The Stability of the Nuclear Lamina Polymer Changes with the Composition of Lamin Subtypes According to Their Individual Binding Strengths

The nuclear lamina, which provides a structural scaffolding for the nuclear envelope, consists largely of a polymer of the intermediate filament lamin proteins. Although different cell types contain distinctive relative amounts of the major lamin subtypes (A, C, B1, and B2), the functions of this variation are not understood. We have investigated the possibility that subtype variation affects lamina stability. We find that homotypic and heterotypic binding interactions of lamin B2 are substantially less resistant to chemical dissociation in vitro than those between the other lamin subtypes, whereas lamin A interactions are the most stable. Surprisingly, removal of the central four-fifths of the rod domain did not substantially weaken the interactions of lamins A and B2, suggesting that other regions also strongly contribute to their binding interactions. In contrast, this rod deletion strongly destabilizes the binding interactions of lamins B1 and C. Consistent with the binding studies, lamins are more readily solubilized by chemical extraction from cells enriched for lamin B2 than from cells enriched for lamin A. This suggests that the distinctive ensemble of heterotypic lamin interactions in a particular cell type affects the stability of the lamin polymer, and, correspondingly, could be relevant to tissue-specific properties of the lamina including its involvement in disease. The nuclear lamina, which provides a structural scaffolding for the nuclear envelope, consists largely of a polymer of the intermediate filament lamin proteins. Although different cell types contain distinctive relative amounts of the major lamin subtypes (A, C, B1, and B2), the functions of this variation are not understood. We have investigated the possibility that subtype variation affects lamina stability. We find that homotypic and heterotypic binding interactions of lamin B2 are substantially less resistant to chemical dissociation in vitro than those between the other lamin subtypes, whereas lamin A interactions are the most stable. Surprisingly, removal of the central four-fifths of the rod domain did not substantially weaken the interactions of lamins A and B2, suggesting that other regions also strongly contribute to their binding interactions. In contrast, this rod deletion strongly destabilizes the binding interactions of lamins B1 and C. Consistent with the binding studies, lamins are more readily solubilized by chemical extraction from cells enriched for lamin B2 than from cells enriched for lamin A. This suggests that the distinctive ensemble of heterotypic lamin interactions in a particular cell type affects the stability of the lamin polymer, and, correspondingly, could be relevant to tissue-specific properties of the lamina including its involvement in disease. The nuclear lamina is a filamentous protein meshwork that lines the inner nuclear membrane. Its core consists of a polymer of the intermediate filament (IF) 1The abbreviations used are: IF, intermediate filament; NE, nuclear envelope; WT, wild-type; TPA, O-tetradecanoylphorbol-13-mysistate acetate. lamin proteins, which binds to chromatin and connects to the inner nuclear membrane via integral membrane proteins (reviewed in Refs. 1Goldman R. Gruenbaum Y. Moir R. Shumaker D. Spann T. Genes Dev. 2002; 16: 533-547Crossref PubMed Scopus (505) Google Scholar and 2Mattout-Drubezki A. Gruenbaum Y. Cell Mol. Life Sci. 2003; 60: 2053-2063Crossref PubMed Scopus (115) Google Scholar). Mutations in lamins and associated membrane proteins cause a variety of debilitating human diseases (laminopathies) including muscular dystrophy, neuropathy, and developmental disorders (reviewed in Refs. 3Mounkes L. Kozlov S. Burke B. Stewart C.L. Curr. Opin. Genet. Dev. 2003; 13: 223-230Crossref PubMed Scopus (170) Google Scholar and 4Worman H.J. Courvalin J.C. Trends Cell Biol. 2002; 12: 591-598Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar). Differences are observed in the relative amounts of the major products of the three lamin genes (encoding lamins A/C, B1, and B2) in different cell types and at different stages of development. Lamins B1 and B2 are expressed throughout development, whereas lamins A and C, which are splice variants differing in their C termini, usually appear only near the time of or following differentiation in specific tissues of chicken and mammals (5Houliston E. Guilly M.N. Courvalin J.C. Maro B. Development. 