Glycosphingolipid metabolic reprogramming drives neural differentiation
2017; Springer Nature; Volume: 37; Issue: 7 Linguagem: Inglês
10.15252/embj.201797674
ISSN1460-2075
AutoresDomenico Russo, Floriana Della Ragione, Riccardo Rizzo, Eiji Sugiyama, Francesco Scalabrì, Kei Hori, Serena Capasso, Lucia Sticco, Salvatore Fioriniello, Roberto De Gregorio, Ilaria Granata, Mario Rosario Guarracino, Vittorio Maglione, Ludger Johannes, Gian Carlo Bellenchi, Mikio Hoshino, Mitsutoshi Setou, Maurizio D’Esposito, Alberto Luini, Giovanni D’Angelo,
Tópico(s)Pluripotent Stem Cells Research
ResumoArticle27 December 2017free access Transparent process Glycosphingolipid metabolic reprogramming drives neural differentiation Domenico Russo Institute of Protein Biochemistry, National Research Council, Naples, Italy Search for more papers by this author Floriana Della Ragione Institute of Genetics and Biophysics, National Research Council, Naples, Italy IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Riccardo Rizzo Institute of Protein Biochemistry, National Research Council, Naples, Italy Search for more papers by this author Eiji Sugiyama International Mass Imaging Center, Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan Search for more papers by this author Francesco Scalabrì Institute of Genetics and Biophysics, National Research Council, Naples, Italy IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Kei Hori Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Search for more papers by this author Serena Capasso Institute of Protein Biochemistry, National Research Council, Naples, Italy Istituto di Ricovero e Cura a Carattere Scientifico-SDN, Naples, Italy Search for more papers by this author Lucia Sticco Institute of Protein Biochemistry, National Research Council, Naples, Italy Search for more papers by this author Salvatore Fioriniello Institute of Genetics and Biophysics, National Research Council, Naples, Italy Search for more papers by this author Roberto De Gregorio Institute of Genetics and Biophysics, National Research Council, Naples, Italy Search for more papers by this author Ilaria Granata High Performance Computing and Networking Institute, National Research Council, Naples, Italy Search for more papers by this author Mario R Guarracino High Performance Computing and Networking Institute, National Research Council, Naples, Italy Search for more papers by this author Vittorio Maglione IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Ludger Johannes Chemical Biology of Membranes and Therapeutic Delivery Unit, Institut Curie, INSERM U 1143, CNRS, UMR 3666, PSL Research University, Paris Cedex 05, France Search for more papers by this author Gian Carlo Bellenchi Institute of Genetics and Biophysics, National Research Council, Naples, Italy Search for more papers by this author Mikio Hoshino Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Search for more papers by this author Mitsutoshi Setou International Mass Imaging Center, Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan Search for more papers by this author Maurizio D'Esposito Institute of Genetics and Biophysics, National Research Council, Naples, Italy IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Alberto Luini Institute of Protein Biochemistry, National Research Council, Naples, Italy Istituto di Ricovero e Cura a Carattere Scientifico-SDN, Naples, Italy Search for more papers by this author Giovanni D'Angelo Corresponding Author [email protected] orcid.org/0000-0002-0734-4127 Institute of Protein Biochemistry, National Research Council, Naples, Italy Istituto di Ricovero e Cura a Carattere Scientifico-SDN, Naples, Italy Search for more papers by this author Domenico Russo Institute of Protein Biochemistry, National Research Council, Naples, Italy Search for more papers by this author Floriana Della Ragione Institute of Genetics and Biophysics, National Research Council, Naples, Italy IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Riccardo Rizzo Institute of Protein Biochemistry, National Research Council, Naples, Italy Search for more papers by this author Eiji Sugiyama International Mass Imaging