Sphingolipids and Membrane Biology as Determined from Genetic Models

Abstract

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.

Introduction

Sphingolipids are essential components of all eukaryotic cell membranes, and are required for the survival of Drosophila, yeast, and mammals [1-4]. The biological and clinical importance of sphingolipid metabolism was revealed by the discovery that diseases such as Gaucher’s disease and Niemann-Pick were caused by defective sphingolipid degradation. However, the lack of genetic model systems that would permit molecular analyses impaired rapid progress in understanding the role of sphingolipids in the etiopathogenesis of these syndromes. In the mean-time, however, in vitro studies began to provide convincing evidence that sphingolipid metabolites function as second messengers [5-9] and that they influence signal-transduction events through their ability to segregate the plasma membrane into microdomains [10-12]. In addition, complex sphingolipids were shown to act as receptors to permit the entry of microorganisms into the cell and to alter the sensitivity of cells to cell-surface ligands [13-15]. Sphingolipid metabolites also facilitate cellular processes such as cell division, differentiation, and cell death [2, 16-18]. The relative concentrations of sphingolipid metabolites are thought to be regulated tightly, because each of them could have a different effect on cells. For example, ceramide and sphingosine have ‘pro-apoptotic’ effects, while sphingosine 1 phosphate (S1P) has a ‘pro-growth’ function [19-21].

These findings have led to a model of sphingolipid function called the ‘sphingolipid rheostat.’ According to this model, enzymes that interconvert active sphingolipids act as switches that control the relative levels of each of metabolite, thereby regulating vital cellular processes [21-23]. The subsequent identification of genes that encode proteins that catalyze these reactions in yeast, data from genome annotation, and the identification of homologous genes in higher eukaryotes have led to the generation of knock-out transgenic animals. Phenotypic analyses of these model systems and the integration of information obtained both in vitro and in vivo are beginning to elucidate the molecular nature of sphingolipid function in membrane biology. Since membrane biology is a prominent contributor to the genesis and/or progress of numerous diseases, including those caused by metabolic disorders, such as diabetes and some cardiac diseases, and by cancer, infections, and genetic mutations, a detailed understanding of how sphingolipids influence membrane biology may prove important for developing both diagnostic and therapeutic approaches.

Most sphingolipids exist as components of membranous structures, and the composition and concentrations of sphingolipids vary among organs and within sub-cellular organelles. Therefore, it seems intuitively likely that the biological functions of sphingolipids would most immediately affect the compartments in which they reside (assuming they are not merely cargo in transit). Membranes and membrane-derived structures participate in a number of cell functions. The cell membrane defines the first physical boundary of the cell and acts as a protective barrier. Because it is selectively permeable, it regulates the movement of small molecules in and out of the cell, thereby regulating the chemical composition of its internal contents. The membrane participates in endo- and exocytosis, signaling between the intracellular and extracellular spaces, and cell-cell interactions, and it functions as a reservoir of signaling lipids. Biochemical experiments suggest that sphingolipids influence these functions.

The roles of sphingolipid metabolites have been studied in vitro and in vivo. In vitro studies have used enzyme inhibitors, structural analogues of substrates, activator molecules, and gene-knock down (RNAi) approaches to this issue. These studies have provided a great deal of information on the kinetic and the mechanistic details of the enzyme reactions. Studies making use of mutant cell lines have provided the primary information on these pathways and have led to the discovery of novel proteins. In vivo approaches have also provided a great deal of insight into the biochemical roles as well as the physiological significance of these proteins and pathways.

Eukaryotic model systems that are commonly used for in vivo studies of sphingolipid function and biochemistry include yeast, Leishmania, Drosophila, Arabidopsis, and mouse. Pioneering work in yeast provided a wealth of information on the genes and enzymes that regulate the sphingolipid metabolic pathway. Phenotype-based genetic screening has elucidated the link between this metabolic pathway and several cellular processes and facilitated a better understanding of how this pathway interacts with signal-transduction cascades [24]. Drosophila is emerging as a model system for studying sphingolipid metabolism, and although a detailed chemical profile of sphingolipids is not available currently, recent studies have attempted to bridge this gap [25]. Finally, mouse models have been used to great advantage to understand the workings of sphingolipids in the mammalian system.

Here, we will first briefly describe the best understood sphingolipids and their metabolism. We will then highlight genetic studies that have addressed the role played by sphingolipids in membrane biology, including in endocytosis, exocytosis, the formation of micro-domains, membrane integrity, and membrane signaling.

Structural Overview and Biosynthesis of Sphingolipids

All sphingolipids contain a Long Chain Base (LCB), commonly referred to as the sphingoid base, as their primary structural element. The ‘sphingoid base’ itself comprises a broad range of 2-amino-1, 3-dihydroxyalkanes or -enes [26]. In yeast there are two types of LCBs, dihydrosphingosine (DHS) and phytosphingosine (PHS), which have a carbon chain length of 18 to 20 (C-18-20). Mammals have sphingosine (predominantly C-18 [17]) as their major sphingoid base, although they also make small amounts of DHS and PHS [27]. In Drosophila and other dipterans, the sphingoid bases are not C-18, but rather C-14 and C-16 [28, 29]. The existence of shorter LCBs correlates well with Drosophila’s requirement for lower ambient temperatures to survive [22]. A long fatty-acid chain attached to an LCB through an amide linkage forms ceramide, which is esterified to form different head groups. The polar head groups of different sphingolipid species show wide biochemical variations.

The de novo biosynthesis of sphingolipids begins in the endoplasmic reticulum (ER), with the condensation of serine with acyl CoA, which forms 3-keto dihydro sphingosine. The subsequent reaction steps—reduction, acylation, and desaturation—lead to the formation of ceramide as depicted in Fig. 1. This general scheme of reactions is essentially the same in yeast, mammals, and Drosophila, although the intermediates in the biosynthetic steps vary in chain length and degree of saturation and hydroxylation, between eukaryotic species [22, 30, 31].

Figure 1.

de novo sphingolipid biosynthetic pathways in yeast, mammals and Drosophila The figure outlines the steps that lead to biosynthesis of major sphingolipids in the three different species.

