A Representative neurons expressing myc - tagged gephyrin or G375D pHluorin - tagged GABAAR γ2 . Surface - expressed γ2 - subunits were immunolabelled with GFP - antibodies in red , pHluorin - ( total γ2 ) are shown in green . B C ( fl . ) of surface - expressed γ2 clusters in myc - gephyrin myc - G375D - expressing dendrites ( n = 20 myc - gephyrin 21 myc - G375D neurons in B 18 myc - gephyrin myc - G375D neurons in C ) D Mean γ2 - pHluorin in myc - gephyrin myc - G375D - expressing dendrites ( n = 20 18 myc - gephyrin myc - G375D expressing neurons ) . E Number of surface γ2 clusters on dendrites normalized to 20 µm segments ( n = 18 myc - gephyrin myc - G375D expressing neurons ) . At least three independent cultures were used for each experiment . Results are expressed as mean ± ; Data were analyzed by t - .

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A Representative neurons expressing myc-tagged gephyrin or G375D and pHluorin-tagged GABAAR γ2. Surface-expressed γ2-subunits were immunolabelled with GFP-antibodies in red, and the pHluorin-fluorescence (total γ2 levels) are shown in green.B Size and C fluorescence (fl.) intensity of surface-expressed γ2 clusters in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 myc-gephyrin and 21 myc-G375D neurons in B and 18 myc-gephyrin and myc-G375D neurons in C)D Mean γ2-pHluorin fluorescence in myc-gephyrin and myc-G375D-expressing dendrites (n = 20 and 18 myc-gephyrin and myc-G375D expressing neurons).E Number of surface γ2 clusters on dendrites normalized to 20 µm segments (n = 18 myc-gephyrin and myc-G375D expressing neurons). At least three independent cultures were used for each experiment. Results are expressed as mean ± SEM; Data were analyzed by t-test.


F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP - gephyrin or GFP - G375D . G mIPSC amplitudes from neurons expressing GFP - gephyrin or GFP - G375D . Numbers show analyzed - numbers . Data were analyzed by one - way ANOVA post - hoc Tukey . H Frequency of mIPSCs from neurons expressing GFP - gephyrin or GFP - G375D . horizontal within boxes indicates median frequency for represented condition ( bottom / top boundary indicates 25th / 75th percentile ) . Top bottom error bars indicate 90th 10th percentiles , respectively . by Kruskal - Wallis followed by post - hoc Mann - Whitney .

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F Representative traces of miniature recordings on cultured hippocampal neurons transfected either with GFP-gephyrin or GFP-G375D.G mIPSC amplitudes from neurons expressing GFP-gephyrin or GFP-G375D. Numbers show analyzed cell-numbers. Data were analyzed by one-way ANOVA and post-hoc Tukey test.H Frequency of the mIPSCs from neurons expressing GFP-gephyrin or GFP-G375D. The horizontal line within the boxes indicates the median frequency for the represented condition (bottom/top boundary indicates the 25th /75th percentile). Top and bottom error bars indicate the 90th and 10th percentiles, respectively. Analysis by Kruskal-Wallis test followed by post-hoc Mann-Whitney test.


A Western blot of HEK293 expressing GFP - tagged gephyrin or G375D using GFP - specific polyclonal gephyrin antibodies . Note that both gephyrin variants are stably expressed show no degradation in . Asterisk in poly - gephyrin blot shows endogenous gephyrin .

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A Western blot of HEK293 cells expressing GFP-tagged gephyrin or G375D using GFP-specific and polyclonal gephyrin antibodies. Note that both gephyrin variants are stably expressed and show no degradation in the cells. Asterisk in the poly-gephyrin blot shows endogenous gephyrin.


B SDS - PAGE of E . coli expressed purified 6His - gephyrins CD spectra . Mean residue ellipticity of gephyrin G375D show profiles with minima at 208 nm 222 nm .

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B SDS-PAGE of E. coli expressed and purified 6His-gephyrins and CD spectra. Mean residue ellipticity of gephyrin and G375D show profiles with minima at 208 nm and 222 nm.


C - exclusion chromatography of purified 6His - gephyrin isolated E - domain variants . Full - gephyrins isolated E - domains were loaded onto a Superose 6 gel filtration column elution of was monitored by measuring absorbance at 280 nm ( mAU , milliabsorbance units ) .

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C Size-exclusion chromatography of purified 6His-gephyrin and isolated E-domain variants. Full-length gephyrins and isolated E-domains were loaded onto a Superose 6 gel filtration column and elution of the protein was monitored by measuring absorbance at 280 nm (mAU, milliabsorbance units).


D Co - immunoprecipitation of HEK293 lysates expressing myc - tagged gephyrin together with GFP - tagged gephyrin or G375D using myc - specific antibodies . Control IgG were used to show specificity of .

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D Co-immunoprecipitation of HEK293 cell lysates expressing myc-tagged gephyrin together with GFP-tagged gephyrin or G375D using myc-specific antibodies. Control IgG were used to show specificity of the assay.


B Representative pulldown - experiment of 6His - tagged gephyrin G375D using immobilized α3 - peptide . 6His - tagged gephyrin variants were expressed in E . coli , purified to homogeneity incubated with α3 - peptides immobilized to NeutrAvidin beads . n = 3 , normalized to 6His - gephyrin . 38 . 9 ± 1 . 5 % G375D were pulled down .

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B Representative pulldown-experiment and quantification of 6His-tagged gephyrin and G375D using immobilized α3-peptide. 6His-tagged gephyrin variants were expressed in E. coli cells, purified to homogeneity and incubated with α3-peptides immobilized to NeutrAvidin beads. n = 3, normalized to 6His-gephyrin. 38.9±1.5% G375D were pulled down.


C Representative SPR sensograms of 6His - gephyrin 6His - G375D binding to immobilized GABAAR α3 - peptide . Response units are plotted against time . Injected gephyrin concentrations were 0 . 5 , 2 , 10 , 25 , 40 µM .

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C Representative SPR sensograms of 6His-gephyrin and 6His-G375D binding to immobilized GABAAR α3-peptide. Response units are plotted against time. Injected gephyrin concentrations were 0.5, 2, 10, 25, 40 µM.


D SPR binding isotherms of a representative experiment as in C . Calculated KD values of 3 . 2 10 µM for gephyrin G375D , respectively , are indicated by vertical lines . experiment was repeated three times with two independently purified batches two sensor chips .

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D SPR binding isotherms of a representative experiment as in C. Calculated KD values of 3.2 and 10 µM for gephyrin and G375D, respectively, are indicated by vertical lines. The experiment was repeated three times with two independently purified protein batches and two sensor chips.


E Representative ITC binding isotherms of loop of GlyR ß - subunit ( GlyR - ßL , residue 378 - 426 ) 6His - tagged gephyrin or G375D . GlyR - ßL - gephyrin binding data ( black ) were fitted in a two - side model , whereas G375D binding could only be fitted to a single binding site . Dissociation constants KD stoichiometry ( N = number of binding sites ) are given for each binding component . n = 5 from two independently purified batches . Results are expressed as mean ± .

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E Representative ITC binding isotherms of the cytoplasmic loop of the GlyR ß-subunit (GlyR-ßL, residue 378-426) and 6His-tagged gephyrin or G375D. The GlyR-ßL-gephyrin binding data (black) were fitted in a two-side model, whereas G375D binding could only be fitted to a single binding site. Dissociation constants KD and stoichiometry (N = number of binding sites) are given for each binding component. n = 5 from two independently purified protein batches. Results are expressed as mean ± SEM.


A Biosynthesis pathway of Moco with stable intermediates . Gephyrin G - E - domain catalyze last two steps as depicted . B In vitro Moco - synthesis using 150 pmol purified 6His - tagged gephyrins . D580A is an activity - deficient gephyrin mutation previously identified in Moco - deficient ( Reiss et al . , 2011 ) . Assays without addition of gephyrin ( - gephyrin ) or molybdenum ( - Mo ) served as internal controls of ( see also ( Belaidi Schwarz , 2013 ) ) . C Experiment as in B but with pre - incubation of 6His - gephyrin / G375D in a 1 : 1 to simulate heterozygous mutation . n = at least 3 with two independently purified batches . Results are expressed as mean ± .

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A Biosynthesis pathway of Moco with all stable intermediates. Gephyrin G- and E-domain catalyze the last two steps as depicted.B In vitro Moco-synthesis assay using 150 pmol purified 6His-tagged gephyrins. D580A is an activity-deficient gephyrin mutation previously identified in Moco-deficient patients (Reiss et al., 2011). Assays without the addition of gephyrin (-gephyrin) or molybdenum (-Mo) served as internal controls of the assay (see also (Belaidi Schwarz, 2013)).C Experiment as in B but with pre-incubation of 6His-gephyrin/G375D in a 1:1 ratio to simulate the heterozygous mutation. n = at least 3 with two independently purified protein batches. Results are expressed as mean ± SEM.


