Figure 4.
LEM domain proteins and NE repair factors accumulate near nuclear polyQ aggregates. (A) Depth-encoded color projection of representative expanded U2OSWT cells expressing polyQ74-NLS, showing low and high stages of aggregation progression. See also Video 5. (B) Sum projection (inverted grayscale) and 3D volumetric render (green) of single aggregates shown in A. (C) Representative image of expanded U2OSWT cell expressing polyQ74-NLS (green), stained for lamin B1 (magenta). (D) 3D volumetric render of lamin B1 disruption shown in C. (E) Sum projection and intensity profile of cell in C, showing polyQ74-NLS fibrils protruding through a lamin B1 disruption (white arrow). (F) Representative images of expanded U2OSWT cell expressing polyQ74-NLS (green) and cGAS (cyan), stained with total membrane stain (mCling; magenta). White arrowheads indicate local nuclear membrane deformation at a nuclear aggregate-induced rupture site (marked by cGAS accumulation). (G) Schematic representation of pre-rupture localization (1) and recruitment of repair factors after NE rupture. Upon NE rupture BAF (2), and LEM domain proteins and CHMP4B (3) are recruited to the gap in the NE. (H–K) Representative images of U2OSWT cells expressing polyQ23-NLS, polyQ74-NLS or polyQ74 (green) immunolabeled with various antibodies (magenta). Graphs show quantification of the percentage of control cells, or cells expressing polyQ23-NLS, polyQ74-NLS or polyQ74 that show nuclear accumulation of BAF (H; ncells = 908, 722, 823, 384, respectively; N = 3), CHMP4B (I; ncells = 313, 306, 269, 121; N = 4,4,4,3), Emerin (J; ncells = 384, 307, 444, 306; N = 3) or LAP2B (K; ncells = 461, 332, 437, 352; N = 3). Dots represent independent replicates. Orange arrowheads (H–K) and zooms (H and I) indicate nuclear accumulation of immunolabeled protein at nuclear aggregates. Orange arrows indicate co-aggregation of CHMP4B around cytosolic aggregate (I). Contrast of GFP signal was adjusted for (H–K) to aid visibility. Scale bars represent 2.5 μm (A), 0.5 μm (B, C, and F), 0.25 μm (Fzoom), 1 μm (E), and 10 μm (H–K). Voxel sides are 600 nm (grid of D). Horizontal bars represent mean. *P < 0.05, ***P ≤ 0.001, assessed by Fischer’s exact test.

LEM domain proteins and NE repair factors accumulate near nuclear polyQ aggregates. (A) Depth-encoded color projection of representative expanded U2OSWT cells expressing polyQ74-NLS, showing low and high stages of aggregation progression. See also Video 5. (B) Sum projection (inverted grayscale) and 3D volumetric render (green) of single aggregates shown in A. (C) Representative image of expanded U2OSWT cell expressing polyQ74-NLS (green), stained for lamin B1 (magenta). (D) 3D volumetric render of lamin B1 disruption shown in C. (E) Sum projection and intensity profile of cell in C, showing polyQ74-NLS fibrils protruding through a lamin B1 disruption (white arrow). (F) Representative images of expanded U2OSWT cell expressing polyQ74-NLS (green) and cGAS (cyan), stained with total membrane stain (mCling; magenta). White arrowheads indicate local nuclear membrane deformation at a nuclear aggregate-induced rupture site (marked by cGAS accumulation). (G) Schematic representation of pre-rupture localization (1) and recruitment of repair factors after NE rupture. Upon NE rupture BAF (2), and LEM domain proteins and CHMP4B (3) are recruited to the gap in the NE. (H–K) Representative images of U2OSWT cells expressing polyQ23-NLS, polyQ74-NLS or polyQ74 (green) immunolabeled with various antibodies (magenta). Graphs show quantification of the percentage of control cells, or cells expressing polyQ23-NLS, polyQ74-NLS or polyQ74 that show nuclear accumulation of BAF (H; ncells = 908, 722, 823, 384, respectively; N = 3), CHMP4B (I; ncells = 313, 306, 269, 121; N = 4,4,4,3), Emerin (J; ncells = 384, 307, 444, 306; N = 3) or LAP2B (K; ncells = 461, 332, 437, 352; N = 3). Dots represent independent replicates. Orange arrowheads (H–K) and zooms (H and I) indicate nuclear accumulation of immunolabeled protein at nuclear aggregates. Orange arrows indicate co-aggregation of CHMP4B around cytosolic aggregate (I). Contrast of GFP signal was adjusted for (H–K) to aid visibility. Scale bars represent 2.5 μm (A), 0.5 μm (B, C, and F), 0.25 μm (Fzoom), 1 μm (E), and 10 μm (H–K). Voxel sides are 600 nm (grid of D). Horizontal bars represent mean. *P < 0.05, ***P ≤ 0.001, assessed by Fischer’s exact test.

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