MARINE‐DERIVED DINOFLAGELLATES IN ANTARCTIC SALINE LAKES: COMMUNITY COMPOSITION AND ANNUAL DYNAMICS1*[link]

The saline lakes of the Vestfold Hills in Antarctica offer a remarkable natural laboratory where the adaptation of planktonic protists to a range of evolving physiochemical conditions can be investigated. This study illustrates how an ancestral marine community has undergone radical simplification leaving a small number of well‐adapted species. Our objective was to investigate the species composition and annual dynamics of dinoflagellate communities in three saline Antarctic lakes. We observed that dinoflagellates occur year‐round despite extremely low PAR during the southern winter, which suggests significant mixotrophic or heterotrophic activity. Only a small number of dominant dinoflagellate species were found in each lake, in contrast to the species‐rich Southern Ocean from which the lake communities are believed to be derived. We verified that the lake species were representatives of the marine polar dinoflagellate community, and not freshwater species. Polarella glacialis Montresor, Procaccini et Stoecker, a bipolar marine species, was for the first time described in a lake habitat and was an important phototrophic component in the higher salinity lakes. In the brackish lakes, we found a new sibling species to the brackish‐water species Scrippsiella hangoei (J. Schiller) J. Larsen, previously observed only in the Baltic Sea.     

Functional dissection of transmembrane domains of human TAP-like (ABCB9)

An ABC transporter, TAP-Like (TAPL), was dissected into its amino-terminal transmembrane domain and the following core domain. When these domains were transiently expressed as tagged proteins with a His6- or Myc-epitope tag, the amino-terminal ones (Met¹-Lys¹⁸²) could not associate with each other, or with the full-length transporter (Met¹-Ala⁷⁶⁶). However, both the core domain (Arg¹⁴¹-Ala⁷⁶⁶) and full-length protein mutually interacted. The amino-terminal domain (Met¹-Arg¹⁴¹) as well as the full-length transporter fused with fluorescent protein GFP was sorted to lysosomal membranes upon their stable expression, as visualized by means of fluorescent microscopy, while the core domain (Arg¹⁴¹-Ala⁷⁶⁶) was broadly distributed in the intra-cellular membranes. These results suggest that the sorting signal for lysosomes is present within the amino-terminal transmembrane domain (Met¹-Arg¹⁴¹) of the TAPL molecule.     

Association of Gap-43 (neuromodulin) with microtubule-associated protein MAP-2 in neuronal cells

Gap-43 (B-50, neuromodulin) is a presynaptic protein implicated in axonal growth, neuronal differentiation, plasticity, and regeneration. Its activities are regulated by its dynamic interactions with various neuronal proteins, including actin and brain spectrin. Recently we have shown that Gap-43 co-localizes with an axonal protein DPYSL-3 in primary cortical neurons. In the present study we provide evidence that Gap-43 co-localizes and potentially interacts with microtubule-associated protein MAP-2 in adult and fetal rat brain, as well as in primary neuronal cultures. Our studies suggest that this interaction may be developmentally regulated.     

Development of an all-in-one technology for glycan profiling targeting formalin-embedded tissue sections

An ultra-sensitive method for glycan analysis targeting small tissue sections (1.5 mm in diameter) is described as an application of a recently-established lectin microarray technology. The developed system achieved a high level of detection of a tissue section consisting of approximately 500 cells for differential profiling, where both N- and O-glycans attached to a pool of glycoproteins are subjected to multiplex analysis with 43 lectins. By using an optimized protocol for differential glycan analysis, sections of adenocarcinoma (n = 28) and normal epithelia (n = 12) of the colon were analyzed in an all-in-one manner. As a result, Wisteria floribunda agglutinin (WFA) was found to clearly differentiate cancerous from normal epithelia with P < 0.0001. The obtained results correlated well with the subsequent histochemical study using biotinylated WFA. Thus, the developed technology proved to be valid for expanding the lectin microarray applications to tissue-based glycomics, and hence, should accelerate a discovery phase of glycan-related biomarkers.     

