Search

Pancreatic β cells secrete insulin in response to glucose elevation to maintain glucose homeostasis. A complex network of inter-organ communication operates to modulate insulin secretion and regulate glucose levels after a meal. Lipids obtained from diet or generated intracellularly are known to amplify glucose-stimulated insulin secretion, however, the underlying mechanisms are not completely understood. Here, we show that a Drosophila secretory lipase, Vaha (CG8093), is synthesized in the midgut and moves to the brain where it concentrates in the insulin-producing cells in a process requiring Lipid Transfer Particle, a lipoprotein originating in the fat body. In response to dietary fat, Vaha stimulates insulin-like peptide release (ILP), and Vaha deficiency results in reduced circulatory ILP and diabetic features including hyperglycemia and hyperlipidemia. Our findings suggest Vaha functions as a diacylglycerol lipase physiologically, by being a molecular link between dietary fat and lipid amplified insulin secretion in a gut-brain axis., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

T helper 17 (T H 17) cells are implicated in autoimmune diseases, and several metabolic processes are shown to be important for their development and function. In this study, we report an essential role for sphingolipids synthesized through the de novo pathway in T H 17 cell development. Deficiency of SPTLC1, a major subunit of serine palmitoyl transferase enzyme complex that catalyzes the first and rate-limiting step of de novo sphingolipid synthesis, impaired glycolysis in differentiating T H 17 cells by increasing intracellular reactive oxygen species (ROS) through enhancement of nicotinamide adenine dinucleotide phosphate oxidase 2 activity. Increased ROS leads to impaired activation of mammalian target of rapamycin C1 and reduced expression of hypoxia-inducible factor 1-alpha and c-Myc-induced glycolytic genes. SPTLCI deficiency protected mice from developing experimental autoimmune encephalomyelitis and experimental T cell transfer colitis. Our results thus show a critical role for de novo sphingolipid biosynthetic pathway in shaping adaptive immune responses with implications in autoimmune diseases.

All living organisms require the division of a cell into daughter cells for their growth and maintenance. During cell division, both genetic and cytoplasmic contents are equally distributed between the two daughter cells. At the end of cell division, cytoplasmic contents and the plasma membrane are physically separated between the two daughter cells via a process known as cytokinesis. Hundreds of proteins and lipids involved in the cytokinetic process have been identified; however, much less is known about the mechanisms by which these molecules regulate cytokinesis, being therefore an intense area of current research. Male meiotic cytokinesis in Drosophila melanogaster testes has been shown to be an excellent model to study cytokinesis in vivo. Currently, several excellent protocols are available to study cytokinesis in Drosophila testes. However, improved methods are required to study cytokinesis under in vitro and ex vivo conditions. Here, we demonstrate a simple method to perform live imaging on individual spermatocyte cysts isolated from adult testes. We evaluate amenability of this in vitro method for treatment with pharmacological agents. We show that cytokinesis is strongly inhibited upon treatment with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis. In addition, we also demonstrate an ex vivo method to perform live imaging on whole mount adult testes on gas permeable membrane chambers. We believe the protocols described here are valuable tools to study cytokinetic mechanisms under various genetic and treatment conditions. Key features • In vitro method to study male meiotic cytokinesis in dissected spermatocyte cysts. • In vitro method allows acute treatment with various pharmacological agents to study cytokinesis. • Ex vivo method to image male meiosis cytokinesis in intact adult testes. • Requires 15-60 min to set up and could be imaged up to 6-12 h., Competing Interests: Competing interestsThe authors declare no conflicts of interest., (©Copyright : © 2024 The Authors; This is an open access article under the CC BY-NC license.)

The plasma membrane of eukaryotic cells is composed of a large number of lipid species that are laterally segregated into functional domains as well as asymmetrically distributed between the outer and inner leaflets. Additionally, the spatial distribution and organization of these lipids dramatically change in response to various cellular states, such as cell division, differentiation, and apoptosis. Division of one cell into two daughter cells is one of the most fundamental requirements for the sustenance of growth in all living organisms. The successful completion of cytokinesis, the final stage of cell division, is critically dependent on the spatial distribution and organization of specific lipids. In this review, we discuss the properties of various lipid species associated with cytokinesis and the mechanisms involved in their polarization, including forward trafficking, endocytic recycling, local synthesis, and cortical flow models. The differences in lipid species requirements and distribution in mitotic vs. male meiotic cells will be discussed. We will concentrate on sphingolipids and phosphatidylinositols because their transbilayer organization and movement may be linked via the cytoskeleton and thus critically regulate various steps of cytokinesis.

