Plasmodium falciparum exports the Golgi marker sphingomyelin synthase into a tubovesicular network in the cytoplasm of mature erythrocytes

This work describes two unusual features of membrane development in a eukaryotic cell. (a) The induction of an extensive network of tubovesicular membranes by the malaria parasite Plasmodium falciparum in the cytoplasm of the mature erythrocyte, and its visualization with two ceramide analogues C5-DMB-ceramide and C6-NBD-ceramide. "Sectioning" of the infected erythrocytes using laser confocal microscopy has allowed the reconstruction of detailed three-dimensional images of this novel membrane network. (b) The stage-specific export of sphingomyelin synthase, a biosynthetic activity concentrated in the Golgi of mammalian cells, to this tubovesicular network. Evidence is presented that in the extracellular merozoite stage the parasite retains sphingomyelin synthase within its plasma membrane. However, intracellular ring- and trophozoite-stage parasites export a substantial fraction (approximately 26%) of sphingomyelin synthase activity to membranes beyond their plasma membrane. Importantly we do not observe synthesis of new enzyme during these intracellular stages. Taken together these results strongly suggest that the export of this classic Golgi enzyme is developmentally regulated in Plasmodium. We discuss the significance of this export and the tubovesicular network with respect to membrane development and function in the erythrocyte cytosol.

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Secretion of Plasmodium falciparum rhoptry protein into the plasma membrane of host erythrocytes.

The Journal of Cell Biology ◽

10.1083/jcb.106.5.1507 ◽

1988 ◽

Vol 106 (5) ◽

pp. 1507-1513 ◽

Cited By ~ 98

Author(s):

T Y Sam-Yellowe ◽

H Shio ◽

M E Perkins

Keyword(s):

Monoclonal Antibody ◽

Plasmodium Falciparum ◽

Plasma Membrane ◽

Final Stage ◽

Trophozoite Stage ◽

Parasite Invasion ◽

Disulfide Linkage ◽

The Matrix ◽

Erythrocytic Cycle ◽

Rhoptry Protein

The rhoptry is an organelle of the malarial merozoite which has been suggested to play a role in parasite invasion of its host cell, the erythrocyte. A monoclonal antibody selected for reactivity with this organelle identifies a parasite synthesized protein of 110 kD. From biosynthetic labeling experiments it was demonstrated that the protein is synthesized midway through the erythrocytic cycle (the trophozoite stage) but immunofluorescence indicates the protein is not localized in the organelle until the final stage (segmenter stage) of intraerythrocytic development. Immunoelectron microscopy shows that the protein is localized in the matrix of the rhoptry organelle and on membranous whorls secreted from the merozoite. mAb recognition of the protein is dithiothreitol (DTT) labile, indicating that the conformation of the epitope is dependent on a disulfide linkage. During erythrocyte reinvasion by the extracellular merozoite, immunofluorescence shows the rhoptry protein discharging from the merozoite and spreading around the surface of the erythrocyte. The protein is located in the plasma membrane of the newly invaded erythrocyte. These studies suggest that the 110-kD rhoptry protein is inserted into the membrane of the host erythrocyte during merozoite invasion.

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Antimalarials increase vesicle pH in Plasmodium falciparum.

The Journal of Cell Biology ◽

10.1083/jcb.101.6.2302 ◽

1985 ◽

Vol 101 (6) ◽

pp. 2302-2309 ◽

Cited By ~ 166

Author(s):

D J Krogstad ◽

P H Schlesinger ◽

I Y Gluzman

Keyword(s):

