Transbilayer phosphatidylethanolamine movements in the yeast plasma membrane

Aminophospholipid movements in the plasma membrane of higher eukaryotic cells seem to be regulated by an ATP-dependent, protein-mediated process. To examine whether similar mechanisms exist in yeast cells, we have analysed phosphatidylethanolamine (PtdEtn) distributions in Saccharomyces cerevisiae (A184D) cells under a variety of conditions, with trinitrobenzenesulfonic acid and fluorescamine as the external membrane probes. The levels of external PtdEtn in the intact cells were reduced to about 50% by pretreatment of the cells with inhibitors of mitochondrial ATP synthesis, ATPase inhibitors or protein-sulfhydryl-group-modifying reagents, or by depletion of the cells of ATP by metabolic starvation. The levels of external PtdEtn could be restored to normal by repletion of the energy-depleted cells with ATP. Furthermore, treatment of the energy-depleted cells with sulfhydryl-modyfying reagents did not cause further reduction in the external PtdEtn levels but decreased the accessibility of PtdEtn to fluorescamine after restoration of the cellular ATP levels to normal in these cells. These results demonstrate an involvement of an ATP-dependent, protein-mediated process(es) in the regulation of the PtdEtn distribution across the plasma-membrane bilayer of yeast cells. The results are discussed with regard to possible models that can generate and maintain the transbilayer phospholipid asymmetry in the yeast plasma membrane

Role of the actin cytoskeleton in regulating the outer phosphatidylethanolamine levels in yeast plasma membrane

Transbilayer phosphatidylethanolamine (PtdEtn) movements in the plasma membrane of Saccharomyces cerevisiae are regulated by an ATP-dependent, protein-mediated process(es). To examine whether this process is influenced by the actin cytoskeleton, we have studied the PtdEtn translocation in S. cerevisiae cells after treatment with microfilament disrupting and microtubule-disrupting agents. PtdEtn translocation was studied by measuring the external PtdEtn levels, using fluorescamine as the external membrane probe, in the ATP-depleted, ATP-depleted and repleted, and N-ethylmaleimide-treated cells. The microfilaments and microtubules were disrupted by treatment with various cytochalasins and colchicine (or benomyl) respectively PtdEtn translocation became abnormal in the cytochalasin-treated cells but not in cells that were treated with microtubule-disrupting agents, such as colchicine or benomyl. These results have been interpreted to suggest that the actin cytoskeleton is involved in regulating the PtdEtn translocase activity in the yeast cell plasma membrane

Probing the role of C-1 ester group in Naja naja phospholipase A<SUB>2</SUB>-phospholipid interactions using butanetriol-containing phosphatidylcholine analogues

To understand the role of the ester moiety of the sn-1 acyl chain in phospholipase A2-glycerophospholipid interactions, we introduced an additional methylene residue between the glycerol C1 and C2 carbon atoms of phosphatidylcholines, and then studied the kinetics of hydrolysis and the binding of such butanetriol-containing phospholipids with Naja naja phospholipase A2. Hydrolysis was monitored by using phospholipids containing a NBD-labelled sn-2 acyl chain and binding was ascertained by measuring the protein tryptophan fluorescence. The hydrolysis of butanetriol-containing phospholipids was invariably slower than that of the glycerol-containing phospholipids. In addition, the enzyme binding with the substrate was markedly decreased upon replacing the glycerol residue with the 1,3,4-butanetriol moiety in phosphatidylcholines. These results have been interpreted to suggest that the sn-1 ester group in glycerophospholipids could play an important role in phospholipase A2-phospholipid interactions

Ancient Leishmania coronin (CRN12) is involved in microtubule remodeling during cytokinesis

