EMBO/SourceData huggingface dataset:
Figure 1A) Volcano plot of proteome analysis. Plotted is the negative log10 of the p-value (y-axis) versus the log2 LFQ ratio of SPPL2c overexpressing and control cells (x-axis) of a given identified protein. A permutation based FDR correction for multiple hypotheses was applied (p = 0.05; s0 = 0.1; dashed line). Open grey circles indicate proteins not-significantly altered in SPPL2c expressing cells, red circles significantly altered proteins after FDR correction. Filled red circles indicate type IV membrane proteins and filled blue circles with red line and blue names type II proteins altered in SPPL2c expressing cells, n=6 biological repl. per cell line.C) Western Blot of selected type IV SPPL2c candidate substrates. Endogenous protein levels of VAPA, VAPB, VAMP8, Syntaxin 8, 18 (Stx8 & Stx18) and Membrin (encoded by the gene Golgi SNAP Receptor Complex Member 2, GOSR2) were analyzed in HEK293 cells ectopically expressing wild type SPPL2c (SPPL2c WT) or a catalytically inactive SPPL2c (SPPL2c D/A). Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. VAPA, VAPB, VAMP8, Stx8 and Membrin protein levels were decreased in SPPL2c expressing cells but not in control or SPPL2c D/A expressing cells. For Stx18 a cleavage product was detected in SPPL2c WT expressing cells. Expression levels of SPPL2c are depicted and Calnexin serves as loading control.
Figure 2A) SPPL2c overexpression causes a decrease on SPPL3 levels. Endogenous levels of SPPL3 were assessed in cells ectopically expressing wild type SPPL2c (SPPL2c WT) or catalytically inactive SPPL2c (SPPL2c D/A) by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. Note that SPPL3 expression is reduced in cells expressing SPPL2c wt. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlB) SPPL2c impairs secretion and maturation of glycan-modifying enzymes. Protein levels of endogenous EXTL3, B4GALT1, OGFOD3 and GnT-V were monitored in lysates of cells treated with control siRNA (siCtr) or with siRNA targeting SPPL3 (siSPPL3) and in cells ectopically expressing either SPPL2c or SPPL3 by Western Blot. In addition, the secreted forms (sEXTL3, sB4GALT1, sOGFOD3, sGnTV,) were analyzed in the corresponding conditioned media using Western Blot. Note that SPPL2c reduces secretion of the glycosyltransferases similar to SPPL3 knock down, but additionally induces a decrease of the mature glycosyltransferases in the cell lysate similar to ectopic expression of SPPL3. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlC) Processing of ectopically expressed GnT-V with an N-terminal Flag- and C-terminal V5-tag (Flag-GnTV-V5) was analyzed in lysates and conditioned media (sup.) of either SPPL2c wt or SPPL2c D/A expressing cells by Western Blot. Non-transfected cells (-) or stably transfected but non-induced SPPL2c wt cells (n/i) served as controls. In addition, maturation of the glycoproteins Nicastrin (NCT), Lamp2 and SPPL2a was monitored by detection of the respective mature (mat) and immature (im) version. Note that secretion of GnT-V is impaired in SPPL2c wt expressing cells and immature glycoproteins accumulate. Data information: Expression levels of the ectopically expressed SPPL proteases are shown and Calnexin serves as loading controlD) SPPL2c induces mislocalization of GnT-V. Ectopically expressed GnT-V was visualized by immunofluorescence staining using the anti-V5 antibody either in control cells (Ctr) or in cells co-expressing either SPPL2c wt or SPPL2c D/A. The ER was stained using the anti-BiP antibody and the Golgi with the anti-TGN46 antibody. While in control cells GnT-V mainly localizes to the Golgi, its localization shifts to more ER-like structures in SPPL2c expressing cells. Scale bar 5 µm.
