Stability and function of a putative microtubule-organizing center in the human parasite Toxoplasma gondii

Abstract

The organization of the microtubule cytoskeleton is dictated by microtubule nucleators or organizing centers. Toxoplasma gondii, an important human parasite, has an array of 22 regularly spaced cortical microtubules stemming from a hypothesized organizing center, the apical polar ring. Here we examine the functions of the apical polar ring by characterizing two of its components, KinesinA and APR1, and show that its putative role in templating can be separated from its mechanical stability. Parasites that lack both KinesinA and APR1 (ΔkinesinAΔapr1) are capable of generating 22 cortical microtubules. However, the apical polar ring is fragmented in live ΔkinesinAΔapr1 parasites and is undetectable by electron microscopy after detergent extraction. Disintegration of the apical polar ring results in the detachment of groups of microtubules from the apical end of the parasite. These structural defects are linked to a diminished ability of the parasite to move and invade host cells, as well as decreased secretion of effectors important for these processes. Together the findings demonstrate the importance of the structural integrity of the apical polar ring and the microtubule array in the Toxoplasma lytic cycle, which is responsible for massive tissue destruction in acute toxoplasmosis.

FIGURE 1:

Cartoon and electron micrograph depicting multiple tubulin-containing cytoskeletal structures (red) in T. gondii, including the 22 cortical microtubules, a pair of intraconoid microtubules, and the 14 fibers that make up the conoid. In its retracted state, the conoid forms a truncated cone that lies basal to the apical polar ring inside the parasite. Treatment of parasites with a calcium ionophore such as A23187 increases the concentration of calcium in the cytoplasm, inducing a change in the pitch of the conoid fibers and protrusion of the conoid through the apical polar ring. Bottom left, cortical microtubules forming during endodyogeny, a process in which daughter parasites assemble inside the mother (mature) parasite. For clarity, only a subset of organelles is shown. The cortical microtubules of the mother parasite are not shown so those in the daughter parasites can be better visualized. ER, endoplasmic reticulum; IMC, inner membrane complex.

FIGURE 2:

KinesinA and APR1 are localized to a ring structure at the apex of the parasite, with KinesinA located apical to APR1. (A) 3D SIM projection of live intracellular parasites ectopically expressing ptubA1-APR1-mCherryFP (red) and KinesinA endogenously tagged with mNeonGreenFP (green). The ring that KinesinA-mNeonGreenFP forms is apical to that formed by APR1-mCherryFP. Insets are shown at 2×. (B) Immunoelectron micrograph of a parasite ectopically expressing EGFP-tagged APR1, extracted with sodium deoxycholate, labeled with anti-GFP antibody and 1.4-nm gold secondary antibody, and silver enhanced. The gold/silver deposits are found in close proximity to the apical polar ring (black arrowhead). (C) Wide-field epifluorescence images of live parasites showing that KinesinA (green) is localized apical to the conoid (labeled by transiently expressing mCherryFP-α1-tubulin [TUBA1, red] from a T. gondii tubulin promoter) when the conoid is retracted (dimethyl sulfoxide [DMSO], parasite treated with vehicle only). When the conoid is protruded in the presence of the calcium ionophore A23187, KinesinA is basal to the conoid. Quantification of the intensity profiles for both the KinesinA and TUBA1 signals along the dashed line is shown below the insets. Note that the parasite in the DMSO panel is replicating, and KinesinA is present in the apical complexes of the two developing daughters (arrows). Insets are enlarged 2×. (D) 3D SIM projection of intracellular KinesinB-mNeonGreenFP endogenously tagged parasites fixed and labeled with anti-ISP1 antibody. The apical boundary of KinesinB-mNeonGreenFP along the cortical microtubules coincides with the base of the apical cap marked by ISP1. Arrows indicate the ISP1 signal in daughters.

FIGURE 3:

KinesinA is recruited to the apical polar ring during an early stage of daughter construction. Montage showing 3D-SIM projections of live, intracellular KinesinA-mNeonGreenFP (green) endogenously tagged parasites transiently expressing mAppleFP-β1-tubulin (TUBB1, red) from a T. gondii tubulin promoter. KinesinA-mNeonGreenFP is localized to the apical polar ring of mature, interphase parasites (A), and in daughters from the initiation of daughter formation (B) to the final stages as the fully assembled daughters emerge from the mother (E), of which only the residual body containing the mother apical polar ring is left. Insets show the apical region of one of the daughter parasites enlarged at 2×.

FIGURE 4:

APR1 is recruited to the apical polar ring at a late stage of daughter construction. Montage showing 3D-SIM projections of live, intracellular APR1-mCherryFP (pseudocolored green) knock-in parasites ectopically expressing EGFP-β1-tubulin (TUBB1, pseudocolored red) from a T. gondii tubulin promoter (Hu, 2003; Wu et al., 2016). APR1-mCherryFP is localized to the apical polar ring of mature, interphase parasites (A) and detected in daughters only during late stages of assembly and emergence from the mother (D, E). Insets show the apical region of one of the daughter parasites enlarged at 2×.

