Erythrocyte membrane alterations induced by Plasmodium simium infection in Saimiri sciureus: relation to Schüffner's dots.

The nature of erythrocyte membrane alterations in Plasmodium simium infections was determined employing light microscopy, carbon replication and transmission electron microscopy. Light microscopy of Giemsa stained preparations shows that infected cells initially acquire a faint stippling (schuffnerization) which becomes pronouced with subsequent parasite development. Enlargement of the host cell usually accompanied stippling. Both phenomena appear to depend on host cell age since infected mature erythrocytes were neither stippled nor enlarged. Carbon replicas show numerous indentations over the outer membrane surface of most infected cells. Their distribution suggests that they account for Schuffner's granules. The surface indentations are manifest as small infundibular which open to the infected cell's surface. Cytoplasmic microvesciles in the infected cell's stroma frequently are observed adjacent or catenated to the surface infundibula. Images suggest their funsion with the surface infundibula thus adding membrane to the cell's surface and accounting for host cell enlargement.     

Plasmodium falciparum: fine structural changes in the cytoskeletons of infected erythrocytes

Following parasitization by Plasmodium falciparum, numerous changes take place in the host erythrocyte membrane. In this study, we used the technique of whole cell mount electron microscopy to determine if the ultrastructure of the erythrocyte cytoskeleton changed following parasitization with knobby and knobless strains of P. falciparum. Using this technique, a network of spectrin filaments (3-10 X 45-120 nm) branching from electron dense junctions (15-25 nm in diameter), the presumed site of bands 4.1 and actin, were visualized. The overall architecture of normal and parasitized erythrocyte cytoskeletons was the same: however, additional patches (35 to 60 nm in size) and aggregates (30 X 150 nm) of electron dense material were present in parasitized skeletons. The ultrastructure of knobby and knobless cytoskeletons was similar, except knobless skeletons usually did not possess the larger aggregates of material. Antigens associated with the erythrocyte cytoskeleton of cells infected with knobby and knobless strains, but not uninfected cells, were demonstrated by indirect immunofluorescence. Results suggest that antigens, associated with the erythrocyte cytoskeleton, may contribute to perturbations in the host erythrocyte membrane.     

Trafficking determinants for PfEMP3 export and assembly under the Plasmodium falciparum-infected red blood cell membrane

During the maturation of intracellular asexual stages of Plasmodium falciparum parasite-encoded proteins are exported into the erythrocyte cytosol. A number of these parasite proteins attach to the host cell cytoskeleton and facilitate transformation of a disk-shaped erythrocyte into a rounded and more rigid infected erythrocyte able to cytoadhere to the vasculature. Knob formation on the surface of infected erythrocytes is critical for this cytoadherence to the host endothelium. P. falciparum proteins have been identified that localize to the parasite-infected erythrocyte membrane: the variant cytoadherence ligand erythrocyte membrane protein 1 (PfEMP1), the knob-associated histidine-rich protein (KAHRP) and the erythrocyte membrane protein 3 (PfEMP3). In this study, we have generated parasites expressing PfEMP3-green fluorescent protein chimeras and identified domains involved in entry to the secretory pathway, export across the parasitophorous vacuolar membrane and attachment to Maurer's clefts and the erythrocyte membrane. Solubility assays, fluorescence photobleaching experiments and immunogold electron microscopy suggest that the exported chimeric proteins are trafficked in a complex rather than in vesicles. This study characterizes elements involved in the tight but transient binding of PfEMP3 to Maurer's clefts and shows that the same elements are necessary for correct assembly under the erythrocyte membrane.     