Ceramide must localize to the lumen of the Golgi apparatus to serve as the substrate for the synthesis of sphingomyelin and complex sphingolipids [32-34]. To do so, ceramide must first traverse the cytosolic compartment. The spontaneous translocation of ceramide across the interbilayer is too slow to be biologically significant, since the t_1/2 for such movement is on the order of days [35]. Thus, ceramide requires active transport from the ER to the Golgi complex. Both vesicular and nonvesicular transport mechanisms can mediate this process [36]. The nonvesicular transport is mediated by the CERT protein in mammals, in an ATP-dependent manner [37, 38]. Recent studies from our laboratory have confirmed that a CERT homolog in Drosophila has the same role, but yeast lacks a CERT homolog [37, 39]. Once transported to the Golgi complex, several different head groups can be added to ceramide to form different classes of complex sphingolipids (Fig. 2). The dominant head groups vary among animal species. In yeast, the major sphingolipids are inositol phosphoceramide (IPC), mannose inositol phosphoceramide, and mannose inositol (P₂) ceramide (M(IP)₂C) and they have inositol phosphate and mannose inositol phosphate(s) as head groups [40]. Mammals have sphingomyelin (SM), with a phosphocholine moiety as the head group, glucosyl ceramide (with glucose as the head group), and gangliosides, which have a head group of several sugar residues linked by various types of glycosidic linkages [41]. In Drosophila, the major sphingolipids are ceramide phosphoethanolamine (CPE), a structural analogue of SM, with phosphoethanolamine as the head group, and glycosphingolipids (GSLs), which have sugar residues as the head group [22].

Figure 2. Core Structure of sphingolipids.

Long chain bases (LCB) form the core structure of sphingolipids. The structure base varies in it chain length (represented by // in the figure), degree of saturation and hydroxylation. N-acylation of LCB with very long chain fatty acid (VLFA) leads to the formation of respective ceramides. Further esterificationof the R-group of the LCB leads to generation of complex sphingolipids. The nature and complexity of the R groups defines the class of complex sphingolipids.

Sphingolipids in endocytosis and exocytosis

Endocytosis is a membrane-mediated phenomenon that permits the entry of substances into cells without passing through the plasma membrane. It requires both membrane-invagination and vesicle-formation steps. Endocytosis is a key process for the cellular uptake of extracellular nutrients. It also regulates membrane dynamics and cellular responses to external stimuli by modulating cell surface-receptor activity [42]. Endocytosed vesicles are dynamic. They are continuously modified as they traverse the cell to their final destination, showing changes in composition, size, and shape, and switching from actin-based transport to microtubule-mobilized structures [43]. In addition, sphingolipids can affect endocytosis by physically altering the local environment or by initiating signaling events.

In yeast, phenotype-based classical genetic screens have led to the isolation of mutants that are defective in different endocytotic steps. Several of these mutants are associated with genes encoding proteins involved in lipid metabolism [44]. One of them, end8, has a mutation in the LCB1 gene, which encodes Lcb 1p, a component of serine palmitoyl transferase, which catalyzes the formation of 3-ketosphinganine [45, 46]. lcb1-100 is a temperature-sensitive yeast line that carries a mutation in the LCB1 gene. At the permissive temperature (24°C), lcb1-100 grows like wild-type yeast, but upon being shifted to the restrictive temperature (37°C), it fails to synthesize sphingolipids de novo and, as a consequence, fail to grow. A further study showed that lcb1-100 exhibits endocytosis defects [47]. This phenotype is rescued by providing medium containing LCB [47], indicating that the defective sphingoid base synthesis is correlated with the faulty endocytosis. Other studies extended this finding by showing that at restrictive temperatures the mutant cells have an abnormal non-polarized cortical distribution of actin filaments associated with the endocytic defects. This is corrected by providing dihydrosphingosine (DHS) or phytosphingosine (PHS), indicating that sphingoid base synthesis is required for proper actin localization. Moreover, the deregulation of actin dynamics has been shown to affect the internalization step in endocytosis [48, 49] (Fig. 3). Subsequent studies indicated that proper sphingoid base synthesis is required to control protein phosphorylation events that are crucial for endocytosis and overexpression of protein kinase C1 (PKC1) and PKH1 or PKH2 (ortholgues of mammalian protein-phosphoinositide dependent protein kinase 1 or PDK1) restores the actin localization defects in the lcb1-100 mutant [50, 51]. These studies suggest that LCB can mediate the activation of Pkh1/2.

Figure 3.

A schematic representation of modulation of endocytosis by sphingolipids, as determined from studies in yeast.

It is also worth mentioning here that a known target of Pkh1 and Pkh2 is the Pkc1-MAP kinase pathway [52, 53], which is involved in maintaining cell-wall integrity in yeast, supporting the idea of a connection between Pkh1/2 and the cell membrane [54, 55]. Furthermore, another study showed that the mutant rvs161Δ (reduced viability upon starvation 161, an amphiphysin homolog) is unable to repolarize actin cytoskeleton following salt stress, and this phenotype was attributed to the failure of the Rvs161p protein to associate with lipid rafts. Mutations that suppress the salt sensitivity of rvs161Δ map to genes required for sphingolipid biosynthesis, thus providing a genetic link between sphingolipids and cytoskeletal organization [56] .

Earlier studies from our laboratory demonstrated that the modulation of sphingolipid biosynthesis in vivo rescues photoreceptor degeneration in a mutant Drosophila defective in endocytosis [57]. Phototransduction in Drosophila is a prototypic guanine nucleotide-binding protein-coupled receptor signaling system and is initiated by the activation of the visual pigment Rhodopsin by incident light. Arrestins bind to light-activated Rhodopsin and assist in its inactivation. They also facilitate the endocytic turnover of light-activated Rhodopsin. In Arrestin mutants, the photoreceptors degenerate because of endocytic defects [58-60]. In these mutants, chronically active rhodopsin leads to increased calcium entry, calcium necrotoxicity, and the defective endocytosis of rhodopsin. The targeted overexpression of neutral ceramidase in the mutant background rescues the photoreceptor degeneration. In addition, ceramidase overexpression rescues retinal degeneration caused by targeted expression of a temperature-sensitive allele of dynamin, UAS-shi^ts1, in photoreceptors suggesting that the modulation of ceramide levels could affect endocytosis [61]. Increased ceramidase expression also facilitates Rhodopsin and membrane turnover. Rhodopsin (Rh1) is required not only for transducing the light signal in the adult retina but also for stabilizing the actin-based cytoskeletal scaffold of developing photoreceptors, which is called the rhabdomere terminal web (RTW). In ninaE¹¹⁷ mutants, which lack Rh1, the RTW is not organized and rhabdomere biogenesis is defective. Newly eclosed flies show inappropriately formed rhabdomeric components that involute into the cytosol [62]. Ceramidase overexpression facilitates the clearing of these inappropriately formed rhabdomeric components by internalization [63]. These results suggest a potential role for sphingolipid metabolites in endocytosis and membrane turnover.