( b ) Parkin is recruited selectively to depolarized mitochondria directs mitophagy . HeLa transfected with HA - Parkin were treated with CCCP for indicated times . Mitochondria were stained by anti - TOM20 ( pseudo coloured ; blue ) a ΔΨm dependent MitoTracker ( red ) . Parkin was stained with anti - HA ( green ) . Without treatment , mitochondria are intact stained by both mitochondrial markers , whereas Parkin is equally distributed in cytoplasm . After 2 h of CCCP treatment , mitochondria are depolarized as shown by loss of MitoTracker . Parkin completely translocates to mitochondria clustering at perinuclear regions . After 24 h of CCCP treatment , massive loss of mitochondria is observed as shown by disappearance of mitochondrial marker . Only Parkin - positive show mitochondrial clustering clearance , in contrast to adjacent untransfected . Scale bars , 10 μm .

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(b) Parkin is recruited selectively to depolarized mitochondria and directs mitophagy. HeLa cells transfected with HA-Parkin were treated with CCCP for the indicated times. Mitochondria were stained by anti-TOM20 (pseudo coloured; blue) and a ΔΨm dependent MitoTracker (red). Parkin was stained with anti-HA (green). Without treatment, mitochondria are intact and stained by both mitochondrial markers, whereas Parkin is equally distributed in the cytoplasm. After 2 h of CCCP treatment, mitochondria are depolarized as shown by the loss of MitoTracker staining. Parkin completely translocates to mitochondria clustering at perinuclear regions. After 24 h of CCCP treatment, massive loss of mitochondria is observed as shown by the disappearance of the mitochondrial marker. Only Parkin-positive cells show mitochondrial clustering and clearance, in contrast to adjacent untransfected cells. Scale bars, 10 μm.


( c ) Pathogenic Parkin mutations abrogate mitochondrial translocation / or clearance . HeLa transfected with Flag - Parkin wild - type or pathogenic mutants were treated with CCCP stained with anti - Flag ( green ) , anti - TOM20 ( red ) Hoechst33342 ( blue ) . Representative merged are shown . Scale bars , 10 μm . Immunostaining of transfected with non - functional cysteine mutants functional Parkin variants is shown in Supplementary Information , Fig . S1a S1b , respectively .

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(c) Pathogenic Parkin mutations abrogate mitochondrial translocation and/or clearance. HeLa cells transfected with Flag-Parkin wild-type or pathogenic mutants were treated with CCCP and stained with anti-Flag (green), anti-TOM20 (red) and Hoechst33342 (blue). Representative merged images are shown. Scale bars, 10 μm. Immunostaining of cells transfected with non-functional cysteine mutants and functional Parkin variants is shown in Supplementary Information, Fig. S1a and S1b, respectively.


( d , e ) on mitochondrial effects , Parkin mutants can be classified into functional non - functional classes . ( d ) of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment , as defined by complete colocalization of Parkin with mitochondrial marker ( n = 3 ) . ( e ) of with uncleared mitochondria after 24 h of CCCP treatment , as shown by complete loss of mitochondrial ( n = 3 ) . Data represent mean ± s . d . , * P ≤ 0 . 05 , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with wild - type . WT , wild - type .

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(d, e) Based on the mitochondrial effects, Parkin mutants can be classified into functional and non-functional classes. (d) Quantification of Parkin translocation to damaged mitochondria after 2 h of CCCP treatment, as defined by complete colocalization of Parkin with the mitochondrial marker (n = 3). (e) Quantification of cells with uncleared mitochondria after 24 h of CCCP treatment, as shown by complete loss of mitochondrial staining (n = 3). Data represent the mean ± s.d., *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 compared with wild-type. WT, wild-type.


Stable Parkin wild - type or pathogenic mutants expressing SH - SY5Y lines were induced with doxycycline ( DOX ) for 72 h treated with CCCP for indicated times . were stained with anti - Parkin ( green ) , anti - TOM20 ( red ) Hoechst33342 ( blue ) . ( a - b ) CCCP treatment of stable SH - SY5Y for 0 h . Unstimulated control ( - DOX ) show relatively low of endogenous Parkin , compared to with induced expression of Parkin wild - type or K161N , R275W G430D mutants ( + DOX ) . Immunostaining ( a ) western blot ( b ) of induced demonstrate comparable of Parkin expression in selected clones . β - Actin served as a loading control .

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Stable Parkin wild-type or pathogenic mutants expressing SH-SY5Y cell lines were induced with doxycycline (DOX) for 72 h and treated with CCCP for the indicated times. Cells were stained with anti-Parkin (green), anti-TOM20 (red) and Hoechst33342 (blue). (a-b) CCCP treatment of stable SH-SY5Y cells for 0 h. Unstimulated control cells (- DOX) show relatively low levels of endogenous Parkin, compared to cells with induced expression of Parkin wild-type or K161N, R275W and G430D mutants (+ DOX). Immunostaining (a) and western blot analysis (b) of the induced cells demonstrate comparable levels of Parkin expression in the selected clones. β-Actin served as a loading control.


( c , d ) After 2 h of CCCP treatment , massive colocalization of wild - type Parkin mitochondrial clusters can be observed in induced when compared with unstimulated controls . Similarly , Parkin K161N R275W mutants translocate to mitochondria , whereas G430D does not .

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(c, d) After 2 h of CCCP treatment, massive colocalization of wild-type Parkin and mitochondrial clusters can be observed in the induced cells when compared with unstimulated controls. Similarly, Parkin K161N and R275W mutants translocate to mitochondria, whereas G430D does not.


( e , f ) After 24 h of CCCP treatment , induced wild - type Parkin - expressing show full clearance of damaged mitochondria . In contrast , pathogenic mutants analysed fail to execute mitophagy , therefore , damaged mitochondria remain uncleared . Representative of three independent experiments are shown . Scale bars , 10 μm ( a , c e ) . ( d , f ) Quantifications of observed mitochondrial effects in Parkin - expressing SH - SY5Y ( n = 3 ) . Data represent mean ± s . d . , * * * P ≤ 0 . 0005 compared with induced wild - type Parkin . WT , wild - type .

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(e, f) After 24 h of CCCP treatment, induced wild-type Parkin-expressing cells show full clearance of damaged mitochondria. In contrast, the pathogenic mutants analysed fail to execute mitophagy and, therefore, damaged mitochondria remain uncleared. Representative images of three independent experiments are shown. Scale bars, 10 μm (a, c and e). (d, f) Quantifications of the observed mitochondrial effects in Parkin-expressing SH-SY5Y cells (n = 3). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with induced wild-type Parkin. WT, wild-type.


( b ) Co - immunoprecipitations of Parkin PINK1 after mitochondrial depolarization . Endogenous Parkin PINK1 were co - immunoprecipitated from neuronal SH - SY5Y after CCCP treatment with specific antibodies .

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(b) Co-immunoprecipitations of Parkin and PINK1 after mitochondrial depolarization. Endogenous Parkin and PINK1 were co-immunoprecipitated from neuronal SH-SY5Y cells after CCCP treatment with specific antibodies.


( c ) Western blot of PINK1 siRNA - transfected HeLa used in d - h demonstrates knockdown efficiency specificity of PINK1 silencing .

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(c) Western blot of PINK1 siRNA-transfected HeLa cells used in d-h demonstrates the knockdown efficiency and specificity of PINK1 silencing.


d - h ) PINK1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , mitochondrial clearance after 24 h , of CCCP treatment . This can be rescued by re - transfection with wild - type PINK1 , but not with a kinase - dead mutant or a PINK1 variant lacking MTS . Control siRNA - or PINK1 siRNA - silenced HeLa were transfected with EGFP - Parkin wild - type PINK1 - V5 wild - type , 3 × KD or ΔMTS mutants . Use of appropriate empty vector controls is depicted . were treated with CCCP stained with anti - TOM20 ( pseudo coloured ; blue ) anti - V5 ( red ) to visualize mitochondrial re - transfected PINK1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive are surrounded by a . Representative of four independent experiments are shown . Scale bars , 10 μm .

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d-h) PINK1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. This can be rescued by re-transfection with wild-type PINK1, but not with a kinase-dead mutant or a PINK1 variant lacking the MTS. Control siRNA- or PINK1 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type and PINK1-V5 wild-type, 3×KD or ΔMTS mutants. Use of appropriate empty vector controls is depicted. Cells were treated with CCCP and stained with anti-TOM20 (pseudo coloured; blue) and anti-V5 (red) to visualize mitochondrial morphology and re-transfected PINK1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative images of four independent experiments are shown. Scale bars, 10 μm.


