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. 2023 Jan 10;12:e85837. doi: 10.7554/eLife.85837

Figure 4. Topological localization of α-syn and DNAJC5 in enlarged endosomes.

(A) DNAJC5 was internalized inside enlarged endosomes. Live U2OS cells expressing mCherry-Rab5Q79L (red) showed circular enlarged endosomes labeled by Rab5 mutant. DNAJC5-HaloTag (green) was visualized by addition of HaloTag Oregon Green Ligand. Representative enlarged endosomes show diffuse (1) or punctate (2 and 3) internalized DNAJC5. Scale bar: 15 μm in overviews and 1 μm in magnified insets. (B) α-syn was excluded from enlarged endosomes. In live U2OS cells, expression of BFP-Rab5Q79L (blue) produced enlarged endosomes of similar morphology compared with mCherry-Rab5Q79L. mNeonGreen-α-syn (mNG-α-syn, green) was expressed both in the nucleus and cytosol. No mNG-α-syn was found inside enlarged endosomes (1–3). Scale bar: 20 μm in overviews and 1 μm in magnified insets. (C) α-syn enters into enlarged endosomes in the presence of DNAJC5. DNAJC5-HaloTag (red) and mNG-α-syn (green) were coexpressed in U2OS cells carrying BFP-Rab5Q79L (blue) mutant and imaged. No mNG-α-syn was internalized in endosome without DNAJC5-HaloTag inside (1). In contrast, mNG-α-syn was found inside endosomes with DNAJC5-HaloTag inside (2 and 3). Scale bar: 10 μm in overviews and 1 μm in magnified insets. (D) α-syn and DNAJC5 co-sedimented in membrane fractionation. HEK293T cell homogenate was sequentially centrifuged at increasing velocity from 3000×g (3k), 25,000×g (25k), and 100,000×g (100k). The 25k membrane fraction had the highest amount of both α-syn and DNAJC5. (E) Quantification of the membrane fractionation results in (D). (F) Proteinase K protection assay of 25k membrane-containing α-syn and DNAJC5. (G) Quantification of the proteinase K protection assay in (F). (H) Quantification of the ratio of α-syn-containing endosomes in control cells (no-DNAJC5 transfection) or cells co-transfected with DNAJC5. More than 100 enlarged endosomes were counted in each group. Error bars represent standard deviations. P value<0.0001, two-tailed t test.

Figure 4—source data 1. Uncropped immunoblot corresponding to Figure 4.

Figure 4.

Figure 4—figure supplement 1. Immunofluorescence (IF) images of endogenous DNAJC5 and enlarged endosomes.

Figure 4—figure supplement 1.

(A) Colocalization between endogenous CD63 (red) and DNAJC5 (green). U2OS cells were cultured and fixed, incubated with corresponding antibodies for IF detection of endogenous CD63 and DNAJC5. Representative colocalized region is shown in magnified insets. Scale bar: 20 μm. (B) CD63 (green) was inside the enlarged endosomes labeled by peripheral mCherry-Rab5Q79L (red). U2OS expressing mCherry-Rab5Q79L were fixed, followed by IF using anti-CD63 antibody. Scale bar: 25 μm.
Figure 4—figure supplement 2. Live-cell images of U2OS cells expressing DNAJC5 L115R mutant and α-syn.

Figure 4—figure supplement 2.

(A) Live-cell imaging of U2OS cells transfected with DNAJC5 (L115R)-HaloTag and mCherry-Rab5Q79L. DNAJC5 (L115R)-HaloTag (green) is diffuse in cytosol and outside of the enlarged endosomes labeled by peripheral mCherry-Rab5Q79L (red). Scale bar: 10 μm in overviews and 1 μm in magnified insets. (B) Live-cell imaging of U2OS cells transfected with mNeonGreen (mNG)-α-syn, DNAJC5 (L115R)-HaloTag, and mCherry-Rab5Q79L. In the condition of coexpression with DNAJC5 (L115R)-HaloTag (red), no mNG-α-syn (green) was found inside of enlarged endosomes labeled by peripheral BFP-Rab5Q79L (blue). Scale bar: 10 μm in overviews and 1 μm in magnified insets.
Figure 4—video 1. Time lapse of movement of internalized α-syn and DNAJC5 in the enlarged endosomes.
Download video file (28MB, mp4)
DNAJC5-HaloTag (red) and mNG-α-syn (green) were coexpressed in U2OS cells carrying BFP-Rab5Q79L (blue) mutant and imaged. Scale bar: 10 μm.