On the mechanism of anaphase A: evidence that ATP is needed for microtubule disassembly and not generation of polewards force

Abstract. As anaphase began, mitotic PtK ~ and newt lung epithelial cells were permeabilized with digitonin in permeabilization medium (PM). Permeabilization stopped cytoplasmic activity, chromosome movement, and cytokinesis within about 3 min, presumably due to the loss of endogenous ATE ATE GTP, or ATP-~-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened. AMPPNP could not restart anaphase A; ATP was ineffective if the spindle was stabilized in PM + DMSO. Cells permeabilized in PM + taxol varied in their response to ATP depending on the stage of anaphase reached: one mid-anaphase cell showed initial movement of chromosomes back to the metaphase plate upon permeabilization but later, anaphase A resumed when ATP was added. Anaphase A was also reactivated by cold PM (,x,16 ~ or PM containing calcium (1-10 mM). Staining of fixed cells with antitubulin showed that microtu-N 'o consensus currently exists regarding the role of ATP during anaphase A (chromosome-to-pole movement) and it is difficult to evaluate the diverse data on this subject. We (Pickett-Heaps and Spurck, 1982; Spurck et al., 1986a, b) have found that the concentration of metabolic inhibitors needed to cause rapid (within 1 min) and completely reversible cessation of cellular activity (presumably by depleting cellular ATP levels) in live cells is critical; 2,4 dinitrophenol (DNP) 1 is effective in both diatoms and mammalian cells only at the seemingly high concentration of 1 mM, and lowering the concentration even to 0.5 mM results in a significantly delayed and often incomplete response. In contrast, far lower concentrations of metabolic inhibitors were used in most previous work (reviewed by Spurck et al., 1986a, b; Hepler and Palevitz, 1986). Furthermore, many who work with animal cell systems use inhibitors of oxidative phosphorylation alone. We (Spurck et al., 1986a) found that glycolysis must also be blocked by usin

Apicoplast and Mitochondrion in Gametocytogenesis of Plasmodium falciparum▿ †

Live cell imaging of human malaria parasites Plasmodium falciparum during gametocytogenesis revealed that the apicoplast does not grow, whereas the mitochondrion undergoes remarkable morphological development. A close connection of the two organelles is consistently maintained. The apicoplast and mitochondrion are not components of the male gametes, suggesting maternal inheritance

The glucans extracted with warm water from diatoms are mainly derived from intracellular chysolaminaran and not extracellular polysaccharides

Several recent studies have employed warm-water treatment of diatom cells to extract nominally bound extracellular polymeric substances. Where examined, the dominant neutral sugar in these extracts was glucose. In the present study, we sought to characterize the structure of the glucose-rich polymers in the water extracts of diatoms and to determine the origin of these polymers. The marine diatoms surveyed were Phaeodactylum tricornutum, Cylindrotheca fusiformis, Craspedostauros australis and Thalassiosira pseudonana. A freshwater species, Pinnularia viridis, was also investigated for the dye labelling experiments. Freshly harvested marine diatoms were extracted with water at 308C for 1 h. Constituent monosaccharide analyses showed that glucose was the dominant neutral sugar (80 - 95 mol% of the total) in the extracts from three marine species, whereas the P. tricornutum extract contained predominantly ribose, galactose and glucose, and was inferred to be enriched in low-molecular-weight components. Linkage analysis of the constituent monosaccharides and proton nuclear magnetic resonance spectroscopy showed that the glucose in these extracts was derived primarily from 1,3-b-D-glucan. Immunocytochemistry with a monoclonal anti-1,3-b-D-glucan antibody confirmed that the glucan was localized in the vacuoles of diatom cells preserved by freeze-substitution. Nearly all diatom cells incubated with a fluorescent dye, DiBAC4(3), during warm water treatment at 308C or 608C incorporated the dye, demonstrating that the membrane integrity of the diatoms was compromised and supporting the contention that intracellular glucan was released during the treatment. In light of these data, the extracellular glucans of diatoms reported in some previous studies are re-interpreted as intracellular chrysolaminaran

Atypical lipid composition in the purified relict plastid (apicoplast) of malaria parasites.

International audienceThe human malaria parasite Plasmodium falciparum harbors a relict, nonphotosynthetic plastid of algal origin termed the apicoplast. Although considerable progress has been made in defining the metabolic functions of the apicoplast, information on the composition and biogenesis of the four delimiting membranes of this organelle is limited. Here, we report an efficient method for preparing highly purified apicoplasts from red blood cell parasite stages and the comprehensive lipidomic analysis of this organelle. Apicoplasts were prepared from transgenic parasites expressing an epitope-tagged triosephosphate transporter and immunopurified on magnetic beads. Gas and liquid chromatography MS analyses of isolated apicoplast lipids indicated significant differences compared with total parasite lipids. In particular, apicoplasts were highly enriched in phosphatidylinositol, consistent with a suggested role for phosphoinositides in targeting membrane vesicles to apicoplasts. Apicoplast phosphatidylinositol and other phospholipids were also enriched in saturated fatty acids, which could reflect limited acyl exchange with other membrane phospholipids and/or a requirement for specific physical properties. Lipids atypical for plastids (sphingomyelins, ceramides, and cholesterol) were detected in apicoplasts. The presence of cholesterol in apicoplast membranes was supported by filipin staining of isolated apicoplasts. Galactoglycerolipids, dominant in plant and algal plastids, were not detected in P. falciparum apicoplasts, suggesting that these glycolipids are a hallmark of photosynthetic plastids and were lost when these organisms assumed a parasitic lifestyle. Apicoplasts thus contain an atypical melange of lipids scavenged from the human host alongside lipids remodeled by the parasite cytoplasm, and stable isotope labeling shows some apicoplast lipids are generated de novo by the organelle itself

Inhibition of Dendritic Cell Maturation by Malaria Is Dose Dependent and Does Not Require Plasmodium falciparum Erythrocyte Membrane Protein 1▿

Red blood cells infected with Plasmodium falciparum (iRBCs) have been shown to modulate maturation of human monocyte-derived dendritic cells (DCs), interfering with their ability to activate T cells. Interaction between Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) and CD36 expressed by DCs is the proposed mechanism, but we show here that DC modulation does not require CD36 binding, PfEMP1, or contact between DCs and infected RBCs and depends on the iRBC dose. iRBCs expressing a PfEMP1 variant that binds chondroitin sulfate A (CSA) but not CD36 were phagocytosed, inhibited lipopolysaccharide (LPS)-induced phenotypic maturation and cytokine secretion, and abrogated the ability of DCs to stimulate allogeneic T-cell proliferation. CD36- and CSA-binding iRBCs showed comparable inhibition. P. falciparum lines rendered deficient in PfEMP1 expression by targeted gene knockout or knockdown also inhibited LPS-induced phenotypic maturation, and separation of DCs and iRBCs in transwells showed that inhibition was not contact dependent. Inhibition was observed at an iRBC:DC ratio of 100:1 but not at a ratio of 10:1. High doses of iRBCs were associated with apoptosis of DCs, which was not activation induced. Lower doses of iRBCs stimulated DC maturation sufficient to activate autologous T-cell proliferation. In conclusion, modulation of DC maturation by P. falciparum is dose dependent and does not require interaction between PfEMP1 and CD36. Inhibition and apoptosis of DCs by high-dose iRBCs may or may not be physiological. However, our observation that low-dose iRBCs initiate functional DC maturation warrants reevaluation and further investigation of DC interactions with blood-stage P. falciparum