( e ) Wild - type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed ( n = 4 ) . ( g ) of results from in e .

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(e) Wild-type Parkin colocalization with impaired mitochondria after 2 h of CCCP treatment was analysed (n = 4). (g) Quantification of results from cells in e.


( f ) Uncleared mitochondria were analysed after 24 h of CCCP treatment ( n = 4 ) . ( h ) of results from in f . Data represent mean ± s . d . , * * P ≤ 0 . 005 , * * * P ≤ 0 . 0005 compared with control siRNA - transfected . Immunostaining of control siRNA - transfected at analysed time points is shown in Supplementary Information , Fig . S2 . WT , wild - type .

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(f) Uncleared mitochondria were analysed after 24 h of CCCP treatment (n = 4). (h) Quantification of results from cells in f. Data represent the mean ± s.d., **P ≤ 0.005, ***P ≤ 0.0005 compared with control siRNA-transfected cells. Immunostaining of control siRNA-transfected cells at all analysed time points is shown in Supplementary Information, Fig. S2. WT, wild-type.


a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa were transfected with Flag - Parkin wild - type or pathogenic mutants . were treated with CCCP before immunostaining of Parkin with anti - Flag ( green ) endogenous poly - ubiquitin with a specific antibody FK1 ( red ) . ( a ) In contrast to pathogenic mutations , wild - type Parkin shows colocalization with poly - ubiquitin after 6 h of CCCP treatment at site of condensed mitochondria . ( b ) After 24 h of CCCP treatment , K161N R275W Parkin mutants colocalize with both poly - ubiquitin mitochondria . In contrast , translocation - deficient Parkin mutants K211N , T240R G430D lack mitochondria - associated poly - ubiquitin signal . Representative are shown . Scale bars , 10 μm . Complementary Parkin of untreated of colocalization of Parkin , mitochondria poly - ubiquitin upon CCCP treatment are shown in Supplementary Information , Fig . S3a , b c , respectively .

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a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP before immunostaining of Parkin with anti-Flag (green) and endogenous poly-ubiquitin with a specific antibody FK1 (red). (a) In contrast to pathogenic mutations, wild-type Parkin shows colocalization with poly-ubiquitin after 6 h of CCCP treatment at the site of condensed mitochondria. (b) After 24 h of CCCP treatment, K161N and R275W Parkin mutants colocalize with both poly-ubiquitin and mitochondria. In contrast, translocation-deficient Parkin mutants K211N, T240R and G430D lack the mitochondria-associated poly-ubiquitin signal. Representative images are shown. Scale bars, 10 μm. Complementary Parkin of untreated cells and quantification of colocalization of Parkin, mitochondria and poly-ubiquitin upon CCCP treatment are shown in Supplementary Information, Fig. S3a, b and c, respectively.


( c , d ) Parkin - directed mitophagy specifically involves linkage of ubiquitin through lysines 27 63 . HeLa were transfected with EGFP - Parkin wild - type ubiquitin lysine variants treated with CCCP before immunostaining with anti - HtrA2 / Omi , as a mitochondrial marker ( pseudo coloured ; blue ) anti - His ( red ) to detect ectopic ubiquitin . Parkin was visualized by its fusion to EGFP ( green ) . ( c ) Note that only wild - type , K27 K63 6 × His - tagged ubiquitin variants are colocalized with condensed mitochondria EGFP - Parkin after 6 h of CCCP treatment . other ubiquitin mutants interfere with Parkin translocation to mitochondria . ( d ) Only co - expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6 × His - tagged wild - type ubiquitin after 24 h of CCCP treatment . Representative are shown . Scale bars , 10 μm . Immunostaining of untreated other ubiquitin variant combinations tested are shown in Supplementary Information , Fig . S4a b , c , respectively . WT , wild - type ; PolyUb , poly - ubiquitin .

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(c, d) Parkin-directed mitophagy specifically involves linkage of ubiquitin through lysines 27 and 63. HeLa cells were transfected with EGFP-Parkin wild-type and ubiquitin lysine variants and treated with CCCP before immunostaining with anti-HtrA2/Omi, as a mitochondrial marker (pseudo coloured; blue) and anti-His (red) to detect ectopic ubiquitin. Parkin was visualized by its fusion to EGFP (green). (c) Note that only wild-type, K27 and K63 6×His-tagged ubiquitin variants are colocalized with condensed mitochondria and EGFP-Parkin after 6 h of CCCP treatment. All other ubiquitin mutants interfere with Parkin translocation to mitochondria. (d) Only co-expression of K27 or K63 ubiquitin recapitulates mitochondrial clearance similarly to 6×His-tagged wild-type ubiquitin after 24 h of CCCP treatment. Representative images are shown. Scale bars, 10 μm. Immunostaining of untreated cells and all other ubiquitin variant combinations tested are shown in Supplementary Information, Fig. S4a and b, c, respectively. WT, wild-type; PolyUb, poly-ubiquitin.


( a , b ) Stable Mito - DsRed ( pseudo coloured ; blue ) - expressing HeLa were transfected with Flag - Parkin wild - type or pathogenic mutants . were treated with CCCP for indicated times before immunostaining with anti - Flag ( green ) anti - p62 ( red ) . ( a ) In contrast to pathogenic mutants , wild - type Parkin colocalizes with p62 at site of clustered mitochondria , after 6 h of CCCP treatment . ( b ) After 24 h of CCCP treatment , wild - type Parkin - transfected show complete loss of mitochondria , whereas K161N R275W Parkin mutants colocalize with both p62 mitochondria . In contrast , translocation - compromised mutants K211N , T240R G430D , lacking mitochondria - associated poly - ubiquitin signal ( Fig . 4b ) , did not colocalize with p62 . Representative of four independent experiments are shown . Data from untreated control of colocalization of Parkin , mitochondria p62 upon CCCP treatment are shown in Supplementary Information , Fig . 5a b , c , respectively .

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(a, b) Stable Mito-DsRed (pseudo coloured; blue)-expressing HeLa cells were transfected with Flag-Parkin wild-type or pathogenic mutants. Cells were treated with CCCP for the indicated times before immunostaining with anti-Flag (green) and anti-p62 (red). (a) In contrast to the pathogenic mutants, wild-type Parkin colocalizes with p62 at the site of clustered mitochondria, after 6 h of CCCP treatment. (b) After 24 h of CCCP treatment, wild-type Parkin-transfected cells show complete loss of mitochondria, whereas K161N and R275W Parkin mutants colocalize with both p62 and mitochondria. In contrast, translocation-compromised mutants K211N, T240R and G430D, lacking the mitochondria-associated poly-ubiquitin signal (Fig. 4b), did not colocalize with p62. Representative images of four independent experiments are shown. Data from untreated control cells and quantification of colocalization of Parkin, mitochondria and p62 upon CCCP treatment are shown in Supplementary Information, Fig. 5a and b, c, respectively.


( c , d ) Control siRNA - or p62 siRNA - silenced HeLa were transfected with EGFP - Parkin wild - type . were treated with CCCP for indicated times stained with anti - TOM20 ( pseudo coloured ; blue ) to visualize mitochondrial anti - p62 ( red ) to monitor silencing efficiency . Parkin wild - type was visualized by EGFP epifluorescence ( green ) . Parkin - positive with cleared mitochondria are surrounded by a . (

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(c, d) Control siRNA- or p62 siRNA-silenced HeLa cells were transfected with EGFP-Parkin wild-type. Cells were treated with CCCP for the indicated times and stained with anti-TOM20 (pseudo coloured; blue) to visualize mitochondrial morphology and anti-p62 (red) to monitor silencing efficiency. Parkin wild-type was visualized by EGFP epifluorescence (green). Parkin-positive cells with cleared mitochondria are surrounded by a line. (


( c - f ) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment ( c - e ) , but completely blocks final clearance after 24 h ( c , d , f ) . ( e , f ) Relative parkin translocation ( e ) mitochondrial clearance ( f ) after p62 knockdown CCCP treatment ( n = 4 ) . Data represent mean ± s . d . , * * * P ≤ 0 . 0005 compared with control siRNA - transfected . Representative of four independent experiments are shown . Scale bars , 10 μm . Immunostainings of control are shown in Supplementary Information , Fig . S5d , e .

