A ) Cell viability analysis by CellTiter - Blue assay of sgCtrl - or sgTSC2 - expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days . Statistical analysis was performed by two - tailed unpaired Student t test . For D10 , error bars represent SD from three independent experiments ( n = 3 ) . For A549 , error bars represent SD of pooled data from two independent experiments with three technical replicates each ( n = 2 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

A) Cell viability analysis by CellTiter-Blue assay of sgCtrl- or sgTSC2-expressing D10 melanoma and A549 lung cancer cells treated with TRAIL at indicated concentrations for three days. Statistical analysis was performed by two-tailed unpaired Student t test. For D10, error bars represent SD from three independent experiments (n = 3). For A549, error bars represent SD of pooled data from two independent experiments with three technical replicates each (n = 2). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


C ) Flow cytometry analysis of TRAIL - R2 surface expression level in multiple sgTSC2 - expressing tumor cell lines compared to their sgCtrl - expressing controls . Statistical analysis was performed by two - way ANOVA with Sidak ' s multiple comparisons test . Error bars represent SD of three biological replicates ( n = 3 ) . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

C) Flow cytometry analysis of TRAIL-R2 surface expression level in multiple sgTSC2- expressing tumor cell lines compared to their sgCtrl-expressing controls. Statistical analysis was performed by two-way ANOVA with Sidak's multiple comparisons test. Error bars represent SD of three biological replicates (n = 3). * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


D ) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2 , TRAIL receptors ( TNFRSF10A and TNFRSF10B ) , or both in a co - culture with MART - 1 T cells . Statistical analysis was performed with a one - way ANOVA , followed by a Tukey post - hoc test . Error bars indicate SD of four biological replicates with different T cell donors . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 .

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

D) In vitro competition assay performed by mixing control and tumor cells with ablation for either TSC2, TRAIL receptors (TNFRSF10A and TNFRSF10B), or both in a co-culture with MART-1 T cells. Statistical analysis was performed with a one-way ANOVA, followed by a Tukey post-hoc test. Error bars indicate SD of four biological replicates with different T cell donors. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001.


B ) TSC2 expression : TRAIL signaling expression ratio for predicting ICB treatment responses in pre - treatment patient samples Combo , anti - PD - 1 plus anti - CTLA - 4 combination treatment . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TSC2 expression: TRAIL signaling expression ratio for predicting ICB treatment responses in pre-treatment patient samples Combo, anti-PD-1 plus anti-CTLA-4 combination treatment. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).


C ) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment . Early during treatment ( EDT ) , anti - PD - 1 + anti - CTLA - 4 , and anti - PD - 1 alone treatment cohorts . Statistical significance was calculated with Student t test for normally distributed data , or with Mann - Whitney test for non - normally distributed data . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ; * * * * P < 0 . 0001 . Partial response and complete response ( PRCR ) are defined as responder ( R ) ; stable disease ( SD ) and progressive disease ( PD ) are defined as non - responder ( NR ) .

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

C) TSC2 immune challenge signature expression in cohorts of patients after ICB treatment. Early during treatment (EDT), anti-PD-1 + anti-CTLA-4, and anti-PD-1 alone treatment cohorts. Statistical significance was calculated with Student t test for normally distributed data, or with Mann-Whitney test for non-normally distributed data. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Partial response and complete response (PRCR) are defined as responder (R); stable disease (SD) and progressive disease (PD) are defined as non-responder (NR).


( A ) RAW 264 . 7 macrophages infected with heat killed ( top panels ) or live ( bottom panels ) S . aureus USA300 ( in red ) were imaged by DIC time - lapse microscopy at 6 h post - infection . The images on the left are of time zero in the image sequence and show the presence of S . aureus . The images on the right are of the same cells and the presence of membrane ruffles and dynamic filopodia are denoted by the yellow arrow heads . Candidate pinosomes are denoted by yellow arrows .

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

(A) RAW 264.7 macrophages infected with heat killed (top panels) or live (bottom panels) S. aureus USA300 (in red) were imaged by DIC time-lapse microscopy at 6 h post-infection. The images on the left are of time zero in the image sequence and show the presence of S. aureus. The images on the right are of the same cells and the presence of membrane ruffles and dynamic filopodia are denoted by the yellow arrow heads. Candidate pinosomes are denoted by yellow arrows.


( B ) RAW 264 . 7 macrophages expressing the lipid biosensor for PI ( 4 , 5 ) P2 , PH - Plcδ - RFP ( in red ) , were left uninfected or were infected with live GFP - expressing S . aureus USA300 ( in green ) for 8 h prior to fixation . The macrophage plasmalemma and extracellular bacteria are indicated by Cy5 - conjugated wheat germ agglutinin ( WGA , in blue ) . As a control , to demonstrate displacement of PH - Plcδ - RFP from the macrophage membrane , transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation . These images are representative of three independent experiments .

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

(B) RAW 264.7 macrophages expressing the lipid biosensor for PI(4,5)P2, PH-Plcδ-RFP (in red), were left uninfected or were infected with live GFP-expressing S. aureus USA300 (in green) for 8 h prior to fixation. The macrophage plasmalemma and extracellular bacteria are indicated by Cy5-conjugated wheat germ agglutinin (WGA, in blue). As a control, to demonstrate displacement of PH-Plcδ-RFP from the macrophage membrane, transfected cells were treated with 10 µM ionomycin in the presence of 1 mM CaCl2 for 20 min prior to fixation. These images are representative of three independent experiments.


( C , D ) Internalization of FITC - Dextran by uninfected ( top row ) and S . aureus ( in blue ) - infected ( bottom row ) RAW 264 . 7 macrophages at 10 h post - infection . These images are representative of three independent experiments . The quantitation of FITC - dextran uptake by either uninfected or infected RAW 264 . 7 and primary bone marrow derived macrophages is shown in ( D ) . For each experiment , macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition . Shown is the mean fluorescence intensity ( MFI ) in arbitrary units ( a . u . ) ± standard deviation where each symbol represents the measurement of a single cell . The data derive from three independent experiments and n . s . indicates the data are not significant as determined by an unpaired two - tailed t - test ( p > 0 . 05 ) .

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

(C, D) Internalization of FITC-Dextran by uninfected (top row) and S. aureus (in blue)-infected (bottom row) RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. The quantitation of FITC-dextran uptake by either uninfected or infected RAW 264.7 and primary bone marrow derived macrophages is shown in (D). For each experiment, macrophages were treated with 1 mM concanamycin A for 30 min prior to image acquisition. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u.) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).


( E ) Potential for co - localization of FITC - dextran ( in green ) with phagocytosed S . aureus expressing mCherry ( in red ) . The micrographs depict representative macrophages infected with S . aureus at 10 h post - infection and the white arrows point to mCherry positive bacteria that are co - localizing with dextran . Macrophages were treated with 1 μM concanamycin A prior to imaging live .

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

(E) Potential for co-localization of FITC-dextran (in green) with phagocytosed S. aureus expressing mCherry (in red). The micrographs depict representative macrophages infected with S. aureus at 10 h post-infection and the white arrows point to mCherry positive bacteria that are co-localizing with dextran. Macrophages were treated with 1 μM concanamycin A prior to imaging live.


( A ) Internalization and processing of DQ ™ Green BSA by uninfected and S . aureus ( in blue ) infected RAW 264 . 7 macrophages at 10 h post - infection . These images are representative of three independent experiments . ( B ) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S . aureus ( in blue ) infected RAW 264 . 7 . Shown is the mean fluorescence intensity ( MFI ) in arbitrary units ( a . u ) ± standard deviation where each symbol represents the measurement of a single cell . The data derive from three independent experiments and n . s . indicates the data are not significant as determined by an unpaired two - tailed t - test ( p > 0 . 05 ) .

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

(A) Internalization and processing of DQ™ Green BSA by uninfected and S. aureus (in blue) infected RAW 264.7 macrophages at 10 h post-infection. These images are representative of three independent experiments. (B) Quantitation of DQ BSA green fluorescence displayed by either uninfected or S. aureus (in blue) infected RAW 264.7. Shown is the mean fluorescence intensity (MFI) in arbitrary units (a.u) ± standard deviation where each symbol represents the measurement of a single cell. The data derive from three independent experiments and n.s. indicates the data are not significant as determined by an unpaired two-tailed t-test (p>0.05).