Cell division, wherein 1 cell divides into 2 daughter cells, is fundamental to all living organisms. Cytokinesis, the final step in cell division, begins with the formation of an actomyosin contractile ring, positioned midway between the segregated chromosomes. Constriction of the ring with concomitant membrane deposition in a specified spatiotemporal manner generates a cleavage furrow that physically separates the cytoplasm. Unique lipids with specific biophysical properties have been shown to localize to intercellular bridges (also called midbody) connecting the 2 dividing cells; however, their biological roles and delivery mechanisms remain largely unknown. In this study, we show that ceramide phosphoethanolamine (CPE), the structural analog of sphingomyelin, has unique acyl chain anchors in Drosophila spermatocytes and is essential for meiotic cytokinesis. The head group of CPE is also important for spermatogenesis. We find that aberrant central spindle and contractile ring behavior but not mislocalization of phosphatidylinositol phosphates (PIPs) at the plasma membrane is responsible for the male meiotic cytokinesis defect in CPE-deficient animals. Further, we demonstrate the enrichment of CPE in multivesicular bodies marked by Rab7, which in turn localize to cleavage furrow. Volume electron microscopy analysis using correlative light and focused ion beam scanning electron microscopy shows that CPE-enriched Rab7 positive endosomes are juxtaposed on contractile ring material. Correlative light and transmission electron microscopy reveal Rab7 positive endosomes as a multivesicular body-like organelle that releases its intraluminal vesicles in the vicinity of ingressing furrows. Genetic ablation of Rab7 or Rab35 or expression of dominant negative Rab11 results in significant meiotic cytokinesis defects. Further, we show that Rab11 function is required for localization of CPE positive endosomes to the cleavage furrow. Our results imply that endosomal delivery of CPE to ingressing membranes is crucial for meiotic cytokinesis., Competing Interests: The authors have declared that no competing interests exist.

Background: DLC1, a tumor suppressor gene that is downregulated in many cancer types by genetic and nongenetic mechanisms, encodes a protein whose RhoGAP and scaffolding activities contribute to its tumor suppressor functions. The role of the DLC1 START (StAR-related lipid transfer; DLC1-START) domain, other than its binding to Caveolin-1, is poorly understood. In other START domains, a key function is that they bind lipids, but the putative lipid ligand for DLC1-START is unknown., Methods: Lipid overlay assays and Phosphatidylserine (PS)-pull down assays confirmed the binding of DLC1-START to PS. Co-immunoprecipitation studies demonstrated the interaction between DLC1-START and Phospholipase C delta 1 (PLCD1) or Caveolin-1, and the contribution of PS to those interactions. Rho-GTP, cell proliferation, cell migration, and/or anchorage-independent growth assays were used to investigate the contribution of PS and PLCD1, or the implications of TCGA cancer-associated DLC1-START mutants, to DLC1 functions. Co-immunoprecipitations and PS-pull down assays were used to investigate the molecular mechanisms underlying the impaired functions of DLC1-START mutants. A structural model of DLC1-START was also built to better understand the structural implications of the cancer-associated mutations in DLC1-START., Results: We identified PS as the lipid ligand for DLC1-START and determined that DLC1-START also binds PLCD1 protein in addition to Caveolin-1. PS binding contributes to the interaction of DLC1 with Caveolin-1 and with PLCD1. The importance of these activities for tumorigenesis is supported by our analysis of 7 cancer-associated DLC1-START mutants, each of which has reduced tumor suppressor function but retains wildtype RhoGAP activity. Our structural model of DLC1-START indicates the mutants perturb different elements within the structure, which is correlated with our experimental findings that the mutants are heterogenous with regard to the deficiency of their binding properties. Some have reduced PS binding, others reduced PLCD1 and Caveolin-1 binding, and others are deficient for all of these properties., Conclusion: These observations highlight the importance of DLC1-START for the tumor suppressor function of DLC1 that is RhoGAP-independent. They also expand the versatility of START domains, as DLC1-START is the first found to bind PS, which promotes the binding to other proteins., (© 2021. The Author(s).)

Serine palmitoyltransferase complex (SPT) mediates the first and rate-limiting step in the de novo sphingolipid biosynthetic pathway. The larger subunits SPTLC1 and SPTLC2/SPTLC3 together form the catalytic core while a smaller third subunit either SSSPTA or SSSPTB has been shown to increase the catalytic efficiency and provide substrate specificity for the fatty acyl-CoA substrates. The in vivo biological significance of these smaller subunits in mammals is still unknown. Here, using two null mutants, a conditional null for ssSPTa and a null mutant for ssSPTb, we show that SSSPTA is essential for embryogenesis and mediates much of the known functions of the SPT complex in mammalian hematopoiesis. The ssSPTa null mutants are embryonic lethal at E6.5 much like the Sptlc1 and Sptlc2 null alleles. Mx1-Cre induced deletion of ssSPTa leads to lethality and myelopoietic defect. Chimeric and competitive bone marrow transplantation experiments show that the defect in myelopoiesis is accompanied by an expansion of the Lin - Sca1 + c-Kit + stem and progenitor compartment. Progenitor cells that fail to differentiate along the myeloid lineage display evidence of endoplasmic reticulum stress. On the other hand, ssSPTb null mice are homozygous viable, and analyses of the bone marrow cells show no significant difference in the proliferation and differentiation of the adult hematopoietic compartment. SPTLC1 is an obligatory subunit for the SPT function, and because Sptlc1 -/- and ssSPTa -/- mice display similar defects during development and hematopoiesis, we conclude that an SPT complex that includes SSSPTA mediates much of its developmental and hematopoietic functions in a mammalian model., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Published by Elsevier Inc.)

