The Ceramide Synthase Subunit Lac1 Regulates Cell Growth and Size in Fission Yeast

Cell division produces two viable cells of a defined size. Thus, all cells require mechanisms to measure growth and trigger cell division when sufficient growth has occurred. Previous data suggest a model in which growth rate and cell size are mechanistically linked by ceramide-dependent signals in budding yeast. However, the conservation of mechanisms that govern growth control is poorly understood. In fission yeast, ceramide synthase is encoded by two genes, Lac1 and Lag1. Here, we characterize them by using a combination of genetics, microscopy, and lipid analysis. We showed that Lac1 and Lag1 co-immunoprecipitate and co-localize at the endoplasmic reticulum. However, each protein generates different species of ceramides and complex sphingolipids. We further discovered that Lac1, but not Lag1, is specifically required for proper control of cell growth and size in Schizosaccharomyces pombe. We propose that specific ceramide and sphingolipid species produced by Lac1 are required for normal control of cell growth and size in fission yeast.

Complex sphingolipids: Vital determinants of drug susceptibility, membrane integrity and pathogenesis of Candida glabrata

Complex Sphingolipids (SLs) are unique to fungi, which apart from being novel drug targets, also appear to act as molecular signals, in diverse biological processes. In this study, we have specifically blocked the key synthesis step of SLs metabolism by disruption of the uncharacterized CgIPT1 gene, which based on homology with other Candida spp., predicted to mediate the conversion of MIPC to M(IP)2C. We followed fusion based PCR homologous recombination method for IPT1 deletion by using dominant markerNAT1. The knockout was selected on a nourseothricin drug plate and confirmed by gene specific PCR and by checking M(IP)2C levels. We observed that the specific accumulation of MIPC or lack of M(IP)2C in C. glabrata displayed increased susceptibility to both imidazole’s (ketoconazole, miconazole and clotrimazole) and triazoles (fluconazole, itraconazole and posaconazole). RNA Sequencing of Cgipt1Δcells revealed no major impact on of expression levels of common MDR determinants albeit a distinct imbalances in expression of lipid homeostasis genes was evident. The Fluorescence Recovery after Photobleaching (FRAP) experiments confirmed that plasma membrane in Cgipt1Δ cells display a reduction in micro-viscosity leading to increase in drug diffusion and susceptibility of Cgipt1Δcells. Interestingly, the Cgipt1Δ also exhibit attenuated virulence in a murine model. Together, our data confirms the relevance of M(IP)2C in governing drug susceptibility and virulence in C. glabrata.

Microdomain protein Nce102 is a local sensor of plasma membrane sphingolipid balance

Sphingolipids are essential building blocks of eukaryotic membranes and important signalling molecules, tightly regulated in response to environmental and physiological inputs. Mechanism of sphingolipid level perception at the plasma membrane remains unclear. In Saccharomyces cerevisiae, Nce102 protein has been proposed to function as sphingolipid sensor as it changes its plasma membrane distribution in response to sphingolipid biosynthesis inhibition. We show that Nce102 redistributes specifically in regions of increased sphingolipid demand, e.g., membranes of nascent buds. Furthermore, we report that production of Nce102 increases following sphingolipid biosynthesis inhibition and Nce102 is internalized when excess sphingolipid precursors are supplied. This suggests that the total amount of Nce102 in the plasma membrane is a measure of the current need for sphingolipids, whereas its local distribution marks sites of high sphingolipid demand. Physiological role of Nce102 in regulation of sphingolipid synthesis is demonstrated by mass spectrometry analysis showing reduced levels of complex sphingolipids and long-chain bases in nce102? deletion mutant. Nce102 behaves analogously in human fungal pathogen Candida albicans, suggesting a conserved principle of local sphingolipid control across species.

