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. 2021 Nov 12;10:e71982. doi: 10.7554/eLife.71982

Figure 4. IDR-driven YBX1 phase separation is required for sorting YBX1 into exosomes.

(A) Representative microscope images from U2OS cells expressing mChery-RAB5Q79L. Confocal micrographs of cells expressing mCherry-RAB5Q79L, alone (upper row) or with EGFP (lower row). Cells are stained with anti-CD63 (upper row) or with anti-GFP (lower row). (B) Confocal micrographs of U2OS cells expressing mChery-RAB5Q79L and YFP-YBX1. (C) Over-expression of YBX1 in U2OS cells increased the secretion of YBX1 in EVs. Immunoblots for the indicated protein markers in cells and high-speed pellet fractions. The numbers under the YBX1 blot represent quantification analysis of endogenous YBX1, YFP-YBX1, and YFP-YBX1-F85A in cells and sedimentable particles by Fiji software. ‘*’ is a non-specific band; Blue arrow represents endogenous YBX1; Red arrow represents fusion YBX1 or YBX1-F85A. (D) IDR-driven YBX1 phase separation is required for YBX1 secretion in EVs. Immunoblots for the indicated protein markers in U2OS cells and high-speed pellet fractions. The numbers under the YFP blot represent quantification analysis of endogenous YBX1 and variants in cells and sedimentable particles by Fiji software. (E) Proteinase K protection assay on high-speed pellet fractions from U2OS cells. Triton X-100 (0.5%) was used to disrupt the membranes. Immunoblots for YBX1, ALIX, Flotillin-2, and CD9 are shown. (F) Proteinase K protection assay on high-speed pellet fractions from U2OS cells expressing YFP-YBX1. Triton X-100 (0.5%) was used to disrupt the membranes. Immunoblots for YBX1, ALIX, Flotillin-2, and CD9 are shown. (G) Schematic showing exosome purification with buoyant density flotation in a sucrose step gradient. (H) Nanoparticle tracking analysis (NTA) quantification of exosomes from cultured U2OS cells. (I) YFP-YBX1 detected in sucrose post-flotation fraction from U2OS cells. Immunoblots for YBX1, ALIX, and CD63 from buoyant exosomes are shown. (J) Schematic showing exosome purification with buoyant density flotation in a linear iodixanol gradient. (K) Immunoblots across the iodixanol gradient from U2OS cells for classical exosome markers CD9, CD63 and ALIX (the left panel). Collection of fractions F15-F17 corresponding to high density vesicles and immunoblots for YBX1 and CD9. The numbers under YBX1 blot and CD9 blot represent quantification analysis of YFP-YBX1-WT or YFP-YBX1-F85A and CD9 in HD vesicles, respectively, by Fiji software. Scale bars, 3 µm.

Figure 4—source data 1. Uncropped Western blot images corresponding to Figure 4C.
Figure 4—source data 2. Uncropped Western blot images corresponding to Figure 4D.
Figure 4—source data 3. Uncropped Western blot images corresponding to Figure 4E.
Figure 4—source data 4. Uncropped Western blot images corresponding to Figure 4F.
Figure 4—source data 5. Uncropped Western blot images corresponding to Figure 4I.
Figure 4—source data 6. Uncropped Western blot images corresponding to Figure 4K.

Figure 4.

Figure 4—figure supplement 1. YBX1 entering into ILVs is dependent on IDR-driven phase separation.

Figure 4—figure supplement 1.

(A) Confocal micrographs of U2OS cells expressing mChery-RAB5Q79L and YFP tagged YBX1 variants. Scale bar, 3 µm. (B) Nanoparticle tracking analysis (NTA) quantification of exosomes from cultured HEK293T cells. (C) Representative electron micrographs of negative stained exosomes purified from 10/40 % sucrose interface. Scale bar is 100 nm.