Lipid droplet de novo formation and fission are linked to the cell cycle in fission yeast.

Cells sequester neutral lipids in bodies called lipid droplets. Thus, the formation and breakdown of the droplets are important for cellular metabolism; unfortunately, these processes are difficult to quantify. Here, we used time-lapse confocal microscopy to track the formation, movement and size changes of lipid droplets throughout the cell cycle in fission yeast Schizosaccharomyces pombe. In theory, the number of lipid droplets in these cells must increase for daughter cells to have the same number of droplets as the parent at a reference point in the cell cycle. We observed stable droplet formation events in G2 phase that were divided evenly between de novo formation of nascent droplets and fission of preexisting droplets. The observations that lipid droplet number is linked to the cell cycle and that droplets can form via fission were both new discoveries. Thus, we scrutinized each fission event for multiple signatures to eliminate possible artifacts from our microscopy. We augmented our time-lapse confocal microscopy with electron microscopy, which showed lipid droplet 'intermediates': droplets shaped like dumbbells that are potentially in transition states between two spherical droplets. Using these complementary microscopy techniques and also dynamic simulations, we show that lipid droplets can form by fission.

Masking of ß(1-3)-glucan in the cell wall of Candida albicans from detection by innate immune cells depends on phosphatidylserine.

The virulence of Candida albicans in a mouse model of invasive candidiasis is dependent on the phospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE). Disruption of the PS synthase gene CHO1 (i.e., cho1Δ/Δ) eliminates PS and blocks the de novo pathway for PE biosynthesis. In addition, the cho1Δ/Δ mutant's ability to cause invasive disease is severely compromised. The cho1Δ/Δ mutant also exhibits cell wall defects, and in this study, it was determined that loss of PS results in decreased masking of cell wall ß(1-3)-glucan from the immune system. In wild-type C. albicans, the outer mannan layer of the wall masks the inner layer of ß(1-3)-glucan from exposure and detection by innate immune effector molecules like the C-type signaling lectin Dectin-1, which is found on macrophages, neutrophils, and dendritic cells. The cho1Δ/Δ mutant exhibits increases in exposure of ß(1-3)-glucan, which leads to greater binding by Dectin-1 in both yeast and hyphal forms. The unmasking of ß(1-3)-glucan also results in increased elicitation of TNF-α from macrophages in a Dectin-1-dependent manner. The role of phospholipids in fungal pathogenesis is an emerging field, and this is the first study showing that loss of PS in C. albicans results in decreased masking of ß(1-3)-glucan, which may contribute to our understanding of fungus-host interactions.

Inhibition of copper uptake in yeast reveals the copper transporter Ctr1p as a potential molecular target of saxitoxin.

Saxitoxin is a secondary metabolite produced by several species of dinoflagellates and cyanobacteria which targets voltage-gated sodium and potassium channels in higher vertebrates. However, its molecular target in planktonic aquatic community members that co-occur with the toxin producers remains unknown. Previous microarray analysis with yeast identified copper and iron-homeostasis genes as being differentially regulated in response to saxitoxin. This study sought to identify the molecular target in microbial cells by comparing the transcriptional profiles of key copper and iron homeostasis genes (CTR1, FRE1, FET3, CUP1, CRS5) in cells exposed to saxitoxin, excess copper, excess iron, an extracellular Cu(I) chelator, or an intracellular Cu(I) chelator. Protein expression and localization of Ctr1p (copper transporter), Fet3p (multicopper oxidase involved in high-affinity iron uptake), and Aft1p (iron regulator) were also compared among treatments. Combined transcript and protein profiles suggested saxitoxin inhibited copper uptake. This hypothesis was confirmed by intracellular Cu(I) imaging with a selective fluorescent probe for labile copper. On the basis of the combined molecular and physiological results, a model is presented in which the copper transporter Ctr1p serves as a molecular target of saxitoxin and these observations are couched in the context of the eco-evolutionary role this toxin may serve for species that produce it.

Paralytic shellfish toxins inhibit copper uptake in Chlamydomonas reinhardtii.

