Nuclear Envelope Breakdown Requires Overcoming the Mechanical Integrity of the Nuclear Lamina

In prophase cells, lamin B1 is the major component of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the nuclear membrane, whereas lamin A/C is dissociated from the scaffold. In vivo fluorescence microscopy studies have shown that, during the G2/M transition, the first gap in the nuclear envelope (NE) appears before lamin B1 disassembly and is caused by early spindle microtubules impinging on the NE. This result suggests that the mechanical tearing of the NE by microtubules plays a central role to the progression of mitosis. To investigate whether this microtubule-induced NE deformation is sufficient for NE breakdown, we assess the mechanical resilience of a reconstituted lamin B1 network. Quantitative rheological methods demonstrate that human lamin B1 filaments form stiff networks that can resist much greater deformations than those caused by microtubules impinging on the NE. Moreover, lamin B1 networks possess an elastic stiffness, which increases under tension, and an exceptional resilience against shear deformations. These results demonstrate that both mechanical tearing of the lamina and biochemical modification of lamin B1 filaments are required for NE breakdown. In prophase cells, lamin B1 is the major component of the nuclear lamina, a filamentous network underlying the nucleoplasmic side of the nuclear membrane, whereas lamin A/C is dissociated from the scaffold. In vivo fluorescence microscopy studies have shown that, during the G2/M transition, the first gap in the nuclear envelope (NE) appears before lamin B1 disassembly and is caused by early spindle microtubules impinging on the NE. This result suggests that the mechanical tearing of the NE by microtubules plays a central role to the progression of mitosis. To investigate whether this microtubule-induced NE deformation is sufficient for NE breakdown, we assess the mechanical resilience of a reconstituted lamin B1 network. Quantitative rheological methods demonstrate that human lamin B1 filaments form stiff networks that can resist much greater deformations than those caused by microtubules impinging on the NE. Moreover, lamin B1 networks possess an elastic stiffness, which increases under tension, and an exceptional resilience against shear deformations. These results demonstrate that both mechanical tearing of the lamina and biochemical modification of lamin B1 filaments are required for NE breakdown. The nuclear lamina is a filamentous meshwork underlying the nucleoplasmic side of the nuclear envelope (NE) 1The abbreviations used are: NE, nuclear envelope; IF, intermediate filament; F-actin, filamentous actin.1The abbreviations used are: NE, nuclear envelope; IF, intermediate filament; F-actin, filamentous actin. (1Aebi U. Cohn J. Buhle L. Gerace L. Nature. 1986; 323: 560-564Crossref PubMed Scopus (675) Google Scholar). The primary components of the nuclear lamina are intermediate filament (IF) lamins and in particular lamin B1, which is constitutively expressed (2Hoger T.H. Zatloukal K. Waizenegger I. Krohne G. Chromosoma (Berl.). 1990; 100: 67-69Crossref PubMed Scopus (0) Google Scholar, 3Zewe M. Hoger T.H. Fink T. Lichter P. Krohne G. Franke W.W. Eur. J. Cell Biol. 1991; 56: 342-350PubMed Google Scholar). Human lamin B1 mutated by deletion of the rod domain (B1Δrod) causes severe alteration of the nuclear lamina organization (4Foisner R. Gerace L. Cell. 1993; 73: 1267-1279Abstract Full Text PDF PubMed Scopus (443) Google Scholar, 5Schirmer E.C. Guan T. Gerace L. J. Cell Biol. 2001; 153: 479-489Crossref PubMed Scopus (95) Google Scholar, 6Furukawa K. Fritze C.E. Gerace L. J. Biol. Chem. 1998; 273: 4213-4219Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar), which suggests that lamin B1 plays a key role in the structural integrity of the