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Coupling between endocytosis and sphingosine kinase 1 recruitment.

Shen H, Giordano F, Wu Y, Chan J, Zhu C, Milosevic I, Wu X, Yao K, Chen B, Baumgart T, Sieburth D, De Camilli P - Nat. Cell Biol. (2014)

Bottom Line: Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface.The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme.Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA [2] Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA [3].

ABSTRACT
Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

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Related in: MedlinePlus

The sphingoid base modifying enzyme, sphingosine kinase 1 (SPHK1) is recruited to the tubular endocytic invaginationsa. Sphingolipid metabolic pathway. SPTLC = serine palmitoyltransferase; SPHK = sphingosine kinase; SGPP = sphingosine-1-phosphate phosphatase; SGPL1 = sphingosine-1-phosphate lyase 1; CerS = ceramide synthase; CDase = ceramidase; SMS = sphingomyelin synthase; SMase = sphingomyelinase; CerK = ceramide kinase. b and c. GFP-tagged SPHK1 cosegregates into the clusters positive for Ruby-tagged endophilin 2 in cells treated with MβCD in b, or treated with SMase in c. d and e. Concentration of both SPHK1 and endophilin 2 on the endocytic tubular intermediates induced by MβCD treatment in a cell transfected with SPHK1-GFP alone in d or both SPHK1-HA and endophilin 2-GFP in e. Single or dual immunogold labeling with 15 nm gold particles for anti-GFP in c and e and 10 nm gold particles for anti-HA in e. f. Selected frames from a time series of a cluster colabeled for SPHK1-GFP and endophilin 2-Ruby (left). Fluorescence intensity of endophilin 2 (red) and SPHK1 (green) positive tubule clusters (n = 15 foci from 6 different cells) showing that recruitment of endophilin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard errors of the mean. g. Selected frames from a time series of a tubule cluster colabeled by SPHK1-GFP and dynamin 2-RFP (left). Fluorescence intensity of dynamin 2 (red) and SPHK1 (green) positive tubule clusters (n = 16 foci from 6 different cells) showing that recruitment of dynamin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard error of the mean. h. Tubule clusters formed after MβCD treatment are positive for fluorescent ceramide (BODIPY-TR-ceramide, top) and fluorescent sphingosine (BODIPY-sphingosine, bottom). i. Confocal images of a cell expressing SPHK1-GFP 2 min (top), 4 min (middle) and 10 min (bottom) after MβCD treatment. SPHK1 strongly labels filament-like structures originated from the clusters. Scale bar: 10 µm in b and c; 200 nm in d and e; 3 µm in h; and 5 µm in i.
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Figure 3: The sphingoid base modifying enzyme, sphingosine kinase 1 (SPHK1) is recruited to the tubular endocytic invaginationsa. Sphingolipid metabolic pathway. SPTLC = serine palmitoyltransferase; SPHK = sphingosine kinase; SGPP = sphingosine-1-phosphate phosphatase; SGPL1 = sphingosine-1-phosphate lyase 1; CerS = ceramide synthase; CDase = ceramidase; SMS = sphingomyelin synthase; SMase = sphingomyelinase; CerK = ceramide kinase. b and c. GFP-tagged SPHK1 cosegregates into the clusters positive for Ruby-tagged endophilin 2 in cells treated with MβCD in b, or treated with SMase in c. d and e. Concentration of both SPHK1 and endophilin 2 on the endocytic tubular intermediates induced by MβCD treatment in a cell transfected with SPHK1-GFP alone in d or both SPHK1-HA and endophilin 2-GFP in e. Single or dual immunogold labeling with 15 nm gold particles for anti-GFP in c and e and 10 nm gold particles for anti-HA in e. f. Selected frames from a time series of a cluster colabeled for SPHK1-GFP and endophilin 2-Ruby (left). Fluorescence intensity of endophilin 2 (red) and SPHK1 (green) positive tubule clusters (n = 15 foci from 6 different cells) showing that recruitment of endophilin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard errors of the mean. g. Selected frames from a time series of a tubule cluster colabeled by SPHK1-GFP and dynamin 2-RFP (left). Fluorescence intensity of dynamin 2 (red) and SPHK1 (green) positive tubule clusters (n = 16 foci from 6 different cells) showing that recruitment of dynamin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard error of the mean. h. Tubule clusters formed after MβCD treatment are positive for fluorescent ceramide (BODIPY-TR-ceramide, top) and fluorescent sphingosine (BODIPY-sphingosine, bottom). i. Confocal images of a cell expressing SPHK1-GFP 2 min (top), 4 min (middle) and 10 min (bottom) after MβCD treatment. SPHK1 strongly labels filament-like structures originated from the clusters. Scale bar: 10 µm in b and c; 200 nm in d and e; 3 µm in h; and 5 µm in i.

