expression in escherichia coli of human lamin as and c...

Expression in Escherichia coli of human lamins A and C: influence of head

and tail domains on assembly properties and paracrystal formation

ROBERT D. MOIR, ANNE D. DONALDSON and MURRAY STEWART*

MRC Laboratory of Molecular Biology, Hills Rd, Cambridge CB2 2QH, UK

* Author for correspondence

Summary

We have expressed in Escherichia coli cDNA corre-sponding to human lamins A and C, together with anumber of fragments produced using site-specificmutagenesis. The proteins produced in this way werecharacterised both biochemically and ultrastructur-ally, and appeared to retain their native confor-mation. Crosslinking showed that all fragmentsformed 4-chain molecular dimers (tetramers') anal-ogous to those formed by intact intermediate fila-ment proteins. Shadowed preparations showed thepresence of rod-like particles that closely resembledthose observed for other intermediate filament pro-

teins and their proteolytically prepared rod domains.Moreover, the expressed lamins and a series offragments in which different domains had beendeleted formed paracrystals similar to those ob-served with native material. Deletion of either the N-or C-terminal non-helical domains altered the solu-bility and aggregation properties of the expressedprotein, indicating that both domains have a role inlamin assembly.

Key words: lamins, expression, assembly, molecularinteractions, structure.

Introduction

Lamins are the principal components of the fibrous laminathat underlies the nuclear envelope of eukaryotic cells(reviewed by Gerace, 1986). The lamina fibres are about10 run in diameter and are arranged in a dense fibrous matthat sometimes shows a remarkable tetragonal order(Aebi et al. 1986; Stewart and Whytock, 1988). Besides astructural role in maintaining nuclear envelope integrity(Newport et al. 1990; Whytock et al. 1990), the lamina mayinfluence interphase chromatin organization, DNA repli-cation and gene expression (Lebkowski and Laemmli,1982; Benevente and Krohne, 1986).

Lamins are found in a wide range of species. Themammalian lamin gene family has a complex pattern ofexpression and, for example, there appear to be at leastfive distinct lamins (termed A to E) in rat liver(Kaufmann, 1989). In mammals, lamins A, B and C havebeen more throughly characterised and cDNAs corre-sponding to each have been cloned (McKeon et al. 1986;Fisher et al. 1986; Hoger et al. 1988; Pollard et al. 1990).Lamins D and E have only recently been described and,although detectable in a number of other somatic tissues,are quantitatively less common than other lamins. In rats,lamin B is expressed in all tissues throughout develop-ment, with the possible exception of sperm (Kaufmann,1989). Recently, two lamin B isoforms have been identifiedin a murine cell line, but the differences in theirexpression patterns have yet to be elucidated (Weber et al.1990). In man, lamins A and C are virtually identical insequence except that lamin A has 90 additional aminoacids at its C terminus and the two proteins probably arisefrom alternative splicing of the same gene (McKeon et al.

Journal of Cell Science 99, 363-372 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

1986). They have have an identical pattern of expression.They are found in many differentiated tissues and theirexpression can be induced in non-expressing cells withmitogens (Kaufmann, 1989) or retinioc acid (Lebel et al.1987). In chickens, a single A-type lamin and two lamin Bisoforms have been cloned (Vorburger et al. 1989; Peter etal. 1989). In Xenopus laevis there are at least five differentlamins with a complex pattern of expression (Stick, 1988)but there appears to be a single lamin gene in Drosophilamelanogaster (Gruenbaum et al. 1988).

Lamin sequences (McKeon et al. 1986; Fisher et al. 1986;Pollard et al. 1990; Hoger et al. 1988; Weber et al. 1990;Krohne etal. 1987; Stick, 1988; Vorberger etal. 1989; Peteret al. 1989; Gruenbaum et al. 1988) show strong homologiesto intermediate filament proteins and indicate a three-domain structural model for lamins in which there is acentral fibrous rod, having an alpha-helical coiled-coilconformation, flanked by non-helical N- and C-terminaldomains (see Steinert and Roop, 1988; or Stewart, 1990, forreviews of intermediate filament protein structure). Thelamin N-terminal domain is small compared with mostother intermediate filament proteins, whereas the laminC-termina] domain is comparatively large. Electronmicroscopy of shadowed single molecules (Aebi et al. 1986)and of paracrystals (Aebi et al. 1986; Parry et al. 1987; Moiret al. 1990) support this model and show a rod-shapedmolecule, about 52 nm long, with a prominent globulardomain at one end. The sequence homology with inter-mediate filament proteins is strongest in the rod domain,where there is a strong heptad repeat of hydrophobicresidues that is characteristic of alpha-helical coiled-coils(Crick, 1953; McLachlan and Stewart, 1975; Stewart et al.1989a). However, when compared with intermediate

363

filament protein sequences from all species except invert-ebrates, the lamins are distinctive in having a 42-residueinsertion located 85-90 residues from the N terminus ofthe rod domain (McKeon et al. 1986; Fisher et al. 1986;Conway and Parry, 1988).