Parasite‐encoded Hsp40 proteins define novel mobile structures in the cytosol of the P. falciparum‐infected erythrocyte

Plasmodium falciparum is predicted to transport over 300 proteins to the cytosol of its chosen host cell, the mature human erythrocyte, including 19 members of the Hsp40 family. Here, we have generated transfectant lines expressing GFP‐ or HA‐Strep‐tagged versions of these proteins, and used these to investigate both localization and other properties of these Hsp40 co‐chaperones. These fusion proteins labelled punctate structures within the infected erythrocyte, initially suggestive of a Maurer's clefts localization. Further experiments demonstrated that these structures were distinct from the Maurer's clefts in protein composition. Transmission electron microscopy verifies a non‐cleft localization for HA‐Strep‐tagged versions of these proteins. We were not able to label these structures with BODIPY–ceramide, suggesting a lower size and/or different lipid composition compared with the Maurer's clefts. Solubility studies revealed that the Hsp40–GFP fusion proteins appear to be tightly associated with membranes, but could be released from the bilayer under conditions affecting membrane cholesterol content or organization, suggesting interaction with a binding partner localized to cholesterol‐rich domains. These novel structures are highly mobile in the infected erythrocyte, but based on velocity calculations, can be distinguished from the ‘highly mobile vesicles’ previously described. Our study identifies a further extra‐parasitic structure in the P. falciparum‐infected erythrocyte, which we name ‘J‐dots’ (as their defining characteristic so far is the content of J‐proteins). We suggest that these J‐dots are involved in trafficking of parasite‐encoded proteins through the cytosol of the infected erythrocyte.     

Sorting of malarial antigens into vesicular compartments within the host cell cytoplasm as demonstrated by immunoelectron microscopy

A double and triple immunogold labeling technique has been applied to demonstrate that several malarial antigens of the erythrocytic stages of Plasmodium falciparum are exported from the parasite into distinct compartments within the host cell cytoplasm. Multiple species of vesicles, each with specifically packaged contents, are consistent with a sorting function of vesicular structures in the Plasmodium infected erythrocyte. During schizogony, two parasite antigens, an S-antigen and a parasitophorous vacuole membrane antigen, QF 116, become packaged into such vesicles and are transported into the erythrocyte cytoplasm. At this stage of parasite development, host cell material is taken in through the parasitophorous vacuole membrane into the vacuolar space surrounding the parasite.     

Multimodal analysis of Plasmodium knowlesi‐infected erythrocytes reveals large invaginations,swelling of the host cell,and rheological defects

The simian parasite Plasmodium knowlesi causes severe and fatal malaria infections in humans, but the process of host cell remodelling that underpins the pathology of this zoonotic parasite is only poorly understood. We have used serial block‐face scanning electron microscopy to explore the topography of P. knowlesi‐infected red blood cells (RBCs) at different stages of asexual development. The parasite elaborates large flattened cisternae (Sinton Mulligan's clefts) and tubular vesicles in the host cell cytoplasm, as well as parasitophorous vacuole membrane bulges and blebs, and caveolar structures at the RBC membrane. Large invaginations of host RBC cytoplasm are formed early in development, both from classical cytostomal structures and from larger stabilised pores. Although degradation of haemoglobin is observed in multiple disconnected digestive vacuoles, the persistence of large invaginations during development suggests inefficient consumption of the host cell cytoplasm. The parasite eventually occupies ~40% of the host RBC volume, inducing a 20% increase in volume of the host RBC and an 11% decrease in the surface area to volume ratio, which collectively decreases the ability of the P. knowlesi‐infected RBCs to enter small capillaries of a human erythrocyte microchannel analyser. Ektacytometry reveals a markedly decreased deformability, whereas correlative light microscopy/scanning electron microscopy and python‐based skeleton analysis (Skan) reveal modifications to the surface of infected RBCs that underpin these physical changes. We show that P. knowlesi‐infected RBCs are refractory to treatment with sorbitol lysis but are hypersensitive to hypotonic lysis. The observed physical changes in the host RBCs may underpin the pathology observed in patients infected with P. knowlesi.     