Although these studies show that ceramidase affects endocytosis, the molecular details still need to be worked out. Given that sphingoid base assembly affects actin dynamics in yeast, it would be interesting to see if a similar mechanism operates in Drosophila. The conversion of ceramide to sphingosine should affect the physical attributes of the membrane locally and could affect endocytosis. Theoretically, the asymmetric generation or removal of ceramides in the leaflets of membranes could cause vesicle aggregation and facilitate fusion and fission reactions [64]. In addition, alterations in sphingolipid composition affect the phase behavior of cell membranes. Such changes could influence lipid mobility and sphingolipids’ access to other lipids, such as phosphoinositides, thereby affecting the interactions of phosphoinositide-binding proteins with the membranes. Future genetic and biochemical studies will be required to address these and other possibilities.

In recent years, studies have also linked sphingolipids to exocytosis. During exocytosis, the secretory vesicles are directed from donor membranes to target membranes. In yeast, this process is mediated in part by membrane-associated proteins called SNAREs, which are encoded by the SNC genes [65-67]. The disruption of genes encoding the SNARE proteins can be detrimental to the organism or even lethal [68, 69].

A temperature-sensitive yeast mutant, snc, which is null for the SNC genes, grows slowly at 30°C, and its growth is blocked at restrictive temperatures due to the failure of secretory vesicles to fuse with the plasma membrane and to other defective secretory events [70]. Interestingly, the suppressor mutations of this phenotype map to the VBM1 and VBM2 genes, which are identical to ELO 2 and 3, whose gene products are involved in the elongation of fatty acids to generate the long-chain fatty acids required for ceramide synthesis [71]. While attempting to understand the role of sphingolipids in the growth and secretion phenotypes of the snc mutant cells (see below) cells, Marash and Gerst found that treating the cells with ceramide precursors or analogs restores normal growth at restrictive temperatures [72]. These results suggest that sphingolipids (ceramide and PHS) regulate the dephosphorylation event required for membrane fusion during exocytosis.

Another study showed that the mutant strain M42A snc2Δ [73], in which methionine 42 in snc1p is changed to alanine and the SNC2 gene is deleted, also has defective exocytosis. The SNC1 and SNC2 genes encode snc1p and snc2p respectively, which are archetypal v-SNARES involved in the exocytosis process [68, 69, 71, 74]. The mutants cease growth at 38°C, and at 37°C they accumulate secretory vesicles and show defective protein secretion. High copy suppressor screening showed that overexpression of the DPL1 gene (encoding dihydrosphingosine phosphate lyase) restores the growth of this mutant at 38°C. Therefore, the enhanced degradation of DHS-1P mediated by the overexpressed DPL1 gene product probably downregulates DHS-1P-mediated signaling, which leads to growth restoration. Alternatively, DHS-1P degradation could induce compositional changes of the plasma membrane that contribute to growth restoration.

In this context, it is worth noting that, in Drosophila sphingosine 1 phosphate lyase (S1P lyase) mutants, pathology of the membrane structure has been invoked as a probable cause of the reduced viability and fertility and of abnormality in the dorsal longitudinal muscle [75]. In addition, Dictyostelium discoideum S1P lyase mutants are resistant to cis-platin treatment, have defects in their ability to form migrating slugs, and show defects in F-actin distribution, again linking sphingolipid metabolism with membrane dynamics [76, 77].

S1P lyase (called SGPL in mouse) has been identified as an immediate early gene (IEG) product of PDGF activation [78]. In a recent report on mouse knock-out lines for twelve of these IEGs, including SGPL, all the lines showed similar phenotypes, in spite of the disparate functions ascribed to the individual proteins. Although SGPL mice are viable at birth, they all die by about 8 wks of age. They show reduced gains in weight and size after birth, compared with normal mice. The SGPL mutants also show hemorrhages and microaneurysms at birth and are anemic by six weeks of age. Their blood contains circulating immature blood cells, probably due to the chronic blood loss. SGPL mutants also have a high blood urea concentration and glomerular and other renal defects, along with an apparent failure in the terminal differentiation of the kidney. The skeletal system shows defects in bone development and in ossification. Finally, fibroblasts from these mice also show reduced cell migration after scratch induction [79]. Additional information about the secretory functions of cell lines from SGPL mutant mice could help shed light on the physiological role of sphingolipids.

Defective protein sorting can also lead to defects in exocytosis. In a study designed to isolate mutants of non-essential genes that are required for the sorting of proteins to the plasma membrane, mutations in four of the ten genes involved in ceramide biosynthesis, elo3, SurP, YPC1, and Ayr1 were identified (the other five genes of the pathway are essential genes and were not evaluated in this screen) [80]. Mutants of elo3, which generates a C26:0 fatty acid, cannot synthesize long-chain fatty acids for ceramide biosynthesis and mis-sort cargo to the vacuole instead of the plasma membrane. A mutation in the SurP protein affects the hydroxylation of the sphingosine backbone, and these mutants accumulate cargo in the trans-Golgi network. Mutants of YPC1 (encoding a ceramidase) show defects in the exit of the cargo protein from the Golgi. Mutation in Ayr1p (which has 3-ketoreductase activity and could therefore be involved in fatty acid elongation and thus contribute to ceramide synthesis), also affects protein targeting to the plasma membrane. It is not clear if the mistargeting of the sphingolipid metabolites that accumulate in these mutants is caused by general deleterious effects on protein sorting or by changes in the membrane composition of vesicles owing to faulty sphingolipid biosynthesis.

Data from Drosophila also provide evidence for sphingolipid involvement in exocytosis. Phenotype-based genetic screening for mutants with defective synaptic function led to the identification of slab [81]. The slab mutants arrest their growth as fully developed embryos and have severely reduced movement. They display defects in synaptic vesicle fusion and trafficking. Ultrastructural studies indicated an increased tethering of the vesicles to the plasma membrane, and impaired neurotransmission at the neuromuscular junctions was also observed. slab turned out to encode neutral ceramidase, which was described previously, supporting a role for sphingolipids in exocytosis [57, 82]. It has also been hypothesized that ceramide might modulate membrane trafficking, depending on its topological location in the membrane [64]. Conceivably, in the absence of SLAB ceramidase, an imbalance in sphingolipid composition could lead to an altered lipid raft environment and the subsequent impairment of exocytosis, which is required for proper NMJ functioning.

