Figure 5A . Transwell migration of MDA - MB - 231 cells treated with control PMOs or RAB13 PMOs ( 191 + 230 ) . Cells reaching the bottom surface after 4hrs were counted . n = 25 fields of view in each of 6 independent experiments . Bars : mean ± s . e . m . . B . Cells expressing Cherry - NLS , and treated with the indicated PMOs , were tracked every 5min for 10hrs to derive average migration speed . n = 65 cells . Bars : mean ± s . e . m . . C . PMO - treated cells were induced to invade through a Matrigel plug . Cell staining intensity in arbitrary units ( a . u . ) was used to quantify relative invasion from n = 4 independent experiments . Bars : mean ± s . e . m . . D . Lifeact - GFP - expressing cells were treated with the indicated PMOs and imaged every minute over 1 hr . Sequential image frames , from a control cell , highlight edge retraction ( red arrowheads ) or protrusion ( yellow arrowheads ) . Corresponding edge velocity is shown , with negative values indicating retraction and positive values indicating extension . Average protrusion and retraction speeds were calculated from n = 11 - 13 cells . Bars : mean ± s . e . m . . See also movies EV2 and EV3 . E . Cells treated with the indicated PMOs or siRNAs were analyzed by western blot ( upper panels ) to detect RAB13 and GAPDH protein levels . Migration speed was assessed as in ( B ) from n = 55 - 78 cells ( bottom graph ) . Bars : mean ± s . e . m . . F . Protrusion and retraction speed of cells treated with the indicated PMOs or siRNAs was assessed as in ( D ) . Graphs show average normalized values from n = 10 - 13 cells imaged for 1 hr . Bars : mean ± s . e . m . . G . Migration speed of RAB13 knockdown cells re - expressing GFP - RAB13 or GFP - RAB13 with a frameshift ( fs ) mutation at the beginning of the RAB13 coding region . 28 - 52 cells were analyzed . Bars : mean ± s . e . m . . Similar results were obtained in two additional independent experiments . Re - expressing cells were identified through GFP fluorescence and cells with similar , low GFP signal were tracked . All cells are expressing cherry - NLS for accurate tracking .

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 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 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 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 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 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

Figure 5A. Transwell migration of MDA-MB-231 cells treated with control PMOs or RAB13 PMOs (191+230). Cells reaching the bottom surface after 4hrs were counted. n=25 fields of view in each of 6 independent experiments. Bars: mean ± s.e.m..B. Cells expressing Cherry-NLS, and treated with the indicated PMOs, were tracked every 5min for 10hrs to derive average migration speed. n=65 cells. Bars: mean ± s.e.m..C. PMO-treated cells were induced to invade through a Matrigel plug. Cell staining intensity in arbitrary units (a.u.) was used to quantify relative invasion from n=4 independent experiments. Bars: mean ± s.e.m..D. Lifeact-GFP-expressing cells were treated with the indicated PMOs and imaged every minute over 1 hr. Sequential image frames, from a control cell, highlight edge retraction (red arrowheads) or protrusion (yellow arrowheads). Corresponding edge velocity is shown, with negative values indicating retraction and positive values indicating extension. Average protrusion and retraction speeds were calculated from n=11-13 cells. Bars: mean ± s.e.m..See also movies EV2 and EV3.E. Cells treated with the indicated PMOs or siRNAs were analyzed by western blot (upper panels) to detect RAB13 and GAPDH protein levels. Migration speed was assessed as in (B) from n=55-78 cells (bottom graph). Bars: mean ± s.e.m..F. Protrusion and retraction speed of cells treated with the indicated PMOs or siRNAs was assessed as in (D). Graphs show average normalized values from n=10-13 cells imaged for 1 hr. Bars: mean ± s.e.m..G. Migration speed of RAB13 knockdown cells re-expressing GFP-RAB13 or GFP-RAB13 with a frameshift (fs) mutation at the beginning of the RAB13 coding region. 28-52 cells were analyzed. Bars: mean ± s.e.m.. Similar results were obtained in two additional independent experiments. Re-expressing cells were identified through GFP fluorescence and cells with similar, low GFP signal were tracked. All cells are expressing cherry-NLS for accurate tracking.


Figure 6A . Widefield images of RAB13 immunofluorescence in MDA - MB - 231 cells treated with control or RAB13 ( 191 + 230 ) PMOs and ratios of peripheral / perinuclear intensity . Scale bars : 10 μm . n = 45 - 50 cells . Bars : mean ± s . e . m . . Similar results were observed in two additional independent experiments . B . Fluorescence images ( projections of confocal slices throughout the cell height ) of cells expressing GFP - RAB13 with the indicated UTRs . Note that in both cases the protein assumes indistinguishable distribution . Scale bars : 10 μm . C . Soluble / particulate fractionation of the indicated cell lines followed by Western blot to detect the indicated proteins . RhoGDI and TfRc serve as soluble and particulate markers , respectively . Graph shows quantitation from n = 3 independent experiments . Bars : mean ± s . e . m . . Active RAB13 ( RAB13 - GTP ) was pulled - down using MICAL - L1 RBD - GST from the indicated PMO - treated cells The amount of endogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted . n = 8 ( D ) , n = 4 ( E ) . Bars : mean ± s . e . m . . E . Active RAB13 ( RAB13 - GTP ) was pulled - down using MICAL - L1 RBD - GST from GFP - RAB13 expressing lines The amount of endogenous or exogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted . n = 8 ( D ) , n = 4 ( E ) . Bars : mean ± s . e . m . .

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

Figure 6A. Widefield images of RAB13 immunofluorescence in MDA-MB-231 cells treated with control or RAB13 (191+230) PMOs and ratios of peripheral/perinuclear intensity. Scale bars: 10 μm. n=45-50 cells. Bars: mean ± s.e.m.. Similar results were observed in two additional independent experiments.B. Fluorescence images (projections of confocal slices throughout the cell height) of cells expressing GFP-RAB13 with the indicated UTRs. Note that in both cases the protein assumes indistinguishable distribution. Scale bars: 10 μm.C. Soluble/particulate fractionation of the indicated cell lines followed by Western blot to detect the indicated proteins. RhoGDI and TfRc serve as soluble and particulate markers, respectively. Graph shows quantitation from n=3 independent experiments. Bars: mean ± s.e.m..Active RAB13 (RAB13-GTP) was pulled-down using MICAL-L1 RBD-GST from the indicated PMO-treated cells The amount of endogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted. n=8 (D), n=4 (E). Bars: mean ± s.e.m..E. Active RAB13 (RAB13-GTP) was pulled-down using MICAL-L1 RBD-GST from GFP-RAB13 expressing lines The amount of endogenous or exogenous RAB13 was measured by quantitative Western and relative levels of active RAB13 are plotted. n=8 (D), n=4 (E). Bars: mean ± s.e.m..


Figure 7A . Immunoprecipitation and Western blot analysis to detect proteins bound to GFP - RAB13 expressed from constructs carrying wt or ∆ PMO RAB13 3 ' UTR . GFP - RAB13 ( T22N ) expresses a nucleotide - free mutant which binds tightly to putative GEFs . RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP - RAB13 . B . Quantification of RABIF binding to RAB13 from experiments as in ( A ) . n = 6 . Bars : mean ± s . e . m . . C . Active RAB13 pulldown assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs . n = 3 . Bars : mean ± s . e . m . . D . Quantification of RABIF - RAB13 PLA signal from cells transfected with the indicated siRNAs . Number of observed cells is indicated within each bar . Bars : mean ± s . e . m . . Similar results were observed in one additional independent experiment . E . Representative RABIF - RAB13 PLA images and quantitations from cells transfected with the indicated PMOs . N > 70 cells . Bars : mean ± s . e . m . . Arrows indicate PLA signal at the cell periphery . Arrowheads indicate PLA signal within the cell body . Boxed regions are magnified in the insets . Scale bars : 15 μm ; 5 μm in insets . F . Representative RABIF - GFP PLA images and quantitations from cells expressing GFP - RAB13 ( T22N ) carrying wt or ∆ PMO RAB13 3 ' UTR . N > 70 cells . Bars : mean ± s . e . m . . Arrows indicate PLA signal at the cell periphery . Arrowheads indicate PLA signal within the cell body . Boxed regions are magnified in the insets . Scale bars : 10 μm ; 4 μm in insets .

