Figure 4B . Representative images of mouse seminiferous tubule ( ST ) sections stained for PLZF ( red ) , SCP1 ( green ) and DAPI ( blue ) at P4 , P6 and P7 . Scale bars : 20 μm . Arrowheads highlight PLZF - SCP1 + ( green ) cells localize to the middle of the ST lumen , which represent differentiated spermatogonia that translocated to the middle of the ST lumen . C . Quantification of ( B ) . Shown is the number of PLZF - SCP1 + cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section , as normalized to the total cell number per ST section . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 30 STs were analyzed for each mouse . D . Quantification of ( B ) . Shown is the total number of SCP1 + cells which translocated inwards the seminiferous tubule lumen per ST section , as normalized to the total cell number per seminiferous tubule section . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 30 STs were analyzed for each mouse . E . Quantification of ( B ) . Shown is the number of PLZF + SCP1 + cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section , as normalized to the total cell number per seminiferous tubule section . Dots ( colors reflect STs from different mice ) represent value per ST , CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice , at least 30 STs were analyzed for each mouse . Data information : In ( C - E ) , data are presented as mean ± SEM . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( the Mann - Whitney test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4B. Representative images of mouse seminiferous tubule (ST) sections stained for PLZF (red), SCP1 (green) and DAPI (blue) at P4, P6 and P7. Scale bars: 20 μm. Arrowheads highlight PLZF- SCP1+ (green) cells localize to the middle of the ST lumen, which represent differentiated spermatogonia that translocated to the middle of the ST lumen. C. Quantification of (B). Shown is the number of PLZF- SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per ST section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. D. Quantification of (B). Shown is the total number of SCP1+ cells which translocated inwards the seminiferous tubule lumen per ST section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. E. Quantification of (B). Shown is the number of PLZF+ SCP1+ cells which translocated inwards the seminiferous tubule lumen per seminiferous tubule section, as normalized to the total cell number per seminiferous tubule section. Dots (colors reflect STs from different mice) represent value per ST, CEP250+/+ n=3 mice, CEP250-/- n=3 mice, at least 30 STs were analyzed for each mouse. Data information: In (C-E), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the Mann-Whitney test).


Figure 5A . Representative images of P4 mouse seminiferous tubule ( ST ) 10 µm cryo - sections stained for PLZF ( red ) , γ - Tubulin ( green ) , PH3 ( magenta ) and DAPI ( blue ) . Arrowheads highlight the centrosomes . Dashed yellow line highlights the basement membrane . Semi - transparent white line , which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane . Anaphase cells were analyzed . Scale bars : 5 µm . B . Representative images of P5 mouse seminiferous tubule 10 µm cryo - sections stained for PLZF ( red ) , γ - Tubulin ( green ) , PH3 ( magenta ) and DAPI ( blue ) . Arrowheads highlight the centrosomes . Dashed yellow line highlights the basement membrane . Semi - transparent white line , which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane . Anaphase cells were analyzed . Scale bars : 5 µm . ( C Quantitation of spindle angle of anaphase GSCs in P4 ST sections ( from A ) . Spindle angle relative to basement membrane for CEP250 + / + and CEP250 - / - ) . In ( C ) , dots ( colors reflect different mice ) represent spindle angle of each GSC . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 cells were analyzed for each mouse . ( D Quantitation of spindle angle of anaphase GSCs in P5 ST sections ( from B ) . for P5 . In ( D ) , dots ( colors reflect different mice ) represent spindle angle of each GSC . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 cells were analyzed in each mouse . E ) Quantitation of spindle angle of anaphase GSCs in P4 ST sections Spindle angle relative to basement membrane for CEP250 + / + and CEP250 - / - ) . In ( E ) , the three dots represent averaged spindle angle per mouse . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 cells were analyzed for each mouse . F ) Quantitation of spindle angle of anaphase GSCs in P5 ST sections for P5 . In ( F ) , the three dots represent averaged spindle angle per mouse . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 10 cells were analyzed in each mouse . Data information : In ( E , F ) , data are presented as mean ± SEM . n . s . , not significant , * P < 0 . 05 , * * * P < 0 . 005 ( the unpaired Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A. Representative images of P4 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. B. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), γ-Tubulin (green), PH3 (magenta) and DAPI (blue). Arrowheads highlight the centrosomes. Dashed yellow line highlights the basement membrane. Semi-transparent white line, which bisects both spindle poles was used to determine spindle angle Ø orientation in relation to the basement membrane. Anaphase cells were analyzed. Scale bars: 5 µm. (C Quantitation of spindle angle of anaphase GSCs in P4 ST sections (from A). Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (C), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. (D Quantitation of spindle angle of anaphase GSCs in P5 ST sections (from B). for P5. In (D), dots (colors reflect different mice) represent spindle angle of each GSC. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse.E) Quantitation of spindle angle of anaphase GSCs in P4 ST sections Spindle angle relative to basement membrane for CEP250+/+ and CEP250-/-). In (E), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed for each mouse. F) Quantitation of spindle angle of anaphase GSCs in P5 ST sections for P5. In (F), the three dots represent averaged spindle angle per mouse. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 10 cells were analyzed in each mouse. Data information: In (E, F), data are presented as mean ± SEM. n.s., not significant, *P<0.05, ***P < 0.005 (the unpaired Student's t-test).


Figure 6B . Representative images of P5 mouse ST . 10 µm cryo - sections stained for PLZF ( red ) , E - cadherin ( green ) and DAPI ( blue ) showing GSCs in interphase . Scale bars : 5 μm . C . Quantification of ( B ) . Percentage of GSCs displaying different polarity status are indicated . CEP250 + / + n = 5 mice , CEP250 - / - n = 4 mice . At least 100 cells were analyzed in each mouse and the average was calculated for each mouse . Data information : In white arrowheads highlight basement membrane ; yellow arrowhead highlights cortical enrichment of E - cadherin towards the basement membrane . In ( C data are presented as mean ± SEM . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( the unpaired Student ' s t - test ) . D . Representative images of P5 mouse ST . 10 µm cryo - sections stained for PLZF ( red ) , E - cadherin ( green ) and PH3 ( magenta ) showing GSCs in prophase . PH3 staining marks the mitotic chromatin . Scale bars : 5 μm . E . Quantification of ( D ) . Percentage of GSCs displaying different polarity status are indicated . CEP250 + / + n = 5 mice , CEP250 - / - n = 4 mice . At least 10 cells were analyzed in each mouse and the average was calculated for each mouse . Data information : In white arrowheads highlight basement membrane ; yellow arrowhead highlights cortical enrichment of E - cadherin towards the basement membrane . In data are presented as mean ± SEM . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( the unpaired Student ' s t - test ) . F . Representative images of P5 mouse ST 10 µm cryo - sections stained for PLZF ( red ) , E - cadherin ( green ) and PH3 ( magenta ) showing GSCs in prometaphase . PH3 staining marks the mitotic chromosome condensation . Scale bars : 5 μm . G . Quantification of ( F ) , percentage of GSCs displaying different polarity status are indicated . CEP250 + / + n = 5 mice , CEP250 - / - n = 4 mice . At least 30 cells were analyzed in each mouse and the average was calculated for each mouse . Data information : In F , white arrowheads highlight basement membrane ; yellow arrowhead highlights cortical enrichment of E - cadherin towards the basement membrane . In G ) , data are presented as mean ± SEM . n . s . , not significant , * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 005 ( the unpaired Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6B. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and DAPI (blue) showing GSCs in interphase. Scale bars: 5 μm. C. Quantification of (B). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 100 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In (C data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).D. Representative images of P5 mouse ST. 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prophase. PH3 staining marks the mitotic chromatin. Scale bars: 5 μm. E. Quantification of (D). Percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 10 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).F. Representative images of P5 mouse ST 10 µm cryo-sections stained for PLZF (red), E-cadherin (green) and PH3 (magenta) showing GSCs in prometaphase. PH3 staining marks the mitotic chromosome condensation. Scale bars: 5 μm. G. Quantification of (F), percentage of GSCs displaying different polarity status are indicated. CEP250+/+ n=5 mice, CEP250-/- n=4 mice. At least 30 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In F, white arrowheads highlight basement membrane; yellow arrowhead highlights cortical enrichment of E-cadherin towards the basement membrane. In G), data are presented as mean ± SEM. n.s., not significant, *P<0.05, **P < 0.01, ***P < 0.005 (the unpaired Student's t-test).