Microdomains and membrane integrity

Studies done in the past decade have recognized the important role of sphingolipids in the organization of localized regions called membrane microdomains (“lipid rafts”). These microdomains have distinguishable phase-separation properties and are enriched in sphingolipids and cholesterol. The preferential distribution of sphingolipids into localized domains occurs because of their unique structural properties: sphingolipids contain OH and amido groups in the ceramide moiety, and these functional groups can act simultaneously both as hydrogen-bond acceptors and as hydrogen-bond donors [83-87]. Hence, sphingolipids can form a compact hydrogen-bonded network. In the case of glycosphingolipids, the sugar residues offer substantial inter-residue binding interactions [88, 89], and polar head group interactions are reported for sphingomyelin as well. In addition, due to favorable Van der Waals interactions, sphingolipids strongly bind cholesterol, which further promotes phase separation [85]. Collectively, these interactions act as the driving force for the lateral segregation of sphingolipids into microdomains. These microdomains are detergent resistant at cold temperatures, and this characteristic is used to isolate microdomains as the detergent-resistant membrane fraction (DRM) [90, 91]. These membrane microdomains can affect cellular functions like calcium homeostasis, endocytosis, and protein sorting and they can influence signaling pathways [92-94].

Some functions of lipid rafts in vivo have been demonstrated in yeast genetic studies. These studies indicate a possible link between lipid raft integrity and the appropriate membrane targeting of proteins. Bagnat et al. studied the sorting of membrane H⁺-ATPase (Pma 1p) to the cell surface as a function of its association with lipid rafts [95]. Pma 1p is an essential plasma membrane protein in yeast that requires an association with lipid rafts to relocate from the Golgi complex to the membrane. In the temperature-sensitive mutant lcb1-100, at restrictive temperatures that impair sphingolipid biosynthesis, the Pma 1p protein fails to reach the cell surface and is degraded. However, this phenotype is rescued at permissive temperatures that coincide with restored sphingolipid biosynthesis [95]. Another study showed that defects in the oligomerization of Pma 1p are attributed to defective sphingolipid biosynthesis, and externally added PHS rescues the phenotype [96]. In addition, in the yeast mutant pma 1-7, in which lipids rafts are perturbed, newly synthesized Pma 1p is mistargeted to the endosomal system for degradation instead of being delivered to the plasma membrane. These studies suggest a model in which the targeting of Pma 1p to the plasma membrane requires its association with lipid rafts, although additional parameters appear to influence it as well [97, 98]

In yeast, sphingolipids are also required for GPI-anchored proteins to associate stably with the membrane. In the lcb1-100 mutant line, GPI-anchored proteins fail to associate with the membrane, behaving instead like peripheral membrane proteins. Furthermore, sphingolipid biosynthesis is required for the transport of GPI-anchored proteins from the ER to the Golgi complex. Exogenously added D-erythro-dihydroceramide partially rescues this phenotype [99].

Studies in Candida albicans (C. albicans) also emphasize a role for lipid microdomains in membrane-mediated events. erg mutants of C. albicans are deficient in ergosterol synthesis and are hypersensitive to antifungal drugs such as fluconazole, ketoconazole, and terbinafine. It was initially believed that the increased plasma membrane fluidity caused by the ergosterol deficiency was responsible for the increased drug sensitivity. However, artificially fluidizing the membrane did not increase the mutant cells’ susceptibility to the drugs, indicating that increased membrane fluidity could not be solely responsible. However, the drug susceptibilities of the erg mutants to squalene synthase were further sensitized by perturbations in the sphingolipid metabolism caused by fumonisin 1B, an inhibitor of ceramide synthase. The authors suggest that a stable interaction between ergosterol and sphingolipid is essential to maintain the stability of sphingolipid-rich microdomains [100].

Later studies with C. albicans mutants in which the ERG1 gene was conditionally disrupted or the gene for IPT1 (the synthase responsible for generating mannosyl di-inositol diphosphoceramide from mannosyl inositol phosphoceramide) was homozygously disrupted [101] showed that the lack of either gene renders the organism more susceptible to the antifungal drugs compared with wild type. In these mutant backgrounds, the authors also analyzed the surface localization of Cdr1p, a major ABC drug efflux protein that functions in the drug resistance mechanism [102, 103]. In the mutant C. albicans, Cdr1p shows impaired surface localization and defective efflux function. While it is not yet certain that Cdr1p associates with lipid rafts, these results support a model in which the drug efflux protein Cdr1p requires a sphingolipid and ergosterol for its proper localization at the membrane, which is required for it to function.

A Leishmania mutant missing one of the two subunits (SPT2) of serine palmitoyltransferase (SPT) is completely deficient in de novo sphingolipid biosynthesis. This mutant is viable, but its growth rate and doubling time in log phase are compromised. Once it reaches stationary phase, it displays a progressively higher incidence of cell-shape abnormalities and loss of viability. In stationary phase, these parasites display defects in membrane structure such as increased vacuolation, alterations in flagellar surface, and the accumulation of small vesicles reminiscent of multivesicular bodies [104]. The stationary-phase parasites also fail to differentiate into the infective metacyclic stage. In a different study, SPT2-mutant Leishmania amastigotes showed a delay in the onset of infection compared with wild-type amastigotes in vivo. In addition, these mutants show defects in membrane trafficking, and a failure of the virulence factor GP63 to associate with lipid rafts, which subsequently leads to defects in the formation of infective extracellular parasites [105].

Although a large body of work and the genetic evidence point to their significance, the definition of the lipid raft as the detergent-resistant membrane fraction is controversial, given that rafts isolated using different detergents show compositional differences [106]. The use of detergents for isolating and defining lipid rafts presents a contradiction since the detergents themselves induce phase separation in vitro [107]. Nevertheless, the studies conducted in genetic model systems provide us with good indirect evidence on the composition of lipid rafts. The study of lipid rafts encompasses multiple disciplines and requires biochemical, biophysical, and cell biological analyses. Genetic studies can provide additional information that would bridge many of the lacunae in our understanding of these intriguing structures and their role in cell function.

