Regulation of neural progenitor cell motility by ceramide and potential implications for mouse brain development.
ABSTRACT: We provide evidence that the sphingolipid ceramide, in addition to its pro-apoptotic function, regulates neural progenitor (NP) motility in vitro and brain development in vivo. Ceramide (N-palmitoyl d-erythro sphingosine and N-oleoyl d-erythro sphingosine) and the ceramide analog N-oleoyl serinol (S18) stimulate migration of NPs in scratch (wounding) migration assays. Sphingolipid depletion by inhibition of de novo ceramide biosynthesis, or ceramide inactivation using an anti-ceramide antibody, obliterates NP motility, which is restored by ceramide or S18. These results suggest that ceramide is crucial for NP motility. Wounding of the NP monolayer activates neutral sphingomyelinase indicating that ceramide is generated from sphingomyelin. In membrane processes, ceramide is co-distributed with its binding partner atypical protein kinase C zeta/lambda (aPKC), and Cdc42, alpha/beta-tubulin, and beta-catenin, three proteins involved in aPKC-dependent regulation of cell polarity and motility. Sphingolipid depletion by myriocin prevents membrane translocation of aPKC and Cdc42, which is restored by ceramide or S18. These results suggest that ceramide-mediated membrane association of aPKC/Cdc42 is important for NP motility. In vivo, sphingolipid depletion leads to ectopic localization of mitotic or post-mitotic neural cells in the embryonic brain, while S18 restores the normal brain organization. In summary, our study provides novel evidence that ceramide is critical for NP motility and polarity in vitro and in vivo.
Project description:The primary cilium is an important sensory organelle, the regulation of which is not fully understood. We found that in polarized Madin-Darby Canine Kidney cells, the sphingolipid ceramide is specifically distributed to a cis-Golgi compartment at the base of the primary cilium. This compartment immunostained for the centrosome marker gamma-tubulin, the Rho type GTPase cell division cycle 42 (Cdc42), and atypical protein kinase Czeta/lambda (aPKC), a kinase activated by ceramide and associated with a polarity protein complex consisting of partitioning defective (Par)6 and Cdc42. Inhibition of ceramide biosynthesis with Fumonisin B1 prevented codistribution of aPKC and Cdc42 in the centrosomal/pericentriolar compartment and severely impaired ciliogenesis. Cilium formation and codistribution of aPKC and Cdc42 were restored by incubation with N-acetyl or N-palmitoyl sphingosine (C2 or C16 ceramide), or the ceramide analog N-oleoyl serinol (S18). Cilium formation was also restored by the glycogen synthase kinase-3beta (GSK-3beta) inhibitor indirubin-3-monoxime, suggesting that regulation of ciliogenesis depends on the inhibition of GSK-3beta by ceramide-activated aPKC. Consistently, inhibition of aPKC with a pseudosubstrate inhibitor prevented restoration of ciliogenesis by C2 ceramide or S18. Our data show for the first time that ceramide is required for primary cilium formation.
Project description:We show here that human embryonic stem (ES) and induced pluripotent stem cell-derived neuroprogenitors (NPs) develop primary cilia. Ciliogenesis depends on the sphingolipid ceramide and its interaction with atypical PKC (aPKC), both of which distribute to the primary cilium and the apicolateral cell membrane in NP rosettes. Neural differentiation of human ES cells to NPs is concurrent with a threefold elevation of ceramide-in particular, saturated, long-chain C16:0 ceramide (N-palmitoyl sphingosine) and nonsaturated, very long chain C24:1 ceramide (N-nervonoyl sphingosine). Decreasing ceramide levels by inhibiting ceramide synthase or neutral sphingomyelinase 2 leads to translocation of membrane-bound aPKC to the cytosol, concurrent with its activation and the phosphorylation of its substrate Aurora kinase A (AurA). Inhibition of aPKC, AurA, or a downstream target of AurA, HDAC6, restores ciliogenesis in ceramide-depleted cells. Of importance, addition of exogenous C24:1 ceramide reestablishes membrane association of aPKC, restores primary cilia, and accelerates neural process formation. Taken together, these results suggest that ceramide prevents activation of HDAC6 by cytosolic aPKC and AurA, which promotes acetylation of tubulin in primary cilia and, potentially, neural processes. This is the first report on the critical role of ceramide generated by nSMase2 in stem cell ciliogenesis and differentiation.
