Apical annuli are specialised sites of post-invasion secretion of dense granules in Toxoplasma
- Department of Biochemistry, University of Cambridge, United Kingdom
Figures
Figure 1 with 1 supplement
TgLMBD3 is a conserved protein in the plasma membrane at apical annuli sites in T. gondii.
(A) Wide-field immunofluorescence assay imaging of cells expressing TgLMBD3-6xHA (magenta) and eGFP-Centrin2 (green) and immunostained inner membrane complex (IMC)1 (blue) in either the …
Figure 1—figure supplement 1
Maximum likelihood phylogenies of the LMBR1 domain-containing proteins from Figure 1.
(A) All LMBR1 domain-containing proteins can be divided into four different orthogroups. Numbers at branches are bootstrap/aLRT SH-like supports for the monophylies of the four orthogroups. …
Figure 2
Apical annuli occur at gaps in the inner membrane complex (IMC).
Three-dimensional structured illumination microscopy (3D-SIM) imaging of immunofluorescence assays of intracellular tachyzoites in host cells. (A) TgLMBD3-6xHA (magenta) and eGFP-Centrin2 (green) …
Figure 3
TgLMBD3 is recruited to the apical annuli late in cytokinesis.
Three-dimensional structured illumination microscopy (3D-SIM) imaging of immunofluorescence assays of intracellular tachyzoites at various stages of daughter cell formation and emergence from the …
Figure 4
Three SNARE proteins likely form a complex at the inner side of the plasma membrane at the apical annuli.
(A) Schematic of the SNARE complex which facilitates fusion of a secretory vesicle with target membrane (Jahn and Scheller, 2006). (B) Wide-field fluorescence microscopy localisation of TgStxPM, TgNP…
Figure 5 with 1 supplement
Depletion of apical annuli plasma membrane proteins impairs replication rates of T. gondii.
(A). Depletion of each apical annuli protein shown in hours of 3-indolacetic acid (IAA) auxin treatment observed by anti-V5 western blots. Histone H3 serves as a loading control, and molecular …
Figure 5—figure supplement 1
Western blot and PCR analysis validating correct epitope-tag integration.
(A) Western blots of cell lysates using anti-v5 antibody shows TgNPSN, TgStxPM, and TgSyp7 were successfully tagged with 3xV5. (B) PCR showing successful integration of epitope tag in the intended …
Figure 6 with 2 supplements
Secretion of dense granule proteins into the parasitophorous vacuole is inhibited when apical annuli membrane proteins are knocked down.
Wide-field immunofluorescence assays of (A) GRA5, (B) GRA1, and (C) GRA2 without (control) or with auxin-induced protein knockdown for the four apical annuli membrane proteins. Assays for GRA5 were …
Figure 6—figure supplement 1
Wide-field images of parasitophorous vacuoles stained for GRA5 after depletion of the four apical annuli proteins.
Immunofluorescence assays for GRA5 without (control) or with auxin-induced protein knockdown for the four apical annuli membrane proteins. Cells were pre-treated with auxin to induce complete annuli …
Figure 6—figure supplement 2
Abundance and distribution of rhoptry and microneme proteins are unaffected when apical annuli membrane proteins are knocked down.
Wide-field immunofluorescence assays of MIC2 and ROP1 without (control) or with auxin-induced protein knockdown for the four apical annuli membrane proteins. Assays for MIC2 and ROP1 were done 24 hr …
Figure 7
Knockdown of apical annuli membrane proteins results in accumulation of dense granule proteins in the parasite.
(A) Volcano plots showing the changes of abundance of cell proteins with three apical annuli proteins depleted over 24 hr of auxin treatment compared to untreated controls (N=3). Black dots …
Additional files
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Supplementary file 1
BioID supplementary data file.
Sheet 1: BioID_significant_changes: Columns show ToxoDB accession number, log2-fold change (logFC) with left and right confidence intervals (CI.L, CI.R), the average log2-abundance value of this protein across treatments and their replicates (AveExpr), the moderated t-statistics value (t), the raw p-value (P.Value), the adjusted p-value (adj.P.Val), and the log-odds that the protein is differentially abundant (B). Sheet 2: Normalised TMT intensity values for three LMBD3-BirA* biological replicates (RUN1-3) each with three parental control samples.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp1-v2.xlsx
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Supplementary file 2
Quantitative proteomics supplementary data file.
Sheets 1–3 give data for the three cell lines: TgLMBD3-mAID-3xV5, mAID-3xV5-TgNPSN, and mAID-3xV5-TgSyp7. Columns show ToxoDB accession number, number of peptides, log2-median-aligned protein abundances for all replicates in knockdown (KD_rep1-rep3) and control treatment (control_rep1-rep3), treatment means and standard deviation (SD), effect size (Cohen’s D), statistical power for two-sided t-test at p=0.01, log2-fold change between treatment and control (logFC), the average log2-abundance value of this protein across treatments and their replicates (AveExpr), the moderated t-statistics value (t), the raw p-value (P.Value), the adjusted p-value (adj.P.Val), the log-odds that the protein is differentially abundant (B), the protein description (Description), and the TAGM-predicted subcellular location according to the ToxoLOPIT map of Barylyuk et al., 2020.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp2-v2.xlsx
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Supplementary file 3
Primers and plasmids for genetic modifications.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp3-v2.xlsx
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Supplementary file 4
Antibodies used for immunofluorescence assays (IFAs) and Western blots.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp4-v2.xlsx
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Supplementary file 5
R markdown file of analytical workflow of quantitative proteomics data.
The markdown file contains the pipeline from the peptide-to-spectrum match (PSM)-level input data obtained from Proteome Discoverer. It provides an overview on structure and quality of the raw data (chunk 1–5), explores missing data structure and protein coverage across experiments (chunk 6–7), aggregates the psm-level data to proteins (chunk 8), and creates the linear model fits using Limma Bayes algorithms (chunk 10). The last two chunks (11+12) create the output data files and Volcano plots submitted with this manuscript.
- https://cdn.elifesciences.org/articles/94201/elife-94201-supp5-v2.pdf
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MDAR checklist
- https://cdn.elifesciences.org/articles/94201/elife-94201-mdarchecklist1-v2.pdf