Figure 7A . Representative images of P5 mouse seminiferous tubule ( ST ) 10 µm cryo - sections stained for PLZF ( red ) , ODF2 ( green ) , NIN ( magenta ) and PH3 ( blue ) showing GSCs in prophase . Scale bars : 2 μm . Yellow arrowheads highlight the two centrosomes . B . Quantification of Centrosome distance in GSCs in prophase at P5 . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 50 cells were analyzed in each mouse ( color of dot indicates mouse ) . Dashed line indicates 4 μm as the threshold of well separated centrosomes . Data information : In ( B ) , data are presented as mean ± SEM . * * * * P < 0 . 001 ( the Mann - Whitney test ) . D . Representative images of P5 mouse seminiferous tubule 10 µm cryo - sections stained for PLZF ( red ) , ODF2 ( green ) , NIN ( magenta ) and PH3 ( blue ) , focusing on PLZF + germ stem cells ( GSCs ) in prometaphase . Scale bars : 2 μm . Yellow lines highlight basement membrane . Yellow arrowheads highlight the mother centrosomes and white arrowheads highlight the daughter centrosomes . E . Quantification of Percentage of prometaphase GSCs which preferentially position their mother centrosome close to the basement membrane in CEP250 + / + and CEP250 - / - testis at P5 . CEP250 + / + n = 3 mice , CEP250 - / - n = 3 mice . At least 50 cells were analyzed in each mouse and the average was calculated for each mouse . Data information : In ( E ) , data are presented as mean ± SEM . * * * * P < 0 . 001 ( the unpaired Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A. Representative images of P5 mouse seminiferous tubule (ST) 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue) showing GSCs in prophase. Scale bars: 2 μm. Yellow arrowheads highlight the two centrosomes.B. Quantification of Centrosome distance in GSCs in prophase at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse (color of dot indicates mouse). Dashed line indicates 4 μm as the threshold of well separated centrosomes. Data information: In (B), data are presented as mean ± SEM. ****P < 0.001 (the Mann-Whitney test).D. Representative images of P5 mouse seminiferous tubule 10 µm cryo-sections stained for PLZF (red), ODF2 (green), NIN (magenta) and PH3 (blue), focusing on PLZF+ germ stem cells (GSCs) in prometaphase. Scale bars: 2 μm. Yellow lines highlight basement membrane. Yellow arrowheads highlight the mother centrosomes and white arrowheads highlight the daughter centrosomes.E. Quantification of Percentage of prometaphase GSCs which preferentially position their mother centrosome close to the basement membrane in CEP250+/+ and CEP250-/- testis at P5. CEP250+/+ n=3 mice, CEP250-/- n=3 mice. At least 50 cells were analyzed in each mouse and the average was calculated for each mouse. Data information: In (E), data are presented as mean ± SEM. ****P < 0.001 (the unpaired Student's t-test).


Figure 1Representative time - lapse image series of exponentially growing M . smegmatis P2P1recA - GFPwt ( C ) and P2P1recA - GFPdes ( D ) reporters . Phase - contrast and green fluorescence are merged . Numbers represent hours . Scale bar , 10 µm . Histograms showing the distributions of single - cell fluorescence ( n = 204 ) averaged over the lifetime of the cell ( right panels ) . Black line indicates fitting of the data with a Lognormal function ( C ) or with the sum of three Gaussians ( D ) . Insets : P2P1recA - GFPwt population ' s geometric mean ( Gµ ) calculated by computing the logarithm of the single - cell fluorescence values , the mean of the logarithms and its antilog , P2P1recA - GFPdes subpopulations ' means ( µ ) , and R2 values . The two distributions show that the destabilized GFPdes is more suitable to monitor the dynamics of recA expression and to detect subpopulations , which are masked by using the stable GFPwt variant . E Spearman ' s correlation between single - cell P2P1recA - GFPdes fluorescence range and VMR of fluorescence over the lifetime of the cell ( n = 204 ) . The data shown are from 3 independent experiments . Dashed lines separate different levels of fluorescence dispersion over the lifetimes of single - cells . Colors denote cells belonging to underdispersed ( gray ) , dispersed ( dark green ) , and highly dispersed ( light green ) subpopulations . F P2P1recA - GFPdes fluorescence range over the lifetime of the cell of subpopulations segregated by fluorescence VMR ( 40 < n < 112 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significant difference by Kruskal - Wallis and Dunn ' s multiple comparison test : * * * * p < 0 . 0001 . G Single - cell time traces of P2P1recA - GFPdes fluorescence expressed as a percent of generation time of subpopulations segregated by fluorescence VMR . The average fluorescence ( solid black lines ) and half of the generation time ( dashed lines ) are indicated . Population statistics ( n = 204 ) : 38 % ( 0 < VMR ≤ 1 ) ; 44 % ( 1 < VMR ≤ 10 ) ; 18 % ( VMR > 10 ) . The data shown are from 3 independent experiments . H Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time . Dark green bars indicate moderately pulsing cells ( n = 165 ) . Light green bars indicate highly pulsing cells ( n = 66 ) . Significance by Mann - Whitney test . The data shown are from 3 independent experiments . Peaks mainly occur between 40 % and 70 % of the generation time .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Representative time-lapse image series of exponentially growing M. smegmatis P2P1recA-GFPwt (C) and P2P1recA-GFPdes (D) reporters. Phase-contrast and green fluorescence are merged. Numbers represent hours. Scale bar, 10 µm. Histograms showing the distributions of single-cell fluorescence (n = 204) averaged over the lifetime of the cell (right panels). Black line indicates fitting of the data with a Lognormal function (C) or with the sum of three Gaussians (D). Insets: P2P1recA-GFPwt population's geometric mean (Gµ) calculated by computing the logarithm of the single-cell fluorescence values, the mean of the logarithms and its antilog, P2P1recA-GFPdes subpopulations' means (µ), and R2 values. The two distributions show that the destabilized GFPdes is more suitable to monitor the dynamics of recA expression and to detect subpopulations, which are masked by using the stable GFPwt variant. E Spearman's correlation between single-cell P2P1recA-GFPdes fluorescence range and VMR of fluorescence over the lifetime of the cell (n = 204). The data shown are from 3 independent experiments. Dashed lines separate different levels of fluorescence dispersion over the lifetimes of single-cells. Colors denote cells belonging to underdispersed (gray), dispersed (dark green), and highly dispersed (light green) subpopulations. F P2P1recA-GFPdes fluorescence range over the lifetime of the cell of subpopulations segregated by fluorescence VMR (40 < n < 112). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001. G Single-cell time traces of P2P1recA-GFPdes fluorescence expressed as a percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 204): 38% (0<VMR≤1); 44% (1<VMR≤10); 18% (VMR>10). The data shown are from 3 independent experiments. H Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Dark green bars indicate moderately pulsing cells (n = 165). Light green bars indicate highly pulsing cells (n = 66). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 40% and 70% of the generation time.


