F Representative spectra of recombinant PDIA1b ' xWT fragments equivalent ALS - linked mutants ( n = 6 ) . Ratios from two peak areas change compared to PDIA1 b ' xWT . Both mutants analyzed show a significant shift in peak position compared to wild - type , but in opposite directions , suggesting that PDIA1D292N shifts equilibrium towards capped version ( x region over binding pocket ) PDIA1R300H mutant towards uncapped version ( Nguyen et al , 2008 ) .

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F Representative fluorescence spectra of recombinant PDIA1b'xWT fragments and the equivalent ALS-linked mutants (n=6). Ratios from the two peak areas and change compared to PDIA1 b'xWT. Both mutants analyzed show a significant shift in peak position compared to wild-type, but in opposite directions, suggesting that PDIA1D292N shifts equilibrium towards the capped version (x region over the binding pocket) and the PDIA1R300H mutant towards the uncapped version (Nguyen et al, 2008).


G activity of recombinant PDIA1 was measured in vitro using a BPTI refolding following by mass spectrometry . percentages of different BPTI species was calculated ( 6H , fully reduced ; 1S , one disulfide bond ; 2S , two disulfide bonds ; 3S , three disulfide bonds ) at time point 2 . 5 min in four independent experiments .

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G The activity of recombinant PDIA1 was measured in vitro using a BPTI refolding assay following by mass spectrometry analysis. The percentages of different BPTI species was calculated (6H, fully reduced; 1S, one disulfide bond; 2S, two disulfide bonds; 3S, three disulfide bonds) at time point 2.5 min in four independent experiments.


H Measurement of H2O2 at ER lumen of living NSC34 . Left panel : Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen ( O2 ) , generating hydrogen peroxide ( H2O2 ) as a product of PDIs activity . Right panel : NSC34 were transiently co - transfected with ER luminal HyPer sensor indicated PDIs . After 48 h 490 / 420 nm was recorded for 2 min under basal conditions . Means derived from per condition ( n = 55 - 74 ) monitored in four independent experiments are shown .

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H Measurement of H2O2 levels at the ER lumen of living NSC34. Left panel: Reduced PDIs can be oxidized by oxidized ERO1Lα that then transfers electrons to molecular oxygen (O2), generating hydrogen peroxide (H2O2) as a product of PDIs activity. Right panel: NSC34 cells were transiently co-transfected with ER luminal HyPer sensor and indicated PDIs. After 48 h the 490/420 nm fluorescence ratio was recorded for 2 min under basal conditions. Means and SEM derived from all cells per condition (n=55-74) monitored in four independent experiments are shown.


I HEK293T were transfected with expression vectors for V5 - tagged wild - type mutant PDIA1 , as well as empty vector . After 48 h , V5 - tagged were immunoprecipitated eluted with V5 peptide . interaction with endogenous ERO1Lα was analyzed by western blot . inputs elutions are shown as control . Right panel : of degree of interaction is presented .

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I HEK293T cells were transfected with expression vectors for V5-tagged wild-type and mutant PDIA1, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous ERO1Lα was analyzed by western blot. The inputs and elutions are shown as control. Right panel: Quantification of the degree of interaction is presented.


A PDI mutants form abnormal disulfide - dependent complexes . NSC34 were transiently transfected with expression vectors for V5 - tagged wild - type mutant ERp57 . After 48 h , differential disulfide - dependent interactions / aggregations of overexpressed PDI variants was assessed under reducing ( + DTT ) non - reducing ( - DTT ) conditions in an 8 % SDS - PAGE . Anti - V5 was used for detection in Western blot .

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A PDI mutants form abnormal disulfide-dependent protein complexes. NSC34 cells were transiently transfected with expression vectors for V5-tagged wild-type and mutant ERp57. After 48 h, differential disulfide-dependent interactions/aggregations of overexpressed PDI variants was assessed under reducing (+DTT) and non-reducing (-DTT) conditions in an 8% SDS-PAGE. Anti-V5 was used for detection in Western blot.


B ALS - linked ERp57 variants were generated as recombinant then analyzed by circular dichroism ( CD ) . Averages for CD spectroscopic scans of recombinant ERp57WT mutants are shown .

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B ALS-linked ERp57 variants were generated as recombinant proteins and then analyzed by circular dichroism (CD). Averages for CD spectroscopic scans of recombinant ERp57WT and mutants are shown.


C A thermal denaturation curve of ERp57WT mutants was performed .

C A thermal denaturation curve of ERp57WT and mutants was performed.


D Representative electrophoresis of Proteinase K treated ERp57 recombinant .

D Representative electrophoresis of Proteinase K treated ERp57 recombinant proteins.


E Surface Plasmon Resonance was performed to monitor affinity of recombinant ERp57 for recombinant CRT P - domain . KD values are expressed as of ERp57WT . Values are derived from four independent experiments . For absolute KD values please see Appendix Table S2 .

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E Surface Plasmon Resonance was performed to monitor the affinity of recombinant ERp57 for recombinant CRT P-domain. KD values are expressed as percentage of ERp57WT. Values are derived from four independent experiments. For absolute KD values please see Appendix Table S2.


FHEK293T NSC34 were transfected with expression vectors for V5 - tagged wild - type mutant ERp57 , as well as empty vector . After 48 h , V5 - tagged were immunoprecipitated eluted with V5 peptide . interaction with endogenous calnexin ( CNX ) calreticulin ( CRT ) was analyzed by Western blot . inputs elutions are shown . Right panel : of degree of interaction is presented .

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FHEK293T NSC34 were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector. After 48 h, V5-tagged proteins were immunoprecipitated and eluted with V5 peptide. The interaction with endogenous calnexin (CNX) and calreticulin (CRT) was analyzed by Western blot. The inputs and elutions are shown. Right panel: Quantification of the degree of interaction is presented.


G gain of an N - glycosylation site of ERp57D217N was predicted after of since change of Asp217 to an Asn creates NXT / S consensus . Neuro2a were transfected with expression vectors for V5 - tagged wild - type mutant ERp57 , as well as empty vector treated with 1 µg / ml tunicamycin ( Tm ) for 20 h to inhibit N - glycosylation . Alternatively , extracts were digested with PNGase F possible removal of N - glycosylation was analyzed by Western blot using anti - V5 antibody .

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G The gain of an N-glycosylation site of ERp57D217N was predicted after the analysis of the protein sequence since the change of Asp217 to an Asn creates the NXT/S consensus sequence. Neuro2a cells were transfected with expression vectors for V5-tagged wild-type and mutant ERp57, as well as empty vector and treated with 1 µg/ml tunicamycin (Tm) for 20 h to inhibit N-glycosylation. Alternatively, protein extracts were digested with PNGase F and the possible removal of the N-glycosylation was analyzed by Western blot using anti-V5 antibody.


A ERp57flox / flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient . of ERp57 in spinal cord were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) mice . HSP90 were determined as a loading control . Right panel : of ERp57 was performed relative to Hsp90 .

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A ERp57flox/flox mice were crossed with Nestin Cre transgenic mice to generate nervous system specific ERp57 deficient animals. The levels of ERp57 protein in the spinal cord were monitored by Western blot. ERp57WT (n=4), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4) mice. HSP90 levels were determined as a loading control. Right panel: Quantification of ERp57 levels was performed relative to Hsp90 levels.


B measurements were performed for indicated time points in ERp57WT ( n = 50 ) , ERp57Nes + / - ( n = 32 ) ERp57Nes - / - ( n = 19 ) mice .

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B Body weight measurements were performed for indicated time points in ERp57WT (n=50), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=19) mice.


C Rotarod performance was performed ERp57WT ( n = 20 ) , ERp57Nes + / - ( n = 15 ) ERp57Nes - / - ( n = 8 ) mice .

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C Rotarod performance was performed ERp57WT (n=20), ERp57Nes+/- (n=15) and ERp57Nes-/- (n=8) mice.


D Hanging performance was assessed in ERp57WT ( n = 41 ) , ERp57Nes + / - ( n = 32 ) ERp57Nes - / - ( n = 12 ) mice .

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D Hanging test performance was assessed in ERp57WT (n=41), ERp57Nes+/- (n=32) and ERp57Nes-/- (n=12) mice.


E Kaplan - Meier survival curve for ERp57Nes - / - mice ( N = 19 ) that prematurely died or had to be sacrificed because of health reasons between ages 22 73 days . Mean survival of this subgroup of was 57 days . ERp57WT ( n = 50 ) ERp57Nes + / - ( n = 32 ) mice are shown as a reference .

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E Kaplan-Meier survival curve for ERp57Nes-/- mice (N = 19) that prematurely died or had to be sacrificed because of health reasons between the ages 22 and 73 days. Mean survival of this subgroup of animals was 57 days. ERp57WT (n=50) and ERp57Nes+/- (n=32) mice are shown as a reference.


F Histological of NeuN GFAP was performed in spinal cord tissue from ERp57WT ERp57Nes - / - mice in three per group using indirect immunofluorescence . nucleus was stained with Hoechst . Representative from one mouse per group are shown . Scale bar : 50 μm .