Sphingolipids such as sphingomyelin are considered to be structural components of the membrane. In this role they are thought to assist in the physiological interactions of membranes with their environment. Several lines of evidence indicate a close link between sphingolipid signaling and aging. Studies indicate that sphingolipids accumulate in several tissues in an age-dependent manner [108, 109], and caloric restriction decreases sphingolipid accumulation by decreasing serine palmitoyltransferase (SPT) activity [110]. Recently, we established a link between sphingolipid biosynthesis, membrane integrity, oxidative stress response, and aging in Drosophila [111]

Sphingolipids are synthesized vectorially. The early biosynthetic reactions that lead to the formation of ceramide take place in the ER, and most of the complex sphingolipids (barring some glucosylceramide) are synthesized in the Golgi complex. This necessitates the transport of ceramide from the ER to the Golgi complex. The transport of ceramide is achieved mostly through an active ATP-dependent non-vesicular mode and by a minor ATP-independent pathway that may include vesicular transport [36, 112-114]. Hanada and his colleagues recently discovered a novel protein that is responsible for the former transport mode. It was identified in studies using a mutant chinese hamster ovary (CHO) cell line (LY-A) that is defective in sphingomyelin synthesis. The LY-A cell line, when grown in sphingolipid-deficient media, synthesizes only 40% of the sphingomyelin synthesized by control CHO cells, and this defect is rescued by transfecting the cells with a cDNA clone from a human library. The responsible protein was named CERT [37].

CERT is a 68-Kda protein that possesses ceramide-transfer activity in vitro [37]. The Drosophila genome encodes a CERT homolog that maps to the left arm of the third chromosome (CG7207) at 63C3 and is named Dcert. Like the mammalian protein, it has an N-terminal PH domain followed by a coiled-coil domain with an FFAT tract and a C-terminal START domain. Drosophila lacking a functional Dcert were isolated in a western blot-based genetic screen and named dcert¹ [111, 115]. Mass spectrometric analyses revealed that these flies had a defective sphingolipid metabolism with total CPE and ceramide levels less than 30% of those in wild-type control flies. These flies have a short life span and live only up to 30 days compared with the 90-day life span of wild-type flies.

In an attempt to understand the possible link between decreased CPE synthesis and reduced life span, we investigated the effect of decreased CPE biosynthesis on the plasma membrane. Drosophila CPE, like mammalian SM, localizes to the membrane and has similar phase-distribution properties. We found that the plasma membrane of the mutant flies has compromised physical properties and is more fluid than normal. Plasma membranes harbor unsaturated fatty acids (UFA) and are vulnerable to attack by reactive oxygen species (ROS), which are constantly generated in cells. The sensitivity of the plasma membrane to ROS is regulated by several factors, including the UFA composition. We hypothesized that a less compact (more fluid) plasma membrane would be more accessible to ROS and hence would suffer increased lipid peroxidation. We found that plasma membrane preparations from the mutant flies were susceptible to ferrous-induced lipid peroxidation in vitro, which translated into increased oxidative damage to the protein in vivo. The mutant flies accumulate larger amounts of oxidatively modified proteins with age than do wild-type flies. They also display an age-dependent reduction in thermal tolerance, increased mitochondrial dysfunction, decreased ATP production, and ineffective glucose utilization. These are some of the common features of premature aging [116]. These studies demonstrate that sphingolipids, acting as structural components of the membrane, can influence the sensitivity of UFAs in the plasma membrane to oxidative stress. Thus, perturbations in the integrity of the plasma membrane can have detrimental effects on the stress response and life span.

It is of interest to note here that a mouse model of Niemann-Pick disease C (Table 1) also shows abnormalities of the plasma membrane. Tissues derived from these mice show increased sphingomyelin content and increased ratios of saturated fatty acid to unsaturated fatty acids. These effects are accompanied by decreased plasma membrane fluidity [117].

Table 1.

Mouse Models of Sphingolipidoses

Mouse Models for the Disease	Enzyme Mutated	Phenotype and Features
Gaucher’s	Glucosylceramide-β-glucosidase	Animals die perinatally with accumulation of glucosylceramide in the lysosomes of the reticuloendothelial system [191].
Tay-Sach’s	α- chain of Hexosaminidase	Late onset behavioral changes Accumulation of GM2 in cerebral cortex, hippocampus, amygdala, hypothalamus, mamillary bodies etc [192].
Sandhoff	β- chain of Hexosaminidase	Show progressive motor incoordination and by 5 months almost gravely ill. Accumulation of PAS positive laden cells throughout the CNS [193].
Fabry Disease	Lysosomal α-galactosidase A	Accumulation of Gb3 in liver and Kidneys. Life span not affected till 80 wks [194, 195].
Krabbe Disease	Galactosylceramide-β-galactosidase	Twitcher mouse, die around 42 days. Accumulation of galactosylceramide and psychosine in the oligodendrocytes and Schwann cells with abundant cell death and dysmyelination of nerves. Increase in latency of neurotransmission [196].
Farber’s Disease	Acid-ceramidase	Embryonic lethal around 2 cell stage [197, 198]
Metachromatic leukodystrophy	Arylsulfatase A	Mice turn deaf because of loss of spiral ganglion cells. Reduced galactosylceramide and cholesterol [199, 200].
Niemann-Pick A	Acid Sphingomyelinase	Mice die between 4 and 6 months of age. Accumulation of sphingomyelin in vesicular structures of macrophages and reticuloendothelial cells. Degeneration of Purkinje cell layer of cerebellum that leads to impaired motor coordination [201].
Niemann-Pick C	NPC protein	Mice die around 10 weeks of age. (Accumulates unesterified cholesterol in many tissues. Age related loss of Purkinje cells in the Cerebellum [202]}.
	Neutral ceramidase	Inability of intestinal cells to metabolize dietary ceramide [203].
Sphingolipid activator proteins	Prosaposin (deletes A, B, C and D)	Symptoms early or late onset. Mice die neonatally or around 6 weeks of age. Hypomyelinated nerve fibers and complex sphingolipid accumulation in brain, liver and kidney
	Saposin A	Milder form compared knockout of all saposins
	Saposin C and D	Die by about 56 days but no myelination defect. However, these mice loose all their Purkinje cells by week 6 and accumulate glucosylceramide and α-hydroxy ceramides in brain and kidney reference [204-206]
GalNAcT	β1,4-N-acetylgalactosaminyltransferase	Wallerian degeneration of the myelinated neurons. Accumulation of GM3/GD3 lack of GM2/GD2
	Glucosylceramide synthase	Die during gastrulation due to enhanced apoptosis in the ectoderm. Conditional KO show loss of axonal branching in Purkinje cells [207-209]

Models of Sphingolipidoses and Membrane Biology

Several human inborn errors of metabolism affect sphingolipid metabolism, chiefly that of complex sphingolipids, which leads to the accumulation of sphingolipids in intracellular compartments. These effects can cause degenerative changes in tissues, with devastating clinical implications.