An Exported Heat Shock Protein 40 Associates with Pathogenesis-Related Knobs in Plasmodium falciparum Infected Erythrocytes

Cell surface structures termed knobs are one of the most important pathogenesis related protein complexes deployed by the malaria parasite Plasmodium falciparum at the surface of the infected erythrocyte. Despite their relevance to the disease, their structure, mechanisms of traffic and their process of assembly remain poorly understood. In this study, we have explored the possible role of a parasite-encoded Hsp40 class of chaperone, namely PFB0090c/PF3D7_0201800 (KAHsp40) in protein trafficking in the infected erythrocyte. We found the gene coding for PF3D7_0201800 to be located in a chromosomal cluster together with knob components KAHRP and PfEMP3. Like the knob components, KAHsp40 too showed the presence of PEXEL motif required for transport to the erythrocyte compartment. Indeed, sub-cellular fractionation and immunofluorescence analysis (IFA) showed KAHsp40 to be exported in the erythrocyte cytoplasm in a stage dependent manner localizing as punctuate spots in the erythrocyte periphery, distinctly from Maurer's cleft, in structures which could be the reminiscent of knobs. Double IFA analysis revealed co-localization of PF3D7_0201800 with the markers of knobs (KAHRP, PfEMP1 and PfEMP3) and components of the PEXEL translocon (Hsp101, PTEX150). KAHsp40 was also found to be in a complex with KAHRP, PfEMP3 and Hsp101 as confirmed by co-immunoprecipitation assay. Our results suggest potential involvement of a parasite encoded Hsp40 in chaperoning knob assembly in the erythrocyte compartment.     

Cellular architecture of Plasmodium falciparum-infected erythrocytes

Plasmodium falciparum is a protozoan parasite that is responsible for the most pathogenic form of human malaria. The particular virulence of this parasite derives from its ability to develop within the erythrocytes of its host and to subvert their function. The intraerythrocytic parasite devours haemoglobin, and remodels its host cell to cause adhesion to blood vessel walls. Ultrastructural studies of P. falciparum have played a major role in defining its cell architecture and in resolving cell biology controversies. Here we review some of the early studies and describe some recent developments in electron microscopy techniques that have revealed information about the organization of the parasite in the blood stage of development. We present images of P. falciparum at different stages of the life cycle and highlight some of the plasmodium-specific organelles, the haemoglobin digestive apparatus and the membrane structures that are elaborated in the host cell cytoplasm to traffic virulence proteins to the erythrocyte surface. We describe methods for whole cell ultrastructural imaging that can provide three-dimensional views of intraerythrocytic development.     

Erythrocyte exit: Out, damned merozoite! Out I say!

A new study has combined video microscopy with fluorescent labeling of host and parasite membranes to follow Plasmodium falciparum merozoites as they exit their host erythrocyte. The result has yielded some arresting images, which make compelling viewing irrespective of whether or not you have an interest in cell motility in general or P. falciparum erythrocyte exit in particular. Moreover, this work injects important new insights into the long-running debate about the biological mechanisms that underpin merozoite release.     

Structural alteration of the membrane of erythrocytes infected with Plasmodium falciparum

Human erythrocytes infected with five strains of Plasmodium falciparum and Aotus erythrocytes infected with three strains of P. falciparum were studied by thin-section and freeze-fracture electron microscopy. All strains of P. falciparum we studied induced electron-dense conical knobs, measuring 30-40 nm in height and 90-100 nm in diameter on erythrocyte membranes. Freeze-fracture demonstrated that the knobs were distributed over the membrane of both human and Aotus erythrocytes. A distinct difference was seen between the intramembrane particle (IMP) distribution over the knobs of human and Aotus erythrocyte membranes. There was no change in IMP distribution in infected human erythrocyte membranes, but infected Aotus erythrocytes showed an aggregation of IMP over the P face of the knobs with a clear zone at the base. This difference in IMP distribution was related only to the host species and not to parasite strains. Biochemical analysis demonstrated that a higher proportion of band 3 was bound to the cytoskeleton of uninfected Aotus erythrocytes than uninfected human erythrocytes after Triton X-100 extraction. This may account for the different effects of P. falciparum infection on IMP distribution in the two different cell types.     