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(c-f) Knockdown of p62 has no influence on Parkin translocation to mitochondria after 6 h of CCCP treatment (c-e), but completely blocks final clearance after 24 h (c, d, f). (e, f) Relative parkin translocation (e) and mitochondrial clearance (f) after p62 knockdown and CCCP treatment (n = 4). Data represent the mean ± s.d., ***P ≤ 0.0005 compared with control siRNA-transfected cells. Representative images of four independent experiments are shown. Scale bars, 10 μm. Immunostainings of control cells are shown in Supplementary Information, Fig. S5d, e.


( g ) Western blot of siRNA - transfected HeLa used in c - f demonstrates efficiency of p62 silencing . β - Actin probing served as a loading control . WT , wild - type .

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(g) Western blot of siRNA-transfected HeLa cells used in c-f demonstrates the efficiency of p62 silencing. β-Actin probing served as a loading control. WT, wild-type.


( a ) Time - course experiment of HeLa transfected with Flag - Parkin wild - type treated with CCCP for indicated periods . Total lysates were prepared , western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 anti - VDAC1 antibodies . β - Actin probing served as a loading control . of detected are endogenously expressed , except Flag - Parkin .

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(a) Time-course experiment of HeLa cells transfected with Flag-Parkin wild-type and treated with CCCP for the indicated periods. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. β-Actin probing served as a loading control. All of the proteins detected are endogenously expressed, except Flag-Parkin.


( b ) Pathogenic Parkin mutants fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . HeLa were transfected with empty vector ( - ) , Flag - Parkin wild - type or pathogenic mutants along with 6 × His - tagged ubiquitin wild - type for 24 h . were treated with CCCP for 6 h subsequently lysates were prepared in urea buffer . Denaturing conditions of Ni - NTA affinity purifications were used to specifically pulldown only covalently modified with 6xHis - tagged ubiquitin . Western blots of lysates purified Ni - NTA agarose elutions were probed with anti - VDAC1 , anti - TOM20 anti - Parkin antibodies . Modification of VDAC1 was observed in expressing Parkin wild - type functional mutant G328E , whereas non - functional mutants did not have this effect . (

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(b) Pathogenic Parkin mutants fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. HeLa cells were transfected with empty vector (-), Flag-Parkin wild-type or pathogenic mutants along with 6×His-tagged ubiquitin wild-type for 24 h. Cells were treated with CCCP for 6 h and subsequently protein lysates were prepared in urea buffer. Denaturing conditions of Ni-NTA affinity purifications were used to specifically pulldown only proteins covalently modified with 6xHis-tagged ubiquitin. Western blots of protein lysates and purified Ni-NTA agarose elutions were probed with anti-VDAC1, anti-TOM20 and anti-Parkin antibodies. Modification of VDAC1 was observed in cells expressing Parkin wild-type and the functional mutant G328E, whereas non-functional mutants did not have this effect. (


( c ) In response to CCCP treatment , Parkin catalyses predominantly K27 - linked poly - ubiquitin chains on endogenous VDAC1 is itself strongly poly - ubiquitylated . HeLa were transfected with an empty vector ( - ) or Flag - Parkin wild - type along with 6 × His - tagged ubiquitin variants for 24 h . Treatment of , preparation of lysates Ni - NTA purification were performed as described in c probed for Parkin , VDAC1 β - actin . WT , wild - type ; Ub , ubiquitin .

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(c) In response to CCCP treatment, Parkin catalyses predominantly K27-linked poly-ubiquitin chains on endogenous VDAC1 and is itself strongly poly-ubiquitylated. HeLa cells were transfected with an empty vector (-) or Flag-Parkin wild-type along with 6×His-tagged ubiquitin variants for 24 h. Treatment of cells, preparation of lysates and Ni-NTA purification were performed as described in c and probed for Parkin, VDAC1 and β-actin. WT, wild-type; Ub, ubiquitin.


( a ) Western blot of parental SH - SY5Y treated with CCCP for indicated times . Time - course experiments of CCCP - treated neuronal reveal increasing modifications of VDAC1 over time . Total lysates were prepared , western blots were probed with specific anti - Parkin , anti - PINK1 , anti - TOM20 anti - VDAC1 antibodies . represent endogenous .

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(a) Western blot analysis of parental SH-SY5Y cells treated with CCCP for the indicated times. Time-course experiments of CCCP-treated neuronal cells reveal increasing modifications of VDAC1 over time. Total lysates were prepared, and western blots were probed with specific anti-Parkin, anti-PINK1, anti-TOM20 and anti-VDAC1 antibodies. All proteins represent endogenous levels.


( b ) Immunoprecipitations of Parkin VDAC1 from parental SH - SY5Y confirm ubiquitylation of both . CCCP treatment of neuronal increases ubiquitylated species of endogenous Parkin VDAC1 over time . Using specific antibodies , Parkin VDAC1 were immunoprecipitated from lysates of parental SH - SY5Y at different time points , western blotted probed with anti - ubiquitin , anti - Parkin anti - VDAC1 antibodies .

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(b) Immunoprecipitations of Parkin and VDAC1 from parental SH-SY5Y cells confirm ubiquitylation of both proteins. CCCP treatment of neuronal cells increases ubiquitylated species of endogenous Parkin and VDAC1 over time. Using specific antibodies, Parkin and VDAC1 were immunoprecipitated from lysates of parental SH-SY5Y cells at different time points, western blotted and probed with anti-ubiquitin, anti-Parkin and anti-VDAC1 antibodies.


( c ) Pathogenic Parkin mutations fail to poly - ubiquitylate endogenous VDAC1 in response to CCCP treatment . Western blot of a time - course experiment with inducible Parkin SH - SY5Y after CCCP treatment . Total lysates were prepared , western blots were probed with specific anti - Parkin anti - VDAC1 antibodies . β - Actin probing served as a loading control in a - c . WT , wild - type ; Ub , ubiquitin .

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(c) Pathogenic Parkin mutations fail to poly-ubiquitylate endogenous VDAC1 in response to CCCP treatment. Western blot analysis of a time-course experiment with inducible Parkin SH-SY5Y cells after CCCP treatment. Total lysates were prepared, and western blots were probed with specific anti-Parkin and anti-VDAC1 antibodies. β-Actin probing served as a loading control in a-c. WT, wild-type; Ub, ubiquitin.


VDAC1 - specific siRNA abrogates Parkin translocation to mitochondria after 2 h , mitochondrial clearance after 24 h , of CCCP treatment . Both effects can be rescued by re - transfection with wild - type VDAC1 . ( a ) Immunofluorescence ( b ) western blot of siRNA - transfected HeLa used in c - g demonstrate partial silencing of endogenous VDAC1 . ( a ) Silenced were stained with anti - HtrA2 / Omi , a mitochondrial marker ( pseudo coloured ; blue ) , anti - VDAC1 ( red ) antibodies . ( b ) Lysates from silenced were subjected to western blot probed with an anti - VDAC1 antibody , while TOM20 β - actin probing served as loading controls .

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VDAC1-specific siRNA abrogates Parkin translocation to mitochondria after 2 h, and mitochondrial clearance after 24 h, of CCCP treatment. Both effects can be rescued by re-transfection with wild-type VDAC1. (a) Immunofluorescence and (b) western blot of siRNA-transfected HeLa cells used in c-g demonstrate partial silencing of endogenous VDAC1. (a) Silenced cells were stained with anti-HtrA2/Omi, a mitochondrial marker (pseudo coloured; blue), and anti-VDAC1 (red) antibodies. (b) Lysates from silenced cells were subjected to western blot analysis and probed with an anti-VDAC1 antibody, while TOM20 and β-actin probing served as loading controls.


( c - e ) Control siRNA - or VDAC1 siRNA - silenced HeLa were transfected with indicated combinations of EGFP - Parkin wild - type , VDAC1 - Flag wild - type or appropriate empty vector controls . were treated with CCCP for 2 h 24 h stained with anti - HtrA2 / Omi ( pseudo coloured ; blue ) anti - Flag ( red ) to visualize mitochondrial re - transfected VDAC1 , respectively . Wild - type Parkin was visualized by EGFP epifluorescence ( green ) . Parkin - positive are surrounded by a . Representative pictures of three independent experiments are shown . Scale bars , 10 μm .

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(c-e) Control siRNA- or VDAC1 siRNA-silenced HeLa cells were transfected with the indicated combinations of EGFP-Parkin wild-type, VDAC1-Flag wild-type or appropriate empty vector controls. Cells were treated with CCCP for 2 h and 24 h and stained with anti-HtrA2/Omi (pseudo coloured; blue) and anti-Flag (red) to visualize mitochondrial morphology and re-transfected VDAC1, respectively. Wild-type Parkin was visualized by EGFP epifluorescence (green). Parkin-positive cells are surrounded by a line. Representative pictures of three independent experiments are shown. Scale bars, 10 μm.