Glycosphingolipids (GSL) are complex sphingolipids that are synthesized from ceramides. They are extended by the sequential addition of sugar moieties and, in mammals, by the addition of sialic acids and in some instances of sulfated galactosylceramides. They have distinct sub-cellular localizations, for example, an apical distribution in the epithelial cells of the intestinal and urinary tracts, suggesting a role in cell polarity [118, 119]. They have also been implicated in vesicular transport along the exocytic and endocytic recycling pathways [120-122]. Mouse models for several human disorders of sphingolipid metabolism have now been generated and used for the molecular dissection of the factors leading to disease states and to evaluate therapeutic strategies.

Gangliosides are complex glycosphingolipids that contain sialic acid. They are most abundant in the central nervous system and are thought to play crucial roles in cellular interactions and the control of cell proliferation [123, 124]. The biosynthesis of gangliosides begins with lactosyl ceramide (Fig. 4). The transfer of sialic acid to lactosyl ceramide forms GM3, which serves as substrate for two different pathways. The product of GD3 synthase action on GM3 is GD3 and that of β1,4-N-acetylgalactosaminyltransferase (also known as GalNAcT EC 2.4.1.92) is GM2 and other complex gangliosides [125].

Figure 4.

Biosynthesis of gangliosides. In this figure we outline the committed steps that lead to the generation of GM3, GM2 and GD2 in mammals.

Table 1 lists some of the knock-out mouse lines for sphingolipid-related genes that have been generated over the last two decades. Many of these mutants manifest at least some of the symptoms seen in human patients, and almost all of them show biochemical defects that are associated with human disorders of sphingolipid metabolism. Broadly speaking, the disease states of the mutant mice result from the accumulation of the sphingolipids upstream of the enzymatic deficiency and result in their death or in tissue dysfunction, particularly of the tissues that show abnormal accumulation of the sphingolipid product(s). Hence, the symptoms manifest as loss of function of the relevant tissue. Although in some cases there is a one-to-one correlation with human symptoms, in others there are not. For example, in the mouse model of Tay-Sach’s disease, the null-mutant mice do not display symptoms like those observed in humans, despite the absence of hexosaminidase A activity. This is because, in mouse, other sialidases can convert GM2 to GA2, which is further degraded by other glycosidases, thus alleviating the symptoms of GM2 accumulation. One note of interest is that many sphingolipidoses result in myelination defects. This is not surprising, given that myelin, the fatty substance that ensheathes vertebrate axons, is rich in sphingolipids, and any impairment in the sphingolipid metabolism can disrupt the synthesis and composition of myelin.

Myelin defects can have a wide variety of consequences. As seen in Table 1, many of the mouse mutants also show defects in myelinated neurons. Galactosyl ceramide is synthesized by the enzyme ceramide galactosyl transferase. Knock-out mice for this gene do not synthesize galactosyl ceramide but can make myelin, possibly by substituting glucosyl ceramide for galactosyl ceramide. However, although the ultrastructural features of the myelin sheath from mutant mice appear normal, myelin function was compromised. The mutant mice exhibit a noticeable tremor at rest that worsens with movement. Electrophysiological data indicated the impaired conduction of nerve impulses, and the action potential parameters suggested that the myelin is defective in its ability to insulate the nerve fiber [126]. Defective sphingolipid metabolism can also affect neuronal function directly. It is interesting to note that while sialyltransferase (GD3 synthase) knockout mice are homozygous viable without clinical defects, a double knockout of β1,4-N-acetylgalactosaminyltransferase and GD3 synthase results in sudden death and susceptibility to seizures, indicating altered membrane properties [125]. GM3 synthase knock-out mice have a hypersensitive insulin response, and progress to hypoglycemia faster than wild-type mice when challenged with parenteral insulin. This result highlights the importance of gangliosides in regulating insulin receptor activity, probably by affecting glycosphingolipid rich micro-domains [15].

Spermatogenesis and fertilization require dynamic membrane remodeling, and sperm membranes have specific lipid requirements for normal function [127, 128]. Therefore, several studies have focused on sperm and fertilization in sphingolipid mutants. Acid sphingomyelinase knock-out mice die by about 6 months of age. They also have aberrations in their sperm membrane. Mature spermatozoa in these mutants show accumulations of sphingomyelin and cholesterol. The mitochondria are depolarized, and the plasma and acrosomal membranes are disrupted. The spermatozoa have an abnormal morphology with kinks and bends and faulty capacitation [129]. Morphologically abnormal sperm has also been detected in heterozygote males with this knock-out mutation [130].

Krabbe disease is caused by the absence of galactocerebrosidase in humans and results in central and peripheral neurodegeneration [131, 132]. The mouse model for Krabbe disease, caused by a defect in the homologous gene, die before the age of mating. Although these mice complete spermiogenesis, they have abnormal sperm. The acrosomes are swollen and the flagellum is angulated. In addition, the acrosomal membranes are redundant, detached from the nucleus, and folded [133].

Homozygous acid ceramidase knockout mice die as embryos and show extensive apoptosis owing to the accumulated ceramide (Table 1). The heterozygotes appear normal, although they too accumulate ceramide over time and show abnormal histopathology. One review points out that the young mice produce a 2:1 ratio of heterozygotes to wild-type progeny from heterozygous intercross matings, but aged mice produced litters with a 10:1 ratio of heterozygotes to wild-type progeny, indicating a reproductive advantage for heterozygotes that increases with age. It is possible that increased ceramide in the heterozygotes leads to secondary changes in the membrane composition of the sperm that favors fertilization [134]. In addition, acrosomal exocytosis is a crucial event in fertilization and is facilitated by ceramide [135].

In ceramide galactosyl transferase mutants, spermatogenesis in the homozygous males is arrested at the late pachytene stage, and the spermatogenic cells degenerate through apoptosis [136].

