Polyglutamine Aggregates Are Inherited by De Novo Generated Neuroblast Cells after Mitosis in <i>D. Melanogaster</i>

<div><p>(A) Expression of Htt-Q128 (red) and Pon-GFP (green) was assessed by confocal laser scanning microscopy in whole embryos (Stage 11, in which anterior is at the top). Occasionally, Htt-Q128 aggregates were observed (inset).</p> <p>(B) During mitosis, the aggregated protein Htt-Q128 is associated with only one of the poles in metaphase, anaphase, and telophase, opposing the Pon-GFP crescent, indicative of asymmetric inheritance to de novo generated neuroblast.</p> <p>(C) Spindle pole–associated aggregates were more clearly visualised after α-tubulin (red) staining in Htt-Q128 (cyan), Pon-GFP (green) neuroblasts. DNA is stained with DAPI (blue).</p></div

Polyglutamine-Expanded Proteins Form Aggresomes in Hamster O23 Cells and Human HEK293 Cells

<div><p>(A) Percentage of cells containing inclusions 24 h after transfection with a fluorescently tagged huntingtin fragment containing a stretch of either 74 (O23: EGFP-HDQ74) or 119 (HEK293: HDQ119-EYFP) glutamines.</p> <p>(B) Fraction of cells showing either aggresome-like inclusions or non–aggresome-like inclusions (nuclear and/or multiple scattered inclusions). Bars represent standard errors of the mean.</p> <p>(C) Aggresome-like inclusions are either close to (upper panel) or co-localise (lower panel) with the centrosomes (decorated with γ-tubulin antibodies) in interphase O23 cells (likewise in HEK293 cells, <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040417#pbio-0040417-sg001" target="_blank">Figure S1</a>).</p> <p>(D) Vimentin microfilaments are redistributed in a cage-like manner around the inclusion, consistent with aggresome morphology. Note that also microtubules (decorated with α-tubulin antibodies) showed partial redistribution to the aggresome.</p> <p>(E) Sequential confocal planes of an aggresome showing both co-localisation with the centrosome and the cage of vimentin. For a full image of this cell see <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0040417#pbio-0040417-sg001" target="_blank">Figure S1</a>B. DNA is stained with DAPI (blue) and only shown in the overlay images. Bars in (C) and (D) represent 10 μm. Bar in (E) represents 2 μm.</p></div

Polyglutamine Aggregates Are Present in Committed Crypt Cells but Absent in Stem Cells in the Small Intestine of SCA3 Patients

<p>(A) Schematic representation of an intestinal crypt for visualisation of the different cell types present in this tissue. Light micrographs of a 4-μm (B) and 1-μm (C) section of a crypt from an SCA3 patient showing positive staining for anti-Musashi antibody. Note that stem cells are localised in between and on both sides of the morphologically recognizable Paneth cells residing at the base of the crypt. (D) A light micrograph of a crypt of a SCA3 patient showing positive staining for the anti-polyglutamine antibody IC2 in some epithelial cells (arrowheads). The asterisks (marked E–J) show representative positions of cells analyzed by subsequent electron microscopy. (E–J) show digitally modified, pseudo-coloured images of electron micrographs indicating the polyglutamine staining in blue. Differentiated epithelial cells (E), transit epithelial cells (F and I), and Paneth cells (J) contain polyglutamine aggregates (arrowheads). Stem cells (G) are negative for polyglutamine aggregates but occasionally contain micro-aggregates (H). Note that also some electron dense material is stained blue by this digital processing. In (G and H), contours are provided in black dashed lines to indicate the stem cells. Bars: D, 20 μm; E and H, 2 μm; F, 1 μm; G, I and J, 5 μm. (K) Quantification of cells with aggregates in the crypts of two SCA3 patients. As double labelling for aggregates and stem cells failed, only the stem cells that were adjacent to the Paneth cells were counted, because these could be easily identified on this basis.</p

Aggresomes Do Not Impair Mitotic Cell Division

<div><p>(A) Quantitative analysis of total mitoses in wild-type HEK293 cells and polyglutamine-expressing HEK293-HDQ119 cells. Bars represent standard error of the mean.</p> <p>(B) Relative fraction of mitoses in each population (diffuse, aggresome-containing, and non–aggresome-containing) of HEK293-HDQ119 cells.</p> <p>(C–G) Representative pictures of fixed O23 (C and D) and HEK293 (E–G) aggresome-containing cells in different mitotic phases. The aggresome is associated with only one of the poles during metaphase, anaphase, and telophase. (C) shows that alignment of chromosomes in metaphase appears normal, and (D and F) show that segregation during anaphase-telophase appears to be normal. Similarly, (C and D) show that positioning of the centrosomes is normal, and (E–F) show that distribution of microtubules and (G) cytokinesis are normal. DNA is stained with DAPI (blue). Bars, 5 μm.</p></div