Center, Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan Search for more papers by this author Francesco Scalabrì Institute of Genetics and Biophysics, National Research Council, Naples, Italy IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Kei Hori Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Search for more papers by this author Serena Capasso Institute of Protein Biochemistry, National Research Council, Naples, Italy Istituto di Ricovero e Cura a Carattere Scientifico-SDN, Naples, Italy Search for more papers by this author Lucia Sticco Institute of Protein Biochemistry, National Research Council, Naples, Italy Search for more papers by this author Salvatore Fioriniello Institute of Genetics and Biophysics, National Research Council, Naples, Italy Search for more papers by this author Roberto De Gregorio Institute of Genetics and Biophysics, National Research Council, Naples, Italy Search for more papers by this author Ilaria Granata High Performance Computing and Networking Institute, National Research Council, Naples, Italy Search for more papers by this author Mario R Guarracino High Performance Computing and Networking Institute, National Research Council, Naples, Italy Search for more papers by this author Vittorio Maglione IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Ludger Johannes Chemical Biology of Membranes and Therapeutic Delivery Unit, Institut Curie, INSERM U 1143, CNRS, UMR 3666, PSL Research University, Paris Cedex 05, France Search for more papers by this author Gian Carlo Bellenchi Institute of Genetics and Biophysics, National Research Council, Naples, Italy Search for more papers by this author Mikio Hoshino Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan Search for more papers by this author Mitsutoshi Setou International Mass Imaging Center, Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan Search for more papers by this author Maurizio D'Esposito Institute of Genetics and Biophysics, National Research Council, Naples, Italy IRCCS INM, Neuromed, Pozzilli, Italy Search for more papers by this author Alberto Luini Institute of Protein Biochemistry, National Research Council, Naples, Italy Istituto di Ricovero e Cura a Carattere Scientifico-SDN, Naples, Italy Search for more papers by this author Giovanni D'Angelo Corresponding Author [email protected] orcid.org/0000-0002-0734-4127 Institute of Protein Biochemistry, National Research Council, Naples, Italy Istituto di Ricovero e Cura a Carattere Scientifico-SDN, Naples, Italy Search for more papers by this author Author Information Domenico Russo1, Floriana Della Ragione2,3, Riccardo Rizzo1, Eiji Sugiyama4, Francesco Scalabrì2,3, Kei Hori5, Serena Capasso1,6, Lucia Sticco1, Salvatore Fioriniello2, Roberto De Gregorio2, Ilaria Granata7, Mario R Guarracino7, Vittorio Maglione3, Ludger Johannes8, Gian Carlo Bellenchi2, Mikio Hoshino5, Mitsutoshi Setou4, Maurizio D'Esposito2,3, Alberto Luini1,6 and Giovanni D'Angelo *,1,6 1Institute of Protein Biochemistry, National Research Council, Naples, Italy 2Institute of Genetics and Biophysics, National Research Council, Naples, Italy 3IRCCS INM, Neuromed, Pozzilli, Italy 4International Mass Imaging Center, Department of Cellular and Molecular Anatomy, Hamamatsu University School of Medicine, Higashi-ku, Hamamatsu, Japan 5Department of Biochemistry and Cellular Biology, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan 6Istituto di Ricovero e Cura a Carattere Scientifico-SDN, Naples, Italy 7High Performance Computing and Networking Institute, National Research Council, Naples, Italy 8Chemical Biology of Membranes and Therapeutic Delivery Unit, Institut Curie, INSERM U 1143, CNRS, UMR 3666, PSL Research University, Paris Cedex 05, France *Corresponding author. Tel: +39 0816132543; E-mail: [email protected] EMBO J (2018)37:e97674https://doi.org/10.15252/embj.201797674 See also: YA Hannun (April 2018) PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Neural development is accomplished by differentiation events leading to metabolic reprogramming. Glycosphingolipid metabolism is reprogrammed during neural development with a switch from globo- to ganglio-series glycosphingolipid production. Failure