Biogenesis of the inner membrane complex is dependent on vesicular transport by the alveolate specific GTPase Rab11B

Abstract

Apicomplexan parasites belong to a recently recognised group of protozoa referred to as Alveolata. These protists contain membranous sacs (alveoli) beneath the plasma membrane, termed the Inner Membrane Complex (IMC) in the case of Apicomplexa. During parasite replication the IMC is formed de novo within the mother cell in a process described as internal budding. We hypothesized that an alveolate specific factor is involved in the specific transport of vesicles from the Golgi to the IMC and identified the small GTPase Rab11B as an alveolate specific Rab-GTPase that localises to the growing end of the IMC during replication of Toxoplasma gondii. Conditional interference with Rab11B function leads to a profound defect in IMC biogenesis, indicating that Rab11B is required for the transport of Golgi derived vesicles to the nascent IMC of the daughter cell. Curiously, a block in IMC biogenesis did not affect formation of sub-pellicular microtubules, indicating that IMC biogenesis and formation of sub-pellicular microtubules is not mechanistically linked. We propose a model where Rab11B specifically transports vesicles derived from the Golgi to the immature IMC of the growing daughter parasites.

Figure 1. Alveolate organisms possess two distinct types of Rab11 homologues.

(A) This tree, which presents the best maximum likelihood (ML) phylogeny out of 12 trees (ln likelihood = −13,028.03), clearly shows the two distinct apicomplexan Rab11 subfamilies Rab11A and Rab11B. While the Rab11B cluster includes several alveolate sequences from outside the Apicomplexa and is well supported by the bootstrap analysis, the apicomplexan Rab11A sequences are situated among the larger diversity of eukaryote Rab11 homologues, usually in close proximity to Rab11 from red algae. The apicomplexan Rab11A cluster is generally unsupported by the bootstrap analysis but does occur as a monophyletic unit in some trees: panel (B) shows the corresponding branch of another ML tree with a slightly lower likelihood (ln likelihood = −13,033.89). Bootstrap support values (100 replicates) for ML and Neighbor-Joining (NJ) analyses are indicated above and below the relevant branches, respectively, where they are greater than 50. GenBank accession numbers are indicated in the figure behind the genus names. Species names: Arabidopsis thaliana, Aspergillus flavus, Babesia bovis, Chlamydomonas reinhardtii, Cryptococcus neoformans, Cryptosporidium hominis, Cryptosporidium parvum, Cyanidioschyzon merolae, Danio rerio, Dictyostelium discoideum, Drosophila melanogaster, Entamoeba histolytica, Gracilaria lemaneiformis, Homo sapiens, Leishmania major, Monosiga brevicollis, Neurospora crassa, Paramecium tetraurelia, Perkinsus marinus, Phaeodactylum tricornutum, Phytophthora infestans, Picea sitchensis, Plasmodium falciparum, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Tetrahymena thermophila, Thalassiosira pseudonana, Theileria parva, Toxoplasma gondii, Trichomonas vaginalis, Trichoplax adhaerens, Trypanosoma cruzi, Ustilago maydis.

Figure 2. Dynamic location of Rab11B between the golgi and the nascent IMC during cell division.

(A) Immunofluorescent analysis of parasites expressing the wild type Rab11B under control of the endogenous promoter (pRab11B_wt). Parasites were double-labelled with anti-myc (red) and anti-IMC1 (green) to visualise Rab11B and IMC1. Rab11B cycles from a location close to the nucleus (interphase parasites in upper panel) to the growing IMC of the daughter parasites (dividing parasites in middle and lower panel) in a cell cycle dependent manner. (B) Immunofluorescent analysis of parasites expressing ddRab11B_wt and the Golgi marker (GRASP-RFP in red) in presence of 0.1 µM Shld-1 for 24 h. Parasites are labelled with anti-myc antibody (green). Rab11B localises to the Golgi at the initial phase of cell division (upper panel and inlet) and accumulates to the nascent IMC of daughter parasites during endodyogeny (see A,C middle panel). After endodyogeny is completed Rab11B again accumulates at the Golgi (lower panel and inlet). (C) Immunofluorescent analysis of parasites expressing the ddRab11B_wt and the α-tubulin marker (mCherry-α-Tubulin in red) in presence of 0.25 µM Shld-1 for 24 h. Parasites are labelled with anti-myc antibody (green). At the onset of endodyogeny (upper panel) Rab11B accumulates to the newly assembled conoid of the daughter cells. Later becomes concentrated along the daughter scaffold throughout endodyogeny (middle and lower panel). Scale bars represent 5 µm.