( C ) Fluorescence - based proliferation assays were performed in conjunction with the probe DQ ™ Green BSA . Processed DQ ™ Green BSA could be found co - localizing with intracellular mCherry - expressing S . aureus ( in red ) and with intracellular S . aureus has replicated ( i . e . is proliferation dye negative ) at 10h post - infection as depicted here . Data information : Scale bars equal 10 μm .

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

(C) Fluorescence-based proliferation assays were performed in conjunction with the probe DQ™ Green BSA. Processed DQ™ Green BSA could be found co-localizing with intracellular mCherry-expressing S. aureus (in red) and with intracellular S. aureus has replicated (i.e. is proliferation dye negative) at 10h post-infection as depicted here. Data information: Scale bars equal 10 μm.


( A ) Uptake of Alexa ® Fluor647 - conjugated transferrin by RAW 264 . 7 macrophages in the presence and absence of PIK - III and Dynasore . The fluorescence micrographs show transferrin ( in blue ) and the macrophage plasmalemma demarcated by TMR - conjugated wheat germ agglutinin ( in red ) . The dashed line indicates the region of the cell for which the line scan analysis , shown graphically on the right , was done . These micrographs are representative images obtained from three independent experiments . Line scan analysis was performed to indicate the distribution of fluorescent transferrin ( blue line ) relative to the plasmalemma ( red line ) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs . Scale bars equal 10 μm .

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

(A) Uptake of Alexa® Fluor647-conjugated transferrin by RAW 264.7 macrophages in the presence and absence of PIK-III and Dynasore. The fluorescence micrographs show transferrin (in blue) and the macrophage plasmalemma demarcated by TMR-conjugated wheat germ agglutinin (in red). The dashed line indicates the region of the cell for which the line scan analysis, shown graphically on the right, was done. These micrographs are representative images obtained from three independent experiments. Line scan analysis was performed to indicate the distribution of fluorescent transferrin (blue line) relative to the plasmalemma (red line) for the DMSO and Dynasore treated cells presented in the fluorescence micrographs. Scale bars equal 10 μm.


( B ) RAW macrophages were pre - treated with vehicle ( DMSO ) or the vps34 inhibitor PIK - III prior to the addition of 10 kDa FITC - dextran ( 100 µg / mL ) for 2 hrs . Prior to imaging live , macrophages were also stained with Cy5 - conjugated WGA . The representative micrographs depict inhibited dextran uptake indicating PIK - III treatment can perturb fluid phase uptake . Scale bars equal 10 µm .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RAW macrophages were pre-treated with vehicle (DMSO) or the vps34 inhibitor PIK-III prior to the addition of 10 kDa FITC-dextran (100 µg/mL) for 2 hrs. Prior to imaging live, macrophages were also stained with Cy5-conjugated WGA. The representative micrographs depict inhibited dextran uptake indicating PIK-III treatment can perturb fluid phase uptake. Scale bars equal 10 µm.


( C ) The intensity of dextran fluorescence was quantified for RAW cells pre - treated with vehicle , PIK - III ( 10 µM ) , Dynasore ( 100 µM ) or PIK - III and Dynasore ( 10 and 100 µM ) respectively . The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments . * indicates p < 0 . 05 , as determined by an ordinary one - way ANOVA with a Bonferroni multiple comparisons test .

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

(C) The intensity of dextran fluorescence was quantified for RAW cells pre-treated with vehicle, PIK-III (10 µM), Dynasore (100 µM) or PIK-III and Dynasore (10 and 100 µM) respectively. The data shown are the mean fluorescence intensity ± standard deviation for each experiment calculated from at least 30 cells from two independent experiments. * indicates p<0.05, as determined by an ordinary one-way ANOVA with a Bonferroni multiple comparisons test.


( A ) The effects of prolonged exposure to gentamicin ( 300 µg / mL ) or vancomycin ( 300 µg / mL ) on the survival of phagocytosed S . aureus USA300 in RAW macrophages is shown . Depicted above the graph is a timeline detailing the treatment regime . The data are presented as the mean fold change in CFU / mL + / - standard deviation for 15 biological replicates of S . aureus , represented by each symbol , from five independent experiments . For each replicate the fold change was calculated by dividing the bacterial count at 10 h post - infection by the counts at 1 . 5 h post - infection . The dashed line indicates a fold change value equal to 1 ( i . e . no change in bacterial burden ) . * * p < 0 . 01 and statistical significance was determined by a one - way ANOVA with a Dunnett ' s post - hoc test .

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

(A) The effects of prolonged exposure to gentamicin (300 µg/mL) or vancomycin (300 µg/mL) on the survival of phagocytosed S. aureus USA300 in RAW macrophages is shown. Depicted above the graph is a timeline detailing the treatment regime. The data are presented as the mean fold change in CFU/mL +/- standard deviation for 15 biological replicates of S. aureus, represented by each symbol, from five independent experiments. For each replicate the fold change was calculated by dividing the bacterial count at 10 h post-infection by the counts at 1.5 h post-infection. The dashed line indicates a fold change value equal to 1 (i.e. no change in bacterial burden). **p<0.01 and statistical significance was determined by a one-way ANOVA with a Dunnett's post-hoc test.


( B ) The ability to protect S . aureus USA300 from intracellular killing by gentamicin is shown . Here , experiments were performed as in ( A ) except during the initial gentamicin treatment ( 100 μg / mL for 1h ) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK - III / Dynasore to inhibit fluid phase uptake . At 1 . 5 h post - infection cells were washed and treated with gentamicin ( 300 µg / mL ) in the presence of either DMSO ( vehicle ) or PIK - III + Dynasore which were maintained throughout the experiment . The data are presented as the mean number of surviving bacteria in CFU / mL at 10 h post - infection + / - standard deviation . These data derive from a total of 15 biological replicates of S . aureus from at least three independent experiments . * * * p < 0 . 001 and statistical significance was determined by an unpaired two - tailed t - test with a Welch ' s correction .

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

(B) The ability to protect S. aureus USA300 from intracellular killing by gentamicin is shown. Here, experiments were performed as in (A) except during the initial gentamicin treatment (100 μg/mL for 1h) to kill extracellular bacteria macrophages were also treated with either DMSO or PIK-III/Dynasore to inhibit fluid phase uptake. At 1.5 h post-infection cells were washed and treated with gentamicin (300 µg/mL) in the presence of either DMSO (vehicle) or PIK-III + Dynasore which were maintained throughout the experiment. The data are presented as the mean number of surviving bacteria in CFU/mL at 10 h post-infection +/- standard deviation. These data derive from a total of 15 biological replicates of S. aureus from at least three independent experiments. *** p<0.001 and statistical significance was determined by an unpaired two-tailed t-test with a Welch's correction.


( C ) The effect of PIK - III / Dynasore treatment on the ability of S . aureus USA300 to replicate within RAW macrophages is shown . After gentamicin treatment at 1 . 5 h , RAW 264 . 7 macrophages having phagocytosed S . aureus were lysed and plated to determine the number of gentamicin protected bacteria . Alternatively , infected samples treated with either DMSO as a vehicle control or with PIK - III / Dynasore which was added at 1 . 5h post - infection after gentamicin . At 10 h post - infection macrophages exposed to DMSO or inhibitors , that were maintained throughout the experiment , were lysed and plated to determine the bacterial burden . The data shown are the mean fold change in CFU / mL at 10 h where the data are normalized to the bacterial burden obtained at 1 . 5 h . Each symbol represents a biological replicate ( n = 8 ) of S . aureus and derive from three independent experiments . The data presented are the mean ± standard deviation . * indicates p < 0 . 05 as determine by an unpaired two - tailed t - test with a Welch ' s correction .

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

(C) The effect of PIK-III/Dynasore treatment on the ability of S. aureus USA300 to replicate within RAW macrophages is shown. After gentamicin treatment at 1.5 h, RAW 264.7 macrophages having phagocytosed S. aureus were lysed and plated to determine the number of gentamicin protected bacteria. Alternatively, infected samples treated with either DMSO as a vehicle control or with PIK-III/Dynasore which was added at 1.5h post-infection after gentamicin. At 10 h post-infection macrophages exposed to DMSO or inhibitors, that were maintained throughout the experiment, were lysed and plated to determine the bacterial burden. The data shown are the mean fold change in CFU/mL at 10 h where the data are normalized to the bacterial burden obtained at 1.5 h. Each symbol represents a biological replicate (n=8) of S. aureus and derive from three independent experiments. The data presented are the mean ± standard deviation. * indicates p<0.05 as determine by an unpaired two-tailed t-test with a Welch's correction.


