( d ) Bafilomycin A1 stabilizes LC3B - interacting Δ133p53α in senescent cells . The same set of cells as in c were treated with bafilomycin A1 ( + , 100 nM for 4 h ) or untreated ( − ) . These cells were used in IP with anti - LC3B antibody , followed by IB with anti - Δ133p53 antibody . Whole - protein lysates without IP were also examined in IB with anti - LC3B antibody . While untreated samples replicate the result shown in c , the differences between early passage and senescent cells become more evident with bafilomycin A1 treatment .

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(d) Bafilomycin A1 stabilizes LC3B-interacting Δ133p53α in senescent cells. The same set of cells as in c were treated with bafilomycin A1 (+, 100 nM for 4 h) or untreated (−). These cells were used in IP with anti-LC3B antibody, followed by IB with anti-Δ133p53 antibody. Whole-protein lysates without IP were also examined in IB with anti-LC3B antibody. While untreated samples replicate the result shown in c, the differences between early passage and senescent cells become more evident with bafilomycin A1 treatment.


( a ) Δ133p53α protein is polyubiquitinated . FLAG - tagged versions of wild - type Δ133p53α ( wt ) and mutant Δ133p53α ( mut ; from c below ) , along with vector control ( − ) , were retrovirally transduced into MRC - 5 fibroblasts . The wild - type Δ133p53α - expressing cells had a pair of STUB1 siRNA - transfected ( + , siRNA no . 1 for 4 days ) and untransfected ( − ) counterparts . These cells were maintained for 8 h under amino acid - and serum - starved conditions with bafilomycin A1 ( 100 nM ) . Protein lysates were used either in IP with anti - ubiquitin ( Ub ) antibody , followed by IB with anti - FLAG antibody ( top ) , or directly in IB for FLAG - Δ133p53α , STUB1 and β - actin ( lower three panels ) . Smear signals indicate polyubiquitinated FLAG - Δ133p53α protein ( Poly - Ub ) . Asterisk indicates a nonspecific band in negative control . These experimental conditions resulted in similar amounts of FLAG - Δ133p53α in the presence and absence of STUB1 knockdown , allowing quantitative analysis of ubiquitination . The relative densitometric values of wild - type FLAG - Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown ( normalized with total FLAG - Δ133p53α ) are shown . (

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

(a) Δ133p53α protein is polyubiquitinated. FLAG-tagged versions of wild-type Δ133p53α (wt) and mutant Δ133p53α (mut; from c below), along with vector control (−), were retrovirally transduced into MRC-5 fibroblasts. The wild-type Δ133p53α-expressing cells had a pair of STUB1 siRNA-transfected (+, siRNA no. 1 for 4 days) and untransfected (−) counterparts. These cells were maintained for 8 h under amino acid- and serum-starved conditions with bafilomycin A1 (100 nM). Protein lysates were used either in IP with anti-ubiquitin (Ub) antibody, followed by IB with anti-FLAG antibody (top), or directly in IB for FLAG-Δ133p53α, STUB1 and β-actin (lower three panels). Smear signals indicate polyubiquitinated FLAG-Δ133p53α protein (Poly-Ub). Asterisk indicates a nonspecific band in negative control. These experimental conditions resulted in similar amounts of FLAG-Δ133p53α in the presence and absence of STUB1 knockdown, allowing quantitative analysis of ubiquitination. The relative densitometric values of wild-type FLAG-Δ133p53α polyubiquitination in the presence and absence of STUB1 knockdown (normalized with total FLAG-Δ133p53α) are shown. (


( b ) Δ133p53α is ubiquitinated at the C - terminal lysine residues . MRC - 5 fibroblasts expressing wild - type FLAG - Δ133p53α in the absence of STUB1 knockdown were used in IP with anti - FLAG antibody , and the resulting immunoprecipitates were analysed using mass spectrometry . Representative MS / MS spectrum of an [ M + 3H ] 3 + peptide from FLAG - Δ133p53α shows sites of ubiquitin modification . The peptide sequence for the spectrum is shown , with the sites of modification marked as asterisks : amino - acid positions 248 and 249 , which correspond to positions 381 and 382 in full - length p53 . The same peptide was also observed in the unmodified state .

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(b) Δ133p53α is ubiquitinated at the C-terminal lysine residues. MRC-5 fibroblasts expressing wild-type FLAG-Δ133p53α in the absence of STUB1 knockdown were used in IP with anti-FLAG antibody, and the resulting immunoprecipitates were analysed using mass spectrometry. Representative MS/MS spectrum of an [M+3H]3+ peptide from FLAG-Δ133p53α shows sites of ubiquitin modification. The peptide sequence for the spectrum is shown, with the sites of modification marked as asterisks: amino-acid positions 248 and 249, which correspond to positions 381 and 382 in full-length p53. The same peptide was also observed in the unmodified state.


( c ) Lysine ( K ) - to - arginine ( R ) substitutions at amino - acid residues 248 and 249 .

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(c) Lysine (K)-to-arginine (R) substitutions at amino-acid residues 248 and 249.


( d ) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation . The same three cells as in a were cultured under amino - acid - and serum - starved conditions for 8 h ( without bafilomycin A1 ) , transfected with STUB1 siRNA ( no . 1 for 4 days ) or untreated , and examined in IBs for FLAG - Δ133p53α , STUB1 , p62 / SQSTM1 and β - actin . The effect of starvation was confirmed by decreased p62 / SQSTM1 in each cell . The upregulation of p62 / SQSTM1 by wild - type and mutant FLAG - Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression .

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

(d) Mutation of the ubiquitination sites renders Δ133p53α resistant to degradation. The same three cells as in a were cultured under amino-acid- and serum-starved conditions for 8 h (without bafilomycin A1), transfected with STUB1 siRNA (no. 1 for 4 days) or untreated, and examined in IBs for FLAG-Δ133p53α, STUB1, p62/SQSTM1 and β-actin. The effect of starvation was confirmed by decreased p62/SQSTM1 in each cell. The upregulation of p62/SQSTM1 by wild-type and mutant FLAG-Δ133p53α is likely associated with enhanced cell proliferation by Δ133p53α overexpression.


( a ) Δ133p53α is colocalized with p62 / SQSTM1 upon autophagy induction . MRC - 5 fibroblasts expressing FLAG - Δ133p53α ( wild - type ) were cultured under amino - acid - and serum - starved conditions for 4 h , and co - immunostained with anti - FLAG antibody ( Δ133p53α , green ) and anti - p62 / SQSTM1 antibody ( red ) . Two representative sets of images , including nuclear staining with 4 ′ , 6 - diamidino - 2 - phenylindole ( DAPI ) and merged images , are shown . Scale bars , 10 μm .

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(a) Δ133p53α is colocalized with p62/SQSTM1 upon autophagy induction. MRC-5 fibroblasts expressing FLAG-Δ133p53α (wild-type) were cultured under amino-acid- and serum-starved conditions for 4 h, and co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-p62/SQSTM1 antibody (red). Two representative sets of images, including nuclear staining with 4′,6-diamidino-2-phenylindole (DAPI) and merged images, are shown. Scale bars, 10 μm.


( b ) Knockdown of p62 / SQSTM1 stabilizes endogenous Δ133p53α . MRC - 5 fibroblasts at late passage ( PDLs 51 ) were transfected with p62 / SQSTM1 siRNA ( p62 ) or control siRNA ( C ) for 3 days , and examined in immunoblots for Δ133p53α and p62 / SQSTM1 expression . The relative expression levels of Δ133p53α ( normalized with β - actin ) are shown .

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(b) Knockdown of p62/SQSTM1 stabilizes endogenous Δ133p53α. MRC-5 fibroblasts at late passage (PDLs 51) were transfected with p62/SQSTM1 siRNA (p62) or control siRNA (C) for 3 days, and examined in immunoblots for Δ133p53α and p62/SQSTM1 expression. The relative expression levels of Δ133p53α (normalized with β-actin) are shown.


( c ) Δ133p53α is colocalized with LC3B upon autophagy induction . MRC - 5 fibroblasts with FLAG - Δ133p53α under starved conditions ( as in a ) were co - immunostained with anti - FLAG antibody ( Δ133p53α , green ) and anti - LC3B antibody ( red ) . Two representative sets of images , including DAPI and merged images , are shown . Scale bars , 10 μm . ( d ) Quantitative analysis of Δ133p53α - LC3B colocalization . The number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 cells . Data ( percent colocalization ) are mean ± s . d . Almost all Δ133p53α signals were colocalized with LC3B , while ~ 80 % of LC3B signals were colocalized with Δ133p53α .

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

(c) Δ133p53α is colocalized with LC3B upon autophagy induction. MRC-5 fibroblasts with FLAG-Δ133p53α under starved conditions (as in a) were co-immunostained with anti-FLAG antibody (Δ133p53α, green) and anti-LC3B antibody (red). Two representative sets of images, including DAPI and merged images, are shown. Scale bars, 10 μm. (d) Quantitative analysis of Δ133p53α-LC3B colocalization. The number of colocalized signals out of total number of Δ133p53α signals or out of total number of LC3B signals was counted in 26 cells. Data (percent colocalization) are mean±s.d. Almost all Δ133p53α signals were colocalized with LC3B, while ~80% of LC3B signals were colocalized with Δ133p53α.


a , Autophagosome formation in the heart indicated by GFP - LC3 . Hearts were isolated from GFP - LC3 transgenic mice at multiple stages , including 18 . 5 day embryos and 0 . 5 h , 3 h , 6 h , 24 h and 2 . 5 day neonates , immediately fixed , cryosectioned , and analysed by fluorescence microscopy . Scale bar , 10 µm .

