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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

SPHK1 is concentrated at synapses and mutations that abolish membrane binding do not rescue synaptic transmission defects of loss-of-function mutantsa. SPHK1-GFP is targeted to presynaptic terminals in cultured hippocampal neurons as shown by colocalization with immunoreactivity for Bassoon, a marker of pre-synaptic nerve terminals. b. SPHK1 is selectively enriched at the presynaptic terminal of photoreceptor cells in mouse retina. Z-projection confocal image of a mouse retina slice containing a SPHK1-GFP expressing rod cell (as a result of electroporation). The retina was fixed and immunostained for the synaptic vesicle marker synaptobrevin 2 to reveal synapses of the outer plexiform layer where the axon ending of the rod cell terminates. c – e. Analysis of worm sphingosine kinase (cSPHK-1). c. The localization of cSPHK-1WT-GFP or of the predicted membrane binding mutant cSPHK-1V268Q-GFP in the cell body (left), ventral (middle) and dorsal nerve cord (right) of motor neurons of C. elegans. d. Quantification of parameters of the fluorescence spots in the axon terminals (intensity, width and density) in wild-type animals expressing WT and mutant cSPHK-1. For cSPHK-1-GFP, n= 321 synapses from 17 worms, and for cSPHK-1V268Q-GFP, n=387 synapses from 23 worms. Each animal is an independent experiment, and data from independent experiments were pooled. Error bars: standard error of the mean. [ns] not significant; [*] P < 0.05; [**] P < 0.005, Student's t-test. e. Time-course of the onset of paralysis of the indicated worm strains upon exposure to the acetylcholine esterase inhibitor aldicarb (1 mM). sphk-1; cSPHK-1 and sphk-1;cSPHK-1V268Q stand for sphk-1  mutants expressing full length worm SPHK-1-GFP or SPHK-1V268Q-GFP cDNA in neurons, respectively. A total of 60 worms were counted from each plate. Percentages were averaged at each time point per genotype and plotted graphically. n = 3 plates. The analysis was repeated two times, with two different transgenic lines. Data shown are from a single experiment. Error bars: standard error of the mean. Scale bar: 10 µm in a, 20 µm in b, and 10 µm in c.
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Figure 6: SPHK1 is concentrated at synapses and mutations that abolish membrane binding do not rescue synaptic transmission defects of loss-of-function mutantsa. SPHK1-GFP is targeted to presynaptic terminals in cultured hippocampal neurons as shown by colocalization with immunoreactivity for Bassoon, a marker of pre-synaptic nerve terminals. b. SPHK1 is selectively enriched at the presynaptic terminal of photoreceptor cells in mouse retina. Z-projection confocal image of a mouse retina slice containing a SPHK1-GFP expressing rod cell (as a result of electroporation). The retina was fixed and immunostained for the synaptic vesicle marker synaptobrevin 2 to reveal synapses of the outer plexiform layer where the axon ending of the rod cell terminates. c – e. Analysis of worm sphingosine kinase (cSPHK-1). c. The localization of cSPHK-1WT-GFP or of the predicted membrane binding mutant cSPHK-1V268Q-GFP in the cell body (left), ventral (middle) and dorsal nerve cord (right) of motor neurons of C. elegans. d. Quantification of parameters of the fluorescence spots in the axon terminals (intensity, width and density) in wild-type animals expressing WT and mutant cSPHK-1. For cSPHK-1-GFP, n= 321 synapses from 17 worms, and for cSPHK-1V268Q-GFP, n=387 synapses from 23 worms. Each animal is an independent experiment, and data from independent experiments were pooled. Error bars: standard error of the mean. [ns] not significant; [*] P < 0.05; [**] P < 0.005, Student's t-test. e. Time-course of the onset of paralysis of the indicated worm strains upon exposure to the acetylcholine esterase inhibitor aldicarb (1 mM). sphk-1; cSPHK-1 and sphk-1;cSPHK-1V268Q stand for sphk-1 mutants expressing full length worm SPHK-1-GFP or SPHK-1V268Q-GFP cDNA in neurons, respectively. A total of 60 worms were counted from each plate. Percentages were averaged at each time point per genotype and plotted graphically. n = 3 plates. The analysis was repeated two times, with two different transgenic lines. Data shown are from a single experiment. Error bars: standard error of the mean. Scale bar: 10 µm in a, 20 µm in b, and 10 µm in c.

