EMBO/soda-nel huggingface dataset:
Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii (2017)
Figure 1

Figure 1. PKA1 is targeted to the inner membrane complex (IMC) by dual acylation of PKAr

A, B Western blot analysis of ΔKu80, PKAc1-3Ty (A) and PKAr-3Ty (B) parasite lysates probed with αTy antibodies. Profilin (αPRF) served as loading control.

C, D Peripheral localization of PKAc1-3Ty (C) and PKAr-3Ty (D) shown by IFA using anti-Ty antibodies. Antibodies against GAP45 and IMC1 were used as markers of plasma membrane and IMC, respectively.

E Plasma membrane stained with an αSAG1 antibody is separated from the IMC stained with anti-Ty after aerolysin treatment, indicating that PKAr-3Ty localises at the IMC.

F The N-terminus putative acylation site of PKAr is essential for its IMC targeting.

G A second copy of DDmyc-PKAc1 is stabilised after 1 hour in presence of Shld-1.

H A second copy of DDmyc-PKAc1 stabilised with Shld-1 is targeted to the IMC in presence of a second copy of PKArWT-Ty. When of a second copy of PKArG2A-Ty is provided, stabilised DDmyc-PKAc1 does not localise at the cell periphery.

Data information: Scale bars = 2 μm.
Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii (2017)
Figure 2

Figure 2. Down-regulation of PKAr or overexpression of PKAc1 blocks parasite division

A Lytic growth of intracellular wild type Toxoplasma gondii tachyzoite stages over a period of 8 days results in plaques within mononolayers of host cells. Destabilisation of PKAr-TyDD leads to the absence of plaque formation.

B A drop in PKAr-TyDD expression is detected 5 hours after Shld-1 removal and a significant parasite growth defect is observed as early as 12 hours later. Profilin (αPRF) served as loading control.

C IFA analysis showed that the down regulation of PKAr-TyDD leads to the abnormal segregation of the apicoplast-associated thioredoxin family protein (ATrx1) 24 hours after Shld-1withdrawal.

D Replication assay of PKAr-TyDD parasites grown for 24 hours +/- Shld-1 indicates that no more than four parasites are observed per vacuole upon PKAr destabilisation. (100 parasites were counted in three independent replicates).

E, F Stabilisation of a second copy of DDmyc-PKAc1 leads to the complete block of cell growth indicated by plaque assay (E) and western blot analysis (F).

G, H Stabilisation of a second copy of DDmyc-PKAc1 leads to the observation of mainly one parasite per vacuole in cells treated with Shld-1 (100 parasites were counted in three independent replicates).

Data information: In C and G, data are presented as mean ± SEM. Scale bars = 2 μm (C and G), 3 mm (A and E).
Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii (2017)
Figure 3

Figure 3. Down-regulation of PKAc1 leads to premature egress and "restless invasion"

A A drop in PKAc1-Ty-iKD expression is detected as early as 24 hours upon ATc treatment with almost no protein detectable after 36 hours.

B Conditional down-regulation of PKAc1-Ty-iKD leads to the absence of plaque formation.

C, D Down-regulation of PKAc1-Ty-iKD upon ATc treatment leads to increased parasite dispersion after 40 hours and premature egress (data is from three independent biological replicates).

E Prematurely egressed PKAc1-Ty-iKD parasites after 24 hours of ATc treatment invade and exit fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle.

Data information: In D, data are presented as mean ± SD. Scale bars = 1 mm (B), 20 μm (C), 2.5 mm (E).
Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii (2017)
Figure 4

Figure 4. Overexpression of a non-cAMP binding PKArG21E isoform prevents release of active PKAc1 causing premature egress and "restless invasion"

A PKAr is predicted to possess two cAMP binding sites. A single glycine to glutamic acid change in the site B of the cAMP binding domain is known to abolish cAMP binding and to prevent activation of the holoenzyme in the presence of cAMP. In PKAr the conserved glycine residue is Gly321.

B Addition of Shld-1 leads to the rapid stabilisation of DDmyc-PKArWT-Ty and DDmyc-PKArG321E-Ty.

C PKAc1 is co-immunoprecipitated with DDmyc-PKArWT-Ty in absence of cAMP but is not recovered in presence of 20 µM cAMP as revealed with [35S]-labeled methionine/cysteine metabolic labelling. Conversely, PKAc1 is co-immunoprecipitated with DDmyc-PKArG321E-Ty in presence or absence of cAMP indicating the G321E substitution prevents the release of active PKAc1 in presence of cAMP. The autoradiogram shown is representative of two independent biological replicates.

