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. 2013 Mar 7;33(2):e00026.
doi: 10.1042/BSR20120123.

ALG-2-interacting Tubby-like Protein Superfamily Member PLSCR3 Is Secreted by an Exosomal Pathway and Taken Up by Recipient Cultured Cells

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ALG-2-interacting Tubby-like Protein Superfamily Member PLSCR3 Is Secreted by an Exosomal Pathway and Taken Up by Recipient Cultured Cells

Tatsutoshi Inuzuka et al. Biosci Rep. .
Free PMC article

Abstract

PLSCRs (phospholipid scramblases) are palmitoylated membrane-associating proteins. Regardless of the given names, their physiological functions are not clear and thought to be unrelated to phospholipid scrambling activities observed in vitro. Using a previously established cell line of HEK-293 (human embryonic kidney-293) cells constitutively expressing human Scr3 (PLSCR3) that interacts with ALG-2 (apoptosis-linked gene 2) Ca²⁺-dependently, we found that Scr3 was secreted into the culture medium. Secretion of Scr3 was suppressed by 2-BP (2-bromopalmitate, a palmitoylation inhibitor) and by GW4869 (an inhibitor of ceramide synthesis). Secreted Scr3 was recovered in exosomal fractions by sucrose density gradient centrifugation. Palmitoylation sites and the N-terminal Pro-rich region were necessary for efficient secretion, but ABSs (ALG-2-binding sites) were dispensable. Overexpression of GFP (green fluorescent protein)-fused VPS4B(E235Q), a dominant negative mutant of an AAA (ATPase associated with various cellular activities) ATPase with a defect in disassembling ESCRT (endosomal sorting complex required for transport)-III subunits, significantly reduced secretion of Scr3. Immunofluorescence microscopic analyses showed that Scr3 was largely localized to enlarged endosomes induced by overexpression of a GFP-fused constitutive active mutant of Rab5A (GFP-Rab5A(Q79L)). Secreted Scr3 was taken up by HeLa cells, suggesting that Scr3 functions as a cell-to-cell transferable modulator carried by exosomes in a paracrine manner.