( D ) The effect of PIK - III + Dynasore or DMSO on the growth of S . aureus USA300 in RPMI with 5 % ( v / v ) FBS is shown . The data are the mean OD600nm + / - standard deviation of four biological replicates of S . aureus for each condition and are representative of three independent experiments .

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

(D) The effect of PIK-III + Dynasore or DMSO on the growth of S. aureus USA300 in RPMI with 5% (v/v) FBS is shown. The data are the mean OD600nm +/- standard deviation of four biological replicates of S. aureus for each condition and are representative of three independent experiments.


( E ) Analysis of the effects of PIK - III / Dynasore treatment on S . aureus replication using a fluorescence based bacterial proliferation assay is shown . The micrographs depict S . aureus USA300 expressing GFP that were co - labelled with proliferation dye . The top row of micrographs depict vehicle treated macrophages where GFP - positive , yet proliferation dye - negative bacteria were present . The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive . The macrophage plasmalemma and extracellular bacteria are demarcated by TMR - WGA ( in red ) . Images were acquired at 10 h post - infection and are representative of three independent experiments . Scale bars equal 10 μm .

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

(E) Analysis of the effects of PIK-III/Dynasore treatment on S. aureus replication using a fluorescence based bacterial proliferation assay is shown. The micrographs depict S. aureus USA300 expressing GFP that were co-labelled with proliferation dye. The top row of micrographs depict vehicle treated macrophages where GFP-positive, yet proliferation dye-negative bacteria were present. The bottom row depicts inhibitor macrophages treated macrophages where the bacteria fail to replicate and remain proliferation dye positive. The macrophage plasmalemma and extracellular bacteria are demarcated by TMR-WGA (in red). Images were acquired at 10 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm.


( F ) The normalized fraction of macrophages containing replicating bacteria is shown . The graph represents the mean ± standard deviation of the number of macrophages that contain replicating bacteria where the data are normalized to the vehicle control condition . Each symbol represents a biological replicate ( n = 6 ) of S . aureus and derive from three independent experiments . * * * * p < 0 . 0001 and statistical significance was determined by an unpaired two - tailed t - test using a Welch ' s correction .

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

(F) The normalized fraction of macrophages containing replicating bacteria is shown. The graph represents the mean ± standard deviation of the number of macrophages that contain replicating bacteria where the data are normalized to the vehicle control condition. Each symbol represents a biological replicate (n=6) of S. aureus and derive from three independent experiments. ****p<0.0001 and statistical significance was determined by an unpaired two-tailed t-test using a Welch's correction.


( A ) Growth of wild - type S . aureus USA300 or a purK mutant . The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI . Additives are indicated on the x axis and were either 5 % ( v / v ) FBS , 50 μM IMP , 10 μM PIK - III and 100 μM Dynasore or an equivalent volume of DMSO . Each symbol represents a biological replicate of S . aureus and the data derive from three independent experiments . Statistical significance was determined by a one - way ANOVA with a Holm - Sidak ' s multiple comparison test . n . s . indicates not significant , * * * indicates p < 0 . 0001 .

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(A) Growth of wild-type S. aureus USA300 or a purK mutant. The data are the mean ± standard deviation of the end point optical density measured at 18 h at 600nm for each strain in RPMI. Additives are indicated on the x axis and were either 5% (v/v) FBS, 50 μM IMP, 10 μM PIK-III and 100 μM Dynasore or an equivalent volume of DMSO. Each symbol represents a biological replicate of S. aureus and the data derive from three independent experiments. Statistical significance was determined by a one-way ANOVA with a Holm-Sidak's multiple comparison test. n.s. indicates not significant, *** indicates p<0.0001.


( B ) The graph depicts the fold change in bacterial burden for RAW 264 . 7 macrophages infected with purK S . aureus . Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK - III and Dynasore which were maintained throughout the infection . The data are the mean ± standard deviation of the burden at 10 h post - infection normalized to the burden at 1 . 5h post - infection prior to the addition of IMP . Each symbol represents a single biological replicate of S . aureus and derive from three independent experiments . Significance was determined by one - way ANOVA with a Turkeys multiple comparison test . * p < 0 . 05 , * * p < 0 . 01 .

(B) The graph depicts the fold change in bacterial burden for RAW 264.7 macrophages infected with purK S. aureus. Macrophages were left untreated or were exposed to 50 μM IMP or 50 μM IMP after pretreatment with PIK-III and Dynasore which were maintained throughout the infection. The data are the mean ± standard deviation of the burden at 10 h post-infection normalized to the burden at 1.5h post-infection prior to the addition of IMP. Each symbol represents a single biological replicate of S. aureus and derive from three independent experiments. Significance was determined by one-way ANOVA with a Turkeys multiple comparison test. *p<0.05, **p<0.01.


( C , D ) The ability of purK S . aureus to grow in an IMP - dependent manner within primary bone marrow - derived macrophages is shown . ( C ) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non - replicating bacteria are both GFP and proliferation dye positive . In contrast , replicating bacteria ( white arrows ) are only GFP positive . The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S . aureus in the presence of 50 μM IMP . Images were acquired at 12 h post - infection and are representative of three independent experiments . Scale bars equal 10 μm . ( D ) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs . Each data point represents a biological replicate ( n = 8 ) and derived from three independent experiments . The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition . * * indicates p < 0 . 01 as determined by an unpaired two - tailed t - test with a Wilcoxon matched pairs test .

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(C, D) The ability of purK S. aureus to grow in an IMP-dependent manner within primary bone marrow-derived macrophages is shown. (C) Fluorescence micrographs depicting purK replication analyzed by fluorescence proliferation assay where non-replicating bacteria are both GFP and proliferation dye positive. In contrast, replicating bacteria (white arrows) are only GFP positive. The top row depicts purK infected BMDMs without IMP whereas the bottom row depicts macrophages carrying purK S. aureus in the presence of 50 μM IMP. Images were acquired at 12 h post-infection and are representative of three independent experiments. Scale bars equal 10 μm. (D) The fraction of macrophages containing replicating bacteria was determined for purK infected BMDMs. Each data point represents a biological replicate (n=8) and derived from three independent experiments. The data shown are the mean ± standard deviation where the data were normalized to the no IMP condition. ** indicates p<0.01 as determined by an unpaired two-tailed t-test with a Wilcoxon matched pairs test.


B Fatty acid composition of B . subtilis WT cells grown in PF medium , and Δbkd grown with MB , IB or depleted for precursor for 90 min ( IBPF ) . For detailed analyses , see Appendix Fig S1B .

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B Fatty acid composition of B. subtilis WT cells grown in PF medium, and Δbkd grown with MB, IB or depleted for precursor for 90 min (IBPF). For detailed analyses, see Appendix Fig S1B.


E Fatty acid composition of E . coli WT and fabA ( Ts ) cells grown at 30 ° C and shifted to different temperatures for 120 min . For detailed analyses , see Appendix Fig S2B and C .

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E Fatty acid composition of E. coli WT and fabA(Ts) cells grown at 30°C and shifted to different temperatures for 120 min. For detailed analyses, see Appendix Fig S2B and C.


A DPH anisotropy of B . subtilis WT or Δbkd cells supplemented either with MB , IB or depleted for precursor ( IBPF ) for the times indicated . High DPH anisotropy indicates low membrane fluidity . B DPH anisotropy of E . coli WT cells grown steady state at 30 ° C , 37 ° C or shifted from 30 ° C to 37 ° C followed by immediate measurement . In addition , DPH anisotropy of fabA ( Ts ) cells grown steady state at 30 ° C or shifted from 30 ° C to 37 ° C followed by measurement at the times indicated .

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A DPH anisotropy of B. subtilis WT or Δbkd cells supplemented either with MB, IB or depleted for precursor (IBPF) for the times indicated. High DPH anisotropy indicates low membrane fluidity. B DPH anisotropy of E. coli WT cells grown steady state at 30°C, 37°C or shifted from 30°C to 37°C followed by immediate measurement. In addition, DPH anisotropy of fabA(Ts) cells grown steady state at 30°C or shifted from 30°C to 37°C followed by measurement at the times indicated. D


A Images of B . subtilis WT and Δbkd cells co - labelled with the membrane potential - sensitive dye DISC3 ( 5 ) and the membrane permeability indicator Sytox Green . Membrane properties were assessed for Δbkd cells grown in the presence of MB , IB or washed precursor - free ( IBPF ) for the times indicated . As controls , WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC ( gABC ) or pore - forming lantibiotic Nisin . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3A - C . B Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 100 - 142 ) depicted in panel A . Median represented by red line . C Images of E . coli WT and fabA ( Ts ) cells co - labelled with the same indicator dyes as in panel A . Membrane properties were assessed for fabA ( Ts ) at 30 ° C and upon transfer to non - permissive 37 ° C for the times indicated . As controls , WT cells were incubated with the pore - forming antibiotic Polymyxin B ( PolyB ) . For cross - correlation between membrane depolarisation and membrane permeabilisation , see Appendix Fig S3D and E . The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background ( see Fig EV1 ) . D Quantification of DISC3 ( 5 ) fluorescence for cells ( n = 76 - 141 ) depicted in panel C . Median represented by red line .