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 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 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 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

Figure 7A. Immunoprecipitation and Western blot analysis to detect proteins bound to GFP-RAB13 expressed from constructs carrying wt or ∆PMO RAB13 3'UTR. GFP-RAB13 (T22N) expresses a nucleotide-free mutant which binds tightly to putative GEFs. RABIF panel is also shown with adjusted contrast to reveal lower binding to wtGFP-RAB13. B. Quantification of RABIF binding to RAB13 from experiments as in (A). n=6. Bars: mean ± s.e.m.. C. Active RAB13 pulldown assay from cells with CRISPR knockdown of RABIF using the indicated sgRNAs. n=3. Bars: mean ± s.e.m..D. Quantification of RABIF-RAB13 PLA signal from cells transfected with the indicated siRNAs. Number of observed cells is indicated within each bar. Bars: mean ± s.e.m.. Similar results were observed in one additional independent experiment.E. Representative RABIF-RAB13 PLA images and quantitations from cells transfected with the indicated PMOs. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 15 μm; 5 μm in insets.F. Representative RABIF-GFP PLA images and quantitations from cells expressing GFP-RAB13(T22N) carrying wt or ∆PMO RAB13 3'UTR. N>70 cells. Bars: mean ± s.e.m.. Arrows indicate PLA signal at the cell periphery. Arrowheads indicate PLA signal within the cell body. Boxed regions are magnified in the insets. Scale bars: 10 μm; 4 μm in insets.


Figure 8B . Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines . Note that even though RABIF binds several - fold more to RAB13 ( T22N ) ( see Figure 7A , B ) it binds similarly to the wild - type and T22N RAB13 RNA , indicating that RABIF binds similarly to nascent RAB13 . After translation , it is likely displaced upon GTP - loading of wild type RAB13 , while it remains more stably bound to the nucleotide - free ( T22N ) form . N = 6 . Bars : mean ± s . e . m . . p - values : * < 0 . 05 by analysis of variance with Dunn ' s multiple comparisons 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

Figure 8B. Quantification of GFP RNA levels associating with RABIF in immunoprecipitation assays from the indicated cell lines. Note that even though RABIF binds several-fold more to RAB13(T22N) (see Figure 7A, B) it binds similarly to the wild-type and T22N RAB13 RNA, indicating that RABIF binds similarly to nascent RAB13. After translation, it is likely displaced upon GTP-loading of wild type RAB13, while it remains more stably bound to the nucleotide-free (T22N) form. N=6. Bars: mean ± s.e.m.. p-values: *<0.05 by analysis of variance with Dunn's multiple comparisons test.


Figure 1 ( A - B ) Mitochondria isolated from ( A ) hTim8aKO HEK cells with and without hTim8aFLAG re - expression , or ( B ) hTim8bKO HEK cells with and without hTim8bFLAG re - expression were compared to mitochondria isolated from control HEK293 cells by SDS - PAGE and immunoblot . Protein expression of the hTim8 protein was induced with tetracycline ( Tet ) for the indicated time . Representative of n = 2 biological replicates . Affinity enrichment mass spectrometry . Mitochondria isolated from ( C ) hTim8aKO HEK cells expressing hTim8aFLAG , ( D ) hTim8bKO HEK cells expressing hTim8bFLAG , ( E ) hTim8aMUT SH cells expressing hTim8aFLAG , ( F ) hTim8bKO SH cells expressing hTim8bFLAG were treated with DSP crosslinker prior to immunoprecipitation with anti - FLAG resin . The log2 fold change in mean LFQ intensity is plotted against unpaired Student ' s t - test p - value ( n = 3 biological replicates ) . Curve indicates significantly enriched proteins . FDR < 0 . 01 ; ( C ) s0 = 5 . 5 , ( D ) s0 = 4 . 5 , ( E ) s0 = 2 , ( F ) s0 = 5 , Functional annotations manually curated and coloured : TIMM family proteins ( purple ) , Complex I subunits and assembly factors ( orange ) , Complex IV subunits and assembly factors ( green ) , MIA import substrates ( blue ) , significantly enriched ( black ) .

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 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

Figure 1(A-B) Mitochondria isolated from (A) hTim8aKO HEK cells with and without hTim8aFLAG re-expression, or (B) hTim8bKO HEK cells with and without hTim8bFLAG re-expression were compared to mitochondria isolated from control HEK293 cells by SDS-PAGE and immunoblot. Protein expression of the hTim8 protein was induced with tetracycline (Tet) for the indicated time. Representative of n=2 biological replicates.Affinity enrichment mass spectrometry. Mitochondria isolated from (C) hTim8aKO HEK cells expressing hTim8aFLAG, (D) hTim8bKO HEK cells expressing hTim8bFLAG, (E) hTim8aMUT SH cells expressing hTim8aFLAG, (F) hTim8bKO SH cells expressing hTim8bFLAG were treated with DSP crosslinker prior to immunoprecipitation with anti-FLAG resin. The log2 fold change in mean LFQ intensity is plotted against unpaired Student's t-test p-value (n=3 biological replicates). Curve indicates significantly enriched proteins. FDR<0.01; (C) s0=5.5, (D) s0=4.5, (E) s0=2, (F) s0=5, Functional annotations manually curated and coloured: TIMM family proteins (purple), Complex I subunits and assembly factors (orange), Complex IV subunits and assembly factors (green), MIA import substrates (blue), significantly enriched (black).


Figure 2Changes in protein abundance of hTim8a and hTim8b interactors in mitochondria isolated from ( B ) hTim8aKO HEK and hTim8bKO HEK , or ( C ) hTim8aMUT SH and hTim8bKO SH cells ; determined by quantitative mass spectrometry as published in Quadrants are labelled with 8a / 8b ↑ or ↓ to indicate the increase or decrease of protein abundances in each knock - out cell line . The log2 fold change in mean LFQ intensity between knock - out and control cells is plotted on each axis . Lines indicate fold change in abundance of ± 1 . 5 . n = 3 biological replicates for each cell line . Functional annotations manually curated and coloured : TIMM family proteins ( purple ) , Complex I subunits and assembly factors ( orange ) , Complex IV subunits and assembly factors ( green ) , MIA import substrates ( blue ) , significantly altered ( black ) .

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

Figure 2Changes in protein abundance of hTim8a and hTim8b interactors in mitochondria isolated from (B) hTim8aKO HEK and hTim8bKO HEK, or (C) hTim8aMUT SH and hTim8bKO SH cells; determined by quantitative mass spectrometry as published in Quadrants are labelled with 8a/8b ↑ or ↓ to indicate the increase or decrease of protein abundances in each knock-out cell line. The log2 fold change in mean LFQ intensity between knock-out and control cells is plotted on each axis. Lines indicate fold change in abundance of ±1.5. n=3 biological replicates for each cell line. Functional annotations manually curated and coloured: TIMM family proteins (purple), Complex I subunits and assembly factors (orange), Complex IV subunits and assembly factors (green), MIA import substrates (blue), significantly altered (black).


Figure 3 ( B - C ) Mitochondria isolated from ( B ) COX17MUT HEK and ( C ) COX17KO SH cells were analysed by quantitative mitochondrial proteomics relative to control cells . The log2 fold change in mean LFQ intensity is plotted against unpaired Student ' s t - test p - value ( n = 3 biological replicates ) . Curve indicates significantly altered proteins ; FDR < 0 . 05 ; s0 = 0 . 5 . Functional annotations manually curated and coloured : Complex I subunits and assembly factors ( orange ) , Complex II subunits and assembly factors ( yellow ) , Complex III subunits and assembly factors ( red ) Complex IV subunits and assembly factors ( green ) , Complex V subunits and assembly factors ( pink ) , TIMM family proteins ( purple ) , significantly altered ( black ) . ( D ) Mitochondria isolated from control cells , COX17MUT HEK and COX17KO SH cells were analysed by BN - PAGE and immunoblotting with the indicated antibodies . COX4I1 - containing complexes indicated as SC : Respiratory supercomplex , CIII2 + CIV : Complex III2 - Complex IV complex , CIV2 : Complex IV dimer , CIV : mature Complex IV , and S2 * : COX4I1 - containing S2 subcomplex . Representative of n = 2 biological replicates . ( F ) Intact cells or mitochondria isolated from hTim8bKO HEK and control HEK293 cells were subject to ICP - MS analysis to measure copper ( Cu ) and iron ( Fe ) content . Abundance is shown as mean ± S . D for metal content per gram of protein . Significance determined by unpaired Student ' s t - test ( n = 3 biological replicates ) . * * , p < 0 . 01 ; ' ns ' indicates not significant , p > 0 . 05 . ( G ) Recombinant COA6 , hTim8a and hTim8b were purified and incubated , in increasing concentrations , with Cu ( I ) and bathocuproine disulfonic acid ( BCS ) . Protein - Cu ( I ) binding outcompetes BCS2 - Cu ( I ) binding and the decrease in BCS2 - Cu ( I ) is shown by absorbance , particularly at 482 nm . n = 1 biological replicates .