Mentions: We examined the localization of GFP-fusions of several enzymes known to regulate sphingoid base levels (Fig. 3a and Supplementary Fig. 3b and c): neutral sphingomyelinase (SMPD3), neutral ceramidase (ASAH2), ceramide kinase (CerK), sphingosine kinase 1 (SPHK1), sphingosine-1-phosphate phosphatase 1 (SGPP1), sphingosine-1-phosphate lyase (SGPL1) and ceramide synthase 1 (CerS1). Strikingly, SPHK1, but not the other enzymes, was strongly enriched at the tubules induced by MβCD (Fig. 3b) or SMase treatment (Fig. 3c), where it colocalized with endophilin 2-Ruby. SPHK1 is a cytosolic kinase that specifically phosphorylates sphingoid bases to sphingoid base-1-phosphate. Interestingly, yeast sphingosine kinase (LCB4) is one of the enzymes that genetically interact with the RVS genes (LCB4 mutations suppress rvs161 and rvs167 mutations)24 (Supplementary Fig. 3a). Mammalian genomes encode two SPHKs, SPHK1 and SPHK2. FLAG-tagged SPHK2 was also present at endophilin foci (Supplementary Fig. 3d), consistent with a redundant function of the two genes in mice33. The enrichment of SPHK1-GFP at the tubular invaginations induced by MβCD was confirmed by EM with anti-GFP immunogold labeling (Fig. 3d). Co-immunogold labeling of cells co-expressing SPHK1-HA and endophilin 2-GFP further confirmed these results (Fig. 3e).


Coupling between endocytosis and sphingosine kinase 1 recruitment.

Shen H, Giordano F, Wu Y, Chan J, Zhu C, Milosevic I, Wu X, Yao K, Chen B, Baumgart T, Sieburth D, De Camilli P - Nat. Cell Biol. (2014)

The sphingoid base modifying enzyme, sphingosine kinase 1 (SPHK1) is recruited to the tubular endocytic invaginationsa. Sphingolipid metabolic pathway. SPTLC = serine palmitoyltransferase; SPHK = sphingosine kinase; SGPP = sphingosine-1-phosphate phosphatase; SGPL1 = sphingosine-1-phosphate lyase 1; CerS = ceramide synthase; CDase = ceramidase; SMS = sphingomyelin synthase; SMase = sphingomyelinase; CerK = ceramide kinase. b and c. GFP-tagged SPHK1 cosegregates into the clusters positive for Ruby-tagged endophilin 2 in cells treated with MβCD in b, or treated with SMase in c. d and e. Concentration of both SPHK1 and endophilin 2 on the endocytic tubular intermediates induced by MβCD treatment in a cell transfected with SPHK1-GFP alone in d or both SPHK1-HA and endophilin 2-GFP in e. Single or dual immunogold labeling with 15 nm gold particles for anti-GFP in c and e and 10 nm gold particles for anti-HA in e. f. Selected frames from a time series of a cluster colabeled for SPHK1-GFP and endophilin 2-Ruby (left). Fluorescence intensity of endophilin 2 (red) and SPHK1 (green) positive tubule clusters (n = 15 foci from 6 different cells) showing that recruitment of endophilin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard errors of the mean. g. Selected frames from a time series of a tubule cluster colabeled by SPHK1-GFP and dynamin 2-RFP (left). Fluorescence intensity of dynamin 2 (red) and SPHK1 (green) positive tubule clusters (n = 16 foci from 6 different cells) showing that recruitment of dynamin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard error of the mean. h. Tubule clusters formed after MβCD treatment are positive for fluorescent ceramide (BODIPY-TR-ceramide, top) and fluorescent sphingosine (BODIPY-sphingosine, bottom). i. Confocal images of a cell expressing SPHK1-GFP 2 min (top), 4 min (middle) and 10 min (bottom) after MβCD treatment. SPHK1 strongly labels filament-like structures originated from the clusters. Scale bar: 10 µm in b and c; 200 nm in d and e; 3 µm in h; and 5 µm in i.
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Related In: Results  -  Collection

