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Nuclear envelope rupture and repair during cancer cell migration

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Science  15 Apr 2016:
Vol. 352, Issue 6283, pp. 353-358
DOI: 10.1126/science.aad7297
  • Fig. 1 Nuclear rupture during migration through confining environments.

    (A) Image sequence of an MDA-MB-231 breast cancer cell that exhibited multiple NE ruptures while moving through 2- × 5-μm2 constrictions. See also movie S1. DIC, differential interference contrast; EGF, epidermal growth factor. Here and in all other figures, red arrows and lines below frames indicate beginning and duration of NE rupture(s). Scale bar, 20 μm. (B) Fluorescence intensity (arbitrary units) of nuclear and cytoplasmic NLS-GFP of the cell in (A), showing loss of nuclear signal and concomitant increase in cytoplasmic fluorescence upon NE rupture, followed by gradual reimport of NLS-GFP into the nucleus. R1 to R3 indicate NE rupture events. (C) NE rupture in an HT1080 fibrosarcoma cell coexpressing NLS-GFP and fluorescently labeled histones (H2B-RFP) migrating inside a collagen matrix (2.5 mg/ml) with MMP inhibitor GM6001. See also movie S2. White arrowheads indicate the minimal nuclear diameter. (Insets, top row) Close-up of nuclear bleb formation (red arrowhead). Scale bar, 10 μm; insets, 2 μm. (D) Minimal nuclear diameter and nuclear and cytoplasmic NLS-GFP fluorescence intensity for the cell in (C). (E) Minimal nuclear diameter in rupturing and nonrupturing HT1080 cells in collagen matrix. ***P < 0.001; n = 159 and 62 cells, respectively. (F) Incidences of NE rupture as a function of constriction sizes in collagen matrices [slope = –1.224; coefficient of determination (R2) = 0.999] and microfluidic devices during a ~12-hour period (slope = –1.219; R2 = 0.974). Regression based on HT1080 and MDA-MB-231 cells; n = 55 to 445 cells per condition. (G and H) Multiphoton image of HT1080 fibrosarcoma cells 5 days after implantation into the mouse dermis. Dashed box in (G) indicates cell with NE rupture and nuclear bleb (arrowhead) shown in (H). Collagen fibers detected by second harmonic generation (SHG); blood vessels visualized by AlexaFluor-750–labeled 70 kD dextran. Scale bar, 20 μm. (I) Fluorescence intensity of cytoplasmic (red) and total (black and gray) NLS-GFP signal in rupturing (red and black) and nonrupturing (gray) cell(s) in the same field of view. (J) Incidence of NE rupture as function of migration mode. **P < 0.01 versus thin and thick strands; n = 22 to 211 cells per condition. Error bars, SE.

  • Fig. 2 Confined migration leads to chromatin protrusions, nuclear fragmentation, and DNA damage.

    (A) Representative image of an MDA-MB-231 cell coexpressing GFP-lamin C and H2B-RFP developing chromatin protrusion (arrowhead) during migration through a microfluidic constriction. Scale bar, 5 μm. (B) HT1080 cell in a collagen matrix (2.5 mg/ml + GM6001) with chromatin protrusion (arrowheads) across the nuclear lamina, stained for lamin A/C (green), DNA (red), and F-actin (turquoise). Scale bars, 10 μm; bottom inset, 2 μm. (C) Percentage of cells with chromatin protrusions as a function of collagen matrix pore size. 2D, unconfined migration on glass slide. **P < 0.01; ***P < 0.0001; n = 50 to 146 cells per condition. (D) Representative image sequence of the formation of chromatin-filled nuclear membrane blebs (white arrowheads) and subsequent nuclear fragmentation in an MDA-MB-231 breast cancer cell coexpressing NLS-GFP (green) and H2B-RFP (red) during migration through consecutive 2- × 5-μm2 constrictions. Insets are indicated by dashed lines. See also movie S6. (E) Percentage of cells with fragmented nuclei before entry into constriction channel, inside the channel, and after exit. ***P < 0.001; n = 9775, 1376, and 3072, respectively. (F) Example of an intact nucleus (left top), a nucleus with a small fragment (arrowhead) (left middle), and a nucleus with a γ-H2AX–positive fragment (left bottom). Scale bar, 10 μm. Percentage of cells with γ-H2AX–positive nuclear fragments before, inside, and after migration through constriction channels (right). ***P < 0.001; n = 1376 to 3072 cells per condition. (G) Percentage of HT1080 cells migrating through 2- × 5-μm2 or 1- × 5-μm2 constrictions that formed 53BP1-RFP foci as a function of nuclear rupture. **P < 0.01, n = 35 cells total. (H) Representative example of formation of 53BP1 foci (arrowheads) in U2OS cell coexpressing NLS-GFP and RFP-53BP1 during migration through 2- × 5-μm2 constriction and NE rupture. Scale bar, 10 μm. Error bars, SE.