Sphingolipids of Asteroidea and Holothuroidea: Structures and Biological Activities

Sphingolipids are complex lipids widespread in nature as structural components of biomembranes. Commonly, the sphingolipids of marine organisms differ from those of terrestrial animals and plants. The gangliosides are the most complex sphingolipids characteristic of vertebrates that have been found in only the Echinodermata (echinoderms) phylum of invertebrates. Sphingolipids of the representatives of the Asteroidea and Holothuroidea classes are the most studied among all echinoderms. In this review, we have summarized the data on sphingolipids of these two classes of marine invertebrates over the past two decades. Recently established structures, properties, and peculiarities of biogenesis of ceramides, cerebrosides, and gangliosides from starfishes and holothurians are discussed. The purpose of this review is to provide the most complete information on the chemical structures, structural features, and biological activities of sphingolipids of the Asteroidea and Holothuroidea classes.

Structural basis for acyl chain control over glycosphingolipid sorting and vesicular trafficking

The complex sphingolipids exhibit a diversity of ceramide acyl chain structures that influence their trafficking and intracellular distributions, but how the cell discerns among the different ceramides to affect such sorting remains unknown. To address mechanism, we synthesized a library of GM1 glycosphingolipids with naturally varied acyl chains and quantitatively assessed their sorting among different endocytic pathways. We found that a stretch of at least 14 saturated carbons extending from C1 at the water-bilayer interface dictated lysosomal sorting by exclusion from endosome sorting tubules. Sorting to the lysosome by the C14*-motif was cholesterol dependent. Perturbations of the C14*-motif by unsaturation enabled GM1 entry into endosomal sorting tubules of the recycling and retrograde pathways independently of cholesterol. Unsaturation occurring beyond the C14*-motif in very long acyl chains rescued lysosomal sorting. These results define a structural motif underlying membrane organization of sphingolipids and implicate cholesterol-sphingolipid nanodomain formation in sorting mechanisms.

Fumonisin B1-Induced Changes in Cotton Fiber Elongation Revealed by Sphingolipidomics and Proteomics

Sphingolipids are essential biomolecules and membrane components, but their regulatory role in cotton fiber development is poorly understood. Here, we found that fumonisin B1 (FB1)—a sphingolipid synthesis inhibitor—could block fiber elongation severely. Using liquid chromatography tandem mass spectrometry (LC-MS/MS), we detected 95 sphingolipids that were altered by FB1 treatment; of these, 29 (mainly simple sphingolipids) were significantly increased, while 33 (mostly complex sphingolipids) were significantly decreased. A quantitative analysis of the global proteome, using an integrated quantitative approach with tandem mass tag (TMT) labeling and LC-MS/MS, indicated the upregulation of 633 and the downregulation of 672 proteins after FB1 treatment. Most differentially expressed proteins (DEPs) were involved in processes related to phenylpropanoid and flavonoid biosynthesis. In addition, up to 20 peroxidases (POD) were found to be upregulated, and POD activity was also increased by the inhibitor. To our knowledge, this is the first report on the effects of FB1 treatment on cotton fiber and ovule sphingolipidomics and proteomics. Our findings provide target metabolites and biological pathways for cotton fiber improvement.

Enhancement of Sphingolipid Synthesis Improves Osmotic Tolerance of Saccharomyces cerevisiae