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F Histological analysis of NeuN and GFAP staining was performed in spinal cord tissue from ERp57WT and ERp57Nes-/- mice in three animals per group using indirect immunofluorescence. The nucleus was stained with Hoechst. Representative images from one mouse per group are shown. Scale bar: 50 μm.


G Stereological of spinal cord from ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 4 ) ERp57Nes - / - ( n = 4 ) mice . Alternate series of sections from spinal cord of mice were either stained for Nissl ( top row ) or processed for immunohistochemistry for cholinergic marker Choline Acetyl Transferase ( ChAT , bottom row ) . nucleoli of motoneurons , as stained in Nissl series , were counted inside motoneurons pools previously defined using adjacent ChAT series ( contours not shown ) . densities of three genotypes are shown on right plots . No significant differences were found between genotypes . Scale bar represents 200 μm 50 μm on large inset , respectively .

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G Stereological analysis of the spinal cord from ERp57WT (n = 4), ERp57Nes+/- (n = 4) and ERp57Nes-/- (n = 4) mice. Alternate series of sections from the spinal cord of the mice were either stained for Nissl (top row images) or processed for immunohistochemistry for the cholinergic cell marker Choline Acetyl Transferase (ChAT, bottom row images). The nucleoli of the motoneurons, as stained in the Nissl series, were counted inside the motoneurons pools previously defined using the adjacent ChAT series (contours not shown). Cell densities of the three genotypes are shown on the right plots. No significant differences were found between the genotypes. Scale bar represents 200 μm and 50 μm on large and inset images, respectively.


H Representative of spinal cord myelination of ERp57WT ERp57Nes - / - mice .

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H Representative images of the spinal cord myelination of ERp57WT and ERp57Nes-/- mice.


A Representative EMG recordings of ERp57WT ( n = 16 ) , ERp57Nes - / + ( n = 12 ) ERp57Nes - / - ( n = 5 ) mice is presented . presence of positive sharp waves ( PSW ) in ERp57Nes + / - ERp57Nes - / - mice indicated muscle denervation .

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A Representative EMG recordings of ERp57WT (n=16), ERp57Nes-/+ (n=12) and ERp57Nes-/- (n=5) mice is presented. The presence of positive sharp waves (PSW) in ERp57Nes+/- and ERp57Nes-/- mice indicated muscle denervation.


B of NMJ muscle morphologies were performed from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) . Quantifications of endplate width , measuring most ventral region of diaphragms every 134 µm considering both sides of innervation profile . Twenty to forty measurements per diaphragm were obtained per .

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B Analysis of NMJ and muscle morphologies were performed from ERp57WT (n=5), ERp57Nes+/- (n=5) and ERp57Nes-/- (n=4). Quantifications of endplate width, measuring the most ventral region of the diaphragms every 134 µm considering both sides of the innervation profile. Twenty to forty measurements per diaphragm were obtained per animal.


C Whole - mounted diaphragms from ERp57WT ERp57Nes - / - mice were co - stained with anti - neurofilament ( red ) α - BTX to reveal postsynaptic densities ( green ) . 3D reconstructions ( lower panel ) of higher magnification . ERp57WT NMJs are fully innervated pretzel - like , whereas ERp57Nes - / - NMJs display less postsynaptic densities an incomplete or aberrantly distributed innervation pattern ( Movies EV4 - 7 ) .

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C Whole-mounted diaphragms from ERp57WT and ERp57Nes-/- mice were co-stained with anti-neurofilament (red) and α-BTX to reveal the postsynaptic densities (green). 3D reconstructions (lower panel) of higher magnification. ERp57WT NMJs are fully innervated pretzel-like, whereas ERp57Nes-/- NMJs display less complex postsynaptic densities and an incomplete or aberrantly distributed innervation pattern (Movies EV4-7).


D Representative 3D reconstructions of z - stacks showing five categories in which NMJs morphologies were grouped : Pretzel , fragmented , O shaped , C shaped , dismantled NMJs . Histogram of distribution frequency of > 200 different NMJ per is shown .

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D Representative 3D reconstructions of z-stacks showing the five categories in which NMJs morphologies were grouped: Pretzel, fragmented, O shaped, C shaped, and dismantled NMJs. Histogram of the distribution frequency of >200 different NMJ per animal is shown.


E Quantitative morphometry of > 60 NMJs in different genotypes analyzed .

E Quantitative morphometry of >60 NMJs in the different genotypes analyzed.


A of ERp57 in muscle were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) mice . HSP90 was determined as loading control .

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A The levels of ERp57 protein in the muscle were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 was determined as loading control.


B Tibialis anterior from ERp57WT ( n = 5 ) , ERp57Nes + / - ( n = 5 ) , ERp57Nes - / - ( n = 4 ) mice were dissected cryosectioned ( 20 µm ) for of several parameters of muscle physiology . Upper panel : hyaluronic acid ( HA ) was performed to assess gross of muscle . Middle panel : Bismarck brown to monitor skeletal fibrosis . Lower panel : Cryosections were then double stained with wheat germ agglutinin ( WGA ) to visualize plasmamembrane nuclei . absence of central nuclei in genotypes suggests that ERp57 deficiency does not affect degeneration / regeneration cycles . are representative of each genotype where acquired in same anatomical region of TA .

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B Tibialis anterior muscles from ERp57WT (n = 5), ERp57Nes+/- (n = 5), and ERp57Nes-/- (n = 4) mice were dissected and cryosectioned (20 µm) for the analysis of several parameters of muscle physiology. Upper panel: hyaluronic acid (HA) staining was performed to assess the gross morphology of the muscle. Middle panel: Bismarck brown staining to monitor skeletal muscles fibrosis. Lower panel: Cryosections were then double stained with wheat germ agglutinin (WGA) to visualize plasmamembrane and nuclei. The absence of central nuclei in all genotypes suggests that ERp57 deficiency does not affect degeneration/regeneration cycles. Images are representative of each genotype and where acquired in the same anatomical region of the TA muscles.


C Tibialis anterior cryosections ( 20 µm ) from ERp57WT , ERp57Nes + / - , ERp57Nes - / - mice were stained for NADH - TR activity to identify oxidative myofibers ( upper panel ) . Representative of transversal cryosections show variable proportions of , intermediate positive in different genotypes : ( middle panel ) NADH - TR positive ( slow - twitch ) negative ( fast - twitch ) myofibers were quantified as of total for every genotype ( lower panel ) mean diameter of slow - fast - twitch muscle was evaluated at microscope using ImageJ software . Data represent average ± of > 300 per . Scale bars represent 50 m in B C .

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C Tibialis anterior cryosections (20 µm) from ERp57WT, ERp57Nes+/-, and ERp57Nes-/- mice were stained for NADH-TR activity to identify oxidative myofibers (upper panel). Representative micrographs of transversal cryosections show variable proportions of light, intermediate and dark positive staining in the different genotypes: (middle panel) NADH-TR positive (slow-twitch) and negative (fast-twitch) myofibers were quantified as percentage of total for every genotype (lower panel) The mean diameter of slow- and fast-twitch muscle fibers was evaluated at the light microscope using the ImageJ software. Data represent the average ± SEM of >300 fibers per animal.Scale bars represent 50 m in B and C.


A of synaptic SV2 , NMDAR2A , PSD95 Synaptophysin in cortex were monitored by Western blot . ERp57WT ( n = 4 ) , ERp57Nes + / - ( n = 5 ) ERp57Nes - / - ( n = 4 ) mice . HSP90 β - Actin were determined as loading controls .

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A The levels of synaptic proteins SV2, NMDAR2A, PSD95 and Synaptophysin in the cortex were monitored by Western blot. ERp57WT (n = 4), ERp57Nes+/- (n = 5) and ERp57Nes-/- (n = 4) mice. HSP90 and β-Actin were determined as loading controls.


B of SV2 monomer ( left panel ) SV2 high molecular species ( right panel ) . β - Actin was used as reference .

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B Quantification of SV2 monomer (left panel) and SV2 high molecular weight species (right panel). β-Actin was used as the reference.


( A ) volume at indicated time points after CT26 transplant in mice treated or not with LPS n ( number of per group ) = 6 .

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(A) Tumor volume at the indicated time points after CT26 cell transplant in mice treated or not with LPS n (number of animals per group)=6.


( B ) volume in mice depleted of DCs or NK before transplant LPS administration n ( number of per group ) = 5 .

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(B) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n (number of animals per group)=5.


( C ) volume in SCIDmice treated or not with LPS n ( number of per group ) = 5 .

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(C) Tumor volume in SCIDmice treated or not with LPS n (number of animals per group)=5.


( D ) volume at indicated time point after repetitive LPS injections as depicted in bottom scheme n ( number of per group ) = 6 .

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(D) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n (number of animals per group)=6.


( E ) of NK within in mice treated or not with LPS depleted or not of DCs n ( number of per group ) ≥ 4 . NT , control ; DT , diphtheria toxin treated to deplete DCs ; LPS , mice treated with LPS . Error bars depict . Statistical significance was determined with a two - tail t . NT , untreated .