Plasmodium Falciparum ◽

Mammalian Cells ◽

Human Fibroblasts ◽

Food Vacuole ◽

Malarial Parasite ◽

Red Cell ◽

Trophozoite Stage ◽

Weak Bases ◽

Erythrocytic Cycle ◽

Initial Ph

The asexual erythrocytic stage of the malarial parasite ingests and degrades the hemoglobin of its host red cell. To study this process, we labeled the cytoplasm of uninfected red cells with fluorescein-dextran, infected those cells with trophozoite- and schizont-rich cultures of Plasmodium falciparum, and harvested them 110-120 h later in the trophozoite stage. After lysis of the red cell cytoplasm with digitonin, the only fluorescence remaining was in small (0.5-0.9 micron) vesicles similar to the parasite's food vacuole. As measured by spectrofluorimetry, the pH of these vesicles was acid (initial pH 5.2-5.4), and they responded to MgATP with acidification and to weak bases such as NH4Cl with alkalinization. These three properties are similar to those obtained with human fibroblasts and suggest that the endocytic vesicles of plasmodia are similar to those of mammalian cells. Each of the antimalarials tested (chloroquine, quinine, and mefloquine) as well as NH4Cl inhibited parasite growth at concentrations virtually identical to those that increased parasite vesicle pH. These results suggest two conclusions: (a) The increases in vesicle pH that we have observed in our digitonin-treated parasite preparation occur at similar concentrations of weak bases and antimalarials in cultures of parasitized erythrocytes, and (b) P. falciparum parasites are exquisitely dependent on vesicle pH during their asexual erythrocytic cycle, perhaps for processes analogous to endocytosis and proteolysis in mammalian cells, and that antimalarials and NH4Cl may act by interfering with these events.

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Plasmodium falciparum-infected erythrocytes utilize a synthetic truncated ceramide precursor for synthesis and secretion of truncated sphingomyelin

Biochemical Journal ◽

10.1042/bj3080335 ◽

1995 ◽

Vol 308 (1) ◽

pp. 335-341 ◽

Cited By ~ 22

Author(s):

I Ansorge ◽

D Jeckel ◽

F Wieland ◽

K Lingelbach

Keyword(s):

Plasmodium Falciparum ◽

Mammalian Cells ◽

Membrane Fraction ◽

Infected Erythrocyte ◽

Brefeldin A ◽

Parasite Development ◽

Phospholipid Content ◽

Unicellular Eukaryote ◽

Fungal Metabolite ◽

Sphingomyelin Synthase

Plasmodium falciparum is an intracellular parasite of human erythrocytes. Parasite development is accompanied by an increase of the phospholipid content of the infected erythrocyte, but it results in a selective decrease of sphingomyelin. We have studied sphingomyelin biosynthesis in infected erythrocytes using as substrate a synthetic radiolabelled ceramide precursor, truncated in both hydrophobic chains. Lysates of infected, unlike those of non-infected, erythrocytes contained sphingomyelin synthase activity, which therefore is of parasite origin. The enzyme activity was associated with a membrane fraction. In contrast to mammalian cells, the parasite did not synthesize detectable levels of glycosphingolipids. In intact infected erythrocytes the ceramide precursor was converted into a correspondingly truncated soluble sphingomyelin which was released into the medium at 37 degrees C. Release of truncated sphingomyelin was inhibited by low temperature (15 degrees C) but not by the fungal metabolite brefeldin A which, however, arrests protein export from the parasite. While membranes of mammalian cells, including the plasma membrane of non-infected erythrocytes, are impermeable to truncated sphingomyelin, the membrane of infected erythrocytes allowed passage of the molecule in both directions. The results obtained with the unicellular eukaryote used here as an experimental model are discussed in comparison with sphingomyelin synthesis and transport in mammalian cells.

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Three-dimensional cell geometry controls excitable membrane signaling in Dictyotelium cells

10.1101/278853 ◽

2018 ◽

Author(s):

Marcel Hörning ◽

Tatsuo Shibata

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Three Dimensional ◽

Reaction Diffusion ◽

Membrane Topology ◽

Membrane Area ◽

Wave Dynamics ◽

Entire Cell ◽

Membrane Signaling ◽

Dimensional Cell

AbstractPhosphatidylinositol (3,4,5)-trisphosphate (PtdInsP3) is known to propagate as waves on the plasma membrane and is related to the membrane protrusive activities in Dictyostelium and mammalian cells. While there have been a few attempts to study the three-dimensional dynamics of these processes, most studies have focused on the dynamics extracted from single focal planes. However, the relation between the dynamics and three-dimensional cell shape remains elusive, due to the lack of signaling information about the unobserved part of the membrane. Here we show that PtdInsP3 wave dynamics are directly regulated by the three-dimensional geometry - size and shape - of the plasma membrane. By introducing an analysis method that extracts the three-dimensional spatiotemporal activities on the entire cell membrane, we show that PtdInsP3 waves self-regulate their dynamics within the confined membrane area. This leads to changes in speed, orientation and pattern evolution, following the underlying excitability of the signal transduction system. Our findings emphasize the role of the plasma membrane topology in reaction-diffusion driven biological systems and indicate its importance in other mammalian systems.

