minimal levels during themalaria parasite bloodphase, which relies solely on aerobic glycolysis(21). Nevertheless, nominal transport is essential,primarily as an electron sink for pyrimidine bio-synthesis (22). We hypothesize that the modestlevels of mitochondrial electron transport duringblood phase offer relaxed selection on cytB—whichis multicopy and easily mutable (3, 23)—allowingrespiration-deficient mutants (8, 24) with reducedatovaquone binding (9) to be readily selectedby drug pressure. However, when thesemutantsswitch to the mosquito phase—which relies onfull aerobic respiration with an active tricarbo-xylic acid cycle (25), robust electron transport(17, 26, 27), andmitochondrial adenosine triphos-phatase activity (28)—the respiration deficits ofthe cytBmutants (8, 24) prevent them from com-pleting their development and generating infectioussporozoites. This results in a block of transmissionof atovaquone resistance genotypes to newhosts—a block that cannot be overcome by outcrossingbecause cytB is maternally inherited.Cytochrome b is thus a rather unique malaria

drug target. Its genetics are constrained by ma-ternal inheritance (10–12, 29), there is no recom-bination ofmitochondrial DNA (23), andmarkedlydifferent selection regimes in the mammalianversus the mosquito hosts (17, 25, 26, 28) all com-bine to restrict theparasite’s options todisseminatemutations conferring resistance to atovaquone,even though they can arise relatively quickly inpatients (2, 3). These constraints likely apply toother cytochrome b targeting drugs currentlyunder development (30–32) and perhaps to drugstargeting the maternally inherited apicoplast(10–12, 29), an endosymbiotic organelle drug tar-get that also has differential activity across thelife cycle.

REFERENCES AND NOTES

1. G. L. Nixon et al., J. Antimicrob. Chemother. 68, 977–985(2013).

2. R. J. Maude, C. Nguon, A. M. Dondorp, L. J. White, N. J. White,Malar. J. 13, 380 (2014).

3. G. Cottrell, L. Musset, V. Hubert, J. Le Bras, J. Clain,Antimicrob. Agents Chemother. 58, 4504–4514 (2014).

4. E. Y. Klein, Int. J. Antimicrob. Agents 41, 311–317 (2013).5. E. A. Ashley et al., N. Engl. J. Med. 371, 411–423 (2014).6. M. Fry, M. Pudney, Biochem. Pharmacol. 43, 1545–1553

(1992).7. I. K. Srivastava, J. M. Morrisey, E. Darrouzet, F. Daldal,

A. B. Vaidya, Mol. Microbiol. 33, 704–711 (1999).8. J. E. Siregar et al., Parasitol. Int. 64, 295–300 (2015).9. D. Birth, W. C. Kao, C. Hunte, Nat. Commun. 5, 4029

(2014).10. A. Creasey et al., Mol. Biochem. Parasitol. 65, 95–98

(1994).11. A. M. Creasey et al., Curr. Genet. 23, 360–364 (1993).12. A. B. Vaidya, J. Morrisey, C. V. Plowe, D. C. Kaslow,

T. E. Wellems, Mol. Cell. Biol. 13, 7349–7357 (1993).13. D. Jacot, R. F. Waller, D. Soldati-Favre, D. A. MacPherson,

J. I. MacRae, Trends Parasitol. 32, 56–70 (2016).14. D. Syafruddin, J. E. Siregar, S. Marzuki, Mol. Biochem. Parasitol.

104, 185–194 (1999).15. J. E. Siregar, D. Syafruddin, H. Matsuoka, K. Kita, S. Marzuki,

Parasitol. Int. 57, 229–232 (2008).16. M. Korsinczky et al., Antimicrob. Agents Chemother. 44,

2100–2108 (2000).17. K. E. Boysen, K. Matuschewski, J. Biol. Chem. 286,

32661–32671 (2011).18. B. Franke-Fayard et al., Mol. Biochem. Parasitol. 137, 23–33

(2004).19. M. R. van Dijk et al., Cell 104, 153–164 (2001).