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 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 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 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

Figure 3(B-C) Mitochondria isolated from (B) COX17MUT HEK and (C) COX17KO SH cells were analysed by quantitative mitochondrial proteomics relative to control cells. The log2 fold change in mean LFQ intensity is plotted against unpaired Student's t-test p-value (n=3 biological replicates). Curve indicates significantly altered proteins; FDR<0.05; s0=0.5. Functional annotations manually curated and coloured: Complex I subunits and assembly factors (orange), Complex II subunits and assembly factors (yellow), Complex III subunits and assembly factors (red) Complex IV subunits and assembly factors (green), Complex V subunits and assembly factors (pink), TIMM family proteins (purple), significantly altered (black).(D) Mitochondria isolated from control cells, COX17MUT HEK and COX17KO SH cells were analysed by BN-PAGE and immunoblotting with the indicated antibodies. COX4I1-containing complexes indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Representative of n=2 biological replicates.(F) Intact cells or mitochondria isolated from hTim8bKO HEK and control HEK293 cells were subject to ICP-MS analysis to measure copper (Cu) and iron (Fe) content. Abundance is shown as mean ±S.D for metal content per gram of protein. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.01; 'ns' indicates not significant, p>0.05.(G) Recombinant COA6, hTim8a and hTim8b were purified and incubated, in increasing concentrations, with Cu(I) and bathocuproine disulfonic acid (BCS). Protein-Cu(I) binding outcompetes BCS2-Cu(I) binding and the decrease in BCS2-Cu(I) is shown by absorbance, particularly at 482 nm. n=1 biological replicates.


Figure 4 ( A ) hTim8aKO HEK , hTim8bKO HEK and control HEK293 cells were pulsed with [ 35S ] - Met / Cys for 2 h in the presence of anisomycin and chased for the indicated times . Isolated mitochondria were analysed by SDS - PAGE , auto - radiography and immunoblot . n = 1 biological replicates . [ 35S ] - COX6C and [ 35S ] - COX7C were incubated with mitochondria isolated from ( B ) hTim8aMUT SH control SH - SY5Y Import proceeded in the presence or absence of mitochondrial membrane potential ( ΔΨ ) for the indicated times . Samples were analysed by BN - PAGE and autoradiography . CBB = Coomassie brilliant blue staining . Representative of n = 2 biological replicates . [ 35S ] - COX6C and [ 35S ] - COX7C were incubated with mitochondria isolated from ( C ) hTim8bKO HEK and control HEK23 cells . Import proceeded in the presence or absence of mitochondrial membrane potential ( ΔΨ ) for the indicated times . Samples were analysed by BN - PAGE and autoradiography . CBB = Coomassie brilliant blue staining . Representative of n = 2 biological replicates . ( F ) Mitochondria isolated from control , hTim8aMUT SH and hTim8bKO HEK cells were analysed by BN - PAGE and immunoblotting . COX4I1 - containing complexes indicated as SC : Respiratory supercomplex , CIII2 + CIV : Complex III2 - Complex IV complex , CIV2 : Complex IV dimer , CIV : mature Complex IV , and S2 * : COX4I1 - containing S2 subcomplex . Levels of COX4I1 present in subcomplex ( * ) was quantified as mean ± S . D percentage of COX4I1 present in mature Complex IV ( CIV ) for each cell line . Significance determined by unpaired Student ' s t - test ( hTim8bKO HEK n = 6 biological replicates ; hTim8aMUT SH n = 4 biological replicates ) . * * , p < 0 . 01 .

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 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 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

Figure 4(A) hTim8aKO HEK, hTim8bKO HEK and control HEK293 cells were pulsed with [35S]-Met/Cys for 2 h in the presence of anisomycin and chased for the indicated times. Isolated mitochondria were analysed by SDS-PAGE, auto-radiography and immunoblot. n=1 biological replicates.[35S]-COX6C and [35S]-COX7C were incubated with mitochondria isolated from (B) hTim8aMUT SH control SH-SY5Y Import proceeded in the presence or absence of mitochondrial membrane potential (ΔΨ) for the indicated times. Samples were analysed by BN-PAGE and autoradiography. CBB = Coomassie brilliant blue staining. Representative of n=2 biological replicates.[35S]-COX6C and [35S]-COX7C were incubated with mitochondria isolated from (C) hTim8bKO HEK and control HEK23 cells. Import proceeded in the presence or absence of mitochondrial membrane potential (ΔΨ) for the indicated times. Samples were analysed by BN-PAGE and autoradiography. CBB = Coomassie brilliant blue staining. Representative of n=2 biological replicates.(F) Mitochondria isolated from control, hTim8aMUT SH and hTim8bKO HEK cells were analysed by BN-PAGE and immunoblotting. COX4I1-containing complexes indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Levels of COX4I1 present in subcomplex (*) was quantified as mean ±S.D percentage of COX4I1 present in mature Complex IV (CIV) for each cell line. Significance determined by unpaired Student's t-test (hTim8bKO HEK n=6 biological replicates; hTim8aMUT SH n=4 biological replicates). **, p<0.01.


Figure 5 ( B - C ) Profile plots of iBAQ intensity for Complex IV subunits across the complexome profile of mitochondria isolated from ( B ) hTim8aMUT SH and control SH - SY5Y cells ; and ( C ) hTim8bKO HEK and control HEK293 cells . Subunits are plotted by Complex IV module and iBAQ intensity relative to the maximum intensity of plotted subunits . Solid line indicates subunits from control cells , dashed line from knock - out cells . Complex IV assemblies are indicated as SC : Respiratory supercomplex , CIII2 + CIV : Complex III2 - Complex IV complex , CIV : mature Complex IV , and S2 * : COX4I1 - containing S2 subcomplex . Representative of n = 2 biological replicates . Data presented as iBAQ intensity , normalised as in ( A ) and expressed as a ratio of the maximum iBAQ intensity .

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

Figure 5(B-C) Profile plots of iBAQ intensity for Complex IV subunits across the complexome profile of mitochondria isolated from (B) hTim8aMUT SH and control SH-SY5Y cells; and (C) hTim8bKO HEK and control HEK293 cells. Subunits are plotted by Complex IV module and iBAQ intensity relative to the maximum intensity of plotted subunits. Solid line indicates subunits from control cells, dashed line from knock-out cells. Complex IV assemblies are indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Representative of n=2 biological replicates. Data presented as iBAQ intensity, normalised as in (A) and expressed as a ratio of the maximum iBAQ intensity.


Figure 6 ( A ) Mitochondria isolated from hTim8aMUT SH cells with and without hTim8bFLAG expression compared to mitochondria from control SH - SY5Y cells were analysed by SDS - PAGE and immunoblotting . Amount of protein after 12 h induction ( lanes 4 and 10 ) was quantified as mean ± S . D percentage of control SH - SY5Y mitochondria . Significance determined by unpaired Student ' s t - test ( n = 3 biological replicates ) . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ; * * * * , p < 0 . 0001 ; ' ns ' indicates not significant , p > 0 . 05 . ( C ) Mitochondria isolated from hTim8aMUT SH cells with and without hTim8bFLAG expression compared to mitochondria isolated from control SH - SY5Y cells analysed by BN - PAGE and immunoblotting . COX4I1 - containing complexes are indicated as SC : Respiratory supercomplex , CIII2 + CIV : Complex III2 - Complex IV complex , CIV2 : Complex IV dimer , CIV : mature Complex IV , and S2 * : COX4I1 - containing S2 subcomplex . Amount of COX4I1 present in mature monomeric Complex IV ( CIV ) was quantified as mean ± S . D percentage of control SH - SY5Y mitochondria . Significance determined by unpaired Student ' s t - test ( n = 3 biological replicates ) . * * , p < 0 . 001 ; * * * , p < 0 . 001 ; ' ns ' indicates not significant , p > 0 . 05 . ( D ) Mitochondria isolated from hTim8bKO HEK cells with and without hTim8aFLAG expression compared to mitochondria isolated from control HEK293 cells by BN - PAGE . hTim8aFLAG expression induced by tetracycline for 8 h . Native protein complexes detected by immunoblot . COX4I1 - containing complexes by immunoblot indicated as SC : Respiratory supercomplex , CIII2 + CIV : Complex III2 - Complex IV complex , CIV2 : Complex IV dimer , CIV : mature Complex IV , and S2 * : COX4I1 - containing S2 subcomplex . Amount of COX4I1 present in mature monomeric Complex IV ( CIV ) was quantified as mean ± S . D percentage of control HEK293 mitochondria . Significance determined by unpaired Student ' s t - test ( n = 3 biological replicates ) . * * , p < 0 . 01 ; ' ns ' indicates not significant , p > 0 . 05 .

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Figure 6(A) Mitochondria isolated from hTim8aMUT SH cells with and without hTim8bFLAG expression compared to mitochondria from control SH-SY5Y cells were analysed by SDS-PAGE and immunoblotting. Amount of protein after 12 h induction (lanes 4 and 10) was quantified as mean ±S.D percentage of control SH-SY5Y mitochondria. Significance determined by unpaired Student's t-test (n=3 biological replicates). *, p<0.05; **, p<0.01; ***, p<0.001; ****,p<0.0001; 'ns' indicates not significant, p>0.05.(C) Mitochondria isolated from hTim8aMUT SH cells with and without hTim8bFLAG expression compared to mitochondria isolated from control SH-SY5Y cells analysed by BN-PAGE and immunoblotting. COX4I1-containing complexes are indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Amount of COX4I1 present in mature monomeric Complex IV (CIV) was quantified as mean ±S.D percentage of control SH-SY5Y mitochondria. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.001; ***, p<0.001; 'ns' indicates not significant, p>0.05.(D) Mitochondria isolated from hTim8bKO HEK cells with and without hTim8aFLAG expression compared to mitochondria isolated from control HEK293 cells by BN-PAGE. hTim8aFLAG expression induced by tetracycline for 8 h. Native protein complexes detected by immunoblot. COX4I1-containing complexes by immunoblot indicated as SC: Respiratory supercomplex, CIII2+CIV: Complex III2-Complex IV complex, CIV2: Complex IV dimer, CIV: mature Complex IV, and S2*: COX4I1-containing S2 subcomplex. Amount of COX4I1 present in mature monomeric Complex IV (CIV) was quantified as mean ±S.D percentage of control HEK293 mitochondria. Significance determined by unpaired Student's t-test (n=3 biological replicates). **, p<0.01; 'ns' indicates not significant, p>0.05.


