Ultrasensitive Trace Sample Proteomics Unraveled the Protein Remodeling during Mesenchymal-Amoeboid Transition.

Deep mining the proteome of trace biological samples is critical for biomedical applications. However, it remains a challenge due to the loss of analytes caused by current sample preparation procedures. To address this, we recently developed a single-pot and miniaturized in-solution digestion (SMID) method for minute sample handling with three streamlined steps and completed within 3 h. The SMID approach outperformed the traditional workflow in substantially saving time, reducing sample loss, and exhibiting extensive applicability for 10-100 000 cell analysis. This user-friendly and high-sensitivity strategy enables ∼5300 proteins and 53 000 peptides to be confidently identified within 1 h of mass spectrometry (MS) time from a small amount of 1000 HeLa cells. In addition, we accurately and robustly detected proteomes in 10 mouse oocytes with excellent reproducibility. We further adopted SMID for the proteome analysis in cell migration under confinement, which induced cells to undergo a mesenchymal-amoeboid transition (MAT). During the MAT, a systematic quantitative proteome map of 1000 HeLa cells was constructed with seven expression profile clusters, which illustrated the application of SMID and provided a fundamental resource to investigate the mechanism of MAT.

Total synthesis of lindbladione, a Hes1 dimerization inhibitor and neural stem cell activator isolated from Lindbladia tubulina.

Lindbladione (1) is a neural stem cell differentiation activator isolated from Lindbladia tubulina by our group. Hes1 dimerization inhibitory activity of lindbladione (1) was discovered using our original fluorescent Hes1 dimer microplate assay. We also found that lindbladione (1) accelerates the differentiation of neural stem cells. We conducted the first total synthesis of lindbladione (1) via Heck reaction of 1-hexene-3-one 7 with iodinated naphthoquinone 12, which was provided by Friedel-Crafts acylation followed by Claisen condensation, in the presence of Pd (II) acetate. Careful deprotection of the benzyl groups of 13 successively provided lindbladione (1). Synthesized lindbladione (1) exhibited potent Hes1 dimer inhibition (IC50 of 2.7 µM) in our previously developed fluorescent Hes1 dimer microplate assay. Synthesized lindbladione (1) also accelerated the differentiation of C17.2 mouse neural stem cells into neurons dose dependently, increasing the number of neurons by 59% (2.5 µM) and 112% (10 µM) compared to the control. These activities are comparable to those of naturally occurring lindbladione (1) isolated from L. tublina.

Region-Based Segmentation and Wiener Pilot-Based Novel Amoeba Denoising Scheme for CT Imaging.

Computed tomography (CT) is one of the most common and beneficial medical imaging schemes, but the associated high radiation dose injurious to the patient is always a concern. Therefore, postprocessing-based enhancement of a CT reconstructed image acquired using a reduced dose is an active research area. Amoeba- (or spatially variant kernel-) based filtering is a strong candidate scheme for postprocessing of the CT image, which adapts its shape according to the image contents. In the reported research work, the amoeba filtering is customized for postprocessing of CT images acquired at a reduced X-ray dose. The proposed scheme modifies both the pilot image formation and amoeba shaping mechanism of the conventional amoeba implementation. The proposed scheme uses a Wiener filter-based pilot image, while region-based segmentation is used for amoeba shaping instead of the conventional amoeba distance-based approach. The merits of the proposed scheme include being more suitable for CT images because of the similar region-based and symmetric nature of the human body anatomy, image smoothing without compromising on the edge details, and being adaptive in nature and more robust to noise. The performance of the proposed amoeba scheme is compared to the traditional amoeba kernel in the image denoising application for CT images using filtered back projection (FBP) on sparse-view projections. The scheme is supported by computer simulations using fan-beam projections of clinically reconstructed and simulated head CT phantoms. The scheme is tested using multiple image quality matrices, in the presence of additive projection noise. The scheme implementation significantly improves the image quality visually and statistically, providing better contrast and image smoothing without compromising on edge details. Promising results indicate the efficacy of the proposed scheme.