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Defining the Morphology and Mechanism of the Hemoglobin Transport Pathway in Plasmodium falciparum-Infected Erythrocytes

Eukaryotic Cell ◽

10.1128/ec.00267-14 ◽

2015 ◽

Vol 14 (4) ◽

pp. 415-426 ◽

Cited By ~ 21

Author(s):

Katharine J. Milani ◽

Timothy G. Schneider ◽

Theodore F. Taraschi

Keyword(s):

Plasmodium Falciparum ◽

Three Dimensional ◽

Parasite Development ◽

Transport Pathway ◽

Trophozoite Stage ◽

Pharmacological Agents ◽

Content Type ◽

Protein Receptor ◽

Morphological Organization ◽

Hemoglobin Degradation

ABSTRACT Hemoglobin degradation during the asexual cycle of Plasmodium falciparum is an obligate process for parasite development and survival. It is established that hemoglobin is transported from the host erythrocyte to the parasite digestive vacuole (DV), but this biological process is not well characterized. Three-dimensional reconstructions made from serial thin-section electron micrographs of untreated, trophozoite-stage P. falciparum -infected erythrocytes (IRBC) or IRBC treated with different pharmacological agents provide new insight into the organization and regulation of the hemoglobin transport pathway. Hemoglobin internalization commences with the formation of cytostomes from localized, electron-dense collars at the interface of the parasite plasma and parasitophorous vacuolar membranes. The cytostomal collar does not function as a site of vesicle fission but rather serves to stabilize the maturing cytostome. We provide the first evidence that hemoglobin transport to the DV uses an actin-myosin motor system. Short-lived, hemoglobin-filled vesicles form from the distal end of the cytostomes through actin and dynamin-mediated processes. Results obtained with IRBC treated with N -ethylmaleimide (NEM) suggest that fusion of hemoglobin-containing vesicles with the DV may involve a soluble NEM-sensitive factor attachment protein receptor-dependent mechanism. In this report, we identify new key components of the hemoglobin transport pathway and provide a detailed characterization of its morphological organization and regulation.

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Three-dimensional architecture of extended synaptotagmin-mediated endoplasmic reticulum–plasma membrane contact sites

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1503191112 ◽

2015 ◽

Vol 112 (16) ◽

pp. E2004-E2013 ◽

Cited By ~ 121

Author(s):

Rubén Fernández-Busnadiego ◽

Yasunori Saheki ◽

Pietro De Camilli

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Mammalian Cells ◽

Electron Tomography ◽

Three Dimensional ◽

Lipid Binding ◽

Dense Layer ◽

Lipid Transfer ◽

C2 Domains ◽

Membrane Contact Sites

The close apposition between the endoplasmic reticulum (ER) and the plasma membrane (PM) plays important roles in Ca2+ homeostasis, signaling, and lipid metabolism. The extended synaptotagmins (E-Syts; tricalbins in yeast) are ER-anchored proteins that mediate the tethering of the ER to the PM and are thought to mediate lipid transfer between the two membranes. E-Syt cytoplasmic domains comprise a synaptotagmin-like mitochondrial-lipid–binding protein (SMP) domain followed by five C2 domains in E-Syt1 and three C2 domains in E-Syt2/3. Here, we used cryo-electron tomography to study the 3D architecture of E-Syt–mediated ER–PM contacts at molecular resolution. In vitrified frozen-hydrated mammalian cells overexpressing individual E-Syts, in which E-Syt–dependent contacts were by far the predominant contacts, ER–PM distance (19–22 nm) correlated with the amino acid length of the cytosolic region of E-Syts (i.e., the number of C2 domains). Elevation of cytosolic Ca2+ shortened the ER–PM distance at E-Syt1–dependent contacts sites. E-Syt–mediated contacts displayed a characteristic electron-dense layer between the ER and the PM. These features were strikingly different from those observed in cells exposed to conditions that induce contacts mediated by the stromal interaction molecule 1 (STIM1) and the Ca2+ channel Orai1 as well as store operated Ca2+ entry. In these cells the gap between the ER and the PM was spanned by filamentous structures perpendicular to the membranes. Our results define specific ultrastructural features of E-Syt–dependent ER–PM contacts and reveal their structural plasticity, which may impact on the cross-talk between the ER and the PM and the functions of E-Syts in lipid transport between the two bilayers.

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