Figure 3. Functional loss of Rab11B results in the disruption of the daughter cell IMCs.

Analysis of parasites expressing an ectopic copy of ddRab11B_wt or ddRab11B_N126I. (A) A Shld-1 concentration gradient with the clonal ddRab11B_wt (upper panel) and ddRab11B_N125I (lower panel) using immunofluorescent analysis. Parasites were double-labelled with anti-myc (red) to visualise Rab11B and anti-IMC1 (green). Parasites expressing ddRab11B_N125I show a severe defect on the IMC formation at low Shld-1 concentration. In contrast, ddRab11B_wt expressing parasites demonstrate a relative weak phenotypic deformation of the IMC at high Shld-1 concentrations. Scale bar represents 5 µm. (B) Growth analysis of the indicated parasites grown on HFF cells for 7 days. In the ddRab11B_wt expressing parasites plaque size is slowly reduced with increasing Shld-1 concentrations (upper panel). In the dd-Rab11B_N125I parasites growth is strongly inhibited at low concentrations of Shld-1 and completely absent at concentrations <0.5 µM Shld-1 (lower panel). Scale bar represents 5 mm. (C) Immunoblot analysis of clonal ddRab11B_wt (left) and ddRab11B_N125I (right) transfectans. Rising levels of the fusion proteins (40 kDa) can be detected in presence of increasing Shld-1 concentrations. For detection of Rab11B the blot was probed with anti-FKBP12 (Affinity BioReagents). MIC8 served as internal loading control. Note that in presence of 0.1 µM Shld-1 ddRab11B_wt is almost fully stabilised, whereas ddRab11B_N125I is barely detectable.

Figure 4. Phenotypic analysis of the IMC formation in dd-Rab11B_N125I expressing parasites.

(A) ddRab11B_N125I parasites co-expressing YFP-MyoA (green) were treated without and with 1 µM Shld-1 for 24 h. For detection of the dominant negative Rab11B, parasites were labelled with anti-myc (red). (B) ddRab11B_N125I parasites co-expressing different YFP-tagged components of the glideosome/IMC (green) were treated without and with Shld-1 for 24 h. In absence of Shld-1 Gap50 can be identified in the IMC of first- and premature second-generation daughter parasites, while Gap45 can only be detected in the mature IMC (upper panel). In presence of Shld-1, early and late components appear comparably affected during IMC assembly. Initial nucleation of the daughter IMC can still take place (white arrow, second panel). MLC1 remains restricted to the pellicle of the first generation mother cell, while only a faint staining of IMC 1 can be detected (white arrow, middle panel). MLC 1 and MyoA only co-localise at the IMC of the mother, while MLC1 also accumulates close to the nucleus of forming daughter cells (white arrow, last panel). Scale bars represent 5 µm.

Figure 5. Fate of the secretory organelles and mitochondrial division in dd-Rab11B_N125I expressing parasites.

(A,B and C) Immunofluorescent analysis of ddRab11B_N125I expressing parasites incubated in absence and presence of Shld-1 for 24 h and stained with the indicated antibodies. In presence of Shld-1 the organelles are still formed, although the organelles lose their apical localisation (see also Figure 7, 8). SAG1 appears to be correctly localised to the plasma membrane of the mother parasite indicating functional transport of GPI-anchored proteins to the surface.(D and E) ddRab11B_N125I parasites co-expressing the mitochondrial marker HSP60-RFP (red) or the apicoplast marker FNR-RFP (red) were treated without and with Shld-1 for 24 h and subsequently labelled with anti-IMC1 (green). In presence of Shld-1 mitochondrial division and apicoplast segregation appeared normal. Scale bars represent 5 µm.