Figure 1C : Movie stills ( first 4 panels in each row ) and maximum intensity projections ( 5th panel in each row ) of a 90 - second video of P . berghei sporozoites undergoing normal circular gliding motility ( top row ) and patch gliding ( bottom row ) . Scale bars : 5 µm .

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Figure 1C: Movie stills (first 4 panels in each row) and maximum intensity projections (5th panel in each row) of a 90-second video of P. berghei sporozoites undergoing normal circular gliding motility (top row) and patch gliding (bottom row). Scale bars: 5 µm.


Figure 2A : Numbers of oocysts in infected midguts of the different lines . Values ( n ) above dot plots indicate numbers of observed midguts . Shown are data from three biological replicates . Numbers above horizontal bars indicate p - values . Data were tested for significance using the Kruskal - Wallis - test . Images show cFluo oocysts in an infected midgut without ( top ) and with ( bottom ) green fluorescence . Scale bar : 200 µm . B : Western blots using an anti - TRAP antibody of the control lines cFluo and S210C in comparison to S210C / Q216C ( open ) and S210C / F224C ( closed ) . Anti - CSP antibody mAb 3D11 was used as loading control , revealing the two expected CSP - bandsC : Surface expression of TRAP mutants on fixed but not permeabilized sporozoites . Differential interference contrast ( DIC ) and immunofluorescence images of representative examples of hemolymph derived sporozoites of the indicated parasite lines stained with anti - TRAP antibodies shown in red Hoechst was used to reveal the sporozoite nuclear DNA shown in blue . Cytoplasmic GFP or staining with anti - CSP antibodies was used to validate the integrity of sporozoites . Scale bar : 5 µm .

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

Figure 2A: Numbers of oocysts in infected midguts of the different lines. Values (n) above dot plots indicate numbers of observed midguts. Shown are data from three biological replicates. Numbers above horizontal bars indicate p-values. Data were tested for significance using the Kruskal-Wallis-test. Images show cFluo oocysts in an infected midgut without (top) and with (bottom) green fluorescence. Scale bar: 200 µm.B: Western blots using an anti-TRAP antibody of the control lines cFluo and S210C in comparison to S210C/Q216C (open) and S210C/F224C (closed). Anti-CSP antibody mAb 3D11 was used as loading control, revealing the two expected CSP-bandsC: Surface expression of TRAP mutants on fixed but not permeabilized sporozoites. Differential interference contrast (DIC) and immunofluorescence images of representative examples of hemolymph derived sporozoites of the indicated parasite lines stained with anti-TRAP antibodies shown in red Hoechst was used to reveal the sporozoite nuclear DNA shown in blue. Cytoplasmic GFP or staining with anti-CSP antibodies was used to validate the integrity of sporozoites. Scale bar: 5 µm.


Figure 3A : Numbers of hemolymph ( HL ) derived sporozoites reveal no difference between the parasite lines . B : Numbers of salivary gland ( SG ) derived sporozoites reveal strong reduction in salivary gland invasion for the closed and open I domain expressing parasite lines . C : Ratios of hemolymph ( HL ) derived versus oocyst - derived sporozoites reveal no difference between the parasite lines . D : Ratios of salivary gland ( SG ) derived versus hemolymph ( HL ) derived sporozoites reveal strong reduction in salivary gland invasion for the closed and open I domain expressing parasite lines .

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Figure 3A: Numbers of hemolymph (HL) derived sporozoites reveal no difference between the parasite lines. B: Numbers of salivary gland (SG) derived sporozoites reveal strong reduction in salivary gland invasion for the closed and open I domain expressing parasite lines. C: Ratios of hemolymph (HL) derived versus oocyst-derived sporozoites reveal no difference between the parasite lines. D: Ratios of salivary gland (SG) derived versus hemolymph (HL) derived sporozoites reveal strong reduction in salivary gland invasion for the closed and open I domain expressing parasite lines.


Figure 4A : Blood stage parasitemia after injection of 10 , 000 hemolymph ( HL ) derived sporozoites of the different lines into C57Bl / 6 mice from day 3 to day 9 post infection . Shown is the mean + / - SEM . Only data of infected mice have been plotted . In shown are pooled data from 3 ( S210C ) , 2 ( S210C / F224C ) , 3 ( S210C / Q216C ) and 1 ( wild type ) biological replicates using 3 or 4 mice , respectively . B : Kaplan Meier plot showing the percentage of parasite free mice until day 9 of infection . , shown are pooled data from 3 ( S210C ) , 2 ( S210C / F224C ) , 3 ( S210C / Q216C ) and 1 ( wild type ) biological replicates using 3 or 4 mice , respectively . D : Blood stage parasitemia after C57Bl / 6 mice were bitten by mosquitoes ( ten mosquitoes / mouse ) infected with the indicated parasite lines at day 0 . Shown is the mean + / - SEM where appropriate . Only data of infected mice have been plotted for all but the S210C / F224C ( closed I domain ) infections , where all mice stayed negative . each condition represents pooled data from 3 biological replicates using 2 - 4 mice , respectively . E : Kaplan Meier plot showing the percentage of parasite free mice after mosquito bites until day 9 of infection . each condition represents pooled data from 3 biological replicates using 2 - 4 mice , respectively . G : Blood stage parasitemia after injection of 10 . 000 salivary gland ( SG ) derived sporozoites of the indicated parasite lines into C57Bl / 6 mice at day 0 . Shown is the mean + / - SEM . Only data of infected mice have been plotted . shown are pooled data from 3 ( S210C ) , 2 ( S210C / Q216C ) and 3 ( wild type ) biological replicates using 2 - 4 mice , respectively . H : Kaplan Meier plot showing the percentage of parasite free mice after salivary gland sporozoite injection until day 9 of infection . shown are pooled data from 3 ( S210C ) , 2 ( S210C / Q216C ) and 3 ( wild type ) biological replicates using 2 - 4 mice , respectively .

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 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 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 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 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 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

Figure 4A: Blood stage parasitemia after injection of 10,000 hemolymph (HL) derived sporozoites of the different lines into C57Bl/6 mice from day 3 to day 9 post infection. Shown is the mean +/- SEM. Only data of infected mice have been plotted. In shown are pooled data from 3 (S210C), 2 (S210C/F224C), 3 (S210C/Q216C) and 1 (wild type) biological replicates using 3 or 4 mice, respectively.B: Kaplan Meier plot showing the percentage of parasite free mice until day 9 of infection. , shown are pooled data from 3 (S210C), 2 (S210C/F224C), 3 (S210C/Q216C) and 1 (wild type) biological replicates using 3 or 4 mice, respectively.D: Blood stage parasitemia after C57Bl/6 mice were bitten by mosquitoes (ten mosquitoes/mouse) infected with the indicated parasite lines at day 0. Shown is the mean +/- SEM where appropriate. Only data of infected mice have been plotted for all but the S210C/F224C (closed I domain) infections, where all mice stayed negative. each condition represents pooled data from 3 biological replicates using 2-4 mice, respectively.E: Kaplan Meier plot showing the percentage of parasite free mice after mosquito bites until day 9 of infection. each condition represents pooled data from 3 biological replicates using 2-4 mice, respectively.G: Blood stage parasitemia after injection of 10.000 salivary gland (SG) derived sporozoites of the indicated parasite lines into C57Bl/6 mice at day 0. Shown is the mean +/- SEM. Only data of infected mice have been plotted. shown are pooled data from 3 (S210C), 2 (S210C/Q216C) and 3 (wild type) biological replicates using 2-4 mice, respectively.H: Kaplan Meier plot showing the percentage of parasite free mice after salivary gland sporozoite injection until day 9 of infection. shown are pooled data from 3 (S210C), 2 (S210C/Q216C) and 3 (wild type) biological replicates using 2-4 mice, respectively.