( f , g ) of d ( f ) e ( g ) . Parkin wild - type colocalization with impaired mitochondria after 2 h ( f ) uncleared mitochondria after 24 h ( g ) of CCCP treatment ( n = 3 ) . Data represent mean ± s . d . , * P ≤ 0 . 05 compared with control siRNA - transfected or with VDAC1 - Flag wild - type - transfected .

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(f, g) Quantification of d (f) and e (g). Parkin wild-type colocalization with impaired mitochondria after 2 h (f) and uncleared mitochondria after 24 h (g) of CCCP treatment (n = 3). Data represent the mean ± s.d., *P ≤ 0.05 compared with control siRNA-transfected or with VDAC1-Flag wild-type-transfected cells.


( A ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . Data information : EtOH : ethanol ; HAM : hydroxylamine ; 2 - BP : 2 -

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(A) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: EtOH: ethanol; HAM: hydroxylamine; 2-BP: 2-bromopalmitate


( B ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . Data information : FL : full ; N : N - terminal domain ; C : C - terminal

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(B) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: FL: full length; N: N-terminal domain; C: C-terminal domain


( C ) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264 . 7 . Data information : PA : palmitic acid ; 17 - ODYA : 17 - octadecanoic acid .

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(C) Click chemistry was applied to detect endogenous cGAS palmitoylation in RAW264.7 cells. Data information: PA: palmitic acid; 17-ODYA: 17-octadecanoic acid.


( D E ) In vitro palmitoylation for indicated . NBD - Palm - CoA : ( N - [ ( 7 - nitro - 2 - 1 , 3 - benzoxadiazol - 4 - yl ) - methyl ] ) palmitoyl - CoA . recombinant cGAS used in was indicated by Coomassie blue .

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(D and E) In vitro palmitoylation assay for the indicated proteins. NBD-Palm-CoA: (N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)-methyl] amino) palmitoyl-CoA. The recombinant cGAS protein used in the assay was indicated by Coomassie blue staining.


( F ) LC - MS / MS of palmitoylated peptides of cGAS .

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(F) LC-MS/MS analysis of palmitoylated peptides of cGAS.


( G ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . Data information : HAM :

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(G) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: HAM: hydroxylamine


( H - I ) THP - 1 were treated with DMSO or 2 - BP ( 50 μM ) ( H ) or palmitic acid ( 100 μM ) ( I ) for 12 h . Six hours after transfection with HT - ( 2 μg / mL ) , cGAMP was extracted quantified by cGAMP ELISA . Data information : 2 - BP : 2 - bromopalmitate Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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(H-I) THP-1 cells were treated with DMSO or 2-BP (50 μM) (H) or palmitic acid (100 μM) (I) for 12 h. Six hours after transfection with HT-DNA (2 μg/mL), cGAMP was extracted and quantified by cGAMP ELISA. Data information: 2-BP: 2-bromopalmitate Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


( J ) THP - 1 were treated with palmitic acid ( 0 , 50 μM , or 100 μM ) for 12 h before a cGAMP bioassay . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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(J) THP-1 cells were treated with palmitic acid (0, 50 μM, or 100 μM) for 12 h before a cGAMP bioassay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h transfected with HT - ( 2 μg / mL ) for 6 h before RT - qPCR of Ifna4 ( K ) , IFNb1 ( L ) expression . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of Ifna4 (K), IFNb1 (L) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


BMDMs were treated with palmitic acid ( 0 , 100 μM , or 200 μM ) for 12 h transfected with HT - ( 2 μg / mL ) for 6 h before RT - qPCR of , Cxcl10 ( M ) Rantes ( N ) expression . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 .

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

BMDMs were treated with palmitic acid (0, 100 μM, or 200 μM) for 12 h and transfected with HT-DNA (2 μg/mL) for 6 h before RT-qPCR analysis of , Cxcl10 (M) and Rantes (N) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.005; ***, P˂0.001


( O ) L929 were treated with BSA palmitic acid ( 100 μM ) transfected with HT - ( 2 μg / mL ) for indicated times before immunoblotting with indicated antibodies .

(O) L929 cells were treated with BSA and palmitic acid (100 μM) and transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies.


( A ) Acyl - RAC of HEK293T transfected with Flag - cGAS 24 h after transfection with indicated shRNA . ( B ) of palmitoylation of Flag - cGAS in ( A ) . Data information :

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

(A) Acyl-RAC assay of HEK293T cells transfected with Flag-cGAS 24 h after transfection with the indicated shRNA. (B) Quantification of palmitoylation levels of Flag-cGAS in (A). Data information:


( C ) Acyl - RAC of HEK293T transfected with indicated plasmids for 24 h . HAM : hydroxylamine .

(C) Acyl-RAC assay of HEK293T cells transfected with the indicated plasmids for 24 h. HAM: hydroxylamine.


( D ) HEK293T were transfected with HA - cGAS ( WT or C474S mutant ) ( 200 ng ) , Flag - STING ( 200 ng ) Myc - ZDHHC18 ( 200 ng ) expression plasmids for 24 h before luciferase reporter assays . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 (

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(D) HEK293T cells were transfected with HA-cGAS (WT or C474S mutant) (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (200 ng) expression plasmids for 24 h before luciferase reporter assays. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 (D


( E F ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with indicated plasmids for 24 h . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; FL : full

O O O O O O O O O O O O O O O O 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 and F) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length


( H ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with plasmids encoding HA - cGAS Flag - ZDHHC18 truncations or GFP for 24 h . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; FL : full

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(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-cGAS and Flag-ZDHHC18 truncations or GFP for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; FL: full length


( J ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with plasmids encoding HA - ZDHHC18 Flag - cGAS truncates or GFP for 24 h . Data information : WCL : whole -

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

(J) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding HA-ZDHHC18 and Flag-cGAS truncates or GFP for 24 h. Data information: WCL: whole-cell lysate


( K ) Immunofluorescence of HA - cGAS ( green ) Flag - ZDHHC18 ( red ) in HT - - stimulated ( or not ) HeLa . Scale bars : 7 μm . ( L ) Colocalization ( merged volume of total cGAS signal ) of cGAS ZDHHC18 in ( K ) . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * * , P ˂ 0 . 005 ; * * * , P ˂ 0 . 001 L ) .

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

(K) Immunofluorescence analysis of HA-cGAS (green) and Flag-ZDHHC18 (red) in HT-DNA-stimulated (or not) HeLa cells. Scale bars: 7 μm. (L) Colocalization (merged volume of total cGAS signal) of cGAS and ZDHHC18 in (K). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; **, P˂0.005; ***, P˂0.001 L).


( M - N ) Comparison of binding free energy ( M ) binding energy decomposition of a per - residue to binding affinity between ZDHHC18 cGAS ( N ) in different complexes .

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(M-N) Comparison of the binding free energy (M) and binding energy decomposition of a per-residue to the binding affinity between ZDHHC18 and cGAS (N) in different complexes.


( A ) Immunofluorescence of GFP - cGAS , nucleus ( DAPI ) , ER ( calnexin ) , Golgi apparatus ( GM130 ) , early endosomes ( EEA1 ) in HeLa . 2 - BP : 2 - bromopalmitate ; ISD : IFN - stimulatory . Arrows indicate cGAS . Scale bars : 10 μm .

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(A) Immunofluorescence analysis of GFP-cGAS, the nucleus (DAPI), the ER (calnexin), the Golgi apparatus (GM130), and early endosomes (EEA1) in HeLa cells. 2-BP: 2-bromopalmitate; ISD: IFN-stimulatory DNA. Arrows indicate cGAS puncta. Scale bars: 10 μm.


( F HEK293T were transfected with indicated plasmids . Biotin - conjugated ISD was transfected into 6 h before harvesting . Lysates were coprecipitated with streptavidin beads then assessed by immunoblot as shown . Data information : WCL : whole - lysate ; IP : immunoprecipitation . Data are representative of at least two independent experiments .

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(F HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; IP: immunoprecipitation. Data are representative of at least two independent experiments.


G ) HEK293T were transfected with indicated plasmids . Biotin - conjugated ISD was transfected into 6 h before harvesting . Lysates were coprecipitated with streptavidin beads then assessed by immunoblot as shown . Data information : WCL : whole - lysate ; Data are representative of at least two independent experiments .

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G) HEK293T cells were transfected with the indicated plasmids. Biotin-conjugated ISD was transfected into cells 6 h before harvesting. Lysates were coprecipitated with streptavidin beads and then assessed by immunoblot analysis as shown. Data information: WCL: whole-cell lysate; Data are representative of at least two independent experiments.


( H ) Confocal microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 − / − ) transfected with FITC - conjugated ISD for 6 h ( left ) . Scale bar : 20 μm .

