Journal: eLife

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.7554/eLife.93877

Figure Lengend Snippet: ( A ) Volcano plot displaying the protein abundance ratios of SPARK-AID parasites treated with IAA or vehicle for 24 hr and adjusted p-values for n = 2 biological replicates. Proteins identified as up- or down-regulated in parasites overexpressing the driver of differentiation (BFD1) are shown in blue and vermilion, respectively. In total, 4474 proteins were quantified, 3847 of which registered more than one peptide. ( B ) Schematic of the phosphoproteomics experiment following SPARK depletion. Intracellular parasites expressing SPARK-AID were treated with 500 µM IAA for 24, 8, 3, or 0 hr and were harvested at 32 hpi simultaneously with the TIR1 parental strain as a control. Samples were multiplexed with tandem mass tags (TMT). The same experimental design was used for SPARKEL-AID proteomics. Each experiment included two biological replicates. ( C ) Average protein abundances of PKG, PKA-R, and PKA-C1 relative to the TIR1 parental strain after the indicated period of SPARK (thick lines) or SPARKEL (dotted lines) depletion. ( D ) Average protein abundances of up-regulated bradyzoite genes relative to the TIR1 parental strain after the indicated period of SPARK (blue lines) or SPARKEL (purple lines) depletion. Up-regulated bradyzoite proteins were defined as those significantly increased in parasites overexpressing BFD1 and two modified Z-scores above the median in the SPARK depletion proteome. Rank-ordered plots of ( E ) Phosphopeptide basal dysregulation score (peptide abundance in the SPARK-AID strain without IAA treatment relative to the TIR1 parental strain) or ( F ) IAA-dependent score (summed peptide ratios relative to the SPARK-AID t 0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. ( G ) Gene ontology (GO) enrichment of phosphoproteins exhibiting SPARK-dependent regulation. Gene ratio is the proportion of proteins with the indicated GO term divided by the total number of proteins. Significance was determined with a hypergeometric test; only GO terms with p<0.05 are shown. Redundant GO terms were removed. Categories discussed in the main text are highlighted with colored text. ( H ) Gaussian mixture modeling of SPARK-dependent peptides identified by more than one PSM revealed seven kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. ( I ) Protein abundances following biotinylation and streptavidin enrichment of samples derived from parasites expressing mNG- or SPARK-TurboID fusion constructs. A pseudocount of 10 4.5 was assigned to proteins identified in only one sample. Point colors correspond to significance thresholds for n = 2 biological replicates. Dotted lines correspond to one median absolute deviation.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Quantitative Proteomics, Phospho-proteomics, Expressing, Control, Modification, Derivative Assay, Construct

Journal: eLife

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.7554/eLife.93877

Figure Lengend Snippet: ( A ) Average protein abundance ratios of SPARK- or SPARKEL-AID parasites treated with IAA for 24 hr relative to the untreated samples. Enriched proteins identified as up-regulated in alkaline-induced bradyzoites are shown in blue. The points corresponding to PKA C1, PKA R, and PKG are highlighted in pink, orange, and green, respectively. ( B, C ) Principal component analysis of the SPARK-AID depletion phosphoproteome. Plots show the three components accounting for the greatest proportion of the variance ( D ) Average abundances of SPARK and SPARKEL peptides detected by mass spectrometry. ( E ) Average protein or phosphopeptide abundance ratios from SPARK-AID parasites treated with IAA for the indicated number of h relative to untreated samples. Values corresponding to PKA C1, PKA R, and PKG are color-coded.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Quantitative Proteomics, Mass Spectrometry, Phospho-proteomics

Journal: eLife

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.7554/eLife.93877

Figure Lengend Snippet: ( A–B ) Average protein and phosphopeptide abundances of PKA C1 ( A ) and PKA R ( B ) following SPARK depletion. ( C ) Schematic of the genetic strategy used to monitor PKA C1 and PKA R abundances following SPARK (or SPARKEL) down-regulation with IAA. ( D ) Immunofluorescence microscopy of parasites expressing SPARK-AID, PKA C1-mNG, and PKA R-mCherry after 0 or 24 hr of IAA treatment to degrade SPARK. Parasites and nuclei were stained with GAP45 and Hoechst 33342, respectively. GAP45 staining and mNG or mCherry staining were normalized to vehicle-treated tagged samples. ( E, F ) Flow cytometry analysis of parasites expressing PKA C1-mNG, PKA R-mCherry, and SPARK-AID or SPARKEL-AID, respectively, after the indicated period of IAA treatment. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. ( G ) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C1 targets, as defined in the text and methods, are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. ( H ) Heat map of the abundance ratios of candidate PKA C1 targets following SPARK depletion. PKA R depletion results in up-regulation of PKA C1, and candidate PKA C1 targets therefore have positive abundance ratios following PKA R down-regulation.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Phospho-proteomics, Immunofluorescence, Microscopy, Expressing, Staining, Flow Cytometry, Derivative Assay

