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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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Acute hydrolysis of plasma membrane sphingomyelin also induces massive endocytic tubular invaginations positive for N-BAR proteinsa. Cell expressing endophilin 2-GFP before (“control”, top) and after (“+SMase”, bottom) sphingomyelinase (SMase) treatment (525 mU/ml) at 37 °C. b. Transmission electron microscopy image of a tubular membrane cluster induced by SMase treatment. Scale bar: 5 µm in a and 200 nm in b.
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Figure 2: Acute hydrolysis of plasma membrane sphingomyelin also induces massive endocytic tubular invaginations positive for N-BAR proteinsa. Cell expressing endophilin 2-GFP before (“control”, top) and after (“+SMase”, bottom) sphingomyelinase (SMase) treatment (525 mU/ml) at 37 °C. b. Transmission electron microscopy image of a tubular membrane cluster induced by SMase treatment. Scale bar: 5 µm in a and 200 nm in b.

Mentions: Cholesterol and sphingomyelin interact within the plasma membrane, and help define its physical properties32. We explored whether perturbing this partnership by hydrolyzing sphingomyelin, rather than by depleting cholesterol, also result in the tubular invaginations. Addition of purified bacterial sphingomyelinase (SMase) to endophilin2-GFP expressing cells produced a redistribution of GFP fluorescence similar to that produced by MβCD treatment and with a similar time-course (Fig. 2a). Likewise, EM revealed also in these cells the presence of clusters of small tubules (Fig. 2b).


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 hydrolysis of plasma membrane sphingomyelin also induces massive endocytic tubular invaginations positive for N-BAR proteinsa. Cell expressing endophilin 2-GFP before (“control”, top) and after (“+SMase”, bottom) sphingomyelinase (SMase) treatment (525 mU/ml) at 37 °C. b. Transmission electron microscopy image of a tubular membrane cluster induced by SMase treatment. Scale bar: 5 µm in a and 200 nm in b.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4230894&req=5

Figure 2: Acute hydrolysis of plasma membrane sphingomyelin also induces massive endocytic tubular invaginations positive for N-BAR proteinsa. Cell expressing endophilin 2-GFP before (“control”, top) and after (“+SMase”, bottom) sphingomyelinase (SMase) treatment (525 mU/ml) at 37 °C. b. Transmission electron microscopy image of a tubular membrane cluster induced by SMase treatment. Scale bar: 5 µm in a and 200 nm in b.
Mentions: Cholesterol and sphingomyelin interact within the plasma membrane, and help define its physical properties32. We explored whether perturbing this partnership by hydrolyzing sphingomyelin, rather than by depleting cholesterol, also result in the tubular invaginations. Addition of purified bacterial sphingomyelinase (SMase) to endophilin2-GFP expressing cells produced a redistribution of GFP fluorescence similar to that produced by MβCD treatment and with a similar time-course (Fig. 2a). Likewise, EM revealed also in these cells the presence of clusters of small tubules (Fig. 2b).

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