A C-type lectin of Caenorhabditis elegans: its sugar-binding property revealed by glycoconjugate microarray analysis

C-type lectins are a family of proteins with an affinity to carbohydrates in the presence of Ca²⁺. In the genome of Caenorhabditis elegans, almost 300 genes encoding proteins containing C-type lectin-like domains (CTLDs) have been assigned. However, none of their products has ever been shown to have carbohydrate-binding activity. In the present study, we selected 6 potential C-type lectin genes and prepared corresponding recombinant proteins. One of them encoded by clec-79 was found to have sugar-binding activity by using a newly developed glycoconjugate microarray based on evanescent-field excited fluorescence. CLEC-79 exhibited affinity to sugars containing galactose at the non-reducing terminal, especially to the Galβ1-3GalNAc structure, in the presence of Ca²⁺. Combined with structural information of the glycans of C. elegans, these results suggest that CLEC-79 preferentially binds to O-glycans in vivo.     

Actin cytoskeleton-dependent dynamics of the human serotonin1A receptor correlates with receptor signaling

Analyzing the dynamics of membrane proteins in the context of cellular signaling represents a challenging problem in contemporary cell biology. Lateral diffusion of lipids and proteins in the cell membrane is known to be influenced by the cytoskeleton. In this work, we explored the role of the actin cytoskeleton on the mobility of the serotonin_1A (5-HT_1A) receptor, stably expressed in CHO cells, and its implications in signaling. FRAP analysis of 5-HT_1AR-EYFP shows that destabilization of the actin cytoskeleton induced by either CD or elevation of cAMP levels mediated by forskolin results in an increase in the mobile fraction of the receptor. The increase in the mobile fraction is accompanied by a corresponding increase in the signaling efficiency of the receptor. Interestingly, with increasing concentrations of CD used, the increase in the mobile fraction exhibited a correlation of ∼0.95 with the efficiency in ligand-mediated signaling of the receptor. Radioligand binding and G-protein coupling of the receptor were found to be unaffected upon treatment with CD. Our results suggest that signaling by the serotonin_1A receptor is correlated with receptor mobility, implying thereby that the actin cytoskeleton could play a regulatory role in receptor signaling. These results may have potential significance in the context of signaling by GPCRs in general and in the understanding of GPCR-cytoskeleton interactions with respect to receptor signaling in particular.     

A peptide containing residues 26-44 of tau protein impairs mitochondrial oxidative phosphorylation acting at the level of the adenine nucleotide translocator

Having confirmed that adenovirus-mediated overexpression of NH₂-tau fragment lacking the first 25 aminoacids evokes a potent neurotoxic effect, sustained by protracted stimulation of NMDA receptors, in primary neuronal cultures we investigated whether and how chemically synthesized NH₂-derived tau peptides, i.e. NH₂-26-44 and NH₂-1-25 fragments, affect mitochondrial function. We tested both fragments on each step of the processes leading to ATP synthesis via oxidative phosphorylation: i) electron flow via the respiratory chain from physiological substrates to oxygen with the activity of each individual complex of the respiratory chain investigated in some detail, ii) membrane potential generation arising from externally added succinate and iii) the activity of both the adenine nucleotide translocator and iv) ATP synthase. Oxidative phosphorylation is not affected by NH₂-1-25 tau fragment, but dramatically impaired by NH₂-26-44 tau fragment. Both cytochrome c oxidase and the adenine nucleotide translocator are targets of NH₂-26-44 tau fragment, but adenine nucleotide translocator is the unique mitochondrial target responsible for impairment of oxidative phosphorylation by the NH₂-26-44 tau fragment, which then exerts deleterious effects on cellular availability of ATP synthesized into mitochondria.     