to execute this glycosphingolipid switch leads to neurodevelopmental disorders in humans, indicating that glycosphingolipids are key players in this process. Nevertheless, both the molecular mechanisms that control the glycosphingolipid switch and its function in neurodevelopment are poorly understood. Here, we describe a self-contained circuit that controls glycosphingolipid reprogramming and neural differentiation. We find that globo-series glycosphingolipids repress the epigenetic regulator of neuronal gene expression AUTS2. AUTS2 in turn binds and activates the promoter of the first and rate-limiting ganglioside-producing enzyme GM3 synthase, thus fostering the synthesis of gangliosides. By this mechanism, the globo–AUTS2 axis controls glycosphingolipid reprogramming and neural gene expression during neural differentiation, which involves this circuit in neurodevelopment and its defects in neuropathology. Synopsis Schematic representation of glycosphingolipid reprogramming circuit in neural differentiation. Globo-series glycosphingolipids inhibit the production of ganglio-series glycosphingolipids. AUTS2 expression is repressed by globo-series glycosphingolipids. AUTS2 activates the promoter of the first and rate limiting enzyme involved in ganglio-series glycosphingolipids production i.e., GM3 synthase by inducing histone acetylation. The globo-AUTS2 axis regulates the expression of neuronal genes during neural differentiation. The decrease of globo-series glycosphingolipids is required for AUTS2 induction and for stem cell differentiation to neural cells. Introduction Glycosphingolipids (GSLs) are glycosylated derivatives of ceramide that are collectively required for embryonic development and have key roles in the modulation of cell response to morphogens (Yamashita et al, 1999; Hakomori, 2008). Glycosphingolipids are synthesised by the stepwise addition of monosaccharides to a specific position on a carbohydrate chain attached to a ceramide backbone. The nature of the added monosaccharide, the linkage point, and the anomeric type of the glycosidic bond all depend on the specific GSL-synthesising enzyme (GSE) involved (D'Angelo et al, 2013a). Thus, different GSEs can act on the same substrate to produce different and, on first approximation, non-inter-convertible GSLs from the same precursor, which results in the fact that vertebrates synthesise GSLs belonging to different metabolic series. The key "decisional" point towards the production of GSLs belonging to specific series is the glycosylation of lactosylceramide (LacCer; D'Angelo et al, 2013a). LacCer is indeed the common substrate of GM2/GA2 synthase (GM2/GA2S) for the production of GA2 (GalNAc-β1-4-LacCer), of GM3 synthase (GM3S) for the production of GM3 (NeuAc-α2-3-LacCer), of Gb3 synthase (Gb3S) for the production of Gb3 (Gal-α1-4-LacCer), and of Lc3 synthase (LC3S) for the production of Lc3 (GlcNAc-β1-3-LacCer; D'Angelo et al, 2013a). GA2, GM3, Gb3, and Lc3 are then precursors for the synthesis of GSLs belonging to the asialo, ganglio, globo/iso-globo, and lacto/neo-lacto-series, respectively (Fig 1A). Figure 1. Schematic representation of GSL metabolism and of neural GSL reprogramming Schematic representation of GSL metabolism (Glc, glucose; Gal, galactose; GlcNAc, N-acetylglucosamine; GalNAc, N-acetylgalactosamine; NeuAc, N-acetylneuraminic acid). Grey circles indicate GSL precursors GlcCer and LacCer, green circles indicate ganglio-series GSLs, red circles indicate globo-series GSLs, cyan circles indicate asialo-series GSLs, and blue circles indicate lacto-series GSLs. Schematic representation of GSL reprogramming in neural differentiation. Stem cells prevalently produce lacto- and globo-series GSLs (left panel), while neurons prevalently produce ganglio- and asialo-series GSLs (right panel). Download figure Download PowerPoint In our previous studies, we have reported that the type of LacCer glycosylation depends on the mode of transport of its precursor glucosylceramide (GlcCer) to dedicated GSE machineries (D'Angelo et al, 2007, 2013b), where the coordinated regulation of GSE expression is the other main contributor towards the synthesis of