Project description:We show that in Madin-Darby canine kidney (MDCK) cells, an apical ceramide-enriched compartment (ACEC) at the base of primary cilia is colocalized with Rab11a. Ceramide and Rab11a vesicles isolated by magnetic sorting contain a highly similar profile of proteins (atypical protein kinase C [aPKC], Cdc42, Sec8, Rab11a, and Rab8) and ceramide species, suggesting the presence of a ciliogenic protein complex associated with ceramide at the ACEC. It is intriguing that C16 and C18 ceramide, although less abundant ceramide species in MDCK cells, are highly enriched in ceramide and Rab11a vesicles. Expression of a ceramide-binding but dominant-negative mutant of aPKC suppresses ciliogenesis, indicating that the association of ceramide with aPKC is critical for the formation of this complex. Our results indicate that ciliogenic ceramide is derived from apical sphingomyelin (SM) that is endocytosed and then converted to the ACEC. Consistently, inhibition of acid sphingomyelinase with imipramine disrupts ACEC formation, association of ciliogenic proteins with Rab11a vesicles, and cilium formation. Ciliogenesis is rescued by the histone deacetylase (HDAC) inhibitor trichostatin A, indicating that ceramide promotes tubulin acetylation in cilia. Taken together, our results suggest that the ACEC is a novel compartment in which SM-derived ceramide induces formation of a ciliogenic lipid-protein complex that sustains primary cilia by preventing deacetylation of microtubules.
Project description:We reported that amyloid ? peptide (A?42) activated neutral SMase 2 (nSMase2), thereby increasing the concentration of the sphingolipid ceramide in astrocytes. Here, we show that A?42 induced mitochondrial fragmentation in wild-type astrocytes, but not in nSMase2-deficient cells or astrocytes treated with fumonisin B1 (FB1), an inhibitor of ceramide synthases. Unexpectedly, ceramide depletion was concurrent with rapid movements of mitochondria, indicating an unknown function of ceramide for mitochondria. Using immunocytochemistry and super-resolution microscopy, we detected ceramide-enriched and mitochondria-associated membranes (CEMAMs) that were codistributed with microtubules. Interaction of ceramide with tubulin was confirmed by cross-linking to N-[9-(3-pent-4-ynyl-3-H-diazirine-3-yl)-nonanoyl]-D-erythro-sphingosine (pacFACer), a bifunctional ceramide analog, and binding of tubulin to ceramide-linked agarose beads. Ceramide-associated tubulin (CAT) translocated from the perinuclear region to peripheral CEMAMs and mitochondria, which was prevented in nSMase2-deficient or FB1-treated astrocytes. Proximity ligation and coimmunoprecipitation assays showed that ceramide depletion reduced association of tubulin with voltage-dependent anion channel 1 (VDAC1), an interaction known to block mitochondrial ADP/ATP transport. Ceramide-depleted astrocytes contained higher levels of ATP, suggesting that ceramide-induced CAT formation leads to VDAC1 closure, thereby reducing mitochondrial ATP release, and potentially motility and resistance to A?42 Our data also indicate that inhibiting ceramide generation may protect mitochondria in Alzheimer's disease.
