Kirsten Heimann

A photosynthetic alveolate closely related to apicomplexan parasites

Many parasitic Apicomplexa, such as Plasmodium falciparum, contain an unpigmented chloroplast remnant termed the apicoplast, which is a target for malaria treatment. However, no close relative of apicomplexans with a functional photosynthetic plastid has yet been described. Here we describe a newly cultured organism that has ultrastructural features typical for alveolates, is phylogenetically related to apicomplexans, and contains a photosynthetic plastid. The plastid is surrounded by four membranes, is pigmented by chlorophyll a, and uses the codon UGA to encode tryptophan in the psbA gene. This genetic feature has been found only in coccidian apicoplasts and various mitochondria. The UGA-Trp codon and phylogenies of plastid and nuclear ribosomal RNA genes indicate that the organism is the closest known photosynthetic relative to apicomplexan parasites and that its plastid shares an origin with the apicoplasts. The discovery of this organism provides a powerful model with which to study the evolution of parasitism in Apicomplexa.

Growth, Lipid Content, Productivity, and Fatty Acid Composition of Tropical Microalgae for Scale-Up Production

Biomass and lipid productivity, lipid content, and quantitative and qualitative lipid composition are critical parameters in selecting microalgal species for commercial scale‐up production. This study compares lipid content and composition, and lipid and biomass productivity during logarithmic, late logarithmic, and stationary phase of Nannochloropsis sp., Isochrysis sp., Tetraselmis sp., and Rhodomonas sp. grown in L1‐, f/2‐, and K‐medium. Of the tested species, Tetraselmis sp. exhibited a lipid productivity of 3.9–4.8 g m−2 day−1 in any media type, with comparable lipid productivity by Nannochloropsis sp. and Isochrysis sp. when grown in L1‐medium. The dry biomass productivity of Tetraselmis sp. (33.1–45.0 g m−2 day−1) exceeded that of the other species by a factor 2–10. Of the organisms studied, Tetraselmis sp. had the best dry biomass and/or lipid production profile in large‐scale cultures. The present study provides a practical benchmark, which allows comparison of microalgal production systems with different footprints, as well as terrestrial systems. Biotechnol. Bioeng. 2010;107: 245–257.

SPECIFIC ISOFORMS OF ACTIN-BINDING PROTEINS ON DISTINCT POPULATIONS OF GOLGI-DERIVED VESICLES

Golgi membranes and Golgi-derived vesicles are associated with multiple cytoskeletal proteins and motors, the diversity and distribution of which have not yet been defined. Carrier vesicles were separated from Golgi membranes, using an in vitro budding assay, and different populations of vesicles were separated using sucrose density gradients. Three main populations of vesicles labeled with β-COP, γ-adaptin, or p200/myosin II were separated and analyzed for the presence of actin/actin-binding proteins. β-Actin was bound to Golgi cisternae and to all populations of newly budded vesicles. Centractin was selectively associated with vesicles co-distributing with β-COP-vesicles, while p200/myosin II (non-muscle myosin IIA) and non-muscle myosin IIB were found on different vesicle populations. Isoforms of the Tm5 tropomyosins were found on selected Golgi-derived vesicles, while other Tm isoforms did not colocalize with Tm5 indicating the association of specialized actin filaments with Golgi-derived vesicles. Golgi-derived vesicles were shown to bind to F-actin polymerized from cytosol with Jasplakinolide. Thus, newly budded, coated vesicles derived from Golgi membranes can bind to actin and are customized for differential interactions with microfilaments by the presence of selective arrays of actin-binding proteins.