D Conditional overexpression of a dominant-negative DDmyc-PKArG321E-Ty that cannot bind cAMP leads a dramatic reduction in plaque formation while over-expression of DDmyc-PKArWT-Ty is well tolerated by the parasite.

E Overexpression of the dominant negative DDmyc-PKArG321E-Ty triggers premature egress from infected host cells and prevents initiation of a new lytic cycle as revealed by IFA. Images are representative of ~80% of the parasite population. Blue = DAPI; red = GAP45; green = GRA3.

F In heavily infected cells, addition of Shld-1 at 32 hours post-inoculation for 4 hours leads to premature egress of DDmyc-PKArG321E-Ty parasites but not of the DDmyc-PKAr-Ty control line as assessed by IFA.

G Quantification of extracellular DDmyc-PKArG321E-Ty parasites released from heavily infected cells after addition of Shld-1 at 32 hours post-inoculation for 4 hours (100 parasites were counted in three independent replicates).

H A non-selective PKA inhibitor, KT5270, significantly enhances the premature egress of DDmyc-PKArWT-Ty parasites in presence of Shld-1 (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test).

I Prematurely egressed DDmyc-PKArG321E-Ty parasites after 4 hours of Shld-1 treatment invade and exit host cells, fully lysing the monolayer of HFF cells while the non-treated parasites invade and initiate a new lytic cycle.

Data information: In C and E-H, data are presented as mean ± SEM. Scale bars = 3 mm (D), 10 μm (E), 5 µm (F) and 2.5 mm (I).
Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii (2017)
Figure 5

Figure 5. Premature egress induced by PKA genetic inhibition is blocked by PKG chemical inhibition.

A GO term enrichment analysis of proteins differentially phosphorylated upon DDmyc-PKArG321E-Ty stabilisation compared with its wild type counterpart DDmyc-PKArWT-Ty. Bonferroni corrected p-values are indicated.

B Upon Shld-1 addition, a putative cGMP-specific PDE, PDE2 was more phosphorylated at Serine 1317 in DDmyc-PKArG321E-Ty parasites compared with DDmyc-PKArWT-Ty parasites suggesting a crosstalk between cAMP and cGMP signaling.

C Chemical inhibition of the cGMP-dependent PKG by Compound 1 and Compound 2 blocks egress induced by 5 µM BIPPO, a PDE inhibitor, or by stabilisation of DDmyc-PKArG321E-Ty in presence of Shld-1. A PKGT761M substitution that renders PKG resistant to both compounds indicates that the block in egress in only mediated by PKG when 0.2 µM Compound 1 is used while the block in egress associated with C2 is not specific to PKG inhibition only (data is from three independent biological replicates).

D Genetic inhibition of PKA triggers MIC2 secretion in intracellular buffer. Anti-GRA1 antibodies were used as a loading control and anti-catalase antibodies served as a lysis control (data is from three independent biological replicates; statistical analysis was done by two-tailed t-test)

Data information: In C and D, data are presented as mean ± SD.

Source data are available online for this figure.
Crosstalk between PKA and PKG controls pH-dependent host cell egress of Toxoplasma gondii (2017)
Figure 7

Figure 7. Cellular localisation of cAMP and cGMP signalling components possibly involved in egress

A-D Localization of PDE1-3Ty (A), PDE2-3Ty (B), GCα-3Ty (C), and PKG-3Ty shown by IFA using an anti-Ty antibody. Antibodies against GAP45, IMC1, cb-GFP, and myc-GAP70 were used as markers of the plasma membrane, IMC, residual body, and apical pole, respectively.

E Western blot analysis indicates that GCα-3Ty is present in three distinct isoforms. A PKG-3Ty western blot also shows three immune reactive isoforms. Catalase or actin were used as loading controls and ΔKu80 as the parental strain.

F PKG-3Ty parasites were solubilised in either PBS or 1% Triton X-100 (TX100) and split into soluble (S) and pellet (P) fractions. Total lysate is also shown.

G, H The signal of Myc-ACβ-iKD followed by IFA (G) or western blot (H) after 48 hours ± ATc.