In general, coronins play an important role in actin-based processes, and are expressed in a variety of eukaryotic cells, including Leishmania. Here, we show that Leishmania coronin preferentially distributes to the distal tip during cytokinesis, and interacts with microtubules through a microtubule-based motor, kinesin K39. We further show that reduction in coronin levels by 40-50% in heterozygous coronin mutants results in generation of bipolar cells (25-30%), specifically in the log phase, owing to unregulated growth of the corset microtubules. Further analysis of bipolar cells revealed that the main cause of generation of bipolar cell morphology is the intrusion of the persistently growing corset microtubules into the other daughter cell corset from the opposite direction. This defect in cytokinesis, however, disappears upon episomal gene complementation. Additionally, our attempts to prepare homozygous mutants were unsuccessful, as only the aneuploid cells survive the selection process. These results indicate that coronin regulates microtubule remodeling during Leishmania cytokinesis and is essentially required for survival of these parasites in culture

A novel form of actin in Leishmania: molecular characterisation, subcellular localisation and association with subpellicular microtubules

To study the occurrence and subcellular distribution of actin in trypanosomatid parasites, we have cloned and overexpressed Leishmania donovani actin gene in bacteria, purified the protein, and employed the affinity purified rabbit polyclonal anti-recombinant actin antibodies as a probe to study the organisation and subcellular distribution of actin in Leishmania cells. The Leishmania actin did not cross react with antimammalian actin antibodies but was readily recognized by the anti-Leishmania actin antibodies in both the promastigote and amastigote forms of the parasite. About 106 copies per cell of this protein (Mr 42.05 kDa) were present in the Leishmania promastigote. Unlike other eukaryotic actins, the oligomeric forms of Leishmania actin were not stained by phalloidin nor were dissociated by actin filament-disrupting agents, like Latrunculin B and Cytochalasin D. Analysis of the primary structure of this protein revealed that these unusual characteristics may be related to the presence of highly diverged amino acids in the DNase I-binding loop (amino acids 40-50) and the hydrophobic plug (amino acids 262-272) regions of Leishmania actin. The subcellular distribution of actin was studied in the Leishmania promastigotes by employing immunoelectron and immunofluorescence microscopies. This protein was present not only in the flagella, flagellar pocket, nucleus and the kinetoplast but it was also localized on the nuclear, vacuolar and cytoplasmic face of the plasma membranes. Further, the plasma membrane-associated actin was colocalised with subpellicular microtubules, while most of the actin present in the kinetoplast colocalised with the k-DNA network. These results clearly indicate that Leishmania contains a novel form of actin which may structurally and functionally differ from other eukaryotic actins. The functional significance of these observations is discussed

Chloroquine encapsulated in malaria-infected erythrocyte-specific antibody-bearing liposomes effectively controls chloroquine-resistant Plasmodium berghei infections in mice

The suitability of liposomes as drug carriers in the treatment of drug-resistant rodent malaria was examined after covalently attaching F(ab')2 fragments of a mouse monoclonal antibody (MAb), MAb F10, raised against the host cell membranes isolated from the Plasmodium berghei-infected mouse erythrocytes, to the liposome surface. The antibody-bearing liposomes thus formed specifically recognized the P. berghei-infected mouse erythrocytes under both in vitro and in vivo conditions. No such specific binding of the liposomes with the infected cells was observed when MAb F10 was replaced by another mouse monoclonal antibody, MAb D2. Upon loading with the antimalarial drug chloroquine, the MAb F10-bearing liposomes effectively controlled not only the chloroquine-susceptible but also the chloroquine-resistant P. berghei infections in mice. The chloroquine delivered in these liposomes intravenously at a dosage of 5 mg/kg of body weight per day on days 4 and 6 postinfection completely cured the animals (75 to 90%) of chloroquine-resistant P. berghei infections. These results indicate that selective homing of chloroquine to malaria-infected erythrocytes may help to cure the chloroquine-resistant malarial infections with low doses of chloroquine

