Architectural remodeling of the tonoplast during fluid-phase endocytosis

During fluid phase endocytosis (FPE) in plant storage cells, the vacuole receives a considerable amount of membrane and fluid contents. If allowed to accumulate over a period of time, the enlarging tonoplast and increase in fluids would invariably disrupt the structural equilibrium of the mature cells. Therefore, a membrane retrieval process must exist that will guarantee membrane homeostasis in light of tonoplast expansion by membrane addition during FPE. We examined the morphological changes to the vacuolar structure during endocytosis in red beet hypocotyl tissue using scanning laser confocal microscopy and immunohistochemistry. The heavily pigmented storage vacuole allowed us to visualize all architectural transformations during treatment. When red beet tissue was incubated in 200 mM sucrose, a portion of the sucrose accumulated entered the cell by means of FPE. The accumulation process was accompanied by the development of vacuole-derived vesicles which transiently counterbalanced the addition of surplus endocytic membrane during rapid rates of endocytosis. Topographic fluorescent confocal micrographs showed an ensuing reduction in the size of the vacuole-derived vesicles and further suggest their reincorporation into the vacuole to maintain vacuolar unity and solute concentration.     

Existence of two parallel mechanisms for glucose uptake in heterotrophic plant cells

The implied existence of two mechanisms for glucose uptake into heterotrophic plant cells was investigated using the fluorescent glucose derivative 2-NBDG (2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose), two membrane impermeable fluorescent markers (3000 mol. wt. dextran-Texas Red (d-TR) and Alexa-488), hexose carrier and endocytic inhibitors (phloridzin and wortmannin-A, respectively), and fluorescent and confocal microscopy. Both phloridzin and wortmannin-A significantly reduced the uptake of 2-NBDG into sycamore cultured cells, which was confirmed by fluorescent microscopy. Phloridzin prevented 2-NBDG uptake exclusively into the cytosol, whereas the wortmannin-A effect was more general, with 2-NBDG uptake into the vacuole being the more affected. Simultaneous incubation of cells in the membrane-impermeable fluorescent probes Alexa-488 and d-TR for 24 h resulted in co-localization of the labelling in the central vacuole and other endosomal compartments. Cytoplasts, cells devoid of vacuoles, were instrumental in demonstrating the transport of 2-NBDG by separate uptake mechanisms. In cytoplasts incubated simultaneously in 2-NBDG and d-TR for 2 h, a green fluorescent cytosol was indicative of transport of hexoses across the plasmalemma, while the co-localization of 2-NBDG and d-TR in internal vesicles demonstrated transport via an endocytic system. The absence of vesicles when cytoplasts were pre-incubated in wortmannin-A authenticated the endocytic vesicular nature of the co-shared 2-NBDG and d-TR fluorescent structures. In summary, uptake of 2-NBDG occurs by two separate mechanisms: (i) a plasmalemma-bound carrier-mediated system that facilitates 2-NBDG transport into the cytosol, and (ii) an endocytic system that transports most of 2-NBDG directly into the vacuole.     

GLUT12 expression and regulation in murine small intestine and human Caco-2 cells

GLUT12 was cloned from the mammary cancer cell line MCF-7, but its physiological role still needs to be elucidated. To gain more knowledge of GLUT12 function in the intestine, we investigated GLUT12 subcellular localization in the small intestine and its regulation by sugars, hormones, and intracellular mediators in Caco-2 cells and mice. Immunohistochemical methods were used to determine GLUT12 subcellular localization in human and murine small intestine. Brush border membrane vesicles were isolated for western blot analyses. Functional studies were performed in Caco-2 cells by measuring α-methyl-d -glucose (αMG) uptake in the absence of sodium. GLUT12 is located in the apical cytoplasm, below the brush border membrane, and in the perinuclear region of murine and human enterocytes. In Caco-2 cells, GLUT12 translocation to the apical membrane and α-methyl- d -glucose uptake by the transporter are stimulated by protons, glucose, insulin, tumor necrosis factor-α (TNF-α), protein kinase C, and AMP-activated protein kinase. In contrast, hypoxia decreases GLUT12 expression in the apical membrane. Upregulation of TNF-α and hypoxia-inducible factor-1α ( HIF-1α) genes is found in the jejunal mucosa of diet-induced obese mice. In these animals, GLUT12 expression in the brush border membrane is slightly decreased compared with lean animals. Moreover, an intraperitoneal injection of insulin does not induce GLUT12 translocation to the membrane, as it occurs in lean animals. GLUT12 rapid translocation to the enterocytes’ apical membrane in response to glucose and insulin could be related to GLUT12 participation in sugar absorption during postprandial periods. In obesity, in which insulin sensitivity is reduced, the contribution of GLUT12 to sugar absorption is affected.     