I Invasion of ACα-KO tachyzoites or Myc-Acβ-iKD after 48 hours ± ATc is not affected. However, addition of ATc for 48 hours reduces the invasion efficiency of ACα-KO/ Myc-Acβ-iKD (data is from three independent biological replicates).

J As for destabilisation of PKAc1-iKD or stabilisation of DDmyc-PKArG321E-Ty, ACα-KO/ Myc-Acβ-iKD tachyzoites treated with ATc for 33 or 40 hours, exit host cells and invade and exit the monolayer of HFF cells leading to lysis while the non-treated parasites invade and initiate a new lytic cycle.

Data information: Scale bars = 2 μm.
Large-scale analysis of Drosophila core promoter function using synthetic promoters (2022)
Figure 3

Figure 3. Combinatorial mutations designed for the motif-rich core promoter region and results for motif knockout.

(A) Motif-wise combinatorial mutations within the core promoter: motif strength and motif position are changed individually. From top to bottom: knockout of motifs (individual or pairwise knockout of motifs, and knockout of all motifs); replacing the original motif with its computationally (XXmotif) derived sequences with different PWM scores (consensus with the highest score), or insertion of the consensus into the motif-less promoter sequences; point mutation of motifs; substitution with functionally or positionally equivalent motifs from other architectures; shift of motif positions; sequence context exchange between different core promoters. The Mec2 motif composition is shown here as an example.

(B-C) Comparison of normalized expression levels between wild-type configuration and motif knockouts for two types of core promoters (developmental: CG8157 (B); constitutive: RpL5 (C)). Upper panels: schematic depiction of the wild-type motif compositions (TTGTT motif in RpL5 is ignored due to its strong overlap with R-INR). Two-sample t-test: ns, not significant, *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; ****p ≤ 0.0001. The middle hinge represents the median. The Interquartile range the difference between the 75th and 25th percentiles. 3-4 biological replicate measurements.

(D) Mean expression fold changes compared to wild-type expressions for individual knockout of motifs in different core promoters. Constitutive and developmental promoters are highlighted in red and green, respectively.

(E-F) Effect of pairwise motif knockout (log2 scale) in core promoters CG7712 (E) and pain (F), respectively. The heatmaps display the mean expression fold changes compared to wild-type expressions for pairwise knockout of motifs compared to individual knockouts (diagonals). Additivity was calculated as the difference between the pairwise effect and the sum of two individual effects, Subadditive (in blue): > 0; superadditive (in yellow): < 0; Additivity values for effects > 3×SDnoise shown in the right lower corner of each pairwise effect.
MITOL promotes cell survival by degrading Parking during mitophagy (2020)
Figure 1

Figure 1. MITOL mediated the K48-linked polyubiquitination of Parkin at K220

A. MITOL interacts with Parkin under CCCP treatment. HeLa cells or HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for 4 h. Lysates of cells were subjected to an IP assay with anti-myc antibody and IB assay with the indicated antibodies.

B. Interaction of endogenous MITOL and Parkin in HeLa cells stably expressing HA-Parkin. HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 12 h and subjected to an IP-IB assay with the indicated antibodies.

C. PINK1 knockdown decreases interaction between MITOL and Parkin. HeLa cells stably expressing HA-Parkin were transfected with PINK1 siRNA or control siRNA and treated with DMSO or CCCP (10 µM) for 4 h. MG132 (30 μM) was added 3 h after each treatment. Lysates of cells were subjected to an IP assay with anti-myc antibody and IB assay with the indicated antibodies.

D. MITOL overexpression enhances the ubiquitination of Parkin. HeLa cells or HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for 8 h. MG132 (30 μM) was added 3 h after each treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies. WT, wild-type; CS, MITOL C65/67S mutant lacking ubiquitin ligase activity.

E. MITOL fails to ubiquitinate the Parkin K220R mutant. HeLa cells were transfected with the indicated vectors and treated with CCCP (10 µM) for 8 h. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies and compared ubiquitination levels of WT, K129R, K220R, and K427R HA-Parkin.

F. Parkin is ubiquitinated by K48-polyubiquitin in mitophagy. HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for 48 h together with MG132 (30 µM) for the final 45 h. Parkin was immunoprecipitated from cell lysates and then performed ubiquitin restriction assay with AMSH, OTUB1, and USP2 using UbiCRest kit. Lysates of cells were subjected to an IB assay with the indicated antibodies. DUBs, deubiquitinating enzyme.

