Representative confocal micrographs ( scale bars are 20 µm ) of caspase 3 / 7 activation in DMKG ( 2 mM ) - treated CTNSWT , CTNS ‑ / - , and CTNSPatient cells for 24 hrs ( n = 3 ) .

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Representative confocal micrographs (scale bars are 20 µm) of caspase 3/7 activation in DMKG (2 mM)-treated CTNSWT, CTNS‑/-, and CTNSPatient cells for 24 hrs (n = 3).


( H , I ) Western blotting and densitometric analyses for LC3 - II / LC3 - I ratio in CTNSWT , CTNS ‑ / - , and CTNSPatient cells cultured in the presence or in the absence of BafA1 ( 25 nM ) and DMKG ( 2 mM ) for 4 hrs , respectively ( n = 3 ) .

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(H, I) Western blotting and densitometric analyses for LC3-II/LC3-I ratio in CTNSWT, CTNS‑/-, and CTNSPatient cells cultured in the presence or in the absence of BafA1 (25 nM) and DMKG (2 mM) for 4 hrs, respectively (n = 3).


( E ) , Proteomic analysis of CTNS - / - cells treated with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide , respectively ( n = 3 ) . AKGDH ; Alpha - ketoglutarate dehydrogenase , GLUD1 ; GLUD2 ; Mitochondrial glutamate dehydrogenase 1 / 2 , IGF2R ; Cation - independent mannose - 6 - phosphate receptor , GSTK1 ; Glutathione S - transferase kappa 1 , COX6B1 ; Cytochrome c oxidase subunit 6B1 , ACACA ; Acetyl - CoA carboxylase 1 - Biotin carboxylase , GLS ; Glutaminase kidney isoform , mitochondrial , CASP3 ; Caspase - 3 , NPC1 ; Niemann - Pick C1 protein .

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(E), Proteomic analysis of CTNS-/- cells treated with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide, respectively (n = 3). AKGDH; Alpha-ketoglutarate dehydrogenase, GLUD1; GLUD2; Mitochondrial glutamate dehydrogenase 1/2, IGF2R; Cation-independent mannose-6-phosphate receptor, GSTK1; Glutathione S-transferase kappa 1, COX6B1; Cytochrome c oxidase subunit 6B1, ACACA; Acetyl-CoA carboxylase 1-Biotin carboxylase, GLS; Glutaminase kidney isoform, mitochondrial, CASP3; Caspase-3, NPC1; Niemann-Pick C1 protein.


( D , E ) Representative confocal micrographs and quantification of DQ - BSA in CTNSWT , and CTNS - / - cells upon treatment with bicalutamide . Scale bars are 20 µm ( n = 8 - 14 quantified images ) .

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(D, E) Representative confocal micrographs and quantification of DQ-BSA in CTNSWT, and CTNS-/- cells upon treatment with bicalutamide. Scale bars are 20 µm (n = 8-14 quantified images).


( F ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in CTNS - / - in the absence of the drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 6 ) , bicalutamide ( n = 6 ) , and a combination of cysteamine and bicalutamide ( n = 6 ) . ( G ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in CTNSPatient in the absence of the drug ( NT ) ( n = 4 ) or upon treatment with cysteamine ( n = 4 ) , bicalutamide ( n = 4 ) , and a combination of cysteamine and bicalutamide ( n = 6 ) .

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(F) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNS-/- in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 6), bicalutamide (n = 6), and a combination of cysteamine and bicalutamide (n = 6). (G) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in CTNSPatient in the absence of the drug (NT) (n = 4) or upon treatment with cysteamine (n = 4), bicalutamide (n = 4), and a combination of cysteamine and bicalutamide (n = 6).


( A ) Immunocytochemistry of patient - derived cystinotic tubuloids ( CNTSPatient - 1 and CTNSPatient - 2 ) and healthy kidney tissue - derived control tubuloids ( CNTSWT - 1 and CTNSWT - 2 ) for PAX8 , TP63 and F - actin . Scale bar = 100 µm .

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(A) Immunocytochemistry of patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) and healthy kidney tissue-derived control tubuloids (CNTSWT-1 and CTNSWT-2) for PAX8, TP63 and F-actin. Scale bar = 100 µm.


( C , D ) Quantification of cystine levels ( nmol / mg protein ) by HPLC - MS / MS in two different patient - derived cystinotic tubuloids in the absence of the drugs ( NT ) or upon treatment with cysteamine ( 100 μM ) , bicalutamide ( 35 μM ) or cysteamine ( 100 μM ) - bicalutamide ( 35 μM ) combination treatment ( n = 3 ) . ( E ) αKG levels measured in patient - derived cystinotic tubuloids ( CNTSPatient - 1 and CTNSPatient - 2 ) in the absence of the drugs ( NT ) or upon treatment with cysteamine , bicalutamide or cysteamine - bicalutamide combination treatment using metabolomics ( n = 3 ) .

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(C, D) Quantification of cystine levels (nmol/mg protein) by HPLC-MS/MS in two different patient-derived cystinotic tubuloids in the absence of the drugs (NT) or upon treatment with cysteamine (100 μM), bicalutamide (35 μM) or cysteamine (100 μM)-bicalutamide (35 μM) combination treatment (n = 3). (E) αKG levels measured in patient-derived cystinotic tubuloids (CNTSPatient-1 and CTNSPatient-2) in the absence of the drugs (NT) or upon treatment with cysteamine, bicalutamide or cysteamine-bicalutamide combination treatment using metabolomics (n = 3).


( I ) Survival rates in ctns - / - zebrafish upon treatment with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide ( n = 40 embryos per group ) .

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(I) Survival rates in ctns-/- zebrafish upon treatment with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).


( K ) Hatching rates in surviving ctns - / - zebrafish evaluated at 72 and 96 hrs post fertilization ( hpf ) with cysteamine , bicalutamide , and a combination of cysteamine and bicalutamide ( n = 40 embryos per group ) .

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(K) Hatching rates in surviving ctns-/- zebrafish evaluated at 72 and 96 hrs post fertilization (hpf) with cysteamine, bicalutamide, and a combination of cysteamine and bicalutamide (n=40 embryos per group).


A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito - QC . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 5 µm in ( A )

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A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito-QC. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 5 µm in (A)


B N2a cells expressing mito - QC and myc - Parkin together with hTau - V5 , hP301L - V5 or an empty vector control . Treatment with CCCP ( 8 µM , 17 h ) induces mitophagy in control cells , as indicated by a decrease in the GFP / mCherry fluorescence ratio , but not in hTau or hP301L cells . Deconvolution ( Classic Maximum Likelihood Estimation ) was applied to images for display only . C Quantification of GFP / mCherry fluorescence intensity per cell . Data were analysed by 2 - way ANOVA , showing significant main effects of CCCP treatment , F ( 1 , 314 ) = 15 . 66 , p < 0 . 0001 , and tau expression , F ( 2 , 314 ) = 12 . 26 , p < 0 . 0001 , and a significant interaction effect , F ( 2 , 314 ) = 6 . 137 , p = 0 . 0024 , n = 40 - 73 cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 10

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

B N2a cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control. Treatment with CCCP (8 µM, 17 h) induces mitophagy in control cells, as indicated by a decrease in the GFP/mCherry fluorescence ratio, but not in hTau or hP301L cells. Deconvolution (Classic Maximum Likelihood Estimation) was applied to images for display only. C Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing significant main effects of CCCP treatment, F (1, 314) = 15.66, p < 0.0001, and tau expression, F (2, 314) = 12.26, p < 0.0001, and a significant interaction effect, F (2, 314) = 6.137, p = 0.0024, n = 40-73 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm


D Cells expressing mito - QC and myc - Parkin together with hTau - V5 , hP301L - V5 or an empty vector control , treated with a mixture of antimycin and oligomycin ( AO , 15 µM , 17 h ) . E Quantification of GFP / mCherry fluorescence intensity per cell . Data were analysed by 2 - way ANOVA , showing no significant main effects of AO treatment , F ( 1 , 212 ) = 0 . 564 , p = 0 . 4534 , or tau expression F ( 2 , 212 ) = 1 . 579 , p = 0 . 2087 , but a significant interaction effect , F ( 2 , 212 ) = 9 . 854 , p < 0 . 0001 , n = 16 - 50 cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * = p < 0 . 01 , * * * * = p < 0 . 0001 for simple effects . Scale bar = 10

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D Cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control, treated with a mixture of antimycin and oligomycin (AO, 15 µM, 17 h). E Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing no significant main effects of AO treatment, F (1, 212) = 0.564, p = 0.4534, or tau expression F (2, 212) = 1.579, p = 0.2087, but a significant interaction effect, F (2, 212) = 9.854, p < 0.0001, n = 16-50 cells/group. Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 10 µm


A Illustration of the image analysis process . The inverted " Union Jack " look - up table ( LUT ) was used to better visualise GFP - Parkin clusters , which were then manually selected as regions of interest ( ROIs ) and the corresponding mitochondrial fluorescence intensity was measured . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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A Illustration of the image analysis process. The inverted "Union Jack" look-up table (LUT) was used to better visualise GFP-Parkin clusters, which were then manually selected as regions of interest (ROIs) and the corresponding mitochondrial fluorescence intensity was measured. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