Figure 5A : Movement type analysis of hemolymph derived sporozoites ( HLS ) of the indicated parasite lines . Top : Classification of attached sporozoites by motility . Bottom : Classification by motility type . n indicates the number of observed sporozoites . Floaters have been omitted in the analysis presented in the top graph and are not included in n . Shown are pooled data from 3 ( S210C ) , 2 ( S210C / F224C ) , 2 ( S210C / Q216C ) and 2 ( wild type ) biological replicates . Shown is the mean + / - SEM . D : Percentage of gliding and non - gliding control hemolymph sporozoites ( HLS ) at increasing concentrations of DTT . n indicates the number of observed sporozoites . Floaters are included in the non - motile fraction . Each condition represents pooled data from 2 biological replicates . Shown is the mean + / - SEM . E : Percentage of gliding hemolymph sporozoites ( HLS ) expressing the open TRAP I domain at increasing concentrations of DTT . n indicates the total number of observed sporozoites . Floaters are included in the non - motile fraction . Shown are pooled data from 3 ( 0mM DTT ) , 2 ( 50mM DTT ) , and 3 biological replicates for concentrations of 100mM DTT in the S210C / Q216C line . For concentrations of 200mM and 330mM DTT in the S210C / Q216C line , data shown represent pooled data from 1 biological replicate . Several comparable screening experiments previously performed showed no productive motility at these concentrations . Shown is the mean + / - SEM . F : Maximum projections ( 1 min time lapse , 3 s per frame ) of single S210C / Q216C sporozoites in presence or absence of 50 mM DTT . A projection from a motile sporozoite of the cFluo line treated with 50 mM DTT is shown as control . Scale bar : 5 µm .

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Figure 5A: Movement type analysis of hemolymph derived sporozoites (HLS) of the indicated parasite lines. Top: Classification of attached sporozoites by motility. Bottom: Classification by motility type. n indicates the number of observed sporozoites. Floaters have been omitted in the analysis presented in the top graph and are not included in n. Shown are pooled data from 3 (S210C), 2 (S210C/F224C), 2 (S210C/Q216C) and 2 (wild type) biological replicates. Shown is the mean +/- SEM.D: Percentage of gliding and non-gliding control hemolymph sporozoites (HLS) at increasing concentrations of DTT. n indicates the number of observed sporozoites. Floaters are included in the non-motile fraction. Each condition represents pooled data from 2 biological replicates. Shown is the mean +/- SEM. E: Percentage of gliding hemolymph sporozoites (HLS) expressing the open TRAP I domain at increasing concentrations of DTT. n indicates the total number of observed sporozoites. Floaters are included in the non-motile fraction. Shown are pooled data from 3 (0mM DTT), 2 (50mM DTT), and 3 biological replicates for concentrations of 100mM DTT in the S210C/Q216C line. For concentrations of 200mM and 330mM DTT in the S210C/Q216C line, data shown represent pooled data from 1 biological replicate. Several comparable screening experiments previously performed showed no productive motility at these concentrations. Shown is the mean +/- SEM.F: Maximum projections (1 min time lapse, 3 s per frame) of single S210C/Q216C sporozoites in presence or absence of 50 mM DTT. A projection from a motile sporozoite of the cFluo line treated with 50 mM DTT is shown as control. Scale bar: 5 µm.


Figure 1A . Genes involved in innate immunity are significantly upregulated in nuo - 6 and isp - 1 worms . Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild - type N2 worms . Results represent counts per million ( CPM ) expressed as a percentage of wild - type of six biological replicates per strain . Data information : Error bars indicate SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a one - way ANOVA with Dunnett ' s multiple comparison testB . Upregulation of innate immunity genes is dependent on the p38 - mediated innate immune signaling pathway including NSY - 1 , SEK - 1 , PMK - 1 and ATF - 7 . Gene expression changes were examined by quantitative RT - PCR of three biological replicates per strain . Data information : Error bars indicate SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a one - way ANOVA with Dunnett ' s multiple comparison testC . Using a fluorescent reporter strain for the innate immunity gene T24B8 . 5 confirms that innate immunity genes are upregulated in the long - lived mitochondrial mutants and that components of the p38 - mediated innate immune signaling pathway are required for their upregulation . Scale bar indicates 250 µM . Three biological replicates per strain were quantified . Data information : Error bars indicate SEM . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance was determined using a two - way ANOVA with Bonferroni post -

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 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 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

Figure 1A. Genes involved in innate immunity are significantly upregulated in nuo-6 and isp-1 worms. Gene expression changes in the mitochondrial mutants were determined by RNA sequencing and compared to wild-type N2 worms. Results represent counts per million (CPM) expressed as a percentage of wild-type of six biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison testB. Upregulation of innate immunity genes is dependent on the p38-mediated innate immune signaling pathway including NSY-1, SEK-1, PMK-1 and ATF-7. Gene expression changes were examined by quantitative RT-PCR of three biological replicates per strain. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a one-way ANOVA with Dunnett's multiple comparison testC. Using a fluorescent reporter strain for the innate immunity gene T24B8.5 confirms that innate immunity genes are upregulated in the long-lived mitochondrial mutants and that components of the p38-mediated innate immune signaling pathway are required for their upregulation. Scale bar indicates 250 µM. Three biological replicates per strain were quantified. Data information: Error bars indicate SEM. *p<0.05, **p<0.01, ***p<0.001. Statistical significance was determined using a two-way ANOVA with Bonferroni post-test


Figure 2A - D . Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay . Both nuo - 6 and isp - 1 long - lived mitochondrial mutants show increased resistance compared to wild - type worms ( A ) . Disruption of components of the p38 - mediated innate immune signaling pathway ( nsy - 1 , sek - 1 , pmk - 1 , atf - 7 ) markedly decreases resistance to PA14 in wild - type ( B ) , nuo - 6 ( C ) and isp - 1 ( D ) worms . All strains were tested in a single parallel experiment . Two biological replicates per strain were performed with a total of at least 175 animals per strain . Data information : * * * p < 0 . 001 . Statistical significance for survival plots was determined with the log - rank test . Significance is indicated between the strain listed on top and all other strains . Data from panel A is repeated in panels B , C , and D for direct comparison .

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

Figure 2A-D. Resistance to bacterial pathogens was tested by exposing worms to Pseudomonas aeruginosa strain PA14 in a slow kill assay. Both nuo-6 and isp-1 long-lived mitochondrial mutants show increased resistance compared to wild-type worms (A). Disruption of components of the p38-mediated innate immune signaling pathway (nsy-1, sek-1, pmk-1, atf-7) markedly decreases resistance to PA14 in wild-type (B), nuo-6 (C) and isp-1 (D) worms. All strains were tested in a single parallel experiment. Two biological replicates per strain were performed with a total of at least 175 animals per strain. Data information: ***p<0.001. Statistical significance for survival plots was determined with the log-rank test. Significance is indicated between the strain listed on top and all other strains. Data from panel A is repeated in panels B, C, and D for direct comparison.


Figure 3A - H . To examine the role of the p38 - mediated innate immune signaling pathway in the long - lifespan of the nuo - 6 and isp - 1 mitochondrial mutants , we crossed the long - lived mitochondrial mutants to worms with mutations in nsy - 1 , sek - 1 , pmk - 1 and atf - 7 ( gof ) . In each case , mutation of genes involved in p38 - mediated innate immunity signaling markedly reduces the lifespan of the long - lived mitochondrial mutant but have little or no impact on wild - type lifespan . In panel G , we utilized pmk - 1 RNAi instead of the pmk - 1 mutation because of the close proximity of isp - 1 and pmk - 1 on the same chromosome . EV indicates empty vector . The lifespan results for panels A , B , D , E , F , and H were performed in a single parallel experiment . Results are from a minimum of three biological replicates per strain . Data information : Statistical significance for survival plots was determined with the log - rank test . p - values indicate significance between red and purple lines . Control strains are shown in multiple panels for direct comparison .

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

Figure 3A-H. To examine the role of the p38-mediated innate immune signaling pathway in the long-lifespan of the nuo-6 and isp-1 mitochondrial mutants, we crossed the long-lived mitochondrial mutants to worms with mutations in nsy-1, sek-1, pmk-1 and atf-7(gof). In each case, mutation of genes involved in p38-mediated innate immunity signaling markedly reduces the lifespan of the long-lived mitochondrial mutant but have little or no impact on wild-type lifespan. In panel G, we utilized pmk-1 RNAi instead of the pmk-1 mutation because of the close proximity of isp-1 and pmk-1 on the same chromosome. EV indicates empty vector. The lifespan results for panels A, B, D, E, F, and H were performed in a single parallel experiment. Results are from a minimum of three biological replicates per strain. Data information: Statistical significance for survival plots was determined with the log-rank test. p-values indicate significance between red and purple lines. Control strains are shown in multiple panels for direct comparison.