specific GSLs. The physiological meaning of GSL diversity and of the existence of systems devoted to control their metabolism is still largely elusive. Nevertheless, metabolic channelling mechanisms are exploited during development where the GSL synthetic flux is switched from one metabolic direction to another. The most important example of developmental GSL remodelling is that operating in neural differentiation (Furukawa et al, 2014). Brain development relies on the execution of morphogenetic programmes that begin at early embryonic stages and are protracted throughout the animal life (Stiles & Jernigan, 2010). A central event in this respect is neural differentiation whereby stem cells are committed to neural fate. In neural differentiation, transcriptional, epigenetic, and metabolic programmes are triggered to yield the different neural cell populations (Hamby et al, 2008; Kim et al, 2014; Qiao et al, 2016). Remarkably, during stem cell differentiation to neural cells, the production of GSLs is shifted from globo-series to ganglio-series (Fig 1B; Liang et al, 2010, 2011). This transition is caused by a coordinated change in the expression of GSEs responsible for the production of GSLs belonging to the two metabolic series (Liang et al, 2010, 2011). Thus, the expression of A4GALT (encoding Gb3S) is suppressed while that of ST3GAL5 (encoding GM3S) is induced in neural differentiation (Liang et al, 2010, 2011). Notably, mutations in GM3S resulting in the absence of ganglio-series GSLs (and metabolic redirection towards globo-series GSL production) cause a severe disease in humans (i.e. GM3S deficiency syndrome, OMIM: #609056) characterised by epilepsy, brain atrophy and impaired psychomotor development (Simpson et al, 2004; Fragaki et al, 2013; Lee et al, 2016). Moreover, genetic manipulations in animal models have highlighted a fundamental role for GSLs in brain function (Jennemann et al, 2005; Yamashita et al, 2005). In spite of this evidence, the mechanisms driving GSL reprogramming and its exact role in neurodevelopment are not known. Here, we provide data indicating that the neural GSL reprogramming is internally controlled. We indeed uncover a circuit whereby the globo-series GSLs negatively modulate the expression of the epigenetic regulator of neural gene expression autism susceptibility candidate 2 (AUTS2). AUTS2, in turn, binds and activates GM3S promoter inducing GM3S expression and thus the synthesis of GM3 and of downstream gangliosides. Importantly, AUTS2 controls the transcription of a wide set of neural genes by stimulating histone acetylation at their promoters (Gao et al, 2014; Oksenberg et al, 2014) and, as a consequence, it is required for proper neurodevelopment (Kalscheuer et al, 2007; Bedogni et al, 2010; Beunders et al, 2013, 2016; Oksenberg et al, 2013; Gao et al, 2014). Here, we show that, by suppressing AUTS2, globo-series GSLs reduce histone acetylation at neuronal gene promoters counteracting their expression and therefore neural differentiation. Based on these data, we propose that the GSL metabolic switch drives neuronal gene expression during neural differentiation. Results Globo-series GSLs inhibit GSL reprogramming and neural differentiation To recapitulate neural differentiation in vitro, murine E14 embryonic stem cells (E14-mESCs) were induced to differentiate to neural cells, as described in Ref. Fico et al (2008). Neural differentiation was evaluated by changes in the levels of stemness (i.e. Nanog and Oct4) and neural (i.e. Tuj-1) markers (Fig 2A). As shown in Fig 2B, the mRNA levels of Gb3S are reduced during differentiation. On the contrary, GSEs devoted to ganglio-series GSL synthesis (including GM3S) increase in this process (Fig 2B). To assess the impact of these transcriptional changes on GSL composition, we used validated anti-GSL antibodies (i.e. anti-Gb4 and anti-Forssman antigen for the globo-series and anti-GT1b for the ganglio-series) in cytofluorimetric assays. We observed that E14-mESCs expose globo-series GSLs at their cell surfaces (i.e. Gb4 and Forssman), while after neural differentiation, the globo-series GSLs are no longer detected and ganglio-series GSLs are