EMBO/SourceData huggingface dataset:
Figure 7A Transmission electron microscopy (TEM) cross sections of WT, dhhc2kd and ∆isp1/3 parasites showing the arrangement of SPM underneath IMC. Approximately 60 recognizable hollow microtubules (green) are associated with IMC (red), distributing evenly around circumference with an interval of 100-120 nm in WT ookinetes. Microtubules lost association with IMC in the ookinetes of dhhc2kd and ∆isp1/3 parasites. Left: one representative cell in each parasite is shown; Right: two areas are magnified to show the details. Lower panel is the quantification of distance between adjacent microtubules associated with IMC. Values are mean ± SD for n=204 (WT), 189 (dhhc2kd), and 211 (∆isp1/3) measurements in 20 cells each group from two independent experiments; Kolmogorov-Smirnov test, ****, P<0.0001.B Apical polar ring and the emanating SPM in WT, dhhc2kd and ∆isp1/3 parasites examined by negative staining and TEM. A dome-like SPM structure radiating from the apical polar ring was observed in early (3 hours) or mature (12 hours) WT ookinetes, but impaired in mutant parasites. n is the number of cells analyzed from three independent experiments. More images in the Appendix Figure S5. Scale bar = 1μm.C SiR-tubulin staining show the SPM structures of WT, dhhc2kd and ∆isp1/3 parasites. Arrowheads point to cell apical. Scale bar = 5 μm.
Figure 8A GST-ISP1 pulls down endogenous β-Tubulin from cell lysate of WT ookinetes. Lower panel indicates the Coomassie blue stain of recombinant GST and GST-ISP1.B Episomally expressed ISP1::HA immunoprecipitated with β-Tubulin in WT ookinetes. Episomal vector as control.C Endogenous ISP1::3V5 immunoprecipitated with β-Tubulin in the TTS ookinetes.D Co-localization of ISP1::HA and β-Tubulin at ookinete periphery in IFA. Scale bar = 5 μm.E Proximity ligation assay (PLA) detecting interaction of ISP1-HA and β-Tubulin at ookinete. Episomal vector as control. Scale bar = 5 μm.F Solubility assay detecting membrane association of α- and β-Tubulins in WT, dhhc2kd, and ∆isp1/3 parasites. GAPDH is a cytosol soluble protein, P28 is a plasma membrane integral protein.G Quantification of α- and β-Tubulins membrane association in F. The membrane association index is defined as the ratio of Carb fraction to Hypo fraction in F, and normalized to that of WT. Values are means ± SEM from three independent tests.H Linear correlation of membrane association index of α-Tubulin and β-Tubulin with the conversion rate to mature ookinete among WT, dhhc2kd and ∆isp1/3 parasites. Horizontal and vertical values are means ± SD.I Membrane association of α- and β-Tubulins in WT ookinete culture after DMSO, 2-BP, or colchicine treatment.J Quantification of α- and β-Tubulins membrane association Values are means ± SEM from three independent repeats.
Figure 1(D-E) Measurements of ciliary cAMP levels using cilia-Pink Flamindo. (D) Exemplary images are shown for cilia (cilia-bPAC or cyto-bPAC cells) expressing the cilia-Pink Flamindo biosensor after light stimulation. Scale bars are indicated; cAMP levels are color-coded using a high-low look-up table. (E) Quantification of data exemplified in (D). The cAMP level is determined as the mean ciliary fluorescence intensity during the first 60 s after light stimulation (measurement interval 10 s); p value for a Kolmogorov-Smirnov test is indicated. Each data point represents an individual cilium. Bars display median and interquartile range; cilia-bPAC: 40 cilia (biological replicates) from n = 14 independent experiments; cyto-bPAC: 31 cilia (biological replicates) from n = 31 independent experiments.