B Representative images of N2a cells expressing GFP - Parkin and hTau , hP301L or empty vector ( Control ) for 24 h , that were then treated with 10 μM CCCP for 4 h . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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B Representative images of N2a cells expressing GFP-Parkin and hTau, hP301L or empty vector (Control) for 24 h, that were then treated with 10 μM CCCP for 4 h. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


C Quantification of Parkin clusters / cell at 24 h post - transfection . Data were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 8 . 18 , p = 0 . 0168 , followed by Dunn ' s post - hoc analysis , n = 33 - 38 cells / group . D Quantification of Parkin clusters / cell at 48 h post - transfection . Data were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 19 . 2 , p < 0 . 0001 , followed by Dunn ' s post - hoc analysis , n = 37 - 55 cells / group cells / group . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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C Quantification of Parkin clusters/cell at 24 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 8.18, p = 0.0168, followed by Dunn's post-hoc analysis, n = 33-38 cells/group. D Quantification of Parkin clusters/cell at 48 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 19.2, p < 0.0001, followed by Dunn's post-hoc analysis, n = 37-55 cells/group cells/group. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


E To assess colocalisation of Parkin clusters and mitochondria at 24 h post - transfection , Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated . Control r = 0 . 616 , p = 0 . 0001 ; hTau r = 0 . 217 , p = 0 . 2771 ; hP301L r = 0 . 548 , p = 0 . 0014 ( n = 27 - 34 cells / group ) . F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post - transfection . Control r = 0 . 434 , p = 0 . 0167 ; hTau r = 0 . 128 , p = 0 . 4941 ; hP301L r = - 0 . 299 , p = 0 . 2794 ( n = 15 - 31 cells / group ) . Note the different intensity scales between ( E ) and ( F ) reflect different bit depths of the images . Data information : Data are given as mean and SEM , * = p < 0 . 05 , * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 μm .

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E To assess colocalisation of Parkin clusters and mitochondria at 24 h post-transfection, Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated. Control r = 0.616, p = 0.0001; hTau r = 0.217, p = 0.2771; hP301L r = 0.548, p = 0.0014 (n = 27-34 cells/ group). F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post-transfection. Control r = 0.434, p = 0.0167; hTau r = 0.128, p = 0.4941; hP301L r = -0.299, p = 0.2794 (n = 15-31 cells/ group). Note the different intensity scales between (E) and (F) reflect different bit depths of the images. Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.


A FACS analysis of cells loaded with Rhodamine 123 . The geometric mean fluorescence was calculated and expressed as % of the empty vector control . A CCCP - treated cohort was included as a positive control , but not included in the statistical analysis . Data were analysed by one - way ANOVA , F ( 2 , 14 ) = 0 . 196 , p = 0 . 8245 . B FACS analysis of cells treated with CCCP after 24 h of tau expression . Data were analysed by one - way ANOVA , F ( 2 , 15 ) = 0 . 434 , p = 0 . 6555 . The DMSO negative control is shown but was not analysed statistically . Data information : Data are given as mean and SEM ( n = 5 - 6 samples / experimental group from two independent experiments ) .

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A FACS analysis of cells loaded with Rhodamine 123. The geometric mean fluorescence was calculated and expressed as % of the empty vector control. A CCCP-treated cohort was included as a positive control, but not included in the statistical analysis. Data were analysed by one-way ANOVA, F (2, 14) = 0.196, p = 0.8245. B FACS analysis of cells treated with CCCP after 24 h of tau expression. Data were analysed by one-way ANOVA, F (2, 15) = 0.434, p = 0.6555. The DMSO negative control is shown but was not analysed statistically. Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).


C Representative dot plots of cells loaded with Rhodamine 123 . The gates ( black line ) were set in unstained cells to distinguish Rhodamine - positive cells ( right half ) from Rhodamine - negative cells ( left half ) . Data information : Data are given as mean and SEM ( n = 5 - 6 samples / experimental group from two independent experiments ) .

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C Representative dot plots of cells loaded with Rhodamine 123. The gates (black line) were set in unstained cells to distinguish Rhodamine-positive cells (right half) from Rhodamine-negative cells (left half). Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).


A Immunostaining for microtubules after transient expression of hTau and hP301L . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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A Immunostaining for microtubules after transient expression of hTau and hP301L. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


B Taxol induces microtubule bundling to a similar extent to tau expression . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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B Taxol induces microtubule bundling to a similar extent to tau expression. Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment ( Mann - Whitney test , U = 22448 , p < 0 . 0001 , n = 224 , 273 cells / group for CCCP only and CCCP + Taxol , respectively ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment (Mann-Whitney test, U = 22448, p < 0.0001, n = 224, 273 cells/group for CCCP only and CCCP + Taxol, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


D Immunoblot analysis of acetylated tubulin ( Ace - Tubulin ) following expression of hTau and hP301L for 24 h ( One - way ANOVA , F ( 2 , 9 ) = 0 . 0432 , p = 0 . 9579 ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

D Immunoblot analysis of acetylated tubulin (Ace-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0432, p = 0.9579). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


E Immunoblot analysis of tyrosinated tubulin ( Tyr - Tubulin ) following expression of hTau and hP301L for 24 h ( One - way ANOVA , F ( 2 , 9 ) = 0 . 0402 , p = 0 . 9608 ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

E Immunoblot analysis of tyrosinated tubulin (Tyr-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0402, p = 0.9608). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide ( JPL ) ( Mann - Whitney test , U = 16069 , p < 0 . 0001 , n = 224 , 199 cells / group for CCCP only and CCCP + JPL , respectively ) . Data information : Data are given as mean and SEM , * * * * = p < 0 . 0001 . Scale bar = 10 µm .

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

F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide (JPL) (Mann-Whitney test, U = 16069, p <0.0001, n = 224, 199 cells/group for CCCP only and CCCP + JPL, respectively). Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.


A Co - immunoprecipitations of V5 - tagged tau and GFP - Parkin . The V5 tag was used to pull down tau and the membrane was probed for V5 ( using an antibody raised in a different species ) and GFP . IP = immunoprecipitation , Flow = flow - through lysate following removal of beads . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Co-immunoprecipitations of V5-tagged tau and GFP-Parkin. The V5 tag was used to pull down tau and the membrane was probed for V5 (using an antibody raised in a different species) and GFP. IP = immunoprecipitation, Flow = flow-through lysate following removal of beads. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


B Maximum projection images of proximity ligation assays ( PLAs ) for tau and endogenous Parkin in cells expressing hTau - GFP , hP301L - GFP or GFP . Inset = negative control ( antibodies to tau and histone H3 , which are not known binding partners ) . C Quantification of PLA signals in maximum projections normalised to the GFP signal . Results were analysed with a Kruskal - Wallis test , χ2 ( 2 ) = 27 . 2 , p < 0 . 0001 , followed by Dunn ' s post hoc analysis to test for differences from the GFP control , n = 17 cells in GFP condition , 14 cells in hTau condition and 27 cells in hP301L condition . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Maximum projection images of proximity ligation assays (PLAs) for tau and endogenous Parkin in cells expressing hTau-GFP, hP301L-GFP or GFP. Inset = negative control (antibodies to tau and histone H3, which are not known binding partners). C Quantification of PLA signals in maximum projections normalised to the GFP signal. Results were analysed with a Kruskal-Wallis test, χ2 (2) = 27.2, p < 0.0001, followed by Dunn's post hoc analysis to test for differences from the GFP control, n = 17 cells in GFP condition, 14 cells in hTau condition and 27 cells in hP301L condition. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


D PLA for Parkin and hP301L in an aged pR5 mice and a littermate control . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 PLA for Parkin and hP301L in an aged pR5 mice and a littermate control. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


F , G PLA for Parkin and ∆ Tau , revealing interactions via the projection domain of tau . Data were analysed with a Mann - Whitney test ( U = 185 , p = 0 . 9224 , n = 21 , 18 cells / group for hTau and ∆ Tau , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, G PLA for Parkin and ∆Tau, revealing interactions via the projection domain of tau. Data were analysed with a Mann-Whitney test (U = 185, p = 0.9224, n = 21, 18 cells/group for hTau and ∆Tau, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


H , I PLA for Parkin and the microtubule - binding domain construct ( MTBD ) . Data were analysed with a t - test ( t = 5 . 226 , p < 0 . 0001 , n = 16 , 17 cells / group for hTau and MTBD , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

H, I PLA for Parkin and the microtubule-binding domain construct (MTBD). Data were analysed with a t-test (t = 5.226, p < 0.0001, n = 16, 17 cells/group for hTau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


J , K Cells expressing tau constructs together with GFP - Parkin ( 24 h ) with and without CCCP treatment . Parkin clusters were counted , illustrating that Parkin translocation was impaired by ∆ Tau , but not MTBD . Data were analysed with a Kruskal - Wallis test χ2 ( 2 ) = 10 . 4 , p = 0 . 0056 , followed by Dunn ' s post - hoc , n = 67 , 42 , 53 cells / group for for hTau , ∆ Tau , and MTBD , respectively . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