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(E) Percent of NK cells within the tumor in mice treated or not with LPS and depleted or not of DCs n (number of animals per group) ≥ 4. NT, control; DT, diphtheria toxin treated animals to deplete DCs; LPS, mice treated with LPS. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in scheme at bottom of panel .

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(A) Explanted tumors at Day 12 after repetitive injections of LPS as depicted in the scheme at the bottom of the panel.


( B ) Detection of blood vessels by immunohistochemistry using anti - CD31 antibody ( brown ) in frozen sections of tumors from mice treated or not with with LPS as in scheme in ( A ) . Nuclei are stained with hematoxylin . Two representative sections are shown , magnification 20X . Left panel scale bars , 500m ; right panel scale bars 100 m . of vessel mean dimensions coverage by vessels is also shown . Three sections from three tumors have been analyzed . Error bars depict . Statistical significance was determined with a two - tail t . NT , untreated .

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(B) Detection of blood vessels by immunohistochemistry using anti-CD31 antibody (brown staining) in frozen sections of tumors from mice treated or not with with LPS as in the scheme in (A). Nuclei are stained with hematoxylin. Two representative sections are shown, magnification 20X. Left panel scale bars, 500m; right panel scale bars 100 m. Quantification of vessel mean dimensions and area coverage by vessels is also shown. Three sections from three tumors have been analyzed. Error bars depict SEM. Statistical significance was determined with a two-tail t test. NT, untreated.


( A , B ) Absolute numbers of IFN - γ + total NK in draining lymph nodes at indicated time points before after LPS administration in mice treated or not with FTY720 ( 25g / mouse ) to reduce ingress of lymphocytes in efferent lymphatics .

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(A,B) Absolute numbers of IFN-γ+ and total NK cells in draining lymph nodes at the indicated time points before and after LPS administration in mice treated or not with FTY720 (25g/mouse) to reduce the ingress of lymphocytes in the efferent lymphatics.


( C ) volume in mice treated or not with LPS / or FTY720 according to scheme .

(C) Tumor volume in mice treated or not with LPS and/or FTY720 according to the scheme.


( D ) of IFN - γ + NK within in mice treated or not with LPS with FTY720 . n ( number of per group ) = 4 . Error bars depict . FTY : FTY720 . Statistical significance was determined with a two - tail t . NT , untreated .

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(D) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with FTY720. n (number of animals per group)=4. Error bars depict SEM. FTY: FTY720. Statistical significance was determined with a two-tail t test. NT, untreated.


( A ) Absolute numbers of NK in one of 4 draining lymph nodes in mice treated or not with anti - CD62L antibody to inhibit NK cel ingress in lymph node from blood . Where indicated mice were challenged with LPS .

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(A) Absolute numbers of NK cells in one of the 4 draining lymph nodes in mice treated or not with the anti-CD62L antibody to inhibit NK cel ingress in the lymph node from blood. Where indicated mice were challenged with LPS.


( B ) Absolute numbers of IFN - γ + NK in one of 4 draining lymph nodes before after LPS administration in mice in which NK entry in lymph node was blocked or not by anti - CD62L treatment .

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(B) Absolute numbers of IFN-γ+ NK cells in one of the 4 draining lymph nodes before and after LPS administration in mice in which NK cell entry in the lymph node was blocked or not by anti-CD62L treatment.


( C ) ( left panel ) Absolute numbers of endogenous adoptively transferred ( CFSE + ) NK in draining lymph node 5 hours after LPS administration ; ( right panel ) of endogenous adoptively transferred NK on total lymphocytes in draining lymph node 5 hours after LPS administration .

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(C) (left panel) Absolute numbers of endogenous and adoptively transferred (CFSE+) NK cells in the draining and contralateral lymph node 5 hours after LPS administration; (right panel) percent of endogenous and adoptively transferred NK cells on total lymphocytes in the draining and contralateral lymph node 5 hours after LPS administration.


( D ) Flow cytometry of LPS draining lymph node from mice that received CFSE labeled at moment of LPS administration . Lymph node were stained with anti - CD3 , anti - DX5 anti - IFN - γ antibodies performed on gated CD3 - DX5high . One representative of 4 independent experiments is shown .

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(D) Flow cytometry analysis of LPS draining and contralateral lymph node from mice that received CFSE labeled cells at the moment of LPS administration. Lymph node cells were stained with anti-CD3, anti-DX5 and anti-IFN-γ antibodies and the analysis performed on gated CD3-DX5high cells. One representative of 4 independent experiments is shown.


( E ) volume in mice treated or not with LPS , where indicated mice received anti - CD62L antibody 12h before LPS treatment .

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(E) Tumor volume in mice treated or not with LPS, where indicated mice received anti-CD62L antibody 12h before LPS treatment.


( F ) of IFN - γ + NK within in mice treated or not with LPS with anti - CD62L antibody .

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(F) Percent of IFN-γ+ NK cells within the tumor in mice treated or not with LPS and with the anti-CD62L antibody.


( A ) Representative image of an NK ( red ) interacting with four DCs ( green ) . ( B , C , D ) Plot of confinement ( CR , black squares ) , normalized NK speed Vthr ( ti ) ( red circles ) digitalized NK - DC , DistD ( ti ) ( green triangles ) as a function of time for NK reported in image . B , C D panels correspond respectively to interaction with DC1 , DC2 DC4 displayed in ( A ) . For reference also normalized Dist ( ti ) is reported in plots ( blue down triangles ) . time periods in which three conditions discussed in text are fulfilled are marked with red striped rectangles in plots . green up - triangles indicate parameter TonToff defined as TonToff = - 1 if Dist ( ti ) < d = 25 µm TonToff = - 1 if Dist ( ti ) d indicate putative interactions according to a more simplified algorithm on DC - NK alone .

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(A) Representative image of an NK cell (red) interacting with four DCs (green). (B, C, D) Plot of the confinement ratio (CR, black squares), the normalized NK speed Vthr(ti) (red circles) and the digitalized NK-DC distance, DistD(ti) (green triangles) as a function of time for the NK cell reported in the image. The B, C and D panels correspond respectively to the interaction with the DC1, DC2 and DC4 displayed in (A). For reference also the normalized distance Dist(ti) is reported in the plots (blue down triangles). The time periods in which all the three conditions discussed in the text are fulfilled are marked with red striped rectangles in the plots. The green up-triangles indicate the parameter TonToff defined as TonToff = -1 if Dist(ti) < d = 25 µm and TonToff = -1 if Dist(ti) d and indicate the putative interactions according to a more simplified algorithm based on the DC-NK cell distance alone.


( A ) NK DC trajectories as they are tracked by Volocity software on 4D volumes collected in lymph nodes on TPE microscope . of shows interaction between an NK ( red ) a DC ( green ) . In top panels NK moves according to a directional random motion while in lower left middle panels NK seems to recognize DC to go back . In right lower panel stable contact DC - NK is clearly visible . ( B ) Time duration of DC - NK contacts in steady state ( - LPS ) inflammatory ( + LPS ) conditions . Bars indicate mean of distribution . of showing long interaction times ( ≥ 15 min ) are shown . ( C ) NK 3D velocities . 3D velocities of NK taking contacts with DCs before ( black ) 5 h ( green ) after LPS treatment .

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(A) NK and DC trajectories as they are tracked by Volocity software on the 4D volumes collected in lymph nodes on the TPE microscope. The sequence of images shows the interaction between an NK cell (red) and a DC (green). In the top panels the NK cell moves according to a directional random motion while in the lower left and middle panels the NK cell seems to recognize the DC and to go back. In the right lower panel the stable contact DC-NK cell is clearly visible. (B) Time duration of the DC-NK contacts in steady state (-LPS) and inflammatory (+LPS) conditions. Bars indicate the mean of the distribution. The percent of cells showing long interaction times (≥ 15 min) are shown. (C) NK cell 3D velocities. 3D velocities of NK cells taking contacts with DCs before (black) and 5 h (green) after LPS treatment.


( A ) Differential interference contrast ( left panels ) confocal microscopy of IL - 18 ( right panels ) in a DC / NK conjugate after 2 hours of coculture in presence of LPS in unstimulated ( NT ) . Representative from 3 experiments are shown , magnification 63X . Scale bars , 4 . 36 m .

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(A) Differential interference contrast images (left panels) and confocal microscopy analysis of IL-18 (right panels) in a DC/NK cell conjugate after 2 hours of coculture in the presence of LPS and in unstimulated cells (NT). Representative images from 3 experiments are shown, magnification 63X. Scale bars, 4.36 m.


( B ) IFN - γ release by NK cultured in presence of supernatants recovered from untreated DCs ( untreated ) or DCs treated O / N with LPS ( LPS ) . Where indicated IL - 18 was added to cultures .

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(B) IFN-γ release by NK cells cultured in the presence of supernatants recovered from untreated DCs (untreated) or DCs treated O/N with LPS (LPS). Where indicated IL-18 was added to the cultures.