20. L. Reininger et al., J. Biol. Chem. 280, 31957–31964(2005).

21. J. I. MacRae et al., BMC Biol. 11, 67 (2013).22. H. J. Painter, J. M. Morrisey, M. W. Mather, A. B. Vaidya, Nature

446, 88–91 (2007).23. M. D. Preston et al., Nat. Commun. 5, 4052 (2014).24. N. Fisher et al., J. Biol. Chem. 287, 9731–9741 (2012).25. H. Ke et al., Cell Reports 11, 164–174 (2015).26. A. Hino et al., J. Biochem. 152, 259–268 (2012).27. T. Q. Tanaka, M. Hirai, Y. Watanabe, K. Kita, Parasitol. Int. 61,

726–728 (2012).28. A. Sturm, V. Mollard, A. Cozijnsen, C. D. Goodman,

G. I. McFadden, Proc. Natl. Acad. Sci. U.S.A. 112, 10216–10223(2015).

29. N. Okamoto, T. P. Spurck, C. D. Goodman, G. I. McFadden,Eukaryot. Cell 8, 128–132 (2009).

30. T. G. Nam et al., ACS Chem. Biol. 6, 1214–1222 (2011).31. A. M. Stickles et al., Am. J. Trop. Med. Hyg. 92, 1195–1201

(2015).32. C. K. Dong et al., Chem. Biol. 18, 1602–1610 (2011).

ACKNOWLEDGMENTS

This work was supported by grants from the National Health andMedical Research Council (Australia); the Australian ResearchCouncil; the Indonesian Ministry of Research, Technology, and HigherEducation; the U.S. National Institutes of Health (grants AI031478 andRR00052); the Japanese Society for Promotion of Science (JSPS)

KAKENHI (grant 23117004 to M. M. and 26253025 to K. K.); andJapanese Science and Technology Agency/Japan InternationalCooperation Agency Science and Technology Research Partnership forSustainable Development (SATREPS; no. 10000284 to K. K.). Weacknowledge support of the Program for Promotion of Basic andApplied Researches for Innovations in Bio-oriented Industry (BRAIN)and the Science and Technology Research Promotion Program forAgriculture, Forestry, Fisheries and Food Industry. J.E.S. was a JSPSPh.D. fellow (RONPAKU program) and an Endeavor Fellow from ScopeGlobal, Australia. A.S. is affiliated with TropIQ Health Sciences(http://tropiq.nl). We thank the Johns Hopkins Malaria ResearchInstitute mosquito and parasite core facilities for help with mosquitorearing and P. falciparum cultures, S. Narulitha of the Eijkman Institutefor Molecular Biology for the novel P. berghei PbM133I atovaquoneresistance mutant, and Walter Reed Army Institute of Research forthe P. falciparum NF54e parasites. Data are archived at figshare(https://figshare.com) under doi 10.4225/49/56DE29B278684.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6283/349/suppl/DC1Materials and MethodsFig. S1References (33–39)

23 November 2015; accepted 10 March 201610.1126/science.aad9279

CELL BIOLOGY

Nuclear envelope rupture and repairduring cancer cell migrationCeline M. Denais,1* Rachel M. Gilbert,1* Philipp Isermann,1* Alexandra L. McGregor,1

Mariska te Lindert,2 Bettina Weigelin,2 Patricia M. Davidson,1 Peter Friedl,2,3,4

Katarina Wolf,2 Jan Lammerding1†

During cancer metastasis, tumor cells penetrate tissues through tight interstitial spaces,which requires extensive deformation of the cell and its nucleus. Here, we investigatedmammalian tumor cell migration in confining microenvironments in vitro and in vivo.Nuclear deformation caused localized loss of nuclear envelope (NE) integrity, which ledto the uncontrolled exchange of nucleo-cytoplasmic content, herniation of chromatinacross the NE, and DNA damage. The incidence of NE rupture increased with cellconfinement and with depletion of nuclear lamins, NE proteins that structurally supportthe nucleus. Cells restored NE integrity using components of the endosomal sortingcomplexes required for transport III (ESCRT III) machinery. Our findings indicate that cellmigration incurs substantial physical stress on the NE and its content and requiresefficient NE and DNA damage repair for cell survival.