J, K Cells expressing tau constructs together with GFP-Parkin (24 h) with and without CCCP treatment. Parkin clusters were counted, illustrating that Parkin translocation was impaired by ∆Tau, but not MTBD. Data were analysed with a Kruskal-Wallis test χ2 (2) = 10.4, p = 0.0056, followed by Dunn's post-hoc, n = 67, 42, 53 cells/ group for for hTau, ∆Tau, and MTBD, respectively. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


L The V5 fluorescence intensity in each cell analysed in ( K ) ( Mann - Whitney test , U = 953 , p = 0 . 2332 , n = 42 , 53 cells / group for ∆ Tau and MTBD , respectively ) . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

L The V5 fluorescence intensity in each cell analysed in (K) (Mann-Whitney test, U = 953, p = 0.2332, n = 42, 53 cells/group for ∆Tau and MTBD, respectively). Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


M Cells were transfected with GFP - Parkin and hTau - V5 , hP301L - V5 or an empty vector control , and treated with CCCP . PLAs were performed for tau and Parkin . Notably , at Parkin clusters ( arrow ) , no interactions between tau and Parkin were evident . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

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

M Cells were transfected with GFP-Parkin and hTau-V5, hP301L-V5 or an empty vector control, and treated with CCCP. PLAs were performed for tau and Parkin. Notably, at Parkin clusters (arrow), no interactions between tau and Parkin were evident. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


N 3D reconstructions in IMARIS , illustrating a lack of PLA signals in Parkin clusters . Data information : Scale bars = 10 μm . Data are given as mean and SEM , * * = p < 0 . 01 , * * * * = p < 0 . 0001 .

N 3D reconstructions in IMARIS, illustrating a lack of PLA signals in Parkin clusters. Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.


Expression pattern of the mito - Rosella biosensor in the nervous system .

Expression pattern of the mito-Rosella biosensor in the nervous system.


B Analysis pipeline of mitochondria in the C . elegans ventral nerve cord . Mitochondria were selected by choosing a threshold in the dsRed channel . This threshold was used to create ROIs around single mitochondria and the corresponding GFP fluorescence intensity was then measured .

O O O O O O O O O O O O O O O O O O O O O O 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 Analysis pipeline of mitochondria in the C. elegans ventral nerve cord. Mitochondria were selected by choosing a threshold in the dsRed channel. This threshold was used to create ROIs around single mitochondria and the corresponding GFP fluorescence intensity was then measured.


C , D Analysis of mitophagy in worms transgenic for mito - Rosella but not tau ( WT ) , that were treated with NaN3 ( 8 mM , 1 h ) or CCCP ( 15 µM , 2 h ) . Data were analysed by unpaired t test , NaN3 t = 5 . 56 , p < 0 . 0001 ; CCCP t = 3 . 92 p = 0 . 0002 . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral

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

C, D Analysis of mitophagy in worms transgenic for mito-Rosella but not tau (WT), that were treated with NaN3 (8 mM, 1 h) or CCCP (15 µM, 2 h). Data were analysed by unpaired t test, NaN3 t = 5.56, p < 0.0001; CCCP t = 3.92 p = 0.0002. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


E , F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment ( unpaired t test , t = 1 . 62 , p = 0 . 1126 ) and CCCP treatment ( Mann - Whitney test , U = 113 , p = 0 . 002 ) . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral

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

E, F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment (unpaired t test, t = 1.62, p = 0.1126) and CCCP treatment (Mann-Whitney test, U = 113, p = 0.002). Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


G , H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 ( Mann - Whitney test , U = 126 , p = 0 . 2711 ) or CCCP ( Mann - Whitney test , U = 375 , p = 0 . 3697 ) treatment . Data information : n = 16 - 25 worms / group for NaN3 and 23 - 39 worms / groups for CCCP experiments . Data are given as mean and SEM . * * * = p < 0 . 001 , * * * * = p < 0 . 0001 . Scale bar = 10 µm . Images are high - magnification of a small number of mitochondria in the ventral

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

G, H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 (Mann-Whitney test, U = 126, p = 0.2711) or CCCP (Mann-Whitney test, U = 375, p = 0.3697) treatment. Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm. Images are high-magnification of a small number of mitochondria in the ventral cord


I Tau expression in worm lysates , as detected using the human tau - specific antibody HT7 .

I Tau expression in worm lysates, as detected using the human tau-specific antibody HT7.


B ) Egg chambers flatten along the coverslip , allowing for follicle cell morphologies to be compared in two dimensions . In ( B ) , three live w1118 ( control ) egg chambers expressing the membrane marker Basigin : : YFP are shown in multiple planes of focus , with a z - reconstruction at the bottom . Because the egg chambers flatten at the coverslip , the basal follicle cell morphologies can be revealed in a single focal plane . To account for differences in follicle cell height between the three egg chambers , which increase in maturity from left to right , separate focal planes are shown for the apical morphologies . A diagram illustrating the position of the three focal planes with respect to the flattened egg chamber is shown in ( B " ) . Scale bars =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Egg chambers flatten along the coverslip, allowing for follicle cell morphologies to be compared in two dimensions. In (B), three live w1118 (control) egg chambers expressing the membrane marker Basigin::YFP are shown in multiple planes of focus, with a z-reconstruction at the bottom. Because the egg chambers flatten at the coverslip, the basal follicle cell morphologies can be revealed in a single focal plane. To account for differences in follicle cell height between the three egg chambers, which increase in maturity from left to right, separate focal planes are shown for the apical morphologies. A diagram illustrating the position of the three focal planes with respect to the flattened egg chamber is shown in (B"). Scale bars =


C - E ) Follicle cell cross - sectional area decreases ( C ) and cell shape becomes more regular ( D ) and hexagonal ( E ) as early - stage egg chambers mature . These measurements were derived from at least three egg chambers from at least three flies per AR range . Bars in ( C ) and ( D ) represent mean and standard deviation . Statistical significance for cell side distributions was calculated using a chi - squared test . Number of cells used for morphological analysis : 1 . 0 - 1 . 2 apical n = 23 , basal n = 27 ; 1 . 2 - 1 . 4 apical n = 114 , basal n = 61 ; 1 . 4 - 1 . 6 , apical n = 160 , basal n = 149 . Vertex counts : 1 . 0 - 1 . 2 apical n = 38 , basal n = 33 ; 1 . 2 - 1 . 4 apical n = 73 , basal n = 70 ; 1 . 4 - 1 . 6 , apical n = 284 , basal n = 259 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Follicle cell cross-sectional area decreases (C) and cell shape becomes more regular (D) and hexagonal (E) as early-stage egg chambers mature. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars in (C) and (D) represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for morphological analysis: 1.0-1.2 apical n=23, basal n=27; 1.2-1.4 apical n=114, basal n=61; 1.4-1.6, apical n=160, basal n=149. Vertex counts: 1.0-1.2 apical n =38, basal n=33; 1.2-1.4 apical n=73, basal n=70; 1.4-1.6, apical n=284, basal n=259.


Egg chamber elongation is unaffected by reduced cell proliferation . String - shRNA expressed for 24 hours alters epithelial shape and cell number ( A Chambers shown in ( A ) have approximately equal widths , with the cross - sectional area corresponding to developmental stage 6 . Scale bars = 20 μm . Number of chambers analyzed : w1118 1 . 0 - 1 . 2 n = 24 , 1 . 2 - 1 . 4 n = 14 , 1 . 4 - 1 . 6 n = 18 String - shRNA 1 . 0 - 1 . 2 n = 11 , 1 . 2 - 1 . 4 n = 10 , 1 . 4 - 1 . 6 n = 15 . Bars represent mean and standard

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

Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell number (A Chambers shown in (A) have approximately equal widths, with the cross-sectional area corresponding to developmental stage 6. Scale bars = 20 μm. Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviation


Egg chamber elongation is unaffected by reduced cell proliferation . String - shRNA expressed for 24 hours alters epithelial shape and cell numbe but not the relationship between egg chamber aspect ratio and cross - sectional area , which is quantified in ( B ) Number of chambers analyzed : w1118 1 . 0 - 1 . 2 n = 24 , 1 . 2 - 1 . 4 n = 14 , 1 . 4 - 1 . 6 n = 18 String - shRNA 1 . 0 - 1 . 2 n = 11 , 1 . 2 - 1 . 4 n = 10 , 1 . 4 - 1 . 6 n = 15 . Bars represent mean and standard

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

Egg chamber elongation is unaffected by reduced cell proliferation. String-shRNA expressed for 24 hours alters epithelial shape and cell numbe but not the relationship between egg chamber aspect ratio and cross-sectional area, which is quantified in (B) Number of chambers analyzed: w1118 1.0-1.2 n=24,1.2-1.4 n=14,1.4-1.6 n=18 String-shRNA 1.0-1.2 n= 11 ,1.2-1.4 n=10,1.4-1.6 n=15. Bars represent mean and standard deviation


C , D ) Cell proliferation is necessary for normal tissue morphology Follicle cell shape regularity ( C ) and polygonality ( D ) measurements in control and String - shRNA egg chambers of different aspect ratios . These measurements were derived from at least three egg chambers from at least three flies per AR range . Bars represent mean and standard deviation . Statistical significance for cell side distributions was calculated using a chi - squared test . Number of cells used for analysis : w1118 1 . 0 - 1 . 2 n = 23 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 ; String - shRNA 1 . 0 - 1 . 2 , n = 6 , 1 . 2 - 1 . 4 n = 19 , 1 . 4 - 1 . 6 n =

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

C, D) Cell proliferation is necessary for normal tissue morphology Follicle cell shape regularity (C) and polygonality (D) measurements in control and String-shRNA egg chambers of different aspect ratios. These measurements were derived from at least three egg chambers from at least three flies per AR range. Bars represent mean and standard deviation. Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells used for analysis: w1118 1.0-1.2 n= 23, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=6, 1.2-1.4 n=19, 1.4-1.6 n=17


Epithelial cell size and shape is affected by decreased proliferation . Representative images of epithelial cells taken from egg chambers with aspect ratios of approximately 1 . 4 ( A ) .