Serine palmitoyltransferase (SPT) long-chain base subunit 1 (SPTLC1) is 1 of the 2 main catalytic subunits of the SPT complex, which catalyzes the first and rate-limiting step of sphingolipid biosynthesis. Here, we show that Sptlc1 deletion in adult bone marrow (BM) cells results in defective myeloid differentiation. In chimeric mice from noncompetitive BM transplant assays, there was an expansion of the Lin- c-Kit+ Sca-1+ compartment due to increased multipotent progenitor production, but myeloid differentiation was severely compromised. We also show that defective biogenesis of sphingolipids in the endoplasmic reticulum (ER) leads to ER stress that affects myeloid differentiation. Furthermore, we demonstrate that transient accumulation of fatty acid, a substrate for sphingolipid biosynthesis, could be partially responsible for the ER stress. Independently, we find that ER stress in general, such as that induced by the chemical thapsigargin or the fatty acid palmitic acid, compromises myeloid differentiation in culture. These results identify perturbed sphingolipid metabolism as a source of ER stress, which may produce diverse pathological effects related to differential cell-type sensitivity.

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. Here, we show an animal model of PSE, in Drosophila , owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase ( cpes )-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, we show the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Adenosine triphosphate (ATP) synthase β, the catalytic subunit of mitochondrial complex V, synthesizes ATP. We show that ATP synthase β is deacetylated by a human nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase, sirtuin 3, and its Drosophila melanogaster homologue, dSirt2. dsirt2 mutant flies displayed increased acetylation of specific Lys residues in ATP synthase β and decreased complex V activity. Overexpression of dSirt2 increased complex V activity. Substitution of Lys 259 and Lys 480 with Arg in human ATP synthase β, mimicking deacetylation, increased complex V activity, whereas substitution with Gln, mimicking acetylation, decreased activity. Mass spectrometry and proteomic experiments from wild-type and dsirt2 mitochondria identified the Drosophila mitochondrial acetylome and revealed dSirt2 as an important regulator of mitochondrial energy metabolism. Additionally, we unravel a ceramide-NAD(+)-sirtuin axis wherein increased ceramide, a sphingolipid known to induce stress responses, resulted in depletion of NAD(+) and consequent decrease in sirtuin activity. These results provide insight into sirtuin-mediated regulation of complex V and reveal a novel link between ceramide and Drosophila acetylome., (© 2014 Rahman et al.)

The coat protein II (COPII)-coated vesicular system transports newly synthesized secretory and membrane proteins from the endoplasmic reticulum (ER) to the Golgi complex. Recruitment of cargo into COPII vesicles requires an interaction of COPII proteins either with the cargo molecules directly or with cargo receptors for anterograde trafficking. We show that cytosolic phosphatidic acid phospholipase A1 (PAPLA1) interacts with COPII protein family members and is required for the transport of Rh1 (rhodopsin 1), an N-glycosylated G protein-coupled receptor (GPCR), from the ER to the Golgi complex. In papla1 mutants, in the absence of transport to the Golgi, Rh1 is aberrantly glycosylated and is mislocalized. These defects lead to decreased levels of the protein and decreased sensitivity of the photoreceptors to light. Several GPCRs, including other rhodopsins and Bride of sevenless, are similarly affected. Our findings show that a cytosolic protein is necessary for transit of selective transmembrane receptor cargo by the COPII coat for anterograde trafficking.