Sucrose synthase controls both intracellular ADP glucose levels and transitory starch biosynthesis in source leaves

The prevailing model on transitory starch biosynthesis in source leaves assumes that the plastidial ADPglucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule, ADPG. However, recent investigations have shown that ADPG linked to starch biosynthesis accumulates outside the chloroplast, presumably in the cytosol. This finding is consistent with the occurrence of an 'alternative' gluconeogenic pathway wherein sucrose synthase (SuSy) is involved in the production of ADPG in the cytosol, whereas both plastidial phosphoglucomutase (pPGM) and AGP play a prime role in the scavenging of starch breakdown products. To test this hypothesis, we have compared the ADPG content in both Arabidopsis and potato wild-type (WT) leaves with those of the starch-deficient mutants with reduced pPGM and AGP. These analyses provided evidence against the 'classical' model of starch biosynthesis, since ADPG levels in all the starch-deficient lines were normal compared with WT plants. Whether or not SuSy is involved in the synthesis of ADPG accumulating in leaves was tested by characterizing both SuSy-overexpressing and SuSy-antisensed transgenic leaves. Importantly, SuSy-overexpressing leaves exhibited a large increase of both ADPG and starch levels compared with WT leaves, whereas SuSy-antisensed leaves accumulated low amounts of both ADPG and starch. These findings show that (i) ADPG produced by SuSy is linked to starch biosynthesis; (ii) SuSy exerts a strong control on the starch biosynthetic process; and (iii) SuSy, but not AGP, controls the production of ADPG accumulating in source leaves.     

Basal leptin regulates amino acid uptake in polarized Caco-2 cells

Leptin is secreted by gastric mucosa and is able to reach the intestinal lumen where its receptors are located in the apical membrane of the enterocytes. We have previously demonstrated that apical leptin inhibits sugar and amino acids uptake in vitro and glucose absorption in vivo. Since leptin receptors are also expressed in the basolateral membrane of the enterocytes, the aim of the present work was to investigate whether leptin acting from the basolateral side could also regulate amino acid uptake. Tritiated Gln and β-Ala were used to measure uptake into Caco-2 cells grown on filters, in the presence of basal leptin at short incubation times (5 and 30 min) and after 6 h of preincubation with the hormone. In order to compare apical and basal leptin effect, Gln and β-Ala uptake was measured in the presence of leptin acting from the apical membrane also in cells grown on filters. Basal leptin (8 mM) inhibited by ~15–30 % the uptake of 0.1 mM Gln and 1 mM β-Ala quickly, after 5 min exposure, and the effect was maintained after long preincubation periods. Apical leptin had the same effect. Moreover, the inhibition was rapidly and completely reversed when leptin was removed from the apical or basolateral medium. These results extend our previous findings and contribute to the vision of leptin as an important hormonal signal for the regulation of intestinal absorption of nutrients.     

Activity of membrane-associated sucrose synthase is regulated by its phosphorylation status in cultured cells of sycamore (Acer pseudoplatanus)

Changes in sucrose synthase (SuSy) activity, protein level and degree of phosphorylation were investigated in plasmalemma and tonoplast of sycamore cells cultured either in the presence of sucrose or after 24 h of starvation. SuSy activity was shown to be higher in the plasmalemma than in the tonoplast of cells cultured in the presence of sucrose. In clear contrast, SuSy was shown to be more active in the tonoplast than in the plasmalemma of starved cells. Western blot analyses on both membrane types did not show noticeable differences in SuSy protein levels under the two different regimes. However, phosphorylation state at the serine moieties of the enzyme was shown to be different in the presence or in the absence of sucrose. Plasmalemma-associated SuSy is not phosphorylated in the presence of sucrose, whereas tonoplast-associated SuSy is phosphorylated under similar conditions. Starvation brought about a reverse in phosphorylation state of membrane-bound SuSy. Whereas plasmalemma-associated SuSy became phosphorylated, tonoplast-associated SuSy was completely de-phosphorylated. Together, the data demonstrate that SuSy is simultaneously present in various cell membranes and also demonstrate a lack of direct relationship between membrane type location, and degree of phosphorylation, but substantiate the relevance of phosphorylation to enzymatic activity.     

Autocrine prostaglandin E2 signaling promotes tumor cell survival and proliferation in childhood neuroblastoma

Background

Prostaglandin E₂ (PGE₂) is an important mediator in tumor-promoting inflammation. High expression of cyclooxygenase-2 (COX-2) has been detected in the embryonic childhood tumor neuroblastoma, and treatment with COX inhibitors significantly reduces tumor growth. Here, we have investigated the significance of a high COX-2 expression in neuroblastoma by analysis of PGE₂ production, the expression pattern and localization of PGE₂ receptors and intracellular signal transduction pathways activated by PGE₂.

Principal Findings

A high expression of the PGE₂ receptors, EP1, EP2, EP3 and EP4 in primary neuroblastomas, independent of biological and clinical characteristics, was detected using immunohistochemistry. In addition, mRNA and protein corresponding to each of the receptors were detected in neuroblastoma cell lines. Immunofluorescent staining revealed localization of the receptors to the cellular membrane, in the cytoplasm, and in the nuclear compartment. Neuroblastoma cells produced PGE₂ and stimulation of serum-starved neuroblastoma cells with PGE₂ increased the intracellular concentration of calcium and cyclic AMP with subsequent phosphorylation of Akt. Addition of 16,16-dimethyl PGE₂ (dmPGE₂) increased cell viability in a time, dose- and cell line-dependent manner. Treatment of neuroblastoma cells with a COX-2 inhibitor resulted in a diminished cell growth and viability that was reversed by the addition of dmPGE₂. Similarly, PGE₂ receptor antagonists caused a decrease in neuroblastoma cell viability in a dose-dependent manner.

Conclusions

These findings demonstrate that PGE₂ acts as an autocrine and/or paracrine survival factor for neuroblastoma cells. Hence, specific targeting of PGE₂ signaling provides a novel strategy for the treatment of childhood neuroblastoma through the inhibition of important mediators of tumor-promoting inflammation.