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

Epithelial cell size and shape is affected by decreased proliferation. Representative images of epithelial cells taken from egg chambers with aspect ratios of approximately 1.4 (A).


Epithelial cell size and shape is affected by decreased proliferation . Cell outlines are marked with Basigin : : YFP . Scale bars = 5 μm . Cells become significantly larger ( B ) Cell area shown on a log4 scale in ( B ) . Number of cells analyzed : w1118 1 . 0 - 1 . 2 n = 14 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 Statistical significance calculated using a Mann - Whitney test . Data information : Bars represent mean and standard deviation .

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

Epithelial cell size and shape is affected by decreased proliferation. Cell outlines are marked with Basigin::YFP. Scale bars = 5 μm. Cells become significantly larger (B) Cell area shown on a log4 scale in (B). Number of cells analyzed: w1118 1.0-1.2 n= 14, 1.2-1.4 n=114, 1.4-1.6 n=160 Statistical significance calculated using a Mann-Whitney test. Data information: Bars represent mean and standard deviation.


Epithelial cell size and shape is affected by decreased proliferation . Cell outlines are marked with Basigin : : YFP . Scale bars = 5 μm . Cells become more aligned with the tissue elongation axis when proliferation is stopped using String - shRNA ( C ) . Number of cells analyzed : w1118 1 . 0 - 1 . 2 n = 14 , 1 . 2 - 1 . 4 n = 114 , 1 . 4 - 1 . 6 n = 160 ; String - shRNA 1 . 0 - 1 . 2 , n = 10 , 1 . 2 - 1 . 4 n = 19 , 1 . 4 - 1 . 6 n = 17 . Statistical significance calculated using a Mann - Whitney test . Data information : Bars represent mean and standard deviation .

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

Epithelial cell size and shape is affected by decreased proliferation. Cell outlines are marked with Basigin::YFP. Scale bars = 5 μm. Cells become more aligned with the tissue elongation axis when proliferation is stopped using String-shRNA (C). Number of cells analyzed: w1118 1.0-1.2 n= 14, 1.2-1.4 n=114, 1.4-1.6 n=160; String-shRNA 1.0-1.2, n=10, 1.2-1.4 n=19, 1.4-1.6 n=17. Statistical significance calculated using a Mann-Whitney test. Data information: Bars represent mean and standard deviation.


D , E ) The follicular epithelium ' thins out ' over the egg chamber equator when proliferation is blocked . Cartoon demonstrates the tissue cell shape observed in the follicular epithelium in control ( w1118 ) and String - shRNA treated egg chambers . Quantification of the phenomenon shown in ( D ) reveals that cells are shorter over the equator than at the anterior pole in elongated egg chambers when proliferation is blocked . Statistical significance calculated using unpaired t - test with Welch ' s correction . Number of cells analyzed : w1118 1 . 0 - 1 . 2 n = 21 , 1 . 2 - 1 . 4 n = 11 , 1 . 4 - 1 . 6 n = 10 ; String - shRNA 1 . 0 - 1 . 2 , n = 9 , 1 . 2 - 1 . 4 n = 11 , 1 . 4 - 1 . 6 n = 13 . Data information : Bars represent mean and standard deviation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, E) The follicular epithelium 'thins out' over the egg chamber equator when proliferation is blocked. Cartoon demonstrates the tissue cell shape observed in the follicular epithelium in control (w1118) and String-shRNA treated egg chambers. Quantification of the phenomenon shown in (D) reveals that cells are shorter over the equator than at the anterior pole in elongated egg chambers when proliferation is blocked. Statistical significance calculated using unpaired t-test with Welch's correction. Number of cells analyzed: w1118 1.0-1.2 n= 21, 1.2-1.4 n=11, 1.4-1.6 n=10; String-shRNA 1.0-1.2, n=9, 1.2-1.4 n=11, 1.4-1.6 n=13. Data information: Bars represent mean and standard deviation.


F ) Recoil is observed when cuts are made in apical cortex with a 2 - photon laser with egg chambers in saggital view . Images show cortex before and after cutting . Scale bars = 10 μm . Yellow arrows indicate edges of ablated region . G ) Recoil is higher when cuts are made at the apical cortex at the equator of egg chambers , rather than at the poles . Quantification of experiment shown in ( F ) . Statistical significance tested by Mann - Whitney test . Number of laser cuts : equator n = 30 , pole n = 20 . Data information Laser ablation experiments performed on tissue from sqhAX3 , sqh : : GFP flies .

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F) Recoil is observed when cuts are made in apical cortex with a 2-photon laser with egg chambers in saggital view. Images show cortex before and after cutting. Scale bars = 10 μm. Yellow arrows indicate edges of ablated region. G) Recoil is higher when cuts are made at the apical cortex at the equator of egg chambers, rather than at the poles. Quantification of experiment shown in (F). Statistical significance tested by Mann-Whitney test. Number of laser cuts: equator n=30, pole n=20. Data information Laser ablation experiments performed on tissue from sqhAX3, sqh::GFP flies.


H ) Recoil is observed when larger cuts are made in apical cortex with a UV laser . Images show cortex before and after cutting . Red lines indicate the lines over which recoil was calculated , parallel to the axes of the egg chamber . Scale bars = 5 μm . Yellow arrows indicate edges of ablated region . I ) Recoil tends to be higher towards the AP ( elongation ) axis compared to the the UD ( rotation ) axis . Graph shows the ratio of recoil speed plotted on a log2 scale in the AP versus the UD axis . Red line at x = 1 indicates equal recoil in both axes . Values above 1 indicate higher recoil speeds in the AP axis lower than 1 indicate higher recoil in the UD axis . AP / UD Ratio Median = 1 . 242 ; Mean = 2 . 1 . Wilcoxon Signed Rank Test to test for significance of the recoil ratio AP / UD shows that the median is significantly different from 1 to p = 0 . 0172 . Number of cuts = 20 . Bar represents median . Laser ablation experiments performed on tissue from sqhAX3 , sqh : : GFP flies .

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

H) Recoil is observed when larger cuts are made in apical cortex with a UV laser. Images show cortex before and after cutting. Red lines indicate the lines over which recoil was calculated, parallel to the axes of the egg chamber. Scale bars = 5 μm. Yellow arrows indicate edges of ablated region. I) Recoil tends to be higher towards the AP (elongation) axis compared to the the UD (rotation) axis. Graph shows the ratio of recoil speed plotted on a log2 scale in the AP versus the UD axis. Red line at x=1 indicates equal recoil in both axes. Values above 1 indicate higher recoil speeds in the AP axis lower than 1 indicate higher recoil in the UD axis. AP/UD Ratio Median = 1.242 ; Mean = 2.1. Wilcoxon Signed Rank Test to test for significance of the recoil ratio AP/UD shows that the median is significantly different from 1 to p = 0.0172. Number of cuts = 20. Bar represents median.Laser ablation experiments performed on tissue from sqhAX3, sqh::GFP flies.