HKH40A, the 8-methoxy analog of WMC79, is a synthetic agent with promising in vitro and in vivo antitumor activity, especially against solid tumors. However, molecular mechanisms underlying its antitumor effects are poorly understood. Here, we report that HKH40A markedly reduces the level of GRP78/BiP protein in cancer cell lines of various origin. In this study, we show that HKH40A not only downregulates transcription of GRP78 but also directly binds to the isolated protein and induces its proteosomal degradation. Knockdown of BiP increased the efficacy of the drug and overexpression of BiP diminished its activity. BiP is generally highly elevated in solid tumors having a pivotal role in cancer cell survival and chemoresistance, and has been suggested as a novel target for therapeutic intervention. We show that reduction of BiP level by HKH40A impairs its function and induces unfolded protein response as evidenced by the activation of IRE1α, ATF6 and PERK. This leads to a series of downstream events, including sustained eIF2α phosphorylation, increased abundance of spliced XBP1 mRNA and protein levels of ATF4 and CHOP. We also demonstrate that HKH40A inhibited tumor formation in an in vivo xenograft tumor model. Collectively, our data show that HKH40A reduces BiP levels and this could have an important role in the activity of HKH40A against cancer cells.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum (ER) to the Golgi complex. Its deficiency in mouse leads to embryonic death at E11.5. CERT deficient embryos die from cardiac failure due to defective organogenesis, but not due to ceramide induced apoptotic or necrotic cell death. In the current study we examined the effect of CERT deficiency in a primary cell line, namely, mouse embryonic fibroblasts (MEFs). We show that in MEFs, unlike in mutant embryos, lack of CERT does not lead to increased ceramide but causes an accumulation of hexosylceramides. Nevertheless, the defects due to defective sphingolipid metabolism that ensue, when ceramide fails to be trafficked from ER to the Golgi complex, compromise the viability of the cell. Therefore, MEFs display an incipient ER stress. While we observe that ceramide trafficking from ER to the Golgi complex is compromised, the forward transport of VSVG-GFP protein is unhindered from ER to Golgi complex to the plasma membrane. However, retrograde trafficking of the plasma membrane-associated cholera toxin B to the Golgi complex is reduced. The dysregulated sphingolipid metabolism also leads to increased mitochondrial hexosylceramide. The mitochondrial functions are also compromised in mutant MEFs since they have reduced ATP levels, have increased reactive oxygen species, and show increased glutathione reductase activity. Live-cell imaging shows that the mutant mitochondria exhibit reduced fission and fusion events. The mitochondrial dysfunction leads to an increased mitophagy in the CERT mutant MEFs. The compromised organelle function compromise cell viability and results in premature senescence of these MEFs.

Sphingolipids are an important class of compounds that regulate signal transduction and other vital cellular processes. Herein, we report sensitive normal and reversed phase LC-MS/MS methods for quantitation of multiple sphingolipid classes. In the normal-phase ESI/MS/MS method, a high content of organic solvents was utilized, which, although it included hexane, ethyl acetate, acetonitrile containing 2% methanol, 1-2% acetic acid, and 5 mM ammonium acetate, resulted in a very efficient electrospray ionization of the ceramides (Cers) and hexosylceramides (MHCers). Three normal-phase LC-MS/MS methods using segmented phases were developed to specifically target Cers, MHCers, or sphingomyelins (SMs). This segmentation scheme increases the number of data points acquired for a given analyte and enhances the sensitivity and specificity of the measurements. Nine separate reversed phase chromatography methods were developed for the three classes of compounds. These assays were used for comparing the levels of Cers, SMs, and MHCers from mouse embryonic fibroblast (pMEF) and human embryonic kidney (HEK293) cells. These findings were then compared with the reported data from RAW264.7 mouse macrophage cells, BHK21 hamster cells, and human plasma and serum samples. The analysis of cell lines, using both normal and reversed phase chromatography, revealed discrimination based on the type of chromatography chosen, while sphingolipid assays of samples containing different amounts of protein showed different results, even after normalizing for protein content. Also, LC/MS/MS profiles were provided for the classes and individual compounds so that they could be used as "molecular profiles" for class or individual sample analysis.

Sphingolipids are important structural components of membranes, and play an equally important role in basic cellular processes as second messengers. Recently, sphingolipids are receiving increasing attention in cancer research. Ceramide is the central molecule that regulates sphingolipid metabolism forming the basic structural backbone of sphingolipids and the precursor of all complex sphingolipids. It is been proposed to be an important regulator of tumor cell death following exposure to stress stimuli. The increase or decrease of ceramide levels leading to change in sensitivity of cancer cells to stress stimuli provides support for a central role of ceramide signaling in cell death. In this review, we have focused on ceramide transfer protein (CERT) as a major regulator of ceramide flux in the cell.

Internalized membrane proteins are either transported to late endosomes and lysosomes for degradation or recycled to the plasma membrane. Although proteins involved in trafficking and sorting have been well studied, far less is known about the lipid molecules that regulate the intracellular trafficking of membrane proteins. We studied the function of sphingosine kinases and their metabolites in endosomal trafficking using Drosophila melanogaster photoreceptors as a model system. Gain- and loss-of-function analyses show that sphingosine kinases affect trafficking of the G protein-coupled receptor Rhodopsin and the light-sensitive transient receptor potential (TRP) channel by modulating the levels of dihydrosphingosine 1 phosphate (DHS1P) and sphingosine 1 phosphate (S1P). An increase in DHS1P levels relative to S1P leads to the enhanced lysosomal degradation of Rhodopsin and TRP and retinal degeneration in wild-type photoreceptors. Our results suggest that sphingosine kinases and their metabolites modulate photoreceptor homeostasis by influencing endolysosomal trafficking of Rhodopsin and TRP.