produced (i.e. GT1b; Fig 2C; Liang et al, 2011). Figure 2. Globo-series GSLs inhibit neural differentiation and GSL reprogramming E14-mESCs in an undifferentiated state (day 0) or induced to differentiate into neurons for 13 days were processed for Western blotting to evaluate the expression of neural (i.e. Tuj-1) and stemness (i.e. Nanog, and Oct4) markers. The mRNA levels of neuronal markers (black); stemness markers (grey); ganglio-series synthesising enzymes (green); globo-series GSL-synthesising enzymes (red) were evaluated in cells treated as in (A). Data are means ± SD of at least three independent experiments. E14-mESCs treated as in (A) were analysed by cytofluorometry with antibodies directed against globo-series GSLs (i.e. Gb4 and Forssman) or the ganglioside GT1b. Cytofluorimetric profiles and normalised mean fluorescence are shown for each antibody at day 0 (grey) and at day 13 (black). Arrows indicate the direction of changes observed during neural differentiation. E14-mESCs were induced to differentiate into neurons over 13 days in presence of the indicated GSLs (25 μM) or vehicle (methanol). Subsequently, cells were processed for Western blotting as in (A). E14-mESCs treated as in (D) were processed for RNA extraction. The mRNA levels for Gb3S and GM3S were evaluated by qPCR. GM3S/Gb3S mRNA ratios are shown. Day 0 (grey); day 13 + vehicle (black); day 13 + Gb3 (red); day 13 + GM3 (green). Data are means ± SD of at least three independent experiments. *P ≤ 0.05. E14-mESCs treated as in (E) were analysed by cytofluorometry after 13 days of differentiation with antibodies directed against globo-series GSLs (i.e. Gb4 and Forssman) or the ganglioside GT1b. Cytofluorimetric profiles and normalised mean fluorescence are shown for each antibody for cells treated with Gb3 (red) or GM3 (green). Red arrows indicate changes induced by Gb3 treatment. E14-mESCs treated with Gb3 or vehicle were stained with ChTxB-Alexa488 (green), ShTxB-Cy3 (red), and DAPI (blue). Dashed lines indicate ShTxB-positive cell perimeters. Scale bar, 50 μm. Download figure Download PowerPoint To evaluate the role of ganglio-series GSL production in neural differentiation, E14-mESCs were treated with different inhibitors of GSL synthesis (Fig EV1A) and induced to differentiate. In agreement with previous reports (Liour & Yu, 2002) in spite of partial inhibition (60%) of GSL synthesis (Fig EV1B) and of reduced ganglio-series GSL levels (~60% reduction in GT1b levels; Fig EV1C), N-butyl-deoxynojirimycin (NB-DNJ; 25 μM; Platt et al, 1994) treated E14-mESCs effectively differentiated to neural cells as judged by levels of stemness and neural markers (Fig EV1D). On the contrary, N-[(1R,2R)-2-hydroxy-1-(4-morpholinylmethyl)-2-phenylethyl]hexadecanamide (PPMP; 2.5 μM) treatment, which resulted in a more sustained (80%) inhibition of GSL production (Fig EV1B) and substantially reduced ganglio-series GSLs levels (~88% reduction in GT1b levels; Fig EV1C), hampered neural differentiation (Fig EV1D). Nonetheless, previous studies have reported that PDMP a compound structurally similar to PPMP inhibits neural differentiation independently on its inhibitory activity on GSL synthesis (Liour & Yu, 2002), and we thus evaluated the effect of additional inhibitors of sphingolipid production (Fumonisin B1 [FB1; 25 μM] and myriocin [Myr; 2.5 μM]) on neural differentiation. As shown in Fig EV1E, FB1 and Myr reduced GT1b levels by ~55% and 85%, respectively, without affecting neural differentiation (Fig EV1F), suggesting that GSL production is largely dispensable for this process. On the same lines, silencing of GM3S (siGM3S; Fig EV1H) did not result in any measurable impairment in the repression of stemness markers or induction of neuronal markers (Fig EV1G and I), indicating that ganglio-series GSL production is in general a result (not a requisite) of neural differentiation in vitro. Click here to expand this figure. Figure EV1. GSL synthesis inhibition does not inhibit neural differentiation Schematic representation of Myriocin, Fumonisin B1, NB-DNJ, PPMP, and GM3S silencing (siGM3S) mediated inhibition of GSL synthesis. Effect of NB-DNJ (25 μM) and PPMP (2.5 