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A hallmark of Alzheimer's, Huntington's and similar diseases is the assembly of proteins into amyloids rather than folding into their native state. There is an increasing appreciation that amyloids, under specific conditions, may be non-pathogenic. Here we show that amyloids form as a normal part of Xenopus oocyte development. Amyloids are detectable in the cytosol and the nucleus using an amyloid binding dye and antibodies that recognize amyloid structure. In the cytosol, yolk platelets are amyloid reactive, as are a number of yet to be characterized particles. In the nucleus, we find particles associated with transcription by RNA polymerase I, II and III and RNA processing contain amyloids. Nuclear amyloids remain intact for hours following isolation; however, RNase treatment rapidly disrupts nuclear amyloids.
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27215327 ???displayArticle.pmcLink???PMC4920187 ???displayArticle.link???Biol Open ???displayArticle.grants???[+]
Fig. 1. Xenopus oocytes contain nuclear and spatially localized cytosolic amyloid particles. Sectioned ovary was probed for amyloids using thioflavin T (thio-T) or antibodies that recognize either oligomeric (A11) or fibrillar (OC) amyloid epitopes. (A-C) Thio-T staining in (A) highlights the difference in amyloid-positive yolk deposition as oocytes develop from stage II on (left) to stage VI (right). B and C show enlargements of the stage II (B) and stage VI (C) oocyte nuclear thio-T staining in A. Scale bars: (A) 500 µm and (B-C) 50 µm. (D,E) Antibody detection of amyloids in stage III oocytes using either an A11 (D) or OC (E) antibody reveals nuclear (dotted circles) and cytosolic staining of particles similar to that found in A-C, but with lower reactivity to yolk platelets. Arrowheads in D and E indicate increased staining in vegetal hemisphere of oocytes. (F) An isotype control antibody-stained sample. Insets in E and F depict increased exposure times to better visualize nuclear staining. Scale bars: 100 µm.
Fig. 2. Isolated Xenopus nuclei (GVs) can be used for combinatorial identification of nuclear particles with thioflavin T and particle specific antibodies. (A) Manual removal of a GV from a stage VI oocyte. Scale bar: 500 µm. (B) Isolated GVs demonstrate amyloid containing particles seconds after thio-T staining. Scale bar: 200 µm. (C) Overlay of nucleolin immunofluorescence (red, D) and thio-T (green, E) staining of an isolated GV. Scale bar: 100 µm. (F) Overlay of images (G, green) stained with thio-T and (H, red) coilin immunofluorescence. A pearl particle is circled with a solid line, a histone locus body circled with a dashed line. Scale bar: 5 µm. (I) Overlay of panels (J, green) stained with thio-T and (K, red) SC35 immunofluorescence. Dashed lines indicate some thio-T-positive SC35-negative particles, dotted lines indicate a thio-T- and SC35-positive particle. The arrowhead points to a nucleolus in I-K. Scale bars: 5 µm.
Fig. 3. Nuclear particles in isolated Xenopus nuclei (GVs) have overlapping but distinctive reactivity to amyloid detecting antibodies and thio-T. Isolated GVs were examined using an anti-oligomeric amyloid antibody A11 and particle identifying antibodies. Panel (A) shows a low magnification view of a GV using A11 (green) and anti-nucleolin (red) antibodies. Arrows point to some of the many nucleolin negative, A11 positive particles. (B-E) are higher magnification images of a single nucleolus with (B) representing the composite of (C) anti-nucleolin (red), (D) A11 antibody (green) and (E) anti-dsDNA (blue) staining. The arrowhead points to an A11 positive sub structure. Scale bars (A) 500µm and (B-E) 10µm. Coilin positive particles, histone locus bodies (F-G) and pearls (H-I) are shown as overlays of coilin (red) and A11 (green) in (F and H). The A11 signal of the histone locus body (G) is higher than that of the pearl (I). Scale bars: 5µm. The composite image of A11 and anti-SC35 used to detect speckles in (J) highlights the variable intensity of the A11 signals. In (J) speckles with low A11 signal are indicated with arrowheads and those with more robust A11 signal with arrows. Scale bar: 50μm.
Fig. 4. Maintenance of nucleolin and thio-T staining of isolated Xenopus nuclei (GVs) is RNA dependent. (A-C) Alexa Fluor 568 Phalloidin-stained (red) stage V-VI Xenopus GVs were left untreated (A) or treated with RNase A (B) or Xrn1 (C) for 30 min in the presence of 50 μM thio-T (green). Scale bars: 100 μm. (D-F) Untreated (D) or RNase A-treated (E) GVs were immunostained for nucleolin (magenta). (F) Dark field image nucleus. Scale bars: 100 μm.
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