Figure 2Color - coded single - cell time traces of the four reporters expressed as percent of the generation time ( B ) . Population coefficients of variation of fluorescence ( % ) are in color - coded boxes . Single - cell VMR of fluorescence ( C ) and fluorescence averaged over the lifetime of the cell ( D ) in exponential phase . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * * p = 0 . 0002 ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments ( n = 105 cells per strain ) . Single - cell fluorescence decreases but variation does not change in strains devoid of positive regulation . Single - cell fluorescence increases and variation decreases in the strain devoid of negative regulation . Color - coded time - traces of fluorescence ( left panel ) averaged for 50 individual cells per strain ( mean ± SD ) treated with MMC ( 0 . 5 μg / ml ) for 2 hours ( gray shading ) . Fluorescence induction during 6 hours from MMC exposure ( right panel ) . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments . Single - cell analysis of independent lineages separated by black vertical lines . Color - coded traces represent single - cell fluorescence of random descendants monitored in fresh 7H9 medium for 24 hours ( n = 20 ) . White and gray vertical bands represents cell divisions . The data shown are from 2 independent experiments . Aligned pulse profiles averaged for each reporter strain ( color - coded as in A ) . Error bars are SD ( 4 < n < 37 ) . The data shown are from 2 independent experiments . Pulses are abrogated in the strain devoid of negative regulation . Analysis of pulse characteristics . Black lines indicate means and SD ( 52 < n < 88 ) . Significance by Kruskal - Wallis nd Dunn ' s multiple comparison test : ns , not significant ; * * * * p < 0 . 0001 . The data shown are from 2 independent experiments . Histogram showing the distribution of the fluorescence peak time of individual cells expressed as a percent of the generation time ( 52 < n < 88 ) . Different strains are color - coded . Statistical analysis by one - way ANOVA and Holm - Sidak ' s multiple comparisons test . Pulses are weaker and fewer in the strain devoid of positive regulation but are not displaced relative to the generation time .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2Color-coded single-cell time traces of the four reporters expressed as percent of the generation time (B). Population coefficients of variation of fluorescence (%) are in color-coded boxes. Single-cell VMR of fluorescence (C) and fluorescence averaged over the lifetime of the cell (D) in exponential phase. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ***p = 0.0002; ****p < 0.0001. The data shown are from 2 independent experiments (n = 105 cells per strain). Single-cell fluorescence decreases but variation does not change in strains devoid of positive regulation. Single-cell fluorescence increases and variation decreases in the strain devoid of negative regulation.Color-coded time-traces of fluorescence (left panel) averaged for 50 individual cells per strain (mean ± SD) treated with MMC (0.5 μg/ml) for 2 hours (gray shading). Fluorescence induction during 6 hours from MMC exposure (right panel). Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.Single-cell analysis of independent lineages separated by black vertical lines. Color-coded traces represent single-cell fluorescence of random descendants monitored in fresh 7H9 medium for 24 hours (n = 20). White and gray vertical bands represents cell divisions. The data shown are from 2 independent experiments.Aligned pulse profiles averaged for each reporter strain (color-coded as in A). Error bars are SD (4 < n < 37). The data shown are from 2 independent experiments. Pulses are abrogated in the strain devoid of negative regulation.Analysis of pulse characteristics. Black lines indicate means and SD (52 < n < 88). Significance by Kruskal-Wallis nd Dunn's multiple comparison test: ns, not significant; ****p < 0.0001. The data shown are from 2 independent experiments.Histogram showing the distribution of the fluorescence peak time of individual cells expressed as a percent of the generation time (52 < n < 88). Different strains are color-coded. Statistical analysis by one-way ANOVA and Holm-Sidak's multiple comparisons test. Pulses are weaker and fewer in the strain devoid of positive regulation but are not displaced relative to the generation time.


Figure 3Spearman ' s correlation between green ( A - D ) or yellow ( E ) fluorescence range and red fluorescence range over the lifetime of the cell ( 105 < n < 153 ) . The data shown are from 2 independent experiments . On the x - axes P2P1recA - GFPdes ( A - D ) or a C - term translational reporter of SSBa fused to mCitrinewt ( E ) . On the y - axes , a C - term translational reporter of RecA fused to mCherrywt ( A and E ) , transcriptional reporters of ruvC ( B ) and radA ( C ) , and mCherrydes expressed from a constitutive control promoter ( D ) . Colors ( A - D ) indicate subpopulations segregated by VMR of P2P1recA - GFPdes : gray ( 0 < VMR ≤ 1 ) , dark green ( 1 < VMR ≤ 10 ) , and light green ( VMR > 10 ) . Colors ( E ) indicate subpopulations segregated by VMR of RecA - mCherrywt : gray ( 0 < VMR ≤ 1 ) , plum ( 1 < VMR ≤ 10 ) , and magenta ( VMR > 10 ) . Pearson ' s correlation between green ( F - I ) or yellow ( J ) fluorescence and red fluorescence , measured 3 hours after MMC exposure ( 203 < n < 314 ) . The data shown are from 2 independent experiments . On the x - axes P2P1recA - GFPdes ( F - I ) or a C - term translational reporter of SSBa fused to mCitrinewt ( J ) . On the y - axes , a C - term translational reporter of RecA fused to mCherrywt ( F and J ) , transcriptional reporters of ruvC ( G ) and radA ( H ) , and mCherrydes expressed from a constitutive control promoter ( I ) . Positive correlations between recA and other DNA - damage markers both under normal growth conditions , where spontaneous DNA - damage events occur at the subpopulation level , and in the presence of an alkylating agent that induces double - strand breaks in the entire population , validate recA as a reliable proxy for DNA damage in single cells .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Spearman's correlation between green (A-D) or yellow (E) fluorescence range and red fluorescence range over the lifetime of the cell (105 < n < 153). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (A-D) or a C-term translational reporter of SSBa fused to mCitrinewt (E). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (A and E), transcriptional reporters of ruvC (B) and radA (C), and mCherrydes expressed from a constitutive control promoter (D). Colors (A-D) indicate subpopulations segregated by VMR of P2P1recA-GFPdes: gray (0<VMR≤1), dark green (1<VMR≤10), and light green (VMR>10). Colors (E) indicate subpopulations segregated by VMR of RecA-mCherrywt: gray (0<VMR≤1), plum (1<VMR≤10), and magenta (VMR>10).Pearson's correlation between green (F-I) or yellow (J) fluorescence and red fluorescence, measured 3 hours after MMC exposure (203 < n < 314). The data shown are from 2 independent experiments. On the x-axes P2P1recA-GFPdes (F-I) or a C-term translational reporter of SSBa fused to mCitrinewt (J). On the y-axes, a C-term translational reporter of RecA fused to mCherrywt (F and J), transcriptional reporters of ruvC (G) and radA (H), and mCherrydes expressed from a constitutive control promoter (I). Positive correlations between recA and other DNA-damage markers both under normal growth conditions, where spontaneous DNA-damage events occur at the subpopulation level, and in the presence of an alkylating agent that induces double-strand breaks in the entire population, validate recA as a reliable proxy for DNA damage in single cells.


Figure 4Representative time - lapse image series of the merodiploid recA reporter strain ( P2P1recA - GFPdes _ RecA - mCherrywt ) cultured in fresh ( A ) or spent ( B ) flow medium ( see Materials and Methods ) . Fresh 7H9 medium is unlabeled ; spent medium ( S ) , ciprofloxacin 1 µg / ml in fresh ( CIP ) or spent ( S - CIP ) medium , and SYTOX 0 . 5 µM ( SX ) are labeled . Phase - contrast ( gray ) , GFPdes ( green ) , and mCherrywt ( magenta ) fluorescence are merged . Fluorescence images of each channel are scaled to the brightest frame . Light - green background in the first 4 frames ( B ) is due to the autofluorescence of spent medium . Cells were imaged at 30 - min intervals and numbers represent hours . Scale bar , 10 µm . Representative time traces of individual lineages of cells fed with fresh medium ( C ) or spent medium ( D ) . Different styles are used for lineages with different fates : death and lysis ( L ) ; SYTOX - positive ( SX + ) ; and survival ( S ) . Line breaks represent division events in the same lineage , where a single random daughter cell is tracked for clarity . Vertical bars represent fresh ( white ) or spent ( dark shading ) 7H9 medium , CIP ( yellow ) , and SX ( blue ) . Comparison of single - cell growth rates prior to CIP exposure for " D " cells that die and " S " cells that survive and regrow after CIP washout ( E ) , and lag time of regrowth ( F ) . Cells were cultured in fresh ( EXP ) or spent ( STAT ) flow medium . Gray and black lines indicate mean ± SD . Significance by Kruskal - Wallis and Dunn ' s multiple comparison test ( E ) and unpaired Mann - Whitney test ( F ) : ns , not significant ; * * * * p < 0 . 0001 ; 6 < n < 84 . The data shown are from 2 independent experiments . Single - cell fate is independent of the growth rate before exposure to the drug . However , cells cultured in spent medium ( STAT ) grow at significantly slower rates , survive at higher frequencies , and recover faster after CIP washout compared to cells cultured in fresh medium ( EXP ) . Representative GFPdes ( G ) and mCherrywt ( H ) fluorescence time - traces for single - cell lineages of cells cultured in fresh medium ( EXP ) and cells cultured in spent medium ( STAT ) , and having different fates . Vertical bars are color - coded as in C and D . Single - cell fate as a function of VMR of P2P1recA - GFPdes ( I ) and RecA - mCherrywt ( J ) fluorescence prior to CIP exposure . Significance by Chi - square test of independence for cells that die ( n = 84 ) and cells that survive ( n = 54 ) . The data shown are from 2 independent experiments . Cells that do not experience DNA damage prior to drug exposure ( RecA - non - pulsing cells ) survive more than cells that do experience DNA damage ( RecA - pulsing cells ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4Representative time-lapse image series of the merodiploid recA reporter strain (P2P1recA-GFPdes_RecA-mCherrywt) cultured in fresh (A) or spent (B) flow medium (see Materials and Methods). Fresh 7H9 medium is unlabeled; spent medium (S), ciprofloxacin 1 µg/ml in fresh (CIP) or spent (S-CIP) medium, and SYTOX 0.5 µM (SX) are labeled. Phase-contrast (gray), GFPdes (green), and mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Light-green background in the first 4 frames (B) is due to the autofluorescence of spent medium. Cells were imaged at 30-min intervals and numbers represent hours. Scale bar, 10 µm.Representative time traces of individual lineages of cells fed with fresh medium (C) or spent medium (D). Different styles are used for lineages with different fates: death and lysis (L); SYTOX-positive (SX+); and survival (S). Line breaks represent division events in the same lineage, where a single random daughter cell is tracked for clarity. Vertical bars represent fresh (white) or spent (dark shading) 7H9 medium, CIP (yellow), and SX (blue).Comparison of single-cell growth rates prior to CIP exposure for "D" cells that die and "S" cells that survive and regrow after CIP washout (E), and lag time of regrowth (F). Cells were cultured in fresh (EXP) or spent (STAT) flow medium. Gray and black lines indicate mean ± SD. Significance by Kruskal-Wallis and Dunn's multiple comparison test (E) and unpaired Mann-Whitney test (F): ns, not significant; ****p < 0.0001; 6 < n < 84. The data shown are from 2 independent experiments. Single-cell fate is independent of the growth rate before exposure to the drug. However, cells cultured in spent medium (STAT) grow at significantly slower rates, survive at higher frequencies, and recover faster after CIP washout compared to cells cultured in fresh medium (EXP).Representative GFPdes (G) and mCherrywt (H) fluorescence time-traces for single-cell lineages of cells cultured in fresh medium (EXP) and cells cultured in spent medium (STAT), and having different fates. Vertical bars are color-coded as in C and D.Single-cell fate as a function of VMR of P2P1recA-GFPdes (I) and RecA-mCherrywt (J) fluorescence prior to CIP exposure. Significance by Chi-square test of independence for cells that die (n = 84) and cells that survive (n = 54). The data shown are from 2 independent experiments. Cells that do not experience DNA damage prior to drug exposure (RecA-non-pulsing cells) survive more than cells that do experience DNA damage (RecA-pulsing cells).


