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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

Acute perturbation of plasma membrane cholesterol induces massive endocytic tubular invaginations positive for N-BAR proteinsa. TIRF images of a COS-7 cell expressing GFP-clathrin light chain (CLC) before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. b. Statistical analysis of tracked clathrin-coated pits before (ctrl) and after (+MβCD) MβCD treatment (pooled results from three independent experiments). Mean lifetime: n=158 (ctrl) and 211 (+MβCD) pits. Mean fluorescence intensity: n=330 (ctrl) and 270 (+MβCD) pits. Error bars: standard error of the mean. [***] P < 0.0001, Student's t-test. Mean clathrin-coated pits number: n=3 (ctrl) and 3 (+MβCD) cells. c. TIRF images of a COS-7 cell expressing GFP-clathrin light chain and endophilin-2 Ruby before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. d. Confocal images of HeLa cells expressing endophilin 2-GFP before (top) and after (bottom) MβCD treatment (bottom). e and f. Selected frames from a time series of the endophilin 2-GFP fluorescence from the cell shown in d. g. Confocal image of a middle section of a HeLa cell expressing endophilin 2-GFP after MβCD treatment. h. Measurement of cellular free cholesterol before and after MβCD treatment. n = 6 dishes Data are pooled from two independent experiments. Error bars: standard error of the mean. i. DiI staining of a cell expressing endophilin 2-GFP and treated with MβCD. j. Transmission electron microscopy image of a tubular membrane cluster formed during MβCD treatment. k. Histogram of tubule diameter. n=61 tubules. l. Anti-GFP immunogold labeling of a cell expressing endophilin 2-GFP and treated with MβCD. m. Average change of the footprint of cells (green) and of the percent areas (relative to the original footprints) occupied by endophilin 2 foci (red) during MβCD treatment. n = 7 cells. Data are pooled from 7 independent experiments. Error bars: standard error of the mean. Scale bar: 3 µm in a, c, and e; 5 µm in i; 10 µm in d and g; 200 nm in j and l.
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Figure 1: Acute perturbation of plasma membrane cholesterol induces massive endocytic tubular invaginations positive for N-BAR proteinsa. TIRF images of a COS-7 cell expressing GFP-clathrin light chain (CLC) before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. b. Statistical analysis of tracked clathrin-coated pits before (ctrl) and after (+MβCD) MβCD treatment (pooled results from three independent experiments). Mean lifetime: n=158 (ctrl) and 211 (+MβCD) pits. Mean fluorescence intensity: n=330 (ctrl) and 270 (+MβCD) pits. Error bars: standard error of the mean. [***] P < 0.0001, Student's t-test. Mean clathrin-coated pits number: n=3 (ctrl) and 3 (+MβCD) cells. c. TIRF images of a COS-7 cell expressing GFP-clathrin light chain and endophilin-2 Ruby before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. d. Confocal images of HeLa cells expressing endophilin 2-GFP before (top) and after (bottom) MβCD treatment (bottom). e and f. Selected frames from a time series of the endophilin 2-GFP fluorescence from the cell shown in d. g. Confocal image of a middle section of a HeLa cell expressing endophilin 2-GFP after MβCD treatment. h. Measurement of cellular free cholesterol before and after MβCD treatment. n = 6 dishes Data are pooled from two independent experiments. Error bars: standard error of the mean. i. DiI staining of a cell expressing endophilin 2-GFP and treated with MβCD. j. Transmission electron microscopy image of a tubular membrane cluster formed during MβCD treatment. k. Histogram of tubule diameter. n=61 tubules. l. Anti-GFP immunogold labeling of a cell expressing endophilin 2-GFP and treated with MβCD. m. Average change of the footprint of cells (green) and of the percent areas (relative to the original footprints) occupied by endophilin 2 foci (red) during MβCD treatment. n = 7 cells. Data are pooled from 7 independent experiments. Error bars: standard error of the mean. Scale bar: 3 µm in a, c, and e; 5 µm in i; 10 µm in d and g; 200 nm in j and l.

Mentions: Acute cholesterol extraction from cells with methyl-β-cyclodextrin (MβCD) results in the perturbation of clathrin-mediated endocytosis accompanied by formation of shallow clathrin-coated pits3, 4. To monitor the dynamics of this effect, we examined live cells expressing GFP-clathrin light chain (CLC) and endophilin 2-Ruby by TIRF microscopy (Fig. 1a–c). Endophilin is an endocytic adaptor recruited at the necks of late stage endocytic clathrin-coated pits, where it coordinates acquisition of bilayer curvature (via its BAR domain) with the recruitment of dynamin and synaptojanin (via its SH3 domain)18–20, two factors required for fission and uncoating respectively.