G. Endogenous MITOL enhances the K48-polyubiquitin chains of Parkin. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for indicated time. MG132 (30 μM) was added 3 h after CCCP treatment. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IP-IB assay with the indicated antibodies. K48-polyubiquitin chains were immunoblotted with K48-specific ubiquitin antibody.
MITOL promotes cell survival by degrading Parking during mitophagy (2020)
Figure 2

Figure 2. MITOL regulates mitophagy through the degradation of Parkin

A. MITOL degrades Parkin through the ubiquitin-proteasome system. HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for 8 h alone or either with Bafilomycin A1 (10 µM) or Lactacystin (10 µM) for the final 5 h. Lysates of cells were subjected to an IB assay with the indicated antibodies.

B. Overexpression of MITOL CS mutant fails to degrade Parkin. HeLa cells stably expressing HA-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for the indicated time. Lysates of cells were subjected to an IB assay with the indicated antibodies. MITOL CS mutant; lacking ubiquitin ligase activity.

C. Parkin K220R mutant is not regulated by MITOL in mitophagy. HeLa cells were transfected with the indicated vectors and treated with DMSO or CCCP (10 μM) for the indicated time. Lysates of cells were subjected to an IB assay with the indicated antibodies.

D. Endogenous MITOL attenuates Parkin in both CHX- and CCCP-treated conditions. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) as indicated times. CHX (30 μM) was added 5 h after CCCP treatment. Lysates of cells were subjected to an IB assay with the indicated antibodies.

E. MITOL regulates mitophagy. MITOL WT HeLa cells, MITOL KO HeLa cells and KO HeLa cells transfected with MITOL WT plasmids 0.5µg (+) and 5µg (++) stably expressing HA‐Parkin and mt‐Keima were treated with CCCP (10µM) for 8h. Then, mKeima was measured at 488 (pH 7) and 561 (pH 4) nm lasers using Flow Cytometer. Percentages of mitophagy were calculated from 30,000 cells in each independent experiment. Data represent the mean ± SD of three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.
MITOL promotes cell survival by degrading Parking during mitophagy (2020)
Figure 3

Figure 3. MITOL translocates to the ER with FKBP38 in the late stage of mitophagy

A. MITOL was not degraded in mitophagy. HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h and subjected to an IB assay with the indicated antibodies.

B. Schematic diagram of EGFP knock-in for N-terminal tagging. MITOL-specific and PITCh-specific sgRNAs expressed from pX330A-MITOL/PITCh (not shown) individually target the MITOL exon 1 locus and the donor vector. This allows for both the cleavage of the genomic locus and the release of the EGFP-containing cassette. MMEJ leads to the repair of double-strand break via the insertion of the EGFP-containing cassette, resulting in endogenously EGFP tagged MITOL.

C. Endogenous MITOL translocates to the ER in later phase of mitophagy. EGFP-MITOL knock in HeLa cells were transfected with HA-Parkin and treated with DMSO or CCCP (10 µM) as indicated times. Cells were fixed, permeabilized and subjected to immunofluorescence analysis with the indicated antibodies. Colocalization was quantified by Manders's coefficient. Means ± SEM of more than 10 cells obtained from three independent experiments. For statistical analysis, a one-way ANOVA with Tukey's multiple comparisons test were performed, ****P < 0.0001. Scale bar represents 1 μm.

D. MITOL interacts with FKBP38. HeLa cells or HeLa cells stably expressing mCherry-Parkin were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for indicated times. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies.

E. ER-localized MITOL is transported from the mitochondria, and is not newly synthesized protein. HeLa cells stably expressing HA-Parkin were co-transfected with vectors for MITOL-KikGR and FLAG-FKBP38. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel. Right panel is schematic experimental model for KikGR in this study.
MITOL promotes cell survival by degrading Parking during mitophagy (2020)
Figure 4

Figure 4. MITOL selectively recognizes phosphorylated form of Parkin

A. Phosphorylated Parkin accumulates in MITOL KO cells. HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for the indicated time and subjected to IB assay with the indicated antibodies. Other cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag). p-Parkin, phosphorylated Parkin.

B. Accumulation of activated Parkin in MITOL KO cells. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with CCCP (10 µM) for 30 h and then performed Duolink in situ proximity ligation (PLA) assay demonstrating the interaction between Parkin and Ubiquitin. Scale bar, 5 μm. Higher magnification images of the boxed regions are shown in the small panel.

