Amoebae Research Articles

Locally excitable Cdc42 signals steer cells during chemotaxis.

Neutrophils and other amoeboid cells chemotax by steering their front towards chemoattractant. While Ras, Rac, Cdc42, and RhoA small GTPases all regulate chemotaxis, it has been unclear how they spatiotemporally control polarization and steering. Using fluorescence biosensors in neutrophil-like PLB-985 cells and photorelease of chemoattractant, we show that local Cdc42 signals, but not those of Rac, RhoA or Ras, precede cell turning during chemotaxis. Furthermore, preexisting local Cdc42 signals in morphologically unpolarized cells predict the future direction of movement upon uniform stimulation. Moreover, inhibition of actin polymerization uncovers recurring local Cdc42 activity pulses, suggesting that Cdc42 has the excitable characteristic of the compass activity proposed in models of chemotaxis. Globally, Cdc42 antagonizes RhoA, and maintains a steep spatial activity gradient during migration, while Ras and Rac form shallow gradients. Thus, chemotactic steering and de novo polarization are both directed by locally excitable Cdc42 signals.

Morphology and life cycle of amoeboflagellate Pharyngomonas sp. (Heterolobosea, Excavata) from hypersaline inland Razval Lake

The morphology, life cycle, feeding and multiplication of amoeboflagellate Pharyngomonas sp., strain RL isolated from hypersaline inland Razval Lake was studied. Life cycle includes flagellate, amoeboid, and cyst stages. Flagellated cells have two anterior flagella and two posterior flagella emerging apically from the anterior end of the cell. The nucleus localizes closely to the base of flagella. The longitudinal groove is on the ventral side of the cell. Flagellated cells have a distinct cytopharynx in the anterior part of the ventral groove. The cytopharynx passes into an intracellular channel reaching the posterior end of the cell. Amoeboid cells have two morphotypes—heterolobosean and lobosean. Cysts are spherical, their wall is smooth. Cell division occurs either at flagellated stage or at amoeboid one. Distinctions of Pharyngomonas sp., strain RL, from Pharyngomonas kirbyi were determined. They include longer flagella and long filamentous branching pseudopodia at the posterior end of amoeboid cells. The morphological distinctions are in agreement with differences in structure of the 18S rRNA gene. The revealed features may be used for differentiation of Pharyngomonas species.

Characteristic Changes of Astrocyte and Microglia in Rat Striatum Induced by 3-NP and MCAO.

Our previous studies had confirmed that both 3-NP and MCAO induced the behavioral defect as well as striatal neuronal injury and loss in experimental rats. This study aimed to examine different response forms of striatal astrocyte and microglia in 3-NP and MCAO rat models. The present results showed that the immunoreaction for GFAP was extremely weak in the lesioned core of striatum, but in the transition zone of 3-NP model and the penumbra zone of MCAO model, GFAP+ cells showed strong hypertrophic and proliferative changes. Statistical analysis for the number, size and integral optical density (IOD) of GFAP+ cells showed significant differences when compared with their controls and compared between the core and the transition zone or the penumbra zone, respectively, but no differences between the 3-NP and MCAO groups. However, Iba-1+ cells showed obvious hypertrophy and proliferation in the injured striatum in the 3-NP and the MCAO models, especially in the transition zone of 3-NP model and the penumbra zone of MCAO model. These Iba-1+ cells displayed two characteristic forms as branching cells with thick processes and amoeboid cells with thin processes. Statistical analysis showed that the number, size and IOD of Iba-1+ cells were significantly increased in the cores and the transition zone of 3-NP group and the penumbra zone of MCAO group than that of the controls, and the immune response of Iba-1 was stronger in the MCAO group than in the 3-NP group. The present results suggested that characteristic responses of astrocyte and microglia in the 3-NP and the MCAO models display their different effects on the pathological process of brain injury.