ABSTRACT To enhance the growth performance of Saccharomyces cerevisiae under osmotic stress, mutant XCG001, which tolerates up to 1.5 M NaCl, was isolated through adaptive laboratory evolution (ALE). Comparisons of the transcriptome data of mutant XCG001 and the wild-type strain identified ELO2 as being associated with osmotic tolerance. In the ELO2 overexpression strain (XCG010), the contents of inositol phosphorylceramide (IPC; t18:0/26:0), mannosylinositol phosphorylceramide [MIPC; t18:0/22:0(2OH)], MIPC (d18:0/22:0), MIPC (d20:0/24:0), mannosyldiinositol phosphorylceramide [M(IP)2C; d20:0/26:0], M(IP)2C [t18:0/26:0(2OH)], and M(IP)2C [d20:0/26:0(2OH)] increased by 88.3 times, 167 times, 63.3 times, 23.9 times, 27.9 times, 114 times, and 208 times at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002. As a result, the membrane integrity, cell growth, and cell survival rate of strain XCG010 increased by 24.4% ± 1.0%, 21.9% ± 1.5%, and 22.1% ± 1.1% at 1.0 M NaCl, respectively, compared with the corresponding values of the control strain XCG002 (wild-type strain with a control plasmid). These findings provided a novel strategy for engineering complex sphingolipids to enhance osmotic tolerance. IMPORTANCE This study demonstrated a novel strategy for the manipulation of membrane complex sphingolipids to enhance S. cerevisiae tolerance to osmotic stress. Elo2, a sphingolipid acyl chain elongase, was related to osmotic tolerance through transcriptome analysis of the wild-type strain and an osmosis-tolerant strain generated from ALE. Overexpression of ELO2 increased the content of complex sphingolipid with longer acyl chain; thus, membrane integrity and osmotic tolerance improved.

Engineering of membrane complex sphingolipids improves osmotic tolerance of Saccharomyces cerevisiae

ABSTRACTIn order to enhance the growth performance of S. cerevisiae under harsh environmental conditions, mutant XCG001, which tolerates up to 1.5M NaCl, was isolated via adaptive laboratory evolution (ALE). Comparisons made via transcriptome data of XCG001 and the wild-type strain identified ELO2 as being associated with osmotic tolerance. Overexpression of ELO2 increased the contents of inositol phosphorylceramide (IPC, t18:0/26:0), mannosylinositol phosphorylceramide (MIPC, t18:0/22:0(2OH)), MIPC (d18:0/22:0), MIPC (d20:0/24:0), mannosyldiinositol phosphorylceramide (M(IP)2C, d20:0/26:0), M(IP)2C (t18:0/26:0(2OH)) and M(IP)2C (d20:0/26:0(2OH)) by 88.3-, 166.9-, 63.3-, 23.9-, 27.9-, 113.8- and 208.1-fold at 1.0 M NaCl, respectively, compared those of strain XCG002. As a result, membrane integrity, cell growth and cell survival of the ELO2 overexpression strain (XCG010) increased by 24.4%, 29% and 22.1% at 1.0 M NaCl, respectively, compared those of strain XCG002. The findings provided a novel strategy for engineering complex sphingolipids to enhance osmotic tolerance.IMPORTANCEThis study demonstrated a novel strategy for manipulation membrane complex sphingolipids to enhance S. cerevisiae tolerance to osmotic stress. Osmotic tolerance was related to sphingolipid acyl chain elongase, Elo2, via transcriptome analysis of the wild-type strain and an osmotic tolerant strain generated from ALE. Overexpression of ELO2 increased complex sphingolipid with longer acyl chain, thus improved membrane integrity and osmotic tolerance.

Sphingolipid metabolism – an ambiguous regulator of autophagy in the brain

In mammals, the brain exhibits the highest lipid content in the body next to adipose tissue. Complex sphingolipids are characteristic compounds of neuronal membranes. Vital neural functions including information flux and transduction occur along these membranes. It is therefore not surprising that neuronal function and survival is dependent on the metabolism of these lipids. Autophagy is a critical factor for the survival of post-mitotic neurons. On the one hand, it fulfils homeostatic and waste-recycling functions and on the other hand, it constitutes an effective strategy to eliminate harmful proteins that cause neuronal death. A growing number of experimental data indicate that several sphingolipids as well as enzymes catalyzing their metabolic transformations efficiently but very differently affect neuronal autophagy and hence survival. This review attempts to elucidate the roles and mechanisms of sphingolipid metabolism with regard to the regulation of autophagy and its consequences for brain physiology and pathology.