C. Phosphorylated Parkin accumulates in the ER fraction in the absence of MITOL. WT or MITOL KO HeLa cells stably expressing HA-Parkin were treated with DMSO or CCCP (10 µM) for 48 h. Lysates of cells were fractionated into cytosolic, mitochondrial and ER fractions, and then subjected to an IB assay with the indicated antibodies. Phospho-Ser65-Parkin is detected by using specific antibody. Right panels are the quantification of IB about Phospho-Ser65-Parkin protein levels of the ER fraction. The data represent the mean ± SD for three independent experiments. For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.

D. Parkin is detected at the ER in the absence of MITOL. HeLa cells were transfected with vectors for KikGR-Parkin. After ultraviolet light (365 nm) exposure to the whole plate, cells were incubated with or without CCCP (10 µM) for indicated time, and monitored for red and green KikGR fluorescence. Scale bar, 10 μm. Higher magnification images of the boxed regions are shown in the small panel.
MITOL promotes cell survival by degrading Parking during mitophagy (2020)
Figure 5

Figure 5. MITOL protects FKBP38 by degrading phosphorylated Parkin for cell survival

A. Ubiquitination of FKBP38 depends on Parkin E3 activity. HeLa cells were transfected with the indicated vectors and treated with DMSO or CCCP (10 µM) for indicated times. MG132 (30 μM) was added 1 h before each treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies. WT, wild-type.

B. Parkin fails to ubiquitinate the FKBP38 K271/273R mutant. HeLa cells were transfected with the indicated vectors and treated with CCCP (10 µM) for 30 h. MG132 (30 μM) was added 5 h after CCCP treatment. Lysates of cells were subjected to an IP-IB assay with the indicated antibodies and compared ubiquitination levels of WT, K271R, K273R, and K271/273R FLAG-FKBP38.

C. Overexpression of WT FKBP38 is degraded in MITOL KO cells in mitophagy. MITOL KO HeLa cells stably expressing HA-Parkin were transfected with WT or K271/273R mutant FLAG-FKBP38 and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with the indicated antibodies.

D. MITOL KO increases degradation of ER targeting FKBP38. HeLa cells stably expressing HA-Parkin were transfected with mitochondrial targeting FLAG-FKBP38 (FKBP38ActA) and ER targeting FLAG-FKBP38 (FKBP38IYFFT), and treated with DMSO or CCCP (10 µM) for 24 h. Lysates of cells were subjected to an IB assay with FLAG antibodies for organelle targeting FKBP38 or indicated antibodies. Another cell lysates were subsequently separated on a Phos-tag gel, followed by IB with anti-Parkin antibodies (Phos-tag).

E-G. MITOL partly prevents CCCP-induced cell death by reducing Parkin-mediated degradation of FKBP38. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were transfected with the indicated vectors, and treated with DMSO or CCCP (10 µM) for 24 h. Cells were stained with propidium iodide (PI) for counting the percentage of dead cells using fluorescence microscopy. Data represent the mean ± SD of five independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01. KR, FKBP38 K271/273R.
MITOL promotes cell survival by degrading Parking during mitophagy (2020)
Figure 6

Figure 6. MITOL deletion causes mislocalization of Bcl-2 and leads to excessive cell death via calcium leak in mitophagy

A. Mislocalization of Bcl-2 is observed in MITOL KO cells in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin were transfected with FLAG-Bcl2 and treated with DMSO or CCCP (10 µM) for the indicated times. Cells were immunostained with indicated antibody. Scale bar, 1 μm.

B. MITOL prevents caspase-dependent cell death in mitophagy. WT or MITOL KO HeLa cells stably expressing HA-Parkin and HeLa cells were treated with DMSO or CCCP (10 µM) with or without Z-VAD-FMK (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.

C. Cell death in mitophagy caused by MITOL deficiency depends on calcium leak. WT and MITOL KO HeLa cells stably expressing HA-Parkin or HeLa cells were treated with DMSO or CCCP (10 µM) with or without BAPTA-AM (10 μM) for 24 h. Cells were stained with propidium iodide (PI) for counting fluorescence microscopy of the percentage of dead cells. Data represent the mean ± SD of three independent experiments (>100 individual cells were counted). For statistical analysis, a one-way ANOVA with Tukey post-test was performed, **P < 0.01.

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