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A Images of B. subtilis WT and Δbkd cells co-labelled with the membrane potential-sensitive dye DISC3(5) and the membrane permeability indicator Sytox Green. Membrane properties were assessed for Δbkd cells grown in the presence of MB, IB or washed precursor-free (IBPF) for the times indicated. As controls, WT cells were measured in the presence of depolarising antimicrobial peptide gramicidin ABC (gABC) or pore-forming lantibiotic Nisin. For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3A-C. B Quantification of DISC3(5) fluorescence for cells (n=100-142) depicted in panel A. Median represented by red line. C Images of E. coli WT and fabA(Ts) cells co-labelled with the same indicator dyes as in panel A. Membrane properties were assessed for fabA(Ts) at 30°C and upon transfer to non-permissive 37°C for the times indicated. As controls, WT cells were incubated with the pore-forming antibiotic Polymyxin B (PolyB). For cross-correlation between membrane depolarisation and membrane permeabilisation, see Appendix Fig S3D and E. The integrity of the diffusion barrier function was additionally studied via ONPG permeability in a ΔlacY background (see Fig EV1). D Quantification of DISC3(5) fluorescence for cells (n=76-141) depicted in panel C. Median represented by red line. Dat


A Images of B . subtilis Δbkd cells stained with membrane dye FM 5 - 95 and expressing GFP fusions of DNA - binding protein HBsu ( top ) , cell division protein FtsZ ( middle ) or cell elongation protein MreB ( bottom ) . Cells were grown with IB or depleted for precursors for 90 min ( IBPF ) . For further examples and additional time points , see Appendix Fig S4A . B Images of E . coli fabA ( Ts ) cells stained with FM 5 - 95 for the outer membrane and with DNA - intercalating dye DAPI ( top ) , or expressing GFP sandwich ( SW ) fusions to the cell division protein FtsZ ( middle ) and the cell elongation protein MreB ( bottom ) , respectively . Depicted are cells grown at 30 ° C or with a temperature shift to 37 ° C for 120 min . For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery , see Figs EV2 , EV3 , EV4 , and S5 . For further examples and additional time points , see Appendix Fig S4B .

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A Images of B. subtilis Δbkd cells stained with membrane dye FM 5-95 and expressing GFP fusions of DNA-binding protein HBsu (top), cell division protein FtsZ (middle) or cell elongation protein MreB (bottom). Cells were grown with IB or depleted for precursors for 90 min (IBPF). For further examples and additional time points, see Appendix Fig S4A. B Images of E. coli fabA(Ts) cells stained with FM 5-95 for the outer membrane and with DNA-intercalating dye DAPI (top), or expressing GFP sandwich (SW) fusions to the cell division protein FtsZ (middle) and the cell elongation protein MreB (bottom), respectively. Depicted are cells grown at 30°C or with a temperature shift to 37°C for 120 min. For a more detailed view on the influence of low membrane fluidity on membrane dissociation of RNase E as well as on the cell division machinery, see Figs EV2, EV3, EV4, and S5. For further examples and additional time points, see Appendix Fig S4B. D


B Images of B . subtilis Δbkd cells grown with IB or without precursor ( IBPF ) . Cells were stained with membrane fluidity - sensitive dye Laurdan and imaged at 460 nm , 520 nm and as the corresponding colour - coded Laurdan GP map . C Images of E . coli fabA ( Ts ) cells grown at 30 ° C or shifted to non - permissive 37 ° C for 120 min . Cells were stained and imaged as described in panel B . For a more pronounced view on domain formation associated with local differences in membrane fluidity , see Appendix Fig S7 , showing E . coli fabA ( Ts ) grown in LB instead of M9 minimal medium with glucose / casamino acids . D Images of B . subtilis Δbkd cells grown and stained with Laurdan ( imaged at 460 nm ) as in panel B , but additionally expressing WALP23 - mCherry . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8A . Co - localisation of membrane dye FM 5 - 95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C . E Images of E . coli fabA ( Ts ) cells grown and stained as in panel C , but additionally expressing WALP23 - mScarlet - I . For corresponding fluorescence intensity correlation between images , see Appendix Fig S8B .

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B Images of B. subtilis Δbkd cells grown with IB or without precursor (IBPF). Cells were stained with membrane fluidity-sensitive dye Laurdan and imaged at 460 nm, 520 nm and as the corresponding colour-coded Laurdan GP map. C Images of E. coli fabA(Ts) cells grown at 30°C or shifted to non-permissive 37°C for 120 min. Cells were stained and imaged as described in panel B. For a more pronounced view on domain formation associated with local differences in membrane fluidity, see Appendix Fig S7, showing E. coli fabA(Ts) grown in LB instead of M9 minimal medium with glucose/casamino acids. D Images of B. subtilis Δbkd cells grown and stained with Laurdan (imaged at 460 nm) as in panel B, but additionally expressing WALP23-mCherry. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8A. Co-localisation of membrane dye FM 5-95 and transmembrane peptide WALP23 is shown in Appendix Figs S9 and S8C. E Images of E. coli fabA(Ts) cells grown and stained as in panel C, but additionally expressing WALP23-mScarlet-I. For corresponding fluorescence intensity correlation between images, see Appendix Fig S8B. Dat


A Time lapse images of E . coli fabA ( Ts ) cells expressing FOF1 a - mNG . For corresponding controls , see Appendix Fig S10 . Cells were grown at 30 ° C and shifted from 30 ° C to non - permissive 37 ° C . See Movie EV2 for full time lapse .

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A Time lapse images of E. coli fabA(Ts) cells expressing FOF1 a-mNG. For corresponding controls, see Appendix Fig S10. Cells were grown at 30°C and shifted from 30°C to non-permissive 37°C. See Movie EV2 for full time lapse.


E Fatty acid composition of E . coli fabA ( Ts ) cells ( same cell batch as in D ) upon growth at 30 ° C , upon depletion of UFA by incubation at 37 ° C for 120 min and upon recovery by oleate supplementation for additional 120 min . For detailed analyses , see Appendix Fig S11B - D .

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E Fatty acid composition of E. coli fabA(Ts) cells (same cell batch as in D) upon growth at 30°C, upon depletion of UFA by incubation at 37°C for 120 min and upon recovery by oleate supplementation for additional 120 min. For detailed analyses, see Appendix Fig S11B-D.


A Representative trajectory maps of individual FOF1 a - mNG molecules in E . coli WT and fabA ( Ts ) cells grown at 30 ° C and upon shift to non - permissive 37 ° C for 120 min . See Movie EV3 ( WT ) and Movie EV4 ( fabA ( Ts ) ) for accumulating trajectory maps of FOF1 a - mNG molecules .

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A Representative trajectory maps of individual FOF1 a-mNG molecules in E. coli WT and fabA(Ts) cells grown at 30°C and upon shift to non-permissive 37°C for 120 min. See Movie EV3 (WT) and Movie EV4 (fabA(Ts)) for accumulating trajectory maps of FOF1 a-mNG molecules.


D Apparent lateral diffusion coefficients ( Dapp ) of FOF1 a - mNG analysed in panel B .

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D Apparent lateral diffusion coefficients (Dapp) of FOF1 a-mNG analysed in panel B.


E . Validation of individual targets by manually curated in vitro ubiquitylation experiments . HA - tagged ( hemagglutinin ) candidate proteins were purified from Hek293T cells via anti - HA immunoprecipitation ( IP ) followed by HA - peptide elution . Candidates were incubated with 20 µM His6 - Ubiquitin , 170 nM E1 , E2s ( 0 . 5 µM each of UbcH5b and Cdc34 ) and 5 mM ATP in the presence or absence of 0 . 1 µM SCFFbxw5 for 2 hours at 37 ° C . Substrates were detected via SDS - PAGE followed by western blotting using anti - HA antibodies for detection .