B ) i ) Planar division orientation is initially random in the follicular epithelium , with a significant bias towards the egg chamber elongation axis evident at stage 6 ( asterisk , p = 0 . 0031 , Wilcoxon Signed Rank Test , Theoretical Mean of 45 ; p = 0 . 0218 when comparison to a randomly generated list of the same size ) . Number of cells analyzed : stage 2 / 3 n = 16 , stage 4 n = 27 stage 5 n = 30 , stage 6 n = 40 . ii ) Quantification of the angle of tissue expansion as calculated by vector addition of the minor and major axis of egg chambers . Number of egg chambers analyzed : stage 2 / 3 n = 10 , stage 4 n = 15 , stage 5 n = 15 , stage 6 n = 10 . iii ) Quantification of cell elongation angles ( as shown in E ) with respect to the chamber elongation axis by stage from live egg chambers . Number of cells analyzed : stage 2 / 3 n = 18 , stage 4 n = 49 stage 5 n = 64 , stage 6 n = 97 . Bars represent mean and standard

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) i) Planar division orientation is initially random in the follicular epithelium, with a significant bias towards the egg chamber elongation axis evident at stage 6 (asterisk, p = 0.0031, Wilcoxon Signed Rank Test, Theoretical Mean of 45; p = 0.0218 when comparison to a randomly generated list of the same size). Number of cells analyzed: stage 2/3 n=16, stage 4 n=27 stage 5 n=30, stage 6 n=40. ii) Quantification of the angle of tissue expansion as calculated by vector addition of the minor and major axis of egg chambers. Number of egg chambers analyzed: stage 2/3 n=10, stage 4 n=15, stage 5 n=15, stage 6 n=10. iii) Quantification of cell elongation angles (as shown in E) with respect to the chamber elongation axis by stage from live egg chambers. Number of cells analyzed: stage 2/3 n=18, stage 4 n=49 stage 5 n=64, stage 6 n=97. Bars represent mean and standard deviation


C ) Planar division orientation at Stage 6 correlates with tissue expansion in Fat2 - shRNA and Pak - shRNA egg chambers . Mann Whitney test w1118 versus Pak shRNA p = 0 . 0094 . Neither the Fat2 - shRNA nor Pak - shRNA distributions are stastically different from random ( Wilcoxon Signed Rank Test , Theoretical Mean of 45 ) . Number of cells analyzed : w1118 n = 97 , Fat2 - shRNA n = 44 , Pak - shRNA n = 39 . Egg chambers analyzed : w1118 n = 40 , Fat2 - shRNA n = 21 , Pak - shRNA n =

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Planar division orientation at Stage 6 correlates with tissue expansion in Fat2-shRNA and Pak-shRNA egg chambers. Mann Whitney test w1118 versus Pak shRNA p = 0.0094. Neither the Fat2-shRNA nor Pak-shRNA distributions are stastically different from random (Wilcoxon Signed Rank Test, Theoretical Mean of 45). Number of cells analyzed: w1118 n =97, Fat2-shRNA n=44, Pak-shRNA n=39. Egg chambers analyzed: w1118 n=40, Fat2-shRNA n=21, Pak-shRNA n=21


D ) Neighboring cells can form new attachments underneath ( basal to ) a mitotic cell that has moved apically . Scale bars = 5 μm . Thin yellow lines in the XZ reconstructions indicate the z - position of the two XY focal

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D) Neighboring cells can form new attachments underneath (basal to) a mitotic cell that has moved apically. Scale bars = 5 μm. Thin yellow lines in the XZ reconstructions indicate the z-position of the two XY focal planes


E ) Workflow for determining follicle shape shapes . Segmentation of an egg chamber with an aspect ratio of 1 . 5 reveals that follicle cell elongation is usually perpendicular to the elongation axis of the egg chamber . Cell outlines were marked with Basigin : : YFP . Scale bars = 5

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E) Workflow for determining follicle shape shapes. Segmentation of an egg chamber with an aspect ratio of 1.5 reveals that follicle cell elongation is usually perpendicular to the elongation axis of the egg chamber. Cell outlines were marked with Basigin::YFP. Scale bars = 5 μm


F ) Live confocal imaging shows that the long axis of a follicle cell at interphase does not predict the orientation of division . Long axis orientations at the timepoints shown were determined as in ( E ) . Scale bars = 5 μm .

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F) Live confocal imaging shows that the long axis of a follicle cell at interphase does not predict the orientation of division. Long axis orientations at the timepoints shown were determined as in (E). Scale bars = 5 μm.


G ) The interphase long axis does not correlate with the division angle in both the follicular epithelium ( FE ) and embyronic neuroectoderm ( ENE ) . n = 10 cells ( FE ) , 18 cells ( ENE )

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G) The interphase long axis does not correlate with the division angle in both the follicular epithelium (FE) and embyronic neuroectoderm (ENE). n=10 cells (FE), 18 cells (ENE)


B , C ) Pins and Mud are symmetric around the entire cell cortex in ( B ) mitotic follicle cells and ( C ) mitotic cells in the ENE . ( B " and C " ) Confocal imaging of immunostained fixed tissue . Scale

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B, C) Pins and Mud are symmetric around the entire cell cortex in (B) mitotic follicle cells and (C) mitotic cells in the ENE. (B" and C") Confocal imaging of immunostained fixed tissue. Scale b


E ) Live imaging reveals a mitotic spindle rotating in the plane of the FE until anaphase at timepoint 0 . Timepoints shown in minutes relative anaphase . Scale bars = 5 μm .

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E) Live imaging reveals a mitotic spindle rotating in the plane of the FE until anaphase at timepoint 0. Timepoints shown in minutes relative anaphase. Scale bars = 5 μm.


Live confocal imaging of Myosin - II shown by Zipper : : YFP ( A at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue - level organization during stage

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Live confocal imaging of Myosin-II shown by Zipper::YFP (A at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6


Live confocal imaging of Myosin - II shown b Spaghetti squash - mCherry ( B ) at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue - level organization during stage 6 . ( B ) The localization Sqh - mCherry is shown relative to adherens junction marked by Shg : :

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Live confocal imaging of Myosin-II shown b Spaghetti squash-mCherry (B) at the apical cortex of follicle cells during egg chamber elongation showing a transition from cell to tissue-level organization during stage 6. (B) The localization Sqh-mCherry is shown relative to adherens junction marked by Shg::GFP


A ) Apical Myo - II localization as marked by Sqh - GFP is disrupted in round ( stage 2 / 3 ) and elongated ( stage 6 ) egg chambers . Red arrows point to the seperation of Myo - II at cell junctions , indicating that actomyosin is no longer correctly linked to adherens junctions . Live confocal imaging . Scale bars = 5 μm .

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A) Apical Myo-II localization as marked by Sqh-GFP is disrupted in round (stage 2/3) and elongated (stage 6) egg chambers. Red arrows point to the seperation of Myo-II at cell junctions, indicating that actomyosin is no longer correctly linked to adherens junctions. Live confocal imaging. Scale bars = 5 μm.


Tissue topology is affected by disruption of Canoe , but not Mud ( D ) Statistical significance for cell side distributions was calculated using a chi - squared test . Number of cells analyzed : w1118 1 . 4 - 1 . 6 n = 284 ; Canoe - shRNA 1 . 4 - 1 . 6 n = 310 ; mud3 / mud4 1 . 4 - 1 . 6 n =

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

Tissue topology is affected by disruption of Canoe, but not Mud (D) Statistical significance for cell side distributions was calculated using a chi-squared test. Number of cells analyzed: w1118 1.4-1.6 n=284; Canoe-shRNA 1.4-1.6 n=310; mud3/mud4 1.4-1.6 n=251


Tissue topology is affected by disruption of Canoe , but not Mud Representative image of a Canoe - shRNA follicle epithelium . Cell outlines are marked with Basigin : : YFP . This egg chamber has no mitotic cells and AR of 1 . 58 . Scale bars = 5

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Tissue topology is affected by disruption of Canoe, but not Mud Representative image of a Canoe-shRNA follicle epithelium. Cell outlines are marked with Basigin::YFP. This egg chamber has no mitotic cells and AR of 1.58. Scale bars = 5 μm


B Representative confocal microscopy of human PBMCs stained with DAPI ( white arrows ) co - cultured with previously labeled UC - MSC with MitoTracker - Green ( top image ) and specific antibody for human CD90 ( bottom image ) .

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B Representative confocal microscopy of human PBMCs stained with DAPI (white arrows) co-cultured with previously labeled UC-MSC with MitoTracker-Green (top image) and specific antibody for human CD90 (bottom image).


C Percentage of mitochondrial transfer ( MitoT ) on total human CD45 + cells after 24 hrs co - culture with MitoTracker - Green labeled bone marrow ( BM ) , menstrual ( Mens ) or umbilical cord ( UC ) - MSCs ( MSC : PBMC ratio 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of BM , Mens and UC - MSCs ) .

C Percentage of mitochondrial transfer (MitoT) on total human CD45+ cells after 24 hrs co-culture with MitoTracker-Green labeled bone marrow (BM), menstrual (Mens) or umbilical cord (UC)-MSCs (MSC:PBMC ratio 1:25) (n=3 biological replicates, with 3 different donors of BM, Mens and UC-MSCs).


D FACS analysis of MitoT on T CD3 + ( n = 4 ) , B CD19 + ( n = 4 ) and natural killer ( NK ) CD56 + ( n = 3 ) cells after 24 hrs co - culture with MitoGreen labeled UC - MSCs ( ratio 1 : 25 ) ( Biological replicates , with different donors of UC - MSCs ) .

D FACS analysis of MitoT on T CD3+ (n=4), B CD19+ (n=4) and natural killer (NK) CD56+ (n=3) cells after 24 hrs co-culture with MitoGreen labeled UC-MSCs (ratio 1:25) (Biological replicates, with different donors of UC-MSCs).


E FACS analysis of MitoT on CD3 + CD4 + or CD3 + CD8 + T cell populations after 24 hrs co - culture with labeled UC - MSCs ( ratio 1 : 25 ) ( n = 3 biological replicates , with 3 different donors of UC - MSCs ) .

