Sphingosine and Sphingosine Kinase 1 Involvement in Endocytic Membrane Trafficking

Abstract

The balance between cholesterol and sphingolipids within the plasma membrane has long been implicated in endocytic membrane trafficking. However, in contrast to cholesterol functions, little is still known about the roles of sphingolipids and their metabolites. Perturbing the cholesterol/sphingomyelin balance was shown to induce narrow tubular plasma membrane invaginations enriched with sphingosine kinase 1 (SphK1), the enzyme that converts the bioactive sphingolipid metabolite sphingosine to sphingosine-1-phosphate, and suggested a role for sphingosine phosphorylation in endocytic membrane trafficking. Here we show that sphingosine and sphingosine-like SphK1 inhibitors induced rapid and massive formation of vesicles in diverse cell types that accumulated as dilated late endosomes. However, much smaller vesicles were formed in SphK1-deficient cells. Moreover, inhibition or deletion of SphK1 prolonged the lifetime of sphingosine-induced vesicles. Perturbing the plasma membrane cholesterol/sphingomyelin balance abrogated vesicle formation. This massive endosomal influx was accompanied by dramatic recruitment of the intracellular SphK1 and Bin/Amphiphysin/Rvs domain-containing proteins endophilin-A2 and endophilin-B1 to enlarged endosomes and formation of highly dynamic filamentous networks containing endophilin-B1 and SphK1. Together, our results highlight the importance of sphingosine and its conversion to sphingosine-1-phosphate by SphK1 in endocytic membrane trafficking.

Keywords: endocytosis; endosome; sphingolipid; sphingosine; sphingosine kinase (SphK); sphingosine-1-phosphate (S1P).

FIGURE 1.

Sphingosine and analogs induce dilated vesicle formation. A, molecular structures of d-erythro-sphingosine and the SphK1 inhibitors SK1-I, N,N-dimethylsphingosine (DMS), PF543, and FTY720. B, phase-contrast images of H460 cells treated for 30 min with vehicle, sphingosine (10 μm), SK1-I (10 μm), PF543 (1 μm), or FTY720 (5 μm). Scale bars = 10 μm.

FIGURE 2.

SK1-I, a sphingosine-like SphK1 inhibitor, induces vesicles in diverse cell types. A–D, phase-contrast images and quantification of vesicle-positive cells following 30-min treatment with vehicle (veh) or SK1-I (10 μm). A, DLD1 colon cancer cells. B, HCT116 colon cancer cells. C, MEFs. D, human breast epithelial MCF10A breast cells. Data are mean ± S.D. **, p ≤ 0.01; ****, p ≤ 0.0001. Scale bars = 10 μm.

FIGURE 3.

Vesicle size is dependent on SphK1. A–C, characterization of Sphk1 knockout MEFs. A, PCR genotyping of wild-type and Sphk1^−/− MEFs. The expected PCR product for WT Sphk1 allele amplification is 291 bp and 503 bp for the Sphk1 knockout neomycin cassette PCR product. B, Sphk1^−/− MEFs showed no activity in a SphK1 isoform-specific activity assay (76). n = 3. C, sphingolipids were extracted from wild-type and Sphk1^−/− MEFs, and levels of S1P, Sph, and ceramide (Cer) were determined by LC-ESI-MS/MS. Data are mean ± S.D. D–G, phase-contrast images (D and F) and vesicle diameter quantitation (E and G) of Sphk1^−/− and WT MEFs treated with vehicle, 10 μm SK1-I for 2.5 h (D and E), or 5 μm FTY720 for 6 h (F and G). Data are mean ± S.D. **, p < 0.01; ***, p < 0.001; ****, p ≤ 0.0001. Scale bars = 10 μm; au, arbitrary units.

FIGURE 4.

Inhibition of SphK1 leads to accumulation of dilated vesicles. A and B, phase-contrast images and quantification of vesicle-positive H460 cells treated with Sph (10 μm) or pretreated with PF543 (1 μm) for 30 min, followed by Sph (10 μm) or SK1-I alone (10 μm) for 3 or 20 h, as indicated. C and D, wild-type and Sphk1^−/− MEFs were treated with 10 μm Sph for the indicated times, and vesicle-positive cells were quantified (D). At least 5 fields/time point were analyzed. Representative phase-contrast images are shown, and vesicle-positive cells were quantitated. Data are mean ± S.D. ns, not significant; ***, p ≤ 0.001; ****, p ≤ 0.0001. Scale bars = 10 μm. veh, vehicle.

FIGURE 5.

Sphingosine- and SK1-I-induced vesicles are derived from the plasma membrane. A and B, phase-contrast and confocal images of live H460 cells pulsed for 15 min with pHrodo-Red-Dextran (0.1 mg/ml) followed by treatment with vehicle, SK1-I (10 μm), or Sph (10 μm) for 30 min as indicated. Arrowheads indicate pHrodo-Red-positive dilated vesicles. Nuclei were labeled with Hoechst. C, confocal images of live HCT116 cells treated for 5 h with vehicle, chloroquine (50 μm), or SK1-I (10 μm), followed by staining with acridine orange (250 nm). D and E, phase-contrast images of WT and Sphk1^−/− MEFs were treated with vehicle or 50 μm chloroquine for 7 h (D), and vesicle diameter was quantified (E). The data in E are mean ± S.D. ns, not significant. Scale bars = 10 μm.

FIGURE 6.