Figure 4B) Processing of endogenous Syntaxin 5 (Stx5) and Syntaxin 6 (Stx6) by SPPL2c wt by Western Blot. Non-transfected cells (-), stably transfected but non-induced SPPL2c wt cells (n/i) and induced cells stably expressing SPPL2c D/A served as controls. Results are derived from one experiment, thus expression (SPPL2c) and loading (Calnexin) control are the same. Note, that Syntaxin 5 is cleaved by SPPL2c while Syntaxin 6 is not.C) Syntaxin 5 protein levels were down regulated and processing and maturation of ectopically expressed GnT-V, as well maturation of the endogenous glycoproteins Nicastrin (NCT) and SPPL2a was analyzed in Western Blot. Calnexin served as loading control and ectopic expression of SPPL2c WT was confirmed using a SPPL2c specific antibody. While GnT-V and Nicastrin maturation were similarly affected by Syntaxin 5 knock down and SPPL2c overexpression, SPPL2a maturation was hardly impaired in Syntaxin 5 knock down cells.D) Syntaxin 5 localization is impaired by overexpression of SPPL2c. Endogenous Syntaxin 5 was visualized in control cells without SPPL2c expression (Ctr) and in cells overexpressing SPPL2c wt or SPPL2c D/A using immunofluorescence. BiP was used as a marker protein of the ER and TGN46 as Golgi marker. White arrow indicating cells with disturbed Golgi and Syntaxin 5 staining. Scale bar 5 µm.E) Syntaxin 5 knock down induces mislocalization of GnT-V similar to SPPL2c overexpression Cells ectopically expressing GnT-V were treated either with non-specific siRNA (siCtr) or with siRNA targeting Syntaxin 5 (siStx5). GnT-V was visualized in immunofluorescence using anti-V5 antibody targeting its C-terminal tag and either BIP was co-stained as ER marker protein or TGN46 as Golgi marker protein. Scale bar 5 µm.
Figure 5After seeding cells were cultured for 72h and expression of catalytically active (wt) or non-active (D/A) SPPL2c was induced by addition of doxycycline for either 24h or 48h as indicated. Non-transfected cells (Ctr) or non-induced SPPL2c cells (n/i) served as controls. The ER was stained with the anti-BiP antibody, the cis/medial-Golgi with the anti-Giantin, the trans-Golgi Network with the anti-TGN46 antibody and all Golgi subcompartments with the CAB45-specific antibody in immunofluorescence. Note that only upon expression of catalytically active SPPL2c the morphologies of ER and cis-Golgi change compared to controls. Scale bar 5 µm.
Figure 6A) Western Blot analysis of SPPL2c in membrane preparations from HEK293 inducibly overexpressing SPPL2c, human testis and mouse testis tissue. For human and mouse tissue, 5 μg protein of each total testis preparation is loaded. The membrane fraction obtained from a confluent 6 cm dish of SPPL2c expressing HEK293 cells is diluted 1:50 (0.1 μg total protein).B) Western Blot analysis of selected glycoproteins in mouse tissues from two wildtype C57BL/6J (Black6) mice. Endogenous Nicastrin (NCT) and SPPL2a were analysed in membrane preparations from testis, kidney, brain, spleen and liver in littermate male mice. Note that immature proteins (im) accumulate in testis compared to other tissues. SPPL2c protein is only detected in testis tissue.C) Analysis of Syntaxin 8 (Stx8) in SPPL2c-/- mice compared to wildtype (wt) littermates. Membrane isolates from whole testis were analyzed for Syntaxin 8 (Stx8) and SPPL2c expression. TMEM192 serves as loading control.D) Syntaxin 8 protein levels were quantified from the respective Western blots by densitometric analysis and normalized to those of TMEM192. Bars represent mean values of n=3 animals ± SD normalized to mean Syntaxin 8 levels of wildtype mice. Statistical significance was calculated applying an unpaired, two-sided Student's t-test. * p≤0.05. Note that Syntaxin 8 is accumulating in SPPL2c-/- mice.E) Real-time qPCR analysis of Syntaxin 8 (Stx8) mRNA levels in testis of SPPL2c-/- and the wildtype mice normalized to that of Tuba1a. The diagram depicts mean values of Stx8 mRNA levels normalized to that of wildtype animals of n=6 individuals from two independent experiments ± SD. For statistical analysis an unpaired, two-sided Student's t-test was performed. ns = not significant.F) Immunohistochemical stainings of cross-sectioned seminiferous tubules of SPPL2c+/+ and SPPL2c-/- mice using anti-Cab45 antibody. Stages VII/VIII are depicted. Higher magnifications are indicated with dashed lines and black arrows indicate pre-acrosomal structures. Scale bar 50 µm.G) Volcano plot depicting the binding affinities of lectins on sperm lysates from SPPL2c-/- mice compared to SPPL2c+/+ (wt). Each circle represents one lectin. The lectins above the dashed line (light blue) have a significant p-value<0.05. Lectins on the left side of the blot demonstrate reduced binding while UEA-I on the right side has increased binding. n=4 animals per genotype, two-sided, unpaired Student's t-Test.