Characterization of the anti-angiogenic properties of arresten, an alpha1beta1 integrin-dependent collagen-derived tumor suppressor

Physiological and pathological turnover of basement membranes liberates biologically active cryptic molecules. Several collagen-derived fragments possess anti-angiogenic activity. Arresten is the 26-kDa non-collagenous domain of type IV collagen α1 chain. It functions as an efficient inhibitor of angiogenesis and tumor growth in mouse models, but its anti-angiogenic mechanism is not completely known. Here we show that arresten significantly increases apoptosis of endothelial cells in vitro by decreasing the amount of anti-apoptotic molecules of the Bcl-family; Bcl-2 and Bcl-xL. Although the pro-apoptotic effect of arresten is endothelial cell specific in vitro, in mouse tumors arresten induced apoptosis both in endothelial and tumor cells. The tumor cell apoptosis is likely an indirect effect due to the inhibition of blood vessel growth into the tumor. The active site of arresten was localized by deletion mutagenesis within the C-terminal half of the molecule. We have previously shown that arresten binds to α1β1 integrin on human umbilical vein endothelial cells. However, the microvascular endothelial cells (MLECs) are more important in the context of tumor vasculature. We show here that arresten binds also to the microvascular endothelial cells via α1β1 integrin. Furthermore, it has no effect on Matrigel neovascularization or the viability of integrin α1 null MLECs. Tumors implanted on integrin α1 deficient mice show no integrin α1 expression in the host-derived vascular endothelium, and thus arresten does not inhibit the tumor growth. Collectively, this data sheds more light into the anti-angiogenic mechanism of arresten.     

Transferrin uptake in Trypanosoma cruzi is impaired by interference on cytostome-associated cytoskeleton elements and stability of membrane cholesterol, but not by obstruction of clathrin-dependent endocytosis

Transferrin uptake by Trypanosoma cruzi epimastigotes occurs mainly through the cytostome/cytopharynx. Here, we present evidences for the association of sterol-rich membrane domains with the transferrin endocytic site. Assays using pharmacological treatments to disrupt clathrin-coated pits and hinder caveolae formation showed no association between transferrin uptake and clathrin-dependent endocytosis, but indicated that cholesterol stability in membrane domains is essential for the endocytosis of transferrin. Furthermore, it was observed a connection between the integrity of cytoskeleton elements at the cytopharynx and the function of the cytostome. Our data show that T. cruzi epimastigotes depend on a specialized pathway for transferrin uptake, which is cholesterol-dependent, clathrin-independent, and closely associated with the structural stability of the cytostome/cytopharynx cytoskeleton.     

Fluxes of dissolved organic carbon,other nutrients and microbial communities in a water-filled treehole ecosystem

Water-filled treeholes are temporally and spatially variable habitats that consist of communities of a limited number of insect orders, namely dipterans and beetles. Since these systems are largely heterotrophic, treeholes are dependent on the surrounding terrestrial ecosystem for their basal energy input. In this study, we observed a cyclic succession of three system states in a water-filled Fagus grandifolia treehole: ‘connected’ during rainfall; ‘isolated’ during periods without rain; and ‘dry’ when no freestanding water was present. During the isolated phase, a rapid, microbially mediated turn-over of nitrogen and sulphate took place, coincident with an accumulation of orthophosphate. Ammonium was the dominant form of nitrogen in the treehole water, and a net decrease in its concentration was observed when the water volume decreased. Normally, nitrate concentration showed only minor fluctuations (0.3–1.3 mg l⁻¹) and concentrations of nitrite were very low (3–18 μg l⁻¹). Concentration of sulphate showed a net decrease, coincident with an increase in sulphide. During the connected phase, the effect of stemflow and throughfall on the nutrient concentrations in the treehole water proved to be variable. Over time, both dilution of, and increase in, treehole water nutrient concentrations were observed. Dissolved organic carbon (DOC) concentration showed a net decrease during the isolated phase. Spring coarse particulate organic matter input into the treehole was variable (0.20–1.74 mg cm⁻² week⁻¹). The observed variability in precipitation inputs as well as fall-in of organic matter underlines the pulsed character of basal energy entering the treehole food-web. DOC concentrations varied mainly with depth (15–57.4 mg l⁻¹) as concentrations were almost three times greater within the detritus than within the water column. Bacterial abundances ranged from 7.3 × 10⁵ to 9.3 × 10⁶ cells ml⁻¹ and did not appear to vary within the water column or in the detrital sediment. Rates of bacterial production increased 24 h after rain events, suggesting that the combined nutrient fluxes due to increased stemflow stimulate the microbial community. Handling editor: J. Cole