1988; 102: 271-278PubMed Google Scholar, 6Lehner C.F. Stick R. Eppenberger H.M. Nigg E.A. J. Cell Biol. 1987; 105: 577-587Crossref PubMed Scopus (143) Google Scholar, 7Rober R.A. Weber K. Osborn M. Development. 1989; 105: 365-378PubMed Google Scholar). These different subtypes are presumed to interact in vivo, since binding of A/C lamins to lamin B1 is observed in blot overlays (8Krohne G. Wolin S.L. McKeon F.D. Franke W.W. Kirschner M.W. EMBO J. 1987; 6: 3801-3808Crossref PubMed Scopus (89) Google Scholar), column binding assays (9Georgatos S.D. Stournaras C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4325-4329Crossref PubMed Scopus (55) Google Scholar), and two-hybrid analysis (10Ye Q. Worman H.J. Exp. Cell Res. 1995; 219: 292-298Crossref PubMed Scopus (62) Google Scholar). However, the binding between other pairs of lamin subtypes has not yet been examined. The observation that different nuclear envelope (NE) diseases preferentially affect certain tissues (3Mounkes L. Kozlov S. Burke B. Stewart C.L. Curr. Opin. Genet. Dev. 2003; 13: 223-230Crossref PubMed Scopus (170) Google Scholar, 4Worman H.J. Courvalin J.C. Trends Cell Biol. 2002; 12: 591-598Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) may relate to the distinctive expression patterns of lamins in different tissues and the specific biochemical properties of each subtype. Similar to other IF proteins, lamins contain a central α-helical “rod” domain composed of ∼48 heptad repeats, flanked by N- and C-terminal “head” and “tail” domains. The rod domain assembles into a parallel, unstaggered coiled-coil homodimer that is stable in 8 m urea. In vitro, dimers of each major lamin subtype can assemble laterally into homotypic filaments by staggered antiparallel interactions (11Moir R.D. Donaldson A.D. Stewart M. J. Cell Sci. 1991; 99: 363-372PubMed Google Scholar, 12Heitlinger E. Peter M. Lustig A. Villiger W. Nigg E.A. Aebi U. J. Struct. Biol. 1992; 108: 74-89Crossref PubMed Scopus (109) Google Scholar, 13Stuurman N. Sasse B. Fisher P.A. J. Struct. Biol. 1996; 117: 1-15Crossref PubMed Scopus (72) Google Scholar). The five heptads at each end of the rod, particularly those at the tail end, have been suggested to be critical for initiating the coiled-coil (14Stuurman N. Heins S. Aebi U. J. Struct. Biol. 1998; 122: 42-66Crossref PubMed Scopus (605) Google Scholar, 15Kammerer R.A. Schulthess T. Landwehr R. Lustig A. Fischer D. Engel J. J. Biol. Chem. 1998; 273: 10602-10608Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Fragments containing these terminal heptads together with either the head or tail can assemble into dimers and tetramers in vitro but not into filaments (14Stuurman N. Heins S. Aebi U. J. Struct. Biol. 1998; 122: 42-66Crossref PubMed Scopus (605) Google Scholar). Both charged and hydrophobic residues on the outer surface of the dimer rod are thought to contribute to stabilizing filaments. Although there are few details on how different subtypes assemble into the lamina in vivo, their resistance to chemical extraction from cells and the low rates of exchange for lamins A and B1 fused to GFP (16Moir R. Yoon M. Khuon S. Goldman R. J. Cell Biol. 2000; 151: 1155-1168Crossref PubMed Scopus (328) Google Scholar) argue that they are stably integrated into a polymeric structure. In this study, we have used biochemical approaches to compare the properties of all four principal mammalian lamin subtypes. We found that each pairing yielded a different binding “strength” as defined by its resistance to chemical dissociation. Interestingly, we found that lamin B2 had much weaker homotypic and heterotypic interactions than the interactions seen among the other lamin subtypes, and, correspondingly, all lamin subtypes were more readily extracted from cultured cells that were induced to express relatively high levels of lamin B2. Paralleling this at the opposite extreme, lamin A interactions were the strongest among the different lamin subtypes, and lamins were less readily extracted from cells expressing high levels of lamin A. Analysis of lamin mutants lacking the middle four-fifths of the rod domain suggested that this region contributes significantly to the homotypic binding interactions of lamins B1, A, and C and that lamin A contains additional binding sites unique to its C terminus that are important for its unusually strong interactions. These results suggest that changes in the ratio of lamins A and B2 during development