Figure 4A , B . To determine the extent to which the p38 - mediated innate immunity pathway is activated in nuo - 6 and isp - 1 mutants , we measured the ratio of phosphorylated PMK - 1 / p38 to total PMK - 1 / p38 by Western blotting . Representative blots are shown ( A ) . There is no difference in the ratio of phosphorylated to total PMK - 1 / p38 in the long - lived mitochondrial mutants compared to wild - type ( B ) . Eight biological replicates of wild - type worms and nine of mitochondrial mutants were quantified . Data information : Error bars indicate SEM . * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison testF . Constitutive activation of atfs - 1 is sufficient to decrease food consumption to the same level as in long - lived mitochondrial mutants . Three biological replicates per strain were measured , each including at least three technical replicates . atfs - 1 ( lof ) allele is gk3094 , atfs - 1 ( gof ) allele is et17 . Data information : Error bars indicate SEM . * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison 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 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 4A,B. To determine the extent to which the p38-mediated innate immunity pathway is activated in nuo-6 and isp-1 mutants, we measured the ratio of phosphorylated PMK-1/p38 to total PMK-1/p38 by Western blotting. Representative blots are shown (A). There is no difference in the ratio of phosphorylated to total PMK-1/p38 in the long-lived mitochondrial mutants compared to wild-type (B). Eight biological replicates of wild-type worms and nine of mitochondrial mutants were quantified. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison testF. Constitutive activation of atfs-1 is sufficient to decrease food consumption to the same level as in long-lived mitochondrial mutants. Three biological replicates per strain were measured, each including at least three technical replicates. atfs-1(lof) allele is gk3094, atfs-1(gof) allele is et17. Data information: Error bars indicate SEM. ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test


Figure 5A . A deletion of daf - 16 completely abolishes the increased resistance to bacterial pathogens in isp - 1 mutants . p - value indicates the significance of difference between red and purple lines . Three biological replicates per strain were performed . Data information : Statistical significance was assessed using log - rank test . B , C . While daf - 16 RNAi effectively decreases the expression of DAF - 16 target genes ( B , sod - 3 , dod - 3 , mtl - 1 , ftn - 1 , icl - 1 , sodh - 1 ) , it does not affect the expression of any of the innate immunity genes ( C , T24B8 . 5 , K08D8 . 5 , F55G11 . 8 , clec - 65 , clec - 67 , dod - 22 , Y9C9A . 8 and C32H11 . 4 ) . This indicates that DAF - 16 is not required for expression of innate immune signaling pathway target genes in isp - 1 mutants . daf - 16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation . RNA was isolated from six biological replicates at the young adult stage of the experimental generation . RNA from the six biological replicates was pooled for RNA sequencing .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. A deletion of daf-16 completely abolishes the increased resistance to bacterial pathogens in isp-1 mutants. p-value indicates the significance of difference between red and purple lines. Three biological replicates per strain were performed. Data information: Statistical significance was assessed using log-rank test.B,C. While daf-16 RNAi effectively decreases the expression of DAF-16 target genes (B, sod-3, dod-3, mtl-1, ftn-1, icl-1, sodh-1), it does not affect the expression of any of the innate immunity genes (C, T24B8.5, K08D8.5, F55G11.8, clec-65, clec-67, dod-22, Y9C9A.8 and C32H11.4). This indicates that DAF-16 is not required for expression of innate immune signaling pathway target genes in isp-1 mutants. daf-16 expression was knocked down using RNAi beginning at the L4 stage of the parental generation. RNA was isolated from six biological replicates at the young adult stage of the experimental generation. RNA from the six biological replicates was pooled for RNA sequencing.


Figure 6A . To examine the role of the ATFS - 1 transcription factor and the mitoUPR response in the enhanced bacterial pathogen resistance in nuo - 6 mutants , we examined the effect of disrupting atfs - 1 in nuo - 6 mutants . Loss of atfs - 1 completely abolishes the increased resistance to bacterial pathogens in nuo - 6 worms . p - value indicates significance of difference between red and purple lines . nuo - 6 ; atfs - 1 contains the gk3094 deletion allele . Three biological replicates per strain were measured . B . To examine the role of ATFS - 1 in the upregulation of innate immunity genes in nuo - 6 mutants , we compared gene expression between nuo - 6 and nuo - 6 ; atfs - 1 mutants with wild - type worms and atfs - 1 mutants as controls . Deletion of atfs - 1 significantly decreases the expression of genes involved in innate immunity in nuo - 6 worms but does not decrease the expression of these genes in wild - type worms . Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype . Results represent counts per million ( CPM ) expressed as a percentage of wild - type . Worms in a wild - type background ( control ) are shown with white bars , while worms with atfs - 1 deletion are shown with blue bars . Data information : Error bars indicate SEM . * * p < 0 . 01 , * * * p < 0 . 001 . Statistical significance in panel A was assessed using log - rank test . Statistical significance in panel B was assessed using a two - way ANOVA with Bonferroni posttest .

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

Figure 6A. To examine the role of the ATFS-1 transcription factor and the mitoUPR response in the enhanced bacterial pathogen resistance in nuo-6 mutants, we examined the effect of disrupting atfs-1 in nuo-6 mutants. Loss of atfs-1 completely abolishes the increased resistance to bacterial pathogens in nuo-6 worms. p-value indicates significance of difference between red and purple lines. nuo-6; atfs-1 contains the gk3094 deletion allele. Three biological replicates per strain were measured. B. To examine the role of ATFS-1 in the upregulation of innate immunity genes in nuo-6 mutants, we compared gene expression between nuo-6 and nuo-6; atfs-1 mutants with wild-type worms and atfs-1 mutants as controls. Deletion of atfs-1 significantly decreases the expression of genes involved in innate immunity in nuo-6 worms but does not decrease the expression of these genes in wild-type worms. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Worms in a wild-type background (control) are shown with white bars, while worms with atfs-1 deletion are shown with blue bars. Data information: Error bars indicate SEM. **p<0.01, ***p<0.001. Statistical significance in panel A was assessed using log-rank test. Statistical significance in panel B was assessed using a two-way ANOVA with Bonferroni posttest.


Figure 7A . Examination of mRNA levels of genes involved in innate immunity in two constitutively active atfs - 1 mutants ( et15 and et17 ) revealed that innate immunity genes are upregulated when ATFS - 1 is activated . This indicates that activation of the mitoUPR can cause activation of genes that are regulated by the p38 - mediated innate immune pathway . Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype . Results represent counts per million ( CPM ) expressed as a percentage of wild - type . Data information : Error bars indicate SEM . * p < 0 . 05 * * p < 0 . 01 , * * * p < 0 . 001 . NS = not significant . Statistical significance was assessed using a one - way ANOVA with Dunnett ' s multiple comparison 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

Figure 7A. Examination of mRNA levels of genes involved in innate immunity in two constitutively active atfs-1 mutants (et15 and et17) revealed that innate immunity genes are upregulated when ATFS-1 is activated. This indicates that activation of the mitoUPR can cause activation of genes that are regulated by the p38-mediated innate immune pathway. Gene expression changes were determined by RNA sequencing of six biological replicates of each genotype. Results represent counts per million (CPM) expressed as a percentage of wild-type. Data information: Error bars indicate SEM. *p<0.05 **p<0.01, ***p<0.001. NS = not significant. Statistical significance was assessed using a one-way ANOVA with Dunnett's multiple comparison test.


Figure 1Human Gut microbial species explored for the existence of subspecies show wide phylogenetic spread according to NCBI taxonomy ( A ) and include Methanobrevibacter smithii , the main archaeal member of the human gut microbiome , as well as representatives of all abundant phyla . Species names are according to NCBI taxonomy , with species cluster ( specI ) identifiers according to Mende et al . , which splits some named species into multiple specI clusters ( Mende et al , 2013 ) . Of 71 investigated species , 44 stratify into subspecies ( highlighted in blue ) . Each species ' average abundance across 2 , 144 human gut metagenomes is proportional to the size of the circles on the cladogram . Bars represent the number of subspecies identified in each , with ' 1 ' indicating no subdivision . The black portion of the bar corresponds to subspecies for which no representative genome sequence is available from NCBI . Geographic enrichments of subspecies are displayed as a heat map ( showing only significant enrichment , FDR corrected Fisher test p - value < 0 . 05 , per country as maximum log - odds ratio across con - specific subspecies ) . Subspecies with a restricted geographic range are predominantl y found in the Chinese and Kazakh population . The 71 investigated species captured an average of 95 . 5 % of sequencing reads that were assigned to any reference genome . The subset of 44 species with identified subspecies accounted for the majority of this abundance ( B ) .

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

Figure 1Human Gut microbial species explored for the existence of subspecies show wide phylogenetic spread according to NCBI taxonomy (A) and include Methanobrevibacter smithii, the main archaeal member of the human gut microbiome, as well as representatives of all abundant phyla. Species names are according to NCBI taxonomy, with species cluster (specI) identifiers according to Mende et al., which splits some named species into multiple specI clusters (Mende et al, 2013). Of 71 investigated species, 44 stratify into subspecies (highlighted in blue). Each species' average abundance across 2,144 human gut metagenomes is proportional to the size of the circles on the cladogram. Bars represent the number of subspecies identified in each, with '1' indicating no subdivision. The black portion of the bar corresponds to subspecies for which no representative genome sequence is available from NCBI. Geographic enrichments of subspecies are displayed as a heat map (showing only significant enrichment, FDR corrected Fisher test p-value < 0.05, per country as maximum log-odds ratio across con-specific subspecies). Subspecies with a restricted geographic range are predominantly found in the Chinese and Kazakh population. The 71 investigated species captured an average of 95.5% of sequencing reads that were assigned to any reference genome. The subset of 44 species with identified subspecies accounted for the majority of this abundance (B).