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

a, Autophagosome formation in the heart indicated by GFP-LC3. Hearts were isolated from GFP-LC3 transgenic mice at multiple stages, including 18.5 day embryos and 0.5 h, 3 h, 6 h, 24 h and 2.5 day neonates, immediately fixed, cryosectioned, and analysed by fluorescence microscopy. Scale bar, 10 µm.


b , Quantification of GFP - LC3 dots in neonatal tissues . Cryosections were prepared from tissues isolated at the indicated times . The ratio of the total area of GFP - LC3 dots to the total cellular area is shown as a percentage . Values represent mean ± s . d . of three mice . Diaph . , diaphragm ; Panc . , pancreas ; Muscle ; gastrocnemius muscle .

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b, Quantification of GFP-LC3 dots in neonatal tissues. Cryosections were prepared from tissues isolated at the indicated times. The ratio of the total area of GFP-LC3 dots to the total cellular area is shown as a percentage. Values represent mean ± s.d. of three mice. Diaph., diaphragm; Panc., pancreas; Muscle; gastrocnemius muscle.


c , Electron microscopic analysis of the heart from wild - type neonates . Typical autophagosomes ( arrows ) and autolysosomes ( arrowhead ) were observed in the heart at 10 h after birth . Scale bar , 1 µm .

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c, Electron microscopic analysis of the heart from wild-type neonates. Typical autophagosomes (arrows) and autolysosomes (arrowhead) were observed in the heart at 10 h after birth. Scale bar, 1 µm.


d , Conversion of LC3 - I to LC3 - II in neonatal hearts . Heart homogenates from five embryos / neonates were pooled and analysed by immunoblotting using an anti - LC3 antibody . The bands representing LC3 - I and LC3 - II are marked with arrows .

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d, Conversion of LC3-I to LC3-II in neonatal hearts. Heart homogenates from five embryos/neonates were pooled and analysed by immunoblotting using an anti-LC3 antibody. The bands representing LC3-I and LC3-II are marked with arrows.


a , Detection of wild - type ( WT ) and targeted alleles . Genomic DNA was prepared from mouse tails and analysed by Southern blot analysis . Restriction enzymes used were HpaI for the 5 ′ probe and SpeI for the 3 ′ probe ( see Supplementary Fig . S2 for the restriction map ) . KO , knockout ( targeted ) allele .

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

a, Detection of wild-type (WT) and targeted alleles. Genomic DNA was prepared from mouse tails and analysed by Southern blot analysis. Restriction enzymes used were HpaI for the 5′ probe and SpeI for the 3′ probe (see Supplementary Fig.S2 for the restriction map). KO, knockout (targeted) allele.


b , Photograph of a representative Atg5 - / - neonate compared to a wild - type littermate .

b, Photograph of a representative Atg5-/- neonate compared to a wild-type littermate.


c , Immunoblot analysis of Atg5 and LC3 . Heart homogenates were prepared from neonates 10 h after birth . MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10 % FBS ( unstarved ) or Hanks ' solution for 2 h ( starved ) , and total cell lysates were prepared . Immunoblot analysis was performed using anti - Atg5 and anti - LC3 antibodies . The position of the Atg12 - Atg5 conjugate on the blot is indicated .

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c, Immunoblot analysis of Atg5 and LC3. Heart homogenates were prepared from neonates 10 h after birth. MEFs were cultured in Dulbeccos modified Eagles medium supplemented with 10% FBS (unstarved) or Hanks' solution for 2 h (starved), and total cell lysates were prepared. Immunoblot analysis was performed using anti-Atg5 and anti-LC3 antibodies. The position of the Atg12-Atg5 conjugate on the blot is indicated.


d , Absence of GFP - LC3 dot formation in Atg5 - / - mouse heart . Various tissues from Atg5 - / - mice expressing GFP - LC3 were analysed for dot formation by fluorescence microscopy . A representative result from a neonatal heart at 3 h after birth is shown .

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

d, Absence of GFP-LC3 dot formation in Atg5-/- mouse heart. Various tissues from Atg5-/- mice expressing GFP-LC3 were analysed for dot formation by fluorescence microscopy. A representative result from a neonatal heart at 3 h after birth is shown.


Neonates were obtained by caesarean delivery . a , b , Successfully resuscitated pups were monitored in a humidified chamber without milk feeding ( a ) or with artificial milk feeding provided every 3 - 6 h through a tube inserted into the stomach ( b ) .

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Neonates were obtained by caesarean delivery. a, b, Successfully resuscitated pups were monitored in a humidified chamber without milk feeding (a) or with artificial milk feeding provided every 3-6 h through a tube inserted into the stomach (b).


a , Plasma and tissue amino acid concentrations . Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions . ' Total ' indicates the sum of the Asp , Thr , Ser , Asn , Glu , Gln , Pro , Gly , Ala , Val , Cys , Met , Ile , Leu , Tyr , Phe , Lys , His and Arg concentrations ; ' Essential ' indicates the sum of Thr , Val , Met , Ile , Leu , Phe , Lys , His and Arg concentrations ; ' BCAA ' indicates the sum of the Val , Ile and Leu concentrations . Tissue amino acid concentrations are expressed as mmol kg - 1 of wet weight . Bars represent mean ± s . d . of three mice . Single and double asterisks indicate a significant difference between mutant and control ( wild - type plus heterozygous ) mice at P 0 . 05 and P 0 . 01 , respectively .

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

a, Plasma and tissue amino acid concentrations. Amino acid concentrations were measured at 0 and 10 h after the caesarean delivery under fasting conditions. 'Total' indicates the sum of the Asp, Thr, Ser, Asn, Glu, Gln, Pro, Gly, Ala, Val, Cys, Met, Ile, Leu, Tyr, Phe, Lys, His and Arg concentrations; 'Essential' indicates the sum of Thr, Val, Met, Ile, Leu, Phe, Lys, His and Arg concentrations; 'BCAA' indicates the sum of the Val, Ile and Leu concentrations. Tissue amino acid concentrations are expressed as mmol kg-1 of wet weight. Bars represent mean ± s.d. of three mice. Single and double asterisks indicate a significant difference between mutant and control (wild-type plus heterozygous) mice at P 0.05 and P 0.01, respectively.


b , Activation of AMPK in Atg5 - / - mice . Hearts were isolated from unfed neonates at 0 h ( lanes 1 , 3 , 5 ) and 10 h ( lanes 2 , 4 , 6 ) , and force - fed mice at 10 h ( lane 7 ) after the caesarean delivery . Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti - AMPK and anti - phospho - AMPK antibodies .

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b, Activation of AMPK in Atg5-/- mice. Hearts were isolated from unfed neonates at 0 h (lanes 1, 3, 5) and 10 h (lanes 2, 4, 6), and force-fed mice at 10 h (lane 7) after the caesarean delivery. Homogenates were prepared in the presence of phosphatase inhibitors and analysed by immunoblotting using anti-AMPK and anti-phospho-AMPK antibodies.


c , ECGs from neonatal mice at the indicated time after caesarean delivery . ST elevation was observed in all Atg5 - / - mice ( n = 4 ) at 8 - 9 h after delivery .

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c, ECGs from neonatal mice at the indicated time after caesarean delivery. ST elevation was observed in all Atg5-/- mice (n = 4) at 8-9 h after delivery.


A . Hcp release assay . HA - tagged Hcp ( HcpHA ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) , ΔtssM cells or ΔtssM cells carrying the AHT - inducible FLAG - tagged tssM - borne plasmid ( tssM + ) treated ( bulgecin ) or not ( NT ) with bulgecin A prior to tssM gene induction . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and the periplasmic TolB protein ( control for cell lysis ) , HcpHA and FLTssM were immunodetected using anti - TolB ( middle panel ) , anti - HA ( lower panel ) and anti - FLAG ( upper panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in duplicate and a representative result is shown .

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

A. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT), ΔtssM cells or ΔtssM cells carrying the AHT-inducible FLAG-tagged tssM-borne plasmid (tssM+) treated (bulgecin) or not (NT) with bulgecin A prior to tssM gene induction. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis), HcpHA and FLTssM were immunodetected using anti-TolB (middle panel), anti-HA (lower panel) and anti-FLAG (upper panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.


B . Hcp release assay . HA - tagged Hcp ( HcpHA ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 wild - type ( WT ) cells before washing cells ( before wash ) and after washing and growth ( after wash ) in absence ( NT ) or presence ( bulgecin ) of bulgecin A . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and the periplasmic TolB protein ( control for cell lysis ) and HcpHA were immunodetected using anti - TolB ( upper panel ) and anti - HA ( lower panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in duplicate and a representative result is shown .

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

B. Hcp release assay. HA-tagged Hcp (HcpHA) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 wild-type (WT) cells before washing cells (before wash) and after washing and growth (after wash) in absence (NT) or presence (bulgecin) of bulgecin A. Proteins were separated by 12.5%-acrylamide SDS-PAGE and the periplasmic TolB protein (control for cell lysis) and HcpHA were immunodetected using anti-TolB (upper panel) and anti-HA (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in duplicate and a representative result is shown.