  • Fig. 3 Molecular sequence of nuclear rupture.

    (A) Nuclear membrane bleb formation (arrowhead) and collapse upon NE rupture. Scale bars, top and bottom rows, 10 μm; inset, 5 μm. (B) Distribution of NE rupture sites along nuclear periphery. Regions are based on the migration direction (black arrow) and denoted in the cartoon. n = 352 cells; *P < 0.05; ***P < 0.001; all comparisons relative to the hypothetical uniform distribution of 33%. (C) Kymograph of nuclear bleb formation (arrowhead) and collapse during subsequent NE rupture, corresponding to the image sequence in Fig. 1C. (D) Nuclear membrane blebs formed at sites of low GFP-lamin B1 intensity and were devoid of GFP-lamin B1 (arrowheads). Intensity along the blue line is quantified in (E). The gray area indicates the local absence of lamin B1. Representative cell out of 178 cells observed. (E) GFP-lamin B1 fluorescence intensity profile. The gray area and arrowhead indicate the section where the nuclear bleb forms. ***P < 0.001; comparing values inside and outside the gray area. (F) Percentage of nuclear blebs containing detectable amounts of lamin A, B1, or B2. ***P < 0.001; compared with the expected value of 100% for the primary nucleus; n = 178 to 199 cells per condition. (G) Incidence of NE rupture after small interfering RNA (siRNA) treatment against lamin A/C, lamin B2, or nontarget (NT) control. *P < 0.05; **P < 0.01; ***P < 0.001; n = 384, 150, and 163 cells for constrictions ≤2 × 5 μm2; n = 166, 68, and 49 for 15- × 5-μm2 constrictions. (H) Blebbistatin treatment reduced NE rupture incidence during migration in constriction channels, but not in 15- × 5-μm2 control channels. DMSO, dimethyl sulfoxide as a control. *P < 0.05; n = 286 and 194 for constrictions ≤2 × 5 μm2; n = 122, and 54 for cells in constrictions 15 × 5 μm2. (I) GFP-lamin A accumulated at sites of NE rupture, forming lamin scars (arrowheads). Gray and black bars under images correspond to line profiles at different times in (J). Scale bar, 10 μm. (J) GFP-lamin A signal intensity along a section of the nuclear rim [blue line in (I)]. (K) GFP-lamin A accumulation increases with the severity of NE rupture. P < 0.0001; n = 46 cells (slope = 1.345, R2 = 0.3945). Error bars, SE.

  • Fig. 4 ESCRT III mediates nuclear envelope repair.

    (A) CHMP4B-GFP was transiently recruited to the site of nuclear membrane damage (arrowhead). See also movie S7. Dashed boxes indicate areas used for measurements in (B). Representative sequence from 12 HT1080 cells total. Scale bar, 10 μm. (B) CHMP4B-GFP fluorescence intensity at rupture site (black), normalized to prerupture intensity, increased after NE rupture, indicated by increase in cytoplasmic NLS-RFP signal (gray). Red arrow indicates time of NE rupture. ΔF/F0, the change of the intensity from the original intensity before stimulation. (C) Recruitment of VPS4B-GFP to sites of NE rupture (arrowhead) in an MDA-MB-231 cell. Dashed boxes indicate areas used for measurements in (D). Representative example from 18 cells total. Scale bar, 10 μm. (D) VPS4B-GFP fluorescence intensity in the cytoplasm (black) increased rapidly after NE rupture, detected by increase in cytoplasmic NLS-RFP signal (gray). Red arrow indicates time of NE rupture. (E) Representative superresolution image (from 12 cells total) of endogenous CHMP4B accumulation at NE rupture site. Lamin A/C accumulation confirmed rupture site (red arrowhead). Blue arrowhead indicates decreased lamin B intensity at the base of the bleb. Scale bars, 5 μm; inset, 1 μm. (F) siRNA-mediated depletion of ESCRT III proteins CHMP7 and CHMP2A in HT1080 cells resulted in an increased duration of NLS-GFP in the cytoplasm after migration induced NE rupture compared with nontarget (NT) controls. ***P < 0.001; n = 137 and 107, respectively. (G) Expression of the dominant-negative mutant VPS4BE235Q-GFP increased the duration of NLS-GFP in the cytoplasm after migration induced NE rupture in HT1080 cells, which indicated impaired nuclear membrane repair. **P < 0.005; n = 17 and 10, respectively. (H) Percentage of HT1080 cells dying after migration induced NE rupture, in the absence or presence of inhibition of ESCRT III by dominant-negative VPS4BE235Q-GFP and/or DNA repair with the ataxia telangiectasia mutated kinase (ATM) inhibitor KU-55933 (ATMi). **P < 0.01, comparing ATMi+/E235Q to ATMi+/WT and ATMi/E235Q; n = 20, 32, 38, 30, respectively. Error bars, means ± SEM.