The nuclear envelope (NE), comprising theinner and outer nuclear membranes, nu-clear pore complexes, and the nuclear lam-ina, presents a physical barrier between thenuclear interior and the cytoplasm that pro-

tects the genome from cytoplasmic componentsand establishes a separate compartment forDNAand RNA synthesis and processing (1). Loss of

NE integrity and nuclear pore selectivity hasbeen linked to the normal aging process and avariety of human diseases, including cancer (2).In cancer progression, key steps of tumor cellinvasion depend upon deformation of the nu-cleus into available spaces within the three-dimensional tissue (3–6). Whereas the cytoplasmof migrating cells can penetrate even submicron-sized pores, the deformation of the large andrelatively rigid nucleus becomes a rate-limitingfactor in migration through pores <25 mm2 incross section (4, 6–10). We hypothesized thatmigration through such tight spaces provides asubstantial mechanical challenge to the integrityof the nucleus. Thus, we investigated whethercell migration through confining spaces inducesNE rupture and compromises DNA integrity

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1Nancy E. and Peter C. Meinig School of Biomedical Engineeringand Weill Institute for Cell and Molecular Biology, CornellUniversity, Ithaca, NY, USA. 2Department of Cell Biology,Radboud University Medical Center, Nijmegen, Netherlands.3Department of Genitourinary Medical Oncology, The Universityof Texas MD Anderson Cancer Center, Houston, TX, USA.4Cancer Genomics Center, Netherlands (CGC.nl).*These authors contributed equally to this work. †Correspondingauthor. E-mail: jan.lammerding@cornell.edu

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and how cells repair such NE ruptures duringinterphase.To model cancer cell invasion with precise

control over cell confinement, we designed a mi-crofluidic device containing constrictions withfixed height and varying widths mimicking in-

terstitial pore sizes (fig. S1, A and B) (11). Wedetected NE rupture using previously establishedfluorescent reporters consisting of green or redfluorescent proteins fused to anuclear localizationsequence (NLS-GFP and NLS-RFP, respectively)that rapidly escape into the cytoplasm when NE

integrity is lost (12–14). Breast cancer, fibrosarcoma,andhuman skin fibroblast cells displayed transientloss of NE integrity, which coincided with thenucleus passing through the constrictions (Fig. 1,A and B; fig. S1, C to L; andmovie S1). NE rupturewas associatedwith transient influx of fluorescently

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Fig. 1. Nuclear rupture during migra-tion through confining environments.(A) Image sequence of an MDA-MB-231breast cancer cell that exhibitedmultiple NE ruptures while movingthrough 2- × 5-mm2 constrictions. Seealso movie S1. DIC, differential interfer-ence contrast; EGF, epidermal growthfactor. Here and in all other figures, redarrows and lines below frames indicatebeginning and duration of NE rupture(s).Scale bar, 20 mm. (B) Fluorescenceintensity (arbitrary units) of nuclear andcytoplasmic NLS-GFP of the cell in (A),showing loss of nuclear signal andconcomitant increase in cytoplasmicfluorescence upon NE rupture, followedby gradual reimport of NLS-GFP intothe nucleus. R1 to R3 indicate NErupture events. (C) NE rupture in anHT1080 fibrosarcoma cell coexpressingNLS-GFP and fluorescently labeledhistones (H2B-RFP) migrating inside acollagen matrix (2.5 mg/ml) with MMPinhibitor GM6001. See also movie S2.White arrowheads indicate the minimalnuclear diameter. (Insets, top row)Close-up of nuclear bleb formation (redarrowhead). Scale bar, 10 mm; insets,2 mm. (D) Minimal nuclear diameter andnuclear and cytoplasmic NLS-GFP fluo-rescence intensity for the cell in (C).(E) Minimal nuclear diameter inrupturing and nonrupturing HT1080cells in collagen matrix. ***P < 0.001;n = 159 and 62 cells, respectively.(F) Incidences of NE rupture as a functionof constriction sizes in collagen matrices[slope = –1.224; coefficient of determi-nation (R2) = 0.999] and microfluidicdevices during a ~12-hour period (slope =–1.219; R2 = 0.974). Regression basedon HT1080 and MDA-MB-231 cells;n = 55 to 445 cells per condition.(G and H) Multiphoton image of HT1080fibrosarcoma cells 5 days after implanta-tion into the mouse dermis. Dashed boxin (G) indicates cell with NE rupture andnuclear bleb (arrowhead) shown in (H).Collagen fibers detected by secondharmonic generation (SHG); bloodvessels visualized by AlexaFluor-750–labeled 70 kD dextran. Scale bar, 20 mm.(I) Fluorescence intensity of cytoplasmic(red) and total (black and gray) NLS-GFPsignal in rupturing (red and black) andnonrupturing (gray) cell(s) in the samefield of view. (J) Incidence of NE ruptureas function of migration mode. **P < 0.01 versus thin and thick strands; n = 22 to 211 cells per condition. Error bars, SE.