Growth of Stenotrophomonas maltophilia and expression of Sme efflux pumps encoding genes in the presence of supernatants from amoebal and bacterial co-cultures: towards the role of amoebal secondary metabolites.

Resistance-Nodulation-Division (RND) efflux pumps are relevant determinants of Stenotrophomonas maltophilia multidrug resistance as they can extrude a broad range of antibiotics and compounds involved in virulence and physiological functions. S. maltophilia, an environmental bacterium, was shown to be associated with amoebae and able to multiply inside them. To explore whether S. maltophilia RND efflux pumps play a role when interacting with amoebae, we evaluated the effect of amoebal culture and co-culture supernatants on the growth of S. maltophilia and the expression of sme efflux pump genes. Acanthamoeba castellanii and Willaertia magna were used as amoebal models and strain S. maltophilia BurE1 as bacterial one. Our data showed that both bacterial growth and sme gene expression were not modified by amoebal culture supernatants. On the contrary, co-culture supernatants negatively impacted the growth of BurE1 and induced the expression of three out of eight efflux pump genes, i.e. smeE, smeN and smeZ. Finally, we evidenced the production of A. castellanii secondary metabolites, putatively belonging to the diterpene family, in the amoebal supernatant and in the co-culture supernatant of A. castellanii and BurE1. Whether these compounds act directly as substrates of the efflux pumps and/or inducers of the sme genes need further investigations.

Amoebae Assemble Synthetic Spherical Particles To Form Reproducible Constructs.

Difflugia are testate amoebae that use particulate inorganic matter to build a protective shell (generally called a test or theca). Difflugia globulosa were grown both in culture containing only naturally occurring theca-building materials and under conditions where synthetic particles were present also. The presence of monodisperse Stöber silica microspheres of 1, 3, and 6 µm in diameter or 4 µm polystyrene spheres dramatically increased the rate of Difflugia growth, and foreign microspheres became the overwhelmingly dominant construction material. Optical and electron microscopy of the 6 µm particle studies revealed that Difflugia construct spherical vase-shaped thecae with strikingly reproducible composition, morphology, and size. Time-lapse photography revealed construction techniques and masonry skills as Difflugia herded particles together, trapped them using phagocytosis, and applied the particles with biocement from inside the developing theca. The reported observations identify taxonomy complications, biomicrofabrication possibilities, and a discrete environmental impact of synthetic particle pollutants.

Light variability and mixotrophy: Responses of testate amoeba communities and shell δ13C values to a peatland shading experiment.

Paleoecological records suggest that growing season length and/or cloudiness may affect peatland carbon accumulation and testate amoeba-based environmental reconstructions, highlighting a need to understand how light intensity affects microbial communities. We shaded plots on two peatlands for two years to examine effects on testate amoeba communities, the relative abundance of mixotrophic and heterotrophic testate amoebae, transfer-function performance, and δ13C values of two species of mixotrophic testate amoebae. Surprisingly, relative abundance of mixotrophic species increased in shade, although compositional changes did not affect transfer-function performance. Shading did not affect δ13C values of Hyalosphenia papilio and Heleopera sphagni, which ranged from -23.5 to -19.6‰ and -23.2 to -19.2‰, respectively. These δ13C values were higher than those of potential food sources and lower than literature-derived values for Chlorella, the zoochlorellae inhabiting mixotrophic testate amoebae. δ13C values thus suggest that these mixotrophic species obtain some carbon from Chlorella, although coupled dietary and isotope studies are needed to quantify this contribution. More research is needed to assess impacts of light variability on peatland microbial communities; however, carbon sources are recorded by δ13C values of testate amoebae, indicating potential for studies of carbon cycling and how mixotrophy varies temporally and spatially.