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E FACS analysis of MitoT on CD3+CD4+ or CD3+CD8+ T cell populations after 24 hrs co-culture with labeled UC-MSCs (ratio 1:25) (n=3 biological replicates, with 3 different donors of UC-MSCs).


F Representative FACS plots of MitoT on CD45 + CD3 + T cells at increasing amounts of UC - MSC .

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F Representative FACS plots of MitoT on CD45+CD3+ T cells at increasing amounts of UC-MSC.


G Percentage of MitoT on CD45 + CD3 + human cells after 24 hrs co - culture with increasing ratios of UC - MSC : PBMCs ( n = 5 biological replicates , with 5 different UC - MSCs and 5 different PBMCs ) .

G Percentage of MitoT on CD45+CD3+ human cells after 24 hrs co-culture with increasing ratios of UC-MSC:PBMCs (n=5 biological replicates, with 5 different UC-MSCs and 5 different PBMCs).


H FACS analysis of MitoT on CD45 + CD3 + T cells after short times of co - culture with UC - MSCs at two different temperatures , at a ratio 1 : 25 ( n = 3 biological replicates , with 3 different UC - MSCs and 3 different PBMCs ) .

H FACS analysis of MitoT on CD45+CD3+ T cells after short times of co-culture with UC-MSCs at two different temperatures, at a ratio 1:25 (n=3 biological replicates, with 3 different UC-MSCs and 3 different PBMCs).


I Mean fluorescence intensity ( MFI ) of MitoTracker - Green + UC - MSCs at baseline or after 72 hours PBMC co - culture . Representative FACS plot ( left panel ) and average of MFI ( n = 4 biological replicates , with 4 different UC - MSCs ) ( right panel ) .

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I Mean fluorescence intensity (MFI) of MitoTracker-Green+ UC-MSCs at baseline or after 72 hours PBMC co-culture. Representative FACS plot (left panel) and average of MFI (n=4 biological replicates, with 4 different UC-MSCs) (right panel).


J Confocal microscopy of human PBMCs after co - culture with MitoTracker - Green stained UC - MSCs . Images showed MSC - mitochondria ( green ) inside the CD3 + T cells ( red ) .

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J Confocal microscopy of human PBMCs after co-culture with MitoTracker-Green stained UC-MSCs. Images showed MSC-mitochondria (green) inside the CD3+ T cells (red).


K qPCR analysis of human β2 - microglobulin ( B2M ) , human specific mitochondrial ( Mito ) and mouse specific ( β - actin ) gene expression levels in UC - MSCs , mouse derived spleen cells or FACS sorted CD45 + mouse cells after 24 hrs co - culture with UC - MSCs ( n = 3 biological replicates ) . n / d = not detected .

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K qPCR analysis of human β2-microglobulin (B2M), human specific mitochondrial (Mito) and mouse specific (β-actin) gene expression levels in UC-MSCs, mouse derived spleen cells or FACS sorted CD45+ mouse cells after 24 hrs co-culture with UC-MSCs (n=3 biological replicates). n/d = not detected.


M FACS analysis of in vivo MitoT from UC - MSCs to Balb / c mouse mesenteric lymph node and spleen cells , 24 hours after intraperitoneal transplantation of 5 * 106 MitoTracker - labeled MSCs or without cells ( control ) ( n = 1 ) .

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M FACS analysis of in vivo MitoT from UC-MSCs to Balb/c mouse mesenteric lymph node and spleen cells, 24 hours after intraperitoneal transplantation of 5*106 MitoTracker-labeled MSCs or without cells (control) (n=1).


B Confocal microscopy images of human CD3 + T cells with endogenous MT - labeled with MitoTracker Red CMXRos ( white arrows , left panel ) or after mitoception with MitoTracker Green labeled - MT from UC - MSCs ( white asterisk , right panel ) .

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B Confocal microscopy images of human CD3+ T cells with endogenous MT-labeled with MitoTracker Red CMXRos (white arrows, left panel) or after mitoception with MitoTracker Green labeled-MT from UC-MSCs (white asterisk, right panel).


C FACS analysis of MitoT on CD3 + human T cells after mitoception with increasing amounts of UC - MSC MT corresponding to the same cell number ratios used in co - culture experiments ( n = 4 biological replicates , with 4 different donors of UC - MSCs ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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C FACS analysis of MitoT on CD3+ human T cells after mitoception with increasing amounts of UC-MSC MT corresponding to the same cell number ratios used in co-culture experiments (n=4 biological replicates, with 4 different donors of UC-MSCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.


D Representative transmission electron microscopy images of FACS - sorted human CD3 + MitoTneg and CD3 + MitoTpos cells following mitoception with UC - MSC MT . Red arrows show human mitochondria .

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D Representative transmission electron microscopy images of FACS-sorted human CD3+MitoTneg and CD3+MitoTpos cells following mitoception with UC-MSC MT. Red arrows show human mitochondria.


Quantification of human MT in sorted CD3 + MitoTneg and MitoTpos cells after mitoception with UC - MSCs , by transmission electron microscopy ( n = 25 independent cells analyzed per group ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

Quantification of human MT in sorted CD3+ MitoTneg and MitoTpos cells after mitoception with UC-MSCs, by transmission electron microscopy (n=25 independent cells analyzed per group). Graph shows mean ± SEM and statistical analysis by Student's t-test.


B Volcano plot analysis derived from FPKM values showing DE genes ( p < 0 . 05 , Student ' s t test ) in sorted MitoTneg and MitoTpos CD3 + cells ( from 4 different donors of PBMCs ) .

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B Volcano plot analysis derived from FPKM values showing DE genes (p<0.05, Student's t test) in sorted MitoTneg and MitoTpos CD3+ cells (from 4 different donors of PBMCs).


C Heatmap depiction of RNA - seq analysis of significantly ( p < 0 . 01 , Student ' s t test ) DE coding genes from sorted MitoTpos and MitoTneg CD3 + cells ( from 4 different donors of PBMCs ) .

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C Heatmap depiction of RNA-seq analysis of significantly (p<0.01, Student's t test) DE coding genes from sorted MitoTpos and MitoTneg CD3+ cells (from 4 different donors of PBMCs).


E Differentially expressed genes from the significantly enriched pathways listed in figure 2D . Highlighted in red are key markers of Treg phenotype .

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E Differentially expressed genes from the significantly enriched pathways listed in figure 2D. Highlighted in red are key markers of Treg phenotype.


F qRT - PCR analysis of genes related to T cell activation after mitoception with UC - MSC MT ( n = 4 biological replicates , with 4 different donors of PBMCs ) . The graph depicts fold expression in CD3 + MitoTpos cells relative to CD3 + MitoTneg cells , which are set at 1 . Graph shows mean ± SEM and statistical analysis by Student ' s t - test ( * p < 0 . 05 ) .

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F qRT-PCR analysis of genes related to T cell activation after mitoception with UC-MSC MT (n=4 biological replicates, with 4 different donors of PBMCs). The graph depicts fold expression in CD3+MitoTpos cells relative to CD3+MitoTneg cells, which are set at 1. Graph shows mean ± SEM and statistical analysis by Student's t-test (*p<0.05).


A Representative bright - field microscopy images of FACS - sorted CD4 + naïve MitoTneg ( upper panel ) and MitoTpos ( lower panel ) cells after 5 days in culture with plain media ( no cytokines added ) . Red arrows are indicative of activation / proliferation clusters .

A Representative bright-field microscopy images of FACS-sorted CD4+ naïve MitoTneg (upper panel) and MitoTpos (lower panel) cells after 5 days in culture with plain media (no cytokines added). Red arrows are indicative of activation/proliferation clusters.


B Representative FACS plots of Treg ( CD127lowCD25 + FoxP3 + ) populations after T cell differentiation on media with or without cytokines and antibody supplements ( anti - CD3 , IL - 2 and TGF - b1 ) .

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B Representative FACS plots of Treg (CD127lowCD25+FoxP3+) populations after T cell differentiation on media with or without cytokines and antibody supplements (anti-CD3, IL-2 and TGF-b1).


C FACS analysis of Treg induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells in plain ( n = 5 ) or differentiation media ( n = 6 ) ( Biological replicates , with different donors of PBMCs ) .

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C FACS analysis of Treg induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain (n=5) or differentiation media (n=6) (Biological replicates, with different donors of PBMCs).


D ICOS expression on FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) and bar graph ( right panel ) of ICOS high population ( n = 4 biological replicates ) .

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D ICOS expression on FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of ICOS high population (n=4 biological replicates).


E CCR7 expression on sorted CD4 + naïve MitoTneg and MitoTpos cells after 5 - 7 days in culture with differentiation media . Representative FACS plot ( left panel ) and bar graph ( right panel ) of CCR7 + population ( n = 4 biological replicates ) .

E CCR7 expression on sorted CD4+ naïve MitoTneg and MitoTpos cells after 5-7 days in culture with differentiation media. Representative FACS plot (left panel) and bar graph (right panel) of CCR7+ population (n=4 biological replicates).


F FACS analysis of Th1 ( CD4 + IFNγ + ) induction after 5 - 7 days differentiation of FACS - sorted CD4 + naïve MitoTneg and MitoTpos cells in plain media ( n = 3 biological replicates ) . n / s = not significant .