or in different cell types could directly influence the dynamic properties of the lamina and its functions as a structural scaffolding for the NE and chromatin. Plasmid Construction—Primers that added 5′ BamHI/NdeI and 3′ NotI sites were used to amplify by PCR the coding sequences of human lamins A, C, and B1 and mouse lamin B2. To produce deletion mutants lacking the middle four-fifths of the rod domain (referred to throughout as Δ), these primers were used in conjunction with internal primers containing HindIII sites that fused nucleotides 207 and 1017 for lamin B1 and the corresponding residues of the other lamins via an added alanine codon. All genes were moved to pET28a (Novagen) for protein expression and pHHS10B (which bears an HA epitope tag) for mammalian transfection. Protein Purification—WT lamins and deletion mutants were purified from inclusion bodies. The proteins were expressed in BL21-(DE3) cells by induction with 0.3 mm isopropyl-1-thio-β-d-galactopyranoside at A595 0.7 for 3 h at 37 °C, collected by centrifugation, and lysed by sonication in 25 mm HEPES, pH 8.0, 0.1 mm MgCl2, 3 mm β-mercaptoethanol containing 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μm leupeptin, and 1 μm pepstatin. The pellets from a 20-min centrifugation at 27,000 × g were washed with 1% Triton X-100 and resuspended in 20 mm HEPES, pH 8.0, 8 m urea, 3 mm β-mercaptoethanol. For further purification, this was incubated with for of the from the pET28a and proteins were with the containing mm were into 20 mm pH 8.0, 8 m urea, mm 1 mm with for lamins and deletion mutants were to or on their for a as in Refs. S.D. Stournaras C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4325-4329Crossref PubMed Scopus (55) Google Scholar and T. L. J. Cell Biol. PubMed Scopus Google Scholar, that was of were with the used in binding assays to of protein homotypic interactions. lamins were to 20 μg/ml and of into binding 25 mm HEPES, pH mm mm β-mercaptoethanol with This was × to significantly lamins and incubated with the at The were washed in the and with of in 25 mm HEPES, pH 8.0, mm mm β-mercaptoethanol column each were with m by 8 m urea. were by with specific to each lamin subtype T. L. J. Cell Biol. PubMed Scopus Google Scholar). Lamins in each were a with and as the of the Although lamins were in with the was as a had been used the of the between and 8 a of m was of lamins was by and purified lamins into binding and at were to centrifugation at × g for to significantly and pellets were and by and lamins in the were the protein with and of cells were with either lamin A or lamin B2 in the pHHS10B to the were at h cells were by washed in and into for were resuspended and incubated on for in 25 mm HEPES, pH mm 3 mm MgCl2, mm 1 mm Triton was by centrifugation at × g in a for and were by and cells were with 1 from a 1 mm in or from a μm in cells were with and all cells were and collected by of cells lysed in were by with lamin and to the of on relative lamin cells were also extracted and as to changes in the relative of each subtype. for directly compare the binding properties of the four major lamin subtypes, lamins and deletion mutants that the middle four-fifths of the rod were from of the rod deletion mutants five heptads from of the rod by that were fused in These were by to a coiled-coil of and for this from analysis of the lamin that a strong α-helical in by and into filaments that had a as filaments T. L. J. Cell Biol. PubMed Scopus Google Scholar). In to the the lamin dimers are to have four coiled-coil by of heptads by The of the rod are the most regions among lamins but the central of the rod, as as the and are also We directly the homotypic and heterotypic binding of lamins A, C, B1, and B2 and the homotypic binding of their mutants an in vitro that preferentially the of interactions. For this from a (9Georgatos S.D. Stournaras C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4325-4329Crossref PubMed Scopus (55) Google Scholar, T. L. J. Cell Biol. PubMed Scopus Google Scholar), each purified lamin was to an in m homotypic at a relatively high lamin each lamin was and of into a at a protein that was low to into and incubated with the lamin the strongly the of the lamins at a low with the lamins in A of was used for each to associated with were and lamins were in the binding containing of urea. were by with lamin by column was or more To of the different lamin subtypes, a of the at which of the lamin had been was used as an of the binding of