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)

Acute perturbation of plasma membrane cholesterol induces massive endocytic tubular invaginations positive for N-BAR proteinsa. TIRF images of a COS-7 cell expressing GFP-clathrin light chain (CLC) before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. b. Statistical analysis of tracked clathrin-coated pits before (ctrl) and after (+MβCD) MβCD treatment (pooled results from three independent experiments). Mean lifetime: n=158 (ctrl) and 211 (+MβCD) pits. Mean fluorescence intensity: n=330 (ctrl) and 270 (+MβCD) pits. Error bars: standard error of the mean. [***] P < 0.0001, Student's t-test. Mean clathrin-coated pits number: n=3 (ctrl) and 3 (+MβCD) cells. c. TIRF images of a COS-7 cell expressing GFP-clathrin light chain and endophilin-2 Ruby before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. d. Confocal images of HeLa cells expressing endophilin 2-GFP before (top) and after (bottom) MβCD treatment (bottom). e and f. Selected frames from a time series of the endophilin 2-GFP fluorescence from the cell shown in d. g. Confocal image of a middle section of a HeLa cell expressing endophilin 2-GFP after MβCD treatment. h. Measurement of cellular free cholesterol before and after MβCD treatment. n = 6 dishes Data are pooled from two independent experiments. Error bars: standard error of the mean. i. DiI staining of a cell expressing endophilin 2-GFP and treated with MβCD. j. Transmission electron microscopy image of a tubular membrane cluster formed during MβCD treatment. k. Histogram of tubule diameter. n=61 tubules. l. Anti-GFP immunogold labeling of a cell expressing endophilin 2-GFP and treated with MβCD. m. Average change of the footprint of cells (green) and of the percent areas (relative to the original footprints) occupied by endophilin 2 foci (red) during MβCD treatment. n = 7 cells. Data are pooled from 7 independent experiments. Error bars: standard error of the mean. Scale bar: 3 µm in a, c, and e; 5 µm in i; 10 µm in d and g; 200 nm in j and l.
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Related In: Results  -  Collection

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Figure 1: Acute perturbation of plasma membrane cholesterol induces massive endocytic tubular invaginations positive for N-BAR proteinsa. TIRF images of a COS-7 cell expressing GFP-clathrin light chain (CLC) before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. b. Statistical analysis of tracked clathrin-coated pits before (ctrl) and after (+MβCD) MβCD treatment (pooled results from three independent experiments). Mean lifetime: n=158 (ctrl) and 211 (+MβCD) pits. Mean fluorescence intensity: n=330 (ctrl) and 270 (+MβCD) pits. Error bars: standard error of the mean. [***] P < 0.0001, Student's t-test. Mean clathrin-coated pits number: n=3 (ctrl) and 3 (+MβCD) cells. c. TIRF images of a COS-7 cell expressing GFP-clathrin light chain and endophilin-2 Ruby before (“control”, left) and after (“+MβCD”, right) 10 mM MβCD treatment at 37 °C. d. Confocal images of HeLa cells expressing endophilin 2-GFP before (top) and after (bottom) MβCD treatment (bottom). e and f. Selected frames from a time series of the endophilin 2-GFP fluorescence from the cell shown in d. g. Confocal image of a middle section of a HeLa cell expressing endophilin 2-GFP after MβCD treatment. h. Measurement of cellular free cholesterol before and after MβCD treatment. n = 6 dishes Data are pooled from two independent experiments. Error bars: standard error of the mean. i. DiI staining of a cell expressing endophilin 2-GFP and treated with MβCD. j. Transmission electron microscopy image of a tubular membrane cluster formed during MβCD treatment. k. Histogram of tubule diameter. n=61 tubules. l. Anti-GFP immunogold labeling of a cell expressing endophilin 2-GFP and treated with MβCD. m. Average change of the footprint of cells (green) and of the percent areas (relative to the original footprints) occupied by endophilin 2 foci (red) during MβCD treatment. n = 7 cells. Data are pooled from 7 independent experiments. Error bars: standard error of the mean. Scale bar: 3 µm in a, c, and e; 5 µm in i; 10 µm in d and g; 200 nm in j and l.
Mentions: Acute cholesterol extraction from cells with methyl-β-cyclodextrin (MβCD) results in the perturbation of clathrin-mediated endocytosis accompanied by formation of shallow clathrin-coated pits3, 4. To monitor the dynamics of this effect, we examined live cells expressing GFP-clathrin light chain (CLC) and endophilin 2-Ruby by TIRF microscopy (Fig. 1a–c). Endophilin is an endocytic adaptor recruited at the necks of late stage endocytic clathrin-coated pits, where it coordinates acquisition of bilayer curvature (via its BAR domain) with the recruitment of dynamin and synaptojanin (via its SH3 domain)18–20, two factors required for fission and uncoating respectively.

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