Vesicle-mediated trafficking of parasite proteins to the host cell cytosol and erythrocyte surface membrane in Plasmodium falciparum infected erythrocytes

During the development of the asexual stage of the malaria parasite, Plasmodium falciparum, the composition, structure and function of the host cell membrane is dramatically altered, including the ability to adhere to vascular endothelium. Crucial to these changes is the transport of parasite proteins, which become associated with or inserted into the erythrocyte membrane. Protein and membrane targeting beyond the parasite plasma membrane must require unique pathways, given the parasites intracellular location within a parasitophorous vacuolar membrane and the lack of organelles and biosynthetic machinery in the host cell necessary to support a secretory system. It is not clear how these proteins cross the parasitophorous vacuolar membrane or how they traverse the erythrocyte cytosol to reach their final destinations. The identification of: (1) a P. falciparum homologue of the protein Sar1p, which is an essential component of the COPII-based secretory system in mammalian cells and yeast and (2) electron-dense, possibly coated, secretory vesicles bearing P. falciparum erythrocyte membrane protein 1 and P. falciparum erythrocyte membrane protein 3 in the host cell cytosol of P. falciparum infected erythrocytes recently provided the first direct evidence of a vesicle-mediated pathway for the trafficking of some parasite proteins to the erythrocyte membrane. The major advance in uncovering the parasite-induced secretory pathway was made by incubating infected erythrocytes with aluminium tetrafluoride, an activator of guanidine triphosphate-binding proteins, which resulted in the accumulation of the vesicles into multiple vesicle strings. These vesicle complexes were often associated with and closely abutted the erythrocyte membrane, but were apparently prevented from fusing by the aluminium fluoride treatment, making their capture by electron microscopy possible. It appears that malaria parasites export proteins into the host cell cytosol to support a vesicle-mediated protein trafficking pathway.     

Electron tomography of the Maurer's cleft organelles of Plasmodium falciparum-infected erythrocytes reveals novel structural features

During intraerythrocytic development, the human malaria parasite, Plasmodium falciparum, establishes membrane-bound compartments, known as Maurer's clefts, outside the confines of its own plasma membrane. The Maurer's compartments are thought to be a crucial component of the machinery for protein sorting and trafficking; however, their ultrastructure is only partly defined. We have used electron tomography to image Maurer's clefts of 3D7 strain parasites. The compartments are revealed as flattened structures with a translucent lumen and a more electron-dense coat. They display a complex and convoluted morphology, and some regions are modified with surface nodules, each with a circular cross-section of approximately 25 nm. Individual 25 nm vesicle-like structures are also seen in the erythrocyte cytoplasm and associated with the red blood cell membrane. The Maurer's clefts are connected to the red blood cell membrane by regions with extended stalk-like profiles. Immunogold labelling with specific antibodies confirms differential labelling of the Maurer's clefts and the parasitophorous vacuole and erythrocyte membranes. Spot fluorescence photobleaching was used to demonstrate the absence of a lipid continuum between the Maurer's clefts and parasite membranes and the host plasma membrane.     

3D reconstruction of Trypanosoma cruzi-macrophage interaction shows the recruitment of host cell organelles towards parasitophorous vacuoles during its biogenesis

Trypanosoma cruzi has a complex life cycle where two infective developmental stages, known as trypomastigote and amastigote, can be found in the vertebrate host. Both forms can invade a large variety of cellular types and induce the formation of a parasitophorous vacuole (PV), that, posteriorly, disassembles and releases the parasites into the host cell cytoplasm. The biogenesis of T. cruzi PVs has not been analyzed in professional phagocytic cells. We investigated the biogenesis of PVs containing trypomastigotes or amastigotes in peritoneal macrophages. We observed the presence of profiles of the endoplasmic reticulum and lysosomes from the host cell near PVs at early stages of interaction in both developmental stages, suggesting that both organelles may participate as possible membrane donors for the formation of the PVs. The Golgi complex, however, was observed only near already formed PVs. Electron microscopy tomography and FIB-SEM microscopy followed by 3D reconstruction of entire PVs containing amastigotes or trypomastigotes confirmed the presence of both endoplasmic reticulum and lysosomes in the initial stages of PV formation. In addition, Golgi complex and mitochondria localize around PVs during their biogenesis. Taken together these observations provide a whole view of the invasion process in a professional phagocytic cell.     