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labeled cytoplasmic proteins into the nucleus (fig.S2) and could also be detected by accumulationof the fluorescently labeled DNA-binding pro-teins’ barrier-to-autointegration factor (BAF) (15)and guanosine 3′,5′-monophosphate–adenosine3′,5′-monophosphate (cyclic GMP-AMP) synthase(cGAS) (16) at sites of NE rupture (fig. S3).We then tested whether NE rupture also oc-

curs during cancer cell migration in biological

environments. Fibrosarcoma cells and skin fibro-blasts exhibited NE rupture during migration infibrillar collagenmatrices (Fig. 1, C andD; fig. S4;and movie S2), with kinetics similar to thoserecorded in the constriction channels (fig. S1M).NE rupture typically occurred when theminimalnuclear diameter, which closely matches the poresize encountered by the cell (6), dropped to 3 mm(Fig. 1, D and E) and so linked NE rupture to cell

movement through narrow spaces. Accordingly,NE ruptures were rare (i.e., below 5% per ~12-hour observation period) for cells migrating onglass, in low-density collagenmatrices, or throughchannels 15 × 5 mm2 wide, in line with previouslyreported rates of spontaneous NE rupture incancer cell lines (13). However, whenmatrix poresizes were reduced to 5 to 20 mm2 and below byincreasing collagen concentration and/or by

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Fig. 2. Confined migration leads to chromatin protrusions, nuclear fragmen-tation, and DNA damage. (A) Representative image of an MDA-MB-231cell coexpressing GFP-lamin C and H2B-RFP developing chromatin protrusion(arrowhead) during migration through a microfluidic constriction. Scale bar,5 mm. (B) HT1080 cell in a collagen matrix (2.5 mg/ml + GM6001) withchromatin protrusion (arrowheads) across the nuclear lamina, stained forlamin A/C (green), DNA (red), and F-actin (turquoise). Scale bars, 10 mm;bottom inset, 2 mm. (C) Percentage of cells with chromatin protrusions asa function of collagen matrix pore size. 2D, unconfined migration on glassslide. **P < 0.01; ***P < 0.0001; n = 50 to 146 cells per condition. (D) Rep-resentative image sequence of the formation of chromatin-filled nuclearmembrane blebs (white arrowheads) and subsequent nuclear fragmenta-tion in an MDA-MB-231 breast cancer cell coexpressing NLS-GFP (green)and H2B-RFP (red) during migration through consecutive 2- × 5-mm2 con-

strictions. Insets are indicated by dashed lines. See also movie S6. (E) Per-centage 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 asmall fragment (arrowhead) (left middle), and a nucleus with a g-H2AX–positive fragment (left bottom). Scale bar, 10 mm. Percentage of cells withg-H2AX–positive nuclear fragments before, inside, and after migration throughconstriction channels (right). ***P < 0.001; n = 1376 to 3072 cells per con-dition. (G) Percentage of HT1080 cells migrating through 2- × 5-mm2 or 1- ×5-mm2 constrictions that formed 53BP1-RFP foci as a function of nuclearrupture. **P < 0.01, n = 35 cells total. (H) Representative example of forma-tion of 53BP1 foci (arrowheads) in U2OS cell coexpressing NLS-GFP and RFP-53BP1 during migration through 2- × 5-mm2 constriction and NE rupture.Scale bar, 10 mm. Error bars, SE.