Figure 5Spearman ' s correlation between single - cell RecA - mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell ( n = 360 ) . The data shown are from 3 independent experiments . Dashed lines separate different levels of dispersion , and colors denote subpopulations that are underdispersed ( gray ) , dispersed ( plum ) , or highly dispersed ( magenta ) . RecA - mCherrywt fluorescence of subpopulations segregated by fluorescence VMR ( 58 < n < 159 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significant difference by Kruskal - Wallis and Dunn ' s multiple comparison test : * * * * p < 0 . 0001 . Single - cell time traces of RecA - mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR . The average fluorescence ( solid black lines ) and half of the generation time ( dashed lines ) are indicated . Population statistics ( n = 360 ) : 40 % ( 0 < VMR ≤ 1 ) ; 44 % ( 1 < VMR ≤ 10 ) ; 16 % ( VMR > 10 ) . The data shown are from 3 independent experiments . Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time . Bars shaded in plum indicate moderately pulsing cells ( n = 185 ) and magenta bars indicate highly pulsing cells ( n = 57 ) . Significance by Mann - Whitney test . The data shown are from 3 independent experiments . Peaks mainly occur between 80 % and 100 % of the generation time . Single - cell size ( E ) and growth rate ( F ) averaged over the lifetime of the cell ( 58 < n < 159 ) . The data shown are from 3 independent experiments . Black lines indicate means ± SD . Asterisks denote significance by Kruskal - Wallis and Dunn ' s multiple comparison test : ns , not significant ; * * p = 0 . 002 ; * * * * p < 0 . 0001 . Cells experiencing more DNA damage ( VMR > 10 ) are slightly larger and grow at slower rates .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Spearman's correlation between single-cell RecA-mCherrywt fluorescence range and fluorescence VMR over the lifetime of the cell (n = 360). The data shown are from 3 independent experiments. Dashed lines separate different levels of dispersion, and colors denote subpopulations that are underdispersed (gray), dispersed (plum), or highly dispersed (magenta). RecA-mCherrywt fluorescence of subpopulations segregated by fluorescence VMR (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significant difference by Kruskal-Wallis and Dunn's multiple comparison test: ****p < 0.0001.Single-cell time traces of RecA-mCherrywt fluorescence expressed as percent of generation time of subpopulations segregated by fluorescence VMR. The average fluorescence (solid black lines) and half of the generation time (dashed lines) are indicated. Population statistics (n = 360): 40% (0<VMR≤1); 44% (1<VMR≤10); 16% (VMR>10). The data shown are from 3 independent experiments.Histogram showing the distribution of the fluorescence peak time expressed as a percent of generation time. Bars shaded in plum indicate moderately pulsing cells (n = 185) and magenta bars indicate highly pulsing cells (n = 57). Significance by Mann-Whitney test. The data shown are from 3 independent experiments. Peaks mainly occur between 80% and 100% of the generation time.Single-cell size (E) and growth rate (F) averaged over the lifetime of the cell (58 < n < 159). The data shown are from 3 independent experiments. Black lines indicate means ± SD. Asterisks denote significance by Kruskal-Wallis and Dunn's multiple comparison test: ns, not significant; **p = 0.002; ****p < 0.0001. Cells experiencing more DNA damage (VMR>10) are slightly larger and grow at slower rates.


Figure 6Representative time - lapse image series of the dual reporter of RecA and rRNA expression in exponential phase . Pre - CIP growing ( PG - S ) and arrested ( PA - S ) survivors and resistant ( R ) cells are indicated by arrows . CIP exposure ( 1 µg / ml , 4X - MIC ) and SX ( 0 . 5 µM ) death assay ( blue line ) are indicated . Phase - contrast ( gray ) , Prrn - GFPdes ( green ) , and RecA - mCherrywt ( magenta ) fluorescence are merged . Fluorescence images of each channel are scaled to the brightest frame . Cells were imaged at 3 - hour intervals and numbers indicate days . Scale bar , 5 µm . Representative time traces of cell lineages with different fates . Vertical bars represent fresh 7H9 medium ( white ) , CIP ( yellow ) , and SX ( blue ) . Comparison of growth rate ( C ) , cell size averaged over the lifetime of the cell ( D ) , microcolony size ( E ) before CIP exposure Gray and black lines indicate mean ± SD . The data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney test : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA - mCherrywt both before and after drug exposure . fluorescence range over the lifetime of the cell before CIP exposure ( F ) , and mean fluorescence during CIP exposure ( G ) of cells that died ( n = 174 ) and cells that survived ( n = 164 ) . Gray and black lines indicate mean ± SD . The data shown are from 2 independent experiments . Significance by unpaired Mann - Whitney test : ns , not significant ; * * p = 0 . 001 ; * * * * p < 0 . 0001 . Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA - mCherrywt both before and after drug exposure . Single - cell fate as a function of VMR of RecA - mCherrywt fluorescence prior to CIP exposure . Significance by Chi - square test of independence : cells that died ( n = 174 ) , cells that survived ( n = 155 ) , and resistant cells ( n = 9 ) . The data shown are from 2 independent experiments . More survivors arise from the subpopulation that does not experience DNA damage before drug exposure .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Representative time-lapse image series of the dual reporter of RecA and rRNA expression in exponential phase. Pre-CIP growing (PG-S) and arrested (PA-S) survivors and resistant (R) cells are indicated by arrows. CIP exposure (1 µg/ml, 4X-MIC) and SX (0.5 µM) death assay (blue line) are indicated. Phase-contrast (gray), Prrn-GFPdes (green), and RecA-mCherrywt (magenta) fluorescence are merged. Fluorescence images of each channel are scaled to the brightest frame. Cells were imaged at 3-hour intervals and numbers indicate days. Scale bar, 5 µm.Representative time traces of cell lineages with different fates. Vertical bars represent fresh 7H9 medium (white), CIP (yellow), and SX (blue).Comparison of growth rate (C), cell size averaged over the lifetime of the cell (D), microcolony size (E) before CIP exposure Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.fluorescence range over the lifetime of the cell before CIP exposure (F), and mean fluorescence during CIP exposure (G) of cells that died (n = 174) and cells that survived (n = 164). Gray and black lines indicate mean ± SD. The data shown are from 2 independent experiments. Significance by unpaired Mann-Whitney test: ns, not significant; **p = 0.001; ****p < 0.0001. Most survivors had a moderate growth defect before exposure to ciprofloxacin and lower expression of RecA-mCherrywt both before and after drug exposure.Single-cell fate as a function of VMR of RecA-mCherrywt fluorescence prior to CIP exposure. Significance by Chi-square test of independence: cells that died (n = 174), cells that survived (n = 155), and resistant cells (n = 9). The data shown are from 2 independent experiments. More survivors arise from the subpopulation that does not experience DNA damage before drug exposure.