Vesicles are dilated late endosomes. A, phase-contrast and confocal images of live H460 cells pretreated for 20 min with NBD-cholesterol (0.25 μm) followed by SK1-I (10 μm) for 30 min. Boxed area, arrowheads indicate NBD-cholesterol-positive vesicles. B, confocal images of live H460 cells expressing Rab7a-RFP pretreated for 15 min with NBD-Sph (0.25 μm) followed by treatment with SK1-I (7.5 μm) for 30 min. Zoom, vesicles positive for NBD-Sph and Rab7a. C, confocal and phase-contrast images of live H460 cells expressing Rab7a-RFP treated with vehicle or SK1-I (10 μm) for 30 min. Zoom, co-localization of Rab7a to vesicles. D, confocal images of live H460 cells expressing Rab7a-RFP treated with Sph (7.5 μm) or SK1-I (7.5 μm) for 8 h. E and F, confocal and phase-contrast images of H460 cells expressing LC3-RFP-GFP treated with SK1-I (7.5 μm) for 1 h (E) and quantification of RFP:GFP fluorescence ratio (F). Zoom, vesicles were not encircled with LC3-RFP-GFP. Arrowheads point to vesicles. In C and E, nuclei were stained with Hoechst. In F, data are mean ± S.D. ns, not significant. Scale bars = 10 μm.

FIGURE 7.

SK1–1 induces internalization of clathrin and caveolin-1. A and B, confocal images of H460 (A) and HCT116 (B) cells treated for 30 min without or with SK1-I (10 μm), followed by immunostaining with anti-clathrin heavy chain (red) or anti-caveolin-1 (green). Zoom, ring-like structures positive for clathrin and caveolin-1 appeared after treatment with SK1-I. C, confocal images of H460 cells transfected with non-targeting siControl or caveolin-1 specific siRNA (siCaveolin-1). Cells were treated for 30 min with vehicle or SK1-I (10 μm), followed by immunostaining with anti-caveolin-1 (red). D and E, quantification of caveolin-1 fluorescence (D) and immunoblots (E) of the cells in C. n = 3. F, quantification of vesicle-positive cells in C. n = 5. In A–C, nuclei were stained with Hoechst. Data are mean ± S.D. **, p ≤ 0.01; ***, p ≤ 0.001. Scale bars = 10 μm; au, arbitrary units.

FIGURE 8.

Effect of endocytosis inhibitors. A, phase-contrast images of live H460 cells incubated for 30 min with vehicle (DMSO), chlorpromazine (CPZ, 5 μg/ml), nocodazole (noc, 200 μm), cytochalasin D1 (cyt-D1, 10 μg/ml), bafilomycin A1 (bafA1, 50 nm), and MβCD (10 mm) in cholesterol-stripping buffer or acidic sphingomyelinase (SMase, 1000 units/ml), followed by treatment with SK1-I (5 μm) for 45 min. B, quantification of vesicle-positive cells in A. C and D, SK1-I increases internalization of CTB. Shown are confocal images (C) and intracellular CTB fluorescence quantification (D) of H460 cells pretreated for 5 min with 0.05 μg/ml Alexa Fluor 647-CTB followed by treatment without or with 10 μm SK1-I for 30 min. Zoom, ring-like structures positive for CTB appeared after treatment with SK1-I. At least 80 cells were quantified for each time point. ns, not significant; *, p ≤ 0.05; **, p ≤ 0.01; ****, p ≤ 0.0001; t test. Data are mean ± S.D. In C, nuclei were stained with Hoechst. Scale bars = 10 μm. veh, vehicle; au, arbitrary units.

FIGURE 9.

Acute removal of plasma membrane cholesterol induces recruitment of endophilin-A2, endophilin-B1, and SphK1 to tubular invaginations. A, live-cell confocal images of H460 cells expressing endophilin-A2-GFP, SphK1-GFP, or endophilin-B1-GFP, as indicated, before and 320 s after 10 mm MβCD treatment at 37 °C. A total of four independent experiments with a minimum of 5 cells/experiment were performed, and representative images are shown. B and C, confocal images of H460 cells expressing endophilin-A2-GFP and V5-tagged SphK1 (B) or endophilin-B1-GFP and V5-tagged SphK1 (C) before and 360 s after 10 mm MβCD treatment at 37 °C and immunostained with anti-V5 (purple). Arrowheads point to foci formed after MβCD treatment where endophilins and V5-tagged SphK1 co-localize. Scale bars = 10 μm.

FIGURE 10.

SK1-I induces recruitment of SphK1, endophilin-A2, and endophilin-B1 to vesicles and dynamic filamentous structures. A–E, live-cell confocal images of H460 cells expressing SphK1-GFP (A and E), endophilin-A2-GFP (B), or endophilin-B1-GFP (C and D) treated with 10 μm SK1-I for 45 min. D, bottom panels, time-lapse (30-s intervals) images of the region outlined in red. Zoom, magnification of SphK1-GFP-positive (A) or endophilin-A2-GFP-positive (B) vesicles and endophilin-B1-GFP positive filaments (C). Arrowheads in C point to filaments that are contiguous with vesicles. Arrowheads in E point to SphK1-positive filaments. Scale bars = 10 μm.

FIGURE 11.

Relocalization of endogenous endophilin-A2 and endophilin-B1 following treatment with SK1-I and sphingosine. A, C, and D, confocal images of H460 cells treated with vehicle (A), 10 μm SK1-I for 30 min (A and D), or 1 μm PF543 for 30 min followed by 10 μm Sph (C) for 30 min. Cells were then immunostained with anti-endophilin-A2 (red) or anti-endophilin-B1 (green), and nuclei were labeled with Hoechst. Arrowheads in A point to filamentous structures. A and C, zoom, magnification of double-positive foci (A, top) and vesicles (A, bottom, and C). B, the diameters of foci and vesicles in A were determined. D, 3D reconstructions of cells treated without or with 10 μm SK1-I for 30 min. A total of 37 0.44-μm slices were collected for each channel. One slice in the right panel was used in A. Data are mean ± S.D. ***, p ≤ 0.001. Scale bars = 10 μm.