Species composition of testate amoebae in Lake Monte Alegre, Ribeirão Preto, SP, Brazil / Composição de amebas testáceas no lago Monte Alegre, Ribeirão Preto, SP, Brasil

We aimed to perform the first survey of testate amoebae community composition in Lake Monte Alegre at São Paulo state, and contribute to mitigate the taxonomic impediment regarding thediversity of testate amoebae in Brazil. Samplings were performed in 20 sampling sites within the lake, inthe limnetic and littoral regions in April 2015, using a 58 μm-meshplankton net. We identified 20 taxa oftestate amoebae belonging to four families: Arcellidae (8 taxa), Centropyxidae (4 taxa), Difflugiidae (7 taxa) and Lesquereusiidae (2 taxa). The littoral region showed the highest number of taxa (n = 20), whereas inthe limnetic region 11 taxa were registered. Therefore, our results evidenced the importance of testateamoebae in aquatic systems, and that further research, taxonomic and/or ecological, in those environmentsshould include these organisms in their investigation. Moreover, we suggest that future research with greater sampling effort is necessary to expand the identification of possible cryptic species in this environment.

O objetivo deste estudo foi realizar o primeiro levantamento da composição da comunidade de amebas testáceas no lago Monte Alegre no Estado de São Paulo e, assim, contribuir para mitigar oimpedimento taxonômico relacionado à diversidade de amebas testáceas no Brasil. As amostragens foramrealizadas em 20 pontos do lago, nas regiões limnética e litorânea, no mês de abril de 2015, com rede deplâncton de 58 μm de abertura de malha. Foram identificados 20 táxons de amebas testáceas, divididos emquatro famílias: Arcellidae (8 táxons), Centropyxidae (4 táxons), Difflugiidae (7 táxons) e Lesquereusiidae(2 táxons). A região litorânea apresentou o maior número de táxons (n = 20), enquanto que na regiãolimnética foram registrados 11 táxons. Diante disso, é possível indicar que os nossos resultados evidenciama importância das amebas testáceas nos sistemas aquáticos e que novos estudos, taxonômicos e/ou ecológicos, nesses ambientes devem incluir estes organismos em suas investigações. Além disso, sugeremsefuturos estudos com maior esforço amostral para ampliar a identificação de possíveis espécies crípticas noambiente.

Natural Products from Social Amoebae.

Natural products are invaluable sources of structural diversity and complexity ideally suited for the development of therapeutic agents. The search for novel bioactive molecules has prompted scientists to explore various ecological niches. Microorganisms have been shown to constitute such an important source. Despite their biosynthetic potential, social amoebae, that is, microorganisms with both a uni- and multicellular lifestyle, are underexplored regarding their secreted secondary metabolome. In this review, we present the structural diversity of amoebal natural products and discuss their biological functions as well as their total syntheses.

Cinderella's helping pigeons of the microbial world: The potential of testate amoebae for identifying cryptotephra.

Cryptotephra (particles <125µm) is a key record for monitoring past and current volcanic activity. However, its extraction from the host sediment and analysis is often long and difficult because of its small size. Finding a simple method to extract cryptotephra from environmental samples would therefore make its analysis much easier. We hypothesized that arcellinid testate amoebae may hold such a potential. These free-living shelled protists are among the earliest microorganisms to colonize volcanic tephra, and build their shell by agglutinating minerals from their environment. We analyzed by X-ray Spectrometry the mineral signature of tephra from the 2011 Puyehue-Cordon Caulle Volcanic Complex (Chile) eruption ash fallout and compared it to that of the shells of 51 individual testate amoebae (three individuals from each of 17 species) from 13 samples collected at different distances from the active vent. The mineral composition of particles within shells closely matched that of similar size class particles from their environment. The capacity of testate amoebae to randomly use mineral grains from their environment makes it possible to use their shells to assess the mineral composition of cryptotephra from soil, peat or sediment samples. Testate amoebae therefore represent the microbial world's version of Cinderella's helping pigeons.

Root of Dictyostelia based on 213 universal proteins.

