TgICMAP1 is a novel microtubule binding protein in Toxoplasma gondii

Abstract

The microtubule cytoskeleton provides essential structural support for all eukaryotic cells and can be assembled into various higher order structures that perform drastically different functions. Understanding how microtubule-containing assemblies are built in a spatially and temporally controlled manner is therefore fundamental to understanding cell physiology. Toxoplasma gondii, a protozoan parasite, contains at least five distinct tubulin-containing structures, the spindle pole, centrioles, cortical microtubules, the conoid, and the intra-conoid microtubules. How these five structurally and functionally distinct sets of tubulin containing structures are constructed and maintained in the same cell is an intriguing problem. Previously, we performed a proteomic analysis of the T. gondii apical complex, a cytoskeletal complex located at the apical end of the parasite that is composed of the conoid, three ring-like structures, and the two short intra-conoid microtubules. Here we report the characterization of one of the proteins identified in that analysis, TgICMAP1. We show that TgICMAP1 is a novel microtubule binding protein that can directly bind to microtubules in vitro and stabilizes microtubules when ectopically expressed in mammalian cells. Interestingly, in T. gondii, TgICMAP1 preferentially binds to the intra-conoid microtubules, providing us the first molecular tool to investigate the intra-conoid microtubule assembly process during daughter construction.

Figure 1. Schematic drawings of T. gondii (Adapted from [6]).

Left and middle: a longitudinal section and a semitransparent projected view of the cytoskeleton of a dividing cell. Right: an enlarged view of the apical portion of the cytoskeleton. The cytoskeleton of T. gondii includes the apical and the basal complexes, and the cortical cytoskeleton, which contains the cortical MTs, and the Inner Membrane Complex (IMC), a set of flattened vesicles with an underlying filamentous protein network. The centrioles/spindle pole assembly is located close to the nucleus and is not shown in the drawings. The apical complex is formed of the conoid, the intra-conoid MTs and three ring-like structures. The conoid fibers are novel tubulin polymers, the structure of which is dramatically different from that of a canonical MT, such as the cortical MTs and the intra-conoid MTs. In the enlarged view of the apical portion of the cytoskeleton (right), EM cross-sections of a single conoid fiber and a canonical MT are shown.

Figure 2. eGFP-TgICMAP1 coats MTs when ectopically expressed in mammalian cells.

Images of mouse embryonic fibroblast cells transiently expressing eGFP-TgICMAP1 (green) labeled with anti-tubulin YL1/2 (red), which show that eGFP-TgICMAP1 binds to a subset of MTs, mainly around the nucleus. Insets: notice that MT segments labeled by eGFP-TgICMAP1 tend to have much weaker staining of tubulin antibody than adjacent segments (arrowheads and brackets). Images are projections of deconvolved 3-D stacks. Insets are single optical sections of indicated regions in images and are at 2× magnification.

Figure 3. TgICMAP1 is a coiled-coil protein and contains a SMC-like coiled-coil domain.

(A) Graphic display of predicted coiled-coil domains in TgICMAP1. Y-axis, probability of formation of coiled-coil domain where values close to 1 indicate regions with a high propensity to form coiled-coil domains. X-axis, TgICMAP1 amino acids. (B) Schematics of full-length TgICMAP1 and the two TgICMAP1 truncation proteins, TgICMAP1^1–474 and TgICMAP1^455–1231.

Figure 4. Ectopically expressed eGFP-TgICMAP1 stabilizes MTs in mammalian cells.

(A) eGFP-TgICMAP1 transfected cells (green) labeled with rat anti-tubulin YL1/2 antibody (red) and mouse anti-acetylated tubulin antibody (cyan). eGFP-TgICMAP1 coated MTs display higher acetylation level compared with adjacent “uncoated” MTs (insets). Insets are at 2× magnification. Scale bar = 10 µm. (B) Graph showing the mean percentages of untransfected (blue bar, “untransfected”); eGFP-TgICMAP1 transfected (green bar, “transfected”); low (yellow bar) and high (dark green bar) eGFP-TgICMAP1 expressing HeLa cells containing high levels of MTs after nocadazole treatment. The error bars indicate standard error of the mean (SEM). “*” : p<0.01, “**”: p<0.001. (C) Representative images of untransfected and transfected cells expressing low and high levels of eGFP-TgICMAP1 after nocodazole treatment. Scale bar = 10 µm.