Figure 1B Volcano plot showing proteins enrichment ( fold change in log10 scale ) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT ( untagged ) ARHGAP45 proteins prior to ( unstimulated ) and at 120 s after ( stimulated ) TCR stimulation . ARHGAP45 interacting proteins with a greater than 2 - fold enrichment and a p - value < 0 . 01 were selected as specific ARHGAP45 interactors ( Dataset EV1 ) and some of them are specified in red . Red lines represent the thresholds set on P - value and enrichment to identify specific ARHGAP45 interactors .

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Figure 1B Volcano plot showing proteins enrichment (fold change in log10 scale) after affinity purification in Jurkat T cells expressing ARHGAP45OST molecules compared to affinity purification in control Jurkat T cells expressing similar levels of WT (untagged) ARHGAP45 proteins prior to (unstimulated) and at 120 s after (stimulated) TCR stimulation. ARHGAP45 interacting proteins with a greater than 2-fold enrichment and a p-value < 0.01 were selected as specific ARHGAP45 interactors (Dataset EV1) and some of them are specified in red. Red lines represent the thresholds set on P-value and enrichment to identify specific ARHGAP45 interactors.


Figure 3B The ratio of CTV / CMTPX - labeled cells present in each organ ( Ro ) was determined and normalized by dividing it with Ri , the ratio of CTV / CMTPX - labeled cells present in the cell mixtures prior to injection . Ro / Ri ratio corresponding to each of the specified mice are shown . Data shown for Arhgap45 - / - mice correspond to 4 independent experiments ( E1 to E4 ) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments ( E1 and E2 ) involving a total of 5 individual mice . The Arhgap45 - / - mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments . Mean and SD are shown . ns , non - significant , * * * * P ≤ 0 . 0001 ; multiple unpaired T tests . C Expression of CCR7 , and LFA - 1 on naïve CD4 + and CD8 + T cells found in LNs of WT and Arhgap45 - / - mice , analyzed by flow cytometry . Gray shaded curves , isotype control staining . D Quantification of data shown in ( C ) . The MFI corresponding to each analyzed mouse is shown together with mean and SD . Each dot represents an individual mouse and CD4 + T cells from at least 4 mice have been analyzed for each condition . Mice analyzed on the same day are represented by similar dots ( see key in bottom right corner ) . ns , non - significant , * * P ≤ 0 . 002 ; multiple unpaired T tests .

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Figure 3B The ratio of CTV/CMTPX-labeled cells present in each organ (Ro) was determined and normalized by dividing it with Ri, the ratio of CTV/CMTPX-labeled cells present in the cell mixtures prior to injection. Ro/Ri ratio corresponding to each of the specified mice are shown. Data shown for Arhgap45-/-mice correspond to 4 independent experiments (E1 to E4) involving a total of 15 individual mice whereas data shown for WT mice correspond to 2 independent experiments (E1 and E2) involving a total of 5 individual mice. The Arhgap45-/- mice analyzed on the same day as the control mice corresponded to the E1 and E2 experiments. Mean and SD are shown. ns, non-significant, **** P ≤ 0.0001; multiple unpaired T tests.C Expression of CCR7, and LFA-1 on naïve CD4+ and CD8+ T cells found in LNs of WT and Arhgap45-/- mice, analyzed by flow cytometry. Gray shaded curves, isotype control staining. D Quantification of data shown in (C). The MFI corresponding to each analyzed mouse is shown together with mean and SD. Each dot represents an individual mouse and CD4+ T cells from at least 4 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). ns, non-significant, ** P ≤ 0.002; multiple unpaired T tests.


Figure 4The cells specified below were loaded into the upper chamber of a Transwell migration device without ( no ) or with the specified chemokines in the lower chamber . The percentage of cells that migrated to the bottom chamber after 2 h ( T cells ) or 4 h ( B cells ) is shown . A Chemotaxis of naïve CD4 + T cells from WT and Arghap45 − / − mice in response to CCL19 , CCL21 and CXCL12 . B Chemotaxis of naïve CD8 + T cells from WT and Arghap45 − / − mice in response to CCL19 , CCL21 and CXCL12 . C Chemotaxis of naïve B cells from WT and Arghap45 − / − mice in response to CCL19 D Adding CCL19 in both the upper and lower chambers ( CCL19 UL ) prevented migration of WT and Arghap45 − / − naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM - 1 ( CCL19 + ICAM - 1 ) did not change the pattern of chemotaxis observed using ' bare ' membrane inserts ( CCL19 ) . E Chemotaxis of naive CD4 + T cells and B cells from WT , Arghap45 − / − , and Arhgap45 ∆ T / ∆ T mice in response to CCL19 . F Chemotaxis of activated CD4 + T cells from WT and Arghap45 − / − mice in response to CCL19 . Data information : In ( A ) to ( F ) each dot represents an individual mouse and as shown CD4 + T cells from 3 to 9 mice have been analyzed for each condition . Mice analyzed on the same day are represented by similar dots ( see key in bottom right corner ) . Mean and SD are shown . * P ≤ 0 . 033 , * * P ≤ 0 . 002 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 ; A , B , C , D , F multiple unpaired T test ; E two - way ANOVA test .

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Figure 4The cells specified below were loaded into the upper chamber of a Transwell migration device without (no) or with the specified chemokines in the lower chamber. The percentage of cells that migrated to the bottom chamber after 2 h (T cells) or 4 h (B cells) is shown. A Chemotaxis of naïve CD4+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. B Chemotaxis of naïve CD8+ T cells from WT and Arghap45−/− mice in response to CCL19, CCL21 and CXCL12. C Chemotaxis of naïve B cells from WT and Arghap45−/− mice in response to CCL19 D Adding CCL19 in both the upper and lower chambers (CCL19 UL) prevented migration of WT and Arghap45−/− naïve T cells whereas the use of Transwell membrane inserts precoated with ICAM-1 (CCL19 + ICAM-1) did not change the pattern of chemotaxis observed using 'bare' membrane inserts (CCL19). E Chemotaxis of naive CD4+ T cells and B cells from WT, Arghap45−/−, and Arhgap45∆T/∆T mice in response to CCL19. F Chemotaxis of activated CD4+ T cells from WT and Arghap45−/− mice in response to CCL19. Data information: In (A) to (F) each dot represents an individual mouse and as shown CD4+ T cells from 3 to 9 mice have been analyzed for each condition. Mice analyzed on the same day are represented by similar dots (see key in bottom right corner). Mean and SD are shown. *P ≤ 0.033, ** P ≤ 0.002, *** P ≤ 0.001, **** P ≤ 0.0001; A, B, C, D, F multiple unpaired T test; E two-way ANOVA test.


Figure 5Analysis of migration patterns of WT and Arghap45 − / − activated T cells on 2D surface coated with ICAM - 1 . Each track represents the migratory path of individual WT and Arghap45 − / − activated T cells recorded over 466 s in a single field of view , Trajectories were plotted to a common starting point , and > 200 T cells were recorded per plot using time - lapse microscopy at 10 x magnification . Analysis of migration patterns of WT and Arghap45 − / − naive T cells on 2D surface coated with ICAM - 1 and CCL21 . Same conditions as in ( A ) .

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Figure 5Analysis of migration patterns of WT and Arghap45−/− activated T cells on 2D surface coated with ICAM-1. Each track represents the migratory path of individual WT and Arghap45−/− activated T cells recorded over 466 s in a single field of view, Trajectories were plotted to a common starting point, and > 200 T cells were recorded per plot using time-lapse microscopy at 10 x magnification. Analysis of migration patterns of WT and Arghap45−/− naive T cells on 2D surface coated with ICAM-1 and CCL21. Same conditions as in (A).


Figure 6A , B Representative kymographs of WT ( A ) and Arghap45 − / − ( B ) naive CD4 + T cells on 2D surface coated with ICAM - 1 and CCL21 .

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Figure 6A, B Representative kymographs of WT (A) and Arghap45−/− (B) naive CD4+ T cells on 2D surface coated with ICAM-1 and CCL21.


Figure 7A Irradiated H - 2 Kb - positive spleen cells isolated from T cell - deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8 + T cells purified from WT OT - I ( WT ) or Arhgap45 − / − OT - I ( Arhgap45 − / − ) mice . Cell proliferation was measured by luminescence after 48 h . Data are representative of 2 independent experiments with 3 mice per genotype ( mean and SEM of triplicate are shown ) .