( C ) - contact dependent activation of NK by DCs depends on IL - 18 . Unstimulated or LPS - activated DCs NK were cocultured in same wells ( DC + NK + LPS ) or separated by a porous ( ( DC + LPS ) / NK ) . Where indicated IL - 18 was added to transwells ( ( DCs + LPS ) / ( NK + IL - 18 ) ) . NK alone were also cultured in presence of three selected cytokines ; IL - 2 IFN - β in upper chamber NK IL - 18 in lower chamber . IFN - γ in supernatant was then measured by ELISA after 8 hours of coculture . ( B , C ) . n ( number of independent experiments ) = 3 . Error bars depict .

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(C) Cell-contact dependent activation of NK cells by DCs depends on IL-18. Unstimulated or LPS-activated DCs and NK cells were cocultured in the same wells (DC + NK + LPS) or separated by a porous membrane ((DC+LPS)/NK). Where indicated IL-18 was added to the transwells ((DCs+LPS)/(NK+IL-18)). NK cells alone were also cultured in the presence of the three selected cytokines; IL-2 and IFN-β in the upper chamber and NK cells and IL-18 in the lower chamber. IFN-γ in the supernatant was then measured by ELISA after 8 hours of coculture. (B, C). n (number of independent experiments)=3. Error bars depict SEM.


A . IFT22 nucleotide - binding experiments . measurements using increasing amounts of TbIFT22 TbIFT22 / 74342 - 401 / 81397 - 450 incubated with mant - labeled GDP ( mant - GDP ) or non - hydrolysable GTP / ATP analogs ( mant - GMPPNP / mant - AMPPNP ) . is plotted as a function of . Data were fitted to a single - site binding equation for determination of dissociation constant ( Kd ) . Kd values standard deviations are calculated from three independent experiments .

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A. IFT22 nucleotide-binding experiments. Fluorescence measurements using increasing amounts of TbIFT22 and TbIFT22/74342-401/81397-450 core complex incubated with mant-labeled GDP (mant-GDP) or non-hydrolysable GTP / ATP analogs (mant-GMPPNP / mant-AMPPNP). The fluorescence intensity is plotted as a function of protein concentration. Data were fitted to a single-site binding equation for determination of the dissociation constant (Kd). Kd values and standard deviations are calculated from three independent experiments.


D . Top : Cartoon representation of IFT22 ( grey ) with positions of two nucleotide - binding mutants highlighted . GTP is shown in stick representation Mg2 + as a ball . D175 ( blue ) is unusual residue binding guanine base while S19 ( pink ) is a conserved residue required for coordination of Mg2 + cation is commonly mutated to an asparagine to prevent nucleotide binding . Bottom : Nucleotide - binding experiments of IFT22 nucleotide - binding mutants D175E , D175A S19N with fluorescently labeled nucleotides . Only S19N mutation ( pink ) abolished IFT22 nucleotide - binding ability completely .

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D. Top: Cartoon representation of IFT22 (grey) with positions of two nucleotide-binding mutants highlighted. GTP is shown in stick representation and Mg2+ as a ball. D175 (blue) is the unusual residue binding the guanine base while S19 (pink) is a conserved residue required for coordination of the Mg2+ cation and is commonly mutated to an asparagine to prevent nucleotide binding. Bottom: Nucleotide-binding experiments of IFT22 nucleotide-binding mutants D175E, D175A and S19N with fluorescently labeled nucleotides. Only the S19N mutation (light pink) abolished IFT22 nucleotide-binding ability completely.


C . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged TbIFT22 ( WT mutants ) untagged CrIFT25 / 27 / 74 / 81 . WT TbIFT22 is able to pull down Cr tetrameric , thus forming a chimeric IFT - B1 pentamer , while both A86R S19N mutant fail to bind .

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C. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged TbIFT22 (WT and mutants) and untagged CrIFT25/27/74/81. WT TbIFT22 is able to pull down the Cr tetrameric complex, thus forming a chimeric IFT-B1 pentamer, while both the A86R and S19N mutant fail to bind the complex.


A . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged IFT74342 - 401 / 81397 - 450 untagged IFT22 ( WT mutants ) . Pulldowns were done from lysates of co - expressed . Lanes 1 - 4 show similar total expression of different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . IFT22S19N ( inactive GTPase mutant ) IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with IFT74342 - 401 / 81397 - 450 in pull - down experiment .

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A. SDS-PAGE gel of a Ni2+-NTA pulldown using His-tagged IFT74342-401/81397-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT74342-401/81397-450 complex in the pull-down experiment.


B . SDS - PAGE gel of a Ni2 + - NTA pulldown using His - tagged IFT7479 - 401 / 811 - 450 untagged IFT22 ( WT mutants ) . Pulldowns were done from lysates of co - expressed . Lanes 1 - 4 show similar total expression of different co - expressed constructs ( input samples ) . Lanes 5 - 8 show pulldown elutions . IFT22S19N ( inactive GTPase mutant ) IFT22A86R mutant ( IFT74 / 81 - binding mutant ) did not interact with IFT7479 - 401 / 811 - 450 in pull - down experiment .

B. SDS-PAGE gel of a Ni2+-NTA pulldown using the His-tagged IFT7479-401/811-450 and untagged IFT22 (WT and mutants). Pulldowns were done from cell lysates of co-expressed proteins. Lanes 1-4 show similar total expression levels of the different co-expressed constructs (input samples). Lanes 5-8 show pulldown elutions. The IFT22S19N (inactive GTPase mutant) and the IFT22A86R mutant (IFT74/81-binding mutant) did not interact with the IFT7479-401/811-450 complex in the pull-down experiment.


C . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - IFT25 / 27 / 74 / 81 with His - tagged IFT22 ( WT S19N mutant ) . nucleotide - binding deficient mutant IFT22S19N interacts with IFT25 / 27 / 74 / 81 in pull - down experiment .

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C. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and S19N mutant). The nucleotide-binding deficient mutant IFT22S19N interacts with IFT25/27/74/81 in the pull-down experiment.


D . SDS - PAGE gel of a Ni2 + - NTA pull - down of full - IFT25 / 27 / 74 / 81 with His - tagged IFT22 ( WT A86R mutant ) . IFT74 / 81 - binding mutant IFT22A86R fails to pull down full - tetrameric .

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D. SDS-PAGE gel of a Ni2+-NTA pull-down of full-length IFT25/27/74/81 complex with His-tagged IFT22 (WT and A86R mutant). The IFT74/81-binding mutant IFT22A86R fails to pull down the full-length tetrameric complex.


A . Western blot of IFT22RNAi + GFP : : IFT22S19N probed with anti - IFT22 antibody ( bottom ) with an anti - paraflagellar rod ( PFR ) as loading control ( top ) .

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A. Western blot analysis of the IFT22RNAi+GFP::IFT22S19N cell line probed with the anti-IFT22 antibody (bottom) and with an anti-paraflagellar rod (PFR) as loading control (top).


B . IFA of indicated trypanosome lines using mAb25 ( marker for axoneme , left panels ) an anti - IFT172 antibody ( marker for IFT , right panels ) . top panels show phase contrast merged with DAPI ( cyan ) that stains mitochondrial . Scale bars correspond to 5 µm .

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B. IFA of the indicated trypanosome cell lines using the mAb25 (marker for the axoneme, left panels) and an anti-IFT172 antibody (marker for IFT, right panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. Scale bars correspond to 5 µm.


C . Kymographs showing movement of GFP : : IFT22S19N in presence ( left ) or absence ( right ) of IFT22 endogenous . Note improved signal - to - noise in absence of endogenous IFT22 . P D mark proximal distal ends of flagellum , respectively . Scale bars are 5 seconds ( time , vertical ) 5 µm ( , horizontal )

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C. Kymographs showing the movement of the GFP::IFT22S19N in the presence (left) or the absence (right) of the IFT22 endogenous protein. Note the improved signal-to-noise ratio in the absence of endogenous IFT22. P and D mark the proximal and distal ends of the flagellum, respectively. Scale bars are 5 seconds (time, vertical) and 5 µm (length, horizontal)


D . Dot plot representation of flagellum in indicated lines conditions . For each condition , 100 flagella were measured statistical was performed with ANOVA . Significant differences are indicated with a star ( P < 0 .

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D. Dot plot representation of flagellum length in the indicated cell lines and conditions. For each condition, 100 flagella were measured and statistical analysis was performed with the ANOVA test. Significant differences are indicated with a star (P<0.0001


A . Western blot of IFT22RNAi + GFP : : IFT22A86R probed with anti - IFT22 antibody ( bottom ) with an anti - BiP as loading control ( top ) .

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A. Western blot analysis of the IFT22RNAi+GFP::IFT22A86R cell line probed with the anti-IFT22 antibody (bottom) and with an anti-BiP as loading control (top).


B . IFA with indicated lines using mAb25 ( marker for axoneme , central panels ) an anti - IFT172 antibody ( marker for IFT , bottom panels ) . top panels show phase contrast merged with DAPI ( cyan ) that stains mitochondrial . arrowheads indicate presence of short flagella stained with Mab25 antibody in IFT22RNAi . Scale bars correspond to 5 µm .