Ceramidases catalyze the conversion of ceramide to sphingosine. They are acylaminohydrolases that catalyze the deacylation of the amide-linked saturated fatty acid from ceramide to generate sphingosine. They also catalyze the reverse reaction of ceramide biosynthesis using sphingosine and fatty acid. In mammals, different proteins catalyze these reactions while individually exhibiting optimal activity over a narrow pH range and have been accordingly called acid, neutral, and alkaline ceramidases. Several genes encode for variants of alkaline ceramidase in mammals. Brainwashing (Bwa) is the only putative alkaline ceramidase homologue present in Drosophila. In this study we have demonstrated that BWA does not exhibit ceramidase activity and that bwa null mutants display no loss of ceramidase activity. Instead, the neutral ceramidase gene CDase encodes the protein that is responsible for all measurable ceramidase activity in Drosophila. Our studies show strong genetic interaction of Bwa with CDase and the Drosophila ceramide kinase gene (DCERK). We show that, although BWA is unlikely to be a ceramidase, it is a regulator of sphingolipid flux in Drosophila. Bwa exhibits strong genetic interaction with other genes coding for ceramide-metabolizing enzymes. This interaction might partly explain its original identification as a ceramidase.

Sphingomyelin (SM) and ceramide-phosphoethanolamines (cer-PEs) are related lipids present in mammals and insects, respectively. Owing to the critical roles that cer-PEs play in eukaryotic cellular function, there is a need to develop methods that provide accurate quantitation of these compounds. Results obtained in this study demonstrate that Drosophila contains cer-PEs with unsaturated sphingoid base cores as well as low levels of cer-PEs that possess saturated sphingoid base cores. Specifically, the method developed in this study enabled the quantitation of picogram amounts of cer-PE containing both unsaturated d14:1(Delta4) and d16:1(Delta4) and saturated d14:0 sphingoid base cores. Using this method, cer-PE compounds with both saturated and unsaturated sphingoid base cores were initially identified by neutral loss scanning, followed by quantitation using selected reaction monitoring (SRM) scans. The SRM scans measured a product ion originating from the sphingoid base backbone, rather than from the head group, increasing the specificity and sensitivity of the quantitation measurement., (Copyright 2010 Elsevier Inc. All rights reserved.)

Phosphoinositide-specific phospholipase C (PLC) is a central effector for many biological responses regulated by G-protein-coupled receptors including Drosophila phototransduction where light sensitive channels are activated downstream of NORPA, a PLCbeta homolog. Here we show that the sphingolipid biosynthetic enzyme, ceramide kinase, is a novel regulator of PLC signaling and photoreceptor homeostasis. A mutation in ceramide kinase specifically leads to proteolysis of NORPA, consequent loss of PLC activity, and failure in light signal transduction. The mutant photoreceptors also undergo activity-dependent degeneration. Furthermore, we show that a significant increase in ceramide, resulting from lack of ceramide kinase, perturbs the membrane microenvironment of phosphatidylinositol 4, 5, bisphosphate (PIP(2)), altering its distribution. Fluorescence image correlation spectroscopic studies on model membranes suggest that an increase in ceramide decreases clustering of PIP(2) and its partitioning into ordered membrane domains. Thus ceramide kinase-mediated maintenance of ceramide level is important for the local regulation of PIP(2) and PLC during phototransduction.

Ceramide transfer protein (CERT) functions in the transfer of ceramide from the endoplasmic reticulum (ER) to the Golgi. In this study, we show that CERT is an essential gene for mouse development and embryonic survival and, quite strikingly, is critical for mitochondrial integrity. CERT mutant embryos accumulate ceramide in the ER but also mislocalize ceramide to the mitochondria, compromising their function. Cells in mutant embryos show abnormal dilation of the ER and degenerating mitochondria. These subcellular changes manifest as heart defects and cause severely compromised cardiac function and embryonic death around embryonic day 11.5. In spite of ceramide accumulation, CERT mutant mice do not die as a result of enhanced apoptosis. Instead, cell proliferation is impaired, and expression levels of cell cycle-associated proteins are altered. Individual cells survive, perhaps because cell survival mechanisms are activated. Thus, global compromise of ER and mitochondrial integrity caused by ceramide accumulation in CERT mutant mice primarily affects organogenesis rather than causing cell death via apoptotic pathways.

Sphingoid bases, such as unsaturated sphingosine (So) and its corresponding dihydro-saturated species sphinganine (Sa), are present in cell samples in low abundance. This fact combined with their low-to-moderate electrospray ionization (ESI) potential, compared to other sphingolipids such as sphingomyelins, limits their detection and quantitation by liquid chromatography-tandem mass spectrometry (LC-MS(2)). To enhance the ESI efficiency of sphingoid bases, a novel procedure to generate stably derivatized analytes that enhance the LC-MS(2) detection of sphingoid bases when analyzed using LC-MS(2) was developed. In this method, a ruthenium complex, [4-(N-succimidyloxycarbonyl propyl)-4'-methyl-2,2'-bipyridine] bis(2,2'-bipyridine) Ru(II) dihexafluorophosphate, is added directly to a cell extract. This complex reacts with and covalently binds to an amino group within the sphingoid bases. The dicationic nature of the ruthenium ion enhances the compound's ionization efficiency resulting in increased LC-MS(2) signals for the derivatized sphingoid bases. Consequently, the detection and quantitation of sphingoid bases are greatly improved.