Figure 2(A) Wild-type (WT) mIMCD-3 cells cultured in a 3D matrix during continuous exposure to DMSO (control) or 10 µM of Forskolin. Exemplary images are shown (n = 5 biological replicates). Quantification of cyst number and area is shown on the right Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment, p values calculated using an unpaired, two-sided Student's t-test are indicated.(D) mIMCD-3 cells stably expressing cilia-bPAC or cyto-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) or in the dark and incubated with 10 µM of Forskolin. Exemplary images are shown (n > 3). (E) Quantification of the data exemplified in (D). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using an unpaired, two-sided Student's t-test are indicated.
Figure 3(D) Immunoblotting of lysates from mIMCD-3 wild-type (WT) cells and mIMCD-3 cells stably expressing cilia-bPAC for S6 ribosomal protein (S6RP) phosphorylation at Ser235/236 (pS6RP). Levels of total S6RP protein (S6RP) and beta-Tubulin have been determined as controls. Cilia-bPAC cells were treated with DMSO (control) or with 10 nM of Rapamycin. All cells were stimulated with light (1 h light/1 h dark, 48 h, 465 nm, 38.8 µW/cm²) (E) Quantification of the pS6RP/S6RP ratio. Data are shown as mean ± S.D., n > 3; p values were calculated using a paired, two-sided Student's t-test. Each data point shows an independent experiment(G) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark, during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubation with 10 nM rapamycin. Light exposure started 2 d later than incubation with 10 nM rapamycin. Exemplary images are shown (n = 3) (H) Quantification of the data exemplified in (G). Data are shown as mean ± S.D., each datapoint corresponds to an independent experiment; p values calculated using a paired, two-sided Student's t-test are indicated
Figure 4(A) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix in the dark or during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm²) and incubated with DMSO (control), 250 µM IBMX, a ubiquitous PDE inhibitor, or 10 µM Rolipram, a PDE4-specific inhibitor. Exemplary images are shown (n = 3). Scale bars are indicated. (B) Quantification of data exemplified in (A). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual experiments.(C) mIMCD-3 cells stably expressing cyto-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO or 10 µM rolipram. Exemplary images are shown (n = 4-5). Scale bars are indicated. (D) Quantification of data exemplified in (C) Data are shown as mean ± S.D., p values calculated using an unpaired Student's t-test are indicated. Data points show individual experiments.(F) Analysis of Pde4 isoform expression (long and short isoforms) in wild-type mIMCD-3 cells, revealed by immunoblotting against PDE4A, PDE4B, PDE4D, and PDE4D.(H) mIMCD-3 cells stably expressing cilia-bPAC, cultured in a 3D matrix during light exposure (1 h light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², 1 day after pharmacological stimulus) and incubation with DMSO (0 µM), 3 µM, or 10 µM of the Pde4-long-isoform activator MR-L8. Exemplary images are shown (n = 3). Scale bars are indicated. (I) Quantification of data exemplified in (H). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data have been normalized to the Light, 0 µM MR-L8 condition (Set to 100%) within each experiment. Data points show individual experiments.
Figure 5(D) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2. Exemplary images are shown (n = 3 biological replicates). (E) Quantification of images exemplified in (D). Data are shown as mean ± S.D., p values calculated using an unpaired, two-sided Student's t-test are indicated. Data points show individual biological replicates. (F) Quantification of the cyst area in PGE2-stimulated mIMCD-3 WT cells in the presence of the PDE4 long isoform-specific activator MR-L8. Data were normalized to PGE2- and control-treated cells and are shown as mean ± S.D. p values calculated using a one-sample t-test compared to 100% are indicated, n = 4 biological replicates.(G) 3D culture of mIMCD-3 WT cells continuously exposed to acetone (control) or 100 nM PGE2, and DMSO (control) or 10 µM of the EP4 inhibitor L161,982. PGE2 treatment started 2 d after seeding. Exemplary images are shown (n = 3). Scale bars are indicated. (H) Quantification for images exemplified in (G), including addition of 3 µM AH6809, an EP2 inhibitor Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated (p value of n/a indicates that the test is not applicable because both conditions showed no variance). Data points show individual experiments and have been normalized to addition of 100 nM PGE2 (set to 100%) within each experiment.(I) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or the EP4-inhibitor L161,982 in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or L161,982). Exemplary images are shown (n = 3). (J) Quantification of images exemplified in (I). Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments and have been normalized to the light/DMSO condition (Set to 100%) within each experiment.