Supplementary Materials

  • Nuclear envelope rupture and repair during cancer cell migration

    Celine M. Denais, Rachel M. Gilbert, Philipp Isermann, Alexandra L. McGregor, Mariska te Lindert, Bettina Weigelin, Patricia Davidson, Peter Friedl, Katarina Wolf, Jan Lammerding

    Materials/Methods, Supplementary Text, Tables, Figures, and/or References

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    • Materials and Methods
    • Figs. S1 to S15
    • Movies S1 to S7
    • Full References

    Images, Video, and Other Other Media

    Movie S1
    Nuclear rupture in microfluidic device. MDA-MB-231 breast cancer cell migrating through 2 Ã- 5 μm2 constrictions in microfluidic device. The cell exhibits repetitive nuclear ruptures during passage through the constriction, visualized by transient escape of NLSGFP from the nucleus into the cytoplasm. Time interval: 10 min.
    Movie S2
    Nuclear bleb formation and rupture during cell migration in collagen matrix. HT1080 fibrosarcoma cell stably transfected with H2B-RFP (red) and NLS-GFP (green) migrating in fibrillary matrix of rat tail collagen (2.5 mg/ml) in the presence of MMP inhibitor GM6001. Images acquired by confocal microscopy at 37°C. Representative still images of this sequence are shown in Figs. 1C and 3C, and fig. S9, A and B. Insets show nuclear bleb formation and retraction at the front of the nucleus. Time interval: 4 min. Scale bars: 10 μm; inset, 2 μm.
    Movie S3
    Nuclear bleb formation and rupture in vivo. HT1080 fibrosarcoma cells coexpressing H2B-RFP (red) and NLS-GFP (green) were transplanted into the mouse dermis in the dorsal skin-fold chamber and tissue-invading cells were monitored by time-lapse multiphoton microscopy. Blood vessels and collagen fibers were visualized by 70 kD dextran-Al750 (magenta) and SHG (cyan), respectively. To increase visibility of the weak cytoplasmic GFP signal, the nuclear rupture event is displayed in gray scale. Red arrowheads mark nuclear bleb formation preceding cytoplasmic leakage of NLS-GFP (red ellipse). The cell at the bottom right also exhibits a nuclear rupture at the beginning of the sequence. Time interval: 3 min.
    Movie S4
    Chromatin herniation in migrating cell in collagen in vitro. HT1080 fibrosarcoma cells stably coexpressing H2B-RFP (red) and GFP-lamin A (green) migrated in rat tail collagen (3.3 mg/ml) in the presence of MMP inhibitor GM6001. Cells were imaged by confocal microscopy at 37°C. Red arrowheads mark chromatin protrusions across the nuclear lamina; white or black arrowheads mark signal loss of lamin A; and green arrowheads mark lamin A scar formation. Rectangle shows zoom-in area. Representative still images are shown in fig. S7D. Time interval: 2.5 min. Scale bar, 10 μm.
    Movie S5
    Chromatin herniation in migrating cell in vivo. HT1080 fibrosarcoma cells coexpressing H2B-RFP (red) and GFP-lamin (green) were injected in the mouse dermis in the dorsal skin-fold chamber and monitored by time-lapse multiphoton microscopy. Collagen fibers were visualized with second-harmonic generation (SHG, blue). Inset: Arrowhead marks chromatin herniation during migration. Time interval: 3 min.
    Movie S6
    Nuclear rupture and fragmentation in breast cancer cells migrating through narrow constrictions. MDA-MB-231 breast cancer cells expressing NLS-GFP and H2BmCherry. Two cells (starting in the bottom center) display repeated nuclear ruptures and eventually nuclear fragmentation after passage through the 2 Ã- 5 μm2 constriction. Note that some cells only express NLS-GFP, without H2B-mCherry. Time interval: 10 min.
    Movie S7
    CHMP4B-GFP transiently localizes to the site of NE rupture. HT1080 cell coexpressing CHMP4B-GFP and NLS-RFP migrating through a 2 Ã- 5 μm2 constriction. Video shows formation of a nuclear bleb (black arrow) prior to nuclear rupture (red arrow) and subsequent rapid and transient recruitment of CHMP4B-GFP to the site of nuclear rupture (white arrowhead). Time interval: 2 min.

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