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

The 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.

Membrane protein SMP-1 is required for normal flagellum function in Leishmania.

Eukaryotic flagella and cilia are surrounded by a membrane that is continuous with, but distinct from, the rest of the plasma membrane. In Leishmania parasites, the inner leaflet of the flagellar membrane is coated with the acylated membrane protein, SMP-1. Here, we provide evidence that SMP-1 stabilizes the flagellar membrane and is required for flagella elongation and function.

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.

Inhibition of malaria parasite development by a cyclic peptide that targets the vital parasite protein SERA5.

The serine repeat antigen (SERA) proteins of the malaria parasites Plasmodium spp. contain a putative enzyme domain similar to that of papain family cysteine proteases. In Plasmodium falciparum parasites, more than half of the SERA family proteins, including the most abundantly expressed form, SERA5, have a cysteine-to-serine substitution within the putative catalytic triad of the active site.

Leishmania adaptor protein-1 subunits are required for normal lysosome traffic, flagellum biogenesis, lipid homeostasis, and adaptation to temperatures encountered in the mammalian host.

The adaptor protein-1 (AP-1) complex is involved in membrane transport between the Golgi apparatus and endosomes. In the protozoan parasite Leishmania mexicana mexicana, the AP-1 mu1 and sigma1 subunits are not required for growth at 27 degrees C but are essential for infectivity in the mammalian host. In this study, we have investigated the function of these AP-1 subunits in order to understand the molecular basis for this loss of virulence.

What generates flux of tubulin in kinetochore microtubules?

We discuss models for production of tubulin flux in kinetochore microtubules. Current models concentrate solely on microtubules and their associated motors and enzymes. For example, in some models the driving force for flux is enzymes at the poles and the kinetochores; in others the driving force is motor molecules that are associated with a stationary spindle matrix.

Actin and myosin inhibitors block elongation of kinetochore fibre stubs in metaphase crane-fly spermatocytes.

We used an ultraviolet microbeam to cut individual kinetochore spindle fibres in metaphase crane-fly spermatocytes. We then followed the growth of the "kinetochore stubs", the remnants of kinetochore fibres that remain attached to kinetochores. Kinetochore stubs elongate with constant velocity by adding tubulin subunits at the kinetochore, and thus elongation is related to tubulin flux in the kinetochore microtubules.

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.

A mitochondrial protein affects cell morphology, mitochondrial segregation and virulence in Leishmania.

The single mitochondrion of kinetoplastids divides in synchrony with the nucleus and plays a crucial role in cell division. However, despite its importance and potential as a drug target, the mechanism of mitochondrial division and segregation and the molecules involved are only partly understood. In our quest to identify novel mitochondrial proteins in Leishmania, we constructed a hidden Markov model from the targeting motifs of known mitochondrial proteins as a tool to search the Leishmania major genome.

Membrane transporters in the relict plastid of malaria parasites.

Malaria parasites contain a nonphotosynthetic plastid homologous to chloroplasts of plants. The parasite plastid synthesizes fatty acids, heme, iron sulfur clusters and isoprenoid precursors and is indispensable, making it an attractive target for antiparasite drugs. How parasite plastid biosynthetic pathways are fuelled in the absence of photosynthetic capture of energy and carbon was not clear.

Characterisation of a Leishmania mexicana knockout lacking guanosine diphosphate-mannose pyrophosphorylase.

In eukaryotes, the enzyme GDP-mannose pyrophosphorylase (GDP-MP) is essential for the formation of GDP-mannose, the donor of activated mannose for all glycosylation reactions. Unlike other eukaryotes, where deletion of GDP-mannose pyrophosphorylase is lethal, deletion of this gene in Leishmania mexicana has no effect on viability, but leads to the generation of avirulent parasites. In this study, we show that the null mutants have a perturbed morphology and cytokinesis, retarded growth and increased adherence to the substratum where they form large colonies.

SMP-1, a member of a new family of small myristoylated proteins in kinetoplastid parasites, is targeted to the flagellum membrane in Leishmania.

The mechanisms by which proteins are targeted to the membrane of eukaryotic flagella and cilia are largely uncharacterized. We have identified a new family of small myristoylated proteins (SMPs) that are present in Leishmania spp and related trypanosomatid parasites. One of these proteins, termed SMP-1, is targeted to the Leishmania flagellum.

Cutaneous leishmaniasis in red kangaroos: isolation and characterisation of the causative organisms.

This is the first report of cutaneous leishmaniasis in kangaroos where infection was acquired within Australia. The diagnosis is based on the clinical criteria used for humans, the lesion histopathology, the detection and isolation of parasites from the lesions, and the analysis of the small subunit ribosomal RNA genes using the polymerase chain reaction. Despite a clear indication that the parasites belong to the genus Leishmania, no assignation to a known Leishmania species could be made using these or other less conserved genetic loci such as the non-transcribed spacer of the mini-exon repeat.

Structure of kinetochore fibres in crane-fly spermatocytes after irradiation with an ultraviolet microbeam: neither microtubules nor actin filaments remain in the irradiated region.