The importance of sphingolipids in membrane biology was appreciated early in the twentieth century when several human inborn errors of metabolism were linked to defects in sphingolipid degradation. The past two decades have seen an explosion of information linking sphingolipids with cellular processes. Studies have unraveled mechanistic details of the sphingolipid metabolic pathways, and these findings are being exploited in the development of novel therapies, some now in clinical trials. Pioneering work in yeast has laid the foundation for identifying genes encoding the enzymes of the pathways. The advent of the era of genomics and bioinformatics has led to the identification of homologous genes in other species and the subsequent creation of animal knock-out lines for these genes. Discoveries from these efforts have re-kindled interest in the role of sphingolipids in membrane biology. This review highlights some of the recent advances in understanding sphingolipids' roles in membrane biology as determined from genetic models.

Neutral ceramidase, a key enzyme of sphingolipid metabolism, hydrolyzes ceramide to sphingosine. These sphingolipids are critical structural components of cell membranes and act as second messengers in diverse signal transduction cascades. Here, we have isolated and characterized functional null mutants of Drosophila ceramidase. We show that secreted ceramidase functions in a cell-nonautonomous manner to maintain photoreceptor homeostasis. In the absence of ceramidase, photoreceptors degenerate in a light-dependent manner, are defective in normal endocytic turnover of rhodopsin, and do not respond to light stimulus. Consistent with a cell-nonautonomous function, overexpression of ceramidase in tissues distant from photoreceptors suppresses photoreceptor degeneration in an arrestin mutant and facilitates membrane turnover in a rhodopsin null mutant. Furthermore, our results show that secreted ceramidase is internalized and localizes to endosomes. Our findings establish a role for a secreted sphingolipid enzyme in the regulation of photoreceptor structure and function.

Ceramide transfer protein (CERT) transfers ceramide from the endoplasmic reticulum to the Golgi complex, a process critical in synthesis and maintenance of normal levels of sphingolipids in mammalian cells. However, how its function is integrated into development and physiology of the animal is less clear. Here, we report the in vivo consequences of loss of functional CERT protein. We generated Drosophila melanogaster mutant flies lacking a functional CERT (Dcert) protein using chemical mutagenesis and a Western blot-based genetic screen. The mutant flies die early between days 10 and 30, whereas controls lived between 75 and 90 days. They display >70% decrease in ceramide phosphoethanolamine (the sphingomyelin analog in Drosophila) and ceramide. These changes resulted in increased plasma membrane fluidity that renders them susceptible to reactive oxygen species and results in enhanced oxidative damage to cellular proteins. Consequently, the flies showed reduced thermal tolerance that was exacerbated with aging and metabolic compromise such as decreasing ATP and increasing glucose levels, reminiscent of premature aging. Our studies demonstrate that maintenance of physiological levels of ceramide phosphoethanolamine by CERT in vivo is required to prevent oxidative damages to cellular components that are critical for viability and normal lifespan of the animal.

Scramblases are a family of single-pass plasma membrane proteins, identified by their purported ability to scramble phospholipids across the two layers of plasma membrane isolated from platelets and red blood cells. However, their true in vivo role has yet to be elucidated. We report the generation and isolation of null mutants of two Scramblases identified in Drosophila melanogaster. We demonstrate that flies lacking either or both of these Scramblases are not compromised in vivo in processes requiring scrambling of phospholipids. Instead, we show that D. melanogaster lacking both Scramblases have more vesicles and display enhanced recruitment from a reserve pool of vesicles and increased neurotransmitter secretion at the larval neuromuscular synapses. These defects are corrected by the introduction of a genomic copy of the Scramb 1 gene. The lack of phenotypes related to failure of scrambling and the neurophysiological analysis lead us to propose that Scramblases play a modulatory role in the process of neurotransmission.

Sphingolipids are important structural components of membranes that delimit the boundaries of cellular compartments, cells and organisms. They play an equally important role as second messengers, and transduce signals across or within the compartments they define to initiate physiological changes during development, differentiation and a host of other cellular events. For well over a century Drosophila melanogaster has served as a useful model organism to understand some of the fundamental tenets of development, differentiation and signaling in eukaryotic organisms. Directed approaches to study sphingolipid biology in Drosophila have been initiated only recently. Nevertheless, earlier phenotypic studies conducted on genes of unknown biochemical function have recently been recognized as mutants of enzymes of sphingolipid metabolism. Genome sequencing and annotation have aided the identification of homologs of recently discovered genes. Here we present an overview of studies on enzymes of the de novo sphingolipid biosynthetic pathway, known mutants and their phenotypic characterization in Drosophila.