Figures

Figure 1
Figure 1. Secretion of Scr3 into culture medium
(A) HEK-293/Scr3 cells and Scr3-expressing ALG-2 knockdown HEK-293 cells (HEK-293/Scr3/ALG-2KD) were cultured for 2 days, and each CM was centrifuged at 10000 g for 15 min. The obtained culture supernatant (Culture Sup) and TCL were subjected to WB using anti-Scr3 mAb (top panel), anti-ALG-2 pAb (middle panel) and anti-GAPDH mAb (bottom panel). (B) CM of HEK-293/Scr3 cells was fractionated by sequential centrifugations at different gravities, and the supernatants and pellets were analysed for Scr3 by WB. S1, supernatant of 1000 g, 10 min; P1, pellets of 1000 g, 10 min; S2, supernatant of centrifugation of S1 at 10000 g, 15 min; P2, pellets of centrifugation of S1 at 10000 g, 15 min; S3, supernatant of centrifugation of S2 at 100000 g, 60 min; P3, pellets of centrifugation of S2 at 100000 g, 60 min. (C) TCL and pellets of centrifugation of S2 at 100000 g for 60 min of each CM-P3 from HEK-293, HEK-293/ALG-2KD, HEK-293/Scr3 and HEK-293/Scr3/ALG-2KD cells were analysed for Scr3, Alix, ALG-2, Rab5B and GAPDH by WB using specific antibodies. Representative data obtained from three (A) and two (B, C) independent experiments are shown.
Figure 2
Figure 2. Scr3 in secreted exosomes
(A) and (B), 100000 g pellet fraction of CM from HEK-293/Scr3 cells (CM-P3) was resuspended in 2.0 M sucrose in PBS and layered step-wise with a sucrose cushion (1.5, 1.0, 0.5 and 0.25 M) as described in the Materials and methods section. The gradient was spun at 113000 g for 18 h and fractions were collected from the top of the gradient. Each fraction was subjected to WB with antibodies against Scr3, Alix, ALG-2 and Rab5B (A) and to sucrose density measurement (B). (C) Aliquots of exosome fractions obtained by the sucrose density gradient centrifugation were used for a protease resistance assay by incubation with or without 0.1 mg/ml trypsin and 1% Triton X-100 for 20 min. Each sample was subjected to WB using anti-Scr3 antibody. Representative data obtained from three independent experiments are shown.
Figure 3
Figure 3. Requirement of N-terminal region in Scr3 for exosomal secretion
(A) The various GFP-fused Scr3 expression constructs used in this study are schematically represented. The N-terminal PRR (pro-rich region) including ABS1 and ABS2, palmitoylation motif and C-terminal α-helix (αH) are indicated. Full-length or mutants of deletion in ABS1 (ΔABS1), ABS2 (ΔABS2), both ABS1 and ABS2 (ΔABS1/2), N-terminal region (ΔN) and Scr conserved domain (ΔC) were prepared as fusion proteins with GFP. The numbers denote the amino acid (a.a.) positions in each construct. (B) HEK-293 cells were transfected with each plasmid that expresses GFP as a negative control or GFP fused with Scr3 at either N-terminus (GFP–Scr3) or C-terminus (Scr3-GFP). TCL and 100000 g pellet fraction (exosome) from each CM of transfected HEK-293 cells were subjected to WB with antibodies against GFP, Scr3 and Alix. (C) HEK-293 cells were transfected with plasmids expressing GFP (ctrl) or GFP-fused with either WT or various deletion mutants of Scr3, and expressed proteins were similarly analysed by WB as shown in (B). Open arrowhead indicates a non-specific or degraded band of Alix in TCL (bottom panel). Representative data obtained from three independent experiments are shown.
Figure 4
Figure 4. Effects of palmitoylation inhibitor on secretion and intracellular localization of Scr3
(A) After HEK-293/Scr3 cells had been cultured for 24 h in the presence of 50 μM 2-BP or a vehicle (0.5% ethanol) as a control (ctrl), TCL and 100000 g pellet fraction (exosome) of each CM were subjected to WB with antibodies against Scr3 and Alix. (B) Either HEK-293 or HEK-293/Scr3 cells were cultured on poly-l-lysine-coated coverglasses in the presence or absence of 2-BP. After fixation, cells were permeabilized with 0.1% Triton X-100 for 5 min and then immuno-stained with anti-Scr3 mAb and anti-TOM20 pAb and stained with TO-PRO-3 for chromosomal DNA. Fluorescent signals of secondary antibodies of Alexa Fluor 488-conjugated anti-mouse IgG (Scr3: a, e, i; excitation at 488 nm) and Cy3-labelled anti-rabbit IgG (TOM20: b, f, j; excitation at 543 nm), and the signals of TO-PRO-3 (c, g, k; excitation at 633 nm) were analysed with a confocal laser-scanning microscope and are represented in black and white. Merged images are shown in colour (d, h, l) for Scr3 (green), TOM20 (red) and TO-PRO-3 (blue). Bars, 10 μm. Representative data obtained from three (A) and two (B) independent experiments are shown.
Figure 5
Figure 5. Effects of palmitoylation site mutation on secretion of Scr3
(A) Amino acid sequence of the palmitoylation site in Scr3 is displayed. Three and four cysteine residues at the positions indicated by bold face were substituted with alanine residues to create mutants designated as Scr3_3CA and Scr3_4CA, respectively. (B) HEK-293 cells were transfected with plasmids that express untagged WT Scr3, Scr3_3CA (3CA) or Scr3_4CA (4CA) or with an empty vector (pIRES1neo) used as a control (ctrl). TCL and 100000 g pellet fraction (exosome) of each CM of transfected HEK-293 cells were subjected to WB using anti-Scr3 mAb and anti-Alix pAb. Representative data obtained from three independent experiments are shown.
Figure 6
Figure 6. Effects of disturbance of endosomal membrane traffic on secretion of Scr3
(A) HEK-293 cells were co-transfected with pIRES1neo/Scr3 and expression plasmids for GFP–VPS4BE235Q, GFP–Rab5Q79L or GFP as a control (ctrl). TCL and 100000 g pellet fraction (exosome) of the CM from the transfected HEK-293 cells were subjected to WB with antibodies against GFP, Scr3 and Alix. (B) Intensities of WB signals of Scr3 in TCLs and exosomal fractions were quantified and relative secretion efficiencies were calculated by using the value of 1.0 for the control (ctrl). Three independent transfection experiments were performed and calculated values were expressed as means±S.E.M. (n=3). Statistically significant differences of effects on Scr3 secretion were evaluated by Student's t test by comparing the secretion efficiency with the control GFP and GFP-fusion proteins (*P<0.05; **P<0.01). (C) HEK-293 cells cultured on poly-l-lysine-coated coverglasses were co-transfected with pIRES1neo/Scr3 and expression plasmids for GFP–VPS4BE235Q, GFP–Rab5AQ79L or GFP as a control. One day after transfection, the cells were processed for immunocytochemistry using anti-Scr3 mAb and Cy3-conjugated goat anti-mouse IgG. Nuclei were stained with TO-PRO-3. Fluorescence signals of GFP (excitation at 488 nm: a, e and i), Cy3 (excitation at 543 nm: b, f and j) and TO-PRO-3 (excitation at 633 nm: c, g, k) were analysed with a confocal laser scanning microscope and are represented in black and white. Merged images are shown in colour (d, h, l) for GFP (green), Scr3 (red) and TO-PRO-3 (blue). Bars, 10 μm. Representative data obtained from three independent experiments are shown.
Figure 7
Figure 7. Effects of ceramide synthesis inhibition and cholesterol depletion on Scr3 secretion
(A) HEK-293/Scr3 cells were cultured in the presence of either 0.16% DMSO (ctrl), 50 μM 2-BP, HPβCD or 5 μM GW4869 (nSMase inhibitor) for up to 24 h. At the indicated time, each aliquot of the culture medium was centrifuged at 10000 g to remove cell debris, and the culture supernatants were subjected to WB using anti-Scr3 mAb. (B) After HEK-293/Scr3 cells had been cultured for 24 h in the presence of reagents used in (A), TCL and 100000 g pellet fraction (exosome) of each CM were subjected to WB with antibodies against Scr3, Alix and ALG-2. Representative data obtained from two (A) and three (B) independent experiments are shown.
Figure 8
Figure 8. Uptake of secreted Scr3 by HeLa
CM of either HEK-293 cells or HEK-293/Scr3 cells that had been cultured for 48 h was centrifuged at 1000 g, and the supernatant (Culture sup) was mixed 1:1 with the fresh medium and used to culture HeLa cells that had been seeded on coverglasses. After 48 h, cells were fixed and triple-stained with anti-Scr3 mAb (a, e), anti-TOM20 pAb (b, f) and TO-PRO-3 (c, g) and then analysed with a confocal laser-scanning microscope as described in the legend to Figure 4. Merged images are shown in (d) and (h) in colour. Bars, 10 μm. Representative data obtained from three independent experiments are shown.

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