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E. Validation of individual targets by manually curated in vitro ubiquitylation experiments. HA-tagged (hemagglutinin) candidate proteins were purified from Hek293T cells via anti-HA immunoprecipitation (IP) followed by HA-peptide elution. Candidates were incubated with 20 µM His6-Ubiquitin, 170 nM E1, E2s (0.5 µM each of UbcH5b and Cdc34) and 5 mM ATP in the presence or absence of 0.1 µM SCFFbxw5 for 2 hours at 37°C. Substrates were detected via SDS-PAGE followed by western blotting using anti-HA antibodies for detection.


E . Co - IP of HA - Kif2a , HA - Kif2b or HA - MCAK with Flag -

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E. Co-IP of HA-Kif2a, HA-Kif2b or HA-MCAK with Flag-Fbxw5


F . In vitro pull - down experiment as in B . Asterisk indicates an unspecific protein from E . coli . Note : Kif2b purification from Sf21 cells was much less efficient and it was therefore not included in the experiment .

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F. In vitro pull-down experiment as in B. Asterisk indicates an unspecific protein from E.coli. Note: Kif2b purification from Sf21 cells was much less efficient and it was therefore not included in the experiment.


D . In vitro ubiquitylation experiment as in B . Blots 1 & 2 : Plk1 or RanGAP1 used as substrates instead of MCAK . Blot 3 : Left : Experiment as in B . Asterisks indicate signals from Plk1 antibody ( same species as anti - Fbxw5 ) . Right : Same reaction except that Cul1 ~ Nedd8 / Rbx1 complexes were replaced by Cul4A ~ Nedd8 / DDB1 / Rbx1 complexes Bottom blots : Same blots as above incubated with anti - Fbxw5 antibodies . Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex .

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D. In vitro ubiquitylation experiment as in B. Blots 1&2: Plk1 or RanGAP1 used as substrates instead of MCAK. Blot 3: Left: Experiment as in B. Asterisks indicate signals from Plk1 antibody (same species as anti-Fbxw5). Right: Same reaction except that Cul1~Nedd8/Rbx1 complexes were replaced by Cul4A~Nedd8/DDB1/Rbx1 complexes Bottom blots: Same blots as above incubated with anti-Fbxw5 antibodies. Autoubiquitylation of Fbxw5 indicates activity of the E3 ligase complex.


G . In vitro ubiquitylation experiment as in B using either wild type , K48R , K63R , KK48 , 63RR or methylated ubiquitin ( Kmethyl , see Fig EV2K ) . Top : Cdc34 used as E2 . Bottom : UbcH5b used as E2 .

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G. In vitro ubiquitylation experiment as in B using either wild type, K48R, K63R, KK48,63RR or methylated ubiquitin (Kmethyl, see Fig EV2K). Top: Cdc34 used as E2. Bottom: UbcH5b used as E2.


B . Quantification of the MCAK / Uba2 signal ratio normalised to each non - targeting control of 4 independent experiments . Error bars indicate standard deviation , asterisks the p - value of a two - tailed unpaired Student ' s t - test comparing each Fbxw5 - directed siRNA sample with the according non - targeting control ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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B. Quantification of the MCAK/Uba2 signal ratio normalised to each non-targeting control of 4 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test comparing each Fbxw5-directed siRNA sample with the according non-targeting control (*P < 0.05, **P < 0.01, ***P < 0.001).


C . RPE - 1 cells were transfected with the indicated siRNAs ( 72 hours total time ) , split on coverslips 24 hours later and serum - starved for the last 24 hours . Cells were fixed in methanol and analysed via immunofluorescence using the indicated antibodies together with Hoechst staining . Left : Maximum intensity projections ( same for all following microscopy images ) of representative images . Arrows indicate ODF2 signal . Scale bar = 5 µm . Note : Middle panel shows an example image of siFw5 - 3 . Right : Quantification of MCAK signals co - localising with ODF2 . Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells . Asterisks indicate p - value of a Mann Whitney test comparing each sample set with the non - targeting control ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

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C. RPE-1 cells were transfected with the indicated siRNAs (72 hours total time), split on coverslips 24 hours later and serum-starved for the last 24 hours. Cells were fixed in methanol and analysed via immunofluorescence using the indicated antibodies together with Hoechst staining. Left: Maximum intensity projections (same for all following microscopy images) of representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Note: Middle panel shows an example image of siFw5-3. Right: Quantification of MCAK signals co-localising with ODF2. Long black line shows mean intensity and error bars indicate standard error of the mean of 3 independent experiments covering in total more than 280 cells. Asterisks indicate p-value of a Mann Whitney test comparing each sample set with the non-targeting control (**P < 0.01, ***P < 0.001).


A . Fluorescence - based pulse - chase experiment using a monoclonal RPE - 1 cell line expressing mNG - MCAK under a doxycycline - inducible promoter . Cells were transfected with the indicated siRNAs ( siFw5 - 1 & 3 together ) and split 24 hours later on Ibidi 8 Well Glass Bottom µ - slides while simultaneously inducing mNG - MCAK expression with 6 ng / ml doxycycline ( pulse ) . 24 hours later , doxycycline was washed out and cells were released into serum - free medium . Cells that have just finished or were about to finish mitosis ( distinguishable for example by the midbody signal of mNG - MCAK ( arrow heads ) ) were selected and imaged over 24 hours , taking an image every 20 min ( chase ) . Left : representative images of selected time points . Scale bar = 10 µm . Arrows indicate centrosomal mNG - MCAK signals , arrowheads mNG - MCAK signals at the midbody . Right : Quantification of centrosomal mNG - MCAK signals . Error bars show standard error of the mean of 3 independent experiments with 25 cells in total . P - value of a two - tailed unpaired Students t - test comparing signal intensity of Fbxw5 knockdown over non - targeting control was mostly below 0 . 01 for time points 0 to 8 hours and below 0 . 05 for time points 9h to 20h . Dark and light blue lines indicate the time points at which mNG - MCAK signals are reduced by 50 % compared to time point 0 for siNT and siFbxw5 samples , respectively .

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

A. Fluorescence-based pulse-chase experiment using a monoclonal RPE-1 cell line expressing mNG-MCAK under a doxycycline-inducible promoter. Cells were transfected with the indicated siRNAs (siFw5-1&3 together) and split 24 hours later on Ibidi 8 Well Glass Bottom µ-slides while simultaneously inducing mNG-MCAK expression with 6 ng/ml doxycycline (pulse). 24 hours later, doxycycline was washed out and cells were released into serum-free medium. Cells that have just finished or were about to finish mitosis (distinguishable for example by the midbody signal of mNG-MCAK (arrow heads)) were selected and imaged over 24 hours, taking an image every 20 min (chase). Left: representative images of selected time points. Scale bar = 10 µm. Arrows indicate centrosomal mNG-MCAK signals, arrowheads mNG-MCAK signals at the midbody. Right: Quantification of centrosomal mNG-MCAK signals. Error bars show standard error of the mean of 3 independent experiments with 25 cells in total. P-value of a two-tailed unpaired Students t-test comparing signal intensity of Fbxw5 knockdown over non-targeting control was mostly below 0.01 for time points 0 to 8 hours and below 0.05 for time points 9h to 20h. Dark and light blue lines indicate the time points at which mNG-MCAK signals are reduced by 50% compared to time point 0 for siNT and siFbxw5 samples, respectively.


D . Same samples as in C , except that cells grown on coverslips were fixed in methanol and analysed by immunofluorescence . Left : Representative images . Arrows indicate ODF2 signal . Scale bar = 5 µm . Right : Quantification . Error bars show standard error of the mean of 3 independent experiments , covering in total more than 150 cells . Asterisks indicate the p - value of a Mann Whitney test ( * * * P < 0 . 001 ) .

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

D. Same samples as in C, except that cells grown on coverslips were fixed in methanol and analysed by immunofluorescence. Left: Representative images. Arrows indicate ODF2 signal. Scale bar = 5 µm. Right: Quantification. Error bars show standard error of the mean of 3 independent experiments, covering in total more than 150 cells. Asterisks indicate the p-value of a Mann Whitney test (***P < 0.001).


B . CHX chase experiment as in A , except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h , released for 9 hours and again arrested for 16h . Following the 2nd release , 50 µg / ml CHX was added as indicated and samples were analysed as in A . Left : representative blots . Asterisks indicate unspecific bands detected by the Fbxw5 antibody . Right : Quantification of MCAK / Uba2 signal intensity ratios normalised to time point zero . Error bars show standard deviation of 6 independent experiments and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) .