Project description:Sphingolipids are bioactive lipids associated with cellular membranes and plasma lipoproteins, and their synthesis and degradation are tightly regulated. We have previously determined that low plasma concentrations of certain ceramide species predict the development of nephropathy in diabetes patients with normal albumin excretion rates at baseline. Herein, we tested the hypothesis that altering the sphingolipid content of circulating lipoproteins can alter the metabolic and signaling pathways in podocytes, whose dysfunction leads to an impairment of glomerular filtration. Cultured human podocytes were treated with lipoproteins from healthy subjects enriched in vitro with C16 ceramide, or D-erythro 2-hydroxy C16 ceramide, a ceramide naturally found in skin. The RNA-Seq data demonstrated differential expression of genes regulating sphingolipid metabolism, sphingolipid signaling, and mTOR signaling pathways. A multiplex analysis of mTOR signaling pathway intermediates showed that the majority (eight) of the pathway phosphorylated proteins measured (eleven) were significantly downregulated in response to C16 ceramide-enriched HDL2 compared to HDL2 alone and hydroxy ceramide-enriched HDL2. In contrast, C16 ceramide-enriched HDL3 upregulated the phosphorylation of four intermediates in the mTOR pathway. These findings highlight a possible role for lipoprotein-associated sphingolipids in regulating metabolic and signaling pathways in podocytes and could lead to novel therapeutic targets in glomerular kidney diseases.
Project description:Genetic models for studying localized cell suicide that halt the spread of pathogen infection and immune response activation in plants include Arabidopsis accelerated-cell-death 11 mutant (acd11). In this mutant, sphingolipid homeostasis is disrupted via depletion of ACD11, a lipid transfer protein that is specific for ceramide 1-phosphate (C1P) and phyto-C1P. The C1P binding site in ACD11 and in human ceramide-1-phosphate transfer protein (CPTP) is surrounded by cationic residues. Here, we investigated the functional regulation of ACD11 and CPTP by anionic phosphoglycerides and found that 1-palmitoyl-2-oleoyl-phosphatidic acid or 1-palmitoyl-2-oleoyl-phosphatidylglycerol (?15 mol %) in C1P source vesicles depressed C1P intermembrane transfer. By contrast, replacement with 1-palmitoyl-2-oleoyl-phosphatidylserine stimulated C1P transfer by ACD11 and CPTP. Notably, "soluble" phosphatidylserine (dihexanoyl-phosphatidylserine) failed to stimulate C1P transfer. Also, none of the anionic phosphoglycerides affected transfer action by human glycolipid lipid transfer protein (GLTP), which is glycolipid-specific and has few cationic residues near its glycolipid binding site. These findings provide the first evidence for a potential phosphoglyceride headgroup-specific regulatory interaction site(s) existing on the surface of any GLTP-fold and delineate new differences between GLTP superfamily members that are specific for C1P versus glycolipid.
Project description:Ceramide is a bioactive sphingolipid with many associated biological outcomes, yet there is a significant gap in our current understanding of how ceramide mediates these processes. Previously, ceramide has been shown to activate protein phosphatase (PP) 1 and 2A. While continuing this line of work, a late fraction from a Mono-Q column was consistently observed to be activated by ceramide, yet PP1 and PP2A were undetectable in this fraction. Proteomic analysis of this fraction revealed the identity of the phosphatase to be PP2C?/PPM1G. This was consistent with our findings that PP2C? 1-eluted in a high salt fraction due to its strongly acidic domain, and 2-was insensitive to okadaic acid. Further characterization was performed with PP2C?, which showed robust activation by C(6)-ceramide. Activation was specific for the erythro conformation of ceramide and the presence of the acyl chain and hydroxyl group at the first carbon. In order to demonstrate more physiological activation of PP2C? by ceramide, phospho-p38? was utilized as substrate. Indeed, PP2C? induced the dephosphorylation of p38? only in the presence of C(16)-ceramide. Taken together, these results show that the PP2C family of phosphatases is activated by ceramide, which may have important consequences in mediating the biological effects of ceramide.