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Figure 7A Irradiated H-2 Kb- positive spleen cells isolated from T cell-deficient mice were pulsed for 2 hours with the N4 agonist OVA peptide and cultured with CD8+ T cells purified from WT OT-I (WT) or Arhgap45−/− OT-I (Arhgap45−/−) mice. Cell proliferation was measured by luminescence after 48 h. Data are representative of 2 independent experiments with 3 mice per genotype (mean and SEM of triplicate are shown).


Figure 1 ( B ) Representative confocal images show the distribution of the P4M2x reporter in HEK293 - AT1 cells before and after the addition of GSK - A1 ( 30 nM ) . Note the signal disappearing from the PM and strongly highlighting the endosomes and Golgi . Scale bars are 20 µ ( C ) Representative result from a BRET experiment detecting PI4P in various endosomes ( Rab7 , Rab5 and Rab11 ) following addition of GSK - A1 . The graph shows normalized BRET values where the averages of triplicate measurements of A1 - treated cells were divided by the averages of triplicates obtained in DMSO - treated controls . Note that these signals originate from thousands of cells . ( D ) Similar BRET experiment showing the Rab7 - pool of PI4P both in control HEK293 - AT1 cells and in two clones of cells with PI4K2A deletion ( # 20 and # 26 ) . Means ± S . E . M . are shown from three experiments performed in triplicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(B) Representative confocal images show the distribution of the P4M2x reporter in HEK293-AT1 cells before and after the addition of GSK-A1 (30 nM). Note the signal disappearing from the PM and strongly highlighting the endosomes and Golgi. Scale bars are 20 µ(C) Representative result from a BRET experiment detecting PI4P in various endosomes (Rab7, Rab5 and Rab11) following addition of GSK-A1. The graph shows normalized BRET values where the averages of triplicate measurements of A1-treated cells were divided by the averages of triplicates obtained in DMSO-treated controls. Note that these signals originate from thousands of cells.(D) Similar BRET experiment showing the Rab7-pool of PI4P both in control HEK293-AT1 cells and in two clones of cells with PI4K2A deletion (#20 and #26). Means ± S.E.M. are shown from three experiments performed in triplicates.


Figure 2 ( A ) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K / O cells after expression of HA - tagged PI4K2A or its kinase - dead mutant . Means ± S . E . M . are shown from three experiments performed in triplicates . ( B ) Western blot analysis shows that the level of PI4K2A expression was closely matched with that of wild - type cells . ( C ) Distribution of GFP - Rab7 in parental ( left panels ) and two clones of PI4K2A K / O cells . Representative pictures show tubulation in the K / O cells reminiscent of those seen in wild - type cells expressing the GTP - locked Rab7Q67L mutant . Scale bars are 10 µm .

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Figure 2(A) BRET measurement of PI4P in Rab7 compartment in parental and PI4K2A K/O cells after expression of HA-tagged PI4K2A or its kinase-dead mutant. Means ± S.E.M. are shown from three experiments performed in triplicates.(B) Western blot analysis shows that the level of PI4K2A expression was closely matched with that of wild-type cells.(C) Distribution of GFP-Rab7 in parental (left panels) and two clones of PI4K2A K/O cells. Representative pictures show tubulation in the K/O cells reminiscent of those seen in wild-type cells expressing the GTP-locked Rab7Q67L mutant. Scale bars are 10 µm.


Figure 3 ( B ) Representative confocal pictures showing these changes in live cells . Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin ( 100 nM ) for 10 min . Note that the Rab7 recruiter itself is dissociating from the membranes after PI ( 4 , 5 ) P2 generation . Left panels show some cells enlarged . Scale bars : 20 µm . ( C ) Quantification of the changes in BRET experiments . Here the BRET sensor contained the PLCδ1PH - fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co - transfected with the Rab7 - targeted FRB ( tagged with iRFP ) and the FKBP12 - fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence ( CFP * ) . Recruitment of the PIP5Kγ but not its kinase - dead version caused an increase in the BRET signal indicating the increased PI ( 4 , 5 ) P2 in this compartment ( means ± S . E . M . , from three separate experiments performed in triplicates ) . ( D ) PI ( 4 , 5 ) P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP - locked form of Rab7 ( Q67L ) and was negligible with the GDP - locked form of Rab7 ( N125I ) in the BRET construct . Also note that the Rab7 wild - type - based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs ( means ± S . E . M . , from three separate experiments performed in triplicates ) . ( E ) The response of PI4K2A K / O cells was significantly reduced Means ± S . E . M . are shown from three experiments performed in triplicates . ( F ) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments ( means ± S . E . M . , n = 3 ) . One - way ANOVA with Dunnett ' s multiple comparisons was used for statistical analysis ( * P = 0 . 0478 and 0 . 0287 for # 20 and # 26 clones , respectively ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Representative confocal pictures showing these changes in live cells. Cells were transfected with the indicated constructs and imaged before and after addition of rapamycin (100 nM) for 10 min. Note that the Rab7 recruiter itself is dissociating from the membranes after PI(4,5)P2 generation. Left panels show some cells enlarged. Scale bars: 20 µm.(C) Quantification of the changes in BRET experiments. Here the BRET sensor contained the PLCδ1PH-fused to Sluc and the Venus targeted with Rab7 expressed from a single vector and it was co-transfected with the Rab7-targeted FRB (tagged with iRFP) and the FKBP12-fused PIP5Kγ in which the CFP was mutated to eliminate its fluorescence (CFP*). Recruitment of the PIP5Kγ but not its kinase-dead version caused an increase in the BRET signal indicating the increased PI(4,5)P2 in this compartment (means ± S.E.M., from three separate experiments performed in triplicates).(D) PI(4,5)P2 increases were larger when the Venus part of the BRET sensor was targeted with the GTP-locked form of Rab7 (Q67L) and was negligible with the GDP-locked form of Rab7 (N125I) in the BRET construct. Also note that the Rab7 wild-type-based BRET signal slowly returned toward the green trace consistent with Rab7 falling off from the membrane both in the BRET and the recruiting constructs (means ± S.E.M., from three separate experiments performed in triplicates).(E) The response of PI4K2A K/O cells was significantly reduced Means ± S.E.M. are shown from three experiments performed in triplicates.(F) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P = 0.0478 and 0.0287 for #20 and #26 clones, respectively).


Figure 4 ( A ) Representative confocal images of HEK293 - AT1 cells , which show that treatment with OSW1 ( 20 nM ) , a drug that inhibits the cholesterol - PI4P transport protein , OSBP , causes accumulation of PI4P in the Golgi and in the endosomes ( upper panels ) . Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K / O cells . Scale bars : 20 µm ( B ) Quantification of these changes by BRET analysis . The PI4P - Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 ( 20 nM ) for the indicated times . The BRET ratios were expressed relative to those of DMSO treated cells . Data information : Means ± S . E . M . are shown from three experiments performed in triplicates . ( C ) Detection of endosomal PI ( 4 , 5 ) P2 in cells pre - treated with OSW1 for 1 h in serum - free medium using confocal microscopy and the PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter . Angiotensin II ( AngII , 100 nM ) ( a Gq and PLC - activating agonist ) was used to liberate the PLCδ1PH - GFP PI ( 4 , 5 ) P2 reporter from the PM , which was necessary to detect the endosomal PI ( 4 , 5 ) P2 . The signal is transient as the AngII receptors show desensitization . Note the reduced signal in the two PI4K2A K / O clones . Scale bars : 20 µm . ( D ) Quantification of the same changes by BRET analysis in wild - type cells using the PI ( 4 , 5 ) P2 sensor with Rab7 - Q67L - targeting . Note the transient signal increase after AngII stimulation only in cells treated with OSW1 . ( E ) This increase is barely detectable in the PI4K2A K / O cells . D Data information : Means ± S . E . M . are shown from three experiments performed in triplicates . The values in ( D ) and ( E ) were measured in the same 96 - well plate at the same time .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A) Representative confocal images of HEK293-AT1 cells, which show that treatment with OSW1 (20 nM), a drug that inhibits the cholesterol-PI4P transport protein, OSBP, causes accumulation of PI4P in the Golgi and in the endosomes (upper panels). Note that the accumulation is much reduced in the endosome but less so in the Golgi in the PI4K2A K/O cells. Scale bars: 20 µm(B) Quantification of these changes by BRET analysis. The PI4P-Rab7 BRET sensor construct was transfected into the respective cell lines and the cells treated with OSW1 (20 nM) for the indicated times. The BRET ratios were expressed relative to those of DMSO treated cells. Data information: Means ± S.E.M. are shown from three experiments performed in triplicates.(C) Detection of endosomal PI(4,5)P2 in cells pre-treated with OSW1 for 1 h in serum-free medium using confocal microscopy and the PLCδ1PH-GFP PI(4,5)P2 reporter. Angiotensin II (AngII, 100 nM) (a Gq and PLC-activating agonist) was used to liberate the PLCδ1PH-GFP PI(4,5)P2 reporter from the PM, which was necessary to detect the endosomal PI(4,5)P2. The signal is transient as the AngII receptors show desensitization. Note the reduced signal in the two PI4K2A K/O clones. Scale bars: 20 µm.(D) Quantification of the same changes by BRET analysis in wild-type cells using the PI(4,5)P2 sensor with Rab7-Q67L-targeting. Note the transient signal increase after AngII stimulation only in cells treated with OSW1. (E) This increase is barely detectable in the PI4K2A K/O cells. D Data information: Means ± S.E.M. are shown from three experiments performed in triplicates. The values in (D) and (E) were measured in the same 96-well plate at the same time.