Hcp release assay . FLAG - tagged Hcp ( HcpFL ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 cells of the indicated strains . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and TolB and HcpFL were immunodetected using anti - TolB ( upper panel ) and anti - FLAG ( lower panel ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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

Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 cells of the indicated strains. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB and HcpFL were immunodetected using anti-TolB (upper panel) and anti-FLAG (lower panel) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


A . Hcp release assay . FLAG - tagged Hcp ( HcpFL ) release was assessed by separating cells ( C ) and cell - free culture supernatant ( S ) fractions from 109 WT , ΔmltE cells or ΔmltE cells producing wild - type ( mltE + ) or E64Q mutant ( mltEE64Q ) VSV - G - tagged MltE ( MltEV ) from arabinose - inducible plasmids . Proteins were separated by 12 . 5 % - acrylamide SDS - PAGE and TolB , HcpFL and MltEV were immunodetected using anti - TolB ( upper panel ) , anti - FLAG ( middle panel ) and anti - VSV - G ( lower panel ) antibodies , respectively . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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

A. Hcp release assay. FLAG-tagged Hcp (HcpFL) release was assessed by separating cells (C) and cell-free culture supernatant (S) fractions from 109 WT, ΔmltE cells or ΔmltE cells producing wild-type (mltE+) or E64Q mutant (mltEE64Q) VSV-G-tagged MltE (MltEV) from arabinose-inducible plasmids. Proteins were separated by 12.5%-acrylamide SDS-PAGE and TolB, HcpFL and MltEV were immunodetected using anti-TolB (upper panel), anti-FLAG (middle panel) and anti-VSV-G (lower panel) antibodies, respectively. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


B . Anti - bacterial activity . E . coli K - 12 prey cells ( W3110 gfp + , kanR ) were mixed with the indicated attacker cells , spotted onto Sci - 1 inducing medium ( SIM ) agar plates and incubated for 4 hours at 37 ° C . The image of a representative bacterial spot and the average and standard deviation ( n = 3 ) of the relative fluorescence of the bacterial mixture ( in arbitrary unit , AU ) are shown in the upper graph . The number of recovered E . coli prey cells ( counted on selective kanamycin medium ) is indicated in the lower graph ( in log10 of colony - forming unit ( cfu ) ) . The black , dark grey and light grey circles indicate values from three independent assays , and the average is indicated by the bar . The experiment was performed in triplicate and a representative result is shown . Asterisks indicate significant differences compared to the wild - type attacker strain ( NS , non significant ; * * * , p < 0 . 001 ) .

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B. Anti-bacterial activity. E. coli K-12 prey cells (W3110 gfp+, kanR) were mixed with the indicated attacker cells, spotted onto Sci-1 inducing medium (SIM) agar plates and incubated for 4 hours at 37°C. The image of a representative bacterial spot and the average and standard deviation (n=3) of the relative fluorescence of the bacterial mixture (in arbitrary unit, AU) are shown in the upper graph. The number of recovered E. coli prey cells (counted on selective kanamycin medium) is indicated in the lower graph (in log10 of colony-forming unit (cfu)). The black, dark grey and light grey circles indicate values from three independent assays, and the average is indicated by the bar. The experiment was performed in triplicate and a representative result is shown. Asterisks indicate significant differences compared to the wild-type attacker strain (NS, non significant; ***, p < 0.001).


A - B . Bacterial two - hybrid assay . BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X - Gal indicator plates . The blue color of the colony reflects the interaction between the two proteins . TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins . The experiment was performed in triplicate and a representative result is shown .

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A-B. Bacterial two-hybrid assay. BTH101 reporter cells producing the indicated proteins or domains fused to the T18 or T25 domain of the Bordetella adenylate cyclase were spotted on X-Gal indicator plates. The blue color of the colony reflects the interaction between the two proteins. TolB and Pal are two proteins known to interact but unrelated to the T6SS or the MltE proteins. The experiment was performed in triplicate and a representative result is shown.


C . Co - immunoprecipitation assay . The solubilized lysates from 2 × 1010 E . coli K - 12 W3110 cells co - producing the indicated FLAG - tagged TssMP variants ( exported in the periplasm ) and VSV - G - tagged MltE protein ( Total , T ) were subjected to immune precipitation on anti - FLAG - coupled agarose beads . The immunoprecipitated material ( IP ) was subjected to 12 . 5 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG ( upper panel , TssM domains ) and anti - VSV - G ( lower panel , MltE ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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C. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 E. coli K-12 W3110 cells co-producing the indicated FLAG-tagged TssMP variants (exported in the periplasm) and VSV-G-tagged MltE protein (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM domains) and anti-VSV-G (lower panel, MltE) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


A . Remazol brilliant blue assay . The absorbance of supernatants from the reaction containing purified and RBB - labelled E . coli peptidoglycan and the indicated protein ( 50 µg ) were measured at = 595 nm after incubation for 0 . 5 or 4 hours at 37 ° C . The results shown are the average and standard deviation from triplicate reactions ( n = 3 ) . Asterisks indicate significant differences compared to the buffer ( NS , non significant ; * * , p < 0 . 01 ; * * * , p < 0 . 001 ) . The supernatant of the reaction after 4 hours of incubation is shown on bottom .

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A. Remazol brilliant blue assay. The absorbance of supernatants from the reaction containing purified and RBB-labelled E. coli peptidoglycan and the indicated protein (50 µg) were measured at =595 nm after incubation for 0.5 or 4 hours at 37°C. The results shown are the average and standard deviation from triplicate reactions (n=3). Asterisks indicate significant differences compared to the buffer (NS, non significant; **, p < 0.01; ***, p < 0.001). The supernatant of the reaction after 4 hours of incubation is shown on bottom.


B . Peptidoglycan hydrolysis . The decrease of the absorbance of the M . luteus peptidoglycan suspension in presence of the indicated protein ( 50 µg ) was measured at = 600 nm at 37 ° C over time . The experiment was performed in triplicate and a representative result is shown .

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

B. Peptidoglycan hydrolysis. The decrease of the absorbance of the M. luteus peptidoglycan suspension in presence of the indicated protein (50 µg) was measured at =600 nm at 37°C over time. The experiment was performed in triplicate and a representative result is shown.


A - B . Co - immunoprecipitation assay . The solubilized lysates from 2 × 1010 EAEC wild - type or ∆ mltE cells producing FLAG - tagged TssM ( FLTssM ) and / or HA - tagged TssJ ( TssJHA , panel A ) or TssL ( TssLHA , panel B ) ( Total , T ) were subjected to immune precipitation on anti - FLAG - coupled agarose beads . The immunoprecipitated material ( IP ) was subjected to 12 . 5 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG ( upper panel , TssM ) and anti - HA ( lower panel , TssJ or TssL ) antibodies . Molecular weight markers ( in kDa ) are indicated on the left . The experiments were performed in triplicate and a representative result is shown .

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A-B. Co-immunoprecipitation assay. The solubilized lysates from 2×1010 EAEC wild-type or ∆mltE cells producing FLAG-tagged TssM (FLTssM) and/or HA-tagged TssJ (TssJHA, panel A) or TssL (TssLHA, panel B) (Total, T) were subjected to immune precipitation on anti-FLAG-coupled agarose beads. The immunoprecipitated material (IP) was subjected to 12.5%-acrylamide SDS-PAGE and immunodetected with anti-FLAG (upper panel, TssM) and anti-HA (lower panel, TssJ or TssL) antibodies. Molecular weight markers (in kDa) are indicated on the left. The experiments were performed in triplicate and a representative result is shown.


C . BS3 cross - linking assay . 2 × 109 cells of the indicated strain producing FLAG - tagged TssM ( with the exception of ∆ tssM cells ) were treated ( + ) or not ( - ) with the BS3 cross - linker agent . After the cross - linking reaction , cells were boiled in Laemmli buffer and total proteins were subjected to 7 % - acrylamide SDS - PAGE and immunodetected with anti - FLAG antibodies . The TssM protein ( FLTssM ) and its complexes ( * , TssM - TssJ ; * * , TssM - TssL ) are indicated on the right as well as the TssM dimer ( arrow ) . Molecular weight markers ( in kDa ) are indicated on the left . The experiment was performed in triplicate and a representative result is shown .

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

C. BS3 cross-linking assay. 2×109 cells of the indicated strain producing FLAG-tagged TssM (with the exception of ∆tssM cells) were treated (+) or not (-) with the BS3 cross-linker agent. After the cross-linking reaction, cells were boiled in Laemmli buffer and total proteins were subjected to 7%-acrylamide SDS-PAGE and immunodetected with anti-FLAG antibodies. The TssM protein (FLTssM) and its complexes (*, TssM-TssJ; **, TssM-TssL) are indicated on the right as well as the TssM dimer (arrow). Molecular weight markers (in kDa) are indicated on the left. The experiment was performed in triplicate and a representative result is shown.


D . Fluorescence microscopy . Recordings showing TssM localization using the chromosomally - encoded sfGFP - tssM fusion in wild - type ( WT ) or ΔmltE cells or ΔmltE cells producing the wild - type ( mltE + ) or catalytic variant ( mltEE64Q ) MltE protein . Scale bars are 1 μm . The experiment was performed in triplicate and a representative result is shown .

