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 .

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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 + / - , 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 .

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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 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 .

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

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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 ) Tumor volume in mice depleted of DCs or NK cells before tumor cell transplant and LPS administration n ( number of animals 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 ) Tumor volume in SCIDmice treated or not with LPS n ( number of animals 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 ) Tumor volume at the indicated time point after repetitive LPS injections as depicted in the bottom scheme n ( number of animals 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 ) 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 .

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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 the scheme at the bottom of the 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 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 .

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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 + 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 .

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

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


( 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) Tumor 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 ) Percent of IFN - γ + NK cells within the tumor in mice treated or not with LPS and with the 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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 ) 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 .

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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 . 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 .

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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 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 .

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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 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 .

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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 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 .

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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 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 .

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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 - 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 .

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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 - 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 .

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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 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 ) .

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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 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 .

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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 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 )

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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 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 .

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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 analysis of the IFT22RNAi + GFP : : IFT22A86R cell line probed with the anti - IFT22 antibody ( bottom ) and 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 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 .

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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 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 )

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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 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 ) .

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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 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 ) .

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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 . 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 ) .

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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 . 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 .

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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 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

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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 . 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 .

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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 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 .

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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 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 ) .

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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 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 .

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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 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 .

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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 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 .

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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 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 ) ) .

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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 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 .

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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 images of WT and Mfn2 KO MEFs co - transfected with plasmids encoding mitochondria - targeted RFP and 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 , 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 .

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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 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 .

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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 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 ) .

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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 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 ) .

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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 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 .

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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 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 .

O O B-CONTROLLED_VAR 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 B-MEASURED_VAR 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 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

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 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 .

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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 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 .

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 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 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 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

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 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 .

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

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 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 .

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

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 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

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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 ) 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 .

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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 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

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 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

(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 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 .

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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 cells from wild - type animals at indicated stages . Representative images 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 , 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 .

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

(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 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 .

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 O B-CONTROLLED_VAR 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 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

(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 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 ) .

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

(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 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 .

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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 all cells , and expressing Atg18IR specifically in GFP - marked cell clones dissected from animals at 2 h after puparium formation . Representative images 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 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 .

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 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

(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 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 .

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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 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 .

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

(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 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 .

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 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

(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 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 .

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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 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 .

(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 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 .

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

(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 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 .

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(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 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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(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 cell ( lacking RFP ) at 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 cell (lacking RFP) at puparium formation. Representative images are shown.


( 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 ) .

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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 ) 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 .

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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 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 .

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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 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 .

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(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 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 .

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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 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 .

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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 ) , and with an Uba1H33 loss - of - function clone cell ( lacking RFP ) at puparium formation analysed by fluorescence microscopy . Representative images 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 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 .

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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 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 .

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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.


( a , b ) E1 - charging assay for Uba1 and Atg7 with either Atg8a ( a ) or ubiquitin ( Ub ; b ) , n = 2 experiments with independently isolated proteins and analyses . Flag - tagged baculoviral - expressed E1s were incubated with either His - tagged Atg8a or Ub in the presence or absence of β - mercaptoethanol , separated by electrophoresis and blotted . Band shift was detected with anti - Flag antibody . Relative molecular mass ( Mr K ) ladders are indicated . Representative images are shown . Relative molecular masses : Flag - Uba1 , 135K ; Flag - Atg7 , 81K ; His - Atg8a , 29K ; Ub , 9K .

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(a,b) E1-charging assay for Uba1 and Atg7 with either Atg8a (a) or ubiquitin (Ub; b), n = 2 experiments with independently isolated proteins and analyses. Flag-tagged baculoviral-expressed E1s were incubated with either His-tagged Atg8a or Ub in the presence or absence of β-mercaptoethanol, separated by electrophoresis and blotted. Band shift was detected with anti-Flag antibody. Relative molecular mass (Mr K) ladders are indicated. Representative images are shown. Relative molecular masses: Flag-Uba1, 135K; Flag-Atg7, 81K; His-Atg8a, 29K; Ub, 9K.


( c ) Midgut protein extracts at puparium formation from wild - type , Atg7 mutant ( Atg7d77 / d14 ) and Atg8 ( Atg8KG07569 ) mutant animals blotted with anti - Atg8 and anti - Tubulin , n = 3 independent biological experiments . Representative images are shown .

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(c) Midgut protein extracts at puparium formation from wild-type, Atg7 mutant (Atg7d77/d14) and Atg8 (Atg8KG07569) mutant animals blotted with anti-Atg8 and anti-Tubulin, n = 3 independent biological experiments. Representative images are shown.