Figure 6. Absence of IMC biogenesis does not interfere with formation of sub-pellicular microtubules.

(A) ddRab11B_N125I parasites co-expressing the Golgi marker GRASP-RFP (red) were treated without and with Shld-1 for 24 h and labelled with the indicated antibodies (green). In presence of Shld-1 no IMC is formed, but nuclear and Golgi division appears normal (B) ddRab11B_N125I parasites co-expressing IMC1-YFP (green) were grown in absence and presence of Shld-1 for 24 h and subsequently labelled with anti-MORN1 (red). In presence of inducer the centrocone close to the nucleus appears normal (arrow head). However, disruption of IMC formation inhibits formation and relocalisation of the posterior polar ring of daughter cells (white arrow). (C and D) ddRab11B_N125I parasites co-expressing mCherry-α-Tubulin (red) were treated without and with inducer for 24 h and labelled with the anti-IMC1 antibody (green) or with anti-MORN1 antibody (green), respectively. Abrogation of IMC formation does not inhibit assembly and growth of the sub-pellicular microtubules (white arrow). Nevertheless, no formation of the basal complex can be observed despite presence of initial daughter formation (see inlet). Scale bar represents 5 µm.

Figure 7. Electron micrographs of sections of T. gondii in fibroblast.

Parasites were grown without (A) or with Shld-1 (B–E) for 36 h. (A) Low power through a parasitophorous vacuole showing a rosette of eight parasites having undergone three cycles of repeated endodyogeny. N – nucleus. (B) Low power of a large spherical parasite that appears to have a number of nuclei (N) but no evidence of daughter formation. (C) Section through a slightly elongated parasite that appears to have a complex nuclear structure (N) with a number of lobes (arrows). (D) Section through a parasite with two nuclei (N) in which four conical structures representing the initiation of daughter formation (D1–4) can be identified. In A–D scale bar represents 1 µm. (E) Detail showing an early daughter from a stage similar to that in D. The apical conoid (C) and longitudinally running sub-pellicular microtubule (MT) can be identified. Note the absence of IMC on the outside of the microtubule although a number of vesicles (arrows) could be identified. (F) Detail of a cross section through an early daughter showing the sub-pellicular microtubules (MT) with overlying electron dense material (arrows) and an absence of the IMC. A – apicoplast. In E and F scale bar represents 100 nm.

Figure 8. Electron micrographs of parasites cultured with Shld-1.

Parasites were cultured for 24 h (C, D) and 36 h (A, B) Prior to Fixation. (A) Low power of sub-spherical parasite showing multiple nuclei (N) and abnormally located conoids (C) and groups of rhoptries (R) and micronemes (MN) within the mother cell cytoplasm. Scale bar represents 1 µm. (B) Detail of the enclosed area in (A) showing the conoid and sub-pellicular microtubules (MT) representing the anterior of a daughter. There are normal appearing rhoptries (R) and micronemes (MN) associate with the apex of the daughter but note the absence of an overlying IMC. Scale bar represents 100 nm. (C) Lower showing two parasites that appear to be undergoing endodyogeny in which the developing daughters (D) can be identified. N – nucleus. Scale bar represents 1 µm. (D) Detail of the enclosed area in C showing the presence of the IMC and underlying microtubules (MT) but note the abnormal gaps and overlapping between the plates of the IMC. Scale bar represents 100 nm.

Figure 9. Overview of the Rab11 functions during endodyogeny.

(A) In interphase parasites Rab11B resides at the Golgi. (B) During the initial phase of endodyogeny duplication of the Golgi and the formation the apical complex takes place and subsequently the nucleation of the IMC. (C) In developing daughter cell Rab11B cycles between Golgi and the nascent IMC of the daughter. (D) In the final step of cytokinesis separation of the two daughter cells requires the formation of novel plasma membrane by Rab11A dependent vesicular transport. The green and black arrows indicate Rab11B and Rab11A mediated transport respectively.