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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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Recruitment of SPHK1 to physiologically occurring endocytic intermediatesa. SPHK1-GFP is localized on Rab5 (RFP-Rab5) positive endosomal structures. b – e. SPHK1 is recruited to very late stage clathrin-coated endocytic pits. b. Confocal image of a COS-7 cell expressing SPHK1-GFP and RFP-clathrin light chain (CLC) showing that SPHK1 is present at a subset of endocytic clathrin-coated pits (arrowheads), but not others (arrows). c. Intensity profile of SPHK1 (green) and clathrin (red) fluorescence along the line drawn in b. d. Selected frames from a time series of a clathrin-coated pit showing that SPHK1 is recruited to the very late stage during clathrin-mediated endocytosis. e. Mean fluorescence intensity measurement of SPHK1-GFP and RFP-CLC. Pooled data from five independent experiments. n = 15 clathrin-coated pits from 5 different cells. Error bars: standard error of the mean. f. SPHK1-GFP is recruited to macropinosomes induced by PDGF stimulation. Neighboring endocytic membranes are also positive for the kinase. g. Double knockdown (DKD) of SPHK1 and SPHK2 in HeLa cells causes defects in transferrin recycling. Fluorescence intensity of Alexa Fluor 594-transferrin in control and DKD cells were calculated from projected Z-stack images. Numbers of cells counted were more than 30 for each time point. The exact n values are included in the Methods. Data are from a single experiment and representative of three different experiments Error bars: standard error of the mean. Scale bar: 10 µm in a, 3 µm in b, and 5 µm in f.
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Figure 4: Recruitment of SPHK1 to physiologically occurring endocytic intermediatesa. SPHK1-GFP is localized on Rab5 (RFP-Rab5) positive endosomal structures. b – e. SPHK1 is recruited to very late stage clathrin-coated endocytic pits. b. Confocal image of a COS-7 cell expressing SPHK1-GFP and RFP-clathrin light chain (CLC) showing that SPHK1 is present at a subset of endocytic clathrin-coated pits (arrowheads), but not others (arrows). c. Intensity profile of SPHK1 (green) and clathrin (red) fluorescence along the line drawn in b. d. Selected frames from a time series of a clathrin-coated pit showing that SPHK1 is recruited to the very late stage during clathrin-mediated endocytosis. e. Mean fluorescence intensity measurement of SPHK1-GFP and RFP-CLC. Pooled data from five independent experiments. n = 15 clathrin-coated pits from 5 different cells. Error bars: standard error of the mean. f. SPHK1-GFP is recruited to macropinosomes induced by PDGF stimulation. Neighboring endocytic membranes are also positive for the kinase. g. Double knockdown (DKD) of SPHK1 and SPHK2 in HeLa cells causes defects in transferrin recycling. Fluorescence intensity of Alexa Fluor 594-transferrin in control and DKD cells were calculated from projected Z-stack images. Numbers of cells counted were more than 30 for each time point. The exact n values are included in the Methods. Data are from a single experiment and representative of three different experiments Error bars: standard error of the mean. Scale bar: 10 µm in a, 3 µm in b, and 5 µm in f.

Mentions: SPHK1 was shown to have a primarily cytosolic localization and to be recruited to the plasma membrane in response to pharmacological stimuli34, 35. SPHK1 was also reported to be localized on endosomes36 and phagosomes37, 38. Our results shown above, i.e., the recruitment of SPHK1 to “exaggerated” early endocytic intermediates induced by experimental perturbations, support the possibility that physiologically occurring early endocytic structures represent one of its sites of action. We revisited the subcellular targeting of SPHK1 under control conditions using SPHK1-GFP and observed an enrichment of this protein both on Rab5-positive early endosomes (Fig. 4a) and on even earlier endocytic intermediates. For example, SPHK1 was present at a subset (62%) of endocytic clathrin-coated pits (Fig. 4b and c), and at such pits the SPHK1-GFP signal peaked at late stages, when the clathrin signal had already started to decay (Fig. 4d and e). As the clathrin signal dims in parallel with the endophilin signal19, 39, even at these physiologically occurring sites SPHK1 recruitment lagged behind the endophilin recruitment, as observed at MβCD-induced endophilin foci. In addition, SPHK1 was also recruited to macropinosomes induced by PDGF stimulation (Fig. 4f) and neighboring membranes.