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D. Fluorescence microscopy. Recordings showing TssM localization using the chromosomally-encoded sfGFP-tssM fusion in wild-type (WT) or ΔmltE cells or ΔmltE cells producing the wild-type (mltE+) or catalytic variant (mltEE64Q) MltE protein. Scale bars are 1 μm. The experiment was performed in triplicate and a representative result is shown.


High - resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope . Images of IMM in living HeLa , L6 , and H1975 cells , stained with 10 - N - nonyl acridine orange ( NAO ) . NAO preferentially binds phospholipids in the IMM , such as cardiolipin . Arrowheads indicate cristae in the IMM . Scale bar = 500 nm . N ≥ 3 independent experiments for each cell type .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Images of IMM in living HeLa, L6, and H1975 cells, stained with 10-N-nonyl acridine orange (NAO). NAO preferentially binds phospholipids in the IMM, such as cardiolipin. Arrowheads indicate cristae in the IMM. Scale bar = 500 nm. N ≥ 3 independent experiments for each cell type.


High - resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope . Mitochondrion cropped from HeLa cell shown in A ( dashed line ) and zoomed in to show cristae , inner boundary membrane ( IBM ) , and matrix ( arrows ) . Scale bar = 100 nm .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondrion cropped from HeLa cell shown in A (dashed line) and zoomed in to show cristae, inner boundary membrane (IBM), and matrix (arrows). Scale bar = 100 nm.


High - resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope . H1975 cells transduced with matrix - targeted - DsRed and stained with NAO . Matrix - targeted Ds - Red differentiates matrix from cristae stained with NAO . Arrowheads point to cristae . Scale bar = 500 nm . N = 1 independent experiment .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. H1975 cells transduced with matrix-targeted-DsRed and stained with NAO. Matrix-targeted Ds-Red differentiates matrix from cristae stained with NAO. Arrowheads point to cristae. Scale bar = 500 nm. N = 1 independent experiment.


High - resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope . The structure of the IMM in Mic13 - KO cells ( HeLa ) , stained with NAO . The number of cristae is decreased in Mic13 - KO compared to control cells shown in panel A , labeled with arrowheads . Scale bar = 500 nm . N = 3 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. The structure of the IMM in Mic13-KO cells (HeLa), stained with NAO. The number of cristae is decreased in Mic13-KO compared to control cells shown in panel A, labeled with arrowheads. Scale bar = 500 nm. N = 3 independent experiments.


High - resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope . Live - cell imaging of the IMM in control and PTPMT1 KD H1975 cells , a model of cardiolipin deficiency . A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO , showing cristae ( arrowheads ) . Scale bars = 500 nm . N = 3 independent experiments . A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO . Note the derangement of the ultrastructure ( arrowheads ) . Scale bars = 500 nm . N = 3 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Live-cell imaging of the IMM in control and PTPMT1 KD H1975 cells, a model of cardiolipin deficiency. A gallery of mitochondria from various control H1975 cells expressing scrambled shRNA and stained with NAO, showing cristae (arrowheads). Scale bars = 500 nm. N = 3 independent experiments. A gallery of mitochondria from various H1975 cells expressing PTPMT1 shRNA and stained with NAO. Note the derangement of the ultrastructure (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.


High - resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope . Mitochondria from HeLa cell , co - stained with NAO and TMRE . Area from dashed white box , zoomed to right , shows the red and green intensities of TMRE and NAO colocalized . Scale bars = 500 nm . N = 3 independent experiments . Mitochondria in L6 myoblast , co - stained with NAO and TMRE . Area from dashed white box , zoomed to right , shows colocalizing NAO and TMRE at the cristae membrane ( arrowheads ) . Scale bars = 500 nm . N = 3 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria from HeLa cell, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows the red and green intensities of TMRE and NAO colocalized. Scale bars = 500 nm. N = 3 independent experiments. Mitochondria in L6 myoblast, co-stained with NAO and TMRE. Area from dashed white box, zoomed to right, shows colocalizing NAO and TMRE at the cristae membrane (arrowheads). Scale bars = 500 nm. N = 3 independent experiments.


High - resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope . Mitochondria in HeLa cells , stained with NAO alone ( top row ) vs . NAO + TMRE ( bottom row ) , and simultaneously excited with 488 - and 561 - nm lasers . Note that mitochondria stained with NAO alone do not emit noticeable fluorescence in the red channel ; only after adding TMRE does strong signal appear in the red channel , showing negligible bleed - through . Scale bars = 500 nm . N = 2 independent experiments .

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High-resolution imaging of mitochondria in live cells using the Airyscan module of Zeiss LSM880 confocal microscope. Mitochondria in HeLa cells, stained with NAO alone (top row) vs. NAO + TMRE (bottom row), and simultaneously excited with 488- and 561-nm lasers. Note that mitochondria stained with NAO alone do not emit noticeable fluorescence in the red channel; only after adding TMRE does strong signal appear in the red channel, showing negligible bleed-through. Scale bars = 500 nm. N = 2 independent experiments.


Time - lapse Airyscan imaging of ΔΨm in living HeLa cell , co - stained with MTG ( ΔΨm - insensitive after loading ) and TMRE ( ΔΨm sensitive ) . Arrowhead points to a flickering event where a mitochondrion depolarizes ( ~ 9 sec ) and repolarizes ( ~ 16 sec ) , showing that heterogeneous signal from TMRE ( but not MTG ) disappears and reappears . Scale bar = 500 nm . N = 4 independent experiments . Quantification of A . Plot shows average TMRE fluorescence intensity ( FI ) of cristae ( dark red line ) vs . matrix ( light red line ) during the time series . The drop in TMRE FI during the depolarization phase of the flickering is the ΔΨm - sensitive component of the TMRE signal . The remaining TMRE FI during depolarization can be considered as the ΔΨm - insensitive portion of TMRE signal . Note that the remaining TMRE FI after depolarization at the cristae and matrix are approximately identical , indicating that differences in TMRE FI between the cristae and matrix prior to depolarization are derived from differences in ΔΨm . N = 4 independent experiments .

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Time-lapse Airyscan imaging of ΔΨm in living HeLa cell, co-stained with MTG (ΔΨm-insensitive after loading) and TMRE (ΔΨm sensitive). Arrowhead points to a flickering event where a mitochondrion depolarizes (~9 sec) and repolarizes (~16 sec), showing that heterogeneous signal from TMRE (but not MTG) disappears and reappears. Scale bar = 500 nm. N = 4 independent experiments. Quantification of A. Plot shows average TMRE fluorescence intensity (FI) of cristae (dark red line) vs. matrix (light red line) during the time series. The drop in TMRE FI during the depolarization phase of the flickering is the ΔΨm-sensitive component of the TMRE signal. The remaining TMRE FI during depolarization can be considered as the ΔΨm-insensitive portion of TMRE signal. Note that the remaining TMRE FI after depolarization at the cristae and matrix are approximately identical, indicating that differences in TMRE FI between the cristae and matrix prior to depolarization are derived from differences in ΔΨm. N = 4 independent experiments.


Live - cell Airyscan image of mitochondrion in HeLa cell , showing different membrane potentials in different mitochondrial regions . Membrane potentials were calculated based on TMRE FI differences between compartments ( FIcomp ) . Regions of interest from cristae and IBM used for ΔΨm calculations are labeled with arrows on the right - hand side of the mitochondrion , using the cytosol as the reference compartment . Labeled with forks on the left - hand side of the mitochondrion are the calculations of ΔΨm between individual cristae and their neighboring IBM . Nernst equation used to calculate different voltages . Scale bar = 500 nm . LUT of mitochondrion in HeLa cell shown in A , color - coding TMRE FIs on scale of white ( most intense ) to blue ( least intense ) : LUT scale shown in lower left - hand corner . Arrowheads indicate cristae . Scale bar = 500 nm . Note that most - intense pixels ( white ) only associate with cristae . Quantification of ΔΨm ( mV ) at cristae and IBM relative to the average ΔΨm of the whole mitochondrion , calculated as in A . Note : ΔΨm at cristae is significantly higher than ΔΨm at IBM , indicating electrochemical boundaries separate these two regions of the IMM . N = 3 independent experiments .

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

Live-cell Airyscan image of mitochondrion in HeLa cell, showing different membrane potentials in different mitochondrial regions. Membrane potentials were calculated based on TMRE FI differences between compartments (FIcomp). Regions of interest from cristae and IBM used for ΔΨm calculations are labeled with arrows on the right-hand side of the mitochondrion, using the cytosol as the reference compartment. Labeled with forks on the left-hand side of the mitochondrion are the calculations of ΔΨm between individual cristae and their neighboring IBM. Nernst equation used to calculate different voltages. Scale bar = 500 nm. LUT of mitochondrion in HeLa cell shown in A, color-coding TMRE FIs on scale of white (most intense) to blue (least intense): LUT scale shown in lower left-hand corner. Arrowheads indicate cristae. Scale bar = 500 nm. Note that most-intense pixels (white) only associate with cristae. Quantification of ΔΨm (mV) at cristae and IBM relative to the average ΔΨm of the whole mitochondrion, calculated as in A. Note: ΔΨm at cristae is significantly higher than ΔΨm at IBM, indicating electrochemical boundaries separate these two regions of the IMM. N = 3 independent experiments.