Sphingolipidoses models in Drosophila

Drosophila glycosphingolipids are synthesized from ceramide but differ from mammalian glycosphingolipids in their complexity, chain length, and the sequence in which sugar residues are added. The core structure of Drosophila glycosphingolipids consists of mannosylglucosylceramide (Manb1-4Glcb1-cer). The first step in the synthesis is catalyzed by glucosylceramide synthase. RNAi knockdown of this enzyme leads to increased apoptosis in Drosophila [137]. The Drosophila glycosyltransferases encoded by egghead (egh) and brainiac (brn) are responsible for the 2^nd and 3^rd steps of GSL sugar chain elongation, respectively. Egghead is a β-4-mannosyltransferase that adds mannose to GlcCer, and Brn is a β3-N-acetylglucosaminyltransferase that elongates the chain by adding GlcNAc to synthesize the glycosphingolipid core structure, GlcNAcβ1-3Manβ1-4Glcβ1-Cer (N3, triglycosylceramide), which is found widely in invertebrates [138, 139]. Both mutants show defects in epithelial morphogenesis during oogenesis and embryogenesis. They demonstrate abnormal neurogenesis, compound egg chambers, and a dorsal appendage fusion phenotype [140, 141]. Their absence leads to the loss of elongated glycosphingolipids and the accumulation of truncated precursor glycosphingolipids [138]. These mutants die as pupae, and those lacking the maternal gene product die as embryos with incorrect specification of epidermal and neural cell fates. The absence of these genes also affects ovarian follicles, which lose their apical-basal polarity and become surrounded by multiple layers of accumulated follicular cells. It has been proposed that the products of these genes are required for mediating the morphogenesis of the follicle cells by regulating germline-follicle cell adhesion [140]. Glycosyltransferase β1,4-N-acetylgalactosaminyltransferase-A (β4GalNAcTA)- and glycosyltransferase β1,4-N-acetylgalactosaminyltransferase-B (β4GalNAcTB)-null mutants have been generated in Drosophila [142]. These two proteins catalyze the fourth step in Drosophila GSL biosynthesis, by adding N-acetylgalactosamine to N3. The null mutants of β4GalNAcTA have an abnormal locomotion phenotype, i.e., larval crawling defects, a reduction in the nerve terminal bouton number in larval neuromuscular junctions, and defects in spontaneous neurotransmitter release, indicating an inefficient secretory function [143]. The β4GalNAcTB mutant, on the other hand, has no apparent phenotypes, but shows abnormal oogenesis in a third of the ovarioles, with degenerative changes from stages 6 to 9 [144]. Fifty percent of the eggs from the remaining ovarioles show a dorsal appendage fusion phenotype, milder than in egg and brn, which is a sign of defective EGFR signaling between the germ-line and the follicle cells with the defect in the oocyte. Studies suggest that N3 is absolutely critical for survival, but its subsequent elongation, though not critical, is still important for the reproductive capability of the flies.

Sphingolipids as ligands for membrane receptors

In addition to their functions as membrane components and as signaling molecules inside cells, sphingolipids, specifically S1P, can act as extracellular ligand. S1P regulates cell migration and trafficking, also acts as a chemo attractant for migrating cells, and modulates permeability barriers (probably by engaging specific receptors on the target cell surface). S1P is a second messenger molecule with both intra- and extra-cellular functions. Its signal is important for cell growth, differentiation, and organismal development. In mammals, the serum concentrations of S1P are fairly high and believed to be derived from red blood cells and platelets, among others [145].

S1P precursors are found in eukaryotes, and sphingosine kinase, S1P, and sphingosine phosphate lyase are found in yeast, humans, and plants. However, a recognizable S1P receptor has been identified only in chordates and vertebrates [146]. Thus, the evolution of S1P into a signaling ligand at the cell surface is of recent origin and might contribute to the sophistication and complexity of these phyla. S1P is generated by the phosphorylation of sphingosine by sphingosine kinase (SphK) [147, 148]. Because recent reviews have dealt extensively with the S1P family receptors we will make only brief mention of them here, focusing on the findings of in vivo studies with an emphasis on membrane biology [149, 150].

Overview of S1P Family of Receptors

Hla and Maciag found that, following PMA stimulation, human endothelial cells produced abundant mRNA for EDG-1 (now recognized as S1P₁), and they suggested a role for this receptor in the differentiation of endothelial cells [151]. Subsequent studies established that at least in chordates and mammals, S1P act as extracellular ligands for a family of G-protein coupled receptors called S1P receptors [152-154]. In mammals, S1P executes most of its extracellular functions by engaging these receptors. There are five members of this family, designated S1P₁ - S1P₅, and knock-out lines are available for four of them [146, 155, 156]. S1P₁, S1P₂, and S1P₃ show a widespread distribution in various cell and tissue types, whereas S1P₄ and S1P₅ apparently are more restricted in their distribution pattern and are found mainly in the hematopoeitic system [157]. Although all these receptors are activated by S1P binding, studies with receptor inhibitors suggest variations in the S1P binding site among these receptors [152, 158, 159]. There are also differences in the coupling of receptor activation to downstream G proteins [160-163]. S1P ₁ couples to G_i, S1P₂ and S1P₃ couple to Gi, Gq, and G₁₃. S1P₄ couples to Gi and G₁₃ and S1P₅ couples to G_i and G₁₂. G₀/G_i mediate the activation of Ras, Rac, ERK, PI3K and AKT. They can facilitate endothelial barrier function, induce vasodilation and cause migration. These signals also activate PKC and PLC to increase intracellular calcium while concomitantly inhibiting cAMP. When coupled to G_q, the S1P receptors activate PLC. G₁₂/₁₃ activate Rho and ROCK that can inhibit migration, decrease the effectiveness of the endothelial barrier and cause vasoconstriction. Many effects of the S1P receptor signaling pathways could occur via the regulation of actin assembly and disassembly. The coupling of S1P₁ or S1P₃ with G₀ or G_i activates the Rac pathway, which can lead to cell polarization, lamellipodium formation and expansion, organization of focal complexes at the frontal edge of the cell, and adhesion. S1P₂ or S1P₃ coupled with G₁₂/₁₃ stimulates Rho/ROCK, which cause the cells to produce stress fibers and to detach from the substrate. Therefore Rac and Rho are utilized in a concerted fashion to affect migration, the formation and dissociation of focal complexes, and cell-cell adhesion. Endothelial cells express S1P₁ and that S1P₁ is critical for the interactions of endothelial cells with mural cells in the maturation of blood vessels, the failure of which leads to hemorrhage and death in S1P₁-null mice [164]. The mechanism is through S1P₁ activation by S1P, which activates the Rac GTPase (via G_i), which utilizes microtubules to transport N-cadherin to the cell surface where it interacts with the mural cells. The S1P₁ expressed on the mural cells is irrelevant for this function. S1P₁ also stabilizes the cadherin-cadherin interaction by promoting the association of α-, β-, and p120-catenin with N-cadherin and the underlying actin filaments. S1P₁ has also been implicated in cardiovascular and neuronal development, angiogenesis, tonicity of blood vessels, vascular barrier functions, and gap-junction formation and maintenance [165-167]. Through its association with the Rho and Rac family of G-proteins, the S1P₁ receptor induces the formation of cortical actin rings, enforces the formation of cell junction assembly, induces stress fibers, and perturbs or strengthens cell-cell junctions. Thus, fundamental aspects of membrane biology are affected by the S1P-S1P₁ receptor interaction, which is mostly mediated through the actin cytoskeleton.