NE (7Gruenbaum Y. Wilson K.L. Harel A. Goldberg M. Cohen M. J. Struct. Biol. 2000; 129: 313-323Crossref PubMed Scopus (159) Google Scholar). Lamins and many other NE proteins are subject to mitotic phosphorylation by the Cdc2 kinase, which diminishes protein-protein interactions required for NE integrity, dispersing structural proteins into the cytoplasm at mitosis (8Ward G.E. Kirschner M.W. Cell. 1990; 61: 561-577Abstract Full Text PDF PubMed Scopus (271) Google Scholar). In particular, a lamin B1 mutant lacking the Cdc2 kinase phosphorylation site prevents NE breakdown (9Heald R. McKeon F. Cell. 1990; 61: 579-589Abstract Full Text PDF PubMed Scopus (448) Google Scholar, 10Peter M. Nakagawa J. Doree M. Labbe J.C. Nigg E.A. Cell. 1990; 61: 591-602Abstract Full Text PDF PubMed Scopus (540) Google Scholar, 11Peter M. Heitlinger E. Haner M. Aebi U. Nigg E.A. EMBO J. 1991; 10: 1535-1544Crossref PubMed Scopus (139) Google Scholar), which suggests that lamin phosphorylation is a prerequisite for NE destabilization before mitosis (12Buendia B. Courvalin J.C. Exp. Cell Res. 1997; 230: 133-144Crossref PubMed Scopus (77) Google Scholar). Nevertheless, there is growing evidence in vitro and in vivo that lamin B1 disassembly is independent of NE breakdown. Lamin B1 solubilization in vitro has been shown to occur without NE breakdown (13Newport J. Spann T. Cell. 1987; 48: 219-230Abstract Full Text PDF PubMed Scopus (236) Google Scholar). Time-resolved fluorescence microscopy reveals that the organization of green fluorescent proteinlamins in the NE of live cells remains intact until NE breakdown and little soluble lamin (<5%) can be detected through fluorescence after recovery before NE breakdown (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Instead, early spindle microtubules cause folds in the NE, which develop into deep invaginations that create an initial hole in the NE (15Georgatos S.D. Pyrpasopoulou A. Theodoropoulos P.A. J. Cell Sci. 1997; 110: 2129-2140PubMed Google Scholar). This hole forms at a specific NE position, which is distal from the centrosomes (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). The non-random position of the hole on the NE surface argues against weakening of the lamina by lamin disassembly prior to NE breakdown, which would create randomly located holes wherever lamin is completely disassembled (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). Based on these observations, it has been speculated that mechanical tearing generated by microtubules play the main role in NE breakdown (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). To investigate whether the microtubule-induced deformation is sufficient for NE breakdown, we assess the viscoelastic properties of lamin B1 to determine the mechanical resilience of a reconstituted lamin B1 network. We focus on lamin B1 because it is the primary component of the NE prior to its breakdown in the G2/M transition whereas lamin A/C is dissociated from the NE (15Georgatos S.D. Pyrpasopoulou A. Theodoropoulos P.A. J. Cell Sci. 1997; 110: 2129-2140PubMed Google Scholar). We use quantitative rheological methods to examine the mechanical response of lamin B1 networks subject to shear and axial deformations similar to those generated by premitotic microtubules. To provide a basis of comparison, we report the mechanical properties of cytoplasmic intermediate filaments vimentin, complex epithelia keratin K5–K14, and simple epithelia keratin K8–K18, as well as F-actin. Our results show that lamin B1 can (even in the absence of auxiliary proteins) form exceptionally resilient structures through strong interfilament interactions. The maximum deformation of the NE generated by early spindle microtubules is much lower than the deformation required to breakdown a lamin B1 network, which suggests that NE breakdown has to involve both