Live - cell Airyscan imaging of mitochondria in cultured primary mouse hepatocytes stained with TMRE . Arrowheads denote cristae . D . Image showing TMRE - labeled mitochondria . E . LUT color - coding of TMRE FI . F . Quantification of ΔΨm differences using Nernst equation as shown in A . Scale bar = 500 nm . N = 2 independent experiments .

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Live-cell Airyscan imaging of mitochondria in cultured primary mouse hepatocytes stained with TMRE. Arrowheads denote cristae. D. Image showing TMRE-labeled mitochondria. E. LUT color-coding of TMRE FI. F. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 2 independent experiments.


Live - cell STED imaging of mitochondria in HeLa cell stained with TMRM . Arrowheads show cristae . Scale bar = 500 nm . G . Image showing TMRM - labeled mitochondrion . H . LUT color - coding of TMRM FI . I . Quantification of ΔΨm differences using Nernst equation as shown in A . Scale bar = 500 nm . N = 4 independent experiments . Note that calculation of ΔΨm differences between the compartments based on images captured by STED and by Airyscan were very similar .

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Live-cell STED imaging of mitochondria in HeLa cell stained with TMRM. Arrowheads show cristae. Scale bar = 500 nm. G. Image showing TMRM-labeled mitochondrion. H. LUT color-coding of TMRM FI. I. Quantification of ΔΨm differences using Nernst equation as shown in A. Scale bar = 500 nm. N = 4 independent experiments. Note that calculation of ΔΨm differences between the compartments based on images captured by STED and by Airyscan were very similar.


Response of ΔΨm to the F1Fo ATP Synthase inhibitor , oligomycin ( 10 µM ) , compared to the uncoupler , FCCP ( 10 µM ) , determined using live - cell Airyscan imaging of HeLa cells co - stained with MTG and TMRE . Quantification of total mitochondrial TMRE FI . N = 3 independent experiments . Quantification of cristae ΔΨm relative to IBM ( ΔΨCr - IBM ) in response to oligomycin compared to FCCP . F1Fo ATP Synthase is exclusively localized to the cristae and significant increase in ΔΨCr - IBM in response to blocking of F1Fo ATP Synthase with oligomycin indicates that differences in TMRE FI between cristae and IBM are driven by oxidative phosphorylation . N = 3 independent experiments .

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

Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Quantification of total mitochondrial TMRE FI. N = 3 independent experiments. Quantification of cristae ΔΨm relative to IBM (ΔΨCr-IBM) in response to oligomycin compared to FCCP. F1Fo ATP Synthase is exclusively localized to the cristae and significant increase in ΔΨCr-IBM in response to blocking of F1Fo ATP Synthase with oligomycin indicates that differences in TMRE FI between cristae and IBM are driven by oxidative phosphorylation. N = 3 independent experiments.


Response of ΔΨm to the F1Fo ATP Synthase inhibitor , oligomycin ( 10 µM ) , compared to the uncoupler , FCCP ( 10 µM ) , determined using live - cell Airyscan imaging of HeLa cells co - stained with MTG and TMRE . Representative Airyscan images showing mitochondria from living HeLa cells stained with MTG and TMRE . Cells were first stained for one hour and only then treated with oligomycin or FCCP . Images show hyperpolarization of cristae under oligomycin ( middle row ; arrowheads ) and depolarization of cristae under FCCP ( bottom row ; arrowheads ) compared to control ( top row ; arrowheads ) . Scale bar = 500 nm . N = 3 independent experiments . Note that the ΔΨm - independent fraction of TMRE staining is less than 5 % of the signal in control conditions .

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

Response of ΔΨm to the F1Fo ATP Synthase inhibitor, oligomycin (10 µM), compared to the uncoupler, FCCP (10 µM), determined using live-cell Airyscan imaging of HeLa cells co-stained with MTG and TMRE. Representative Airyscan images showing mitochondria from living HeLa cells stained with MTG and TMRE. Cells were first stained for one hour and only then treated with oligomycin or FCCP. Images show hyperpolarization of cristae under oligomycin (middle row; arrowheads) and depolarization of cristae under FCCP (bottom row; arrowheads) compared to control (top row; arrowheads). Scale bar = 500 nm. N = 3 independent experiments. Note that the ΔΨm-independent fraction of TMRE staining is less than 5% of the signal in control conditions.


Zoomed - in region of FCCP - treated mitochondria in C . Increased contrast was used to visualize TMRE to compensate for TMRE loss induced by FCCP . These images show diminished heterogeneity of ΔΨCr - IBM values .

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

Zoomed-in region of FCCP-treated mitochondria in C. Increased contrast was used to visualize TMRE to compensate for TMRE loss induced by FCCP. These images show diminished heterogeneity of ΔΨCr-IBM values.


The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied Representative images of mitochondria in control ( top row ) vs . Mic13 - KO ( bottom row ) HeLa cells . Note that TMRE FI in Mic13 - KO mitochondria is distributed more evenly along the IMM , so that ΔΨCr - IBM is decreased . Scale bar = 500 nm . N = 3 independent experiments . Quantification of A , showing deletion of Mic13 in HeLa cells results in significant decrease in ΔΨCr - IBM . While Mic13 - KO cells show a substantial decrease in cristae number , the loss of cristae does not appear absolute , making it feasible to compare TMRE FI at cristae relative to IBM . N = 3 independent experiments .

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

The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied Representative images of mitochondria in control (top row) vs. Mic13-KO (bottom row) HeLa cells. Note that TMRE FI in Mic13-KO mitochondria is distributed more evenly along the IMM, so that ΔΨCr-IBM is decreased. Scale bar = 500 nm. N = 3 independent experiments. Quantification of A, showing deletion of Mic13 in HeLa cells results in significant decrease in ΔΨCr-IBM. While Mic13-KO cells show a substantial decrease in cristae number, the loss of cristae does not appear absolute, making it feasible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments.


The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied by disrupting cristae using Mic10 - and Mic60 - deficient cells . Mic60 support cristae formation . Mic10 is essential for CJ formation , and , in its absence , cristae tend to remain as vesicles detached from IBM . Live - cell Airyscan imaging of TMRE was used in all figure panels and models . Representative images of Hap1 control ( top row ) vs . Mic60 KO ( middle row ) and Mic10 KO ( bottom row ) ; Mic60 - KO mitochondria show very few cristae structures . TMRE staining along the IMM is relatively homogeneous , indicating decreased ΔΨCr - IBM . In general , Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM ( arrowheads ) as compared to control cells . Deletion of Mic10 induces detachment of cristae from IBM ( see Figure 8 ) . Except for the detached cristae vesicles , the TMRE staining is homogeneously distributed n Mic10 - KO cells , indicating decreased ΔΨCr - IBM . Scale bar = 500 nm . N = 3 independent experiments . Quantification of C , showing Mic60 KO in Hap1 cells results in significant decrease in ΔΨCr - IBM . While Mic60 - KO cells show a marked loss of normal cristae structures , depletion of cristae does not appear absolute , making it possible to compare TMRE FI at cristae relative to IBM . N = 3 independent experiments . Quantification of C , showing Mic10 KO in Hap1 cells results in a significant decrease in ΔΨCr - IBM . Note : quantification of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM . N = 3 independent experiments .

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

The role of cristae structure as well as CJ formation and sealing on the generation of the difference in ΔΨm between cristae and IBM were studied by disrupting cristae using Mic10- and Mic60-deficient cells. Mic60 support cristae formation. Mic10 is essential for CJ formation, and, in its absence, cristae tend to remain as vesicles detached from IBM. Live-cell Airyscan imaging of TMRE was used in all figure panels and models. Representative images of Hap1 control (top row) vs. Mic60 KO (middle row) and Mic10 KO (bottom row); Mic60-KO mitochondria show very few cristae structures. TMRE staining along the IMM is relatively homogeneous, indicating decreased ΔΨCr-IBM. In general, Mic10 KO in Hap1 cells results in decreased TMRE signal intensity at cristae relative to IBM (arrowheads) as compared to control cells. Deletion of Mic10 induces detachment of cristae from IBM (see Figure 8). Except for the detached cristae vesicles, the TMRE staining is homogeneously distributed n Mic10-KO cells, indicating decreased ΔΨCr-IBM. Scale bar = 500 nm. N = 3 independent experiments. Quantification of C, showing Mic60 KO in Hap1 cells results in significant decrease in ΔΨCr-IBM. While Mic60-KO cells show a marked loss of normal cristae structures, depletion of cristae does not appear absolute, making it possible to compare TMRE FI at cristae relative to IBM. N = 3 independent experiments. Quantification of C, showing Mic10 KO in Hap1 cells results in a significant decrease in ΔΨCr-IBM. Note: quantification of ΔΨm at cristae relative to IBM was performed only on cristae structures that appeared to maintain attachment to IBM. N = 3 independent experiments.


Studies show that Opa1 interacts with MICOS complex , promoting closure of CJs . Thus , we tested whether Opa1 was required to maintain the difference in ΔΨm between cristae and IBM . Live - cell Airyscan images of MEF control ( top row ) vs . Opa1 KO ( bottom row ) , co - stained with NAO and TMRE . Arrowheads indicate decreased intensity of TMRE FI at cristae compared to IBM . Note the more even distribution of TMRE staining , indicating the cristae and IBM are relatively equipotential . Scale bar = 500 nm . Quantification of G , showing Opa1 - KO MEFs have significantly decreased ΔΨCr - IBM . N = 3 independent experiments .