Substratum adhesion and gliding in a diatom are mediated by extracellular proteoglycans

Abstract.Diatoms are unicellular microalgae encased in a siliceous cell wall, or frustule. Pennate diatoms, which possess bilateral symmetry, attach to the substratum at a slit in the frustule called the raphe. These diatoms not only adhere, but glide across surfaces whilst maintaining their attachment, secreting a sticky mucilage that forms a trail behind the gliding cells. We have raised monoclonal antibodies to the major cell surface proteoglycans of the marine raphid diatom Stauroneis decipiens Hustedt. The antibody StF.H4 binds to the cell surface, in the raphe and to adhesive trails and inhibits the ability of living diatoms to adhere to the substratum and to glide. Moreover, StF.H4 binds to a periodate-insensitive epitope on four frustule-associated proteoglycans (relative molecular masses 87, 112, and >200 kDa). Another monoclonal antibody, StF.D5, binds to a carbohydrate epitope on the same set of proteoglycans, although the antibody binds only to the outer surface of the frustule and does not inhibit cell motility and adhesion.

Comparative effects of herbicides on photosynthesis and growth of tropical estuarine microalgae

Pulse amplitude modulation (PAM) fluorometry is ideally suited to measure the sub-lethal impacts of photosystem II (PSII)-inhibiting herbicides on microalgae, but key relationships between effective quantum yield [Y(II)] and the traditional endpoints growth rate (micro) and biomass increase are unknown. The effects of three PSII-inhibiting herbicides; diuron, hexazinone and atrazine, were examined on two tropical benthic microalgae; Navicula sp. (Heterokontophyta) and Nephroselmis pyriformis (Chlorophyta). The relationships between Y(II), micro and biomass increase were consistent (r2 > or =0.90) and linear (1:1), validating the utility of PAM fluorometry as a rapid and reliable technique to measure sub-lethal toxicity thresholds of PSII-inhibiting herbicides in these microalgae. The order of toxicity (EC50 range) was: diuron (16-33 nM) > hexazinone (25-110 nM) > atrazine (130-620 nm) for both algal species. Growth rate and photosynthesis were affected at diuron concentrations that have been detected in coastal areas of the Great Barrier Reef.

Additive toxicity of herbicide mixtures and comparative sensitivity of tropical benthic microalgae

Natural waters often contain complex mixtures of unknown contaminants potentially posing a threat to marine communities through chemical interactions. Here, acute effects of the photosystem II-inhibiting herbicides diuron, tebuthiuron, atrazine, simazine, and hexazinone, herbicide breakdown products (desethyl-atrazine (DEA) and 3,4-dichloroaniline (3,4-DCA)) and binary mixtures, were investigated using three tropical benthic microalgae; Navicula sp. and Cylindrotheca closterium (Ochrophyta) and Nephroselmis pyriformis (Chlorophyta), and one standard test species, Phaeodactylum tricornutum (Ochrophyta), in a high-throughput Maxi-Imaging-PAM bioassay (Maxi-IPAM). The order of toxicity was; diuron > hexazinone > tebuthiuron > atrazine > simazine > DEA > 3,4-DCA for all species. The tropical green alga N. pyriformis was up to 10-fold more sensitive than the diatoms tested here and reported for coral symbionts, and is recommended as a standard tropical test species for future research. All binary mixtures exhibited additive toxicity, and the use of herbicide equivalents (HEq) is therefore recommended in order to incorporate total-maximum-load measures for environmental regulatory purposes.

Development of the flagellar apparatus during the cell cycle in unicellular algae

SummaryRecent evidence has shown that algal cells acquire different flagella and a heterogeneous basal apparatus through the prolonged development of these structures over more than one cell cycle. A system for numbering algal flagella and basal bodies, which is based on developmental studies, is discussed along with the various means by which the flagellar/basal body developmental cycle can be determined. We review the information now available on development of the separate components of the flagellar apparatus-this comes particulary from the Chlorophyta and the Chromophyta-and attempt to elucidate any information which may help in phylogenetic comparisons. New data is provided on developmental changes in the cartwheel part of the basal body and basal body-associated connecting fibrils in green algae.