Figure 6(F) 3D culture of mIMCD-3 cells stably expressing cilia-bPAC, incubated with DMSO (control) or 20 µM Celecoxib, a COX-2-inhibitor, in the dark or during light exposure (1 light/1 h dark, 9 days, 465 nm, 38.8 µW/cm², started 2 d after pre-incubation with DMSO or Celecoxib). Exemplary images are shown (n = 3). Scale bars are indicated. (G) Quantification of images exemplified in (F) and from 3D cultures incubated with 1 μM celecoxib. Data are shown as mean ± S.D., p values calculated using a paired, two-sided Student's t-test are indicated. Data points show individual experiments.
Figure 7(A) Wild-type (WT), cilia-bPAC, or cyto-bPAC (magenta) mIMCD-3 cells were stimulated by light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²), and treated with DMSO (control) or 50 µM Ciliobrevin-D, a dynein-inhibitor. Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. B) Quantification of the ciliary pCREB signal in cilia-bPAC and cyto-bPAC cells. Data are shown as mean ± S.D., n > 3. Each data point represents an independent experiment and corresponds to the median of > 73 cilia (biological replicates); p values for an unpaired, two-sided Student's t-test with Welch's correction are indicated.(C) mIMCD-3 Ift27-/- cells with or without stable cilia-bPAC expression (magenta) were stimulated with light (for 1 h prior to fixation, 465 nm, 38.8 µW/cm²). Cells were labeled with DAPI (blue) to label the DNA, an ARL13B antibody (white) to label cilia, and a phospho-specific (Ser133) CREB antibody (pCREB, green). Arrows indicate the direction and the length of the shift of the respective fluorescence channel. The box indicates the position of the magnified view shown on the right of each panel. (D) Quantification of the ciliary pCREB signal. Data are shown as mean ± S.D., n > 3. Each data point represents the median of > 90 cilia from the same experiment; p values are indicated for an unpaired, two-sided Student's t-test with Welch's correction.
Figure 3B. Validation of the TNF-RSC interacting proteins. A549 cells were stimulated with FLAG-TNF-α for the indicated time and the TNF-RSCs were isolated as described in Figure 3A. TNF-α-stimulation-dependent interaction of the indicated proteins was confirmed by isolating the TNF-RSC and blotting with the indicated antibodies. The amount of the analyzed proteins in the input material used for the TNF-RSC isolation is also shown.C. Quantification of ubiquitin linkage peptides in the TNF-RSC. The box plot shows the relative abundance of K48, K63 and linear ubiquitin linkage-specific peptides in the TNF-RSCs isolated from TNF-α-stimulated (15 min) and control cells quantified by SILAC-based MS.E. The list of proteins enriched in SPATA2pull-down. The table shows proteins that were enriched in GFP-SPATA2pull-down. The columns under "Unstimulated" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from unstimulated cells, whereas the columns under "Stimulated (TNF-α 15 min)" shows SILAC ratios of the indicated proteins in SPATA2 pull-downs from cells stimulated with TNF-α for 15 minutes.F. Validation of TNF-α-stimulation-independent association of SPATA2 with CYLD. GFP-SPATA2 was immunoprecipitated from unstimulated and TNF-α-stimulated cells, and probed with CYLD antibody.G. HOIP interacts with SPATA2. FLAG-HOIP expressing A20.2J cells were SILAC labeled and FLAG-HOIP was immunoprecipitated from unstimulated cells and associated proteins were quantified using LC-MS/MS. The table contains proteins that were enriched in HOIPpull-down compared to control pull-down.H. Validation of SPATA2 interaction with HOIP. GFP-tagged RNF31 was purified from unstimulated or TNF-a-stimulated (15 min) HCT116 cells and the immunoprecipitates were blotted with SPATA2, CYLD and GFP (HOIP) antibodies. The lower panels show expression of the indicated proteins in the input. The asterisk indicates unspecific band.