In an important study, Lee et al. showed that the engagement of S1P₁ (and probably S1P₃) leads to the recruitment of VE-Cadherin, and α-, β-, and γ-catenin to cell-cell junctions as well as a dramatic increase in actin stress fibers and cortical actin structures in endothelial cells [168]. They showed it also promotes the homophilic binding of intercellular cadherins and the attachment of these structures to the cytoskeleton. Rac localization at the cell-cell contact area also increases. Several lines of evidence now indicate that S1P activates the Rac pathway through G-protein coupled receptor activation, resulting in the cortical assembly of actin, and that this process of cytoskeletal reorganization is an important event in angiogenesis [168-170] [171]. Lee et al., also found that the S1P-induced assembly of focal contact sites was Rho dependent on three different extracellular matrices. Interestingly, Rac was not required for the initial focal contact assembly and cell spreading. (Rho can be activated in non-adherent human umbilical vein endothelial cells (HUVECs), whereas Rac requires cell adhesion.) Direct measurement showed that both S1P₁ and S1P₃ can activate both Rho and Rac. In addition, integrin alpha-5 beta 3 and 1 were shown to be required for HUVEC migration and morphogenesis in coordination with S1P₁ and S1P₃ activation. This pioneering study laid the groundwork for future studies on S1P receptor knock-out mouse lines.

Thus S1P receptors, activated by binding S1P, control several aspects of endothelial cell function that are important for angiogenesis [172, 173].

Mouse models

S1P₁ knock-out mouse embryos are normal up to E11.5. On E12.5, the embryos exhibit abnormal yolk sacs, are edematous, and lack blood. They die between E12.5 and E14. These findings suggest that the S1P₁ expressed on these cells may play a role in regulating the adhesion of endothelial cells and pericytes [174]. S1P is also believed to be an important signaling molecule in the regulation of cell-cell junction formation, by organizing actin into a strong cortical ring and reinforcing cell-matrix adherence [168, 175]. Thus, S1P is likely to regulate vascular permeability by maintaining the integrity of the vascular barrier.

Lymphocyte egress (a form of chemotaxis) is thought to be mediated by S1P₁ and probably by S1P₄ [145, 176]. Convincing evidence from studies with S1P₁-deficient mice have demonstrated that ligation of the S1P₁ receptor by S1P is critical for the re-entry of T lymphocytes from secondary lymphoid organs to peripheral tissues, where inflammation takes place [176, 177]. S1P increases intracellular calcium by activating the S1P₂ receptor and induces smooth muscle cell contraction [178, 179].

S1P₂ receptor-null mice are apparently normal at birth, with no anatomical defects, but between weeks 3 and 7, 59% of them are affected by sporadic seizures that are occasionally lethal (14% die) [180]. Interestingly, a mutation in a similar gene in zebrafish leads to embryonic lethality [181]. Therefore S1P₂ receptors could affect either microscopic development or membrane excitability by affecting intracellular calcium. The S1P₃ receptor is highly expressed in the heart, lung, spleen, kidney, intestine, diaphragm, and certain cartilaginous tissues. Mice null for this gene do not show any overt phenotype. S1P₄ is expressed mostly in immune compartments, and no knock-out has been reported. S1P₅ is expressed in the CNS, and knock-outs show no obvious phenotype.

The concerted action of S1P₁, S1P₂, and S1P₃ is required for endothelial barrier function [149, 171, 182, 183]. S1P₃ receptors play a prominent role in the regulation (slowing) of the heart rate [184]. The S1P-mediated signaling pathway also assumes importance in driving the migration of cells. Likewise, the S1P₂ and S1P₃ receptors, functioning in concert with the S1P₁ receptor, participate in the formation of an intact embryonic vasculature, as demonstrated by the hemorrhage caused by their simultaneous genetic deletion [185].

Sphingosine Kinases and S1P

Serum S1P must be mostly derived from the action of Sphingosine Kinase 1 (SphK1), since SphK1 null mice have substantially lower levels of circulating S1P [186]. Also, unlike what was previously thought, SPHK1 does not have a determining role in inflammatory responses [187]. Furthermore, the loss of either SPHK1 or SPHK2 alone does not lead to developmental defects in mice. SphK1 knock-out mice are viable, fertile, and have no obvious developmental abnormalities [165, 186]. SphK2^-/- mice are viable and sub-fertile [188]. Two recent reports have shown that SPHK2 is involved in the phosphorylation of the immune modulator FTY720 (a sphingosine analog) and that SPHK2 null mice do not respond to this drug by developing lymphopenia, as normal mice do [188, 189]. In addition, circulating S1P (generated mostly by SphK1) and intrinsic mast cell SPHK2 together determine the anaphylactic response, a phenomenon that involves degranulation, which is a specialized form of exocytosis. SphK2-null mice show defective PKC translocation, NF-κB activation, and calcium influx, which are all part of the regular anaphylactic response [190]. SphK1^-/- SphK2^-/- double mutants die as embryos, although the embryos appear normal until E9.5. By E11.5 and E12.5, however, all the embryos exhibit cranial hemorrhage and none survive beyond E13.5. S1P is undetectable in embryonic homogenates and no accumulation of sphingosine is observed. The vascular defects in these double-knock out embryos are prominent: they exhibit a poorly developed dorsal aortal wall, and the endothelial cells in all the blood vessels are severely vacuolated. The embryos also show mesenchymal defects, cranial neural tube defects, and disrupted cell-cell junctions [165]. These phenotypes are similar to those seen in S1P₁ receptor knock-out mice.

Conclusions

Biochemical studies have implicated sphingolipids in membrane-mediated processes, and studies in yeast have further strengthened this concept. In the last fifteen years, animal knock-out lines for sphingolipid genes and receptors have become available. Most studies have concentrated on gross phenotypic analyses and the functional consequences of these mutations. In some of these mutant lines, however, the focus has now shifted from phenotypic observations to cell biological studies. In the near future, the molecular changes for all the available sphingolipid-related knock-out lines will be dissected. These studies will greatly elucidate how sphingolipids contribute to membrane biology and intracellular signaling.