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blocking the cells’ ability to cleave collagen fibersand widen pores by addition of a matrix metal-loprotease (MMP) inhibitor, the incidence of NErupture increased 10-fold and more (fig. S5, Aand B). Similarly, reducing the pore size of themicrofluidic channels to <20 mm2 increased NErupture more than 10-fold (fig. S5, C and D).Irrespective of the experimental model, the in-cidence of NE rupture increased exponentiallywith decreasing pore size (Fig. 1F) and reached

>90% when the nuclear height was confined to3 mm (fig. S5E). Imaging HT1080 fibrosarcomacells invading the collagen-rich mouse dermisin live tumors after orthotopic implantation con-firmed that migration-induced NE rupture alsooccurs in vivo, particularly in individually dissem-inating cells (Fig. 1, G to J; fig. S6; and movie S3).NE rupture was less prevalent in cells moving asmulticellular collective strands (Fig. 1J and fig.S6), which typically follow linear tracks of least

resistance and undergo less pronounced nucleardeformations (3, 7).NE rupture in vitro and in vivo was often ac-

companied by protrusion of chromatin throughthe nuclear lamina (Fig. 2, A and B; fig. S7, A to I;and movies S4 and S5). The incidence of such“chromatin herniations” increased significantlywith decreasing pore sizes (Fig. 2C and fig. S7H).In severe cases, small pieces of the nucleus werepinched off from the primary nucleus as cells

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Fig. 3. Molecular sequence ofnuclear rupture. (A) Nuclearmembrane bleb formation (arrow-head) and collapse upon NE rup-ture. Scale bars, top and bottomrows, 10 mm; inset, 5 mm.(B) Distribution of NE rupture sitesalong nuclear periphery. Regionsare based on the migration direc-tion (black arrow) and denoted inthe cartoon. n = 352 cells; *P <0.05; ***P < 0.001; all comparisonsrelative to the hypothetical uniformdistribution of 33%. (C) Kymographof nuclear bleb formation (arrow-head) and collapse during subse-quent NE rupture, corresponding tothe image sequence in Fig. 1C.(D) Nuclear membrane blebsformed at sites of low GFP-lamin B1intensity and were devoid of GFP-lamin B1 (arrowheads). Intensityalong the blue line is quantified in(E). The gray area indicates thelocal absence of lamin B1. Repre-sentative cell out of 178 cellsobserved. (E) GFP-lamin B1 fluo-rescence intensity profile. The grayarea and arrowhead indicate thesection where the nuclear blebforms. ***P < 0.001; comparingvalues inside and outside the grayarea. (F) Percentage of nuclearblebs containing detectableamounts of lamin A, B1, or B2.***P < 0.001; compared with theexpected value of 100% for theprimary nucleus; n = 178 to 199 cellsper condition. (G) Incidence of NErupture after small interfering RNA(siRNA) treatment against laminA/C, lamin B2, or nontarget (NT)control. *P < 0.05; **P < 0.01;***P < 0.001; n = 384, 150, and163 cells for constrictions ≤2 ×5 mm2; n = 166, 68, and 49 for15- × 5-mm2 constrictions.(H) Blebbistatin treatment reducedNE rupture incidence during migra-tion in constriction channels, butnot in 15- × 5-mm2 control channels.DMSO, dimethyl sulfoxide as a control. *P < 0.05; n = 286 and 194 for constrictions ≤2 × 5 mm2; n = 122, and 54 for cells in constrictions 15 × 5 mm2. (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 differenttimes in (J). Scale bar, 10 mm. (J) GFP-lamin A signal intensity along a section of the nuclear rim [blue line in (I)]. (K) GFP-lamin A accumulation increaseswith the severity of NE rupture. P < 0.0001; n = 46 cells (slope = 1.345, R2 = 0.3945). Error bars, SE.

