Anomalies in nuclear morphology have been linked to aging-related diseases and malignant transformation, although the mechanism responsible for this connection remains unclear. By expressing dominant-negative TER94 (TER94K2A) mutants in Drosophila photoreceptors, we show disruption of VCP (valosin-containing protein, TER94 ortholog in human), an AAA (ATPase associated with various cellular activities) ATPase essential for ubiquitin-dependent segregation or degradation of proteins, causes an age-dependent nuclear size increase. Loss of VCP function leads to accumulations of MDC1 (mediator of DNA damage checkpoint protein 1), a key DNA damage response gene, and increased γH2AV (an indicator of DNA damage), linking excessive DNA damages and associated responses to this enlarged nuclei defect. Indeed, MDC1-overexpression, similar to TER94K2A, increases γH2AV staining and nuclear size. Moreover, TER94 negatively influences MDC1 level and could interact with MDC1, suggesting that MDC1 is a VCP substrate. MDC1 accumulation increases p53 stability, and this VCP-MDC1-p53 connection is functional, as removal of p53 function suppresses the ability of TER94K2A and MDC1 overexpression to increase nuclear size. While p53 is capable of generating multiple isoforms, our genetic evidence suggests that the p53A isoform specifically contributes to this TER94K2A-associated nuclear size increase. Combining our results with a previous report that p53A expression disrupts autophagic flux, we propose that the failure of removing MDC1 in TER94K2A-expressing cells stabilizes p53A, which blocks autophagy and likely hinders the removal of nuclear content, resulting in aberrant nuclear size increase.
Article
VCP functions to maintain nuclear size by managing DNA damage-induced MDC1–p53–autophagy axis
https://doi.org/10.21203/rs.3.rs-49185/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 12 Jul, 2021
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Developmental changes in the morphology and size of outer photoreceptor cell nuclei. a Time-course analysis of the change in nuclear morphology and size from 70% pupal to the 8-day-old adult stage. Pupal or adult eyes expressing LacZ (control) or TER94K2A by Rh1-GAL4 are stained with phalloidin (red) and anti-Lamin (green) antibody. b Quantification of the nuclear area of R1-R6 from flies of indicated genotypes. 39-162 nuclei from ≥ 2 independent eyes are measured in each group. The horizontal lines shown in b represent mean ± SE. ns, not significant, ***p<0.001 (one-way ANOVA with Bonferroni’s multiple comparison test). Scale bar: 5 μm (a).
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Loss of TER94 function increases the nuclear size without altering the overall cell volume. a Confocal images of 3-day-old adult retinas co-expressing CD4-mCherry with LacZ (control) or TER94K2A under the control of Rh4-GAL4 stained with phalloidin (red), anti-mCherry (green), and anti-Lamin (blue) antibodies to visualize the rhabdomeres, the cytoplasmic membrane, and the nuclear envelope, respectively. The anti- Lamin and anti-mCherry signals are shown separately from the merged images and converted to black and white for comparison. b Quantification of the cellular or nuclear volume of R7 cells labeled by mCherry from flies co-expressing LacZ or TER94K2A. The cellular or nuclear volume are manually outlined according to the mCherry or anti-Lamin signals respectively as shown in (a) and measured by Image J. 7-8 R7 cells from ≥ 4 eyes are measured in each group. c Confocal images of 5-day-old adult retinas expressing LacZ or TER94K2A under the control of Rh1-GAL4 stained with phalloidin (red) and anti-Lamin (green) antibody. d Quantification of the rhabdomeral or nuclear area of R1-R6 nuclei from flies expressing LacZ or TER94K2A. 37-80 nuclei (≥ 2 independent eyes) are measured in each group. The lines represent mean (black) ± SE (red).***p<0.001 (student’s t test, two-tailed). Scale bars: 20 μm (a), 5 μm (d).
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TER94 dysfunction induces nuclear size increase in cone cells and indirect flight muscle cells. a Confocal images of late pupal retinas co-expressing GFP NLS with LacZ (control) or TER94K2A in cone cells under the control of Spa-GAL4 stained with phalloidin (red) and anti-Lamin (magenta) antibody to visualize the rhabdomeres and the nuclear envelope, respectively. GFP-NLS indicates the cone cell nuclei in the retina. B Adult indirect flight muscles expressing LacZ or TER94K2A by Mhc-GAL4; tub-GAL80ts are stained with phalloidin (red), anti-Lamin (magenta), and anti-TER94 (green) antibodies. Flies raised at 18°C are transferred to 30°C after eclosion for 3 weeks to induce transgenes expression. Scale bar: 10 μm (a), 20 μm (b).