Figure 5 ( A ) BRET experiment in parental HEK293 - AT1 cells in OSW1 - pretreated cells after RNAi mediated knock - down of PIP5Kβ or PIP5Kγ . ( means ± S . E . M . from three experiments , each performed in triplicates ) . Note the reduced response in the PIP5Kγ knock - down cells and the slight reduction in the case of PIP5Kβ knock - down . ( B ) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A ( means ± S . E . M . , n = 3 ) . One - way ANOVA with Dunnett ' s multiple comparisons was used for statistical analysis ( * P = 0 . 0241 ; * * P = 0 . 0088 ) . ( ( C ) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time . ( D ) Increased appearance of GFP - LC3 positive vesicles in PI4K2A K / O cells and reduced acidification of the LC3 - positive vesicles using the GFP - mCherry - LC3 reporter . Cells were transfected with the indicated constructs for one day and observed by confocal microscopy . Scale bars : 20 µm . ( E ) Quantification of the LC3 positive vesicles . ( n = 67 , 34 , 26 , 13 , 30 , 27 for the various groups , respectively from left to right ) . * * * designates P < 0 . 0001 using unpaired t - test . The same data set was also analyzed with the non - parametric Mann - Whitney test showing a P = 0 . 0004 and 0 . 0012 for # 20 and # 26 clones relative to control , respectively . ( F ) Co - localization analysis using the Pearson coefficient ( n = 31 , 21 and 14 cells analyzed for the different groups from left to right ) . * * * designates P < 0 . 0001 using unpaired t - test . The same data set was also analyzed with the non - parametric Mann - Whitney test showing a P value < 0 . 0001 for both groups .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) BRET experiment in parental HEK293-AT1 cells in OSW1-pretreated cells after RNAi mediated knock-down of PIP5Kβ or PIP5Kγ. (means ± S.E.M. from three experiments, each performed in triplicates). Note the reduced response in the PIP5Kγ knock-down cells and the slight reduction in the case of PIP5Kβ knock-down. (B) Areas below the curves calculated from the time of rapamycin addition for each of the three separate experiments shown in panel A (means ± S.E.M., n=3). One-way ANOVA with Dunnett's multiple comparisons was used for statistical analysis (* P=0.0241; **P=0.0088). ( (C) These reductions are proportional to the reduction in PIP5Kγ levels as assessed by Western blot analysis of cell lysates obtained from cells treated with siRNAs at the same time.(D) Increased appearance of GFP-LC3 positive vesicles in PI4K2A K/O cells and reduced acidification of the LC3-positive vesicles using the GFP-mCherry-LC3 reporter. Cells were transfected with the indicated constructs for one day and observed by confocal microscopy. Scale bars: 20 µm. (E) Quantification of the LC3 positive vesicles. (n = 67, 34, 26, 13, 30, 27 for the various groups, respectively from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P = 0.0004 and 0.0012 for #20 and #26 clones relative to control, respectively. (F) Co-localization analysis using the Pearson coefficient (n = 31, 21 and 14 cells analyzed for the different groups from left to right). *** designates P < 0.0001 using unpaired t-test. The same data set was also analyzed with the non-parametric Mann-Whitney test showing a P value < 0.0001 for both groups.


Figure 6 ( A , B ) Distribution of mCherry - GFP - LC3 in HEK293 - AT1 cells ( without starvation ) after treatment with control siRNA ( A ) or siRNA for PIP5Kγ ( PIP5K1C ) . Scale bars : 20 µm . Note the massive tubulation of the LC3 compartment in some of the knocked - down cells ( B , lower images , scale bar : 10 µm ) . ( C ) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi . For statistical analysis the unpaired t - test was used ( n = 50 and 45 cells for control a PIP5Kγ knock down groups , respectively ; * * * P < 0 . 0001 ) . ( D ) Representative confocal images of live HEK293 - AT1 cells expressing GFP - Rab7 treated with control or PIP5Kγ RNAi . Note the tubulation in the PIP5Kγ RNAi - treated cells . Scale bars : 10 µm .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(A, B) Distribution of mCherry-GFP-LC3 in HEK293-AT1 cells (without starvation) after treatment with control siRNA (A) or siRNA for PIP5Kγ (PIP5K1C). Scale bars: 20 µm. Note the massive tubulation of the LC3 compartment in some of the knocked-down cells (B, lower images, scale bar: 10 µm). (C) Comparison of Pearson coefficients from cells treated with control or PIP5Kγ RNAi. For statistical analysis the unpaired t-test was used (n = 50 and 45 cells for control a PIP5Kγ knock down groups, respectively; *** P< 0.0001). (D) Representative confocal images of live HEK293-AT1 cells expressing GFP-Rab7 treated with control or PIP5Kγ RNAi. Note the tubulation in the PIP5Kγ RNAi-treated cells. Scale bars: 10 µm.


Figure 7 ( A ) HEK293 - AT1 cells were transfected with PLEKHM1 - GFP - Rab7 along with CFP - FKBP - PIP5Kγ and iRFP - FRB - Rab7 constructs for one day and examined live by confocal microscopy . Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes ( lower row images ) . ( B ) This response is reduced in PI4K2A K / O cells . ( Data information : Scale bars : 20 µm . ( C ) Quantification of these changes by BRET analysis where the full - length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct . For PIP5Kγ recruitment a mutant dark CFP ( W66A ) - FKBP - PIP5Kγ was used with an iRFP - FRB - Rab7 . BRET values were normalized to those of DMSO treated cells . Comparisons were made for parental ( blue ) and PI4K2A K / O ( green and orange ) cells . ( D ) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct . D Data information Means ± S . E . M . are shown from three experiments performed in triplicates .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7(A) HEK293-AT1 cells were transfected with PLEKHM1-GFP-Rab7 along with CFP-FKBP-PIP5Kγ and iRFP-FRB-Rab7 constructs for one day and examined live by confocal microscopy. Representative cells show reduced PLEKHM1 localization when PIP5Kγ was recruited to the Rab7 endosomes (lower row images). (B) This response is reduced in PI4K2A K/O cells. ( Data information: Scale bars: 20 µm.(C) Quantification of these changes by BRET analysis where the full-length PLEKHM1 was fused to Sluc and Venus was fused to Rab7 in the BRET construct. For PIP5Kγ recruitment a mutant dark CFP(W66A)-FKBP-PIP5Kγ was used with an iRFP-FRB-Rab7. BRET values were normalized to those of DMSO treated cells. Comparisons were made for parental (blue) and PI4K2A K/O (green and orange) cells. (D) Similar BRET analysis where the PLEKHM1 was replaced by either RILP or Vps35 in the BRET construct. D Data information Means ± S.E.M. are shown from three experiments performed in triplicates.