Figure 1(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP, panels d, e, and f) for 30 min and then were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) in the continuous presence of dbcAMP. Cells without any treatment were used as control (−dbcAMP, panels a, b, and c). Cells were immediately analyzed by confocal microscopy. A quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown in the right panel (n = 100 cells/condition, * p<0.05 paired Student's t-test). These data are representative of three independent experiments.(B) GFP-LC3 CHO cells were incubated with complete medium in the absence (−dbcAMP) or presence of dbcAMP (+dbcAMP) and treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv) with (lower panel) or without (upper panel) bafilomycin A1 to block lysosomal degradation. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of three independent experiments. (C) The band intensities of two independent experiments were quantificated with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).
Figure 2(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM H89, a PKA inhibitor, in the presence (panels a, b, and c) or absence (panels d, e, and f) of 1 mM dbcAMP. Subsequently, they were incubated for 2 h in starvation medium (Stv, panels b and e) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels c and f) in full nutrient media. Cells without any treatment were used as control (Ctr, panels a and d). Cells were finally analyzed by confocal microscopy. Images are representative of three independent experiments.(B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. ** p<0.01 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.(C) GFP-LC3 CHO cells were preincubated for 30 min in the presence or absence of 10 µM H89, and then they were incubated for 2 h with 1 mM dbcAMP to verify the activity of H89. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-phosphoCREB and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantified with the Adobe Photoshop program (lower panel). These data are representative of two independent experiments.
Figure 3(A) GFP-LC3 CHO cells were preincubated with 10 µM 8-pCPT-2′-O-Me-cAMP (8-pCPT) for 30 min and then they were treated for 4 h with 10 µg/ml of α-hemolysin (Hla) or subjected to starvation conditions (Stv). Cells without any treatment were used as control. Cells were immediately analyzed by confocal microscopy. Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions is shown in the right panel (n = 50 cells/condition). These data are representative of two independent experiments.(B) CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (left panel) or myc-ΔEPAC (right panel). Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (C) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions (n = 20 cells/condition). These data are representative of two independent experiments
Figure 4(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (left panel) or GFP-Rap2b ΔAAX (right panel). Twenty-four hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) in full nutrient media, or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of three independent experiments.(B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions is shown. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.(C) CHO cells were transfected with GFP-Rap2b wt or GFP-Rap2b ΔAAX and incubated with complete medium without (Ctr) or with 10 µg/ml of α-hemolysin (Hla) for 4 h, with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h in the absence or presence of bafilomycin A1. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.(D) Quantification of the Western blot bands intensities of two independent experiments with the Adobe Photoshop program.
Figure 5(A) HeLa cells were cotransfected with Rap2b siRNA and GFP-LC3 (right panel) or irrelevant siRNA and GFP-LC3 (left panel). Forty-eight hours after transfection, they were incubated for 2 h in starvation medium (Stv), with 50 ng/µl of rapamycin (Rapa) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments.(B) Quantification of the percentage of cells presenting LC3 puncta upon incubation under the different conditions. Data correspond to two independent experiments (n = 50 cells/condition).(C) Upper panel: The knockdown of Rap2b was determined by Western blot as indicated in Materials and Methods. Lower panel: The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).(D) HeLa cells were cotransfected with GFP-LC3 and Rap2b siRNA or an irrelevant siRNA and incubated for 4 h with complete medium in the absence (Ctr) or presence of 10 µg/ml of α-hemolysin (Hla), with 50 ng/µl of rapamycin (Rapa) or subjected to starvation conditions (Stv) for 2 h. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody, and subsequently developed with an enhanced chemiluminescence detection kit. These data are representative of two independent experiments.(E) The band intensities of two independent experiments were quantified with the Adobe Photoshop program, and normalized against tubulin. * p<0.05 (paired Student's t-test).
Figure 6cAMP cannot inhibit the autophagy induced by the toxin in cells overexpressing the Rap2b negative mutant.(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b ΔAAX. Twenty-four hours after transfection they were incubated for 2 h in starvation medium (Stv) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla) in the presence (right panels) or absence of 1 mM dbcAMP (left panels). Cells without any treatment were used as control (Ctr). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. These data are representative of two independent experiments.
Figure 7(A) GFP-LC3 CHO cells were preincubated for 30 min with 10 µM of calpeptin (a calpain inhibitor, panels b, f, and j), with 1 mM dbcAMP (panels d, h and l), or with Calpeptin+dbcAMP (panels c, g, and k). Afterwards, they were incubated for 2 h in starvation medium (Stv, panels e, f, g, and h) or treated for 4 h with 10 µg/ml of α-hemolysin (Hla, panels i, j, k, and l). Cells without any treatment were used as control (Ctr, panels a, b, c and d). Cells were analyzed by confocal microscopy. Images are representative of two independent experiments. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation with the different conditions. * p<0.05 (paired Student's t-test, n = 100 cells/condition). These data are representative of two independent experiments.(C) Upper panel: CHO cells were preincubated for 30 min with 10 µM of calpeptin, with 1 mM dbcAMP, or with calpeptin+dbcAMP, and then they were incubated for 4 h in complete medium in the presence (Hla) or absence (Ctr) of α-hemolysin. Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-LC3 and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. Lower panel: Quantification of the band intensities with the Adobe Photoshop program. * p<0.05 (paired Student's t-test). These data are representative of three independent experiments.