F FACS analysis of Th1 (CD4+IFNγ+) induction after 5-7 days differentiation of FACS-sorted CD4+ naïve MitoTneg and MitoTpos cells in plain media (n=3 biological replicates). n/s= not significant.


Representative proliferation FACS histogram of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos cells ( ratio 1 : 1 ) after 5 - 7 days of CD4 + naïve cell activation with Treg induction / differentiation media ( anti - CD3 , IL - 2 and TGF - b1 ) . PBMC alone represents positive control of proliferation and PBMC co - culture with MitoTneg correspond to the inhibition control with induced Treg cells .

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Representative proliferation FACS histogram of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells (ratio 1:1) after 5-7 days of CD4+ naïve cell activation with Treg induction/differentiation media (anti-CD3, IL-2 and TGF-b1). PBMC alone represents positive control of proliferation and PBMC co-culture with MitoTneg correspond to the inhibition control with induced Treg cells.


C Percentage of immunosuppression of CTV - stained PBMC co - cultured with MitoTneg or MitoTpos cells after 5 - 7 days of CD4 + naïve cell activation with differentiation media ( ratio MitoT cells : PBMC ) ( n = 3 biological replicates , with different donors of PBMCs ) . Graph shows mean ± SEM and statistical analysis by Student ' s t - test .

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C Percentage of immunosuppression of CTV-stained PBMC co-cultured with MitoTneg or MitoTpos cells after 5-7 days of CD4+ naïve cell activation with differentiation media (ratio MitoT cells:PBMC) (n=3 biological replicates, with different donors of PBMCs). Graph shows mean ± SEM and statistical analysis by Student's t-test.


B Representative FACS plots of mitoception ( % of MitoTracker + ) in CD4 + or CD8 + T cells from mitocepted PBMC compared to control PBMC , before intravenous injection .

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B Representative FACS plots of mitoception (% of MitoTracker+) in CD4+ or CD8+T cells from mitocepted PBMC compared to control PBMC, before intravenous injection.


C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) . Control group in grey , were untreated mice ( n = 6 mice ) . * p < 0 . 05 by Mann Whitney t - test .

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C Percent of mouse weight after GVHD induction with IV injection of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group). Control group in grey, were untreated mice (n=6 mice). * p<0.05 by Mann Whitney t-test.


D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount ( 12x106 cells ) of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 mice per group ) ( p = 0 . 0218 by log - rank test ) .

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D Kaplan Meier plot showing percent survival of GVHD mice after transplantation of an equal amount (12x106 cells) of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 mice per group) (p=0.0218 by log-rank test).


E FACS analysis of human Treg ( CD4 + CD25 + FoxP3 + ) cell engraftment in mouse spleen following transplantation of PBMC - MitoT or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

E FACS analysis of human Treg (CD4+CD25+FoxP3+) cell engraftment in mouse spleen following transplantation of PBMC-MitoT or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


F FACS analysis of human pro - inflammatory T helper1 ( CD4 + IFNγ + , left panel ) and T cytotoxic ( CD8 + IFNγ + , right panel ) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

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F FACS analysis of human pro-inflammatory T helper1 (CD4+IFNγ+, left panel) and T cytotoxic (CD8+IFNγ+, right panel) cell engraftment in mouse spleen following transplantation of mitocepted or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC - MitoT , PBMC and non - transplanted control samples , stained with Masson ' s trichrome staining . Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts . Arrowheads represents a reduced number of Paneth cells .

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G Representative histopathologic images of GVHD in liver and small intestine sections obtained from PBMC-MitoT, PBMC and non-transplanted control samples, stained with Masson's trichrome staining. Asterisk showed increased periportal collagen deposits in the liver and increased piknotic cells in the small intestine crypts. Arrowheads represents a reduced number of Paneth cells.


H - I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) .

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H-I Semiquantitative scoring system for pathologic damage in the liver and small intestine of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).


J Quantification of Paneth cells per crypt , from at least 50 crypts per PBMC - MitoT ( n = 8 mice ) , PBMC transplanted ( n = 5 mice ) or non - transplanted control ( n = 3 ) mouse .

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J Quantification of Paneth cells per crypt, from at least 50 crypts per PBMC-MitoT (n=8 mice), PBMC transplanted (n=5 mice) or non-transplanted control (n=3) mouse.


Representative histopathologic images of GVHD in lung sections obtained from PBMC - MitoT , PBMC and non - transplanted control , stained with Masson ' s trichrome staining .

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Representative histopathologic images of GVHD in lung sections obtained from PBMC-MitoT, PBMC and non-transplanted control, stained with Masson's trichrome staining.


L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC ( n = 8 independent mice ) or control PBMC transplanted mice ( n = 5 independent mice ) .

L Semiquantitative scoring system for pathologic damage in lung of mitocepted PBMC (n=8 independent mice) or control PBMC transplanted mice (n=5 independent mice).


M FACS analysis of total human CD4 + cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells ( n = 3 , with a total of 25 and 27 mice per group , respectively ) .

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M FACS analysis of total human CD4+ cell engraftment in mouse lungs following transplantation of mitocepted PBMC or control PBMC cells (n=3, with a total of 25 and 27 mice per group, respectively).


( C ) Top panel : Representative images show PS labeling in the WT dLGN at P6 and P13 . RGC inputs were labeled with CTB488 . Bottom panel : Enlarged images of the boxed region in top panels . Circles indicate PS + RGC inputs . Arrows point to the enlarged PS + RGC inputs in the insets . ( D ) Quantification of the percentage of PS + RGC inputs in total RGC inputs at P6 and P13 .

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(C) Top panel: Representative images show PS labeling in the WT dLGN at P6 and P13. RGC inputs were labeled with CTB488. Bottom panel: Enlarged images of the boxed region in top panels. Circles indicate PS+ RGC inputs. Arrows point to the enlarged PS+ RGC inputs in the insets. (D) Quantification of the percentage of PS+ RGC inputs in total RGC inputs at P6 and P13.


( E ) Co - labeling of vGlut2 and PSVue in the dLGN at P6 . Circles indicate colocalized vGlut2 and PSVue .

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(E) Co-labeling of vGlut2 and PSVue in the dLGN at P6. Circles indicate colocalized vGlut2 and PSVue.


( F ) Co - labeling of Homer1 and PSVue in the dLGN at P6 . Circles indicate colocalized Homer1 and PSVue .

(F) Co-labeling of Homer1 and PSVue in the dLGN at P6. Circles indicate colocalized Homer1 and PSVue.


( G ) Orthogonal sections showed a triple positive PS + / CTB488 + / CD68 + RGC inputs inside of microglia .

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(G) Orthogonal sections showed a triple positive PS+/CTB488+/CD68+ RGC inputs inside of microglia.


( H ) Orthogonal sections showed a triple positive PS + / vGlut2 + / CD68 + synapse inside of microglia .

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(H) Orthogonal sections showed a triple positive PS+/vGlut2+/CD68+ synapse inside of microglia.


( I ) A representative image of microglia ( upper - left ) is surface rendered ( bottom - left ) . The white arrow points to cells which might be apoptotic cells labeled by PSVue . RGC inputs and PSVue outside or inside of microglia are shown in the two right panels . Scare bar , 20 µm . ( J ) Quantification of the percentage of PS + and PS - RGC inputs outside of microglia in total inputs . N = 4 . ( K ) Quantification of the percentage of engulfed PS + and PS - RGC inputs in total engulfed inputs . N = 4 .

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(I) A representative image of microglia (upper-left) is surface rendered (bottom-left). The white arrow points to cells which might be apoptotic cells labeled by PSVue. RGC inputs and PSVue outside or inside of microglia are shown in the two right panels. Scare bar, 20 µm.(J) Quantification of the percentage of PS+ and PS- RGC inputs outside of microglia in total inputs. N = 4. (K) Quantification of the percentage of engulfed PS+ and PS- RGC inputs in total engulfed inputs. N=4.


( D ) The binding experiment using flow cytometry show that only the GAIN domain binds to PS , similar as Annexin V binding .

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(D) The binding experiment using flow cytometry show that only the GAIN domain binds to PS, similar as Annexin V binding.


( E ) In the competition experiment , AF647 channel shows that GAIN domain binds 60 . 1 % of the PS + Ba / F3 cells ( left ) . Correspondingly , FITC channel reveals 39 . 7 % Annexin V binding to PS + Ba / F3 cells ( right ) .

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(E) In the competition experiment, AF647 channel shows that GAIN domain binds 60.1% of the PS+ Ba/F3 cells (left). Correspondingly, FITC channel reveals 39.7% Annexin V binding to PS+ Ba/F3 cells (right).


( G ) Direct binding of hFc - tagged GPR56 GAIN proteins to PS and other phospholipids . HFc was used as a negative control , and GAS6 as a positive control .

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(G) Direct binding of hFc-tagged GPR56 GAIN proteins to PS and other phospholipids. HFc was used as a negative control, and GAS6 as a positive control.


( B ) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice .

(B) Gpr56 transcript levels in isolated microglia from Gpr56 S4 mice.