Dictyostelia are common soil microbes that can aggregate when starved to form multicellular fruiting bodies, a characteristic that has also led to their long history of study and widespread use as model systems. Ribosomal RNA phylogeny of Dictyostelia identified four major divisions (Groups 1-4), none of which correspond to traditional genera. Group 1 was also tentatively identified as sister lineage to the other three Groups, although not consistently or with strong support. We tested the dictyostelid root using universal protein-coding genes identified by exhaustive comparison of six completely sequenced dictyostelid genomes, which include representatives of all four major molecular Groups. A set of 213 genes are low-copy number in all genomes, present in at least one amoebozoan outgroup taxon (Acanthamoeba castellanii or Physarum polycephalum), and phylogenetically congruent. Phylogenetic analysis of a concatenation of the deduced protein sequences produces a single topology dividing Dictyostelia into two major divisions: Groups 1+2 and Groups 3+4. All clades in the tree are fully supported by maximum likelihood and Bayesian inference, and all alternative roots are unambiguously rejected by the approximately unbiased (AU) test. The 1+2, 3+4 root is also fully supported even after deleting clusters with strong individual support for this root, or concatenating all clusters with low support for alternative roots. The 213 putatively ancestral amoebozoan proteins encode a wide variety of functions including 21 KOG categories out of a total of 25. These comprehensive analyses and consistent results indicate that it is time for full taxonomic revision of Dictyostelia, which will also enable more effective exploitation of its unique potential as an evolutionary model system.

Pore-forming toxins from pathogenic amoebae.

Some amoeboid protozoans are facultative or obligate parasites in humans and bear an enormous cytotoxic potential that can result in severe destruction of host tissues and fatal diseases. Pathogenic amoebae produce soluble pore-forming polypeptides that bind to prokaryotic and eukaryotic target cell membranes and generate pores upon insertion and oligomerization. This review summerizes the current knowledge of such small protein toxins from amoebae, compares them with related proteins from other species, focuses on their three-dimensional structures, and gives insights into divergent activation mechanisms. The potential use of pore-forming toxins in biotechnology will be briefly outlined.

C-terminal fragment of amebin promotes actin filament bundling, inhibits acto-myosin ATPase activity and is essential for amoeba migration.

Amebin [formerly termed as ApABP-FI; Sobczak et al. (2007) Biochem. Cell Biol. 85] is encoded in Amoeba proteus by two transcripts, 2672-nt and 1125-nt. A product of the shorter transcript (termed as C-amebin), comprising C-terminal 375 amino-acid-residue fragment of amebin, has been expressed and purified as the recombinant GST-fusion protein. GST-C-amebin bound both to monomeric and filamentous actin. The binding was Ca(2+)-independent and promoted filament bundling, as revealed with the transmission electron microscopy. GST-C-amebin significantly decreased MgATPase activity of rabbit skeletal muscle acto-S1. Removal with endoproteinase ArgC of a positively charged C-terminal region of GST-amebin containing KLASMWEQ sequence abolished actin-binding and bundling as well as the ATPase-inhibitory effect of C-amebin, indicating that this protein region was involved in the interaction with actin. Microinjection of amoebae with antibody against C-terminus of amebin significantly affected amoebae morphology, disturbed cell polarization and transport of cytoplasmic granules as well as blocked migration. These data indicate that amebin may be one of key regulators of the actin-cytoskeleton dynamics and actin-dependent motility in A. proteus.

Reducing the impact of PCR-mediated recombination in molecular evolution and environmental studies using a new-generation high-fidelity DNA polymerase.

PCR-mediated recombination can greatly impact estimates of diversity, both in environmental studies and in analyses of gene family evolution. Here we measure chimera (PCR-mediated recombinant) formation by analyzing a mixture of eight partial actin sequences isolated from the amoeba Arcella hemisphaerica amplified under a variety of conditions that mimic standard laboratory situations. We further compare a new-generation proofreading processivity-enhanced polymerase to both a standard proofreading enzyme and previously published results. Proofreading polymerases are preferred over other polymerases in instances where evolutionary inferences must be made. Our analyses reveal that reducing the initial template concentration is as critical as reducing the number of cycles for decreasing chimera formation and improving accuracy. Furthermore, assessing the efficiency of recovery of original haplotypes demonstrates that multiple PCR reactions are required to capture the actual genetic diversity of a sample. Finally, the experiments confirm that processivity-enhanced polymerases enable a substantial decrease in PCR-mediated recombination through reducing starting template concentration, without compromising the robustness of PCR reactions.