Life Cycle-Dependent Cytoskeletal Modifications in Plasmodium falciparum Infected Erythrocytes

Plasmodium falciparum infection of human erythrocytes is known to result in the modification of the host cell cytoskeleton by parasite-coded proteins. However, such modifications and corresponding implications in malaria pathogenesis have not been fully explored. Here, we probed the gradual modification of infected erythrocyte cytoskeleton with advancing stages of infection using atomic force microscopy (AFM). We reported a novel strategy to derive accurate and quantitative information on the knob structures and their connections with the spectrin network by performing AFM–based imaging analysis of the cytoplasmic surface of infected erythrocytes. Significant changes on the red cell cytoskeleton were observed from the expansion of spectrin network mesh size, extension of spectrin tetramers and the decrease of spectrin abundance with advancing stages of infection. The spectrin network appeared to aggregate around knobs but also appeared sparser at non-knob areas as the parasite matured. This dramatic modification of the erythrocyte skeleton during the advancing stage of malaria infection could contribute to the loss of deformability of the infected erythrocyte.     

Localization of a parasite encoded protein to erythrocyte cytoplasmic vesicles of Plasmodium falciparum-infected cells

Intracellular development of the malarial parasite results in substantial modifications of the membrane and cytoskeleton of the erythrocyte host cell. Two related Plasmodium falciparum-encoded proteins of 50 kDa and 43 kDa (Pf 50/43), identified by reactivity with a single mAb, were demonstrated to be localized to the erythrocyte cytoplasm of parasite-infected cells. Immunofluorescence and immunoelectron microscopy using mAb.7E11 demonstrated the Pf 50/43 is localized in the membrane of the vesicles in the erythrocyte cytoplasm, vesicles which correspond to Maurer's clefts. Solubility properties of the proteins suggest they are integral membrane proteins. By immunofluorescence, Pf 50/43 is shown to colocalize with actin which has a highly modified organization in the infected erythrocyte. Pf 50/43 is located exclusively in the vesicles, is not transported to the erythrocyte membrane or secreted. It is proposed the vesicles may play a role in transport of molecules across the erythrocyte cytoplasm, between the parasite and the external erythrocyte plasma membrane.     

From Voxels to Knowledge: A Practical Guide to the Segmentation of Complex Electron Microscopy 3D-Data

Modern 3D electron microscopy approaches have recently allowed unprecedented insight into the 3D ultrastructural organization of cells and tissues, enabling the visualization of large macromolecular machines, such as adhesion complexes, as well as higher-order structures, such as the cytoskeleton and cellular organelles in their respective cell and tissue context. Given the inherent complexity of cellular volumes, it is essential to first extract the features of interest in order to allow visualization, quantification, and therefore comprehension of their 3D organization. Each data set is defined by distinct characteristics, e.g., signal-to-noise ratio, crispness (sharpness) of the data, heterogeneity of its features, crowdedness of features, presence or absence of characteristic shapes that allow for easy identification, and the percentage of the entire volume that a specific region of interest occupies. All these characteristics need to be considered when deciding on which approach to take for segmentation.The six different 3D ultrastructural data sets presented were obtained by three different imaging approaches: resin embedded stained electron tomography, focused ion beam- and serial block face- scanning electron microscopy (FIB-SEM, SBF-SEM) of mildly stained and heavily stained samples, respectively. For these data sets, four different segmentation approaches have been applied: (1) fully manual model building followed solely by visualization of the model, (2) manual tracing segmentation of the data followed by surface rendering, (3) semi-automated approaches followed by surface rendering, or (4) automated custom-designed segmentation algorithms followed by surface rendering and quantitative analysis. Depending on the combination of data set characteristics, it was found that typically one of these four categorical approaches outperforms the others, but depending on the exact sequence of criteria, more than one approach may be successful. Based on these data, we propose a triage scheme that categorizes both objective data set characteristics and subjective personal criteria for the analysis of the different data sets.     