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

Studies show that Opa1 interacts with MICOS complex, promoting closure of CJs. Thus, we tested whether Opa1 was required to maintain the difference in ΔΨm between cristae and IBM. Live-cell Airyscan images of MEF control (top row) vs. Opa1 KO (bottom row), co-stained with NAO and TMRE. Arrowheads indicate decreased intensity of TMRE FI at cristae compared to IBM. Note the more even distribution of TMRE staining, indicating the cristae and IBM are relatively equipotential. Scale bar = 500 nm. Quantification of G, showing Opa1-KO MEFs have significantly decreased ΔΨCr-IBM. N = 3 independent experiments.


Live - cell Airyscan images of laser - induced depolarization time series of Mic10 KO Hap1 cell stained with TMRE . Mitochondrion exposed to rapid , high 2 - photon laser power in region of white box , arrow ( ~ 0 . 3 sec ) ; large , hyperpolarized vesicle ( arrowhead ) remains polarized after the rest of the mitochondrion loses ΔΨm , indicating electrochemical discontinuity between hyperpolarized vesicles and the rest of the mitochondrion , including the IBM . Scale bar = 500 nm . N = 2 independent experiments .

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

Live-cell Airyscan images of laser-induced depolarization time series of Mic10 KO Hap1 cell stained with TMRE. Mitochondrion exposed to rapid, high 2-photon laser power in region of white box, arrow (~0.3 sec); large, hyperpolarized vesicle (arrowhead) remains polarized after the rest of the mitochondrion loses ΔΨm, indicating electrochemical discontinuity between hyperpolarized vesicles and the rest of the mitochondrion, including the IBM. Scale bar = 500 nm. N = 2 independent experiments.


Live - cell Airyscan image of time series showing hyperpolarized vesicles ( arrowheads ; region cropped from dashed white box ) inside mitochondria lacking both Opa1 and Drp1 . Scale bar = 500 nm . N = 2 independent experiments . Free movement of hyperpolarized vesicles within the matrix indicate their detachment from the IBM , representing an extreme case of cristae independency within one mitochondrion ( see Movie EV 1 for time series ) .

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

Live-cell Airyscan image of time series showing hyperpolarized vesicles (arrowheads; region cropped from dashed white box) inside mitochondria lacking both Opa1 and Drp1. Scale bar = 500 nm. N = 2 independent experiments. Free movement of hyperpolarized vesicles within the matrix indicate their detachment from the IBM, representing an extreme case of cristae independency within one mitochondrion (see Movie EV 1 for time series).


Laser - induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM , visualized using live - cell Airyscan microscopy . Representative images of laser - induced depolarization time series from L6 myoblast , stained with ΔΨm - dependent dye , Rho123 . Images show LUT color - coded for Rho123 FI . White and blue pixels represent most and least intense ΔΨm , respectively . See legend of LUT colors on the right . Note that at the first time point ( ~ 0 . 15 sec ) white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2 - photon laser ( ≤ 5 msec ) , inducing depolarization . Ensuing frames show wavelike depolarization away from site of perturbation ( arrowheads ) . Scale bar = 500 nm . N = 3 independent experiments . ( See Movie EV 2 for time series . ) Profile plot showing Rho123 pixel intensity as function of distance along mitochondrion in A . Note that zero value in the X axis corresponds to top area of the mitochondrion shown in A , near site of 2 - photon laser exposure ( box ) . Red line corresponds to frame immediately before depolarization ( ~ 0 . 15 sec ) ; green , blue , purple , and dark purple lines correspond to ~ 0 . 30 , ~ 0 . 60 , ~ 1 . 05 , and ~ 2 . 10 sec , respectively , after initial depolarization , showing wavelike dissipation of ΔΨm over time .

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

Laser-induced mitochondrial membrane depolarization in L6 myoblasts shows gradual depolarization of elements along the IMM, visualized using live-cell Airyscan microscopy. Representative images of laser-induced depolarization time series from L6 myoblast, stained with ΔΨm-dependent dye, Rho123. Images show LUT color-coded for Rho123 FI. White and blue pixels represent most and least intense ΔΨm, respectively. See legend of LUT colors on the right. Note that at the first time point (~0.15 sec) white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Ensuing frames show wavelike depolarization away from site of perturbation (arrowheads). Scale bar = 500 nm. N = 3 independent experiments. (See Movie EV 2 for time series.) Profile plot showing Rho123 pixel intensity as function of distance along mitochondrion in A. Note that zero value in the X axis corresponds to top area of the mitochondrion shown in A, near site of 2-photon laser exposure (box). Red line corresponds to frame immediately before depolarization (~0.15 sec); green, blue, purple, and dark purple lines correspond to ~0.30, ~0.60, ~1.05, and ~2.10 sec, respectively, after initial depolarization, showing wavelike dissipation of ΔΨm over time.


Laser - induced mitochondrial membrane depolarization in living human skin fibroblasts co - stained with TMRE and MTG and imaged with Airyscan , showing gradual depolarization of elements along the IMM . Time - lapse images of merged green ( MTG ) and red ( TMRE ) channels following laser - induced mitochondrial membrane depolarization . Note that , at the first time point ( 0 . 3 sec ) , white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2 - photon laser ( ≤ 5 msec ) , inducing depolarization . Depolarization is marked by the dissipation of the ΔΨm - dependent dye ( TMRE ) while the green ( MTG ) signal persists . Scale bar = 1 µm . N = 4 independent experiments . TMRE channel from C , showing gradual of the loss of TMRE along the IMM . Note that although depolarization has largely completed at the area near the phototoxic stimulus already at 0 . 9 sec , some specific regions at the very top maintain ΔΨm while others depolarize . Quantification of ΔΨm using TMRE FI from C - D . Measurement of TMRE pixel intensities immediately after laser - induced depolarization at site distal ( ≥ 10 µm ) vs . proximal ( ≤ 1 µm ) to box . N = 4 independent experiments ; see specific p values in panel . Quantification of C - D . Percentage of mitochondria that depolarize in a wavelike ( i . e . , gradual ) vs . instantaneous manner after laser - induced depolarization . Note : imaging at high temporal resolution ( ~ 100 - 500 msec / frame ) reveals wavelike depolarizations predominate , suggesting the ΔΨm is composed of multiple , disparate electrochemical domains along the IMM . The time scale of propagation of depolarization is slower than the propagation of electrical phenomena . N = 4 independent experiments .

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

Laser-induced mitochondrial membrane depolarization in living human skin fibroblasts co-stained with TMRE and MTG and imaged with Airyscan, showing gradual depolarization of elements along the IMM. Time-lapse images of merged green (MTG) and red (TMRE) channels following laser-induced mitochondrial membrane depolarization. Note that, at the first time point (0.3 sec), white arrow points to box marking area of mitochondrion exposed to phototoxic pulse of 2-photon laser (≤5 msec), inducing depolarization. Depolarization is marked by the dissipation of the ΔΨm-dependent dye (TMRE) while the green (MTG) signal persists. Scale bar = 1 µm. N = 4 independent experiments. TMRE channel from C, showing gradual of the loss of TMRE along the IMM. Note that although depolarization has largely completed at the area near the phototoxic stimulus already at 0.9 sec, some specific regions at the very top maintain ΔΨm while others depolarize. Quantification of ΔΨm using TMRE FI from C-D. Measurement of TMRE pixel intensities immediately after laser-induced depolarization at site distal (≥ 10 µm) vs. proximal (≤ 1 µm) to box. N = 4 independent experiments; see specific p values in panel. Quantification of C-D. Percentage of mitochondria that depolarize in a wavelike (i.e., gradual) vs. instantaneous manner after laser-induced depolarization. Note: imaging at high temporal resolution (~100-500 msec/frame) reveals wavelike depolarizations predominate, suggesting the ΔΨm is composed of multiple, disparate electrochemical domains along the IMM. The time scale of propagation of depolarization is slower than the propagation of electrical phenomena. N = 4 independent experiments.


We calculated the ΔΨCr - IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence , using live - cell Airyscan imaging . Stability of cristae ΔΨCr - IBM over time . Representative images from time series of HeLa cells co - stained with MTG and TMRE . Top sequence shows merged green ( MTG ) and red ( TMRE ) channels . Bottom sequence shows color - coded LUT of TMRE signal of the same sequence shown above . White arrowheads in the image point to specific cristae that were measured over time . Scale bar = 500 nm . N = 5 independent experiments . Quantification of A , showing average ΔΨCr - IBM of Cristae " A - D " over time . Note that Cristae C and D maintain more than double the ΔΨCr - IBM of Cristae A and B . Error bars indicate STDEV . Quantification of A , showing standard deviation in ΔΨCr - IBM ( mV ) of individual cristae over time compared to the standard deviation in ΔΨCr - IBM ( mV ) among different cristae along a single mitochondrion . N = 5 independent experiments . Note that there is less variability in ΔΨCr - IBM of a single crista over time within the same mitochondrion compared to the variability of ΔΨCr - IBM among cristae over time , indicating cristae possess unique bioenergetic properties .