Paralytic shellfish toxins are secondary metabolites produced by several species of dinoflagellates and cyanobacteria. Known targets of these toxins, which typically occur at detrimental concentrations during harmful algal blooms, include voltage-gated ion channels in humans and other mammals. However, the effects of the toxins on the co-occurring phytoplankton community remain unknown. The present study examined the molecular mechanisms of the model photosynthetic alga Chlamydomonas reinhardtii in response to saxitoxin exposure as a means of gaining insight into the phytoplankton community response to a bloom. Previous work with yeast indicated that saxitoxin inhibited copper uptake, so experiments were designed to examine whether saxitoxin exhibited a similar mode of action in algae. Expression profiling following exposure to saxitoxin or a copper chelator produced similar profiles in copper homeostasis genes, notably induction of the cytochrome c6 (CYC6) and copper transporter (COPT1, CTR1) genes. Cytochrome c6 is used as an alternative to plastocyanin under conditions of copper deficiency, and immunofluorescence data showed this protein to be present in a significantly greater proportion of saxitoxin-exposed cells compared to controls. Live-cell imaging with a copper-sensor probe for intracellular labile Cu(I) confirmed that saxitoxin blocked copper uptake. Extrapolations of these data to phytoplankton metabolic processes along with the copper transporter as a molecular target of saxitoxin based on existing structural models are discussed.

Regulation of iNOS gene transcription by IL-1ß and IFN-γ requires a coactivator exchange mechanism.

The proinflammatory cytokines IL-1ß and IFN-γ decrease functional islet ß-cell mass in part through the increased expression of specific genes, such as inducible nitric oxide synthase (iNOS). Dysregulated iNOS protein accumulation leads to overproduction of nitric oxide, which induces DNA damage, impairs ß-cell function, and ultimately diminishes cellular viability. However, the transcriptional mechanisms underlying cytokine-mediated expression of the iNOS gene are not completely understood. Herein, we demonstrated that individual mutations within the proximal and distal nuclear factor-κB sites impaired cytokine-mediated transcriptional activation. Surprisingly, mutating IFN-γ-activated site (GAS) elements in the iNOS gene promoter, which are classically responsive to IFN-γ, modulated transcriptional sensitivity to IL-1ß. Transcriptional sensitivity to IL-1ß was increased by generation of a consensus GAS element and decreased correspondingly with 1 or 2 nucleotide divergences from the consensus sequence. The nuclear factor-κB subunits p65 and p50 bound to the κB response elements in an IL-1ß-dependent manner. IL-1ß also promoted binding of serine-phosphorylated signal transducer and activator of transcription-1 (STAT1) (Ser727) but not tyrosine-phosphorylated STAT1 (Tyr701) to GAS elements. However, phosphorylation at Tyr701 was required for IFN-γ to potentiate the IL-1ß response. Furthermore, coactivator p300 and coactivator arginine methyltransferase were recruited to the iNOS gene promoter with concomitant displacement of the coactivator CREB-binding protein in cells exposed to IL-1ß. Moreover, these coordinated changes in factor recruitment were associated with alterations in acetylation, methylation, and phosphorylation of histone proteins. We conclude that p65 and STAT1 cooperate to control iNOS gene transcription in response to proinflammatory cytokines by a coactivator exchange mechanism. This increase in transcription is also associated with signal-specific chromatin remodeling that leads to RNA polymerase II recruitment and phosphorylation.

Regulation of the CCL2 gene in pancreatic ß-cells by IL-1ß and glucocorticoids: role of MKP-1.

Release of pro-inflammatory cytokines from both resident and invading leukocytes within the pancreatic islets impacts the development of Type 1 diabetes mellitus. Synthesis and secretion of the chemokine CCL2 from pancreatic ß-cells in response to pro-inflammatory signaling pathways influences immune cell recruitment into the pancreatic islets. Therefore, we investigated the positive and negative regulatory components controlling expression of the CCL2 gene using isolated rat islets and INS-1-derived ß-cell lines. We discovered that activation of the CCL2 gene by IL-1ß required the p65 subunit of NF-κB and was dependent on genomic response elements located in the -3.6 kb region of the proximal gene promoter. CCL2 gene transcription in response to IL-1ß was blocked by pharmacological inhibition of the IKKß and p38 MAPK pathways. The IL-1ß-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids. Moreover, multiple synthetic glucocorticoids inhibited the IL-1ß-stimulated induction of the CCL2 gene. Induction of the MAP Kinase Phosphatase-1 (MKP-1) gene by glucocorticoids or by adenoviral-mediated overexpression decreased p38 MAPK phosphorylation, which diminished CCL2 gene expression, promoter activity, and release of CCL2 protein. We conclude that glucocorticoid-mediated repression of IL-1ß-induced CCL2 gene transcription and protein secretion occurs in part through the upregulation of the MKP-1 gene and subsequent deactivation of the p38 MAPK. Furthermore, the anti-inflammatory actions observed with MKP-1 overexpression were obtained without suppressing glucose-stimulated insulin secretion. Thus, MKP-1 is a possible target for anti-inflammatory therapeutic intervention with preservation of ß-cell function.