Project description:In kidney MDCK and intestinal Caco-2 epithelial cells, glucosylceramide (GlcCer) and sphingomyelin (SPH) synthesized from the short-chain sphingolipid analogue N-6-[7-nitro-2,1,3-benzoxadiazol-4-yl]aminodecanoyl (C6-NBD)-ceramide are delivered to the cell surface with apical/basolateral polarities of 2-4 and 0.6-0.9 respectively. We have tested how variations in the lipid backbone affect these polarities. First, the C6-NBD moiety was replaced by a bare [14C]octanoyl chain or by the even more bulky fluorophores 8-bimanoylthio-octanoyl (C8-bimane) and 8-diethylaminocoumarin-octanoyl (C8-DECA). In addition, the sphingosine in C6-NBD-ceramide was changed in stereoconfiguration (L-threo) or saturation (dihydro). In all cases, GlcCer and SPH were produced and appeared on the cell surface at 37 degrees C, as assayed by back-exchange. The apical/basolateral polarity of the delivery of GlcCer was variable, but always exceeded 1. GlcCer was apically enriched over SPH (2-6 times for MDCK and 3-9 times for Caco-2). Even GlcCer synthesized from a highly water-soluble truncated ceramide (octanoyl-D-erythro-sphingosine analogue with C8 backbone) was enriched apically by a factor of greater than or equal to 2 both in absolute polarity and compared with SPH. Sphingolipid sorting was quantitatively but not qualitatively affected by dramatic changes in the lipid backbone.
Project description:Targeting cell motility, which is required for dissemination and metastasis, has therapeutic potential for ovarian cancer metastasis, and regulatory mechanisms of cell motility need to be uncovered for developing novel therapeutics. Invasive ovarian cancer cells spontaneously formed protrusions, such as lamellipodia, which are required for generating locomotive force in cell motility. Short interfering RNA screening identified class II phosphatidylinositol 3-kinase C2? (PI3KC2?) as the predominant isoform of PI3K involved in lamellipodia formation of ovarian cancer cells. The bioactive sphingolipid ceramide has emerged as an antitumorigenic lipid, and treatment with short-chain C6-ceramide decreased the number of ovarian cancer cells with PI3KC2?-driven lamellipodia. Pharmacological analysis demonstrated that long-chain ceramide regenerated from C6-ceramide through the salvage/recycling pathway, at least in part, mediated the action of C6-ceramide. Mechanistically, ceramide was revealed to interact with the PIK-catalytic domain of PI3KC2? and affect its compartmentalization, thereby suppressing PI3KC2? activation and its driven cell motility. Ceramide treatment also suppressed cell motility promoted by epithelial growth factor, which is a prometastatic factor. To examine the role of ceramide in ovarian cancer metastasis, ceramide liposomes were employed and confirmed to suppress cell motility in vitro. Ceramide liposomes had an inhibitory effect on peritoneal metastasis in a murine xenograft model of human ovarian cancer. Metastasis of PI3KC2? knocked-down cells was insensitive to treatment with ceramide liposomes, suggesting specific involvement of ceramide interaction with PI3KC2? in metastasis suppression. Our study identified ceramide as a bioactive lipid that limits PI3KC2?-governed cell motility, and ceramide is proposed to serve as a metastasis-suppressor lipid in ovarian cancer. These findings could be translated into developing ceramide-based therapy for metastatic diseases.
Project description:Stem cell differentiation is accompanied by a gradual cellular morphogenesis and transcriptional changes. Identification of morphological regulators that control cell behavior during differentiation could shed light on how cell morphogenesis is coupled to transcriptional changes during development. By analyzing cellular behavior during differentiation of mouse embryonic stem cells (ESCs), we uncover a role of Borg5 (binder of Rho guanosine 5'-triphosphatase 5) in regulating trophectoderm (TE) cell morphogenesis. We report that differentiation of ESCs toward TE is accompanied by enhanced actin protrusion and cell motility that require upregulation of Borg5. Borg5 interacts with both Cdc42 and atypical protein kinase C (aPKC) and functions downstream of Cdc42 to enhance TE cell motility. Borg5 is required for the sorting of differentiating TE to the outside of ESCs in vitro. In developing embryos, Borg5 protein localizes to cell-cell contacts and the cytoplasm after compaction. It exhibits higher levels of expression in outer cells than in inner cells in morula and blastocysts. Reduction of Borg5 disrupts aPKC localization and inhibits blastocyst formation. Since Cdx2 and Borg5 facilitate each other's expression as ESCs differentiate toward TE, we propose that cell morphogenesis is coupled with transcriptional changes to regulate TE differentiation. Our studies also demonstrate the utility of ESCs in identifying morphological regulators important for development.