Figure 8(A) GFP-LC3 CHO cells were preincubated with 1 mM dybutiryl cAMP (+dbcAMP) for 30 min and then were infected for 4 h in the presence of db cAMP with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−) +pHla). Cells without any treatment were used as control (−dbcAMP). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods, and immediately visualized by confocal microscopy. Images are representative of two independent experiments.. (B) Quantification of the percentage of cells presenting LC3 puncta (i.e., stimulated cells) upon incubation in the different conditions. * p<0.05, ** p<0.01 (paired Student's t-test, n = 50 cells/condition). These data are representative of two independent experiments.(C) Quantification of the number of CFU/ml of CHO cells infected for 3 h with the wt strain of S. aureus, the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid in the absence or presence of cAMP. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.
Figure 9CHO cells were cotransfected with GFP-LC3 and myc-EPAC wt (upper panel) or myc-ΔEPAC (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. Images are representative of three independent experiments.B) Quantification of the percentage of cells presenting LC3 puncta upon the infection. These data are representative of three independent experiments.(C) Quantification of the percentage of bacteria decorated with EPAC in cells infected with S. aureus. ** p<0.01 (paired Student's t-test, n = 20 cells/condition). These data are representative of three independent experiments.
Figure 10(A) CHO cells were cotransfected with RFP-LC3 and GFP-Rap2b wt (upper panel) or GFP-Rap2b ΔAAX (lower panel). Twenty-four hours after transfection, they were infected for 4 h with the wt strain of S. aureus (wt), the mutant deficient for α-hemolysin (Hla−), or the Hla(−) mutant expressing an α-hemolysin plasmid (Hla(−)+pHla). The nucleus and the bacteria were labeled with TOPRO as indicated in Materials and Methods and immediately visualized by confocal microscopy. These data are representative of three independent experiments.(B) Quantification of the percentage of infected cells presenting LC3 puncta upon the infection. * p<0.05, *** p<0.001 (paired Student's t-test, n = 50 cells/condition). These data are representative of three independent experiments.(C) Quantification of the percentage of bacteria decorated with Rap2b wt upon the infection. ** p<0.01 (paired Student's t-test, n = 30 cells/condition). These data are representative of three independent experim
Figure 11(A) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Afterwards, cells were lysed with sample buffer and the samples were subjected to Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. A quantification of the bands intensities with the Adobe Photoshop program is shown in the lower panel. These data are representative of two independent experiments.B) HeLa cells were incubated for 4 h with complete medium in the presence (Hla) or absence (Ctr) of 10 µg/ml α-hemolysin or they were infected for 4 h with the wt strain of S. aureus (wt) or the α-hemolysin deficient mutant (Hla−). Cells were disrupted and whole cell lysates were subjected to pull-down assays using GST-Ral-GDS-RBD-sepharose. The levels of GTP-bound Rap2b were determined as was described in Materials and Methods by Western blot analysis using a rabbit anti-Rap2b and the corresponding HRP-labeled secondary antibody. The bands were subsequently developed with an enhanced chemiluminescence detection kit. The band intensities were quantificated with the Adobe Photoshop program is shown in the lower panel. * p<0.05 (paired Student's t-test). These data are representative of two independent experiments.
f1(a) Confocal analysis of the subcellular localization of HA-PLD1 (green) and endogenous LC3 (red) in HeLa cells in normal medium and under nutrient deprivation conditions. Left panels: normal medium condition (Nm); middle panels: 1 h with nutrient-deprived medium (1 h St); right panels: 2 h with nutrient-deprived medium (2 h St). Insets denote high magnification of the selected area. Scale bar, 5 μm.(b) Immuno-gold EM micrographs of HeLa cells transfected with GFP-PLD1 and subjected to 1 h of nutrient deprivation in the presence (St+B) or absence (St) of bafilomycin (50 nM). Insets denote high magnification of the selected area. Scale bar, 500 nm. Right panel: Histogram showing morphometric analysis of the relative distribution of GFP-PLD1-positive gold particles on the outer (arrows) and inner (arrowheads) membranes of the autophagosomal-like structures. Values denote means±s.e.m. (n=14). **P0.01.