( D ) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16 . 5 neocortex . Arrows indicate cortical ectopias that are shown in insets . ( E ) Quantification of ectopia size per section . ( F ) The distribution of ectopia from rostral to caudal cortex .

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(D) Representative images of Nissl staining of Gpr56 null and Gpr56 S4 E16.5 neocortex. Arrows indicate cortical ectopias that are shown in insets. (E) Quantification of ectopia size per section. (F) The distribution of ectopia from rostral to caudal cortex.


( G ) . Myelin basic protein ( MBP ) staining of corpus callosum in P28 controls , Gpr56 null and Gpr56 S4 mice .

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(G). Myelin basic protein (MBP) staining of corpus callosum in P28 controls, Gpr56 null and Gpr56 S4 mice.


( H ) Quantification of MBP intensity in corpus callosum .

(H) Quantification of MBP intensity in corpus callosum.


( I ) Gpr56 transcript levels in WT microglia at P5 and P14 .

(I) Gpr56 transcript levels in WT microglia at P5 and P14.


( J ) An overview of vGlut2 and Homer1 staining in dLGN at P10 . The yellow outline indicates the dLGN core , and the dotted boxes show where synapses are quantified .

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(J) An overview of vGlut2 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.


K ) Representative images of vGlut2 / Homer1 staining in the dLGN of control , Gpr56 null , and Gpr56 S4 brains at P10 . Arrows pointing to the enlarged synapse in the insets . L ) Relative vGlut2 / Homer1 synapse density in dLGN . N = 10 ( Ctrl ) , N = 6 ( Gpr56 null ) , N = 3 ( Gpr56 S4 ) .

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K) Representative images of vGlut2/Homer1 staining in the dLGN of control, Gpr56 null, and Gpr56 S4 brains at P10. Arrows pointing to the enlarged synapse in the insets. L) Relative vGlut2/Homer1 synapse density in dLGN. N=10 (Ctrl), N=6 (Gpr56 null), N=3 (Gpr56 S4).


( N ) Representative images and surface rendered microglia ( green ) in which CTB + ( red ) RGC inputs were engulfed . ( O ) Quantification of the percentage of engulfed RGC inputs in controls , Gpr56 null and Gpr56 S4 microglia .

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(N) Representative images and surface rendered microglia (green) in which CTB+(red) RGC inputs were engulfed. (O) Quantification of the percentage of engulfed RGC inputs in controls, Gpr56 null and Gpr56 S4 microglia.


( A ) Gpr56 is deleted in CKO microglia . RNAscope shows Gpr56 transcripts co - localize with microglia ( arrow ) in controls , but not in CKO , and is absent in all cell types in the global Gpr56 KO ( Gpr56 null ) . Arrowhead indicates non - microglia cells expressing Gpr56 . RNAscope was performed in the prefrontal cortex of P30 mice . Scale bar , 20 µm . ( B ) Quantification of Gpr56 fluorescence signals in ( A ) . N = 20 - 50 cells for each .

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(A) Gpr56 is deleted in CKO microglia. RNAscope shows Gpr56 transcripts co-localize with microglia (arrow) in controls, but not in CKO, and is absent in all cell types in the global Gpr56 KO (Gpr56 null). Arrowhead indicates non-microglia cells expressing Gpr56. RNAscope was performed in the prefrontal cortex of P30 mice. Scale bar, 20 µm. (B) Quantification of Gpr56 fluorescence signals in (A). N = 20-50 cells for each.


( C ) Confocal images of vGlut2 ( green ) labeling retinal ganglion cell ( RGC ) presynaptic terminals and Homer1 ( red ) for post - synapses in the dLGN of CKO and controls at P10 . Overlapped vGlut2 and Homer1 are quantified as synapses , indicated by white circles . Arrows pointing to the synapse enlarged in the upper right - hand inset .

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(C) Confocal images of vGlut2 (green) labeling retinal ganglion cell (RGC) presynaptic terminals and Homer1 (red) for post-synapses in the dLGN of CKO and controls at P10. Overlapped vGlut2 and Homer1 are quantified as synapses, indicated by white circles. Arrows pointing to the synapse enlarged in the upper right-hand inset.


( D ) A time course of dLGN synapse density ( vGlut2 + / Homer1 + ) between WT and controls .

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(D) A time course of dLGN synapse density (vGlut2+/Homer1+) between WT and controls.


( E ) Representative super - resolution images and 3D - reconstructed images of vGlut2 and Homer1 staining of P8 dLGN . Green objects represent surface rendering of vGlut2 + presynaptic terminals . Red spots are rendered Homer1 + post - synapses . Magenta spots represent Homer1 + post - synapses within a distance of 300nm from the nearest vGlut2 + surface .

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(E) Representative super-resolution images and 3D-reconstructed images of vGlut2 and Homer1 staining of P8 dLGN. Green objects represent surface rendering of vGlut2+ presynaptic terminals. Red spots are rendered Homer1+ post-synapses. Magenta spots represent Homer1+ post-synapses within a distance of 300nm from the nearest vGlut2+ surface.


( F ) Quantification of Homer1 + spots adjacent to vGlut2 surface within 100nm , 200nm , and 300nm , respectively . N = 3 .

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(F) Quantification of Homer1+ spots adjacent to vGlut2 surface within 100nm, 200nm, and 300nm, respectively. N = 3.


( G ) Western blot of vGlut2 and Homer1 using microdissected dLGN tissue from P8 mice . ( H , I ) Quantification of vGlut2 and Homer1 expression in CKOs and controls . N = 3 , * p = 0 . 027 . ( J ) Quantification of synapse density ( vGlut2 + / Homer1 + ) in the dLGN of both male and female iCKO mice at P10 . N = 3 for male , N = 4 for female .

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(G) Western blot of vGlut2 and Homer1 using microdissected dLGN tissue from P8 mice. (H, I) Quantification of vGlut2 and Homer1 expression in CKOs and controls. N = 3, *p = 0.027. (J) Quantification of synapse density (vGlut2+/Homer1+) in the dLGN of both male and female iCKO mice at P10. N = 3 for male, N = 4 for female.


( K ) An overview of vGlut1 and Homer1 staining in dLGN at P10 . The yellow outline indicates the dLGN core , and the dotted boxes show where synapses are quantified .

(K) An overview of vGlut1 and Homer1 staining in dLGN at P10. The yellow outline indicates the dLGN core, and the dotted boxes show where synapses are quantified.


( L ) Representative images of vGlut1 + ( green ) presynaptic terminals and Homer1 + ( red ) postsynapses in the dLGN of CKO and controls at P10 . Arrows pointing to the synapse enlarged in inset . ( M ) Quantification of synapse density ( vGlut1 / homer1 ) at P10 in the dLGN of CKO mice and controls . N = 3 ( WT ) , N = 5 ( CKO ) , p = 0 . 053 .

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(L) Representative images of vGlut1+ (green) presynaptic terminals and Homer1+ (red) postsynapses in the dLGN of CKO and controls at P10. Arrows pointing to the synapse enlarged in inset. (M) Quantification of synapse density (vGlut1/homer1) at P10 in the dLGN of CKO mice and controls. N=3 (WT), N=5 (CKO), p = 0.053.


( A ) Representative images show hippocampal vGlut2 and Homer1 immunostaining . White box outlines the region of interest and yellow box shows the regions where confocal images were taken .

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(A) Representative images show hippocampal vGlut2 and Homer1 immunostaining. White box outlines the region of interest and yellow box shows the regions where confocal images were taken.


( B ) Confocal images of synaptic immunostaining in CA1 striatum lacunosum - moleculare ( str . lac / mol ) at P10 . Arrows pointing to the enlarged synapse in the insets . ( C , D ) Quantification of synapse density in CA1 str . lac / mol in iCKO versus control at P10 ( C ) , and CKO versus control at P21 ( D ) . At P10 , N ( Ctrl ) = 4 , N ( iCKO ) = 3 , p = 0 . 02 . At P21 , N = 8 , p = 0 . 005 .

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(B) Confocal images of synaptic immunostaining in CA1 striatum lacunosum-moleculare (str. lac/mol) at P10. Arrows pointing to the enlarged synapse in the insets. (C, D) Quantification of synapse density in CA1 str. lac/mol in iCKO versus control at P10 (C), and CKO versus control at P21 (D). At P10, N(Ctrl) = 4, N(iCKO) = 3, p = 0.02. At P21, N = 8, p = 0.005.


( E ) Representative images show hippocampal vGlut1 and Homer1 immunostaining .

(E) Representative images show hippocampal vGlut1 and Homer1 immunostaining.


( F ) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10 . ( G , H ) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 ( G ) , and CKO versus control at P21 ( H ) . At P10 , N = 5 , p = 0 . 83 . At P21 , N = 5 , p = 0 . 89 .

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(F) Confocal images of synaptic immunostaining in CA1 striatum radiatum at P10. (G, H) Quantification of synapse density in CA1 striatum radiatum in iCKO versus control at P10 (G), and CKO versus control at P21 (H). At P10, N = 5, p = 0.83. At P21, N = 5, p = 0.89.