A screen for Drosophila synaptic dysfunction mutants identified slug-a-bed (slab). The slab gene encodes ceramidase, a central enzyme in sphingolipid metabolism and regulation. Sphingolipids are major constituents of lipid rafts, membrane domains with roles in vesicle trafficking, and signaling pathways. Null slab mutants arrest as fully developed embryos with severely reduced movement. The SLAB protein is widely expressed in different tissues but enriched in neurons at all stages of development. Targeted neuronal expression of slab rescues mutant lethality, demonstrating the essential neuronal function of the protein. C(5)-ceramide applied to living preparations is rapidly accumulated at neuromuscular junction (NMJ) synapses dependent on the SLAB expression level, indicating that synaptic sphingolipid trafficking and distribution is regulated by SLAB function. Evoked synaptic currents at slab mutant NMJs are reduced by 50-70%, whereas postsynaptic glutamate-gated currents are normal, demonstrating a specific presynaptic impairment. Hypertonic saline-evoked synaptic vesicle fusion is similarly impaired by 50-70%, demonstrating a loss of readily releasable vesicles. In addition, FM1-43 dye uptake is reduced in slab mutant presynaptic terminals, indicating a smaller cycling vesicle pool. Ultrastructural analyses of mutants reveal a normal vesicle distribution clustered and docked at active zones, but fewer vesicles in reserve regions, and a twofold to threefold increased incidence of vesicles linked together and tethered at the plasma membrane. These results indicate that SLAB ceramidase function controls presynaptic terminal sphingolipid composition to regulate vesicle fusion and trafficking, and thus the strength and reliability of synaptic transmission.

Transgenic expression of ceramidase suppresses retinal degeneration in Drosophila arrestin and phospholipase C mutants. Here, we show that expression of ceramidase facilitates the dissolution of incompletely formed and inappropriately located elements of rhabdomeric membranes in ninaE(I17) mutants lacking the G protein receptor Rh1 in R1-R6 photoreceptor cells. Ceramidase expression facilitates the endocytic turnover of Rh1. Although ceramidase expression aids the removal of internalized rhodopsin, it does not affect the turnover of Rh1 in photoreceptors maintained in dark, where Rh1 is not activated and thus has a slower turnover and a long half-life. Therefore, the phenotypic consequence of ceramidase expression in photoreceptors is caused by facilitation of endocytosis. This study provides mechanistic insight into the sphingolipid biosynthetic pathway-mediated modulation of endocytosis and suppression of retinal degeneration.

Mutations in proteins of the Drosophila phototransduction cascade, a prototypic guanine nucleotide-binding protein-coupled receptor signaling system, lead to retinal degeneration and have been used as models to understand human degenerative disorders. Here, modulating the sphingolipid biosynthetic pathway rescued retinal degeneration in Drosophila mutants. Targeted expression of Drosophila neutral ceramidase rescued retinal degeneration in arrestin and phospholipase C mutants. Decreasing flux through the de novo sphingolipid biosynthetic pathway also suppressed degeneration in these mutants. Both genetic backgrounds modulated the endocytic machinery because they suppressed defects in a dynamin mutant. Suppression of degeneration in arrestin mutant flies expressing ceramidase correlated with a decrease in ceramide levels. Thus, enzymes of sphingolipid metabolism may be suitable targets in the therapeutic management of retinal degeneration.

Phosphoinositides function as important second messengers in a wide range of cellular processes. Inositol polyphosphate 1-phosphatase (IPP) is an enzyme essential for the hydrolysis of the 1-phosphate from either Ins(1,4)P2 or Ins(1,3,4)P3. This enzyme is Li+ sensitive, and is one of the proposed targets of Li+ therapy in manic-depressive illness. Drosophila ipp mutants accumulate IP2 in their system and are incapable of metabolizing exogenous Ins(1,4)P2. Notably, ipp mutants demonstrate compensatory upregulation of an alternative branch in the inositol-phosphate metabolism tree, thus providing a means of ensuring continued availability of inositol. We demonstrate that ipp mutants have a defect in synaptic transmission resulting from a dramatic increase in the probability of vesicle release at larval neuromuscular junctions. We also show that Li+ phenocopies this effect in wild-type synapses. Together, these results support a role for phosphoinositides in synaptic vesicle function in vivo and mechanistically question the "lithium hypothesis."

We have developed an assay using permeabilized cells to monitor fragmentation of the Golgi complex that occurs during mitosis. Golgi stacks, in permeabilized interphase normal rat kidney (NRK) cells, upon incubation with mitotic extracts undergo extensive fragmentation, and the fragmented Golgi membranes are dispersed throughout the cytoplasm. We find that the continued presence of p34cdc2, the mitosis initiation kinase, is not necessary for Golgi fragmentation. Instead, fragmentation depends on cytosolic mitogen-activated protein kinase kinase 1 (MEK1 or MAPKK1). However, the known cytoplasmic substrates for MEK1, ERK1, and ERK2 are not required for this process. Interestingly, we find a Golgi-associated ERK, which we propose as the likely target for MEK1 in Golgi fragmentation.