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

B. CHX chase experiment as in A, except that cells were treated with the indicated siRNAs for 48h and arrested with 200 mM thymidine for 16h, released for 9 hours and again arrested for 16h. Following the 2nd release, 50 µg/ml CHX was added as indicated and samples were analysed as in A. Left: representative blots. Asterisks indicate unspecific bands detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal intensity ratios normalised to time point zero. Error bars show standard deviation of 6 independent experiments and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).


C . Extracts of RPE - 1 cells treated with the indicated siRNAs for 72 hours , grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO ( - ) or 10 µM MG - 132 ( + ) for the very last 6 hours . Left : Representative blots . Asterisk indicates an unspecific band detected by the Fbxw5 antibody . Right : Quantification of MCAK / Uba2 signal ratio normalised to non - targeting control with DMSO of 6 independent experiments . Error bars indicate standard deviation , asterisks the p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) .

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

C. Extracts of RPE-1 cells treated with the indicated siRNAs for 72 hours, grown asynchronously or arrested with the indicated compounds for the last 24 hours and treated either with DMSO (-) or 10 µM MG-132 (+) for the very last 6 hours. Left: Representative blots. Asterisk indicates an unspecific band detected by the Fbxw5 antibody. Right: Quantification of MCAK/Uba2 signal ratio normalised to non-targeting control with DMSO of 6 independent experiments. Error bars indicate standard deviation, asterisks the p-value of a two-tailed unpaired Student's t-test (**P < 0.01).


A . Polyclonal RPE - 1 cells expressing mNG - MCAK under a doxycycline - inducible promoter were seeded on coverslips , induced with 20 ng / ml doxycycline and grown for 24 hours . Subsequently , cells were washed twice with PBS and incubated for another 24 hours in serum - free medium without doxycycline . Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining . Left : Representative images . Scale bar = 10 µm . Right : Quantification of ciliated cells . Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * * P < 0 . 01 ) .

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

A. Polyclonal RPE-1 cells expressing mNG-MCAK under a doxycycline-inducible promoter were seeded on coverslips, induced with 20 ng/ml doxycycline and grown for 24 hours. Subsequently, cells were washed twice with PBS and incubated for another 24 hours in serum-free medium without doxycycline. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 5 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (**P < 0.01).


C . RPE - 1 cells were treated with the indicated siRNA for 48 hours followed by serum - starvation for another 24 hours . Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining . Left : Representative images . Note : Left panel shows an example image of siFw5 - 1 . Scale bar = 10 µm . Right : Quantification of ciliated cells . Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p - value of a two - tailed unpaired Student ' s t - test ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

C. RPE-1 cells were treated with the indicated siRNA for 48 hours followed by serum-starvation for another 24 hours. Cells were then fixed in methanol and subjected to IF using the indicated antibodies together with Hoechst staining. Left: Representative images. Note: Left panel shows an example image of siFw5-1. Scale bar = 10 µm. Right: Quantification of ciliated cells. Error bars show standard deviation of 4 independent experiments covering more than 200 cells and asterisks indicate p-value of a two-tailed unpaired Student's t-test (*P < 0.05, **P < 0.01, ***P < 0.001).


( B ) The inherent elastic response of a lipid membrane ( POPC ) was studied by enforcing indentations via the isolated SNARE C - termini in the presence or absence of a short linker peptide , or the linker plus a fluorescent protein tag ( FP ) . We define " relative indentation " as the change in C - terminus ( Nyv1 ) - C - terminus ( Vam3 ) distance that occurs when the SNARE complex performs mechanical work . It reflects the depth of the formed well upon squeezing . The slope of the obtained work profile estimates the required force ( thin black line ) . Perforation ( red arrow ) requires a force of 80 pN for the wild - type C - termini . The plateau within the work profile indicates that the membrane gives up its elastic response ( perforation transition ) . C - terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime . Furthermore , if the unstructured peptide - spacer would be alpha - helical - and thus much shorter - indentation forces would still be only slightly affected .

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

(B) The inherent elastic response of a lipid membrane (POPC) was studied by enforcing indentations via the isolated SNARE C-termini in the presence or absence of a short linker peptide, or the linker plus a fluorescent protein tag (FP). We define "relative indentation" as the change in C-terminus (Nyv1) - C-terminus (Vam3) distance that occurs when the SNARE complex performs mechanical work. It reflects the depth of the formed well upon squeezing. The slope of the obtained work profile estimates the required force (thin black line). Perforation (red arrow) requires a force of 80 pN for the wild-type C-termini. The plateau within the work profile indicates that the membrane gives up its elastic response (perforation transition). C-terminal tags oppose such a perforation transition but do not significantly affect the forces observed within the indentation regime. Furthermore, if the unstructured peptide-spacer would be alpha-helical - and thus much shorter - indentation forces would still be only slightly affected.


( B ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting .

O 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) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.


( C ) In vivo vacuole morphology was assayed by FM4 - 64 staining . Scale bar : 5 µm . ( D ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

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

(C) In vivo vacuole morphology was assayed by FM4-64 staining. Scale bar: 5 µm. (D) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( E ) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8 - EGFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Scale bar : 5 µm . ( F ) Fusion activity : vacuole fusion was assayed by measuring the percentage of co - localization of the two Pho8 - EGFP and Pho8 - mCherry signals . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments .

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

(E) Vacuoles were isolated from BJ3505 strains expressing the indicated tagged versions of Vam3 and Nyv1 and Pho8-EGFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Scale bar: 5 µm. (F) Fusion activity: vacuole fusion was assayed by measuring the percentage of co-localization of the two Pho8-EGFP and Pho8-mCherry signals. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments.


( A ) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and / or Nyv1 . 10 µg of the organelles were analyzed by SDS - PAGE and Western blotting using antibodies to Vti1 , Vam3 and Nyv1 .

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

(A) Vacuoles were isolated from BJ3505 cells expressing the indicated versions of Vam3 and/or Nyv1. 10 µg of the organelles were analyzed by SDS-PAGE and Western blotting using antibodies to Vti1, Vam3 and Nyv1.


( B ) Localization in vivo . The indicated strains were grown logarithmically in synthetic complete ( SC ) medium . After 16 h at 30 ° C , when the final OD of each culture was about 1 , the cells were observed by confocal fluorescence and differential interference ( DIC ) microscopy . Scale bar : 5 µm .

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

(B) Localization in vivo. The indicated strains were grown logarithmically in synthetic complete (SC) medium. After 16 h at 30°C, when the final OD of each culture was about 1, the cells were observed by confocal fluorescence and differential interference (DIC) microscopy. Scale bar: 5 µm.


( A ) In vivo morphology . Cells expressing the indicated SNARE variants were grown in SC medium ( 16 h , 30 ° C ) , stained with FM4 - 64 and their relative morphology was assessed by fluorescence and DIC microscopy . Scale bar : 5 µm . The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

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

(A) In vivo morphology. Cells expressing the indicated SNARE variants were grown in SC medium (16 h, 30°C), stained with FM4-64 and their relative morphology was assessed by fluorescence and DIC microscopy. Scale bar: 5 µm. The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


( B ) and ( C ) SNARE activation on isolated vacuoles . Vacuoles were isolated from a strain co - expressing NYV1 - S9 - EGFP and VAM3 - S9 - mCitrine and from an isogenic wild - type . 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP - regenerating system and recombinant , purified Sec18 / NSF ( rSec18 , 50 µg / ml ) . After 10 min of incubation at 27 ° C , the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1 . Co - immunoprecipitated proteins analyzed by SDS - PAGE and Western blotting . The histograms provide quantifications of the band intensities as the means and s . d . from three independent experiments .

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

(B) and (C) SNARE activation on isolated vacuoles. Vacuoles were isolated from a strain co-expressing NYV1-S9-EGFP and VAM3-S9-mCitrine and from an isogenic wild-type. 150 µg of the organelles were incubated in fusion reactions in the presence or absence of an ATP-regenerating system and recombinant, purified Sec18/NSF (rSec18, 50 µg/ml). After 10 min of incubation at 27°C, the vacuoles were solubilized and immunoprecipitated with antibodies to Nyv1. Co-immunoprecipitated proteins analyzed by SDS-PAGE and Western blotting. The histograms provide quantifications of the band intensities as the means and s.d. from three independent experiments.


( A ) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS - PAGE and Western blotting .

(A) Vacuoles were isolated from the indicated yeast strains and 30 µg of proteins analyzed by SDS-PAGE and Western blotting.


( B ) Vacuole morphology was assessed for the indicated cells as in Fig . 3C . Scale bar : 5 µm .