TGF-β-Induced Transcription Sustains Amoeboid Melanoma Migration and Dissemination

SummaryCell migration underlies metastatic dissemination of cancer cells, and fast “amoeboid” migration in the invasive fronts of tumors is controlled by high levels of actomyosin contractility. How amoeboid migration is regulated by extracellular signals and sustained over time by transcriptional changes is not fully understood. Transforming growth factor β (TGF-β) is well known to promote epithelial-to-mesenchymal transition (EMT) and contribute to metastasis, but melanocytes are neural crest derivatives that have undergone EMT during embryonic development. Surprisingly, we find that in melanoma, TGF-β promotes amoeboid features such as cell rounding, membrane blebbing, high levels of contractility, and increased invasion. Using genome-wide transcriptomics, we find that amoeboid melanoma cells are enriched in a TGF-β-driven signature. We observe that downstream of TGF-β, SMAD2 and its adaptor CITED1 control amoeboid behavior by regulating the expression of key genes that activate contractile forces. Moreover, CITED1 is highly upregulated during melanoma progression, and its high expression is associated with poor prognosis. CITED1 is coupled to a contractile-rounded, amoeboid phenotype in a panel of 16 melanoma cell lines, in mouse melanoma xenografts, and in 47 human melanoma patients. Its expression is also enriched in the invasive fronts of lesions. Functionally, we show how the TGF-β-SMAD2-CITED1 axis promotes different steps associated with progression: melanoma detachment from keratinocytes, 2D and 3D migration, attachment to endothelial cells, and in vivo lung metastatic initial colonization and outgrowth. We propose a novel mechanism by which TGF-β-induced transcription sustains actomyosin force in melanoma cells and thereby promotes melanoma progression independently of EMT.

Nebela kivuense Gauthier-Lièvre et Thomas, 1961 (Amoebozoa, Arcellinida), Missing for a Half-century; Found 11,500 km from "home"

In 1961, the testate amoeba Nebela kivuense Gauthier-Lievre et Thomas, 1961 was described for the first and only time from an area near Lake Edward in the Democratic Republic of the Congo (at 0.002° N Latitude). The lack of recent reports of this species, despite exhaustive surveys of the testate amoebae fauna of the major continents of the world, suggested that N. kivuense was a rare species perhaps endemic to a small, local equatorial region of the African continent. This paper reports its rediscovery from two wetland-conifer forest ecosystems in southern Ontario, Canada (at 44° N Latitude), thus changing dramatically our previous perception of its very restricted global distribution. This has implications for the idea held by many students of biogeography that there is a special category of microscopic protists that contains truly rare species and their rarity, perhaps together with specific habitat requirements and tolerances, limits opportunities for dispersal around the world. The N. kivuense story is a clear example of the dangers of inferring endemism from rarity.

Crawling and Gliding: A Computational Model for Shape-Driven Cell Migration.

Cell migration is a complex process involving many intracellular and extracellular factors, with different cell types adopting sometimes strikingly different morphologies. Modeling realistically behaving cells in tissues is computationally challenging because it implies dealing with multiple levels of complexity. We extend the Cellular Potts Model with an actin-inspired feedback mechanism that allows small stochastic cell rufflings to expand to cell protrusions. This simple phenomenological model produces realistically crawling and deforming amoeboid cells, and gliding half-moon shaped keratocyte-like cells. Both cell types can migrate randomly or follow directional cues. They can squeeze in between other cells in densely populated environments or migrate collectively. The model is computationally light, which allows the study of large, dense and heterogeneous tissues containing cells with realistic shapes and migratory properties.

Proteomic analysis of theSimkania‐containing vacuole: the central role of retrograde transport

Simkania negevensis is an obligate intracellular bacterial pathogen that grows in amoeba or human cells within a membrane-bound vacuole forming endoplasmic reticulum (ER) contact sites. The membrane of this Simkania-containing vacuole (SnCV) is a critical host-pathogen interface whose origin and molecular interactions with cellular organelles remain poorly defined. We performed proteomic analysis of purified ER-SnCV-membranes using label free LC-MS(2) to define the pathogen-containing organelle composition. Of the 1,178 proteins of human and 302 proteins of Simkania origin identified by this strategy, 51 host cell proteins were enriched or depleted by infection and 57 proteins were associated with host endosomal transport pathways. Chemical inhibitors that selectively interfere with trafficking at the early endosome-to-trans-Golgi network (TGN) interface (retrograde transport) affected SnCV formation, morphology and lipid transport. Our data demonstrate that Simkania exploits early endosome-to-TGN transport for nutrient acquisition and growth.