FIGURE 5:

Generation of endogenously tagged KinesinA-mNeonGreenFP, APR1-mCherryFP knock-in, knockout, and complemented parasites and assessment of their plaquing efficiency. (A) Left, schematic for generating endogenously tagged KinesinA and ΔkinesinA parasites and Southern blotting strategy. RHΔku80 parasites (parental) were used to generate 3′ endogenously tagged KinesinA-mNeonGreenFP parasites (endogenously tagged (KinesinA-mNG)) via single-crossover homologous recombination. mNG, mNeonGreenFP coding sequence; (dup.), kinesinA sequence partially duplicated as a result of the single-crossover homologous recombination event. The endogenously tagged parasites were then transiently cotransfected with a plasmid expressing both Cas9 nuclease and a guide RNA specific for the 5′ end of the kinesinA genomic locus and a repair plasmid containing the 5′ and 3′ flanking regions of kinesinA but lacking its coding sequence in between, to generate a knockout via double-crossover homologous recombination. The mNeonGreenFP(–) parasite population was enriched using FACS before subcloning the knockout parasites. Positions of the Cas9 cleavage site, restriction sites, CDS probe (blue), and probe annealing downstream of the kinesinA genomic locus in the 3′ flanking region (red) used in Southern blotting analysis (right) and the corresponding DNA fragment sizes expected are indicated as shown. Right, Southern blotting analysis of the kinesinA locus in RHΔku80 (WT), KinesinA-mNeonGreenFP endogenously tagged (KinesinA-mNG), ΔkinesinA, ΔkinesinA:APR1-mCherryFP (ΔkinesinA:APR1-mC), and ΔkinesinAΔapr1 parasites generated as described. For the CDS probe, the expected parasite genomic DNA fragment sizes after NcoI digestion are 8225 base pairs for the parental (i.e., wild-type kinesinA locus) and 5879 base pairs for the endogenously tagged line. The expected DNA fragment sizes for the downstream probe are 8225 base pairs for the parental, 10,133 base pairs for the endogenously tagged, and 5151 base pairs for the knockout. (B) Left, schematic for generating APR1-mCherryFP knock-in and Δapr1 parasites and Southern blotting strategy. RHΔku80 parasites (parental) were used to generate APR1-mCherryFP knock-in parasites (knock-in (APR1-mC)) via double-crossover homologous recombination. The knock-in parasites were then transiently transfected with a plasmid expressing Cre recombinase to excise the knock-in expression cassette between the two LoxP sites, and mCherryFP(–) parasites were enriched using FACS before subcloning the knockout parasites. Positions of the restriction sites, CDS probe (blue), and the probe annealing upstream of the apr1 coding sequence (red) used in Southern blotting analysis and the corresponding DNA fragment sizes expected are indicated as shown. Right, Southern blotting analysis of the apr1 locus in RHΔku80 (WT), APR1-mCherryFP knock-in, Δapr1, Δapr1:KinesinA-mNeonGreenFP (Δapr1:KinesinA-mNG), and ΔkinesinAΔapr1 parasites generated as described. For the CDS probe, the expected parasite genomic DNA fragment sizes after HindIII digestion are, for the CDS probe, 4858 base pairs for the parental (i.e., wild-type apr1 locus) and 6444 base pairs for the knock-in. The expected DNA fragment sizes for the upstream probe are the same as for the CDS probe for the parental and knock-in and 1876 base pairs for the knockout. (C) Plaques formed by RHΔku80 (WT), KinesinA-mNeonGreenFP endogenously tagged (KinesinA-mNG), and APR1-mCherry knock-in (APR1-mC) parasites, knockout (ΔkinesinA, Δapr1, ΔkinesinAΔapr1 (ΔΔ)), and Δapr1:APR1-mCherryFP (Δapr1:APR1-mC) and ΔkinesinAΔapr1:APR1-mCherryFP (ΔΔ:APR1-mC) complemented parasite lines. HFF monolayers were infected with an equal number of each line of parasites, grown for 7 d at 37°C, and then fixed and stained with crystal violet. Host cells that remained intact absorbed the crystal violet staining, whereas regions of host cells lysed by the parasites (plaques) are clear. The box outlined in red is enlarged 2× in the inset to visualize the very small plaques formed by the ΔkinesinAΔapr1 parasites. Inset table, quantification of the number and size of plaques (mean ± SE) produced by the T. gondii lines as measured in three independent biological replicates. The cytolytic efficiency (CE) is defined as the total area of the host cell monolayer that has been lysed by a T. gondii line divided by the total area lysed by WT and expressed as a percentage. (D) Localization of KinesinA-mNeonGreenFP (endogenous tagging) in live Δapr1 parasites and APR1-mCherryFP (knock-in) in live ΔkinesinA parasites.