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

(B) Vacuole morphology was assessed for the indicated cells as in Fig. 3C. Scale bar: 5 µm.


( C ) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated . Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [ 65 ] but ensures that trans - SNARE complexes can only form in one orientation , between the Q - SNARE in DKY6281 and the R - SNARE in BJ3505 . The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay . In parallel , identical samples were incubated either incubated on ice , which prevents fusion , or in the presence of 0 . 5 % Triton X - 100 , which allows fusion - independent access of the maturase Pep4 to pro - ALP and controls for the levels of these two reporter enzymes in the samples .

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

(C) Vacuoles were isolated from BJ3505 and DKY6281 cells with SNAREs tagged or deleted as indicated. Note that presence of Nyv1 on only one fusion partner still permits efficient fusion [65] but ensures that trans-SNARE complexes can only form in one orientation, between the Q-SNARE in DKY6281 and the R-SNARE in BJ3505. The vacuoles were used in standard fusion reactions and content mixing was measured via the alkaline phosphatase assay. In parallel, identical samples were incubated either incubated on ice, which prevents fusion, or in the presence of 0.5% Triton X-100, which allows fusion-independent access of the maturase Pep4 to pro-ALP and controls for the levels of these two reporter enzymes in the samples.


( D ) Using strains from C , lipid and content mixing ( left and right panel , respectively ) were performed in parallel in the presence or absence of ATP . Anti - Vam3 antibody ( 3 µg ) was added to some reactions in order to inhibit trans - SNARE pairing and fusion . Means and the s . d . are shown for three independent experiments .

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

(D) Using strains from C, lipid and content mixing (left and right panel, respectively) were performed in parallel in the presence or absence of ATP. Anti-Vam3 antibody (3 µg) was added to some reactions in order to inhibit trans-SNARE pairing and fusion. Means and the s.d. are shown for three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( A ) Protein levels on isolated vacuoles from these strains were compared as in Fig . 3B .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (A) Protein levels on isolated vacuoles from these strains were compared as in Fig. 3B.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( B ) Vacuole morphology was assessed as in Fig . 3C . Scale bar : 5 µm . ( C ) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells . Values represent the means and s . d . from three independent experiments .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (B) Vacuole morphology was assessed as in Fig. 3C. Scale bar: 5 µm. (C) The cells were grouped into three categories according to the number of vacuoles visible per 100 cells. Values represent the means and s. d. from three independent experiments.


Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig . 3A but the spacer was extended ( S34 ) by an additional 25 - amino acid sequence ( SGGGGSGGGGSGGGGSGGGGAAAGG ) to the previous short spacer . ( D ) Fusion activity : vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8 - mCFP or Pho8 - mCherry . 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy . Arrows indicate examples of fusion products . Means s . d . are shown for at least 100 stained vacuoles from 3 experiments . Scale bar : 5 µm . ( E ) Vacuole fusion was assayed by measuring the percentage of co - localisation of the two Pho8 - mCFP and Pho8 - mCherry signals .

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

Vam3 and Nyv1 were tagged with mCitrine and EGFP as shown Fig. 3A but the spacer was extended (S34) by an additional 25-amino acid sequence (SGGGGSGGGGSGGGGSGGGGAAAGG) to the previous short spacer. (D) Fusion activity: vacuoles were isolated from BJ3505 strains expressing the indicated versions of Vam3 and Nyv1 and Pho8-mCFP or Pho8-mCherry. 10 µg of vacuoles were incubated in standard fusion reactions in the presence or absence of ATP and analyzed by confocal microscopy. Arrows indicate examples of fusion products. Means s.d. are shown for at least 100 stained vacuoles from 3 experiments. Scale bar: 5 µm. (E) Vacuole fusion was assayed by measuring the percentage of co-localisation of the two Pho8-mCFP and Pho8-mCherry signals.


( A ) Yeast strain expressing a plasmid with Vph1 - GFP was grown in SC - URA medium for 16 h at 30 ° C . The mobility of Vph1 - GFP in the absence ( upper panels ) or presence ( lower panel ) of CPZ ( 150 µM ) was assessed by FRAP . ( B ) The histogram shows the GFP signals in the bleached area during the FRAP procedure . ( C ) The half - time is shown . Means and s . d . are shown for 20 cells of 3 independent experiments .

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

(A) Yeast strain expressing a plasmid with Vph1-GFP was grown in SC-URA medium for 16 h at 30°C. The mobility of Vph1-GFP in the absence (upper panels) or presence (lower panel) of CPZ (150 µM) was assessed by FRAP. (B) The histogram shows the GFP signals in the bleached area during the FRAP procedure. (C) The half-time is shown. Means and s.d. are shown for 20 cells of 3 independent experiments.


( D ) Yeast cells were grown in SC medium for 16 h at 30 ° C . After staining with FM4 - 64 , cells were treated with CPZ ( 150 µM ) . Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition . Scale bar : 5 µm .

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(D) Yeast cells were grown in SC medium for 16 h at 30°C. After staining with FM4-64, cells were treated with CPZ (150 µM). Vacuole morphology was assessed by confocal microscopy before and after 5 and 20 min of CPZ addition. Scale bar: 5 µm.


( E ) Fusion activity . Vacuoles were isolated from BJ cells carrying Nyv1 - S9 - EGFP and DKY cells deleted for Nyv1 and carrying Vam3 - S9 - mCitrine or from wild - type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine ( CPZ , 150 µM ) and anti - Vam3 ( 3 µg ) . Content mixing was assayed via alkaline phosphatase activity . Means and s . d . are shown for three independent experiments .

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(E) Fusion activity. Vacuoles were isolated from BJ cells carrying Nyv1-S9-EGFP and DKY cells deleted for Nyv1 and carrying Vam3-S9-mCitrine or from wild-type cells and incubated in standard fusion reactions in the presence or absence of chlorpromazine (CPZ, 150 µM) and anti-Vam3 (3 µg). Content mixing was assayed via alkaline phosphatase activity. Means and s.d. are shown for three independent experiments.


( A ) OCR of wt and gas - 1 mutant nematodes ( normalized to nematode size as given by the COPAS Biosort ( Time of Flight - TOF ) ) . One representative curve shown ( mean ± SEM ; n = 3 ) * * * * p value < 0 . 0001 ; Kruskal - Wallis Test , Dunn ' s post hoc correction .

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(A) OCR of wt and gas-1 mutant nematodes (normalized to nematode size as given by the COPAS Biosort (Time of Flight -TOF)). One representative curve shown (mean ± SEM; n=3) ****p value< 0.0001; Kruskal-Wallis Test, Dunn's post hoc correction.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( B ) age - 1 and age - 1 ; gas - 1 mutants animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (B) age-1 and age-1; gas-1 mutants animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


Representative curves show the lifespan of wt and gas - 1 ( fc21 ) nematodes compared to ( C ) daf - 16 and daf - 16 ; age - 1 ; gas - 1 mutant animals . Average median lifespans ± SEM from all replicates indicated beneath representative curves .

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Representative curves show the lifespan of wt and gas-1(fc21) nematodes compared to (C) daf-16 and daf-16; age-1; gas-1 mutant animals. Average median lifespans ± SEM from all replicates indicated beneath representative curves.


( D ) Volcano plot of differentially phosphorylated proteins in gas - 1 ( fc21 ) mutants compared to wt . Cutoffs : 20 % FDR and | log2 ( foldchange ) | > 0 . 345 ( in red ) .

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(D) Volcano plot of differentially phosphorylated proteins in gas-1(fc21) mutants compared to wt. Cutoffs: 20% FDR and |log2(foldchange)| >0.345 (in red).


( F ) Lifespan analysis of mev - 1 ( kn1 ) nematodes compared to wt , age - 1 ( hx546 ) and age - 1 ( hx546 ) ; mev - 1 ( kn1 ) animals . ( G )

(F) Lifespan analysis of mev-1(kn1) nematodes compared to wt, age-1(hx546) and age-1(hx546); mev-1(kn1) animals. (G)


( G ) ATP measurements in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to wt ) , n = 4 . Error bars : Mean ± SEM , * p value < 0 . 05 , * * * p value = 0 . 001 , Kruskal - Wallis Test , Dunn ' s post hoc correction .

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(G) ATP measurements in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (normalized to wt), n=4. Error bars: Mean ± SEM, *p value <0.05, ***p value =0.001, Kruskal-Wallis Test, Dunn's post hoc correction.


