 - PH at the cell plate normalized vs . the intensity at the apical plasma membrane ( wild type , n = 43 cells , 34 roots ; mpk4 - 2 , n = 32 cells , 28 roots ) . Right bottom , intensity of mRFPFAPP1 - PH at the TGN normalized vs . the intensity at the apical plasma membrane ( wild type , n = 40 cells , 29 roots ; mpk4 - 2 , n = 26 cells , 23 roots ) . * , a significant difference ( p < 0 . 05 ) according to a two - tailed Student ' s test .

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Left panels, The distribution of mRFPFAPP1-PH was observed at the cell plate at the end of cytokinesis in root meristem cells of wild type controls (top) and mpk4-2 mutants (bottom). Intensity profiles were recorded as indicated by the dashed lines. Scale bars, 10 µm. Right top, intensity of mRFPFAPP1-PH at the cell plate normalized vs. the intensity at the apical plasma membrane (wild type, n = 43 cells, 34 roots; mpk4-2, n = 32 cells, 28 roots). Right bottom, intensity of mRFPFAPP1-PH at the TGN normalized vs. the intensity at the apical plasma membrane (wild type, n = 40 cells, 29 roots ; mpk4-2, n = 26 cells, 23 roots). *, a significant difference (p<0.05) according to a two-tailed Student's test.


A . Growth curves of the library ( black ) compared to two different controls under increasing selective pressures . DSB ( double - stranded break ) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK . n = 3 for each

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A. Growth curves of the library (black) compared to two different controls under increasing selective pressures. DSB (double-stranded break) Negative Control is a cassette designed to introduce a stop codon at the unrelated gene galK. n=3 for each curve


B . Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non - Target gRNA . n = 3 . C . Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non - Target gRNA . n =

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B. Growth of the reconstructed LysP T33F mutant compared to wild type cells transformed with a Non-Target gRNA. n=3. C. Growth of the reconstructed LysP Q219I mutant compared to wild type cells transformed with a Non-Target gRNA. n=3


D . Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants . Quantification was performed using LC - MS , as described in the methods section . n = 3 . A two - sample student t - test assuming unequal variances was performed to calculate statistical significance . Concentrations are reported as fold - change relative to the wild - type control

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D. Absolute quantification of intracellular lysine concentration in wild type and the reconstructed DapF mutants. Quantification was performed using LC-MS, as described in the methods section. n=3. A two-sample student t-test assuming unequal variances was performed to calculate statistical significance. Concentrations are reported as fold-change relative to the wild-type control samples


E . DapF assay showing kinetics of the wild type , G210D and M260Y mutants . Assay was performed as described in the methods section . n =

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E. DapF assay showing kinetics of the wild type, G210D and M260Y mutants. Assay was performed as described in the methods section. n=5


F . Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT , DapF G210D and DapF M260Y

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F. Differential gene expression quantified via qPCR for the dapF and lysA genes on a WT, DapF G210D and DapF M260Y backgrounds


C . Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control

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C. Absolute quantification of intracellular lysine levels in wild type and the reconstructed LysR S36R mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples


F . Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant . Quantification was performed using LC - MS , as described in the methods section . n = 2 . Concentrations are reported as fold - change relative to the wild - type control

F. Absolute quantification of intracellular lysine levels in wild type and the reconstructed ArgP E246 mutant. Quantification was performed using LC-MS, as described in the methods section. n=2. Concentrations are reported as fold-change relative to the wild-type control samples


( G ) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group ( numbered 1 - 4 ) was detected by anti - IgG secondary antibody .

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(G) Liver proteins were resolved by PAGE and the binding of serum antibodies from 4 individual mice from each group (numbered 1-4) was detected by anti-IgG secondary antibody .


Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . After 2 weeks , 7 μm spleen sections were stained with Red : Alexa - Fluor - 594 - CD4 and Green : FITC - B220 ( D ) or FITC - GL7 ( E ) . Scale bar represent 100 nm .

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Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. After 2 weeks, 7 μm spleen sections were stained with Red:Alexa-Fluor-594-CD4 and Green: FITC-B220 (D) or FITC-GL7 (E). Scale bar represent 100 nm.


Male C57BL / 6 Nr4a2 cKO or wildtype littermate C57BL / 6 mice were immunised twice with NP - KLH emulsified in alum . Serum was assessed for level of IgG antibodies binding NP - 31 and NP - 4 ( F ) .

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Male C57BL/6 Nr4a2 cKO or wildtype littermate C57BL/6 mice were immunised twice with NP-KLH emulsified in alum. Serum was assessed for level of IgG antibodies binding NP-31 and NP-4 (F).


( A ) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3 + CD4 + Th cells against CD127 ( left ) and intracellular Foxp3 ( right )

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(A) PBMC were isolated from healthy volunteers and stained for flow cytometry to identify NRP1 expression in CD3+CD4+ Th cells against CD127 (left) and intracellular Foxp3 (right)


NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . Representative staining is shown for CD4 + CD3 + Th cells ( B ) , and summary data for CD3 + CD4 + Th cells ( C ) Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test .

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NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. Representative staining is shown for CD4+CD3+ Th cells (B), and summary data for CD3+CD4+ Th cells (C) Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.


NRP1 - expressing cells amongst CD4 + CD3 + Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers ( healthy controls , HC ) and 10 SLE patients . summary data for CD3 + CD4 + CD127hi conventional cells ( D ) . Plots show individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * p < 0 . 001 , * * * * p < 0 . 0001 Unpaired Student ' s T test .

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NRP1-expressing cells amongst CD4+CD3+ Th cells were measured by flow cytometry in PBMC from 18 healthy volunteers (healthy controls, HC) and 10 SLE patients. summary data for CD3+CD4+CD127hi conventional cells (D). Plots show individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ***p<0.001, ****p<0.0001 Unpaired Student's T test.


( E ) Individuals were divided into NRP1 - high subjects ( NRP1 + cells > 1 . 3 % in Th cells ) and NRP1 - low subjects ( NRP1 + cells ≤ 1 . 35 % in Th cells ) . Numbers of HC and SLE falling into each group are shown . * * * p < 0 . 001 Chi squared test with Yates ' correction .

(E) Individuals were divided into NRP1-high subjects (NRP1+ cells >1.3% in Th cells) and NRP1-low subjects (NRP1+ cells≤1.35% in Th cells). Numbers of HC and SLE falling into each group are shown. *** p<0.001 Chi squared test with Yates' correction.


( F , G ) NRP1 - expressing cells amongst CD3 + CD4 + CD45RO - cells were measured , representative staining ( F ) and summary data ( G ) . The plot shows individual subjects , HC , n = 18 ; SLE , n = 10 ; with mean ± SD indicated by error bars ; * * * * p < 0 . 0001 Unpaired Student ' s T test .

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(F, G) NRP1-expressing cells amongst CD3+CD4+CD45RO- cells were measured, representative staining (F) and summary data (G). The plot shows individual subjects, HC, n=18; SLE, n=10; with mean ± SD indicated by error bars; ****p<0.0001 Unpaired Student's T test.