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B. IFA with the indicated cell lines using the mAb25 (marker for the axoneme, central panels) and an anti-IFT172 antibody (marker for IFT, bottom panels). The top panels show the phase contrast images merged with DAPI (cyan) that stains nuclear and mitochondrial DNA. The arrowheads indicate the presence of short flagella stained with the Mab25 antibody in the IFT22RNAi cells. Scale bars correspond to 5 µm.


C . Sections of IFT22RNAi + GFP : : IFT22rescue ( top panels ) or IFT22RNAi + GFP : : IFT22A86R ( bottom panels ) were analysed by transmission electron microscopy . Sections through flagellar pocket , transition zone flagellum are shown . Scale bars are 500 nm ( flagellar pocket sections ) or 200 nm ( transition zone flagellum sections ) . arrow indicates an endocytic vesicle budding off flagellar pocket whereas black one points at an IFT train . ( Axo = axoneme , TZ = transition zone , BB = basal )

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C. Sections of IFT22RNAi+GFP::IFT22rescue (top panels) or IFT22RNAi+GFP::IFT22A86R cells (bottom panels) were analysed by transmission electron microscopy. Sections through the flagellar pocket, the transition zone and the flagellum are shown. Scale bars are 500 nm (flagellar pocket sections) or 200 nm (transition zone and flagellum sections). The white arrow indicates an endocytic vesicle budding off the flagellar pocket whereas the black one points at an IFT train. (Axo = axoneme, TZ = transition zone, BB = basal body)


E . Naïve MCF7 MCF7aTT were exposed to indicated stress conditions for 3 h , viability was assessed by dead with trypan blue . bar graphs represent of trypan blue excluding . ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) .

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E. Naïve MCF7 cells and MCF7aTT cells were exposed to the indicated stress conditions for 3 h, and cell viability was assessed by staining dead cells with trypan blue. The bar graphs represent the percentage of trypan blue excluding cells. (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).


A . Whole lysates of MCF7 MCF7aTT exposed to heat shock / or proteasome ( MG132 , 20 μM , 1h ) HSP70 ( VER - 155008 , 50 μM , 1h ) inhibitors were prepared as described in Materials Methods . Total lysate , soluble fractions insoluble fractions were stained by SYPRO Ruby ( left ) quantified ( right ) . ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) .

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A. Whole lysates of MCF7 and MCF7aTT cells exposed to heat shock and/or proteasome (MG132, 20 μM, 1h) and HSP70 (VER-155008, 50 μM, 1h) inhibitors were prepared as described in Materials and Methods. Total lysate, soluble fractions and insoluble fractions were stained by SYPRO Ruby (left) and quantified (right). (means +/- SD, n = 3, numbers above the bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli).


C . soluble insoluble fractions shown in ( A ) were assayed for level of K48 - linked polyubiquitin , polyubiquitylated were quantified ( blue graph , means + / - , n = 4 , numbers above bars indicate p values ) . data for bulk obtained in ( A ) were overlaid onto graph for comparison ( red , means + / - , n = 3 ) .

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C. The soluble and insoluble fractions shown in (A) were assayed for the level of K48-linked polyubiquitin, and polyubiquitylated proteins were quantified (blue line graph, means +/- SEM, n = 4, numbers above the bars indicate p values). The data for bulk protein obtained in (A) were overlaid onto the graph for comparison (red line, means +/- SEM, n = 3).


B . same experiment as described in ( A ) but with MCF7 exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) . Means + / - , n = 2 . C . Lysate from MCF7 exposed to MG132 ( 20 μM , 1 h ) or puromycin ( 100 μg / ml , 10 min ) as indicated were subjected to immunoprecipitation with anti - puromycin antibodies , followed by assaying for co - precipitation of K48 - ubiquitylated . Actin is shown as a reference .

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B. The same experiment as described in (A) but with MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min). Means +/- SEM, n = 2. C. Lysate from MCF7 cells exposed to MG132 (20 μM, 1 h) or puromycin (100 μg/ml, 10 min) as indicated were subjected to immunoprecipitation with anti-puromycin antibodies, followed by assaying for co-precipitation of K48-ubiquitylated proteins. Actin is shown as a reference.


B . Same experiment as in ( A ) with thermotolerant MCF7aTT incubated with HSP70 inhibitor VER - 155008 ( 50 μM , 1h ) or MG132 ( 20 μM , 1h ) . signal obtained for ribosomal RPS19 RPL7 are shown for reference , documenting residual polysomes despite flat UV trace . Right panels are quantifications of K48 - ub ( means + / - , n = 2 ) , black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity . bar graphs represent P / M ratios ( means + / - SD , n = 3 , numbers above bars indicate p values calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) . C . Sucrose density gradient fractions described in ( B ) were assayed for partitioning of HSPA1 HSPH1 by immunoblotting . HSPA1 data were quantified in a graph ( means + / - , n = 4 ) . D

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

B. Same experiment as in (A) with thermotolerant MCF7aTT cells incubated with the HSP70 inhibitor VER-155008 (50 μM, 1h) or MG132 (20 μM, 1h). The signal obtained for ribosomal protein RPS19 and RPL7 are shown for reference, documenting residual polysomes despite the flat UV trace. Right panels are quantifications of K48-ub (means +/- SEM, n = 2), black vertical lines indicate splicing of lanes that were run on different gels due to limitations in lane capacity. The bar graphs represent the P/M ratios (means +/- SD, n = 3, numbers above bars indicate p values calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli). C. Sucrose density gradient fractions described in (B) were assayed for partitioning of HSPA1 and HSPH1 by immunoblotting. HSPA1 data were quantified in a line graph (means +/- SEM, n = 4). D


D . lysate was spun through a 30 % sucrose cushion to pellet polysomes . Nascent were released from polysomes by dissociating 80S ribosomes with EDTA ( see Appendix Figures S3 ) . Soluble insoluble fractions were prepared by centrifugation assayed for level of K48 - linked polyubiquitin . of K48 - linked polyubiquitin were quantified ( means + / - SD , n = 3 ) . Immunoblots within black squares were run on same gel but irrelevant lanes were removed as indicated by lines .

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D. Cell lysate was spun through a 30% sucrose cushion to pellet polysomes. Nascent proteins were released from polysomes by dissociating 80S ribosomes with EDTA (see Appendix Figures S3). Soluble and insoluble fractions were prepared by centrifugation and assayed for the level of K48-linked polyubiquitin. The levels of K48-linked polyubiquitin were quantified (means +/- SD, n = 3). Immunoblots within black squares were run on the same gel but irrelevant lanes were removed as indicated by white lines.


D . Total lysates were prepared from naïve MCF7 thermoresistant MCF7aTT , puromycin labeled peptides were pull down as illustrated in scheme . Precipitates were assayed for presence of K48 - linked polyubiquitin , chaperones , proteasome subunits by immunoblotting .

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D. Total cell lysates were prepared from naïve MCF7 and thermoresistant MCF7aTT cells, and puromycin labeled peptides were pull down as illustrated in the scheme. Precipitates were assayed for the presence of K48-linked polyubiquitin, chaperones, and proteasome subunits by immunoblotting.


E . Whole lysates of KYSE150 KYSE150HSPH1 - / - exposed to indicated conditions ( aTT scheme heat shock ) were fractionated into soluble insoluble fractions as described in Materials Methods . Insoluble fractions were assayed for level of K48 - linked polyubiquitin ( left panel ) . Total of HSPA1 HSPH1 are shown for reference . K48 ubiquitin signals were quantified displayed in a bar graph ( means + / - SD , n = 4 ) .

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E. Whole lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were fractionated into soluble and insoluble fractions as described in Materials and Methods. Insoluble fractions were assayed for the level of K48-linked polyubiquitin (left panel). Total levels of HSPA1 and HSPH1 are shown for reference. K48 ubiquitin signals were quantified and displayed in a bar graph (means +/- SD, n = 4).


F . Lysates of KYSE150 KYSE150HSPH1 - / - exposed to indicated conditions ( aTT scheme heat shock ) were obtained polysomes were isolated by centrifugation through a 30 % sucrose cushion . Whole lysates ( left panel ) polysomal pellets ( right panel ) were assayed by immunoblotting for of HSPH1 , HSPA1 proteasome subunits .

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F. Lysates of cells KYSE150 and KYSE150HSPH1-/- cells exposed to the indicated temperature conditions (aTT scheme and heat shock) were obtained and polysomes were isolated by centrifugation through a 30% sucrose cushion. Whole cell lysates (left panel) and the polysomal pellets (right panel) were assayed by immunoblotting for the levels of HSPH1, HSPA1 and proteasome subunits.


A . Representative of tissue sections derived from oral squamous carcinomas ( OSCC ) esophageal squamous carcinomas ( ESCC ) stained with indicated antibodies by immunohistochemistry ( ) . Top panels show routine H & E . Black squares point to regions magnified in adjacent panels to right .

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A. Representative micrographs of tissue sections derived from oral squamous cell carcinomas (OSCC) and esophageal squamous cell carcinomas (ESCC) stained with the indicated antibodies by immunohistochemistry (IHC). Top panels show routine H&E staining. Black squares point to regions magnified in the adjacent panels to the right.