Mentions: To determine whether the properties that mediate the recruitment of SPHK1 to the tubular invaginations have a physiological significance, we next investigated whether perturbation of the hydrophobic patch rescues the absence of SPHK1 in a functional assay. In neurons, SPHK1 is preferentially targeted to nerve terminals, a compartment highly specialized for the endocytic recycling of synaptic vesicles, as shown by immunocytochemistry47 and by the accumulation of SPHK1-GFP in presynaptic terminals of cortical neurons in vitro47 (and Fig. 6a), or of retinal photoreceptors in vivo (Fig. 6b). The single SPHK1 in C. elegans, cSPHK-1, is also concentrated at synaptic sites along axons, and its absence results in lower sensitivity to aldicarb, indicating a defect in neurotransmitter secretion10, 48. Interestingly, cSPHK-1 is primarily localized at periactive zones of synapses, which are sites of high endocytic activity10. In cSPHK-1, leucine 194 of the hydrophobic patch is replaced by another hydrophobic amino acid, valine 268. Supporting a critical importance of the patch, cSPHK-1V268Q-GFP did not exhibit the same highly punctate localization as cSPHKWT-GFP in axons (Fig. 6c and d). Importantly, while cSPHK-1WT-GFP (Fig. 6e) and even human SPHK1 (Supplementary Fig. 6) rescued neurotransmission in cSPHK-1 mutant worms, cSPHK-1V268Q-GFP did not (Fig. 6e).


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)