Phospholipase C (PLC) is the focal point for two major signal transduction pathways: one initiated by G protein-coupled receptors and the other by tyrosine kinase receptors. Active PLC hydrolyzes phosphatidylinositol bisphosphate (PIP2) into the two second messengers inositol 1,4,5-trisphosphate (InsP3) and diacyl glycerol (DAG). DAG activates protein kinase C, and InsP3 mobilizes calcium from intracellular stores via the InsP3 receptor. Changes in [Ca2+]i regulate the function of a wide range of target proteins, including ion channels, kinases, phosphatases, proteases, and transcription factors (Berridge, 1993). In the mouse, there are three InsP3R genes, and type 1 InsP3R mutants display ataxia and epileptic seizures (Matsumoto et al., 1996). In Drosophila, only one InsP3 receptor (InsP3R) gene is known, and it is expressed ubiquitously throughout development (Hasan and Rosbash, 1992; Yoshikawa et al., 1992; Raghu and Hasan, 1995). Here, we characterize Drosophila InsP3R mutants and demonstrate that the InsP3R is essential for embryonic and larval development. Interestingly, maternal InsP3R mRNA is sufficient for progression through the embryonic stages, but larval organs show asynchronous and defective cell divisions, and imaginal discs arrest early and fail to differentiate. We also generated adult mosaic animals and demonstrate that phototransduction, a model PLC pathway thought to require InsP3R, does not require InsP3R for signaling.

Genes from Drosophila melanogaster have been identified that encode proteins homologous to Orc2p and Orc5p of the Saccharomyces cerevisiae origin recognition complex (ORC). The abundance of the Drosophila Orc2p homolog DmORC2 is developmentally regulated and is greatest during the earliest stages of embryogenesis, concomitant with the highest rate of DNA replication. Fractionation of embryo nuclear extracts revealed that DmORC2 is found in a tightly associated complex with five additional polypeptides, much like the yeast ORC. These studies will enable direct testing of the initiator-based model of replication in a metazoan.

An unusual intermediate bound to the enzyme was detected in the interaction of thiosemicarbazide with sheep liver serine hydroxymethyltransferase. This intermediate had absorbance maxima at 464 and 440 nm. Such spectra are characteristic of resonance stabilized intermediates detected in the interaction of substrates and quasi-substrates with pyridoxal phosphate enzymes. An intermediate of this kind has not been detected in the interaction of thiosemicarbazide with other pyridoxal phosphate enzymes. This intermediate was generated slowly (t 1/2 = 4 min) following the addition of thiosemicarbazide (200 microM) to sheep liver serine hydroxymethyltransferase (5 microM). It was bound to the enzyme as evidenced by circular dichroic bands at 464 and 440 nm and the inability to be removed upon Centricon filtration. The kinetics of interaction revealed that thiosemicarbazide was a slow binding reversible inhibitor in this phase with a k(on) of 11 M-1 s-1 and a k(off) of 5 x 10(-4) s-1. The intermediate was converted very slowly (k = 4 x 10(-5) s-1) to the final products, namely the apoenzyme and the thiosemicarbazone of pyridoxal phosphate. A minimal kinetic mechanism involving the initial conversion to the intermediate absorbing at longer wavelengths and the conversion of this intermediate to the final product, as well as, the formation of pyridoxal phosphate-thiosemicarbazone directly by an alternate pathway is proposed.

The mechanism of interaction of methoxyamine with sheep liver serine hydroxymethyltransferase (EC 2.1.2.1) (SHMT) was established by measuring changes in enzyme activity, visible absorption spectra, circular dichroism and fluorescence, and by evaluating the rate constant by stopped-flow spectrophotometry. Methoxyamine can be considered as the smallest substituted aminooxy derivative of hydroxylamine. It was a reversible noncompetitive inhibitor (Ki = 25 microM) of SHMT similar to O-amino-D-serine. Like in the interaction of O-amino-D-serine and aminooxyacetic acid, the first step in the reaction was very fast. This was evident by the rapid disappearance of the enzyme-Schiff base absorbance at 425 nm with a rate constant of 1.3 x 10(3) M-1 sec-1 and CD intensity at 430 nm. Concomitantly, there was an increase in absorbance at 388 nm (intermediate I). The next step in the reaction was the unimolecular conversion (1.1 x 10(-3) sec-1) of this intermediate to the final oxime absorbing at 325 nm. The identity of the oxime was established by its characteristic fluorescence emission at 460 nm when excited at 360 nm and by high performance liquid chromatography. These results highlight the specificity in interactions of aminooxy compounds with sheep liver serine hydroxymethyltransferase and that the carboxyl group of the inhibitors enhances the rate of the initial interaction with the enzyme.