A new flagellated dispersion stage in Paraphysoderma sedebokerense, a pathogen of Haematococcus pluvialis.

The blastocladialean fungus Paraphysoderma sedebokerense Boussiba, Zarka and James is a devastating pathogen of the commercially valuable green microalga Haematococcus pluvialis, a natural source of the carotenoid pigment astaxanthin. First identified in commercial Haematococcus cultivation facilities, P. sedebokerense is hypothesised to have a complex life cycle that switches between a vegetative and a resting phase depending on favourable or unfavourable growth conditions. Rather unusually for blastocladialean fungi, P. sedebokerense was described as lacking flagellated zoospores and only propagating via aplanosporic amoeboid cells. However, during repeated microscopic observation of P. sedebokerense cultivated in optimal conditions, we detected fast-swimming, transiently uniflagellated zoospores which rapidly transform into infectious amoeboid swarmers, the existence of which suggests a closer than previously thought relatedness of P. sedebokerense to its sister genera Physoderma and Urophlyctis. Additionally, we found some morphological and physiological differences between amoeboid swarmers and discuss hypotheses about their significance. These amoeboid and flagellated propagules are key to the dissemination of P. sedebokerense and are probably also the life stages most vulnerable to adverse environmental conditions. They are therefore a prime target for the development of disease management protocols in industrial cultivation facilities, a goal which requires a detailed understanding of their physiology.Electronic supplementary materialThe online version of this article (doi:10.1007/s10811-015-0700-8) contains supplementary material, which is available to authorized users.

Molecular and Morphological Snapshot Characterisation of the Protist Communities in Contrasting Alpine Glacier Forefields

Phagotrophic protist diversity in oligotrophic soils such as alpine glacier forefields is still poorly studied. Combining morphologic observations with molecular-based analyses, we assessed the diversity of major phagotrophic protist groups in two contrasting glacier forefields in the Swiss Alps (Tiefen glacier forefield, siliceous bedrock, and Wildstrubel glacier forefield, calcareous bedrock), at sites differing in soil development. Ciliates and heterotrophic flagellates could be detected with both approaches, while amoebae could be observed only microscopically. Soils from Tiefen and Wildstrubel glacier forefields harboured distinctly different ciliate, flagellate and amoebae communities. The ciliate clone libraries from the Tiefen glacier forefield were dominated by Oligohymenophorea-related sequences while those from the Wildstrubel glacier forefield were dominated by Spirotrichea-related sequences. Testate amoebae morphospecies of the genera Corythion , Cryptodifflugia , Euglypha and Tracheleuglypha were restricted to the Tiefen glacier forefield, while Centropyxis and Trinema to the Wildstrubel one. No ciliate sequences and only a few ciliate and testate amoebae morphospecies could be retrieved from unvegetated soils of both glacier forefields. The ciliate and testate amoebae community detected at unvegetated sites were a subset of the community developed at vegetated sites. Overall, our results suggest that alpine glacier forefields are colonised by a diverse community of phagotrophic protists which seems to be shaped by bedrock geology and vegetation cover.

A Description of Cochliopodium megatetrastylus n. sp. Isolated from a Freshwater Habitat

Cochliopodium megatetrastylus n. sp. is described based on light microscopy, fine structure and molecular genetic evidence. Amoebae are broadly oval to somewhat triangular during locomotion with average length of 37 μm and breadth of 50 μm, and surrounded by a hyaloplasm margin, somewhat narrow when at rest but more expanded during locomotion (~ 5–10 μm at the anterior). Sparsely occurring subpseudopodia, barely emergent from the hyaloplasm, are blunt and finger-like, occasionally becoming adhesive laterally or at the posterior. Cysts develop after 2–3 weeks in culture and are round with a distinct margin, decreasing in size from 20 to 5 μm during maturation. The granuloplasm contains refractile crystals. A vesicular nucleus (~ 6 μm), containing a nucleolus (2–3 μm), is variable in shape from somewhat lenticular in section to irregularly rounded with undulating or lobed margins. Surface scales (~ 0.3 μm in height) have an apical deeply concave funnel-like collar (~ 0.15 μm deep), without a spine, composed of radial fine rays and concentric filaments forming a finemesh, supported on four non-cross-linked styles (~ 0.2 μm apart) attached to a round to broadly angular base plate (0.6–1 μm) with a fine gridtexture. Cysts are rounded and enclosed by an organic wall bearing remnants of the scales on its outer surface. Both concatenated analysis of SSU-rDNA and COI genes and comparative morphologies support the designation of Cochliopodium megatetrastylus n. sp. as a new species.