mechanical tearing of the lamina and biochemical modifications of lamin filaments. Lamin B1 Purification and Assembly—Unless specified, all reagents were purchased from Sigma Chemical Company. Purification of human lamin B1 is based on published protocols (5Schirmer E.C. Guan T. Gerace L. J. Cell Biol. 2001; 153: 479-489Crossref PubMed Scopus (95) Google Scholar, 16Karabinos A. Schunemann J. Meyer M. Aebi U. Weber K. J. Mol. Biol. 2003; 325: 241-247Crossref PubMed Scopus (62) Google Scholar). Briefly, Escherichia coli BL21/DE3 containing a human lamin B1 expression plasmid was grown at 37 °C in 200 ml of SOB medium (20 g of Tryptone, 5 g of yeast extract, 0.5 g of NaCl, 0.186 g of KCl, and 20 mm MgCl2 per liter H2O) supplemented with 50 μg/ml kanamycin. Human lamin B1 cDNA was a generous gift from E. C. Schirmer (The Scripps Research Institute). Expression was induced with 1 mm isopropyl-thio-β-d-galactopyranoside at A595 0.7 for 4 h at 37 °C. Cells were lysed by sonication in phosphate-buffered saline with 1.5 mm β-mercaptoethanol and protease inhibitor. After a 7-min centrifugation at 10,000 × g, the pellet was washed with 0.2% Triton X-100 and resuspended in 20 mm Tris, pH 8.0, 300 mm NaCl, 8 m urea, 3 mm β-mercaptoethanol, 0.2 mm phenylmethylsulphonyl fluoride. The solution was then incubated with nickel-nitrilotriacetic acid resin (Qiagen, Gaithersburg, MD) for 45 min and eluted in fractions with the same buffer containing a 0–200 mm imidazole gradient. The eluate was dialyzed in 20 mm Tris, pH 8.0, 8 m urea, 3 mm β-mercaptoethanol, 0.2 mm phenylmethylsulphonyl fluoride and was applied to a Mono Q column (Bio-Rad) eluted with NaCl gradient from 0–1 M. The lamin-rich fraction was examined by SDS-PAGE and further dialyzed into 20 mm Tris, pH 8.0, 8 m urea, 2 mm dithiothreitol, 0.2 mm phenylmethylsulphonyl fluoride for storage. The protein purity was verified to be >99% by SDS-PAGE assay followed by densitometry. For filament assembly, lamin in 8 m urea at 1 mg/ml was dialyzed against 20 mm Tris, pH 8.8, 1 mm EDTA, 1 mm dithiothreitol, 0.2 mm phenylmethylsulphonyl fluoride (5Schirmer E.C. Guan T. Gerace L. J. Cell Biol. 2001; 153: 479-489Crossref PubMed Scopus (95) Google Scholar) for 16 h at 4 °C. 150 mm NaCl was added to the solution to induce lamin assembly. Alternatively, dialysis in steps (which is for instance required for keratin (17Yamada S. Wirtz D. Coulombe P.A. J. Struct. Biol. 2003; 143: 45-55Crossref PubMed Scopus (50) Google Scholar)) did not affect the structural and mechanical outcomes of lamin B1 networks (data not shown). Actin and Vimentin Purification and Assembly—Actin was prepared from chicken breast (18MacLean-Fletcher S.D. Pollard T.D. J. Cell Biol. 1980; 85: 414-428Crossref PubMed Scopus (231) Google Scholar) with an extra step of gel filtration by Sephacryl S-300 (Sigma) (19Tseng Y. Fedorov E. McCaffery J.M. Almo S.C. Wirtz D. J. Mol. Biol. 2001; 310: 351-366Crossref PubMed Scopus (112) Google Scholar). Purified actin was stored as Ca2+-actin in continuous dialysis at 4 °C against buffer G (0.2 mm ATP, 0.5 mm dithiothreitol, 0.2 mm CaCl2, 1 mm sodium azide, and 2 mm Tris-HCl, pH 8.0). Mg2+-actin filaments were generated by adding 0.1 volume of 10× KMEI (500 mm KCl, 10 mm MgCl2, 10 mm EGTA, 100 mm imidazole, pH 7.0) polymerizing salt to 0.9 volume of G-actin in buffer G. The purification of human vimentin is based on a published protocol (20Moir R.D. Donaldson A.D. Stewart M. J. Cell Sci. 1991; 99: 363-372PubMed Google Scholar) using a Mono Q column (Bio-Rad). Human vimentin cDNA was a generous gift from R. D. Goldman (Northwestern University Medical School). To form filaments, vimentin that was solubilized in 1 mm EDTA, 0.1 mm EGTA, 0.1 mm dithiothreitol, and 5 mm Tris-HCl, pH 8.4, was dialyzed into filament buffer (0.1 mm dithiothreitol, 160 mm NaCl, and 25 mm Tris-HCl, pH 7.5) at 