A number of important lamin functions have beenlocalised to denned regions of the sequence. For example,Loewinger and McKeon (1988) expressed human lamin Amutants in CHO cells and identified a putative nuclearlocalisation signal that has similarities to that of the SV40large T-antigen. The motif CaaX (a is an aliphatic residueand X is variable) is found at the C terminus of lamins Aand B. This sequence is also at the C terminus of rasproteins and, during their maturation, the last threeresidues are cleaved, an isoprenyl group is added to thesulfur atom of the cysteine, and the carboxy terminus isO-methylated (Gutierrez et al. 1989; Hancock et al. 1989).This cysteine residue is essential for ras protein binding tomembranes and their transforming function (Willumsenet al. 1984a, b). Alteration of the equivalent sequence in thelamins also decreases affinity of the proteins for themembranes of the nuclear envelope (Holtz et al. 1989;Krohne et al. 1989). Thus, human lamin A cDNA withaltered CaaX sequences transfected into tissue culturecells (Holtz et al. 1989) and lamins injected into Xenopuslaevis oocytes (Krohne et al. 1989) are not targetedcorrectly to the nuclear envelope. Lamin A and lamin Bhad previously been shown to be isoprenylated (Beck et al.1990; Wolda and Glomset, 1988) and lamin B is methyl-ated (Chelsky et al. 1987). Interestingly, as part of thenormal maturation process, the last 18 residues of laminA, including the modified cysteine, are removed after theprotein has been inserted into the nuclear envelope(Weber et al. 1989). The function of this proteolytic event isunknown. Finally, lamin disassembly during mitosiscorrelates with phosphorylation of these proteins (Gerace,1986). The serine residues probably involved have beenidentified using mitotic cell extracts or cdc2 kinase tophosphorylate expressed or endogenous lamins (Ward andKirschner, 1990; Peter et al. 1990) and by transfection ofmutant lamin cDNAs into CHO cells (Heald and McKeon,1990). The serine residues, whose phosphorylation pro-motes disassembly, immmediately flank the rod domain.

In order to study more precisely the molecular basis forthe assembly properties of lamins and to explore theinteractions between lamins and other components of thenucleus (such as nuclear pores and chromatin), we haveexpressed the cDNAs for human lamins A and C (McKeonet al. 1986; Fisher et al. 1986) in Escherichia coli. Thisexpression not only permits the production of largequantities of material but also enables specific mutantproteins to be constructed using site-specific mutagenesis.Although we have previously expressed human lamin CcDNA using the pLcII vector system (Moir et al. 1990), thematerial was obtained as a fusion with part of thebacteriophage lambda ell protein, which we were unableto remove using proteases such as factor X or thrombinbecause of the lability of the lamins to digestion. Tocircumvent these difficulties, we have instead used thebacteriophage T7 expression system (Studier et al. 1990)and describe here the production of substantial quantitiesof human lamins A and C, together with specific molecularfragments obtained using site-specific mutagenesis of thelamin cDNA. The expressed material closely resemblednative lamins in a number of key properties anddetermination of the solubility properties of mutantscoupled with electron-microscope examination of the

aggregates they form has enabled us to assess thecontribution of the different domains to the interactionbetween these molecules in paracrystals and filaments.

Materials and methods

DNA cloning and microbiological manipulationsRestriction endonucleases, bacteriophage T4 DNA ligase and theKlenow fragment of DNA polymerase I were obtained from NewEngland Biolabs (Beverly, USA) or Boehringer Mannheim(Mannheim, FRG). Bacteriophage T7 DNA polymerase (Seque-nase I) was obtained from United States Biochemical (Cleveland,USA) and radioisotopes from Amersham (Amersham, UK).Oligonucleotides were synthesised by Terry Smith and Jan Fogg(MRC Laboratory of Molecular Biology, Cambridge) using anApplied Biosystems synthesiser. Human lamin A and C cDNAwas a generous gift from Drs Frank McKeon and Marc Kirschner(UCSF, California, USA). Site-specific mutagenesis and othercloning methods were performed essentially as described (Quin-lan et al. 1989) except that the Sequenase system (United StatesBiochemical, Cleveland, Ohio) was used to sequence some of themutants.