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

We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Stability of cristae ΔΨCr-IBM over time. Representative images from time series of HeLa cells co-stained with MTG and TMRE. Top sequence shows merged green (MTG) and red (TMRE) channels. Bottom sequence shows color-coded LUT of TMRE signal of the same sequence shown above. White arrowheads in the image point to specific cristae that were measured over time. Scale bar = 500 nm. N = 5 independent experiments. Quantification of A, showing average ΔΨCr-IBM of Cristae "A-D" over time. Note that Cristae C and D maintain more than double the ΔΨCr-IBM of Cristae A and B. Error bars indicate STDEV. Quantification of A, showing standard deviation in ΔΨCr-IBM (mV) of individual cristae over time compared to the standard deviation in ΔΨCr-IBM (mV) among different cristae along a single mitochondrion. N = 5 independent experiments. Note that there is less variability in ΔΨCr-IBM of a single crista over time within the same mitochondrion compared to the variability of ΔΨCr-IBM among cristae over time, indicating cristae possess unique bioenergetic properties.


We calculated the ΔΨCr - IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence , using live - cell Airyscan imaging . Representative live - cell Airyscan image from time series showing merged green ( MTG ) and red ( TMRE ) channels . Area marked by dashed box is shown in the time series in E . Scale bar = 500 nm . Time series of cropped region from D , showing tip of mitochondrion ( arrowhead ) maintaining ΔΨm while neighboring region of same mitochondrion depolarizes . The capacity of some cristae to maintain polarity , while adjacent cristae transiently depolarize , indicates cristae can exhibit a significant amount of electrochemical autonomy . N = 3 independent experiments . ( See Movies EV 3 , 4 for time series . ) Zoomed - in LUT of mitochondrion from E ( ~ 2 sec ) , showing some cristae remaining polarized ( red ) while adjacent cristae depolarize ( blue ) . ( See Movie EV 5 for time series . ) Quantification of D - F , showing that during a flickering event some cristae maintain their membrane potentials while other cristae depolarize , resulting in large differences in ΔΨCr - IBM along the individual mitochondrion . N = 3 independent experiments .

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

We calculated the ΔΨCr-IBM between neighboring cristae over time as a surrogate measure of cristae individuality and functional independence, using live-cell Airyscan imaging. Representative live-cell Airyscan image from time series showing merged green (MTG) and red (TMRE) channels. Area marked by dashed box is shown in the time series in E. Scale bar = 500 nm. Time series of cropped region from D, showing tip of mitochondrion (arrowhead) maintaining ΔΨm while neighboring region of same mitochondrion depolarizes. The capacity of some cristae to maintain polarity, while adjacent cristae transiently depolarize, indicates cristae can exhibit a significant amount of electrochemical autonomy. N = 3 independent experiments. (See Movies EV 3, 4 for time series.) Zoomed-in LUT of mitochondrion from E (~2 sec), showing some cristae remaining polarized (red) while adjacent cristae depolarize (blue). (See Movie EV 5 for time series.) Quantification of D-F, showing that during a flickering event some cristae maintain their membrane potentials while other cristae depolarize, resulting in large differences in ΔΨCr-IBM along the individual mitochondrion. N = 3 independent experiments.


D ) Percentage of zone 1 - 3 uninfected hepatocytes day 5 post plating ( + / - s . d . ) of 4 different donors ( blue dots ) . For each donor , > 500 cells were characterized for the final percentages . Kruskal Wallis test ( p = 0 . 0002 ) followed by Dunn ' s multiple comparisons test where Z1 / hGK + + / hGS - versus Z2 / hGK + / hGS - has a p value of 0 . 0140 ( the other comparisons of Z1 / hGK + + / hGS - vs Z3 / hGK - / hGS + and Z2 / hGK + / hGS - vs Z3 / hGK - / hGS + were non - significant ) .

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

D) Percentage of zone 1-3 uninfected hepatocytes day 5 post plating (+/- s.d.) of 4 different donors (blue dots). For each donor, >500 cells were characterized for the final percentages. Kruskal Wallis test (p = 0.0002) followed by Dunn's multiple comparisons test where Z1/hGK++/hGS- versus Z2/hGK+/hGS- has a p value of 0.0140 (the other comparisons of Z1/hGK++/hGS- vs Z3/hGK-/hGS+ and Z2/hGK+/hGS- vs Z3/hGK-/hGS+ were non-significant).


C : Expression of typical liver stage protein markers : Circumsporozoite protein ( CSP ) top left ; Exported Protein 2 ( EXP2 ) top right ; Glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) bottom left ; and Merozoite surface protein 1 ( MSP1 ) bottom right . Scale bar is 25 µm .

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

C: Expression of typical liver stage protein markers: Circumsporozoite protein (CSP) top left; Exported Protein 2 (EXP2) top right; Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) bottom left; and Merozoite surface protein 1 (MSP1) bottom right. Scale bar is 25 µm.


D ) Percentage schizonts ( from total number of schizonts in a well + / - s . d . ) located in the different hepatocyte sub - populations by NF54 , NF135 , NF175 on day 3 p . i . . Each dot represents a biological replicate . For each parasite line , at least 100 schizonts were characterized from 2x 96 wells for the final percentage . The values from three biological replicates were used in a RM ANOVA test followed by a Tukey ' s multiple comparison test . Percentage of schizonts in Z1 / hGK + + / hGS - hepatocytes is not different between the three parasite strains . Percentage of schizonts in Z2 / hGK + / hGS - for NF54 is significantly different from NF135 ( p = 0 . 0015 ) and NF175 ( p = 0 . 0015 ) . Percentage of schizonts in Z3 / hGK - / hGS + hepatocytes for NF54 is significantly different from NF135 ( p = 0 . 0002 ) and NF175 ( p = 0 . 0 . 0002 ) . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

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

D) Percentage schizonts (from total number of schizonts in a well +/- s.d.) located in the different hepatocyte sub-populations by NF54, NF135, NF175 on day 3 p.i.. Each dot represents a biological replicate. For each parasite line, at least 100 schizonts were characterized from 2x 96 wells for the final percentage. The values from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Percentage of schizonts in Z1/hGK++/hGS- hepatocytes is not different between the three parasite strains. Percentage of schizonts in Z2/hGK+/hGS- for NF54 is significantly different from NF135 (p = 0.0015) and NF175 (p = 0.0015). Percentage of schizonts in Z3/hGK-/hGS+ hepatocytes for NF54 is significantly different from NF135 (p = 0.0002) and NF175 (p = 0.0.0002). Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


F ) Size of schizonts in Z1 / hGK + + / hGS - , Z2 / hGK + / hGS - and Z3 / hGK - / hGS + hepatocytes on day 3 post invasion for the different Pf strains . For each parasite line , at least 100 schizonts were measured from each biological replicate . The median values + / - s . d . ( as the sizes within the population is not normally distributed ) from three biological replicates were used in a RM ANOVA test followed by a Tukey ' s multiple comparison test . Data information : * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 .

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

F) Size of schizonts in Z1/hGK++/hGS-, Z2/hGK+/hGS- and Z3/hGK-/hGS+ hepatocytes on day 3 post invasion for the different Pf strains. For each parasite line, at least 100 schizonts were measured from each biological replicate. The median values +/- s.d. (as the sizes within the population is not normally distributed) from three biological replicates were used in a RM ANOVA test followed by a Tukey's multiple comparison test. Data information: * p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001


hGS interaction with NF135 ( A ) , NF175 ( B ) schizonts on days 3 ( top ) , 5 ( middle ) and 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) and phalloidin ( grey ) . The U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

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

hGS interaction with NF135 (A), NF175 (B) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


hGS interaction with NF54 ( C ) schizonts on days 3 ( top ) , 5 ( middle ) and 7 ( bottom ) p . i . . hGS ( green ) , PfHSP70 ( red ) , DAPI ( blue ) and phalloidin ( grey ) . The U panel is composed an uninfected zone 3 hepatocyte on day 3 p . i . . Scale bar is 25 µm .

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

hGS interaction with NF54 (C) schizonts on days 3 (top), 5 (middle) and 7 (bottom) p.i.. hGS (green), PfHSP70 (red), DAPI (blue) and phalloidin (grey). The U panel is composed an uninfected zone 3 hepatocyte on day 3 p.i.. Scale bar is 25 µm.


D ) Median sizes ( + / - s . d . ) of NF135 ( green ) , NF175 ( purple ) and NF54 ( blue ) schizonts divided based on the presence ( solid line ) or absence ( dashed line ) of hGS staining pattern based on high content fluorescence microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates . A two - way RM ANOVA was performed on the median followed by a Tukey ' s multiple comparisons test . The significant differences between hGS + and hGS - population within a strain is showed with asterisks ( * p < 0 . 05 ; * * p < 0 . 005 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 ) . See Appendix Fig S1 for the raw measurements .

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

D) Median sizes (+/- s.d.) of NF135 (green), NF175 (purple) and NF54 (blue) schizonts divided based on the presence (solid line) or absence (dashed line) of hGS staining pattern based on high content fluorescence microscopy from three different liver donors where each donor has at least 100 schizonts were measured for each of three biological replicates. A two-way RM ANOVA was performed on the median followed by a Tukey's multiple comparisons test. The significant differences between hGS+ and hGS- population within a strain is showed with asterisks (* p <0.05; ** p < 0.005; *** p <0.001; **** p <0.0001). See Appendix Fig S1 for the raw measurements.