Figure 4B. The predicted PUB domain in the N-terminus of SPATA2 interacts with CYLD, but not with LUBAC or other TNF-RSC components. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells that were stimulated with TNF-α for 15 minutes, and the enriched proteins were quantified using SILAC-based MS.C. Validation of SPATA2 N-terminus interaction with CYLD. GFP-SPATA2 and the indicated deletion mutants were purified from A549 cells as described in Figure 5A, and association of CYLD was analyzed by immunoblotting. The lower panels show controls.D. The USP domain of CYLD interacts with SPATA2. STREP-FLAG-CYLD and the indicated deletion mutants were purified from A549 cells using Step-Tactin sepharose and blotted with SPATA2 antibody. The lower panels show controls.E. The C-terminus of SPATA2 interacts with HOIP. A549 cells were transiently transfected with GFP-tagged SPATA2, SPATA2 D200 and SPATA2 200*, and SPATA2 and the mutants were immunoprecipitated using GFP-trap beads. The immunoprecipitates were immunoblotted with HOIP, CYLD and GFP (HOIP) antibodies. The lower panels show input control, and the * shows unspecific band.
Figure 5B. Effect of SPATA2 knockdown on association of proteins with TNF-RSC. The list shows SILAC ratios of TNF-RSC-associated proteins in control cells and SPATA2 knockdown cells. The proteins that showed reduced association with the TNF-RSC after SPATA2 knockdown are highlighted red.C. Validation of SPATA2-dependent association of CYLD with the TNF-RSC. The TNF-RSC was purified under the indicated conditions and association of SPATA2, CYLD, RIPK1 and HOIP with the TNF-RSC was analyzed by immunoblotting. The input controls show expression of the analyzed proteins in cell lysates. The asterisk denotes unspecific bands.
Figure 6A. SPATA2 regulates TNF-a-induced NF-κB activation. HEK293T cells were transfected with the SPATA2 siRNA (SMARTpool or with to individual siRNA) or a non-targeting control siRNA, and NF-kB activation was monitored with luciferase-based NF-kB reporter assays. Error bars specify the standard error of the mean (SEM) of three independent experiments. p** < 0.05 (two-tailed, unpaired Student´s t-test).B. Knockdown of SPATA2 enhances expression of the NF-κB target genes. HEK293T cells were transfected with SPATA2 siRNA or a non-targeting control siRNA and the expression of the indicated mRNAs was measured in cells stimulated with TNF-a for 0, 3 and 6 hours with qPCR.C. SPATA2 is involved in TNF-α-induced necroptosis. SPATA2 was knocked down in L929 cells using RNAi, and cells were pretreated with DMSO, Z-VAD, or Z-VAD+NEC1 for 1 hour followed by treatment with TNF-α for 2.5 hours. Cell viability was assessed with propidium iodide staining and flow cytometry. Knockdown of SPATA2 mRNA was verified using real time PCR. p** < 0.05 (two-tailed, unpaired Student´s t-test).D. Knockdown of SPATA2 reduces phosphorylation of MLKL in TNF-α-induced necroptosis. TNF-α-dependent necroptosis was induced in L929 as described in Figure 6C. Phosphorylation of MLKL was analyzed using S358 phospho-specific antibody.
Figure 1(a) Representative 2D μ-CT scans of RasV12; ctrl at day 6, RasV12; scrib-/- larvae at day 6 and day 10. Muscle (green), fat body (blue) and eye-antennal discs/tumor (red) are outlined.Scale bar: 1mm. Anterior (A), Right (R).