Vesicle budding on Golgi membranes: Regulation by G proteins and myosin motors

One of the main functions of the Golgi complex is to generate transport vesicles for the post-Golgi trafficking of proteins in secretory pathways. Many different populations of vesicles are distinguished by unique sets of structural and regulatory proteins which participate in vesicle budding and fusion. Monomeric and heterotrimeric G proteins regulate vesicle budding and secretory traffic into and out of the Golgi complex. An inventory of G protein alpha subunits associated with Golgi membranes highlights their diverse involvement and potential for coupling Golgi trafficking, through various signal transduction pathways, to cell growth or other more specialized cell functions. Cytoskeletal proteins are now also known to associate specifically with the Golgi complex and Golgi-derived vesicles. Amongst these, conventional and unconventional myosins are recruited to vesicle membranes. Several roles in vesicle budding and vesicle trafficking can be proposed for these actin-based motors.

Mechanism of Cytotoxicity and Cellular Uptake of Lipophilic Inert Dinuclear Polypyridylruthenium(II) Complexes

The accumulation, uptake mechanism, cytotoxicity, cellular localisation of—and mode of cell death induced by—dinuclear ruthenium(II) complexes ΔΔ/ΛΛ‐[{Ru(phen)2}2{μ‐bbn}]4+ (Rubbn), where phen is 1,10‐phenanthroline, bbn is bis[4(4′‐methyl‐2,2′‐bipyridyl)]‐1,n‐alkane (n=2, 5, 7, 10, 12 or 16), and the corresponding mononuclear complexes containing the bbn ligands, were studied in L1210 murine leukaemia cells. Cytotoxicity increased with linker chain length, and the ΔΔ‐Rubb16 complex displayed the highest cytotoxicity of the series, with an IC50 value of 5 μM, similar to that of carboplatin in the L1210 murine leukaemia cell line. Confocal microscopy and flow cytometry studies indicated that the complexes accumulate in the mitochondria of L1210 cells, with the magnitude of cellular uptake and accumulation increasing with linking chain length in the bbn bridge of the metal complex. ΔΔ‐Rubb16 entered the L1210 cells by passive diffusion (with a minor contribution from protein‐mediated active transport), inducing cell death via apoptosis. Additionally, metal‐complex uptake in leukaemia cells was approximately 16‐times that observed in healthy B cells highlighting that the bbn series of complexes may have potential as selective anticancer drugs.

The GRIP domain is a specific targeting sequence for a population of trans-Golgi network derived tubulo-vesicular carriers.

Vesicular carriers for intracellular transport associate with unique sets of accessory molecules that dictate budding and docking on specific membrane domains. Although many of these accessory molecules are peripheral membrane proteins, in most cases the targeting sequences responsible for their membrane recruitment have yet to be identified. We have previously defined a novel Golgi targeting domain (GRIP) shared by a family of coiled‐coil peripheral membrane Golgi proteins implicated in membrane trafficking. We show here that the docking site for the GRIP motif of p230 is a specific domain of Golgi membranes. By immuno‐electron microscopy of HeLa cells stably expressing a green fluorescent protein (GFP)‐p230GRIP fusion protein, we show binding specifically to a subset of membranes of the trans‐Golgi network (TGN). Real‐time imaging of live HeLa cells revealed that the GFP‐p230GRIP was associated with highly dynamic tubular extensions of the TGN, which have the appearance and behaviour of transport carriers. To further define the nature of the GRIP membrane binding site, in vitro budding assays were performed using purified rat liver Golgi membranes and cytosol from GFP‐p230GRIP‐transfected cells. Analysis of Golgi‐derived vesicles by sucrose gradient fractionation demonstrated that GFP‐p230GRIP binds to a specific population of vesicles distinct from those labelled for β‐COP or γ‐adaptin. The GFP‐p230GRIP fusion protein is recruited to the same vesicle population as full‐length p230, demonstrating that the GRIP domain is solely proficient as a targeting signal for membrane binding of the native molecule. Therefore, p230 GRIP is a targeting signal for recruitment to a highly selective membrane attachment site on a specific population of trans‐Golgi network tubulo‐vesicular carriers.