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(H) Confocal microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18−/−) transfected with FITC-conjugated ISD for 6 h (left). Scale bar: 20 μm.


( C ) Cross - correlated motions of Cα atoms of WT palmitoylated cGAS .

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(C) Cross-correlated motions of the Cα atoms of the WT and palmitoylated cGAS proteins.


( D ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with plasmids encoding Flag - cGAS , HA - cGAS , Myc - ZDHHC18 ( WT or CS mutant ) for 24 h . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; Data are representative of at least two independent experiments .

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

(D) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with plasmids encoding Flag-cGAS, HA-cGAS, and Myc-ZDHHC18 (WT or CS mutant) for 24 h. Data information: WCL: whole-cell lysate; IP: immunoprecipitation; Data are representative of at least two independent experiments.


( E ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with indicated plasmids followed by addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Data information : WCL : whole - lysate ; IP : immunoprecipitation ; 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments .

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(E) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with the indicated plasmids followed by the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Data information: WCL: whole-cell lysate; IP: immunoprecipitation; 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments.


( F ) FRET of HeLa transfected with indicated plasmids with addition of DMSO , 2 - BP ( 50 μM ) or palmitic acid ( 100 μM ) . Scale bar : 8 μm . ( G ) FRET efficiency of cGAS - GFP cGAS - mCherry in ( F ) . Data information : 2 - BP : 2 - bromopalmitate . Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 (

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

(F) FRET assay of HeLa cells transfected with the indicated plasmids with the addition of DMSO, 2-BP (50 μM) or palmitic acid (100 μM). Scale bar: 8 μm. (G) FRET efficiency of cGAS-GFP and cGAS-mCherry in (F). Data information: 2-BP: 2-bromopalmitate. Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 (G


( H ) Immunoprecipitation ( with an anti - Flag antibody ) immunoblot of HEK293T transfected with Flag - cGAS HA - cGAS ( WT , C405S , C409S C474S ) expression plasmids . Data information : WCL : whole - lysate ; IP : immunoprecipitation Data are representative of at least two independent experiments .

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(H) Immunoprecipitation (with an anti-Flag antibody) and immunoblot analysis of HEK293T cells transfected with Flag-cGAS and HA-cGAS (WT, C405S, C409S and C474S) expression plasmids. Data information: WCL: whole-cell lysate; IP: immunoprecipitation Data are representative of at least two independent experiments.


( I ) FRET of HeLa transfected with indicated plasmids . Scale bar : 8 μm . ( J ) FRET efficiency of cGAS - GFP cGAS - mCherry in ( I ) . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; * , P ˂ 0 . 01 . * * * , P ˂ 0 . 001 J ) .

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(I) FRET assay of HeLa cells transfected with the indicated plasmids. Scale bar: 8 μm. (J) FRET efficiency of cGAS-GFP and cGAS-mCherry in (I). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; *, P˂0.01. ***, P˂0.001 J).


( A ) HEK293T ( 5 × 105 ) were transfected with HA - cGAS ( 200 ng ) , Flag - STING ( 200 ng ) Myc - ZDHHC18 ( WT or CS mutant ) expression plasmids ( 0 , 50 , 100 , or 200 ng ) for 24 h before luciferase reporter assays were conducted . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in DHHC motif of ZDHHC18 data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 . 001 (

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

(A) HEK293T cells (5×105) were transfected with HA-cGAS (200 ng), Flag-STING (200 ng) and Myc-ZDHHC18 (WT or CS mutant) expression plasmids (0, 50, 100, or 200 ng) for 24 h before luciferase reporter assays were conducted. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18 All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001 (A


( B ) HEK293T ( 1 × 106 ) were transfected with HA - cGAS ( 500 ng ) , Flag - STING ( 500 ng ) Myc - ZDHHC18 ( 500 ng ) expression plasmids for 24 h . TBK1 phosphorylation was detected by immunoblotting . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in DHHC motif of

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

(B) HEK293T cells (1×106) were transfected with HA-cGAS (500 ng), Flag-STING (500 ng) and Myc-ZDHHC18 (500 ng) expression plasmids for 24 h. TBK1 phosphorylation was detected by immunoblotting. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18


( C D ) HEK293T were transfected with indicated plasmids , amount of cGAMP in lysates was quantified by LC - MS / MS . Data information : ZDHHC18 ( CS ) : a catalytic mutant with a cysteine - to - serine substitution in DHHC motif of ZDHHC18 ; LC - MS / MS : liquid chromatography - tandem mass spectrometry ;

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

(C and D) HEK293T cells were transfected with the indicated plasmids, and the amount of cGAMP in the lysates was quantified by LC-MS/MS. Data information: ZDHHC18(CS): a catalytic mutant with a cysteine-to-serine substitution in the DHHC motif of ZDHHC18; LC-MS/MS: liquid chromatography-tandem mass spectrometry;


L929 stably transfected with control shRNA or ZDHHC18 shRNA knockdown efficiency of ZDHHC18 is shown by RT - qPCR ( E ) results . Data information : NC : negative control . data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA The knockdown efficiency of ZDHHC18 is shown by RT-qPCR (E) results. Data information: NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting ( F ) results . Data information : NC : negative control .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA efficiency of ZDHHC18 is shown by western blotting (F) results. Data information: NC: negative control.


L929 stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - ( 2 μg / mL ) for indicated times before RT - qPCR of Ifna4 ( G ) , expression . Data information : HT - : herring testis ; NC : negative control . data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of Ifna4 (G), expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


L929 stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - ( 2 μg / mL ) for indicated times before RT - qPCR of IFNb1 ( H ) , Cxcl10 ( I ) expression . Data information : HT - : herring testis ; NC : negative control . data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; ns , not significant ; * * , P ˂ 0 . 01 ; * * * , P ˂ 0 .

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L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before RT-qPCR analysis of IFNb1 (H), and Cxcl10 (I) expression. Data information: HT-DNA: herring testis DNA; NC: negative control. All data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; ns, not significant; **, P˂0.01; ***, P˂0.001


( J ) L929 stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT - ( 2 μg / mL ) for indicated times before immunoblotting with indicated antibodies . Data information : HT - : herring testis ; NC : negative control .

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(J) L929 cells stably transfected with control shRNA or ZDHHC18 shRNA were transfected with HT-DNA (2 μg/mL) for the indicated times before immunoblotting analysis with the indicated antibodies. Data information: HT-DNA: herring testis DNA; NC: negative control.


( A ) Survival of Zdhhc18 + / + Zdhhc18 - / - mice ( n = 8 each , 6 - 8 old ) intranasally injected with VACV ( 5 × 107 PFU per mouse ) monitored daily for survival for 13 days using Kaplan - Meier curves a log - rank ( Mantel - Cox ) . * * * , P ˂ 0 . 001 .

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(A) Survival analysis of Zdhhc18+/+ and Zdhhc18-/- mice (n = 8 each, 6-8 weeks old) intranasally injected with VACV (5 × 107 PFU per mouse) and monitored daily for survival for 13 days using Kaplan-Meier curves and a log-rank (Mantel-Cox) test. ***, P˂0.001.


( B ) Representative photomicrographs of H & E - stained liver kidney tissue sections from mice ( Zdhhc18 + / + or Zdhhc18 - / - ) intravenously injected with HSV - 1 - FS ( 5 × 107 PFU per mouse ) or not after 2 days . Scale bars : 100 μm ( liver ) , 200 μm ( kidney ) . Data information : PFU : plaque - forming units ; H & E : hematoxylin - eosin . Data are representative of at least two independent experiments .

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(B) Representative photomicrographs of H&E-stained liver and kidney tissue sections from mice (Zdhhc18+/+ or Zdhhc18-/-) intravenously injected with HSV-1-FS (5 × 107 PFU per mouse) or not after 2 days. Scale bars: 100 μm (liver), 200 μm (kidney). Data information: PFU: plaque-forming units; H&E: hematoxylin-eosin. Data are representative of at least two independent experiments.


( C BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , viral titers in supernatants were quantified by plaque . Data information : Data are representative of at least two independent experiments .

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(C BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments.


D ) BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with HSV - 1 ( MOI = 2 ) for 8 h , viral titers in supernatants were quantified by plaque . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 . 0001 (

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D) BMDMs (Zdhhc18+/+ or Zdhhc18-/-) were infected with HSV-1 (MOI=2) for 8 h, and viral titers in the supernatants were quantified by plaque assay. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001 (D


( E Frequency of GFP + among infected ( E ) . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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(E Frequency of GFP+ cells among infected cells (E). Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


F ) Microscopy of BMDMs ( Zdhhc18 + / + or Zdhhc18 - / - ) infected with GFP - HSV - 1 ( MOI = 1 ) for 10 h . Scale bar : 200 μm . Data information : Data are representative of at least two independent experiments .