Figure 2(a) Quantification of tumor growth of RasV12; ctrl day 6 (n=15) RasV12; scrib-/- tumors, day 6 (n=15), day 7 (n=5), day 8 (n=4), day 9 (n=5) and day 10 (n=5).(b) Quantifications of muscle volume, of larvae carrying tumors of RasV12; ctrl (n=15), and RasV12; scrib-/- at day 6(n=15), day 7 (n=5), day 8 (n=5), day 9 (n=5) and dat 10 (n=5).(c) Quantification of width of Dorsal Oblique 3 (DO3) muscle in larvae carrying tumors of RasV12; ctrl at day 6 (n=29) and RasV12; scrib-/-at days 6 (n=29) and day 10 (n=31).(d) Quantification of adipose tissue volume in larvae carrying tumors of RasV12; ctrl at day 6 and RasV12; scrib-/-at days 6 and day 10.(e) Representative confocal images of adipose tissue in RasV12; ctrl and RasV12; scrib-/- tumors bearing animals at indicated ages. Lipid droplets are highlighted with Lipid Tox staining. Scale bar: 50μm(f) Quantification of dry weight of RasV12; ctrl (n=9), RasV12; scrib-/- day 6 (n=15) and day 10 (n=18) tumor bearing larvae excluding the tumor weight.(g) Quantification of larval motility measure by crawling distance and crawling pattern in RasV12; ctrl (n=14), RasV12; scrib-/- day 6 (n=15), day 8 (n=16) and day 10 (n=14), each colored line represents a single larva.(h) Coomassie feeding assay to asses food intake of RasV12; scrib-/- over time, three repeated measurements of an average food intake of 20 larvae.
Figure 3(a-e) Cartoon (top) illustrates the genotypes of the larvae, eye-antennal disc (EAD, circle: tumor cells in green and microenvironment cell in black), systemic cells are illustrated as a square (wild type cells in light brown and atg13 mutant cells in light orange) at indicated ages. Cartoon (left) illustrates the structure of cephalic complex attached to mouth hook. Representative images of Larva (image of larva using the backlight of microscope), cephalic complex (green highlights the GFP-labelled tumor clones), muscle (Phalloidin in green stains actin and Hoechst in blue stains nucleus) and adipose tissue (Lipid Tox in red stains for lipid droplets, Hoechst stains nucleus) from top to bottom. (a) RasV12; ctrl tumor bearing animal at day 6. (b) RasV12; scrib-/- tumor bearing animal at day 6. (c) RasV12; scrib-/- tumor bearing animal at day 8. (d) RasV12, scrib-/-, atg13-/-//atg13-/- at day 8. (e) RasV12, scrib-/-, atg13-/-//atg13-/- animal complemented with transgenic atg13, rescuing the tumor growth, at day 8.(f) Quantification of area of space occupied with lobes of fat body within larval segments 4 to 8 (shown in yellow dashed line in 2a), of animals bearing tumors at day 6 RasV12; ctrl (n=45), RasV12; scrib-/- (n=30), RasV12, scrib-/-, atg13-/-//atg13-/- (n=30) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=29) and at day 10 RasV12; scrib-/- (n=40), RasV12, scrib-/-, atg13-/-//atg13-/- (n=25) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=28).(g) Quantification of tumor volumes day 6 RasV12; ctrl (n=7), RasV12; scrib-/- (n=11) and day 8 RasV12; scrib-/- (n=13), RasV12, scrib-/-, atg13-/-//atg13-/- (n=9) and ey3.5-atg13; RasV12, scrib-/-, atg13-/-//atg13-/- (n=9).(h) Quantification of Ventral Longitudinal muscle 4 (VL4) (shown in yellow dashed line in 2a) of third segment of larvae carrying at day 6 RasV12; ctrl (n=15), RasV12; scrib-/- (n=14), and at day8 RasV12; scrib-/- (n=11), RasV12, scrib-/-, atg13-/-//atg13-/- (n=12) and ey3.5-atg13; RasV12; scrib-/-, atg13-/-//atg13-/- (n=14).(i) Quantification of dry weight excluding tumor of RasV12; scrib- -, atg13-/-//atg13-/-at day 6 (n=8) and day 10 (n=7).(j) Quantification of larval motility measured by crawling distance and crawling pattern for RasV12; scrib-/-, atg13-/-//atg13-/- at day 6 (n=15) and at day 8 (n=15), each colored line represents a single larva.