37 °C (21Herrmann H. Haner M. Brettel M. Muller S.A. Goldie K.N. Fedtke B. Lustig A. Franke W.W. Aebi U. J. Mol. Biol. 1996; 264: 933-953Crossref PubMed Scopus (263) Google Scholar). Rheology—The mechanical properties of lamin B1 networks were evaluated using a strain-controlled, cone-and-plate rheometer (ARES-100, TA Instruments, Piscataway, NJ) (19Tseng Y. Fedorov E. McCaffery J.M. Almo S.C. Wirtz D. J. Mol. Biol. 2001; 310: 351-366Crossref PubMed Scopus (112) Google Scholar, 22Tseng Y. Wirtz D. Biophys. J. 2001; 81: 1643-1656Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). Shear deformations of controlled amplitude and frequency were applied via precise dynamic rotations of the lower plate; the stress induced within the network was measured through a sensitive torque transducer connected to the upper cone. Lamin B1 solutions were supplemented with 150 mm NaCl and immediately loaded in the space between the cone and plate. Once the cone was set into position, 0.5 mg/ml phosphatidylcholine dissolved in chloroform was applied to the air-water interface to eliminate the interfacial localization of lamins (17Yamada S. Wirtz D. Coulombe P.A. J. Struct. Biol. 2003; 143: 45-55Crossref PubMed Scopus (50) Google Scholar). The dead time is estimated to be 5 min before rheological measurements. Gelation was monitored by measuring the time-dependent elasticity of the network. Elasticity was measured by applying two oscillatory deformations of 1% amplitude at a frequency of 1 rad/sec until a was the viscoelastic of the and were by the and components of the measured oscillatory stress induced within the network by the applied amplitude (19Tseng Y. Fedorov E. McCaffery J.M. Almo S.C. Wirtz D. J. Mol. Biol. 2001; 310: 351-366Crossref PubMed Scopus (112) Google Scholar, 22Tseng Y. Wirtz D. Biophys. J. 2001; 81: 1643-1656Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The viscoelastic of lamin networks was by the The response of the lamin networks to shear was by applying oscillatory deformations at 1 and from 0.5 to For all rheological the of the solutions was at 25 °C to within 0.1 °C. of Lamin B1 the mechanical properties of human lamin B1 networks in buffer using quantitative rheological methods as a of time after of and at of lamin B1 were to gel in the space between the cone and of the and elastic and of the were by 1% oscillatory deformations. The elastic and the and components of the stress induced in the networks by the amplitude of the applied deformation of and Scholar, P.A. L. S. Wirtz D. Cell Biol. 2000; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). To networks were subject to a of oscillatory deformations of 1% amplitude until the elasticity of the network a first 2 h are shown in The elastic of the network the of the network, an measured by the of the network. The of a its viscoelastic P.A. L. S. Wirtz D. Cell Biol. 2000; 10: Full Text Full Text PDF PubMed Scopus Google for which from a rheological of to a and for a stiff to a reveals that lamin B1 networks a The of estimated as the of the time required for of the from to for lamin B1 between 1 and that the elasticity of lamin B1 networks lamin B1 networks to oscillatory deformations of between and 100 we measured the dynamic and of lamin B1 networks The frequency of was which that for the lamin lamin B1 in solution little for time as as These results show that a rheological of lamin networks as Lamin B1 under to 1 h prior to NE breakdown, the is subject to mechanical the NE folds and deep invaginations caused by early spindle microtubules before the disassembly of lamin B1 filaments, which after NE breakdown (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar, S.D. Pyrpasopoulou A. Theodoropoulos P.A. J. Cell Sci. 1997; 110: 2129-2140PubMed Google Scholar). To the response of lamin B1 to mechanical as those by premitotic lamin B1 