Figure 2Subspecies identified for M . smithii and B . vulgatus / dorei are shown in principle coordinate ( PCoA ) projections of the between - sample distances based on single nucleotide variations ( see Methods ) . The first principal coordinate ( PC ) explains over 70 % of the variation in both cases ( panels A and C ) . Reference genomes have been projected into the same PCoA plots ( marked with ' x ' in A and C , see Methods ) . The numbers adjacent to the placed genomes correspond to those shown in parentheses next to NCBI taxonomy identifiers ( leaves ) on the phylogenetic trees in B and D , respectively . The sample density for each subspecies is highlighted by the histogram above and the total number of samples in which the species could be quantified is indicated in headers . Quantification of the frequency of each subspecies ( bottom plot in A and C ) reveals that for M . smithii only one sample has two subspecies co - occurring in one individual , while all the others have a single dominating one . In contrast , for B . vulgatus / dorei , co - occurrence is more commonly observed . Phylogenies reconstructed from the reference genomes ( NCBI taxonomic identifiers , see Methods ) are fully consistent with the SNP - based clustering . The representative genome for each species , relative to which genomic variants were called , is highlighted with a box . ( E ) In B . vulgatus / dorei , subspecies composition within each individual is generally stable over time , with a change of the dominant subspecies being rare even over a period of up to 1 , 000 days . An exception ( highlighted by purple line ) is seen for an individual , in which one dominant subspecies is replaced by another one after antibiotic treatment . The right hand panel summarizes subspecies frequency changes , underlining remarkable stability over 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 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

Figure 2Subspecies identified for M. smithii and B. vulgatus/dorei are shown in principle coordinate (PCoA) projections of the between-sample distances based on single nucleotide variations (see Methods). The first principal coordinate (PC) explains over 70% of the variation in both cases (panels A and C). Reference genomes have been projected into the same PCoA plots (marked with 'x' in A and C, see Methods). The numbers adjacent to the placed genomes correspond to those shown in parentheses next to NCBI taxonomy identifiers (leaves) on the phylogenetic trees in B and D, respectively. The sample density for each subspecies is highlighted by the histogram above and the total number of samples in which the species could be quantified is indicated in headers. Quantification of the frequency of each subspecies (bottom plot in A and C) reveals that for M. smithii only one sample has two subspecies co-occurring in one individual, while all the others have a single dominating one. In contrast, for B. vulgatus/dorei, co-occurrence is more commonly observed. Phylogenies reconstructed from the reference genomes (NCBI taxonomic identifiers, see Methods) are fully consistent with the SNP-based clustering. The representative genome for each species, relative to which genomic variants were called, is highlighted with a box.(E) In B. vulgatus/dorei, subspecies composition within each individual is generally stable over time, with a change of the dominant subspecies being rare even over a period of up to 1,000 days. An exception (highlighted by purple line) is seen for an individual, in which one dominant subspecies is replaced by another one after antibiotic treatment. The right hand panel summarizes subspecies frequency changes, underlining remarkable stability over time.


Figure 3Three subspecies of Eubacterium rectale ( A ) are color - coded in PCoA plots . In most individuals we observe dominance of one subspecies at a time ( see frequency plots below PCoAs ) . four of Eubacterium eligens ( B ) are color - coded in PCoA plots . In most individuals we observe dominance of one subspecies at a time ( see frequency plots below PCoAs ) . ( D ) Grouping E . rectale individuals from the Danish ( DK ) and Swedish ( SE ) studies , based on this deletion shows a significant ( * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ) increase in BMI and blood fasting insulin levels as well as a decrease in overall community diversity in individuals who are predominantly colonised by the flagellum carrying subspecies ( see Methods ) . The same trend is observed for E . eligens subspecies in the same studies , though not statistically significant ( n . s . ) .

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 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 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

Figure 3Three subspecies of Eubacterium rectale (A) are color-coded in PCoA plots. In most individuals we observe dominance of one subspecies at a time (see frequency plots below PCoAs).four of Eubacterium eligens (B) are color-coded in PCoA plots. In most individuals we observe dominance of one subspecies at a time (see frequency plots below PCoAs).(D) Grouping E. rectale individuals from the Danish (DK) and Swedish (SE) studies, based on this deletion shows a significant (*p < 0.05, **p < 0.01, ***p < 0.001) increase in BMI and blood fasting insulin levels as well as a decrease in overall community diversity in individuals who are predominantly colonised by the flagellum carrying subspecies (see Methods). The same trend is observed for E. eligens subspecies in the same studies, though not statistically significant (n.s.).


Figure 1C . Confirmation of sensor localization to different cellular compartments . Green , HyPer7 ; blue , DAPI stainD . - H . Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A ( a complex III inhibitor , red curve and data points ) or ethanol as control ( black line and data points ) . HEK293 cells were grown either with glucose or galactose as carbon source as indicated . Solid line represents mean , points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells . Data information : As most of the data were not normal distributed , instead of a t - test , a Wilcoxon / Mann - Whitney - U - test was performed and samples were compared in pairs . * * , p ≤ 0 . 01 , * * * , p ≤ 0 .

O B-PANEL_START 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

Figure 1C. Confirmation of sensor localization to different cellular compartments. Green, HyPer7; blue, DAPI stainD.-H. Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A (a complex III inhibitor, red curve and data points) or ethanol as control (black line and data points). HEK293 cells were grown either with glucose or galactose as carbon source as indicated. Solid line represents mean, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. **, p ≤ 0.01, ***, p ≤ 0.001


Figure 2A - D . Response of HyPer7 probes targeted to indicated compartments to reoxygenation after hypoxia ( red curve and data points ) or to continued hypoxia ( black line and data points ) . HEK293 cells were grown either with glucose or galactose as carbon source . Solid line represents mean , points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells . E . Model . Cells grown on galactose - containing medium exhibit increased production of H2O2 and thus also increased detection of cytosolic H2O2 indicating H2O2 gradients of different steepness around mitochondria . Data information : As most of the data were not normal distributed , instead of a t - test , a Wilcoxon / Mann - Whitney - U - test was performed and samples were compared in pairs . * * * , p ≤ 0 .

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

Figure 2A-D. Response of HyPer7 probes targeted to indicated compartments to reoxygenation after hypoxia (red curve and data points) or to continued hypoxia (black line and data points). HEK293 cells were grown either with glucose or galactose as carbon source. Solid line represents mean, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. E. Model. Cells grown on galactose-containing medium exhibit increased production of H2O2 and thus also increased detection of cytosolic H2O2 indicating H2O2 gradients of different steepness around mitochondria. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs.***, p ≤ 0.001


Figure 3C . Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO - expressing HEK293 cells with 2 mM D - Ala ( cells generated with the Flp - In T - REX - system ) . HEK293 cells were grown either with glucose ( black curve and data points ) or galactose ( red curve and data points ) as carbon source . Solid line represents average , points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells . Data information : As most of the data were not normal distributed , instead of a t - test , a Wilcoxon / Mann - Whitney - U - test was performed and samples were compared in pairs . * * * , p ≤ 0 .

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

Figure 3C. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing HEK293 cells with 2 mM D-Ala (cells generated with the Flp-In T-REX-system). HEK293 cells were grown either with glucose (black curve and data points) or galactose (red curve and data points) as carbon source. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001


Figure 4A . Response of HyPer7 probes targeted to indicated compartments to treatment with increasing amounts of exogenous H2O2 ( 2 , 4 and 8 µM H2O2 ; 20 mM DTT ) . HEK293 cells were grown either with glucose ( black curve and data points ) or galactose ( red curve and data points ) as carbon source . As most of the data were not normal distributed , instead of a t - test , a Wilcoxon / Mann - Whitney - U - test was performed and samples were compared in pairs . * * * , p ≤ 0 .

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

Figure 4A. Response of HyPer7 probes targeted to indicated compartments to treatment with increasing amounts of exogenous H2O2 (2, 4 and 8 µM H2O2; 20 mM DTT). HEK293 cells were grown either with glucose (black curve and data points) or galactose (red curve and data points) as carbon source. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001


Figure 5A . Protein levels in cells grown on glucose and galactose . Cell lysates were analyzed by quantitative mass spectrometry ( n = 4 biological replicates Levels of antioxidative enzymes ( especially peroxiredoxins , PRDX in blue ) are not changed except for cytosolic thioredoxin reductase ( TXNRD1 ) . B . Protein levels in cells grown on glucose and galactose . Cell lysates were analyzed by immunoblot ( n = 2 biological replicates ) . Cytosolic thioredoxin reductase ( TXNRD1 ) is increased by more than two - fold in cells grown on galactose compared to glucose - grown cells . The dotted line depicts 1 , meaning no change in protein levels .