( d ) Midguts from control Atg310 / wild - type ( Atg3 / + ) and Atg310 / Df ( 3L ) cat mutant ( Atg3 / Df ) animals at puparium formation analysed by differential interference contrast microscopy . Representative images are shown . ( e ) Cell size quantification ( μm2 ) from d , n = 11 ( Atg3 / + ) and n = 9 ( Atg3 / Df ) animal intestines per genotype with 5 cells measured per intestine .

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(d) Midguts from control Atg310/wild-type (Atg3/+) and Atg310/Df(3L)cat mutant (Atg3/Df) animals at puparium formation analysed by differential interference contrast microscopy. Representative images are shown. (e) Cell size quantification (μm2) from d, n = 11 (Atg3/+) and n = 9 (Atg3/Df) animal intestines per genotype with 5 cells measured per intestine.


( f ) Midguts dissected from early third instar larvae that mis - express Atg1GS10797 ( Atg1 ) and Atg3IR only in the DsRed - marked cell clone and analysed by fluorescence and differential interference contrast ( DIC ) microscopy . Representative images are shown .

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(f) Midguts dissected from early third instar larvae that mis-express Atg1GS10797 (Atg1) and Atg3IR only in the DsRed-marked cell clone and analysed by fluorescence and differential interference contrast (DIC) microscopy. Representative images are shown.


( g ) Midguts dissected from animals expressing Atg4DN specifically in DsRed - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( h ) Cell size quantification ( μm2 ) from g , n = 10 animal intestines per genotype with 1 - 5 cells measured per intestine .

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(g) Midguts dissected from animals expressing Atg4DN specifically in DsRed-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (h) Cell size quantification (μm2) from g, n = 10 animal intestines per genotype with 1-5 cells measured per intestine.


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

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


( j ) Midguts dissected from animals expressing UbIR specifically in GFP - marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy . Representative images are shown . ( k ) Cell size quantification ( μm2 ) from ( j ) , n = 12 animal intestines per genotype with 1 - 5 cells measured per intestine .

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(j) Midguts dissected from animals expressing UbIR specifically in GFP-marked clones of cells at puparium formation and analysed by fluorescence and differential interference contrast microscopy. Representative images are shown. (k) Cell size quantification (μm2) from (j), n = 12 animal intestines per genotype with 1-5 cells measured per intestine.


( l , m ) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 ( Atg1 ) mis - expression in either a wild - type GFP - positive cell clone ( l ) or a Uba1H33 loss - of - function MARCM mutant cell clone ( GFP - positive ; m ) and analysed by fluorescence and differential interference contrast microscopy . Control wild - type and heterozygous Uba1H33 / wild - type cells have no GFP . Representative images are shown . ( n ) Quantification ( μm2 ) from l and m , n = 22 ( control ) , n = 16 ( Atg1 ) and n = 6 ( Atg1 + Uba1H33 ) animal intestines per genotype with 1 - 5 cells measured per intestine . Quantification data for control cells ( l , m ) were pooled because they are genetically identical . Quantification is shown as mean ± s . d . NS , not significant . Scale bars , 20 μm .

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(l,m) Midguts dissected from early third instar larvae that exhibit Atg1GS10797 (Atg1) mis-expression in either a wild-type GFP-positive cell clone (l) or a Uba1H33 loss-of-function MARCM mutant cell clone (GFP-positive; m) and analysed by fluorescence and differential interference contrast microscopy. Control wild-type and heterozygous Uba1H33/wild-type cells have no GFP. Representative images are shown. (n) Quantification (μm2) from l and m, n = 22 (control), n = 16 (Atg1) and n = 6 (Atg1 +Uba1H33) animal intestines per genotype with 1-5 cells measured per intestine. Quantification data for control cells (l,m) were pooled because they are genetically identical. Quantification is shown as mean ± s.d. NS, not significant. Scale bars, 20 μm.


( a , b ) Midguts dissected from either early third instar larvae ( a ) or at puparium formation ( b ) that express GFP - labelled mitochondria in enterocytes ( larger nuclei ) and contain Uba1H33 loss - of - function mutant cell clones ( lacking RFP ) . Wild - type control cells possess stronger RFP and heterozygous cells have weaker RFP . Representative images are shown .

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(a,b) Midguts dissected from either early third instar larvae (a) or at puparium formation (b) that express GFP-labelled mitochondria in enterocytes (larger nuclei) and contain Uba1H33 loss-of-function mutant cell clones (lacking RFP). Wild-type control cells possess stronger RFP and heterozygous cells have weaker RFP. Representative images are shown.


( c ) Midguts dissected at puparium formation that express Uba1IR ( GFP in nucleus and cytoplasm ) and stained with ATP synthase complex V ( ATPV ) to detect mitochondria in all cells . Representative images are shown .

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(c) Midguts dissected at puparium formation that express Uba1IR (GFP in nucleus and cytoplasm) and stained with ATP synthase complex V (ATPV) to detect mitochondria in all cells. Representative images are shown.