Engineering an artificial amoeba propelled by nanoparticle-triggered actin polymerization.

We have engineered an amoeba system combining nanofabricated inorganic materials with biological components, capable of propelling itself via actin polymerization. The nanofabricated materials have a mechanism similar to the locomotion of the Listeria monocytogenes, food poisoning bacteria. The propulsive force generation utilizes nanoparticles made from nickel and gold functionalized with the Listeria monocytogenes transmembrane protein, ActA. These Listeria-mimic nanoparticles were in concert with actin, actin binding proteins, ATP (adenosine triphosphate) and encapsulated within a lipid vesicle. This system is an artificial cell, such as a vesicle, where artificial nanobacteria and actin polymerization machinery are used in driving force generators inside the cell. The assembled structure was observed to crawl on a glass surface analogously to an amoeba, with the speed of the movement dependent on the amount of actin monomers and ATP present.

The cyst wall carbohydrate composition of Balamuthia mandrillaris.

Balamuthia mandrillaris is an opportunistic cyst-producing amoeba that can cause rare, but fatal, Balamuthia amoebic encephalitis (BAE). Cysts are resistant to harsh environmental conditions and many antimicrobial compounds and thus can contribute to BAE recurrence. However, little is known of cyst wall synthesis, cyst wall composition, or how encystment is induced. In this study, we examined the carbohydrate composition of the cyst wall. The major components were mannose (20.9 mol%) and glucose (79.1 mol%), with trace amounts of galactose present in the cyst wall samples analysed. The linkage analysis showed cyst wall carbohydrates with apparently linear and branching saccharides and suggested the presence of cellulose. These components may play an important protective role by creating a permeability barrier around the cyst.

Physical model for membrane protrusions during spreading.

During cell spreading onto a substrate, the kinetics of the contact area is an observable quantity. This paper is concerned with a physical approach to modeling this process in the case of ameboid motility where the membrane detaches itself from the underlying cytoskeleton at the leading edge. The physical model we propose is based on previous reports which highlight that membrane tension regulates cell spreading. Using a phenomenological feedback loop to mimic stress-dependent biochemistry, we show that the actin polymerization rate can be coupled to the stress which builds up at the margin of the contact area between the cell and the substrate. In the limit of small variation of membrane tension, we show that the actin polymerization rate can be written in a closed form. Our analysis defines characteristic lengths which depend on elastic properties of the membrane-cytoskeleton complex, such as the membrane-cytoskeleton interaction, and on molecular parameters, the rate of actin polymerization. We discuss our model in the case of axi-symmetric and non-axi-symmetric spreading and we compute the characteristic time scales as a function of fundamental elastic constants such as the strength of membrane-cytoskeleton adherence.

Cubic membranes: a structure-based design for DNA uptake.

Cubic membranes are soft three-dimensional crystals found within cell organelles in a variety of living systems, despite the aphorism of Fedorov: 'crystallization is death'. They consist of multi-bilayer lipid-protein stacks, folded onto anticlastic surfaces that resemble triply periodic minimal surfaces, forming highly swollen crystalline sponges. Although cubic membranes have been observed in numerous cell types and under different pathophysiological conditions, knowledge about the formation and potential function(s) of non-lamellar, cubic structures in biological systems is scarce. We report that mitochondria with this cubic membrane organization isolated from starved amoeba Chaos carolinense interact sufficiently with short segments of phosphorothioate oligonucleotides (PS-ODNs) to give significant ODNs uptake. ODNs condensed within the convoluted channels of cubic membrane by an unknown passive targeting mechanism. Moreover, the interaction between ODNs and cubic membrane is sufficient to retard electrophoretic mobility of the ODN component in the gel matrix. These ODN-cubic membrane complexes are readily internalized within the cytoplasm of cultured mammalian cells. Transmission electron microscopic analysis confirms ODNs uptake by cubic membranes and internalization of ODN-cubic membrane complexes into the culture cells. Cubic membranes thus may offer a new, potentially benign medium for gene transfection.