FIGURE 6:

Parasite invasion, motility, and microneme secretion are impaired and the parasite shape is altered when KinesinA and APR1 are absent. (A) Representative wide-field epifluorescence images of invasion by the indicated T. gondii lines. Parasites that are intracellular (i.e., have invaded) are labeled green, and parasites that did not invade are labeled both green and red (i.e., yellow). See also Table 1 for quantification of invasion assays. (B) Wide-field epifluorescence images showing trail deposition of the RHΔku80 (WT) and ΔkinesinAΔapr1 parasites on coated dishes, as labeled by an antibody that recognizes TgSAG1. Images are representative of results from three independent biological replicates. (C) The pellet and secreted fractions (supernatant) of parental, knockout, and complemented parasite lines upon ethanol stimulation, as probed by antibodies against MIC2. Western blots are representative of three independent biological replicates. The pellet fraction contains the full length and the supernatant contains the shed ectodomain of MIC2. The numbers on the left indicate molecular masses in kilodaltons. The pellet and secreted fractions were cropped from different regions of the same Western blot. (D) The maximum length and width for live, extracellular RHΔku80 (WT), ΔkinesinA, Δapr1, ΔkinesinAΔapr1 (ΔΔ), and APR1-mCherryFP complemented ΔkinesinAΔapr1 (ΔΔ:APR1-mC) parasites were measured using DIC imaging (representative set of images shown). The experiment was performed in triplicate; values are the mean ± SE (micrometers) from 100 parasites per parasite line in each experiment; unpaired Student’s t test, *p < 0.0001 compared with the wild type. Length (L) and width (W) are in measured in micrometers. Scale bar, 5 μm.

FIGURE 7:

ΔkinesinAΔapr1 parasites are still capable of making 22 cortical microtubules, but the loss of KinesinA and APR1 impairs the stability of the apical polar ring. (A) Wide-field epifluorescence images of extracellular ΔkinesinAΔapr1 parasites that were briefly sonicated and labeled with anti-tubulin antibodies to count the number of splayed cortical microtubules. Three examples, each with 22 cortical microtubules, are shown. (B) 3D-SIM projections of live, intracellular RHΔku80 (WT) and ΔkinesinAΔapr1 parasites stably expressing RNG1 endogenously tagged with mCherryFP (RNG1, red) and transiently expressing mEmeraldFP-α1-tubulin (TUBA1, green) from a T. gondii tubulin promoter. Insets are shown at 3×.

FIGURE 8:

The apical polar ring is unstable in parasites lacking KinesinA when extracted by detergent. (A) Transmission electron micrographs of negatively stained, whole-mount Triton X-100–extracted RHΔku80 (WT), ΔkinesinA, Δapr1, ΔkinesinAΔapr1, and ΔkinesinAΔapr1 complemented with APR1-mCherryFP (ΔkinesinAΔapr1:APR1-mC) and Δtlap2Δspm1Δtlap3 parasites. See also Supplemental Figures S3–S7 for additional electron micrographs. Images are representative of at least two independent biological replicates. Retracted, parasites with a retracted conoid; extended, parasites with an extended conoid. Red arrowheads, apical polar ring; white arrowheads, preconoidal rings; black arrows, cortical microtubules; white arrows, intraconoid microtubules; black arrowheads, a thin line of electron-dense material between the roots of the cortical microtubules; C, conoid. Scale bar, 200 nm. (B) Wide-field epifluorescence images of extracellular RHΔku80 (WT), ΔkinesinA, Δapr1, ΔkinesinAΔapr1 (ΔΔ), and ΔkinesinAΔapr1 complemented with APR1-mCherryFP (ΔΔ:APR1-mC) parasites, extracted with 10 mM sodium deoxycholate, and subsequently fixed with 3.7% (vol/vol) formaldehyde and labeled with a mixture of mouse anti–α- and β-tubulin antibodies. Extracted ΔkinesinAΔapr1 parasites were also labeled with rabbit anti-IMC1 antibody (green) to show that the dissociated cortical microtubules (red) appear to be free of parasite cortex. Images are representative of three independent biological replicates. Scale bar, 5 μm.

FIGURE 9:

MIC2-containing micronemal vesicles are aligned with cortical microtubules, and their distribution is altered in parasites whose cortical microtubule organization has been perturbed. 3D-SIM half-volume projections of fixed, intracellular (A) RHΔku80 (WT), (B) ΔkinesinAΔapr1, and (C) Δtlap2Δspm1Δtlap3 parasites that had been either incubated in low-temperature conditions (cold-treated) or not (37°C) before fixation and labeling with a rabbit anti–Tg-β2-tubulin (TUBB2) antibody (red; Morrissette and Sibley, 2002b) and a mouse anti-MIC2 antibody (green; Carruthers et al., 2000). Arrows indicate MIC2 signal aligned in tracks in cold-treated Δtlap2Δspm1Δtlap3 parasites. Note that the anti–Tg-β2-tubulin antibody does not label the conoid under these conditions, presumably due to antigen accessibility issues (see also Liu et al., 2016). Half-volume rather than full-volume projections are presented for optimal visualization of the localization of MIC2-containing vesicles relative to the cortical microtubules. Insets are shown at 3×. Scale bar, 2 μm.