A ) The total hGS signal within intracellular NF135 and B ) NF175 schizonts on day 3 and 5 . Each graph represents a biological replicate where each dot represents an individual schizont .

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

A) The total hGS signal within intracellular NF135 and B) NF175 schizonts on day 3 and 5. Each graph represents a biological replicate where each dot represents an individual schizont.


A ) Normalized percentage + / - s . d . of intracellular schizont size of NF54 ( blue ) and NF135 ( green ) after treatment of GS inhibitors at different concentrations of AIP . Median size of the schizonts were normalized to that of H2O - treated NF135 ( which is set at 100 % ) . AIP was added one day post invasion and kept on for 48 hours . Each dot represents a biological replicate where ≥ 100 schizonts were measured on day 5 p . i . . A two - way ANOVA was performed followed by a Tukey ' s multiple comparison test with the * * * * < 0 . 0001 to compare conditions within the same parasite strain . A two - way ANOVA was performed followed by a Sidak ' s multiple comparisons test with * * * * < 0 . 0001 to compare conditions between parasite strains ) .

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

A) Normalized percentage +/- s.d. of intracellular schizont size of NF54 (blue) and NF135 (green) after treatment of GS inhibitors at different concentrations of AIP. Median size of the schizonts were normalized to that of H2O-treated NF135 (which is set at 100%). AIP was added one day post invasion and kept on for 48 hours. Each dot represents a biological replicate where ≥100 schizonts were measured on day 5 p.i.. A two-way ANOVA was performed followed by a Tukey's multiple comparison test with the **** < 0.0001 to compare conditions within the same parasite strain. A two-way ANOVA was performed followed by a Sidak's multiple comparisons test with **** <0.0001 to compare conditions between parasite strains).


B ) Size difference between the GS positive ( closed circle ) and negative ( open circle ) NF135 schizonts after treatment of AIP . Each dot represents the median of a biological replicate where ≥ 100 schizonts were measured . Three biological replicates were performed with the median from each plotted + / - s . d . . A two - way ANOVA was performed followed by a Dunnett ' s multiple comparison test with * * * = 0 . 0002 and * * * * = 0 . 0001 .

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

B) Size difference between the GS positive (closed circle) and negative (open circle) NF135 schizonts after treatment of AIP. Each dot represents the median of a biological replicate where ≥100 schizonts were measured. Three biological replicates were performed with the median from each plotted +/- s.d.. A two-way ANOVA was performed followed by a Dunnett's multiple comparison test with *** = 0.0002 and **** = 0.0001.


( a ) Top : immunoblots for the indicated proteins from two independent experiments . Bottom : quantification of changes in total LRRK2 ( n = 6 independent experiments ) . SH - SY5Y cells were untreated or incubated with 5 μM lactacystin ( Lact ) , 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin ( NL ) for 9 . 5 h .

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

(a) Top: immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of changes in total LRRK2 (n = 6 independent experiments). SH-SY5Y cells were untreated or incubated with 5 μM lactacystin (Lact), 10 mM 3MA or 20 mM NH4Cl and 100 μM leupeptin (NL) for 9.5 h.


( b ) Top : representative immunoblots for the indicated proteins from two independent experiments . Bottom : quantification of LRRK2 relative to that in untreated samples ( n = 3 or 4 independent experiments ) . Brain slices from mouse cortex , midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 ° C .

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

(b) Top: representative immunoblots for the indicated proteins from two independent experiments. Bottom: quantification of LRRK2 relative to that in untreated samples (n = 3 or 4 independent experiments). Brain slices from mouse cortex, midbrain or cerebellum were incubated without additions or with NL or 3MA for 2 h at 37 °C.


( c , d ) Homogenate ( Hom ) and lysosomal fractions ( Lys ) isolated from SH - SY5Y cells ( c ) or the indicated mouse brain regions ( d ) , immunoblotted for the indicated proteins .

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(c,d) Homogenate (Hom) and lysosomal fractions (Lys) isolated from SH-SY5Y cells (c) or the indicated mouse brain regions (d), immunoblotted for the indicated proteins.


( e ) HEK293 cells expressing GFP - tagged LRRK2 and transduced with lentivirus control ( Ctr ) or carrying shRNA against LAMP - 2A ( L2A ( − ) ) , incubated or not with NL for 12 h and immunoblotted for the indicated proteins .

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

(e) HEK293 cells expressing GFP-tagged LRRK2 and transduced with lentivirus control (Ctr) or carrying shRNA against LAMP-2A (L2A(−)), incubated or not with NL for 12 h and immunoblotted for the indicated proteins.


( f ) Immunoblots for LRRK2 and LAMP - 2A of homogenates and lysosome - enriched fractions isolated from control fibroblasts ( Ctr ) or fibroblasts stably interfered for LAMP - 2A ( L2A ( − ) ) , maintained in the presence or absence of serum . Actin and LAMP - 1 ( L - 1 ) are shown as loading controls of homogenate and lysosomes , respectively . All values are mean + s . e . m . ; * P 0 . 05 , unpaired t - test . Full - length blots and gels in Supplementary Figure 12 .

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

(f) Immunoblots for LRRK2 and LAMP-2A of homogenates and lysosome-enriched fractions isolated from control fibroblasts (Ctr) or fibroblasts stably interfered for LAMP-2A (L2A(−)), maintained in the presence or absence of serum. Actin and LAMP-1 (L-1) are shown as loading controls of homogenate and lysosomes, respectively. All values are mean + s.e.m.; *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.


( b ) Binding , association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers , untreated or pretreated with protease inhibitors ( PI ) and incubated with LRRK2 . Inset : representative immunoblot . LAMP - 2A is shown as lysosomal loading control . Input ( Inp ) , one - fifth of LRRK2 added . n = 7 independent experiments . Uptake : difference between LRRK2 associated with lysosomes with and without PI .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Binding, association and uptake calculated from quantification of LRRK2 in immunoblots of lysosomes from starved rat livers, untreated or pretreated with protease inhibitors (PI) and incubated with LRRK2. Inset: representative immunoblot. LAMP-2A is shown as lysosomal loading control. Input (Inp), one-fifth of LRRK2 added. n = 7 independent experiments. Uptake: difference between LRRK2 associated with lysosomes with and without PI.


( c ) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes . Values are percentage of the degradation without LRRK2 ( n = 5 or 6 independent experiments ) .

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

(c) Effect of increasing concentrations of LRRK2 on the degradation of a radiolabeled pool of cytosolic proteins by intact lysosomes. Values are percentage of the degradation without LRRK2 (n = 5 or 6 independent experiments).


( d ) Association of GAPDH to rat liver lysosomes in the presence of increasing concentrations of LRRK2 . Values are percentage of the GAPDH associated with lysosomes in the absence of LRRK2 ( n = 4 - 6 independent experiments in duplicate ) . Inset : representative immunoblot . Inp : 1 μg of

(d) Association of GAPDH to rat liver lysosomes in the presence of increasing concentrations of LRRK2. Values are percentage of the GAPDH associated with lysosomes in the absence of LRRK2 (n = 4-6 independent experiments in duplicate). Inset: representative immunoblot. Inp: 1 μg of GAPDH


( e , f ) Association of LRRK2 and RNase A ( e ) or LRRK2 and GAPDH ( f ) with PI - treated rat liver lysosomes incubated with increasing concentrations of GAPDH ( e ) or RNase A ( f ) or ovalbumin ( Ov ) . Left : representative immunoblots . Right : protein association expressed as a percentage of the value when incubated with lysosomes alone ( n = 5 or 6 independent experiments ) . Inp : RNase A ( 0 . 4 μg ) , LRRK2 ( 0 . 04 μg ) or GAPDH ( 1 μg ) .

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

(e,f) Association of LRRK2 and RNase A (e) or LRRK2 and GAPDH (f) with PI-treated rat liver lysosomes incubated with increasing concentrations of GAPDH (e) or RNase A (f) or ovalbumin (Ov). Left: representative immunoblots. Right: protein association expressed as a percentage of the value when incubated with lysosomes alone (n = 5 or 6 independent experiments). Inp: RNase A (0.4 μg), LRRK2 (0.04 μg) or GAPDH (1 μg).


( g , h ) Association of LRRK2 to lysosomes incubated with GAPDH ( g ) or RNase A ( h ) at 4 ° C ( n = 3 independent experiments ) .

(g,h) Association of LRRK2 to lysosomes incubated with GAPDH (g) or RNase A (h) at 4 °C (n = 3 independent experiments).


( i , j ) Immunoblots of brain lysosomes incubated with LRRK2 and increasing concentrations of GAPDH ( i ) or RNase A ( j ) . All values are mean + s . e . m . ; differences with lysosomes incubated with the protein alone were significant at * P 0 . 05 , unpaired t - test . Full - length blots and gels in Supplementary Figure 12 .

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

(i,j) Immunoblots of brain lysosomes incubated with LRRK2 and increasing concentrations of GAPDH (i) or RNase A (j). All values are mean + s.e.m.; differences with lysosomes incubated with the protein alone were significant at *P 0.05, unpaired t-test. Full-length blots and gels in Supplementary Figure 12.