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. 2012 Jan-Feb;3(1):88-100.
doi: 10.4161/nucl.18954.

Transient nuclear envelope rupturing during interphase in human cancer cells

Jesse D Vargas  1 , Emily M Hatch, Daniel J Anderson, Martin W Hetzer
Affiliations

Transient nuclear envelope rupturing during interphase in human cancer cells

Jesse D Vargas et al. Nucleus. 2012 Jan-Feb.

Abstract

Neoplastic cells are often characterized by specific morphological abnormalities of the nuclear envelope (NE), which have been used for cancer diagnosis for more than a century. The NE is a double phospholipid bilayer that encapsulates the nuclear genome, regulates all nuclear trafficking of RNAs and proteins and prevents the passive diffusion of macromolecules between the nucleoplasm and the cytoplasm. Whether there is a consequence to the proper functioning of the cell and loss of structural integrity of the nucleus remains unclear. Using live cell imaging, we characterize a phenomenon wherein nuclei of several proliferating human cancer cell lines become temporarily ruptured during interphase. Strikingly, NE rupturing was associated with the mislocalization of nucleoplasmic and cytoplasmic proteins and, in the most extreme cases, the entrapment of cytoplasmic organelles in the nuclear interior. In addition, we observed the formation of micronuclei-like structures during interphase and the movement of chromatin out of the nuclear space. The frequency of these NE rupturing events was higher in cells in which the nuclear lamina, a network of intermediate filaments providing mechanical support to the NE, was not properly formed. Our data uncover the existence of a NE instability that has the potential to change the genomic landscape of cancer cells.

Keywords: cancer; genomic instability; live-imaging; nuclear envelope; nuclear permeability barrier.

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Figures

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Figure 1. Nuclear envelope rupture during interphase. (A) U2OS cells transiently transfected with GFP3-NLS and imaged every 3 min for 36 h show transient interphase rupturing of the NE followed by recovery of GFP3-NLS into the nucleus. (B) Dynamics of a rupture event. U2OS cells expressing GFP-NLS were imaged every 30s to capture NERDI in high temporal resolution. GFP intensity was normalized by setting the maximum and minimum intensity for each cell to 1 and 0, respectively. Curve fittings of individual interphase NE ruptures were plotted (lines) along with raw data (points). Data was fit using the equation: Y = IF(X < X0,Y0,IF(X < X1,Y0-S*X,Bottom+(Top-Bottom)/(1+10^((Log50-X)*HillSlope)))) where: X0 is the point of inflection between the plateau and the spilling event, Y0 is the plateau value, X1 is the initial point of recovery, S is the slope of spilling, Bottom is the lower plateau for recovery, Top is the upper plateau of recovery, Log50 is the point of 50% recovery, and HillSlope is the linear rate of recovery. (C) Representative images of HeLa cervical and SJSA osteosarcoma cancer cell lines demonstrating spilling in diverse cancer cell types. (D) U2OS cells transiently transfected with GFP3-NLS and imaged every 3 min show localized nuclear deformation and cytoplasmic GFP signal originating from the site of deformation.
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Figure 2. Reduced lamin levels accentuate nuclear ruptures. (A) Frequency of NERDI in U2OS cells after treatment with two rounds of knock down by siRNA directed against: the 3 lamin genes, LBR and Lap2b, Nup93, or Nup107, compared with reporter only (none), scrambled siRNA, or non-transfected IMR90 controls (p = 0.02 3LamKD vs Scram. siRNA). Cells were imaged for a period of 36 h and frequencies represent the proportion of cells that experience an interphase NE rupture at least once over the course of the experiment. (B) Transmission electron microscopy (TEM) of U2OS stably reduced for lamin B1 expression by shRNA and stained with tannic acid to enhance lamin visualization (solid arrows). Characteristic NE herniations exhibit reduced tannic acid staining (open arrows). (C) Dynamics of a rupture in U2OS cells treated with 3 lamin siRNA. U2OS cells transfected with GFP-NLS and 3 lamin siRNA pool were imaged every 30s to capture NERDI in high temporal resolution. Curve fittings, as in Figure 1B, of individual interphase NE ruptures are plotted (lines) along with raw data (points) and show the dynamics of the event. (D) Recovery half-lives of rupture were obtained from each curve and averaged for control and 3 lamin siRNA treated cells with measured half-lives of ~6 and ~12 min, respectively. (E) Left: U2OS cells stably reduced for lamin B1 expression by shRNA and expressing the GFP3-NLS reporter were transiently transfected with either mCherry alone or human lamin B2 tagged with mCherry (mCherry-LmnB2). Frequency of NERDI was analyzed in transfected cells imaged for 36 h. Cells with mCherry-LmnB2 aggregates were excluded from analysis. Right: Average number of spills per cell was determined for cells transfected with either mCherry or mCherry-LmnB2 and imaged for 36 h. For both, n ≥ 340 cells over 2 experiments. Error bars are standard error and the difference in percent cells with spilling nuclei is significant (p ≤ 0.01) by student’s t-test.
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Figure 3. Mislocalization of nuclear and cytoplasmic components. (A) U2OS cells stably reduced for lamin B1 expression by shRNA, expressing GFP3-NLS reporter and stained with antibodies against eIF4AIII (red), tubulin (white), and for DNA with Hoechst (blue) show characteristic NE herniations (top arrows), cytoplasmic localization of eIF4AIII during an interphase NE rupture (middle panel, arrows) with corresponding nuclear influx of soluble tubulin (middle, solid vs. open arrows) contrasted from mitotic NEBD with characteristic condensed DNA and tubulin spindle (bottom panel, arrows). Zoom panel shows linear brightness increase for visualization of diffuse nuclear tubulin. (B) U2OS cells treated as in part A stained for UPF1 (red) and tubulin (white). UPF1 is present in the nuclear interior during an interphase rupture event (top panel, arrows) contrasted from mitotic NEBD by chromatin structure and mitotic spindle (bottom panels, arrows). C) Time series images of NERDI in U2OS cell showing NE deformation and rupture and chromatin dynamics during the event. Nuclear integrity was monitored with GFP3-NLS (green) and chromatin with H2B-mCherry (red) reporters. GFP3-NLS diffuses throughout cytoplasm during NE rupture; H2B-mCherry spills into cytoplasm but is contained to a localized area just beyond the NE boundary.
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Figure 4. Consequences of interphase nuclear rupture. (A) U2OS cells expressing GFP3-NLS (green) and pTurboRFP-mito (red), and stained for DNA with Hoechst (blue) show nuclear mitochondria in confocal slice (top), maximum intensity projection (middle) and 3D reconstruction with nuclear mitochondria indicated (arrows). (B) TEM of U2OS cells stably reduced for lamin B1 expression by shRNA showing cytoplasmic bodies enclosed within the nucleus (i and ii) contrasted from nuclear invaginations with characteristic NE double membrane and ribosome decoration (iii). (C) Proposed model for Nuclear Envelope Rupture During Interphase (NERDI) in cancer cells. Reduced lamin expression leads to a weakened NE that distends outward, eventually rupturing with a mixing of nuclear and cytoplasmic components.
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