Interplay between motility and cell-substratum adhesion in amoeboid cells.

The effective migration of amoeboid cells requires a fine regulation of cell-substratum adhesion. These entwined processes have been shown to be regulated by a host of biophysical and biochemical cues. Here, we reveal the pivotal role played by calcium-based mechanosensation in the active regulation of adhesion resulting in a high migratory adaptability. Using mechanotactically driven Dictyostelium discoideum amoebae, we uncover the existence of optimal mechanosensitive conditions-corresponding to specific levels of extracellular calcium-for persistent directional migration over physicochemically different substrates. When these optimal mechanosensitive conditions are met, noticeable enhancement in cell migration directionality and speed is achieved, yet with significant differences among the different substrates. In the same narrow range of calcium concentrations that yields optimal cellular mechanosensory activity, we uncovered an absolute minimum in cell-substratum adhesion activity, for all considered substrates, with differences in adhesion strength among them amplified. The blocking of the mechanosensitive ion channels with gadolinium-i.e., the inhibition of the primary mechanosensory apparatus-hampers the active reduction in substrate adhesion, thereby leading to the same undifferentiated and drastically reduced directed migratory response. The adaptive behavioral responses of Dictyostelium cells sensitive to substrates with varying physicochemical properties suggest the possibility of novel surface analyses based on the mechanobiological ability of mechanosensitive and guidable cells to probe substrates at the nanometer-to-micrometer level.

Case Reports and Prevention Measures of Waterborne Protozoan Infections in Public Swimming Pools and Other Recreational Waters

Apart from notifiable infectious diseases, Taiwan Centers of Disease Control (TCDC) helps the diagnosis of other infectious diseases for hospitals if necessary. TCDC also cooperates with local health bureaus and research institutions for the environmental surveillance in order to reduce and prevent the occurrence of infectious diseases. Here we discuss the surveillance of recreational water and report the cases of granulomatous amebic encephalitis (GAE) and primary amoebic meningoencephalitis (PAM) caused by free-living amoebae (FLA) in Taiwan since 2006. Guidelines or regulations for correct usage of recreational water and methods for disinfection of water and facilities are introduced. We hope people therefore realize the transmission route and related information of waterborne protozoan infections and know the infection risk of usage of public swimming pools and other recreational waters to prevent the occurrence of disease.

Abstract 4141: Hypoxia-induced transition from collective to amoeboid single cell dissemination in epithelial cancer cells

Abstract Tumor hypoxia, by elevating hypoxia-inducible factors (HIFs), is an established inducer of epithelial-to-mesenchymal transition (EMT) and subsequent cancer cell invasion and metastasis. Using epithelial carcinoma spheroids in 3D fibrillar collagen, we characterized the invasion patterns as well as cellular and molecular mechanisms of hypoxia-induced cancer cell migration modes. While epithelial cancer cells show collective invasion under normoxic conditions, hypoxia or pharmacological stabilization of HIF-1 using the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG) induced EMT-like detachment and migration of single cells. Besides mesenchymal movement, most epithelial cells converted to amoeboid migration with characteristic actin-rich filopodal or distinctive blebby protrusions towards the direction of migration. Whereas mesenchymal migrating cells moved with low velocities in a directionally persistent manner, amoeboid migrating cells generated a broad spectrum of low to high velocities, but with less persistent invasion paths. Sub-cellular molecular analysis showed typical amoeboid features particularly in hypoxic blebby single cell migration, including low to lacking collagen degradation along the migration path and insensitivity to broad-spectrum matrix metalloproteinase (MMP)-inhibitor GM6001, non-focalized cortical actin cytoskeleton within blebs that interacted with collagen structures and low-beta1 integrin expression with lack of integrin focalization. Thus, tumor hypoxia induces a diversity of single-cancer cell invasion modes, including blebby amoeboid migration, thereby enhancing predominantly MMP- and integrin-independent amoeboid dissemination in parallel to EMT induction. Citation Format: Steffi Lehmann, Veronika A M te Boekhorst, Julia Odenthal, Liying Jiang, Sjoerd van Helvert, Peter Friedl. Hypoxia-induced transition from collective to amoeboid single cell dissemination in epithelial cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4141. doi:10.1158/1538-7445.AM2015-4141

The cholesterol-binding protein NPC2 restrains recruitment of stromal macrophage-lineage cells to early-stage lung tumours.