Figure 1 ( A ) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin - actin in solution . ( B ) Time - lapse TIRF microscopy of single filament growth from Alexa488phalloidin - stabilized seeds ( green ) in the presence of 5μM profilin - actin . ( C ) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF - and lipid - coated microspheres with profilin - actin , CP and Arp2 / 3 in solution . ( D ) Widefield epifluorescence images of indicated network components of bead - attached dendritic networks after kinetic arrest at indicated times . Biochemical conditions are matching ( B ) with the exception of the additional presence of 200nM CP and 50nM Arp2 / 3 ( E ) Scheme of dendritic network growth from square WAVE1 - micropatterns on a 2 - dimensional coverslip with profilin - actin , CP and Arp2 / 3 in solution . ( F ) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth . Conditions are as in ( D )

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1(A) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin-actin in solution. (B) Time-lapse TIRF microscopy of single filament growth from Alexa488phalloidin-stabilized seeds (green) in the presence of 5μM profilin-actin.(C) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF- and lipid-coated microspheres with profilin-actin, CP and Arp2/3 in solution. (D) Widefield epifluorescence images of indicated network components of bead-attached dendritic networks after kinetic arrest at indicated times.Biochemical conditions are matching (B) with the exception of the additional presence of 200nM CP and 50nM Arp2/3 (E) Scheme of dendritic network growth from square WAVE1-micropatterns on a 2-dimensional coverslip with profilin-actin, CP and Arp2/3 in solution. (F) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth. Conditions are as in (D)


Figure 2 ( B ) TIRF microscopy of actin monomers ( blue , 1uM total , 30 % Cy5 labeled ) in the presence of equimolar profilin recruited to a WAVE1 pattern ( magenta ) . ( C ) TIRF microscopy of actin polymerization in - ( blue arrows ) and outside ( red arrows ) of WAVE1 - micropatterns ( magenta ) in the presence of 1uM profilin - actin visualized by a filament - binding probe ( 5nM Alexa488 - UTRNN ) . ( D ) Kymograph of a growing filament barbed end entering a WAVE1 micropattern . Conditions as in ( C ) ( G ) Surface mCherry - NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1 , N - WASP or dark mCherry control constructs in 1uM profilin - actin .

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Figure 2(B) TIRF microscopy of actin monomers (blue, 1uM total, 30% Cy5 labeled) in the presence of equimolar profilin recruited to a WAVE1 pattern (magenta).(C) TIRF microscopy of actin polymerization in- (blue arrows) and outside (red arrows) of WAVE1-micropatterns (magenta) in the presence of 1uM profilin-actin visualized by a filament-binding probe (5nM Alexa488-UTRNN).(D) Kymograph of a growing filament barbed end entering a WAVE1 micropattern. Conditions as in (C)(G) Surface mCherry-NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin.


Figure 3 ( B ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) and representative kymographs ( lower panel ) from multi - color TIRF imaging of actin polymerization on WAVE1 - coated coverslips . Conditions are 1uM actin , 5nM Alexa488 - UTRNN . ( E ) TIRF microscopy of actin polymerization from 2uM actin in the absence ( upper panel ) or presence of near - saturating amounts of WAVE1 WCA . ( F ) Barbed end polymerization rates as a function of actin and WAVE1 - actin ratios . The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on - rates as indicated ( see Methods ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of actin polymerization on WAVE1-coated coverslips. Conditions are 1uM actin, 5nM Alexa488-UTRNN.(E) TIRF microscopy of actin polymerization from 2uM actin in the absence (upper panel) or presence of near-saturating amounts of WAVE1 WCA.(F) Barbed end polymerization rates as a function of actin and WAVE1-actin ratios. The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on-rates as indicated (see Methods).


Figure 4 ( B ) Widefield epifluorescence images ( Alexa488 - actin ) of bead - attached dendritic networks following kinetic arrest after 10 min of network growth for spike - in experiments ( i . e . 60 % his10 - darkCherry - PWCA , 40 % his10 - Cherry - W ; molar percentage ) using indicated WAVE1 variants . Conditions are 5μM profilin - actin , 200nM CP and 50nM Arp2 / 3 . For all conditions , actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction . ( C ) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control ( N = 57 - 73 beads per condition ) . Errors bars represent SD . ( D ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) and representative kymographs ( lower panel ) from multi - color TIRF imaging of polymerization from profilin - actin on WAVE1 - coated coverslips . Conditions are 2µM actin , 3µM profilin , 5nM Alexa488 - UTRNN . ( E ) Average barbed end polymerization rates for indicated WAVE1 variants . Errors bars represent SD .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O

Figure 4(B) Widefield epifluorescence images (Alexa488-actin) of bead-attached dendritic networks following kinetic arrest after 10 min of network growth for spike-in experiments (i.e. 60% his10-darkCherry-PWCA, 40% his10-Cherry-W; molar percentage) using indicated WAVE1 variants. Conditions are 5μM profilin-actin, 200nM CP and 50nM Arp2/3. For all conditions, actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction.(C) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control (N= 57-73 beads per condition). Errors bars represent SD.(D) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of polymerization from profilin-actin on WAVE1-coated coverslips. Conditions are 2µM actin, 3µM profilin, 5nM Alexa488-UTRNN.(E) Average barbed end polymerization rates for indicated WAVE1 variants. Errors bars represent SD.


Figure 5 ( A ) The weighted mass average of WAVE1 PRD ( 15 μM ) determined by sedimentation equilibrium ultracentrifugation with LatB - stabilized monomers ( 15 μM ) and various profilin concentrations as indicated . The lower dashed line indicates the expected mass of WAVE1 PRD ; the 5 - 7 kDa upshift is due to non - specific actin binding ( WAVE1 PRD [ null ] , black symbols ) . The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD [ B ] in the presence of 90 μM profilin alone . ( C ) FRET efficiencies for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P _ CA = grey ) as a function of LatB - stabilized , quencher - ( Atto540Q - ) labeled actin monomers concentration . Lines are fits to a single site binding curve . ( D ) WH2 occupancies ( left axis , Methods ) and FRET efficiencies ( right axis ) for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P * WCA ( null ) = cyan , P _ CA = grey ) as a function of profilin concentration . Concentration of LatB - stabilized , Atto540Q - actin monomers was constant ( 175nM ) at all conditions . Lines are fits to a competitive inhibition model ( see Methods ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone.(C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve.(D) WH2 occupancies (left axis, Methods) and FRET efficiencies (right axis) for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P*WCA(null)=cyan, P_CA=grey) as a function of profilin concentration. Concentration of LatB-stabilized, Atto540Q-actin monomers was constant (175nM) at all conditions. Lines are fits to a competitive inhibition model (see Methods).


Figure 2 , Scaled reduced cost analysis for uptake rates of amino acids at different growth rates . Colour represents the scaled reduced cost value . A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate .

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Figure 2, Scaled reduced cost analysis for uptake rates of amino acids at different growth rates. Colour represents the scaled reduced cost value. A higher value indicates a greater impact of a given increase in the amino acid uptake on growth rate.


Figure 4 , Protein fold changes of the CcpA mutant over wild type . Log2 fold changes were calculated from LFQ values p values were calculated based on 2 - sided two sample t - tests with a FDR threshold of 0 . 05 and S 0 equal to 1 ( Tusher et al , 2001 ) . Proteins outside the significance lines ( FDR = 0 . 05 , S 0 = 1 ) are significantly changed in expression . Example sets of proteins are highlighted and are labeled with their gene names .

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Figure 4, Protein fold changes of the CcpA mutant over wild type. Log2 fold changes were calculated from LFQ values p values were calculated based on 2-sided two sample t-tests with a FDR threshold of 0.05 and S0 equal to 1 (Tusher et al, 2001). Proteins outside the significance lines (FDR = 0.05, S0 = 1) are significantly changed in expression. Example sets of proteins are highlighted and are labeled with their gene names.


Figure 1The differentially expressed RBPs during embryoid body ( EB ) differentiation , neural differentiation and cardiac differentiation , which are classified as different clusters . The right line plots show the dynamic expression pattern of each cluster , gray lines represent the expression of all genes in individual cluster , while the red line represents the average expression level . The number on the graph indicates the number of RBPs in the cluster . The expression value was firstly z - score scaled before plotting . EBD4 : embryoid body at day 4 ; EBD8 : embryoid body at day8 ; NSC : Neural Stem Cells ; NPC : Neural Progenitor Cell ; MES : mesodermal cells ; CP : Cardiac Precursors ; CM : Cardiomyocytes . Heatmap showing the expression pattern of the 29 RBPs during EB differentiation .

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Figure 1The differentially expressed RBPs during embryoid body (EB) differentiation, neural differentiation and cardiac differentiation, which are classified as different clusters. The right line plots show the dynamic expression pattern of each cluster, gray lines represent the expression of all genes in individual cluster, while the red line represents the average expression level. The number on the graph indicates the number of RBPs in the cluster. The expression value was firstly z-score scaled before plotting. EBD4: embryoid body at day 4; EBD8: embryoid body at day8; NSC: Neural Stem Cells; NPC: Neural Progenitor Cell; MES: mesodermal cells; CP: Cardiac Precursors; CM: Cardiomyocytes.Heatmap showing the expression pattern of the 29 RBPs during EB differentiation.