ADF/cofilin-driven actin dynamics in early events of Leishmania cell division

ADF/cofilin is an actin-dynamics-regulating protein that is required for several actin-based cellular processes such as cell motility and cytokinesis. A homologue of this protein has recently been identified in the protozoan parasite Leishmania, which has been shown to be essentially required in flagellum assembly and cell motility. However, the role of this protein in cytokinesis remains largely unknown. We show here that deletion of the gene encoding ADF/cofilin in these organisms results in several aberrations in the process of cell division. These aberrations include delay in basal body and kinetoplast separation, cleavage furrow progression and flagellar pocket division. In addition to these changes, the intracellular trafficking and actin dynamics are also adversely affected. All these abnormalities are, however, reversed by episomal complementation. Together, these results indicate that actin dynamics regulates early events in Leishmania cell division

Trafficking activity of myosin XXI is required in assembly of Leishmania flagellum

Actin-based myosin motors have a pivotal role in intracellular trafficking in eukaryotic cells. The parasitic protozoan organism Leishmania expresses a novel class of myosin, myosin XXI (Myo21), which is preferentially localized at the proximal region of the flagellum. However, its function in this organism remains largely unknown. Here, we show that Myo21 interacts with actin, and its expression is dependent of the growth stage. We further reveal that depletion of Myo21 levels results in impairment of the flagellar assembly and intracellular trafficking. These defects are, however, reversed by episomal complementation. Additionally, it is shown that deletion of the Myo21 gene leads to generation of ploidy, suggesting an essential role of Myo21 in survival of Leishmania cells. Together, these results indicate that actin-dependent trafficking activity of Myo21 is essentially required during assembly of the Leishmania flagellum

An unconventional form of actin in protozoan hemoflagellate, Leishmania

Leishmania actin was cloned, overexpressed in baculovirusinsect cell system, and purified to homogeneity. The purified protein polymerized optimally in the presence of Mg2+ and ATP, but differed from conventional actins in its following properties: (i) it did not polymerize in the presence of Mg2+ alone, (ii) it polymerized in a restricted range of pH 7.0-8.5, (iii) its critical concentration for polymerization was found to be 3-4-fold lower than of muscle actin, (iv) it predominantly formed bundles rather than single filaments at pH 8.0, (v) it displayed considerably higher ATPase activity during polymerization, (vi) it did not inhibit DNase-I activity, and (vii) it did not bind the F-actin-binding toxin phalloidin or the actin polymerization disrupting agent Latrunculin B. Computational and molecular modeling studies revealed that the observed unconventional behavior of Leishmania actin is related to the diverged amino acid stretches in its sequence, which may lead to changes in the overall charge distribution on its solvent-exposed surface, ATP binding cleft, Mg2+ binding sites, and the hydrophobic loop that is involved in monomer-monomer interactions. Phylogenetically, it is related to ciliate actins, but to the best of our knowledge, no other actin with such unconventional properties has been reported to date. It is therefore suggested that actin in Leishmania may serve as a novel target for design of new antileishmanial drugs

Leishmania actin binds and nicks kDNA as well as inhibits decatenation activity of type II topoisomerase

Leishmania actin (LdACT) is an unconventional form of eukaryotic actin in that it markedly differs from other actins in terms of its filament forming as well as toxin and DNase-1-binding properties. Besides being present in the cytoplasm, cortical regions, flagellum and nucleus, it is also present in the kinetoplast where it appears to associate with the kinetoplast DNA (kDNA). However, nothing is known about its role in this organelle. Here, we show that LdACT is indeed associated with the kDNA disc in Leishmania kinetoplast, and under in vitro conditions, it specifically binds DNA primarily through electrostatic interactions involving its unique DNase-1-binding region and the DNA major groove. We further reveal that this protein exhibits DNA-nicking activity which requires its polymeric state as well as ATP hydrolysis and through this activity it converts catenated kDNA minicircles into open form. In addition, we show that LdACT specifically binds bacterial type II topoisomerase and inhibits its decatenation activity. Together, these results strongly indicate that LdACT could play a critical role in kDNA remodeling