C . Kaplan - Meier plots showing correlation between HSPH1 mRNA expression level survival of with ESCC . expression data was obtained from visualized with KM Plotter ( www . kmplot . com , ( Nagy et al , 2018 ) using Pan Cancer algorithm at default settings .

C. Kaplan-Meier plots showing the correlation between HSPH1 mRNA expression level and the survival of patients with ESCC. The expression data was obtained from and visualized with KM Plotter (www.kmplot.com, (Nagy et al, 2018) using the Pan Cancer algorithm at default settings.


D . KYSE150 KYSE150HSPH1 - / - were injected into nude mice , xenograft growth was monitored over time ( left panel ; mean volume + / - , n = 4 with 5 per n ) . Mean weights at end of experiments were determined ( means + / - SD , n = 4 with 5 per n , number above bar graph represent p values , * p < 0 . 05 , * * p < 0 . 01 ( calculated with two - stage linear step - up procedure of Benjamini , Krieger Yekutieli ) ) .

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D. KYSE150 and KYSE150HSPH1-/- cells were injected into nude mice, and xenograft tumor growth was monitored over time (left panel; mean tumor volume +/- SEM, n = 4 with 5 animals per n). Mean tumor weights at the end of the experiments were determined (means +/- SD, n = 4 with 5 animals per n, number above the bar graph represent p values, * p<0.05, **p<0.01 (calculated with the two-stage linear step-up procedure of Benjamini, Krieger and Yekutieli)).


( A ) Representative of WT , Mfn1 KO Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria - targeted RFP ( mt - RFP ) . Scale bar : 5 μm .

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(A) Representative images of WT, Mfn1 KO and Mfn2 KO MEFs transfected for 48 h with a plasmid encoding mitochondria-targeted RFP (mt-RFP). Scale bar: 5 μm.


( A ) Representative of WT Mfn2 KO MEFs co - transfected with plasmids encoding mitochondria - targeted RFP ER - targeted GFP . Scale bar : 5 μm .

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(A) Representative images of WT and Mfn2 KO MEFs co-transfected with plasmids encoding mitochondria-targeted RFP and ER-targeted GFP. Scale bar: 5 μm.


( B ) After 48 h , were fixed degree of colocalization between ER mitochondria on red green pixel overlap was analyzed using Mander ' s coefficient ( n = 30 analyzed in 3 independent experiments ) . Data are presented as mean ± . * p < 0 . 05 , two - Student ' s t - .

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(B) After 48 h, the cells were fixed and the degree of colocalization between ER and mitochondria based on red and green fluorescent pixel overlap was analyzed using Mander's coefficient (n= 30 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. *p<0.05, two-tailed Student's t-test.


I - K ) Mitochondrial Ca2 + was determined in WT Mfn2 KO MEFs loaded with Rhod2 . effect of ( I ) caffeine - induced ( 20 mM ) , ( J ) IP3R activation with ATP - induced ( 100 μM ) ( K ) histamine - induced ( 100 μM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 30 from 3 independent experiments ) . Data are presented as mean ± .

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I-K) Mitochondrial Ca2+ was determined in WT and Mfn2 KO MEFs loaded with Rhod2. The effect of (I) caffeine-induced (20 mM), (J) IP3R activation with ATP-induced (100 μM) and (K) histamine-induced (100 μM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM.


WT Mfn2 KO were transfected with plasmids encoding : GFP indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( R ) MMP was analyzed ( n = 90 analyzed in 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding: GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (R) MMP was analyzed (n= 90 cells analyzed in 3 independent experiments).


WT Mfn2 KO were transfected with plasmids encoding GFP indicated combination of non - targeting siRNA ( siCT ) , siRNA targeting MCU ( siMCU ) , ChiMERA or control ( indicated as WT or Mfn2 alone ) plasmids . ( S ) ATP of GFP + was analyzed after 24 h treatment with 2DG ( 10 mM ) ( n = 3 independent experiments ) .

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WT and Mfn2 KO cells were transfected with plasmids encoding GFP and the indicated combination of non-targeting siRNA (siCT), siRNA targeting MCU (siMCU), ChiMERA or control (indicated as WT or Mfn2 alone) plasmids. (S) ATP levels of GFP+ cells was analyzed after 24 h treatment with 2DG (10 mM) (n= 3 independent experiments).


WT Mfn2 KO MEFs were co - transfected with plasmids encoding : mt - RFP ER - Mfn2 or control ( CT ) plasmid . After 48 h , were fixed . ( C ) Representative . Scale bar = 5 μm .

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WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: mt-RFP and ER-Mfn2 or control (CT) plasmid. After 48 h, the cells were fixed. (C) Representative images. Scale bar= 5 μm.


WT Mfn2 KO MEFs were co - transfected with plasmids encoding : J - N ) GFP ER - Mfn2 or control ( CT ) plasmid . ( J ) MMP was determined . values were normalized to surrounding untransfected . ( n = 90 analyzed in 3 independent experiments ) . ( K ) After 24 h , transfected were sorted plated for another 24 h , after which were treated with 2 - DG ( 10 mM ) for 6 h , ATP were measured ( n = 3 independent experiments ) . ( L ) Oxygen consumption was measured ( M ) RCR ( N ) SRC was calculated ( n = 4 independent experiments ) . Data are presented as mean ± . Data information : * p < 0 . 05 , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn2 KO MEFs were co-transfected with plasmids encoding: J-N) GFP and ER-Mfn2 or control (CT) plasmid. (J) MMP was determined. The values were normalized to surrounding untransfected cells. (n= 90 cells analyzed in 3 independent experiments). (K) After 24 h, the transfected cells were sorted and plated for another 24 h, after which cells were treated with 2-DG (10 mM) for 6 h, and ATP levels were measured (n= 3 independent experiments). (L) Oxygen consumption was measured and (M) RCR and (N) SRC was calculated (n= 4 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : A ) mt - RFP , ER - GFP plasmids expressing indicated . Mitochondria - ER colocalization was analyzed using Mander ' s coefficient ( n = 15 analyzed in 3 independent experiments ) . Data are presented as mean ± . B ) GFP ERMITO - RLuc indicated expression vectors . After 24 h , transfected were sorted plated for another 24 h , after which RLuc was analyzed ( n = 3 independent experiments ) . Data are presented as mean ± . C ) Data information : red dashed indicates WT results . * p < 0 . 05 versus Mfn1 / 2 DKO , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn1/2 double KO were transfected with: A) mt-RFP, ER-GFP and plasmids expressing the indicated proteins. Mitochondria-ER colocalization was analyzed using Mander's coefficient (n= 15 cells analyzed in 3 independent experiments). Data are presented as mean ± SEM. B) GFP and ERMITO-RLuc and indicated expression vectors. After 24 h, the transfected cells were sorted and plated for another 24 h, after which RLuc was analyzed (n= 3 independent experiments). Data are presented as mean ± SEM. C) Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : C ) GFP indicated plasmids . After 48 h , mitochondrial Ca2 + was determined ( n = 30 from 3 independent experiments ) . Data are presented as mean ± . Data information : red dashed indicates WT results . * p < 0 . 05 versus Mfn1 / 2 DKO , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn1/2 double KO were transfected with: C) GFP and the indicated plasmids. After 48 h, mitochondrial Ca2+ was determined (n= 30 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : D ) LAR - GECO1 indicated plasmids . After 48 h ER Ca2 + was released with caffeine ( 20 mM ) as indicated ( n = 25 - 27 from 3 independent experiments ) . Data are presented as mean ± . Data information : red dashed indicates WT results . * p < 0 . 05 versus Mfn1 / 2 DKO , one - way ANOVA followed by Tukey ' s post hoc . Note that experiments were performed at same time , so values of WT Mfn2 KO transfected with control plasmid are same but figures have been split in two for sake of linearity .

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WT and Mfn1/2 double KO were transfected with: D) LAR-GECO1 and the indicated plasmids. After 48 h ER Ca2+ was released with caffeine (20 mM) as indicated (n= 25-27 cells from 3 independent experiments). Data are presented as mean ± SEM. Data information: The red dashed line indicates the WT cells results. *p<0.05 versus Mfn1/2 DKO cells, one-way ANOVA followed by Tukey's post hoc test. Note that experiments were performed at the same time, so the values of WT and Mfn2 KO transfected with control plasmid are the same but the figures have been split in two for the sake of linearity.


WT Mfn1 / 2 double KO were transfected with : E ) GFP indicated plasmid . After 48 h , effect of caffeine - induced ( 20 mM ) ER Ca2 + release on mitochondrial Ca2 + was measured ( n = 25 from 3 independent experiments ) . Data are presented as mean ± .

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WT and Mfn1/2 double KO were transfected with: E) GFP and the indicated plasmid. After 48 h, the effect of caffeine-induced (20 mM) ER Ca2+ release on mitochondrial Ca2+ was measured (n= 25 cells from 3 independent experiments). Data are presented as mean ± SEM.