The tumour microenvironment is known to play an integral role in facilitating cancer progression at advanced stages, but its function in some pre-cancerous lesions remains elusive. We have used the V600EBRAF-driven mouse lung model that develop premalignant lesions to understand stroma–tumour interactions during pre-cancerous development. In this model, we have found that immature macrophage-lineage cells (IMCs) producing PDGFA, TGFβ and CC chemokines are recruited to the stroma of premalignant lung adenomas through CC chemokine receptor 1 (CCR1)-dependent mechanisms. Stromal IMCs promote proliferation and transcriptional alterations suggestive of epithelial–mesenchymal transition in isolated premalignant lung tumour cells ex vivo, and are required for the maintenance of early-stage lung tumours in vivo. Furthermore, we have found that IMC recruitment to the microenvironment is restrained by the cholesterol-binding protein, Niemann-Pick type C2 (NPC2). Studies on isolated cells ex vivo confirm that NPC2 is secreted from tumour cells and is taken up by IMCs wherein it suppresses secretion of the CCR1 ligand CC chemokine 6 (CCL6), at least in part by facilitating its lysosomal degradation. Together, these findings show that NPC2 secreted by premalignant lung tumours suppresses IMC recruitment to the microenvironment in a paracrine manner, thus identifying a novel target for the development of chemopreventive strategies in lung cancer.

Regulation of microtubule dynamics by DIAPH3 influences amoeboid tumor cell mechanics and sensitivity to taxanes.

Taxanes are widely employed chemotherapies for patients with metastatic prostate and breast cancer. Here, we show that loss of Diaphanous-related formin-3 (DIAPH3), frequently associated with metastatic breast and prostate cancers, correlates with increased sensitivity to taxanes. DIAPH3 interacted with microtubules (MT), and its loss altered several parameters of MT dynamics as well as decreased polarized force generation, contractility, and response to substrate stiffness. Silencing of DIAPH3 increased the cytotoxic response to taxanes in prostate and breast cancer cell lines. Analysis of drug activity for tubulin-targeted agents in the NCI-60 cell line panel revealed a uniform positive correlation between reduced DIAPH3 expression and drug sensitivity. Low DIAPH3 expression correlated with improved relapse-free survival in breast cancer patients treated with chemotherapeutic regimens containing taxanes. Our results suggest that inhibition of MT stability arising from DIAPH3 downregulation enhances susceptibility to MT poisons, and that the DIAPH3 network potentially reports taxane sensitivity in human tumors.

Eukaryotic Microbial Communities Associated with Rock- dwelling Foliose Lichens: A Functional Morphological and Microecological Analysis

Lichens are widely recognized as important examples of a fungal-algal or fungal-cyanophyte symbiosis; and in some cases they are a major food source for some animal grazers such as caribou ( Rangifer tarandus ), especially in the Arctic during winter. However, relatively little is known about the ecology of their co-associated bacterial and protistan communities. This is one of the first reports of an analysis of microbial communities associated with rock-dwelling foliose lichens ( Flavoparmelia sp.), including a more detailed analysis of the microbial communities associated with segments of the shield-like, radially arranged lobes. Samples were taken from lichens on granite boulders beneath an oak and maple tree stand on the Lamont-Doherty Earth Observatory Campus, Palisades, N.Y. The bacteria and protist members of the lichen associated microbial communities are comparable to recently reported associations for foliose lichens growing on tree bark at the same locale, including the presence of large myxomycete plasmodial amebas, heterotrophic nanoflagellates, and naked and testate amebas. To obtain evidence of possible differences in the microecology of different portions of each radial lobe, three segments of the radial lobe in the shield-like lichen were sampled: 1) inner, more mature, central segment; 2) middle section linking the central and peripheral segments; and 3) outer, peripheral, usually broader, less closely attached segment. The mean densities (number/g) and biomasses (µg/g) of bacteria and heterotrophic nanoflagellates were highest in the older central segment and lowest in the peripheral segment of the radial lobes, especially when expressed on moist weight basis. Large myxomycete plasmodial amebas were typically located in the outermost segment of the radial lobe. The proportion of vannellid amebas ( Vannella spp. and Cochliopodium spp.) were significantly more abundant in the samples of the inner lobes compared to non-vannellid amebas that were more prevalent in the outer lobes. The outer segment of the thallus lobe was typically more spongiose and absorbed more water per unit weight (based on a wet/dry-weight ratio) than the innermost segment. In general, patterns of densities and taxonomic composition of bacteria and eukaryotic microbes intergraded from the inner most segment to the outer part of each lobe – indicating a possible microecological gradient, coincident with the age-related and morphological radial gradations of the lobe. Overall, the evidence shows that the radial variation in the morphology and age-related variables of the three lobe segments may affect the microenvironment of the lobe segments and hence influence the organization of the microbial communities within each segment.