μM) treatment on sphingolipid synthesis in E14-mESCs. E14-mESC control, treated with NB-DNJ or PPMP, was pulse-labelled as for 8 h with [3H]-sphingosine. Sphingomyelin (SM) and GSL synthesis in control (grey), NB-DNJ (cyan)-, and PPMP (orange)-treated cells are expressed as percentage of control ± SD from three independent experiments. **P ≤ 0.01. E14-mESCs induced to differentiate into neurons over 13 days in the presence of NB-DNJ (25 μM) or PPMP (2.5 μM) were analysed by cytofluorometry with antibodies directed against the ganglioside GT1b. Cytofluorimetric profiles are shown for cells treated with NB-DNJ (cyan), or PPMP (orange) or with vehicle (grey). Arrows indicate the direction of changes induced by each treatment. E14-mESCs were kept in an undifferentiated state (day 0) or induced to differentiate into neurons over 13 days in the presence of NB-DNJ (25 μM), PPMP (2.5 μM), or vehicle (methanol). Subsequently, cells were lysed and lysates were processed for SDS–PAGE and immunoblotting using antibodies against the stemness markers Oct-4, and Nanog and the neuronal marker Tuj-1. E14-mESCs induced to differentiate into neurons in the presence of Myriocin (2.5 μM) or Fumonisin B1 (FB1) (25 μM) were analysed as in (C). Cytofluorimetric profiles are shown for cells treated with Myriocin (red), or FB1 (purple) or with vehicle (grey). Arrows indicate the direction of changes induced by treatments. E14-mESCs in an undifferentiated state (day 0) or induced to differentiate into neurons in presence of Myriocin (2.5 μM), Fumonisin B1 (FB1) (25 μM), or vehicle (methanol). Subsequently, cells were lysed and lysates were processed as in (D). E14-mESCs induced to differentiate into neurons and subjected every 3 days to GM3S silencing by transfecting specific siRNA for murine GM3S mRNA (siGM3S) were analysed by cytofluorometry as in (C). Cytofluorimetric profiles are shown for cells treated with non-targeting siRNA (grey), or with specific siGM3S (green). Arrows indicate the direction of changes induced by treatments. E14-mESCs were induced to differentiate into neurons and transfected every 3 days with siGM3S were subjected to RNA extraction and RT–PCR to evaluate the efficiency of murine GM3S silencing. Data are means ± SD from two independent experiments. E14-mESCs treated as in (G) were lysed, and lysates were processed for SDS–PAGE and immunoblotting as in (D). Download figure Download PowerPoint The role of the decrease in globo-series GSLs in neural differentiation was then evaluated. To this end, different exogenous GSLs were administered to E14-mESCs during differentiation. As shown in Fig 2D, treatment with globo-series GSLs (i.e. Gb3 and Gb4) specifically inhibited the induction of the neural marker Tuj-1 and the reduction in the stemness markers. Furthermore, while control E14-mESCs differentiated into distinct neural and glial cell populations, Gb3-treated E14-mESCs failed to effectively differentiate in any of them (Appendix Fig S1), indicating that globo-series GSLs counteract neural commitment. Gb3 administration also inhibited GSL metabolic reprogramming as it attenuated both the GM3S up-regulation and the Gb3S down-regulation, which resulted in ~60% decrease in the GM3S/Gb3S mRNA ratio (Fig 2E). As a consequence, the levels of the ganglio-GSL GT1b were strongly decreased in Gb3-treated cells while that of Gb4 and Forsmann remained high after the induction of neural differentiation (Fig 2F). Along similar lines, by exploiting the capability of B-subunits of Shiga toxin (ShTxB) and Cholera toxin (ChTxB) to bind to Gb3 and the ganglio-GSL GM1, respectively (Heyningen, 1974; Jacewicz et al, 1986), we found that the more exogenous Gb3 individual cells took up, the less GM1 was displayed at their cell surfaces (Fig 2G), suggesting that globo-series GSLs suppress ganglio-series GSL production in individual cells. Single-cell variation in GSL production in non-neural cells Similar to what observed in neural differentiation, non-neural cells are reported to produce ganglio- and globo-GSLs in a mutual exclusive fashion when evaluated at the single-cell level (Majoul et al, 2002). Thus, ganglio- or globo-series GSL levels in