O O O O O O B-CONTROLLED_VAR 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

F) Microscopy of BMDMs (Zdhhc18+/+ or Zdhhc18-/-) infected with GFP-HSV-1 (MOI=1) for 10 h. Scale bar: 200 μm. Data information: Data are representative of at least two independent experiments.


( G ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for indicated times before immunoblot .

O O O O O B-CONTROLLED_VAR 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

(G) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before immunoblot analysis.


( H I ) MEFs ( Zdhhc18 + / + or Zdhhc18 - / - ) were infected with VACV ( 1 : 200 ) for indicated times before RT - qPCR of Cxcl10 ( H ) Ifnb1 ( I ) expression . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

O O O O O O B-CONTROLLED_VAR 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 B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 and I) MEFs (Zdhhc18+/+ or Zdhhc18-/-) were infected with VACV (1:200) for the indicated times before RT-qPCR analysis of Cxcl10 (H) and Ifnb1 (I) expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


( J ) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA ( cocktail ) for 48 h then infected with VACV ( 1 : 200 ) for indicated times before RT - qPCR of IFNB1 expression . Data information : Data are representative of at least two independent experiments . Mean ± from triplicates of technical replicates , unpaired t ; Ns : no significance ; * , P ˂ 0 . 05 ; * * , P ˂ 0 . 01 ; * * * * , P ˂ 0 .

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

(J) PBMCs from healthy people were transfected with scrambled siRNA or ZDHHC18 siRNA (cocktail) for 48 h and then infected with VACV (1:200) for the indicated times before RT-qPCR analysis of IFNB1 expression. Data information: Data are representative of at least two independent experiments. Mean ± SEM from triplicates of technical replicates, unpaired t test; Ns: no significance; *, P˂0.05; **, P˂0.01; ****, P˂0.0001


( C ) Quantitative real time PCR showing head enrichment of dEsyt ; X - axis indicates wild type tissues from which was isolated Y - axis indicates dEsyt transcript level expression normalized to loading control ( RP49 - Ribosomal 49 ) , n = 3 replicates ( biological ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantitative real time PCR analysis showing head enrichment of dEsyt; the X-axis indicates the wild type tissues from which RNA was isolated and Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).


( D ) Quantitative real time PCR showing dEsyt transcript level expression in head samples from wild type sOD . Y - axis indicates dEsyt transcript level expression normalized to loading control ( RP49 - Ribosomal 49 ) , n = 3 replicates ( biological ) .

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

(D) Quantitative real time PCR analysis showing dEsyt transcript level expression in head RNA samples from wild type and sOD. Y-axis indicates the dEsyt transcript level expression normalized to the loading control (RP49- Ribosomal protein 49), n=3 replicates (biological).


( A ) Representative ERG trace from reared 0 - 1 day old flies of indicated genotype stimulated by 2s flash of green . duration of stimulating is shown . Y - axis shows amplitude of ERG response . X - axis shows duration of recording .

O O O O O O O O O O O O O O O O O O O O O O O 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) Representative ERG trace from dark reared 0-1 day old flies of the indicated genotype stimulated by 2s flash of green light. The duration of the stimulating light is shown. Y-axis shows the amplitude of the ERG response. X-axis shows the duration of the recording.


( B ) Graph showing ERG amplitude normalized to . Y - axis shows of ERG amplitude of flies to their . X - axis indicates genotypes . Each data point represents an fly tested . Error bar represents s . e . m .

O O O O O O O O O O O O O O O O O O O O O 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) Graph showing ERG amplitude normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.


( C ) of mean of deep pseudopupil formed by PIP2 probe PH - GFP in one day old flies of indicated genotypes ( n = 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 O

(C) Quantification of the mean fluorescence intensity of the deep pseudopupil formed by the PIP2 probe PH-GFP in the one day old flies of the indicated genotypes (n=10).


( D ) Deep pseudopupil imaging of PIP2 in microvillar of photoreceptors . of PH - GFP probe is depicted . protocol used is shown with red illumination periods shown as red bars flashes of blue ( a - f ) used for image capture depicted . Representative deep pseudopupil acquired at specified time points are depicted . Genotypes as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.


( E ) Graph quantifying recovery kinetics of pseudopupil with time , X - axis represents genotypes , Y - axis represent normalised to of first image . ( n = 10 biological replicates ) . Error bars represent s . e . m .

(E) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalised to the intensity of the first image. (n=10 biological replicates). Error bars represent s.e.m.


( F ) of time course of retinal degeneration . 10 ommatidia from 5 separate flies of each genotype were scored plotted . Y - axis is number of intact rhabdomeres / ommatidium . maximum value possible is 7 . X - axis is of flies post eclosion .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the time course of retinal degeneration. 10 ommatidia from 5 separate flies of each genotype were scored and plotted. Y-axis is the number of intact rhabdomeres/ommatidium. The maximum value possible is 7. X-axis is the age of the flies post eclosion.


( G ) Representative optical neutralization showing rhabdomere integrity of indicated genotypes . Rearing conditions of flies are indicated on top . ( * CL - Constant )

O O O O 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) Representative optical neutralization images showing rhabdomere integrity of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CL- Constant Light)


( H ) Representative ON showing rescue of rhabdomere structural integrity in dEsytKO mutants on expressing dEsyt : : mCherry transgene . Genotypes of flies are indicated on left . Rearing conditions of flies are indicated on top .

O O O O O O O O O O O O O O O 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) Representative ON images showing the rescue of rhabdomere structural integrity in dEsytKO mutants on expressing the dEsyt::mCherry transgene. Genotypes of the flies are indicated on the left. Rearing conditions and the age of the flies are indicated on top.


( A ) ( i ) Representative optical neutralization ( ON ) showing rhabdomere structure of indicated genotypes . Rearing conditions of flies are indicated on top . ( * CD - Constant ) . ( ii ) showing a single ommatidium from photoreceptors of rdgB9 rdgB9 ; dEsytKO mutants reared in 12 hr L / D cycle ( / ) for 2 days . Scale bar : 1µm ( asterisk indicates degenerated rhabdomere ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) (i) Representative optical neutralization (ON) images showing rhabdomere structure of the indicated genotypes. Rearing conditions and the age of the flies are indicated on top. (*CD- Constant Dark). (ii) TEM images showing a single ommatidium from the photoreceptors of rdgB9 and rdgB9;dEsytKO mutants reared in 12 hr L/D cycle ( / ) for 2 days. Scale bar: 1µm (asterisk indicates degenerated rhabdomere).


( B ) of time course taken for retinal degeneration . 10 ommatidia were scored from 5 flies of each genotype plotted .

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

(B) Quantification of the time course taken for retinal degeneration. 10 ommatidia were scored from 5 flies of each genotype and plotted.


( C ) Representative ERG traces showing duration of , X - axis indicates time in msec Y - axis indicates average ERG amplitude in mV .

O O O 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) Representative ERG traces showing the duration of light pulse, X-axis indicates time in msec and Y-axis indicates the average ERG amplitude in mV.


( D ) Graph showing ERG amplitude of rdgB9 rdgB9 ; dEsytKO normalized to . Y - axis shows of ERG amplitude of flies to their . X - axis indicates genotypes . Each data point represents an fly tested . Error bar represents s . e . m .

O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph showing ERG amplitude of rdgB9 and rdgB9;dEsytKO normalized to body weight. Y-axis shows the ratio of ERG amplitude of individual flies to their body weight. X-axis indicates genotypes. Each data point represents an individual fly tested. Error bar represents s.e.m.


( E ) Deep pseudopupil imaging of PIP2 in microvillar of photoreceptors . of PH - GFP probe is depicted . protocol used is shown with red illumination periods shown as red bars flashes of blue ( a - f ) used for image capture depicted . Representative deep pseudopupil acquired at specified time points are depicted . Genotypes as indicated .

(E) Deep pseudopupil imaging of PIP2 levels in the microvillar membrane of photoreceptors. The fluorescence of the PH-GFP probe is depicted. The protocol used is shown with red light illumination periods shown as red bars and flashes of blue light (a-f) used for image capture depicted. Representative deep pseudopupil images acquired at specified time points are depicted. Genotypes as indicated.


( F ) of mean of PIP2 probe PH - GFP from deep pseudopupil formed by one day old flies of indicated genotypes ( n = 10 biological replicates ) .

O O O O O O O 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) Quantification of the mean fluorescence intensity of the PIP2 probe PH-GFP from the deep pseudopupil formed by one day old flies of the indicated genotypes (n=10 biological replicates).