We studied chromosome movement after kinetochore microtubules were severed. Severing a kinetochore fibre in living crane-fly spermatocytes with an ultraviolet microbeam creates a kinetochore stub, a birefringent remnant of the spindle fibre connected to the kinetochore and extending only to the edge of the irradiated region. After the irradiation, anaphase chromosomes either move poleward led by their stubs or temporarily stop moving.

Diatom gliding is the result of an actin-myosin motility system.

Diatoms are a group of unicellular microalgae that are encased in a highly ornamented siliceous cell wall, or frustule. Pennate diatoms have bilateral symmetry and many genera possess an elongated slit in the frustule called the raphe, a feature synonymous with their ability to adhere and glide over a substratum, a process little understood. We have used cytoskeleton-disrupting drugs to investigate the roles of actin, myosin, and microtubules in diatom gliding or motility.

Traction fibre: toward a "tensegral" model of the spindle.

Most current hypotheses of mitotic mechanisms are based on the "PAC-MAN" paradigm in which chromosome movement is generated and powered by disassembly of kinetochore microtubules (k-MTs) by the kinetochore. Recent experiments demonstrate that this model cannot explain force generation for anaphase chromosome movement [Pickett-Heaps et al., 1996: Protoplasma 192:1-10].

Ultraviolet microbeam irradiations of epithelial and spermatocyte spindles suggest that forces act on the kinetochore fibre and are not generated by its disassembly.

Ultraviolet (UV) microbeam irradiations of crane-fly spermatocyte and newt epithelial spindles severed kinetochore fibres (KT-fibres), creating areas of reduced birefringence (ARBs): the remnant KT-fibre consists of two "stubs," a pole-stub attached to the pole and a KT-stub attached to the kinetochore. KT-stubs remained visible but pole-stubs soon became undetectable [Forer et al., 1996].

The amyloid precursor protein of Alzheimer's disease is found on the surface of static but not activity motile portions of neurites.

We have previously found that the amyloid precursor protein (APP) of Alzheimer's disease is present on the surface of rat cortical neurons in culture, in a segmental pattern which first becomes evident after 24 hours and is fully developed by five days. As APP has previously been reported to have a short half-life in neuronal cell lines, and has been shown to contain binding sites for various extracellular matrix components within its extracellular domain, we hypothesized that APP would be associated with portions of neurites undergoing rapid structural change, such as growth cones. To test this hypothesis, we observed selected neurons by video time-lapse differential interference microscopy on 24-hour-old primary rat neuronal cultures for up to 45 minutes, followed by fixation and immunocytochemistry to ascertain surface APP distribution on those same neurons.

The effects of diazepam on mitosis and the microtubule cytoskeleton. I. Observations on the diatoms Hantzschia amphioxys and Surirella robusta.

The effects of diazepam (DZP) on mitosis and the microtubule (MT) cytoskeleton in the live diatoms Hantzschia amphioxys and Surirella robusta were followed using time-lapse video microscopy. Similarly treated cells were fixed and later examined for immunoflouresence staining of MTs or for transmission electron microscopy. DZP treatment (250 microM) had no effect on interphase cells but affected mitosis, resulting in the majority of prometaphase and metaphase chromosomes releasing from one or both spindle poles and collecting irregularly along the central spindle.

UV-microbeam irradiations of the mitotic spindle: spindle forces and structural analysis of lesions.

Mitotic PtK1 spindles were UV irradiated (285 nm) during metaphase and anaphase between the chromosomes and the pole. The irradiation, a rectangle measuring 1.4 x 5 microns parallel to the metaphase plate, severed between 90 and 100% of spindle microtubules (MTs) in the irradiated region.

UV microbeam irradiations of the mitotic spindle. II. Spindle fiber dynamics and force production.

Metaphase and anaphase spindles in cultured newt and PtK1 cells were irradiated with a UV microbeam (285 nM), creating areas of reduced birefringence (ARBs) in 3 s that selectively either severed a few fibers or cut across the half spindle. In either case, the birefringence at the polewards edge of the ARB rapidly faded polewards, while it remained fairly constant at the other, kinetochore edge. Shorter astral fibers, however, remained present in the enlarged ARB; presumably these had not been cut by the irradiation.

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

As anaphase began, mitotic PtK1 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 ATP. ATP, GTP, or ATP-gamma-S added in the PM 4-7 min later restarted anaphase A while kinetochore fibers shortened.

Microtubule dynamics in the spindle. II. A thermodynamic and kinetic description.

We have previously presented a model for the assembly and disassembly of mitotic spindle microtubules (MTs) (Pickett-Heaps et al., 1986). In this paper, we describe the thermodynamics of such spindle MT assembly and present equations to describe the polymerization kinetics of different classes of spindle MTs.

Microtubule dynamics in the spindle. Theoretical aspects of assembly/disassembly reactions in vivo.

The mitotic spindle contains several classes of microtubules (MTs) whose lengths change independently during mitosis. Precise control over MT polymerization and depolymerization during spindle formation, anaphase chromosome movements, and spindle breakdown is necessary for successful cell division. This model proposes the site of addition and removal of MT subunits in each of four classes of spindle MTs at different stages of mitosis, and suggests how this addition and removal is controlled.