the easy-to-manipulate HeLa cells were evaluated using fluorescently labelled ShTxB and ChTxB. As previously reported for other cell lines (Majoul et al, 2002), individual HeLa cells show mutual exclusion in the expression of either Gb3 or GM1 (Fig 3A). Nonetheless, while a dependence of Gb3 or GM1 production on cell cycle phase was found in other cell lines with GM1 being produced predominantly by cells in G0/G1 phase and Gb3 by cells in G2/M phase (Majoul et al, 2002), when the G2/M phase marker phospho-histone H3 (p-H3) was evaluated in ShTxB- and ChTxB-positive HeLa cells, no significant enrichment was found (Fig 3B). Moreover, even though local crowding in cell populations (i.e. local cell density) impacts on lipid composition (Frechin et al, 2015) and GSL production (Snijder et al, 2009), when the distribution of ShTxB- and ChTxB-positive HeLa cells was considered along with cell crowding, local cell density failed to account for mutually exclusive toxin binding (Fig 3C). Figure 3. GSL production in non-neural cells at the single-cell level HeLa cells were fixed and stained with ChTxB-Alexa488 (green), ShTxB-Cy3 (red), and DAPI (blue). Acquired confocal images were segmented by CellProfiler software (Shannon et al, 2003), as detailed in the Appendix. Mean ChTxB- and ShTxB-associated fluorescence intensity was calculated for each cell. Cells with ChTxB or ShTxB fluorescence intensity ≥ 15% maximal recorded fluorescence intensity were considered ChTxB-positive (green squares), ShTxB-positive (red squares), or double-positive (green-edged red squares), with double-negative cells represented by empty squares (central-low panel). Scale bar, 100 μm. Upper right: ChTxB versus ShTxB fluorescence mean intensity for 10,767 individual cells as a scatter plot. Bottom right: as a measure of the degree of clustering of ChTxB (or ShTxB)-positive cells, the colony factor (defined in Appendix Methods) was calculated for 272 ChTxB-positive and 2,334 ShTxB-positive cells. The mean crowding factors ± 3 × SEM for ChTxB-positive cells (green rectangle) and ShTxB-positive cells (red rectangle) are indicated. The experimentally obtained colony factors were then compared with the randomly expected colony factors, as defined in Appendix Methods (lower right panel, grey rectangles). *P ≤ 0.05. Left panels: HeLa cells were fixed and subsequently stained with ChTxB-Alexa488 (green), ShTxB-Cy3 (red), anti-phospho-Ser10 histone-H3 (pH3) as a marker of G2/M phase cells (blue) and DAPI (not shown). Insets show examples of pH3+/ChTxB+ and pH3+/ShTxB+ cells, which indicate that both ChTxB positivity and ShTxB positivity are compatible with G2/M phase cells. Acquired confocal images were segmented using the CellProfiler software (Shannon et al, 2003). Middle panel: mean pH3-, ChTxB-, and ShTxB-associated fluorescence intensities were calculated for 4,051 cells. Individual cells showing an associated ChTxB or ShTxB fluorescence intensity ≥20% (pH3) and ≥15% (ShTxB, ChTxB) maximal recorded fluorescence intensity were considered pH3-, ChTxB-, and ShTxB-positive. The percentages of pH3+ cells in the total population (black column) and in the ShTxB+ (red) and ChTxB+ (green) populations are reported. Right panels: the ChTxB and ShTxB associated mean intensities in the pH3+ and pH3− cell subpopulations. Scale bar, 100 μm. The image dataset obtained in (A) was used to correlate ChTxB and ShTxB staining with local cell density (LCD; defined as the number of cells within a 50 × 50-pixel [69.19 × 69.19 μm] square drown around each cell) for each individual cell imaged. ChTxB- and ShTxB-positive cell distribution obtained from a representative image (left) and the LCD heatmap (right) obtained for the same image. Schematic representation of the GSL synthetic pathway in HeLa cells. GCS, GlcCer synthase; LCS, LacCer synthase; Gb3S, Gb3 synthase; GM3S, GM3 synthase; SMS1, sphingomyelin synthase 1; CERT, ceramide transfer protein; FAPP2, four phosphate adaptor protein 2. Transcriptional profile of GSEs and accessory factors (i.e. CERT, FAPP2) in ChTxB+ HeLa cells (isolated as reported in the Appendix), evaluated
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