( G ) Graph quantifying recovery kinetics of pseudopupil with time , X - axis represents genotypes , Y - axis represent normalized to of first image . ( n = 10 biological replicates ) .

O O O O O O O O O O O O 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) Graph quantifying the recovery kinetics of the fluorescent pseudopupil with time, X-axis represents the genotypes, Y-axis represent intensity normalized to the intensity of the first image. (n=10 biological replicates).


( A ) Confocal showing localization of endogenous dEsyt in photoreceptors of one day old reared flies probed with antibody against dEsyt . Scale bar : 5µm . dEsytKO shows no when probed with dEsyt antibody .

O O O O O O O O O O O O O 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) Confocal images showing the localization of endogenous dEsyt protein in photoreceptors of one day old dark reared flies probed with antibody against dEsyt. Scale bar: 5µm. dEsytKO shows no staining when probed with dEsyt antibody.


( B ) Confocal showing localization of exogenously expressed dEsyt : : mCherry expressed using eye specific Rh1 - Gal4 in one day old reared flies . Rh1 - Gal4 is shown as a control . A single ommatidium is shown . Scale bar : 5 µm . Phalloidin marks F - actin highlights rhabdomeres R1 - R7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Confocal images showing the localization of exogenously expressed dEsyt::mCherry protein expressed using the eye specific Rh1-Gal4 in one day old dark reared flies. Rh1-Gal4 is shown as a control. A single ommatidium is shown. Scale bar: 5 µm. Phalloidin marks F-actin staining and highlights rhabdomeres R1-R7.


( C ) Western blot from head extracts of wild type dEsytKO probed with antibody against RDGB . Rearing conditions , of flies genotype is indicated on top of blot . Tubulin was used as loading control .

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

(C) Western blot from the head extracts of wild type and dEsytKO probed with the antibody against RDGB. Rearing conditions, age of the flies and genotype is indicated on top of the blot. Tubulin was used as the loading control.


( D , E ) Confocal showing localization of RDGB in wild type dEsytKO photoreceptors of flies which are ( D ) 1 day old - reared ( E ) 6 days old - exposed to constant illumination . For ( D ) ( E ) RDGB visualized using an antibody against endogenous . Rhabdomeres are outlined using phalloidin which marks F - actin . Scale bar : 5 µm .

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

(D, E) Confocal images showing the localization of RDGB in wild type and dEsytKO photoreceptors of flies which are (D) 1 day old-dark reared and (E) 6 days old- exposed to constant illumination. For (D) and (E) RDGB visualized using an antibody against the endogenous protein. Rhabdomeres are outlined using phalloidin which marks F-actin. Scale bar: 5 µm.


( A ) of a single ommatidium from wild type photoreceptors of flies reared in ( i ) day 1 ( D1 ) ( iii ) day 14 ( D14 ) , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from same ommatidium ( ii ) day 1 ( iv ) day 14 . Scale bar : 200 nm . Origin boxes for magnification shown . Arrow in ( Aiv ) indicates SMC forming an MCS with microvillar PM . M - Microvillar plasma , S - Sub microvillar cisternae , MCS - Contact Site , P - Pigment granules . ( B ) of number of MCS per wild type photoreceptor of day 1 day 14 old flies reared in ( from figure 6A ) , Y - axis indicates number of MCS / photoreceptor . n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) for ( i ) . n = 9 photoreceptors from 3 separate flies ( R7 ) in ( ii ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM images of a single ommatidium from wild type photoreceptors of flies reared in dark (i) day 1 (D1) and (iii) day 14 (D14), scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day 1 (iv) day 14. Scale bar: 200 nm. Origin boxes for magnification shown. Arrow in (Aiv) indicates the SMC forming an MCS with the microvillar PM. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per wild type photoreceptor of day 1 and day 14 old flies reared in dark (from figure 6A), Y-axis indicates the number of MCS/photoreceptor. n=30 photoreceptors from 3 separate flies (R1-R6) for (i). n=9 photoreceptors from 3 separate flies (R7) in (ii).


( C ) of a single ommatidium from norpAP24 photoreceptors of flies reared in ( i ) day 1 ( iii ) day 14 , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) . Magnified image showing a single photoreceptor from same ommatidium ( ii ) day1 ( iv ) day 14 . Scale bar : 200 nm . Arrows indicate SMC forming an MCS with microvillar PM . ( D ) of number of MCSs per photoreceptor ( from figure 6C ) , X - axis indicates genotype of flies Y - axis indicates number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 )

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(C) TEM images of a single ommatidium from norpAP24 photoreceptors of flies reared in dark (i) day 1 and (iii) day 14, scale bar: 1µm (red asterisk indicates R7 photoreceptor). Magnified image showing a single photoreceptor from the same ommatidium (ii) day1 (iv) day 14. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCSs per photoreceptor (from figure 6C), X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7)


( E ) of a single ommatidium from ( i ) wild type ( iii ) dEsytKO photoreceptors of 0 - 1 day old flies reared in , scale bar : 1µm ( red asterisk indicates R7 photoreceptor ) ( ii , iv ) Magnified image showing a single photoreceptor from ommatidium image shown on left . Scale bar : 200 nm . Arrows indicate SMC forming an MCS with microvillar PM . ( F ) of number of MCS per photoreceptor of wild type day 1 v / s dEsytKO day 1 old flies reared in , X - axis indicates genotype of flies Y - axis indicates number of MCS / photoreceptor ( i ) n = 30 photoreceptors from 3 separate flies ( R1 - R6 ) ( ii ) n = 9 photoreceptors from 3 separate flies ( R7 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM images of a single ommatidium from (i) wild type and (iii) dEsytKO photoreceptors of 0-1 day old flies reared in dark, scale bar: 1µm (red asterisk indicates R7 photoreceptor) (ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (F) Quantification of the number of MCS per photoreceptor of wild type day 1 v/s dEsytKO day 1 old flies reared in dark, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor (i) n=30 photoreceptors from 3 separate flies (R1-R6) (ii) n=9 photoreceptors from 3 separate flies (R7).


( A ) of a single ommatidium from photoreceptors of ( i ) dEsytKO - day 1 ( iii ) dEsytKO - day 14 flies reared in , scale bar : 1 µm except for ( Ai ) : 2 µm ( red asterisk indicates R7 photoreceptor ) . ( A - ii , iv ) Magnified image showing a single photoreceptor from ommatidium image shown on left . Scale bar : 200 nm . Boxes represent origin boxes for magnification . M - Microvillar plasma , S - Sub microvillar cisternae , MCS - Contact Site , P - Pigment granules . ( B ) of number of MCS per photoreceptor , X - axis indicates genotype of flies Y - axis indicates number of MCS / photoreceptor . n = 9 photoreceptors from 3 separate flies for R7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM images of a single ommatidium from photoreceptors of (i) dEsytKO- day 1 (iii) dEsytKO- day 14 flies reared in dark, scale bar: 1 µm except for (Ai): 2 µm (red asterisk indicates R7 photoreceptor). (A-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Boxes represent the origin boxes for magnification. M- Microvillar plasma membrane, S- Sub microvillar cisternae, MCS- Membrane Contact Site, P- Pigment granules. (B) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. n=9 photoreceptors from 3 separate flies for R7.


( C ) of a single ommatidium from photoreceptors of ( i ) norpAP24 ; dEsytKO - day 1 ( iii ) norpAP24 ; dEsytKO - day 14 flies reared in , scale bar : 1 µm ( red asterisk indicates R7 photoreceptor ) . ( C - ii , iv ) Magnified image showing a single photoreceptor from ommatidium image shown on left . Scale bar : 200 nm . Arrows indicate SMC forming an MCS with microvillar PM . ( D ) of number of MCS per photoreceptor , X - axis indicates genotype of flies Y - axis indicates number of MCS / photoreceptor . ( i ) n = 30 photoreceptors from 3 separate flies for R1 - R6 . ( ii ) n = 9 photoreceptors from 3 separate flies for R7 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TEM images of a single ommatidium from photoreceptors of (i) norpAP24;dEsytKO- day 1 (iii) norpAP24;dEsytKO- day 14 flies reared in dark, scale bar: 1 µm (red asterisk indicates R7 photoreceptor). (C-ii, iv) Magnified image showing a single photoreceptor from the ommatidium image shown on the left. Scale bar: 200 nm. Arrows indicate the SMC forming an MCS with the microvillar PM. (D) Quantification of the number of MCS per photoreceptor, X-axis indicates the genotype and age of the flies and Y-axis indicates the number of MCS/photoreceptor. (i) n=30 photoreceptors from 3 separate flies for R1-R6. (ii) n=9 photoreceptors from 3 separate flies for R7.