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Figure 5A. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by quantitative mass spectrometry (n = 4 biological replicates Levels of antioxidative enzymes (especially peroxiredoxins, PRDX in blue) are not changed except for cytosolic thioredoxin reductase (TXNRD1).B. Protein levels in cells grown on glucose and galactose. Cell lysates were analyzed by immunoblot (n = 2 biological replicates). Cytosolic thioredoxin reductase (TXNRD1) is increased by more than two-fold in cells grown on galactose compared to glucose-grown cells.The dotted line depicts 1, meaning no change in protein levels.


Figure 6B . Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A ( AntA , red curve and data points ) or ethanol as control ( black curve and data points ) . HeLa cells were grown with galactose as carbon source . Solid line represents average , points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells . Data information : As most of the data were not normal distributed , instead of a t - test , a Wilcoxon / Mann - Whitney - U - test was performed and samples were compared in pairs . * * * , p ≤ 0 .

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Figure 6B. Response of HyPer7 probes targeted to indicated compartments to incubation with antimycin A (AntA, red curve and data points) or ethanol as control (black curve and data points). HeLa cells were grown with galactose as carbon source. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. ***, p ≤ 0.001


Figure 7B . Peroxiredoxin levels in peroxiredoxin 1 ( PRDX1 KO ) and peroxiredoxin 2 ( PRDX2 KO ) knockout cells . quantitative proteomics ( n = 4 , technical replicates ) The right subpanel of B lists the identified proteins belonging to cellular antioxidative systems which are not altered for PRDX1 KO or PRDX2 KO , respectively . In both quantitative proteomics experiments 4403 proteins were detected in total . D . Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO - expressing cell lines with 4 mM D - Ala ( black , wild type ; red , PRDX1 KO ; blue , PRDX2 KO ; cell generated with the piggyBAC system ) . Cells were grown in glucose - containing medium . Solid line represents average , points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells . Data information As most of the data were not normal distributed , instead of a t - test , a Wilcoxon / Mann - Whitney - U - test was performed and samples were compared in pairs . * , p ≤ 0 . 05 , * * , p ≤ 0 . 01 , * * * , p ≤ 0 .

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

Figure 7B. Peroxiredoxin levels in peroxiredoxin 1 (PRDX1 KO) and peroxiredoxin 2 (PRDX2 KO) knockout cells. quantitative proteomics (n = 4, technical replicates) The right subpanel of B lists the identified proteins belonging to cellular antioxidative systems which are not altered for PRDX1 KO or PRDX2 KO, respectively. In both quantitative proteomics experiments 4403 proteins were detected in total.D. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1 KO; blue, PRDX2 KO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001


Figure 8B . Peroxiredoxin levels in peroxiredoxin 1 and 2 double knockout cells ( PRDX1 / 2 DKO ) . quantitative proteomics ( B ) . The right subpanel in ( B ) lists all detected but not altered proteins belonging to cellular antioxidative systems . E . Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO - expressing cell lines with 4 mM D - Ala ( black , wild type ; red , PRDX1 / 2 DKO ; cell generated with the piggyBAC system ) . Cells were grown in glucose - containing medium . Solid line represents average , points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells . Data information : Statistical analysis was only performed to compare the PRDX1 / 2 DKO with the wildtype . As most of the data were not normal distributed , instead of a t - test , a Wilcoxon / Mann - Whitney - U - test was performed and samples were compared in pairs . * , p ≤ 0 . 05 , * * , p ≤ 0 . 01 , * * * , p ≤ 0 .

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 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

Figure 8B. Peroxiredoxin levels in peroxiredoxin 1 and 2 double knockout cells (PRDX1/2 DKO). quantitative proteomics (B).The right subpanel in (B) lists all detected but not altered proteins belonging to cellular antioxidative systems.E. Response of HyPer7 probes targeted to indicated compartments to treatment of mtDAO-expressing cell lines with 4 mM D-Ala (black, wild type; red, PRDX1/2 DKO; cell generated with the piggyBAC system). Cells were grown in glucose-containing medium. Solid line represents average, points colored in the lighter version of the respective color are the corresponding ratios measured in individual cells. Data information: Statistical analysis was only performed to compare the PRDX1/2 DKO with the wildtype. As most of the data were not normal distributed, instead of a t-test, a Wilcoxon/Mann-Whitney-U-test was performed and samples were compared in pairs. *, p ≤ 0.05, **, p ≤ 0.01, ***, p ≤ 0.001


Figure 2 ( A ) The graphs depict the Aβ profiles generated by WT or mutant PSEN1 MEF cell lines transduced with adenovirus encoding APPC99 . ELISA quantified secreted Aβ37 , Aβ38 , Aβ40 and Aβ42 peptide levels are shown as % of total Aβ ( sum of the four peptides ) . The blue , orange , green and purple dotted lines indicate the levels of Aβ37 , Aβ38 , Aβ40 and Aβ42 peptides in the WT Aβ profile , respectively . Data are presented as mean ± SD , N ≥ 3 independent experiments . ( B ) GSEC processivity levels for the cleavage of APPC99 determined in WT and mutant PSEN1 / GSEC cell lines . The mutant Aβ ( 37 + 38 ) / ( 40 + 42 ) ratios are shown as % of the WT cell line . Data presented as mean ± SD , N ≥ 3 independent experiments . As reference , a dotted line was drawn to indicate 10 % ( WT levels ) . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) ; * * P < 0 . 01 , * * * P < 0 . 001 , * * * * P < 0 . 0001 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(A) The graphs depict the Aβ profiles generated by WT or mutant PSEN1 MEF cell lines transduced with adenovirus encoding APPC99. ELISA quantified secreted Aβ37, Aβ38, Aβ40 and Aβ42 peptide levels are shown as % of total Aβ (sum of the four peptides). The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively. Data are presented as mean ± SD, N ≥ 3 independent experiments.(B) GSEC processivity levels for the cleavage of APPC99 determined in WT and mutant PSEN1/GSEC cell lines. The mutant Aβ(37+38)/(40+42) ratios are shown as % of the WT cell line. Data presented as mean ± SD, N ≥ 3 independent experiments. As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05); **P < 0.01, ***P < 0.001, ****P < 0.0001.


Figure 3 ( A ) Aβ profiles generated by WT or mutant PSEN1 MEFs lines treated with 1 µM GSM III . The chemical structure of the bicyclic heterocycle GSM III is depicted . Data are presented as mean ± SD , N ≥ 3 independent experiments . The blue , orange , green and purple dotted lines indicate the levels of Aβ37 , Aβ38 , Aβ40 and Aβ42 peptides in the WT Aβ profile , respectively . ( B ) Fold change in GSEC processivity of APPC99 estimated by the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio and presented as % of the WT response . The data are presented as mean ± SD , N ≥ 3 . The symbol # marks mutations that fully abolished the GSEC response towards GSM III , As reference , a dotted line was drawn to indicate 10 % ( WT levels ) . One - way ANOVA followed by Dunnett ' s post - hoc test with comparison to WT was used to determine statistical significance ( P < 0 . 05 ) . * * P < 0 . 0001 , * * * * P < 0 . 0001 ( F ( DFn , DFd ) : F ( 32 , 101 ) = 112 . 5 .

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 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

Figure 3(A) Aβ profiles generated by WT or mutant PSEN1 MEFs lines treated with 1 µM GSM III. The chemical structure of the bicyclic heterocycle GSM III is depicted. Data are presented as mean ± SD, N ≥ 3 independent experiments. The blue, orange, green and purple dotted lines indicate the levels of Aβ37, Aβ38, Aβ40 and Aβ42 peptides in the WT Aβ profile, respectively.(B) Fold change in GSEC processivity of APPC99 estimated by the Aβ(37+38)/(40+42) ratio and presented as % of the WT response. The data are presented as mean ± SD, N ≥ 3. The symbol # marks mutations that fully abolished the GSEC response towards GSM III, As reference, a dotted line was drawn to indicate 10% (WT levels). One-way ANOVA followed by Dunnett's post-hoc test with comparison to WT was used to determine statistical significance (P < 0.05). **P < 0.0001, ****P < 0.0001 (F(DFn, DFd): F (32, 101) = 112.5.


Figure 4 ( D ) Aβ profiles generated by WT or mutant PSEN1 cell lines overexpressing APPC99 and treated with increasing concentrations of GSM III . Data are presented as mean ± SD , N = 2 independent experiments . ( E ) GSEC processivity estimated by the Aβ ( 37 + 38 ) / ( 40 + 42 ) ratio was determined for the dose - response analysis ( presented in panel D ) and shown as % of DMSO . Data are presented as mean ± SD .

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Figure 4(D) Aβ profiles generated by WT or mutant PSEN1 cell lines overexpressing APPC99 and treated with increasing concentrations of GSM III. Data are presented as mean ± SD, N = 2 independent experiments.(E) GSEC processivity estimated by the Aβ(37+38)/(40+42) ratio was determined for the dose-response analysis (presented in panel D) and shown as % of DMSO. Data are presented as mean ± SD.