B ) Representative Western blots of indicated during neuronal maturation in vitro ( n = 3 independent experiments ) . C ) Representative Western blots of indicated of WT Mfn2 KO neurons at DIV7 ( n = 3 independent experiments ) . D

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B) Representative Western blots of the indicated proteins during the neuronal maturation in vitro (n= 3 independent experiments). C) Representative Western blots of the indicated proteins of WT and Mfn2 KO neurons at DIV7 (n= 3 independent experiments). D


D ) neurons of tamoxifen - inducible Mfn2 KO mice were transfected at DIV3 with ER - GFP , mt - RFP plus indicated plasmids . Tamoxifen - treated ( for 72 h ) vehicle - treated neurons ( WT ; red dashed ) were fixed mitochondria - ER co - localization was analyzed using Mander ' s coefficient ( n = 15 neurons analyzed in 3 independent experiments ) . Data are presented as mean ± . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc . Scale bar : 500 μm .

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D) Primary neurons of tamoxifen-inducible Mfn2 KO mice were transfected at DIV3 with ER-GFP, mt-RFP plus the indicated plasmids. Tamoxifen-treated (for 72 h) and vehicle-treated neurons (WT; red dashed line) were fixed and mitochondria-ER co-localization was analyzed using Mander's coefficient (n= 15 neurons analyzed in 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm.


( F ) neurite measurement representative tracings ( G ) of cortical neurons of WT Mfn2 KO mice co - transfected at DIV4 with GFP indicated plasmids . After 72 h , neurons were fixed , immunofluorescence with anti - GFP antibodies was performed neurite was measured ( n = 30 neurons from 3 independent experiments ) . Data are presented as mean ± . Data information : * p < 0 . 05 versus WT ; # p < 0 . 05 versus Mfn2 KO neurons , one - way ANOVA followed by Tukey ' s post - hoc . Scale bar : 500 μm . E

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(F) neurite length measurement and representative tracings (G) of primary cortical neurons of WT and Mfn2 KO mice co-transfected at DIV4 with GFP and the indicated plasmids. After 72 h, the neurons were fixed, immunofluorescence with anti-GFP antibodies was performed and neurite length was measured (n= 30 neurons from 3 independent experiments). Data are presented as mean ± SEM. Data information: *p<0.05 versus WT; #p<0.05 versus Mfn2 KO neurons, one-way ANOVA followed by Tukey's post-hoc test. Scale bar: 500 μm. E


( a ) Representative differential interference contrast microscopy of midgut from wild - type at early third instar larval ( Early 3rd ) , late third instar larval ( Late 3rd ) at puparium formation ( prepupal , WPP ) stages .

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(a) Representative differential interference contrast microscopy images of midgut cells from wild-type animals at the early third instar larval (Early 3rd), late third instar larval (Late 3rd) and at puparium formation (white prepupal, WPP) stages.


( b ) Autophagy detected by formation of mCherry - Atg8a punctate spots in midgut from wild - type at indicated stages . Representative are shown .

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(b) Autophagy detected by formation of mCherry-Atg8a punctate spots in midgut cells from wild-type animals at indicated stages. Representative images are shown.


( c ) Midguts from control Atg18KG03090 / wild - type ( Atg18 / + ) , n = 14 , Atg18KG03090 / Df ( 3L ) 6112 mutant ( Atg18 / Df ) , n = 11 , at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( d ) Wild - type , control ( Atg18KG03090 / + ) Atg18 mutant ( Atg18 / Df ) midgut ( μm2 ) at indicated stages ; n = 10 intestines per genotype with 5 measured per intestine per stage .

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(c) Midguts from control Atg18KG03090/wild-type (Atg18/+), n = 14, and Atg18KG03090/Df(3L)6112 mutant (Atg18/Df), n = 11, animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (d) Wild-type, control (Atg18KG03090/+) and Atg18 mutant (Atg18/Df) midgut cell size quantification (μm2) at indicated stages; n = 10 animal intestines per genotype with 5 cells measured per intestine per stage.


( e ) Differential interference contrast of midgut from Atg2EP3697 / wild - type control ( Atg2 / + ) Atg2EP3697 / Df ( 3L ) 6091 mutant ( Atg2 / Df ) at puparium formation . Representative are shown . ( f ) ( μm2 ) from e , n = 12 ( Atg2 / + ) n = 7 ( Atg2 / Df ) intestines per genotype with 5 measured per intestine .

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(e) Differential interference contrast images of midgut cells from Atg2EP3697/wild-type control (Atg2/+) and Atg2EP3697/Df(3L)6091 mutant (Atg2/Df) animals at puparium formation. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 12 (Atg2/+) and n = 7 (Atg2/Df) animal intestines per genotype with 5 cells measured per intestine.


( g - j ) Representative of intestine 2 h after puparium formation . ( g , i ) Control Atg18KG03090 / wild - type ( Atg18 / + , g ) control Atg2EP3697 / wild - type ( Atg2 / + , i ) with an enlarged image showing a double - structure ( arrowhead ) that surrounds a mitochondrion endoplasmic reticulum . Arrows indicate autolysosomes . ( h , j ) Atg18KG03090 / Df ( 3L ) 6112mutant ( Atg18 / Df , h ) Atg2EP3697 / Df ( Atg2 / Df , j ) mutant lacking autophagic structures . is shown as mean ± s . d . Scale bars , 20 μm ( a - c e ) 1 μm ( g - j ) .

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(g-j) Representative TEM images of intestine cells 2 h after puparium formation. (g,i) Control Atg18KG03090/wild-type (Atg18/+, g) and control Atg2EP3697/wild-type (Atg2/+, i) cells with an enlarged image showing a double-membrane structure (arrowhead) that surrounds a mitochondrion and endoplasmic reticulum. Arrows indicate autolysosomes. (h,j) Atg18KG03090/Df(3L)6112mutant (Atg18/Df, h) and Atg2EP3697/Df (Atg2/Df, j) mutant cells lacking autophagic structures. Quantification is shown as mean ± s.d. Scale bars, 20 μm (a-c and e) and 1 μm (g-j).


( a ) Midguts dissected from expressing Atg18IR specifically in DsRed - marked clones of at puparium formation analysed by differential interference contrast ( DIC ) microscopy . Representative are shown . ( b ) ( μm2 ) from a , n = 12 intestines per genotype with 1 - 5 measured per intestine .

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(a) Midguts dissected from animals expressing Atg18IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Quantification (μm2) from a, n = 12 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts expressing mCherry - Atg8a in , expressing Atg18IR specifically in GFP - marked clones dissected from at 2 h after puparium formation . Representative are shown .

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(c) Midguts expressing mCherry-Atg8a in all cells, and expressing Atg18IR specifically in GFP-marked cell clones dissected from animals at 2 h after puparium formation. Representative images are shown.


( d ) Midguts dissected from at puparium formation that contain an Atg1Δ3D loss - of - function mutant clone ( lacking GFP ) analysed by differential interference contrast microscopy . Wild - type ( + / + ) control possesses stronger GFP heterozygous Atg1Δ3D / wild - type ( Δ3D / + ) have weaker GFP . Representative are shown . ( e ) ( μm2 ) from d , n = 7 ( + / + ) , n = 8 ( Δ3D / + ) n = 8 ( Δ3D / Δ3D ) intestines per genotype with 1 - 5 measured per intestine .

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(d) Midguts dissected from animals at puparium formation that contain an Atg1Δ3D loss-of-function mutant cell clone (lacking GFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cell possesses stronger GFP and heterozygous Atg1Δ3D/wild-type (Δ3D/+) cells have weaker GFP. Representative images are shown. (e) Quantification (μm2) from d, n = 7 (+/+), n = 8 (Δ3D/+) and n = 8 (Δ3D/Δ3D) animal intestines per genotype with 1-5 cells measured per intestine.


( f ) Midguts dissected from expressing mCherry - Atg8a in , expressing Atg1IR specifically in GFP - marked clones of at 2 h after puparium formation . Representative are shown .

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(f) Midguts dissected from animals expressing mCherry-Atg8a in all cells, and expressing Atg1IR specifically in GFP-marked clones of cells at 2 h after puparium formation. Representative images are shown.


( g - j ) Representative cut views from three - dimensional reconstitution from wild - type ( g , h ) Atg1 knockdown ( i , j ) at early third instar larval stage ( g , i ) at puparium formation ( h , j ) . ( k ) of volume ( μm3 ) from g - j , n = 5 intestines per genotype with 1 - 5 measured per intestine per stage . Black columns : early third instar larval stage . columns : puparium formation stage . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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(g-j) Representative cut views from three-dimensional reconstitution images from wild-type (g,h) and Atg1 knockdown (i,j) cells at early third instar larval stage (g,i) and at puparium formation (h,j). (k) Quantification of cell volume (μm3) from g-j, n = 5 animal intestines per genotype with 1-5 cells measured per intestine per stage. Black columns: early third instar larval stage. White columns: puparium formation stage. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


( a ) Midguts dissected from at puparium formation that contain Vps34Δm22 loss - of - function mutant clones ( lacking GFP ) analysed by differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control possess stronger GFP heterozygous Vps34Δm22 / wild - type ( Δm22 / + ) have weaker GFP . Representative are shown . ( b ) ( μm2 ) from a , n = 14 intestines per genotype with 1 - 5 measured per intestine .