Figure 5. TgICMAP1 binds MTs in vitro.

Recombinant 6xHis-eGFP-TgICMAP1 (green) (A) or 6xHis-eGFP (B) (green) were incubated with in vitro polymerized MTs, spun onto coverslips, and stained with mouse-anti-tubulin B-5-1-2 (red). 6xHis GFP-TgICMAP1 protein bound along the MT lattice. No MT binding was observed in 6xHis-eGFP control samples. Scale bars = 10 µm.

Figure 6. TgICMAP1 localizes to the intra-conoid MTs in T. gondii.

(A) In T. gondii, eGFP-TgICMAP1 (green) localized to the apical complex of the parasite (inset), in a region narrower than the width of the conoid marked by mCherryFP-TubA1 (red). eGFP-TgICMAP1 also labeled the centriole/spindle pole area, which also incorporated mCherryFP-TubA1 (arrows). Inset is at 3× magnification. Scale bar = 2 µm. (B) Images of a parasite expressing mCherryFP-TubA1 (red) fixed and permeablized with methanol and labeled with anti-TgICMAP1 (green) show the prominent apical complex localization of endogenous TgICMAP1, which is similar to that of eGFP-TgICMAP1, but a specific localization in the centriole/spindle pole area could not be confirmed. The punctate labeling in the parasite body is likely to be the combined result of labeling for TgICMAP1 in the cytoplasmic pool and non-specific labeling, as some spots are seen outside the parasite. Inset is at 3× magnification. Scale bar = 2 µm. (C) Western blot of T. gondii total cell lysate showing that anti-TgICMAP1 antibody recognizes a ∼135 kDa protein doublet (arrowheads) consistent with the predicted size of TgICMAP1. The blot was stripped and reprobed with mouse-anti-tubulin B-5-1-2 to use α-tubulin as a loading control. (D) Left: Two EM images of Ca²⁺ ionophore–treated, deoxycholate-extracted RH parasites, which were immunogold-labeled with anti-TgICMAP1 antibody and negatively stained with phosphotungstic acid. Scale bars = 0.5 µm. Middle: Higher magnification of the apical complex regions of the parasites shown in left panels, showing that the intra-conoid MTs (arrows and arrowheads) are decorated with gold particles, recapitulating the distribution of fluorescence in Figure 6 A and B. Scale bars = 0.2 µm. Right: schematic diagram of the T. gondii apical complex. (E and F) Parasites were extracted with deoxycholic acid, and subsequently fixed and stained with anti-tubulin B-5-1-2 (red) and anti-TgICMAP1 (green). The cortical cytoskeleton of the parasite in E was mostly destroyed with the cortical MTs splaying around the apical complex (white arrow). In (F) the cortical cytoskeleton of the parasite was more intact and a ring-like structure stained with anti-tubulin antibody can be observed at the basal end of the parasite (red arrowheads). Scale bars = 2 µm.

Figure 7. The first 474 amino acids of TgICMAP1 are important for intra-conoid MT association in T. gondii.

Interphase parasites expressing eGFP fusions (green) of full-length TgICMAP1 (A), TgICMAP1^1–474 (B), and TgICMAP1^455–1231 (C) were labeled with anti-IMC1 antibody (red). EGFP-TgICMAP1^1–474 is localized to the intra-conoid MTs (green arrows). However, it displays stronger localization to the basal complex (inset) in comparison to eGFP-TgICMAP1. Its cytoplasmic pool is also more prominent than that of eGFP-TgICMAP1. eGFP -TgICMAP1^455–1231 is localized predominantly to the nucleus and cytosol. The insets are at 2.5× magnification. Scale bars = 2 µm.

Figure 8. Daughter intra-conoid MTs are assembled during daughter formation within the mother cell.

eGFP-TgICMAP1 expression (green) and anti-IMC1 staining (red) in a dividing parasite show that eGFP-TgICMAP1 is present at the intra-conoid MTs of both the mother (green arrow) and the daughter parasites (green arrowhead) as well as the duplicated centriole/spindle pole assembly (white arrowheads). Scale bar = 2 µm.