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passed through narrow constrictions (Fig. 2Dandmovie S6), which resulted in an elevated andpersistent fraction of cells with fragmented nuclei(Fig. 2E and fig. S7J). Furthermore, cells thathad passed through microfluidic constrictionshad more nuclear fragments positive for g-H2AX,a marker of DNA double-strand breaks (17), thancells that had not yet entered the constrictions(Fig. 2F), consistent with recent reports that loss

of NE integrity in micronuclei can cause DNAdamage (14) and chromothripsis (18). Intenseg-H2AX staining could also be found at chroma-tin protrusions (fig. S8A). To confirm that DNAdamagewas caused bymigration-inducednucleardeformation and NE rupture, we performed live-cell imaging on cells coexpressing NLS-GFP andfluorescently labeled 53BP1 (RFP-53BP1), anothermarker of DNA damage (19, 20). NE rupture, and

even severe nuclear deformation alone, resultedin rapid formation of new RFP-53BP1 foci as cellssqueezed through the constrictions (Fig. 2, G andH; and fig. S8, B to G), consistent with previousreports of increased activation of DNA damageresponse genes after compression-induced chro-matin herniation and NE rupture (21).To gain further insights into the biophysical

processes underlying NE rupture, we analyzed

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Fig. 4. ESCRT III mediates nuclearenvelope repair. (A) CHMP4B-GFPwas transiently recruited to the site ofnuclear membrane damage (arrow-head). See also movie S7. Dashedboxes indicate areas used for mea-surements in (B). Representativesequence from 12 HT1080 cells total.Scale bar, 10 mm. (B) CHMP4B-GFPfluorescence intensity at rupture site(black), normalized to preruptureintensity, increased after NE rupture,indicated by increase in cytoplasmicNLS-RFP signal (gray). Red arrow indi-cates time of NE rupture. DF/F0, thechange of the intensity from the origi-nal intensity before stimulation. (C)Recruitment of VPS4B-GFP to sites ofNE rupture (arrowhead) in an MDA-MB-231 cell. Dashed boxes indicateareas used for measurements in (D).Representative example from 18 cellstotal. Scale bar, 10 mm. (D) VPS4B-GFPfluorescence intensity in the cytoplasm(black) increased rapidly after NE rup-ture, detected by increase in cyto-plasmic NLS-RFP signal (gray). Redarrow indicates time of NE rupture.(E) Representative superresolution image(from 12 cells total) of endogenousCHMP4B accumulation at NE rupturesite. Lamin A/C accumulation con-firmed rupture site (red arrowhead).Blue arrowhead indicates decreasedlamin B intensity at the base of thebleb. Scale bars, 5 mm; inset, 1 mm.(F) siRNA-mediated depletion ofESCRT III proteins CHMP7 and CHMP2Ain HT1080 cells resulted in anincreased duration of NLS-GFP in thecytoplasm after migration induced NErupture compared with nontarget (NT)controls. ***P < 0.001; n = 137 and 107,respectively. (G) Expression of thedominant-negative mutantVPS4BE235Q-GFP increased the dura-tion of NLS-GFP in the cytoplasm aftermigration induced NE rupture inHT1080 cells, which indicated impairednuclear membrane repair. **P < 0.005;n = 17 and 10, respectively. (H) Per-centage of HT1080 cells dying aftermigration induced NE rupture, in theabsence or presence of inhibition ofESCRT 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.