Migratory behaviour of Physarum polycephalum microplasmodia

The motility of amoeboid cells of the plasmodial slime mould Physarum polycephalum was studied experimentally. Analysis of their trajectories and of their mean square displacements reveal two characteristic types of behaviour that depend on the time interval τ between any pair of points along the trajectory. Whereas free migration of cells is observed for time intervals τ > 300 s, at short time intervals (of up to τ ≈ 100 s) the motility is due to changes in the cell shape induced by the peristaltic pumping of protoplasm though the cell. Freely migrating cells display persistent random motion with very long persistence times of up to ≈ 5500 s. Superdiffusive motion typically lasts for ≈ 18000 s, independently of the intensity of illumination by green light. Whereas symmetric velocity distributions are found for short time intervals τ, the typical velocity distributions from freely migrating cells show an asymmetry, which reflects the long-lasting persistent motions. We observed that high propagation velocities are correlated with both, episodes of straight motion and an elongated cell shape. Finally, the mean squared displacement of trajectories where the cells avoided crossing their own slime trails were compared to those of freely migrating cells.

Cytotoxicity effects of alkoxy substituted thiourea derivatives towards Acanthamoeba sp.

In this study, the effort was to design and synthesize five new members of alkoxy substituted thiourea derivatives (3a–3e) featuring general formula of A-ArC(O)NHC(S)NHAr-D in which A represents the methoxy group and D as –OCnH2n+1 (alkoxyl group, where n=6,7,8,9, and 10) have been successfully designed, prepared, characterized, and evaluated for anti-amoebic activities. They were spectroscopically characterized by 1H and 13C Nuclear Magnetic Resonance (NMR), Fourier Transform Infrared (FT-IR) spectroscopy, and Ultraviolet–visible (UV–vis) spectroscopy analysis. In turn, they were used to investigate the cytotoxicity effect on Acanthamoeba sp. at their IC50 values and membrane permeability. Compounds 3a and 3b revealed to have good activity towards Acanthamoeba sp. compared to other compounds of 3c, 3d, and 3e. The observation under fluorescence microscopy by AOPI (Acridine-orange/Propidium iodide) staining indicated that treated amoeba cells by 3a–3e show loss of their membrane permeability.

Analysis of the shapes of hemocytes of Callista brevisiphonata in vitro (Bivalvia, Veneridae).

Fractal formalism in conjunction with linear methods of image analysis is suitable for the comparative analysis of such "irregular" shapes (from the point of view of classical Euclidean geometry) as flattened amoeboid cells of invertebrates in vitro. Cell morphology of in vitro spreading hemocytes from the bivalve mollusc Callista brevisiphonata was analyzed using correlation, factor and cluster analysis. Four significantly different cell types were identified on the basis of 36 linear and nonlinear parameters. The analysis confirmed the adequacy of the selected methodology for numerical description of the shape and the adequacy of classification of nonlinear shapes of spread hemocytes belonging to the same species. Investigation has practical significance for the description of the morphology of cultured cells, since cell shape is a result of summation of a number of extracellular and intracellular factors.

T Cells Have a Light Touch

T Cells Have a Light Touch