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Show All Figures
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Figure 3: The sphingoid base modifying enzyme, sphingosine kinase 1 (SPHK1) is recruited to the tubular endocytic invaginationsa. Sphingolipid metabolic pathway. SPTLC = serine palmitoyltransferase; SPHK = sphingosine kinase; SGPP = sphingosine-1-phosphate phosphatase; SGPL1 = sphingosine-1-phosphate lyase 1; CerS = ceramide synthase; CDase = ceramidase; SMS = sphingomyelin synthase; SMase = sphingomyelinase; CerK = ceramide kinase. b and c. GFP-tagged SPHK1 cosegregates into the clusters positive for Ruby-tagged endophilin 2 in cells treated with MβCD in b, or treated with SMase in c. d and e. Concentration of both SPHK1 and endophilin 2 on the endocytic tubular intermediates induced by MβCD treatment in a cell transfected with SPHK1-GFP alone in d or both SPHK1-HA and endophilin 2-GFP in e. Single or dual immunogold labeling with 15 nm gold particles for anti-GFP in c and e and 10 nm gold particles for anti-HA in e. f. Selected frames from a time series of a cluster colabeled for SPHK1-GFP and endophilin 2-Ruby (left). Fluorescence intensity of endophilin 2 (red) and SPHK1 (green) positive tubule clusters (n = 15 foci from 6 different cells) showing that recruitment of endophilin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard errors of the mean. g. Selected frames from a time series of a tubule cluster colabeled by SPHK1-GFP and dynamin 2-RFP (left). Fluorescence intensity of dynamin 2 (red) and SPHK1 (green) positive tubule clusters (n = 16 foci from 6 different cells) showing that recruitment of dynamin precedes SPHK1 (right). Pooled data from 6 independent experiments. Error bars: standard error of the mean. h. Tubule clusters formed after MβCD treatment are positive for fluorescent ceramide (BODIPY-TR-ceramide, top) and fluorescent sphingosine (BODIPY-sphingosine, bottom). i. Confocal images of a cell expressing SPHK1-GFP 2 min (top), 4 min (middle) and 10 min (bottom) after MβCD treatment. SPHK1 strongly labels filament-like structures originated from the clusters. Scale bar: 10 µm in b and c; 200 nm in d and e; 3 µm in h; and 5 µm in i.
Mentions: We examined the localization of GFP-fusions of several enzymes known to regulate sphingoid base levels (Fig. 3a and Supplementary Fig. 3b and c): neutral sphingomyelinase (SMPD3), neutral ceramidase (ASAH2), ceramide kinase (CerK), sphingosine kinase 1 (SPHK1), sphingosine-1-phosphate phosphatase 1 (SGPP1), sphingosine-1-phosphate lyase (SGPL1) and ceramide synthase 1 (CerS1). Strikingly, SPHK1, but not the other enzymes, was strongly enriched at the tubules induced by MβCD (Fig. 3b) or SMase treatment (Fig. 3c), where it colocalized with endophilin 2-Ruby. SPHK1 is a cytosolic kinase that specifically phosphorylates sphingoid bases to sphingoid base-1-phosphate. Interestingly, yeast sphingosine kinase (LCB4) is one of the enzymes that genetically interact with the RVS genes (LCB4 mutations suppress rvs161 and rvs167 mutations)24 (Supplementary Fig. 3a). Mammalian genomes encode two SPHKs, SPHK1 and SPHK2. FLAG-tagged SPHK2 was also present at endophilin foci (Supplementary Fig. 3d), consistent with a redundant function of the two genes in mice33. The enrichment of SPHK1-GFP at the tubular invaginations induced by MβCD was confirmed by EM with anti-GFP immunogold labeling (Fig. 3d). Co-immunogold labeling of cells co-expressing SPHK1-HA and endophilin 2-GFP further confirmed these results (Fig. 3e).

Bottom Line: Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface.The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme.Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

View Article: PubMed Central - PubMed

Affiliation: 1] Howard Hughes Medical Institute, Program in Cellular Neuroscience, Neurodegeneration and Repair, Yale University School of Medicine, New Haven, Connecticut 06510, USA [2] Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA [3].

ABSTRACT
Genetic studies have suggested a functional link between cholesterol/sphingolipid metabolism and endocytic membrane traffic. Here we show that perturbing the cholesterol/sphingomyelin balance in the plasma membrane results in the massive formation of clusters of narrow endocytic tubular invaginations positive for N-BAR proteins. These tubules are intensely positive for sphingosine kinase 1 (SPHK1). SPHK1 is also targeted to physiologically occurring early endocytic intermediates, and is highly enriched in nerve terminals, which are cellular compartments specialized for exo/endocytosis. Membrane recruitment of SPHK1 involves a direct, curvature-sensitive interaction with the lipid bilayer mediated by a hydrophobic patch on the enzyme's surface. The knockdown of SPHKs results in endocytic recycling defects, and a mutation that disrupts the hydrophobic patch of Caenorhabditis elegans SPHK fails to rescue the neurotransmission defects in loss-of-function mutants of this enzyme. Our studies support a role for sphingosine phosphorylation in endocytic membrane trafficking beyond the established function of sphingosine-1-phosphate in intercellular signalling.

Show MeSH
Related in: MedlinePlus