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the timing and location of NE rupture in regardto nuclear deformation, local membrane curva-ture, NE composition, and cytoskeletal forces.NE rupture occurred predominantly (~76%) atthe leading edge of the nucleus (Fig. 3, A and B)and was almost always (89.9 ± 2.14%, n = 198cells) preceded by, or coincided with, the for-mation of nuclearmembrane protrusions (“blebs”)as the nuclei moved through the constrictions(Fig. 1, C and H; Fig. 3, A and C; and fig. S9).Nuclearmembrane blebs typically (96.1 ± 1.38%,n = 178 blebs) formed at sites where the nuclearlamina signal, particularly the lamin B1 network,was weak or absent (Fig. 3, D to F, and fig. S10,A and B). These findings indicate that blebsform when segments of the nuclear membranedetach from the nuclear lamina and bulge intothe cytoplasm. Depletion of the lamins A/C andlamin B2 significantly increased the likelihoodof NE rupture (Fig. 3G and fig. S10C), whichtogether with previous studies (4, 12, 13, 21–23)suggest that lamins are important for stabilizingthe NE.Consistent with previous observations

(13, 21, 22, 24, 25), the expanding nuclear blebswere devoid of GFP-lamin B1 (Fig. 3, D to F) andnuclear pores (fig. S10D), and initially containedlittle or no GFP-lamin A and B2 (fig. S10, A andB). Upon NE rupture the blebs retracted and col-lapsed (Fig. 1, C andH; Fig. 3, A and C; fig. S9; andfig. S10, E to H), which suggested that hydrostaticpressure was released from the fluid-filled blebs.Although the NE contains pores, recent studieshave shown that the NE can provide an effectivebarrier to support intracellular pressure gradients(26, 27). Nuclear pressurization could arise fromactomyosin contraction at the rear of the nucleusthat is required tomove the nucleus through tightspaces (5, 6), which effectively mimicks cellularcompression experiments (21, 22). Supporting thisidea, treatment with low concentrations of bleb-bistatin, a myosin II inhibitor, significantly de-creased the incidence of nuclear rupture (Fig. 3H)without inhibiting the ability of cells to migratethrough larger channels (fig. S10I)The transient nature of NE rupture suggests

that cells can efficiently restore nuclear mem-brane integrity during interphase. We observedrapid (<2 min) accumulation of GFP-lamin A atthe site of rupture that correlated with the sever-ity of nuclear rupture and that often persisted forhours (Fig. 3, I to K, and fig. S11). Subsequentruptureswithin the same cell occurred at distinctsites (fig. S11A), which implied local protection bythese “lamin scars.” Two recent reports haveshown that members of the endosomal sortingcomplexes required for transport III (ESCRT III)family are involved in resealing the nuclear mem-brane during late anaphase (28, 29). To assesswhether ESCRT proteins have a similar functionin interphase NE repair, we generated GFP-fusionconstructs of the ESCRT III subunit CHMP4B,involved in recruiting other ESCRT III proteinsand facilitating membrane scission, and theESCRT III–associated VPS4B, which is requiredfor disassembly and recycling of ESCRT III pro-teins (30). UponNE rupture induced by confined

migration or laser ablation, CHMP4B-GFP andVPS4B-GFP rapidly (in ≤2min) formed transientfoci at the site of nuclear membrane damage(Fig. 4, A toD; fig. S12; fig. S13, A to C; andmovieS7). Superresolutionmicroscopy confirmed recruit-ment of endogenous ESCRT III proteins to sitesof NE rupture into complexes ≤160 nm in size(Figs. 4E and fig. S13, D to H). Recruitment of theESCRT III machinery was independent of micro-tubules (fig. S14). Depletion of the ESCRT IIIsubunit CHMP2A, CHMP7, or ectopic expres-sion of a dominant-negative VPS4B mutant (GFP-VPS4BE235Q) that prevents ESCRT III subunitrecycling significantly increased the time requiredfor nucleo-cytoplasmic recompartmentalization(Figs. 4, F and G, and fig. S13, I to N), whichindicated a crucial role of ESCRT III proteins inrestoring nuclearmembrane integrity. To assessthe functional relevance of NE repair, we quan-tified cell viability afterNE rupture. Under normalconditions, the vast majority (>90%) of cells sur-vived even repeated NE rupture (Figs. 1A and4H and fig. S4A). Inhibiting either ESCRT III–mediated NE repair or DNA damage repairpathways alone did not reduce cell viability, butinhibition of both NE and DNA repair sub-stantially increased cell death after NE rupture(Fig. 4H).Taken together, our studies demonstrate that