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)

Recruitment of SPHK1 to physiologically occurring endocytic intermediatesa. SPHK1-GFP is localized on Rab5 (RFP-Rab5) positive endosomal structures. b – e. SPHK1 is recruited to very late stage clathrin-coated endocytic pits. b. Confocal image of a COS-7 cell expressing SPHK1-GFP and RFP-clathrin light chain (CLC) showing that SPHK1 is present at a subset of endocytic clathrin-coated pits (arrowheads), but not others (arrows). c. Intensity profile of SPHK1 (green) and clathrin (red) fluorescence along the line drawn in b. d. Selected frames from a time series of a clathrin-coated pit showing that SPHK1 is recruited to the very late stage during clathrin-mediated endocytosis. e. Mean fluorescence intensity measurement of SPHK1-GFP and RFP-CLC. Pooled data from five independent experiments. n = 15 clathrin-coated pits from 5 different cells. Error bars: standard error of the mean. f. SPHK1-GFP is recruited to macropinosomes induced by PDGF stimulation. Neighboring endocytic membranes are also positive for the kinase. g. Double knockdown (DKD) of SPHK1 and SPHK2 in HeLa cells causes defects in transferrin recycling. Fluorescence intensity of Alexa Fluor 594-transferrin in control and DKD cells were calculated from projected Z-stack images. Numbers of cells counted were more than 30 for each time point. The exact n values are included in the Methods. Data are from a single experiment and representative of three different experiments Error bars: standard error of the mean. Scale bar: 10 µm in a, 3 µm in b, and 5 µm in f.
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Figure 4: Recruitment of SPHK1 to physiologically occurring endocytic intermediatesa. SPHK1-GFP is localized on Rab5 (RFP-Rab5) positive endosomal structures. b – e. SPHK1 is recruited to very late stage clathrin-coated endocytic pits. b. Confocal image of a COS-7 cell expressing SPHK1-GFP and RFP-clathrin light chain (CLC) showing that SPHK1 is present at a subset of endocytic clathrin-coated pits (arrowheads), but not others (arrows). c. Intensity profile of SPHK1 (green) and clathrin (red) fluorescence along the line drawn in b. d. Selected frames from a time series of a clathrin-coated pit showing that SPHK1 is recruited to the very late stage during clathrin-mediated endocytosis. e. Mean fluorescence intensity measurement of SPHK1-GFP and RFP-CLC. Pooled data from five independent experiments. n = 15 clathrin-coated pits from 5 different cells. Error bars: standard error of the mean. f. SPHK1-GFP is recruited to macropinosomes induced by PDGF stimulation. Neighboring endocytic membranes are also positive for the kinase. g. Double knockdown (DKD) of SPHK1 and SPHK2 in HeLa cells causes defects in transferrin recycling. Fluorescence intensity of Alexa Fluor 594-transferrin in control and DKD cells were calculated from projected Z-stack images. Numbers of cells counted were more than 30 for each time point. The exact n values are included in the Methods. Data are from a single experiment and representative of three different experiments Error bars: standard error of the mean. Scale bar: 10 µm in a, 3 µm in b, and 5 µm in f.
Mentions: SPHK1 was shown to have a primarily cytosolic localization and to be recruited to the plasma membrane in response to pharmacological stimuli34, 35. SPHK1 was also reported to be localized on endosomes36 and phagosomes37, 38. Our results shown above, i.e., the recruitment of SPHK1 to “exaggerated” early endocytic intermediates induced by experimental perturbations, support the possibility that physiologically occurring early endocytic structures represent one of its sites of action. We revisited the subcellular targeting of SPHK1 under control conditions using SPHK1-GFP and observed an enrichment of this protein both on Rab5-positive early endosomes (Fig. 4a) and on even earlier endocytic intermediates. For example, SPHK1 was present at a subset (62%) of endocytic clathrin-coated pits (Fig. 4b and c), and at such pits the SPHK1-GFP signal peaked at late stages, when the clathrin signal had already started to decay (Fig. 4d and e). As the clathrin signal dims in parallel with the endophilin signal19, 39, even at these physiologically occurring sites SPHK1 recruitment lagged behind the endophilin recruitment, as observed at MβCD-induced endophilin foci. In addition, SPHK1 was also recruited to macropinosomes induced by PDGF stimulation (Fig. 4f) and neighboring membranes.

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