the between used this to homotypic interactions We found that the of the lamin B2 homotypic were at relatively low of In contrast, were to the lamin A lamin C and lamin B1 homotypic interactions m was used to the of urea, only a of lamins was However, more lamin B2 was of the as with lamin B1 in to lamin B1, lamin B2 homotypic interactions are substantially more to by chemical that hydrophobic as as interactions the coiled-coil dimer by the lamin rod is not by high or we that the of lamin B2 to by these results from of binding between we the possibility that the tail domain of lamin B2 is or that the of the tail domain of lamin B2, than its binding is by the However, we in this that the domain of the lamin B2 tail C. B. J. J. G. Courvalin J. Worman S. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar) is as in to the regions of lamin B1 and lamin A as the region of lamin B1 is to lamin A as To that the lamins were not significantly or during the of the the lamins were for their were into the and incubated as in the column binding were to centrifugation, and the of was We found that the of all of the lamin the binding by that was and that the proteins are interactions of lamin B1 with a of A/C lamins were to be than the lamin B1 homotypic interactions (9Georgatos S.D. Stournaras C. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4325-4329Crossref PubMed Scopus (55) Google Scholar), an observation we in However, by lamins A and C we further that the lamin B1 heterotypic with lamin A is than its with lamin C we the binding interactions of lamin B2 by this we found that the heterotypic interactions of lamin lamin and lamin were substantially weaker than the homotypic lamin A and lamin B1 they were than the homotypic lamin B2 interactions of the and to lamin mutants are to the of the and that in the dimers and could be used to the relative of the central four-fifths of the rod more lamin regions for interactions central rod and rod interactions with and rod end interactions. We found that the a strong homotypic that was to the homotypic of lamin A interactions between lamin A and lamin which be to be to the in the of the rod, were relatively strong not In contrast, the stability of lamin C homotypic interactions to extraction was by the deletion of the central four-fifths of the rod from a of m to m m for lamin lamins A and C are in their termini, which are by different of lamin C and residues of lamin the binding stability of the mutants that the unique C-terminal region of lamin A a to the relatively strong lamin A homotypic interactions. of the central four-fifths of the rod had a on the binding stability of lamin The binding “strength” of the was with the strong homotypic B1, The of m between the lamin B1 and was to the m observed for lamin C. was than that of the B2 This the possibility that the central four-fifths of the rod domain in lamin B2 has a influence on the lamin B2 homotypic binding interactions. we the possibility that results from with lamin B2 not seen with the other lamin subtypes that the lamin is more for binding interactions than lamin of on the of the in in vitro binding assays that a lamina enriched in lamin B2 in be less stable than enriched in lamin A. different lamin subtypes into the the relative of lamin B2 in the nuclear lamina of cells the to chemical extraction of all lamin subtypes, whereas the relative of lamin A their To this lamin B2 or lamin A was into mammalian cultured and the cells were by for changes in the of each lamin subtype extracted a containing high and which hydrophobic and interactions. The of all lamin subtypes that were extracted was in of cells with lamin A as with whereas the of extracted lamins B1 and B2 was in cells with lamin B2, as with cells significantly the of expressed lamins on the of the since only of the cells in the were cells were lysed at all lamins were at the nuclear as seen by the changes in the of the lamin are not to its or all lamins their yet their was also cells yielded levels of solubilized lamins from to by the of solubilized lamins from the was to the solubilized in a of the The results from four that lamin B2 destabilizes the lamins to chemical whereas lamin A an to the relative levels of lamins in the NE by we used a cell lamin levels are the cells are induced to 1987; PubMed Google Scholar). of cells with for induction the relative of lamin B2, whereas with for induction A/C lamins A. M. D. D. Exp. Cell Res. 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