( H ) OCR of gas - 1 and age - 1 ; gas - 1 mutant nematodes ( normalized to TOF ) . Left panel : One representative curve ( Mean ± SEM ) . Right panel : Basal respiration in wt , gas - 1 and age - 1 ; gas - 1 animals , pooled from 3 biological replicates , * * * * p value < 0 . 0001 , ns = non - significant , Kruskal - Wallis Test , Dunn ' s post hoc correction .

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(H) OCR of gas-1 and age-1; gas-1 mutant nematodes (normalized to TOF). Left panel: One representative curve (Mean ± SEM). Right panel: Basal respiration in wt, gas-1 and age-1; gas-1 animals, pooled from 3 biological replicates, ****p value <0.0001, ns= non-significant, Kruskal-Wallis Test, Dunn's post hoc correction.


( I ) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms . Scale bar : 2 μm . ( J ) Left panel : Percentage of FRAP . Each point : mean recovery of ~ 30 bleached regions ( 2 regions per animal , 15 animals per condition ) ± SEM . Right panel : values of the maximum ( total ) recovery per strain ± SEM , * * * p value < 0 . 001 , * * * * p value < 0 . 0001 , one - way ANOVA , Tukey ' s post - hoc correction .

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(I) STED images of representative mitochondrial morphology in muscle cells of L4 mutant worms. Scale bar: 2 μm. (J) Left panel: Percentage of FRAP. Each point: mean recovery of ~30 bleached regions (2 regions per animal, 15 animals per condition) ± SEM. Right panel: values of the maximum (total) recovery per strain ± SEM, ***p value <0.001, **** p value <0.0001, one-way ANOVA, Tukey's post-hoc correction.


( K ) Representative lifespan of age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and fzo - 1 RNAi bacteria . Average median lifespans ± SEM from all replicates indicated beneath representative curves . For all panels : wt = wild type , a = age - 1 , g = gas - 1 , a ; g = age - 1 ; gas - 1 .

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(K) Representative lifespan of age-1; gas-1 nematodes fed with empty vector (EV) and fzo-1 RNAi bacteria. Average median lifespans ± SEM from all replicates indicated beneath representative curves. For all panels: wt= wild type, a= age-1, g= gas-1, a; g= age-1; gas-1.


( A ) Left panel : Venn Diagram of the number of genes significantly dysregulated in age - 1 ; gas - 1 versus wt ( green ) and gas - 1 versus wt ( red ) . Right panel : Scatter - plots of the expression of genes significantly downregulated ( left ) or upregulated ( right ) in gas - 1 and age - 1 ; gas - 1 mutants compared to wt . In black : expression levels of genes that are significantly up - or down - regulated in gas - 1 mutants compared to wt . In red : expression levels of the same genes in age - 1 ; gas - 1 mutants compared to wt .

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(A) Left panel: Venn Diagram of the number of genes significantly dysregulated in age-1; gas-1 versus wt (green) and gas-1 versus wt (red). Right panel: Scatter-plots of the expression of genes significantly downregulated (left) or upregulated (right) in gas-1 and age-1; gas-1 mutants compared to wt. In black: expression levels of genes that are significantly up- or down-regulated in gas-1 mutants compared to wt. In red: expression levels of the same genes in age-1; gas-1 mutants compared to wt.


( C ) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative

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(C) Representative lifespans of the indicated strains with average median lifespans ± SEM from all replicates indicated beneath representative curves


( D ) Volcano plot of TMT / iTRAQ - based quantitative proteomic analysis . In red , proteins significantly dysregulated in age - 1 ; gas - 1 compared to gas - 1 ( FDR < 20 % ; | log2 ( fold change ) | > 0 . 47 ) . Dashed lines indicate cutoffs . Proteins involved in carbohydrate ( green ) and lipid ( blue ) metabolism are labeled .

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(D) Volcano plot of TMT/iTRAQ-based quantitative proteomic analysis. In red, proteins significantly dysregulated in age-1; gas-1 compared to gas-1 (FDR< 20%; |log2 (fold change)| >0.47). Dashed lines indicate cutoffs. Proteins involved in carbohydrate (green) and lipid (blue) metabolism are labeled.


( H ) Left panel : Glucose quantification in wt , gas - 1 and age - 1 ; gas - 1 nematode lysates with NMR , n = 7 , ns = non - significant , Mann Whitney U test . Right panel : Quantification of total carbohydrates in the indicated strains with phenol - sulfuric assay . Error bars : mean ± SEM , 2 biological replicates ( 5 technical replicates each ) , normalized to wt and number of animals used . * * p value < 0 . 01 , ns = non - significant , Mann - Whitney U test .

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(H) Left panel: Glucose quantification in wt, gas-1 and age-1; gas-1 nematode lysates with NMR, n=7, ns= non-significant, Mann Whitney U test. Right panel: Quantification of total carbohydrates in the indicated strains with phenol-sulfuric assay. Error bars: mean ± SEM, 2 biological replicates (5 technical replicates each), normalized to wt and number of animals used. **p value < 0.01, ns= non-significant, Mann-Whitney U test.


( C ) Immunoblot analysis of phospho - AAK - 2 in wt , age - 1 , gas - 1 and age - 1 ; gas - 1 mutant nematodes ( actin loading control ) . Numbers correspond to band densitometries across 3 biological replicates .

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(C) Immunoblot analysis of phospho-AAK-2 in wt, age-1, gas-1 and age-1; gas-1 mutant nematodes (actin loading control). Numbers correspond to band densitometries across 3 biological replicates.


( D ) Representative lifespan of age - 1 ; gas - 1 double mutant and age - 1 ; aak - 2 ; gas - 1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves .

(D) Representative lifespan of age-1; gas-1 double mutant and age-1; aak-2; gas-1 triple mutant nematodes with average median lifespans ± SEM from all replicates indicated beneath representative curves.


( E ) Lifespan assay of gas - 1 mutant animals compared to gas - 1 ; AAK - 2 overexpressing ( O / E ) nematodes and their wt counterparts . Inset : immunoblot analysis of phospho - AAK - 2 in gas - 1 ( g ) and gas - 1 ; AAK - 2 O / E ( g ; a O / E ) animals ( actin loading control ) , showing the overexpressed protein ( lower band ) and the endogenous one ( upper band ) .

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(E) Lifespan assay of gas-1 mutant animals compared to gas-1; AAK-2 overexpressing (O/E) nematodes and their wt counterparts. Inset: immunoblot analysis of phospho-AAK-2 in gas-1 (g) and gas-1; AAK-2 O/E (g; a O/E) animals (actin loading control), showing the overexpressed protein (lower band) and the endogenous one (upper band).


( F ) Left panel : Immunoblot analysis of phospho - KIN - 1 substrates in young adult nematodes . Numbers correspond to band densitometries across 2 biological replicates . Right panel : immunoblot analysis of phospho - KIN - 1 substrates in age - 1 ; gas - 1 animals grown on bacteria carrying an empty vector ( EV ) or expressing kin - 1 RNAi . Two different kin - 1 RNAi clones ( g1 , b5 ) were used in this experiment . Numbers correspond to band densitometries of the presented immunoblot . Dashed rectangles : band corresponding to downregulated KIN - 1 phospho - substrate .

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(F) Left panel: Immunoblot analysis of phospho-KIN-1 substrates in young adult nematodes. Numbers correspond to band densitometries across 2 biological replicates. Right panel: immunoblot analysis of phospho-KIN-1 substrates in age-1; gas-1 animals grown on bacteria carrying an empty vector (EV) or expressing kin-1 RNAi. Two different kin-1 RNAi clones (g1, b5) were used in this experiment. Numbers correspond to band densitometries of the presented immunoblot. Dashed rectangles: band corresponding to downregulated KIN-1 phospho-substrate.


( G ) Representative lifespan assay of wt and age - 1 ; gas - 1 nematodes fed with empty vector ( EV ) and kin - 1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves .

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(G) Representative lifespan assay of wt and age-1; gas-1 nematodes fed with empty vector (EV) and kin-1 RNAi bacteria with average median lifespans ± SEM from all replicates indicated beneath representative curves.


ATP measurements in : ( H ) age - 1 ( hx546 ) ; gas - 1 ( fc21 ) ( a ; g ) and age - 1 ( hx546 ) ; aak - 2 ( ok524 ) ; gas - 1 ( fc21 ) ( a ; aa ; g ) mutant nematodes ;

ATP measurements in: (H) age-1(hx546); gas-1(fc21) (a; g) and age-1(hx546); aak-2(ok524); gas-1(fc21) (a; aa; g) mutant nematodes;