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(a) Midguts dissected from animals at puparium formation that contain Vps34Δm22 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Vps34Δm22/wild-type (Δm22/+) cells have weaker GFP. Representative images are shown. (b) Quantification (μm2) from a, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts dissected from expressing Atg8aIR specifically in GFP - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( d ) ( μm2 ) from c , n = 10 intestines per genotype with 1 - 5 measured per intestine .

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(c) Midguts dissected from animals expressing Atg8aIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (d) Cell size quantification (μm2) from c, n = 10 animal intestines per genotype with 1-5 cells measured per intestine.


( e ) Midguts dissected from expressing Atg12IR specifically in DsRed - marked clones of at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( f ) ( μm2 ) from e , n = 14 intestines per genotype with 1 - 5 measured per intestine .

(e) Midguts dissected from animals expressing Atg12IR specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (f) Cell size quantification (μm2) from e, n = 14 animal intestines per genotype with 1-5 cells measured per intestine.


( g - i ) Midguts dissected from early third instar larvae that mis - express Atg1GS10797 ( Atg1 ; g , GFP in nucleus cytoplasm ) , both Atg1 + Atg8aIR ( h ) , or both Atg1 + Atg7IR ( i ) stained to detect Discs large ( green ) on cortex of . Representative are shown . ( g ' i ' ) DIC of midguts expressing mCherry - Atg8a in , Atg1 ( g ' ) , Atg1 + Atg7IR ( i ' ) in GFP - marked clones of . mCherry - Atg8a are shown in insets . Representative are shown . is shown as mean ± s . d . Scale bars , 20 μm .

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(g-i) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1; g, GFP in nucleus and cytoplasm), both Atg1 +Atg8aIR (h), or both Atg1 +Atg7IR (i) and stained to detect Discs large (green) on the cortex of all cells. Representative images are shown. (g' and i') DIC images of midguts expressing mCherry-Atg8a in all cells, Atg1 (g'), and Atg1+Atg7IR (i') in GFP-marked clones of cells. mCherry-Atg8a puncta are shown in insets. Representative images are shown. Quantification is shown as mean ± s.d. Scale bars, 20 μm.


( a ) Midguts dissected from at puparium formation that contain Atg7d4 loss - of - function mutant clones ( lacking GFP ) analysed by differential interference contrast ( DIC ) microscopy . Wild - type ( + / + ) control possess stronger GFP heterozygous Atg7d4 / wild - type ( d4 / + ) have weaker GFP . Representative are shown . ( b ) ( μm2 ) from a , n = 8 intestines per genotype with 1 - 5 measured per intestine .

(a) Midguts dissected from animals at puparium formation that contain Atg7d4 loss-of-function mutant cell clones (lacking GFP) and analysed by fluorescence and differential interference contrast (DIC) microscopy. Wild-type (+/+) control cells possess stronger GFP and heterozygous Atg7d4/wild-type (d4/+) cells have weaker GFP. Representative images are shown. (b) Cell size quantification (μm2) from a, n = 8 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts expressing mCherry - Atg8a in , with Atg7d4 loss - of - function clone ( lacking GFP ) at 2 h after puparium formation . Representative are shown .

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(c) Midguts expressing mCherry-Atg8a in all cells, and with Atg7d4 loss-of-function clone cells (lacking GFP) at 2 h after puparium formation. Representative images are shown.


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( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) with an Atg7d4 loss - of - function clone ( lacking RFP ) at puparium formation . Representative are shown .

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(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei) with an Atg7d4 loss-of-function clone cell (lacking RFP) at puparium formation. Representative images are shown.


( g , h ) Representative of intestine 2 h after puparium formation . Arrows indicate autolysosomes . ( g , h ) Control ( Atg7d30 / d14 ; g ) Atg7 mutant ( Atg7d77 / d14 ; h ) both contain autophagic structures . ( h , right ) Enlarged Atg7 mutant image of a double - autophagosome ( arrowhead ) surrounding a mitochondrion . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm ( a , c , d f ) 1 μm ( g , h ) .

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(g,h) Representative TEM images of intestine cells 2 h after puparium formation. Arrows indicate autolysosomes. (g,h) Control (Atg7d30/d14; g) and Atg7 mutant (Atg7d77/d14; h) cells both contain autophagic structures. (h, right) Enlarged Atg7 mutant cell image of a double-membrane autophagosome (arrowhead) surrounding a mitochondrion. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm (a,c,d and f) and 1 μm (g,h).


( a ) of mitochondria numbers from Atg18 control ( Atg18KG03090 / wild type ) mutant ( Atg18KG03090 / Df ( 3L ) 6112 ) or Atg7 control ( Atg7d30 / d14 ) mutant ( Atg7d77 / d14 ) . Mitochondria were quantified from 2 distinct 25 μm2 regions per from 2 per from at least 3 different per genotype .

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(a) Quantification of mitochondria numbers from Atg18 control (Atg18KG03090/wild type) and mutant (Atg18KG03090/Df(3L)6112) or Atg7 control (Atg7d30/d14) and mutant (Atg7d77/d14) TEM images. Mitochondria were quantified from 2 distinct 25 μm2 regions per cell from 2 cells per animal from at least 3 different animals per genotype.


( b , c ) Midguts dissected from either early third instar larvae ( b ) or at puparium formation ( c ) that express GFP - labelled mitochondria in contain an Atg1Δ3D loss - of - function mutant clone ( lacking RFP ) . Wild - type control possess stronger RFP heterozygous have weaker RFP . Representative are shown . is shown as mean ± s . d . NS , not significant . Scale bars , 0 μm .

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(b,c) Midguts dissected from either early third instar larvae (b) or at puparium formation (c) that express GFP-labelled mitochondria in all cells and contain an Atg1Δ3D loss-of-function mutant cell clone (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 0 μm.


( a ) Midguts dissected from expressing Uba1IR specifically in GFP - marked clones of at puparium formation analysed by differential interference contrast ( DIC ) microscopy . Representative are shown . ( b ) ( μm2 ) from a ; n = 16 intestines per genotype with 1 - 5 measured per intestine .

(a) Midguts dissected from animals expressing Uba1IR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown. (b) Cell size quantification (μm2) from a; n = 16 animal intestines per genotype with 1-5 cells measured per intestine.


( c ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function mutant clones ( lacking RFP ) analysed by differential interference contrast microscopy . Wild - type ( + / + ) control possess stronger RFP heterozygous Uba1H33 / wild - type ( H33 / + ) have weaker RFP . Representative are shown . ( d ) ( μm2 ) from c , n = 8 ( + / + ) , n = 11 ( H33 / + ) n = 11 ( H33 / H33 ) intestines per genotype with 1 - 5 measured per intestine .

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(c) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function mutant cell clones (lacking RFP) and analysed by fluorescence and differential interference contrast microscopy. Wild-type (+/+) control cells possess stronger RFP and heterozygous Uba1H33/wild-type (H33/+) cells have weaker RFP. Representative images are shown. (d) Quantification (μm2) from c, n = 8 (+/+), n = 11 (H33/+) and n = 11 (H33/H33) animal intestines per genotype with 1-5 cells measured per intestine.


( e ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function MARCM mutant clones ( GFP - positive ) that also have mCherry - Atg8a expressed in analysed by microscopy . Control wild - type heterozygous have no GFP . Representative are shown .

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(e) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that also have mCherry-Atg8a expressed in all cells and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.


( f ) Midguts expressing GFP - Atg5 in enterocytes ( larger nuclei ) , with an Uba1H33 loss - of - function clone ( lacking RFP ) at puparium formation analysed by microscopy . Representative are shown .

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(f) Midguts expressing GFP-Atg5 in enterocytes (larger nuclei), and with an Uba1H33 loss-of-function clone cell (lacking RFP) at puparium formation analysed by fluorescence microscopy. Representative images are shown.


( g ) Midguts dissected from at puparium formation that contain Uba1H33 loss - of - function MARCM mutant clones ( GFP - positive ) that are stained with p62 antibody analysed by microscopy . Control wild - type heterozygous have no GFP . Representative are shown .

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(g) Midguts dissected from animals at puparium formation that contain Uba1H33 loss-of-function MARCM mutant cell clones (GFP-positive) that are stained with p62 antibody and analysed by fluorescence microscopy. Control wild-type and heterozygous cells have no GFP. Representative images are shown.


( h ) Midguts dissected from expressing Dts7 , a dominant - sensitive mutant of β2 subunit of proteasome , specifically in GFP - marked at puparium formation analysed by differential interference contrast microscopy . Representative are shown . ( i ) ( μm2 ) from h , n = 6 intestines per genotype with 1 - 5 measured per intestine . is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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(h) Midguts dissected from animals expressing Dts7, a dominant temperature-sensitive mutant of the β2 subunit of the proteasome, specifically in GFP-marked cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (i) Cell size quantification (μm2) from h, n = 6 animal intestines per genotype with 1-5 cells measured per intestine. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.