networks were subject to deformations of frequency of 1 and amplitude between 0.5 and of the elasticity of lamin B1 was independent of at deformation at intermediate the network under and at deformations the network under and The maximum by to the with lamin B1 and lamin B1 networks show the network under To further investigate the of shear deformations on lamin B1 oscillatory deformations of the of frequency 1 and deformation amplitude were applied to the and the induced stress was monitored as a of time to a amplitude of was a that a with a maximum stress that from shear to the For of a which that little within the network during shear the network is much elastic than For deformation a with a major because the network during the shear This that the stress than with the in of in lamin networks L. J. Coulombe P.A. Wirtz D. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). Lamin against we the mechanical resilience of the deformation that lamin can before its breakdown. The resilience of lamin B1 the amplitude at which to was and independent of In of of deformations in premitotic cells in J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google that the shear deformation of the NE by spindle microtubules not in This is because the deformations of the NE occur the lamina that the NE is subject to both shear and axial deformations. We a maximum axial resilience between and whereas the axial deformation of the NE in vivo was to be (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). the deformations that the lamin network can before breakdown are much than deformations caused by microtubules prior to NE breakdown. Lamin B1 networks are elastic are and resilient than networks Lamin B1 much than other network of major lamin B1 networks mechanical properties that are similar to those of networks of keratin K8–K18, keratin K5–K14, and vimentin all cytoplasmic intermediate filaments with well structural (17Yamada S. Wirtz D. Coulombe P.A. J. Struct. Biol. 2003; 143: 45-55Crossref PubMed Scopus (50) Google Scholar, P.A. U. P. M. J. Cell Biol. 1991; PubMed Scopus Google Scholar, A. M. H. Franke W.W. J. Cell Biol. Google Scholar). To investigate whether mechanical tearing of the NE by spindle microtubules impinging on the is the required for NE breakdown, we the viscoelastic properties of lamin B1, the primary component of NE quantitative rheological we measured the mechanical properties of lamin B1 networks subject to of shear at Lamin B1 form and resilient tension, lamin B1 networks a the elasticity of the network under The maximum shear that lamin B1 can before breakdown is greater than the deformation caused by spindle microtubules impinging on the NE in mechanical tearing of the lamin lamina is not sufficient to induce NE breakdown. of the of in lamin B1 the in elasticity is with a of the is the in lamin similar of is for the A. J. S.C. Wirtz D. Biophys. J. Full Text Full Text PDF PubMed Scopus Google Scholar). that the elastic of networks of as whereas as and as 1998; Scopus Google Scholar). rheological that lamin B1 in solution are (which the of the is of the same of as which is the of cytoplasmic intermediate filaments N. L. R. T. H. Aebi U. J. J. Mol. Biol. PubMed Scopus Google Scholar, C. J.M. Biophys. J. 2003; 85: Full Text Full Text PDF PubMed Scopus Google Scholar). the of this would provide lamin B1 with a to and form stiff P. Wirtz D. Y. E. Scholar, Wirtz D. Cell Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). Lamin B1 networks under shear which from the that are for and resilience against shear deformations. shear lamin B1 filaments would be to against the by with other filaments, an that is interactions were lamin B1 filaments would be to the stress by for lamin B1 these interactions which prevents stress and as well as mechanical to actin the of other components in the prophase lamina, as and (which with lamin B1 (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google further the resilience and of lamin B1 to Lamin the two major and M. S.C. Pollard T.D. J. Cell Biol. PubMed Scopus Google Scholar, J. Y. Wirtz D. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar), lamin B1 has a to interactions and form in the absence of auxiliary proteins (1Aebi U. Cohn J. Buhle L. Gerace L. Nature. 1986; 323: 560-564Crossref PubMed Scopus (675) Google Scholar, J. Spann T. Cell. 1987; 48: 219-230Abstract Full Text PDF PubMed Scopus (236) Google Scholar, 16Karabinos A. Schunemann J. Meyer M. Aebi U. Weber K. J. Mol. Biol. 2003; 325: 241-247Crossref PubMed Scopus (62) Google Scholar, C. Krohne G. Eur. J. Cell Biol. 1991; Google Scholar, E. M. Lustig A. Nigg E.A. Aebi U. J. Struct. Biol. 108: PubMed Scopus Google Scholar, R.D. Spann H. Goldman R.D. J. Cell Biol. 2000; PubMed Scopus Google Scholar). These interactions the of and and lamin B1 with a to form elastic The and of elastic and of lamin B1 filament are similar to those with reconstituted networks of cytoplasmic intermediate filaments, vimentin, complex keratin and simple keratin (17Yamada S. Wirtz D. Coulombe P.A. J. Struct. Biol. 2003; 143: 45-55Crossref PubMed Scopus (50) Google Scholar, L. S. Wirtz D. Coulombe P.A. Cell Biol. 2001; PubMed Scopus Google Scholar). The strong of not by the two other major from strong interactions between P.A. L. S. Wirtz D. Cell Biol. 2000; 10: Full Text Full Text PDF PubMed Scopus Google Scholar). This lamin B1 networks to possess shear that of and We measured the properties of the in vivo Y. I. Wirtz D. J. Cell Sci. PubMed Scopus Google Scholar). These show an elastic that is much than the and an elasticity similar to that with reconstituted lamin B1 spindle microtubules on the NE, the NE is subject to both shear and axial deformations. can the axial resilience from the measured shear we stress and a we the deformation at to the shear deformation at by the of the is to be for and we it between an and a to that the maximum axial deformation of lamina before breakdown is much than that of the NE before in (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). The of Lamin the of a before NE elastic of lamin B1 deformations of the lamina by spindle microtubules as in the resilience of lamin its be the within the lamin rod domain that the lamin filament networks than cytoplasmic networks L. S. Wirtz D. Coulombe P.A. Cell Biol. 2001; PubMed Scopus Google Scholar), a that we in the The resilience of lamin networks from both the of lamin against deformation N. L. R. T. H. Aebi U. J. J. Mol. Biol. PubMed Scopus Google Scholar, C. J.M. Biophys. J. 2003; 85: Full Text Full Text PDF PubMed Scopus Google Scholar) as well as the of lamin filaments to a to a by other intermediate filaments. The maximum deformation that a lamin B1 network can before breakdown is much greater than the deformations generated by spindle microtubules impinging on the NE. These results with the that lamin B1 after NE breakdown that NE by mechanical tearing of the lamina occur until the lamin B1 network is by biochemical of the between lamin B1 filaments would the mechanical resilience of the lamina would not affect lamin and it would not be detected by in fluorescence after recovery fluorescence (14Beaudouin J. Gerlich D. Daigle N. Eils R. Ellenberg J. Cell. 2002; 108: 83-96Abstract Full Text Full Text PDF PubMed Scopus (348) Google Scholar). The of lamin interfilament interactions before NE breakdown be by premitotic phosphorylation of lamin B1 by proteins that are to be The lamina many components lamin B1, nuclear actin and protein C. S. R. Sci. U. S. A. 2003; 100: PubMed Scopus Google Scholar), which form filamentous structures that further the resilience of We E. C. Schirmer (The Scripps Research for generous gift of human lamin B1 with