Figure 4(a) Changes in groups (carbohydrates, amino acids and fatty acids) of storage metabolites measured in circulating haemolymph with progressing wasting, shown as log2 changes measured by LC-MS and calculated per larvae relative to RasV12; ctrl at day 6.(b) Volcano plot showing autophagy-dependent changes to amounts of circulating metabolites at day 6. X-axis shows the log2 of fold change of RasV12;atg13 -/-scrib-/- // atg13 -/- vs. RasV12; scrib-/- , y-axis shows -log10 p-value, calculated by t-test. Metabolite names are shown for metabolites with log2(FC) >± 1 and/or -log10(P) < 2. Green points indicate log2(FC) >± 1, blue indicate -log10(P) < 2 and red indicates for above both thresholds.(c) Autophagy-driven wasting releases metabolites into circulation of 113 reliably detected metabolites, those showing significant differences in any of the three comparisons are shown. Color indicates the log2(fold change) difference and the numbers show the p-value of the comparison. The statistical test to define significance was FDR-adjusted t-test p-value < 0.05
Figure 5(a) The amount of glycogen in the whole larvae measured by biochemical assay (n=4), normalized to RasV12; ctrl at day 6, and per number of larvae.(b-e) Representative confocal images of muscle and adipose tissue of larvae carrying RasV12; ctrl, RasV12; scrib-/- and RasV12; scrib-/-, atg13-/-//atg13-/- showing glycogen levels (white) at day 6 and day 8.(g) Sources of carbon incorporated into tumor biomass were differentiated by changing the isotopic carbon content of the food source 25 hours before measuring the total carbon content and isotopic ratio of the tumor. A similar, but independent experiment is reported in Holland P., et al., 2021 BMC Biol.
Figure 1HSATIII lncRNA-dependent localization of m6A-related factors. Control and HSATIII knockdown HeLa cells were exposed to thermal stress (42°C for 2 h and recovery for 1 h at 37°C) and stained by HSATIII-FISH and immunofluorescence using an anti-WTAP antibody (B) or anti-YTHDC1 antibody (C). The nuclei were stained with DAPI. Scale bar: 10 µm.YTHDC1 accumulation within nSBs was arrested by METTL3 knockdown. HeLa cells were stained as described in Figure 1C. Scale bar: 10 µm. Box plot of relative intensities of YTHDC1 to HSATIII within nSBs in individual nuclei (n=30). The mean is indicated with X. The first and third quartiles are the ends of the box, the median is indicated with a vertical line in the box, and the minimum and maximum except for the outliers are the ends of the whiskers. The outliers are indicated with open circles. P-values (Kruskal-Wallis test, followed by Dunn's multiple comparison test) are shown above.
Figure 2RNA-MS analysis of HSATIII lncRNAs digested by RNase T1. Base peak chromatogram (BPC, top panel) and extracted chromatograms (XIC, lower panels) for the RNase T1-digested fragments of the isolated HSATIII lncRNAs. XICs detecting for the negatively-charged ions of AAUGp tetramer without modification (second panel), with one methylation (third panel), and two methylations (bottom panel) . The sequence, mass to charge ratio (m/z) and charge state are indicated. NL; normalization level.