Journal: eLife

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.7554/eLife.93877

Figure Lengend Snippet: ( A ) Transcript abundances for PKG, PKA C1, and PKA C3 were compared by qRT-PCR in TIR1 and SPARK-AID, treated with IAA for 0, 3, or 24 hr. Expression levels were normalized to the parental TIR1 strain across n=4 biological replicates. No comparisons yielded significant p-values using a Kruskall-Wallis test. ( B ) Overlap between the SPARK and PP1 phosphoproteomes. Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PP1 targets, as defined in the text and methods, are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. PP1 proteome data was obtained from .

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Quantitative RT-PCR, Expressing, Phospho-proteomics, Derivative Assay

Journal: eLife

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.7554/eLife.93877

Figure Lengend Snippet: ( A ) Protein and phosphopeptide abundances of PKG following SPARK depletion. ( B ) A23187-stimulated egress assays performed at different concentrations of compound 1 after TIR1 and SPARK-AID parasites had been treated with IAA for 24 hr. Curves were fit to the average values of six replicates and were compared with an extra sum of squares F test. ( C ) The IC50 values of each strain for compound 1; each point represents a biological replicate (n = 6). Significance was assessed with a two-tailed t -test. ( D ) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKG targets are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. ( E ) Heat map of the abundance ratios of candidate PKG targets following SPARK depletion.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Phospho-proteomics, Two Tailed Test, Derivative Assay

Journal: eLife

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.7554/eLife.93877

Figure Lengend Snippet: ( A ) Protein abundances from immunopurified PKA C3-mNG-AID lysates or an untagged control strain from n = 2 biological replicates. Dotted lines correspond to one modified z-score. Only proteins quantified by greater than one peptide are shown. Proteins identified in only one IP were assigned a pseudo-abundance of 10 4.5 . Point colors correspond to significance thresholds. ( B ) Immunoblot of parasites expressing SPARK-V5-AID-HA and PKA C3-mNG after 24 hr of IAA treatment. ALD1 serves as a loading control. Band intensity normalized to the dual-tagged strain is shown in ( C ) from three replicates. ( D ) Flow cytometry analysis of SPARK-AID parasites expressing PKA C3-mNG treated with IAA for the indicated number of h. Traces are representative of two biological replicates. The dotted line centers the mode of the vehicle-treated sample. Traces were normalized by unit area. ( E–F ) Fixed, intracellular SPARK-AID/PKA C3-mNG parasites visualized by immunofluorescence microscopy using the mNG epitope after the indicated period of IAA treatment ( E ). The mNG signal was internally normalized to the SPARK-AID parental strain. Quantification of the PKA C3 signal intensity of three replicates is shown in ( F ). ( G ) Phosphopeptide IAA-dependent score (summed peptide ratios relative to the PKA C3-AID t 0 peptide abundance). Dotted lines correspond to 3.5 modified Z scores. Colored points correspond to phosphosites discussed in the main text. ( H ) Gaussian mixture modeling of PKA C3-dependent peptides identified by more than one PSM revealed three kinetically resolved clusters. Individual peptides or the median ratios in each cluster are depicted by light and dark gray lines, respectively. Clusters are numbered according to their discussion in the main text. ( I ) Violin plots displaying the distribution of phosphopeptide abundance values following SPARK depletion. The distributions of candidate PKA C3 targets are shown in green. The distributions and p-values (KS test) were derived from the overlapping subset of phosphopeptides identified in each dataset. ( J ) Heat map of the abundance ratios of candidate PKA C3 targets following SPARK depletion. ( K ) Heat map of the abundance ratios of dense granule proteins following SPARK depletion, as discussed in the text. Figure 7—source data 1. This file contains source data that was used to generate the blot in . mNG, LICOR. Figure 7—source data 2. This file contains source data that was used to generate the blot in . V5, LICOR. Figure 7—source data 3. This file contains source data that was used to generate the blot in . ALD1, LICOR.

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Control, Modification, Western Blot, Expressing, Flow Cytometry, Immunofluorescence, Microscopy, Phospho-proteomics, Derivative Assay

Journal: eLife

Article Title: SPARK regulates AGC kinases central to the Toxoplasma gondii asexual cycle

doi: 10.7554/eLife.93877

Figure Lengend Snippet:

Article Snippet: Resuspended TMT10plex-labeled samples were enriched with the High-Select TiO2 Phosphopeptide Enrichment Kit (Thermo Fisher Scientific A32993).

Techniques: Biomarker Discovery, Plasmid Preparation, Sample Prep, Phospho-proteomics, Peptide Fractionation, Magnetic Beads, Reverse Transcription, SYBR Green Assay, Software