Mononeme: a new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease - PubMed (original) (raw)

Mononeme: a new secretory organelle in Plasmodium falciparum merozoites identified by localization of rhomboid-1 protease

Subhash Singh et al. Proc Natl Acad Sci U S A. 2007.

Abstract

Compartmentalization of proteins into subcellular organelles in eukaryotic cells is a fundamental mechanism of regulating complex cellular functions. Many proteins of Plasmodium falciparum merozoites involved in invasion are compartmentalized into apical organelles. We have identified a new merozoite organelle that contains P. falciparum rhomboid-1 (PfROM1), a protease that cleaves the transmembrane regions of proteins involved in invasion. By immunoconfocal microscopy, PfROM1 was localized to a single, thread-like structure on one side of the merozoites that appears to be in close proximity to the subpellicular microtubules. PfROM1 was not found associated with micronemes, rhoptries, or dense granules, the three identified secretory organelles of invasion. Release of merozoites from schizonts resulted in the movement of PfROM1 from the lateral asymmetric localization to the merozoite apical pole and the posterior pole. We have named this single thread-like organelle in merozoites, the mononeme.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Anti-HA antibodies specifically recognize the HA-PfROM1 protein in transgenic HA-PfROM1 3D7 parasites. (A) Western blot analysis of mature parasite extract probed with anti-HA antibody. Lane 1 shows transgenic HA-PfROM1–3D7 parasite protein extract and lane 2 shows parental 3D7 protein extract. Numbers on the left indicate positions of molecular weight markers in kilodaltons. The arrowhead shows the position of HA-PfROM1 detected in the transgenic parasite extract. (B) IFA analysis of HA-PfROM1 in a developing schizont (4 nuclei stage). (C) Merozoites in a segmenter probed with anti-HA rat mAb 3F10 (green) and the nuclear stain (blue, Upper) or with anti-AMA1 (red, Lower). Note in the individual merozoites anti-HA mAb staining appears to be localized on one side of the nucleus, with characteristic bulbous region (arrowheads) that is toward the apical end of the merozoites. The stalk region runs along one side of the nucleus. (Scale bar, 1 μm.)

Fig. 2.

Fig. 2.

HA-PfROM1 is not localized to known apical secretory organelles: rhoptries, micronemes, and dense granules. Staining with antibodies against different secretory organelle markers (red) is shown in the left-hand column. (A) AMA1, a micronemal marker; (B) EBA175RII, another micronemal marker; (C) RAP2, a rhoptry bulb marker; (D) RON4, a rhoptry neck marker; (E) RESA, a dense granule marker. Corresponding costaining with rat anti-HA mAb 3F10 (green) together with the overlay between the red and green channels is shown in the subsequent columns. The right-hand column shows the corresponding differential interference contrast microscopy (DIC) images. (Scale bar, 1 μm.)

Fig. 3.

Fig. 3.

HA-PfROM1 does not localize with markers of different known subcellular organelles. Staining with antibodies against different subcellular organelle markers is shown in the left-hand column. (A) MSP1, a merozoite surface marker; (B) Bip, an ER marker; (C) ERD2, a _cis_-Golgi marker; (D) ACP, an apicoplast marker; (E) cytochrome c, a mitochondrial marker. Corresponding costaining with rat anti-HA mAb 3F10 (A and C–E) or mouse anti-HA mAb 2C16 (B) together with the overlay between the red and green channels is shown in the subsequent columns. The right-hand column shows the corresponding DIC images. (Scale bar, 1 μm.)

Fig. 4.

Fig. 4.

Quantitative assessment of the colocalization between HA-PfROM1 and microtubule staining. (A) Staining with mouse anti-HA mAb 2C16 and rat anti-HA mAb 3F10. (B–D) Staining with mouse anti-microtubule mAb DM1A and rat anti-HA mAb of mature segmenters, immature schizonts, and free merozoites, respectively. Scatter 2D plots of voxel intensities in red and green channels observed for the respective pictures are shown in the right-hand column. Intensity threshold in both the channels was automatically determined by excluding dark pixels by using the algorithm in “Coloc” function of the image analysis software, Imaris (Bitplane) as described in ref. . Pearson's correlation coefficient was determined for voxel intensity correlation between the red and green channels and is displayed in the lower-right-hand corner. (Scale bar, 1 μm.)

Fig. 5.

Fig. 5.

Asymmetric organization of organelles confers a lateral polarity in P. falciparum merozoites. Staining with antibodies against different subcellular organelle markers is shown in the left-hand column. (A) α-Tubulin antibodies staining the merozoite subpellicular microtubules. (B) Bip, an ER marker. (C) ERD2, a _cis_-Golgi marker. (D) ACP, an apicoplast marker. (E) Cytochrome c, a mitochondrial marker. Corresponding costaining with rat anti-HA mAb 3F10 (A and C–E) or mouse anti-HA mAb 2C16 (B) together with nuclear staining with DAPI (blue) and an overlay of all channels is shown in subsequent columns. The right-hand column shows a digital zoom of merozoites showing that the red and green staining occurs on the same side of the nucleus. (Scale bar, 1 μm.)

Fig. 6.

Fig. 6.

HA-PfROM1 is secreted on the merozoite surface and concentrates at the posterior pole of the merozoite. Staining of free merozoites with antibodies against a micronemal marker (A) or with a rhoptry marker at the apical end (B) is shown in the left-hand column (red). Corresponding costaining with rat anti-HA mAb 3F10 (green) together with the overlay between the red and green channels is shown in subsequent columns. The right-hand column shows the corresponding DIC image. Arrow heads point to the merozoite posterior pole. (Scale bar, 1 μm.)

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