cell migration through confining spaces, as fre-quently encountered during cancer cell invasion,can challenge the integrity of the NE and DNAcontent, which could promote DNA damage,aneuploidy, and genomic rearrangements, and—in the absence of efficient repair—cell death(31). We propose a biophysical model in whichcytoskeletal-generated nuclear pressure resultsin the formation and eventual rupture of nuclearmembrane blebs at sites of high membrane cur-vature and weakness in the underlying nuclearlamina (fig. S15). These events could be particu-larly prominent in cells with reduced levels oflamins, whose expression is deregulated inmanycancers and often correlates with negative out-comes (32, 33). Although NE rupture, and re-sulting genomic instability, may promote cancerprogression, it may also represent a particularweakness of metastatic cancer cells and an op-portunity to develop novel antimetastatic drugsby specifically targeting these cells, for example,by blocking NE repair and inhibiting DNA dam-age repair.

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ACKNOWLEDGMENTS

The authors thank G. Lahav, A. Loewer, M. Zwerger, M. Smolka,S. Emr, N. Buchkovich, B. Burke, H. Worman, S. Young, L. Fong,D. Discher, J. Broers, J. Goedhart, H. Yu, and W. Zipfel for cells,reagents, and helpful discussion. The authors acknowledgeE. Wagena for mouse surgery and cell transfection, C. Paus forgeneration of in vitro collagen data, and K. Zhang, E. Ghazoul,D. Huang, G. Fedorchak, G.-J. Bakker, and F. Ahmadpour for helpwith image analysis. The authors are grateful to M. Piel andcolleagues for sharing their unpublished data and manuscript.This work was supported by awards from NIH (R01 HL082792and R01 NS059348); the Department of Defense BreastCancer Research Program (BC102152 and BC150580); NSF(CBET-1254846); the National Cancer Institute, NIH, throughthe Cornell Center on the Microenvironment and Metastasis(U54 CA143876); and an NSF Graduate Research Fellowshipto A.L.M (DGE-1144153). This work was further supported by theNetherlands Science Organization (NWO-VIDI 917.10.364 toK.W. and NWO-VICI 918.11.626 to P.F.) and the Cancer GenomicsCenter, Netherlands. This work was performed in part at theCornell NanoScale Science and Technology Facility, which issupported by NSF (grant ECCS-15420819). Imaging was performedin part through the Cornell University Biotechnology ResourceCenter, which is supported by New York Stem Cell Foundation(NYSTEM) (CO29155), NIH (S10OD018516), and NSF (1428922),and by the Radboud University Medical Center MicroscopicImaging Center and Preclinical Animal Imaging Center (PRIME).Additional experimental details and data are available in thesupplementary materials.

SUPPLEMENTARY MATERIALS

www.sciencemag.org/content/352/6283/353/suppl/DC1Materials and MethodsFigs. S1 to S15Movie Captions S1 to S7References (34–43)Movies S1 to S7

26 October 2015; accepted 25 February 2016Published online 24 March 201610.1126/science.aad7297

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Nuclear envelope rupture and repair during cancer cell migration

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

originally published online March 24, 2016DOI: 10.1126/science.aad7297 (6283), 353-358.352Science 

, this issue pp. 359 and 353; see also p. 295Scienceduring interphase, assisted by components of the ESCRT III membrane-remodeling machinery.ruptures when they move through tight spaces (see the Perspective by Burke). The nuclear envelope reseals rapidly

show that migrating immune and cancer cells experience frequent and transitory nuclear envelopeet al. and Denais et al.cytosol and the nucleus. Maintaining nuclear envelope integrity during interphase is considered crucial. However, Raab

The nuclear envelope segregates genomic DNA from the cytoplasm and regulates protein trafficking between theRepairing tears in the nuclear envelope

ARTICLE TOOLS http://science.sciencemag.org/content/352/6283/353

MATERIALSSUPPLEMENTARY http://science.sciencemag.org/content/suppl/2016/03/23/science.aad7297.DC1

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