SPHK1 is concentrated at synapses and mutations that abolish membrane binding do not rescue synaptic transmission defects of loss-of-function mutantsa. SPHK1-GFP is targeted to presynaptic terminals in cultured hippocampal neurons as shown by colocalization with immunoreactivity for Bassoon, a marker of pre-synaptic nerve terminals. b. SPHK1 is selectively enriched at the presynaptic terminal of photoreceptor cells in mouse retina. Z-projection confocal image of a mouse retina slice containing a SPHK1-GFP expressing rod cell (as a result of electroporation). The retina was fixed and immunostained for the synaptic vesicle marker synaptobrevin 2 to reveal synapses of the outer plexiform layer where the axon ending of the rod cell terminates. c – e. Analysis of worm sphingosine kinase (cSPHK-1). c. The localization of cSPHK-1WT-GFP or of the predicted membrane binding mutant cSPHK-1V268Q-GFP in the cell body (left), ventral (middle) and dorsal nerve cord (right) of motor neurons of C. elegans. d. Quantification of parameters of the fluorescence spots in the axon terminals (intensity, width and density) in wild-type animals expressing WT and mutant cSPHK-1. For cSPHK-1-GFP, n= 321 synapses from 17 worms, and for cSPHK-1V268Q-GFP, n=387 synapses from 23 worms. Each animal is an independent experiment, and data from independent experiments were pooled. Error bars: standard error of the mean. [ns] not significant; [*] P < 0.05; [**] P < 0.005, Student's t-test. e. Time-course of the onset of paralysis of the indicated worm strains upon exposure to the acetylcholine esterase inhibitor aldicarb (1 mM). sphk-1; cSPHK-1 and sphk-1;cSPHK-1V268Q stand for sphk-1  mutants expressing full length worm SPHK-1-GFP or SPHK-1V268Q-GFP cDNA in neurons, respectively. A total of 60 worms were counted from each plate. Percentages were averaged at each time point per genotype and plotted graphically. n = 3 plates. The analysis was repeated two times, with two different transgenic lines. Data shown are from a single experiment. Error bars: standard error of the mean. Scale bar: 10 µm in a, 20 µm in b, and 10 µm in c.
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Figure 6: SPHK1 is concentrated at synapses and mutations that abolish membrane binding do not rescue synaptic transmission defects of loss-of-function mutantsa. SPHK1-GFP is targeted to presynaptic terminals in cultured hippocampal neurons as shown by colocalization with immunoreactivity for Bassoon, a marker of pre-synaptic nerve terminals. b. SPHK1 is selectively enriched at the presynaptic terminal of photoreceptor cells in mouse retina. Z-projection confocal image of a mouse retina slice containing a SPHK1-GFP expressing rod cell (as a result of electroporation). The retina was fixed and immunostained for the synaptic vesicle marker synaptobrevin 2 to reveal synapses of the outer plexiform layer where the axon ending of the rod cell terminates. c – e. Analysis of worm sphingosine kinase (cSPHK-1). c. The localization of cSPHK-1WT-GFP or of the predicted membrane binding mutant cSPHK-1V268Q-GFP in the cell body (left), ventral (middle) and dorsal nerve cord (right) of motor neurons of C. elegans. d. Quantification of parameters of the fluorescence spots in the axon terminals (intensity, width and density) in wild-type animals expressing WT and mutant cSPHK-1. For cSPHK-1-GFP, n= 321 synapses from 17 worms, and for cSPHK-1V268Q-GFP, n=387 synapses from 23 worms. Each animal is an independent experiment, and data from independent experiments were pooled. Error bars: standard error of the mean. [ns] not significant; [*] P < 0.05; [**] P < 0.005, Student's t-test. e. Time-course of the onset of paralysis of the indicated worm strains upon exposure to the acetylcholine esterase inhibitor aldicarb (1 mM). sphk-1; cSPHK-1 and sphk-1;cSPHK-1V268Q stand for sphk-1 mutants expressing full length worm SPHK-1-GFP or SPHK-1V268Q-GFP cDNA in neurons, respectively. A total of 60 worms were counted from each plate. Percentages were averaged at each time point per genotype and plotted graphically. n = 3 plates. The analysis was repeated two times, with two different transgenic lines. Data shown are from a single experiment. Error bars: standard error of the mean. Scale bar: 10 µm in a, 20 µm in b, and 10 µm in c.
Mentions: To determine whether the properties that mediate the recruitment of SPHK1 to the tubular invaginations have a physiological significance, we next investigated whether perturbation of the hydrophobic patch rescues the absence of SPHK1 in a functional assay. In neurons, SPHK1 is preferentially targeted to nerve terminals, a compartment highly specialized for the endocytic recycling of synaptic vesicles, as shown by immunocytochemistry47 and by the accumulation of SPHK1-GFP in presynaptic terminals of cortical neurons in vitro47 (and Fig. 6a), or of retinal photoreceptors in vivo (Fig. 6b). The single SPHK1 in C. elegans, cSPHK-1, is also concentrated at synaptic sites along axons, and its absence results in lower sensitivity to aldicarb, indicating a defect in neurotransmitter secretion10, 48. Interestingly, cSPHK-1 is primarily localized at periactive zones of synapses, which are sites of high endocytic activity10. In cSPHK-1, leucine 194 of the hydrophobic patch is replaced by another hydrophobic amino acid, valine 268. Supporting a critical importance of the patch, cSPHK-1V268Q-GFP did not exhibit the same highly punctate localization as cSPHKWT-GFP in axons (Fig. 6c and d). Importantly, while cSPHK-1WT-GFP (Fig. 6e) and even human SPHK1 (Supplementary Fig. 6) rescued neurotransmission in cSPHK-1 mutant worms, cSPHK-1V268Q-GFP did not (Fig. 6e).

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