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TER94 dysfunction-caused nuclear expansion 993 is not linked to a failure in ERAD or apoptosis activation. a Time-course analysis of the change in the nuclear size and the signal of ERAD reporter, CD3δ-YFP, from 1-day-old to 6-day-old adult stage. Adult eyes co-expressing CD3δ-YFP with LacZ (control) or TER94K2A by Rh1-GAL4 are stained with phalloidin (red) and anti-Lamin (magenta) antibody. b Time-course analysis of the change in the nuclear size and the signal of apoptotic reporter, cleaved PARP, from 2-day-old to 8-day-old adult stage. Adult eyes co-expressing CD8-PARP-Venus with LacZ or TER94K2A by Rh1-GAL4 are stained with anti-cleaved PARP (green) and anti-Lamin (magenta) antibodies. The signals of Venus and anti-cleaved PARP are used to visualize the expression of the probe and caspase activation, respectively. c 5-day-old adult eyes co-expressing GFP-NLS with LacZ or TER94K2A by Rh4-GAL4 are stained with anti-cleaved caspase 3 (green) and anti-Lamin (magenta) antibodies. GFP-NLS marks the R7 cells with transgenes expression. The white arrows point out the cleaved caspase 3 staining in a small population of GFP-NLS-positive R7 cells. Scale bar: 10 μm (a-c).
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LC3 and ref(2)p accumulate in TER94 dysfunction cells. 5-day1012 old adult retinas expressing LacZ (control) or TER94K2A by Rh1-GAL4 are stained with phalloidin (red), anti-Lamin (magenta), and anti-LC3 (left, green) or anti-ref(2)p (right, green) antibodies. Scale bar: 10 μm.
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Overexpressing TER94WT suppresses Derlin-1-induced nuclear expansion phenotype. a Adult eyes expressing LacZ (control), Derlin-1, or co-expressing Derlin-1 with TER94WT by Rh1-GAL4 are stained with phalloidin (red), anti-Lamin (green) and anti-TER94 (magenta) antibodies. The anti-Lamin and anti-TER94 signals are shown separately from the merged images for comparison. b Quantification of the relative anti-TER94 intensity inside versus outside the nucleus in tissues of indicated genotypes. The analyzing range is randomly chosen by drawing a line across two nuclei in each group as the white lines shown in (a). The intensity of anti-Lamin and anti-TER94 signals along the lines is measured by Image J and the peaks of anti-Lamin signals are used to define the borders of the nuclei. 15 random analyzing regions from 3 independent eyes are measured in each group. c Quantification of the nuclear area of R1-R6 from flies of indicated genotypes. 15 nuclei from 3 independent eyes are measured in each group. The lines shown in (b, c) represent mean (black) ± SE (red). **p<0.01, ***p<0.001 (one-way ANOVA with Bonferroni’s multiple comparison test). Scale bar: 10 μm (a).
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The nuclear expansion induced by TER94 dysfunction is not coupled to cell-cycle re-entry. a EdU cell proliferation assay. Images of 3rd instar Canton S (CS) larval eye discs (left panel) containing proliferating cells incubated with S2 medium (negative control) or 20 μM EdU (positive control) for 1.5 hrs prior to fixation. The right panel shows adult retinas expressing LacZ or TER94K2A by Rh1-GAL4 subjected to the EdU incorporation assay before (0-day, freshly-eclosed) and after (2-day) the nuclear expansion occurs. The anti-Lamin (red) antibody is used to mark the nuclear envelope and the Alexa Fluor® 488 azide (green) is used to detect the incorporated EdU. b Examination of the cell-cycle status with Fly-FUCCI, a fluorescent cell-cycle indicator composed of GFP-E2F11–230 and mRFP1-NLS-CycB1–266. This technique differentially labels cells in the G1 (green; degradation of mRFP-tagged CycB), S (red; degradation of GFP-tagged E2F), and G2/M (yellow) phases. GMR>Fly-FUCCI larval eye discs (upper panel) show the cells behind the morphogenetic furrow in the S, G2/M, and G1 phases are labeled by green, yellow, and red fluorescence, respectively, as the corresponding arrows indicated. Rh1>LacZ or Rh1>TER94K2A adult retinas (lower panel) co-expressing Fly-FUCCI before (1-day) and after (5-day) the nuclear expansion are stained with anti-Lamin (magenta) antibody to visualize the nuclear envelope. c Confocal micrographs of larval and adult eyes expressing S/G2/M-Green, a cell-cycle indicator labeling cells in the S, G2, or M phases of cell cycle with green fluorescence. Larval eye discs (upper panel) expressing S/G2/M-Green by GMR-GAL4 are included as a positive control. Adult retinas (lower panel) co-expressing S/G2/M-Green with LacZ or TER94K2A under the control of Rh1-GAL4 are stained with phalloidin (red) and anti-Lamin antibody (magenta). Scale bar: 20 μm (a-c eye discs), 10 μm (a-c adult retinas).
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A genetic screen targeting DDR genes for ability to modify TER94 dysfunction-induced nuclear expansion. a Confocal images of 5-day-old adult retinas co-expressing TER94K2A with LacZ (control) or the indicated RNAi constructs by Rh1-GAL4 stained with phalloidin (red), anti-Lamin (magenta), and anti-ubiquitin-conjugates (FK2, green) antibodies. b Quantification of the nuclear area of R1-R6 from flies expressing the indicated genes by Rh1-GAL4. 90-117 nuclei (from ≥ 2 independent eyes) are measured in each group. The lines represent mean (black) ± SE (red). ***p<0.001 as compared to the control (Rh1>LacZ)(one-way ANOVA with Bonferroni’s multiple comparison test). Scale bar: 10 μm (a).