Erythrocyte remodeling in Plasmodium berghei infection: the contribution of SEP family members

The malaria parasite Plasmodium largely modifies the infected erythrocyte through the export of proteins to multiple sites within the host cell. This remodeling is crucial for pathology and translocation of virulence factors to the erythrocyte surface. In this study, we investigated localization and export of small exported proteins/early transcribed membrane proteins (SEP/ETRAMPs), conserved within Plasmodium genus. This protein family is characterized by a predicted signal peptide, a short lysine-rich stretch, an internal transmembrane domain and a highly charged C-terminal region of variable length. We show here that members of the rodent Plasmodium berghei family are components of the parasitophorous vacuole membrane (PVM), which surrounds the parasite throughout the erythrocytic cycle. During P. berghei development, vesicle-like structures containing these proteins detach from the PVM en route to the host cytosol. These SEP-containing vesicles remain associated with the infected erythrocyte ghosts most probably anchored to the membrane skeleton. Transgenic lines expressing the green fluorescent protein appended to different portions of sep-coding region allowed us to define motifs required for protein export. The highly charged terminal region appears to be involved in protein-protein interactions.     

小鼠海马锥体细胞树突棘形态的电镜三维重建

大多数神经元的复杂三维结构是很难直接观察的。激光扫描共聚焦显微镜技术结合染料标记技术可以重建神经元的三维形态,但精细结构的识别需要电子显微镜。利用透射电子显微镜技术,可以得到连续超薄组织切片的高分辨率图像,结合计算机支持的三维重建技术就可进一步获得神经细胞精细结构的三维信息。通过电镜三维重建技术对未成熟和成熟小鼠海马锥体细胞树突棘的形态进行了观察和分析,并对其关键步骤的操作技巧进行了重点说明。实验结果为进一步利用成像技术研究树突棘的结构、功能和可塑性提供了重要信息。     

Host Cell and Malarial Targets for Docetaxel (Taxotere™) during the Erythrocytic Development of Plasmodium falciparum

The microtubular stabilizing agent docetaxel (Taxotere™) is known to inhibit the intraerythrocytic development of Plasmodium falciparum. To investigate the mechanism(s) of inhibition, we analyzed the structural organization of the mitotic spindle by immunofluorescence and electron microscopy. When 30 μM docetaxel was applied for five hours on ring forms, alterations in the mitotic spindles leading to abnormal nuclear divisions were observed. At the trophozoite- and schizont-stage, docetaxel pulses prevent mitosis by stabilizing microtubular structures associated with the mitotic apparatus, giving abnormal spindles. However, this inhibition did not interfere with parasite DNA synthesis indicating the absence of a checkpoint that couples exit from mitosis with proper spindle assembly as observed in higher eukaryotic cells. In parallel, intraerythrocytic concentration of docetaxel was measured in parasitized erythrocytes, after incubation of cells with ³H-docetaxel for five hours. It was found to be 14-fold increased at the ring-stage of infected erythrocytes compared to normal ones, 170-fold increased at the trophozoite-stage and 1,500-fold increased at the schizont-stage. Our data show that, even though the overall intracellular concentration of docetaxel is low in docetaxel-pulsed rings, the agent might be sufficient to disturb the spindle organization. However, the existence of targets for docetaxel other than mitotic spindle microtubules. i.e. erythrocyte membrane components, could interfere with mitotic spindle formation     

Expression of the parasite protein Pc90 in plasma membranes of erythrocytes infected with Plasmodium chabaudi

Erythrocytes infected with the malaria parasite Plasmodium chabaudi contain the neo-protein Pc90 in their plasma membrane. We investigate origin, membrane disposition, and intraerythrocytic traffic of this Pc90. Metabolic labeling of P.-infected erythrocytes, combined with cell fractionation as well as Western blot analysis and immunoprecipitation using a Pc90-recognizing monoclonal antibody, show that Pc90 is synthesized by early to mid trophozoites and is transported without any apparent processing steps to the erythrocyte membrane. Based upon the inaccessibility of Pc90 from the outside in intact erythrocytes and the water solubility of membrane-associated Pc90, it is concluded that Pc90 is localized on the cytoplasmic face of the host erythrocyte membrane. Immunoelectron microscopy using a Pc90-specific monoclonal antibody and the occurrence of soluble Pc90 in host cell cytosol indicate that the Pc90 is transported in both a 'vesicle-bound' and a 'free' form through the erythrocyte cytoplasm.