[Heat shock protein of HSP70 family revealed in some contemporary freshwater Amoebae and in Acanthamoeba sp. from cysts isolated from permafrost samples].

A heat shock protein of HSP70 family was revealed for the first time in trophozoites of Acanthamoeba sp. (strain Am8) excysted from cysts previously isolated from samples of permafrost aging 30,000-35,000 years. The constitutive level of this HSP, shown by immunnoblotting in unstressed trophozoites of the ancient acanthamoebae, much surpassed that in unstressed cells of the three examined species of contemporary freshwater amebae of the genus Amoeba.

Characterization of a cDNA of peroxiredoxin II responding to hydrogen peroxide and phagocytosis in Amoeba proteus.

Phagocytic cells have defense systems against reactive oxygen species generated as the first non-specific defense mechanism against invading pathogens or microorganisms. We cloned a cDNA encoding a 21.69-kDa protein in Amoeba proteus homologous to 2-Cys peroxiredoxin (Prx-Ap). In the disk inhibition assay using H2O2 as an oxidizing agent, Escherichia coli overproducing Prx-Ap showed better viability than did E. coli transformed with pBluescript II SK for control. Monoclonal antibodies (mAb) produced against Prx-Ap reacted with a 22.5-kDa protein and several minor proteins. In Western blot analysis, levels of the 22.5-kDa protein in amoebae treated with 2-mM H2O2 for 1 h increased about 2-fold over those in control cells. Immunofluorescence scattered throughout the cytoplasm also increased after H2O2 treatment. In Northern blot analysis using the cDNA as a probe, the level of transcripts also changed with H2O2 treatment. When amoebae were fed with Tetrahymena, the intensity of immunofluorescence increased from 15 min and persisted until 2 h after phagocytosis. These results suggest that the 22.5-kDa protein of A. proteus is a Prx protein and that it has an antioxidant property responding to phagocytosis.

Characterization of Amoeba proteus myosin VI immunoanalog.

Amoeba proteus, the highly motile free-living unicellular organism, has been widely used as a model to study cell motility. However, molecular mechanisms underlying its unique locomotion and intracellular actin-based-only trafficking remain poorly understood. A search for myosin motors responsible for vesicular transport in these giant cells resulted in detection of 130-kDa protein interacting with several polyclonal antibodies against different tail regions of human and chicken myosin VI. This protein was binding to actin in the ATP-dependent manner, and immunoprecipitated with anti-myosin VI antibodies. In order to characterize its possible functions in vivo, its cellular distribution and colocalization with actin filaments and dynamin II during migration and pinocytosis were examined. In migrating amoebae, myosin VI immunoanalog localized to vesicular structures, particularly within the perinuclear and sub-plasma membrane areas, and colocalized with dynamin II immunoanalog and actin filaments. The colocalization was even more evident in pinocytotic cells as proteins concentrated within pinocytotic pseudopodia. Moreover, dynamin II and myosin VI immunoanalogs cosedimented with actin filaments, and were found on the same isolated vesicles. Blocking endogenous myosin VI immunoanalog with anti-myosin VI antibodies inhibited the rate of pseudopodia protrusion (about 19% decrease) and uroidal retraction (about 28% decrease) but did not affect cell morphology and the manner of cell migration. Treatment with anti-human dynamin II antibodies led to changes in directionality of amebae migration and affected the rate of only uroidal translocation (about 30% inhibition). These results indicate that myosin VI immunoanalog is expressed in protist Amoeba proteus and may be involved in vesicle translocation and cell locomotion.