Expression and purification of proteinWe expressed the lamin proteins in E. coli using the T7 RNApolymerase-based pET vectors described by Studier et al. (1990).For convenience of cloning, we inserted the lamin cDNAs into thetarget plasmid containing the T7 promoter (pET-1) at the Ncolsite, which resulted in three additional amino acid residues beingadded to their N terminus (Gly-Ser-Met). The T7 RNA polym-erase gene is located in the bacterial chromosome under control ofthe lac promoter and is induced with isopropyl-/3-D-thiogalacto-side (IPTG). Coulombe and Fuchs (1990) have also used thiscloning strategy for expression of cytokeratdns. We found itnecessary to include a second plasmid expressing lysozyme(pLysE) to inactivate the small quantities of T7 RNA polymeraseresulting from the leakiness of the lac promoter, as has beendescribed for this expression system (Studier et al. 1990). We grewthe bacterial strain BS21CDE21) carrying the two plasmids to anabsorbance of 0.5-0.6 at 600 run before inducing expression with0.4 mM IPTG, and then grew the bacteria for two to three hoursprior to harvesting. The different lamin fragments were par-titioned between soluble and insoluble (inclusion bodies) phasesto varying extents. Protein in inclusion bodies was isolated andpurified using the standard protocol of Nagai and Thogereen(1987). Bacterial cell pellets were homogenised or sonicated in50 mM Tris-HCl, pH8.0, 25% sucrose, lmM EDTA. Lysozymefrom the pLysE plasmid was sufficient to promote lysis. Twovolumes of detergent buffer (26mM Tris-HCl, pH7.4, lmMEDTA, 0.2 M NaCl, 1% sodium deoxycholate, 1% NP40) wereadded and the inclusion bodies sedimented by centrifugatdon(20 000#, 4°C). The crude inclusion body preparations werewashed three times with 0.5% Triton X-100, 1-5 mM EDTA, toremove residual membrane-bound proteins. Phenylmethylsul-phonyl fluoride (PMSF, 0.2 mM) and pepstatin A (0.2 /JM) wereincluded in all solutions to inhibit proteolysis. The inclusionbodies were dissolved in 8 M urea, 20 mM Tris-HCl or NaHepes,pH8.0, 2mM EDTA and lmM dithiothreitol (DTT). The proteinwas further purified using either the Mono S (cation exchange) orMono Q (anion exchange) preparative columns of a PharmaciaFPLC system using the same urea buffer with a 0 to 1 M NaClgradient. In some cases, the majority of the protein was not ininclusion bodies and was solubilised by sonicating the bacterialcell pellet in 2 M urea, 20 mM Tris-HCl or Hepes, pH 8.0, 1 mMEDTA, 1 mM DTT and purified by ion exchange using the samebuffer and a 0 to 1M NaCl gradient. Proteins were stored frozen in8 M urea, 25 mM Tris-HCl, 1 mM DTT, pH8, and, when required,were renatured by dialysis against 0.3 to 0.5 M NaCl, 25 mMTris-HCl, 1 mM DTT, pH 8, at room temperature. Sometimes anintermediate dialysis step against 2 M urea was employed, butthis was generally not necessary to obtain fully renatured protein.

364 R. D. Moir et al.

Biochemical methodsSDS-polyacrylamide gel electrophoresis was carried out asdescribed by Laemmli (1970) using 7.5% to 17.5% gradientminigels. Protein concentration was determined using the BioRadprotein assay kit (based on the Bradford (1976) dye-binding assay)and by absorbance at 280 nm. The extinction coefficient for laminA was determined to be 0.2 by refractometry using a Zeissrefractometer and we have also used this value for the otherfragments (there is only one tryptophan in the lamin sequence, inthe non-helical C-terminal domain). Proteins (100/Jgml"1) werecrosslinked with llmM dimethyl suberimidate (Pierce ChemicalCompany) in 20 mM NaHepes, pH8.0, 250 mM NaCl, lmM DTTfor one hour at room temperature. The reactions were stopped byadding an equal volume of 100 mM Tris-HCl, pH 8.0, 1 M glycineand incubating at room temperature for 10 min before precipitat-ing the samples with chloroform/methanol (1:4, v/v) for gelelectrophoresis.

Aggregate formationWe measured the solubility of the fragments by both dialysis anddilution into appropriate buffers. The absolute solubility atapproximate physiological conditions was measured by dialysingsamples in urea at about 5mgml"1 into 20mM Tris-HCl, pH8.0,300mM NaCl, lmM DTT, then into 20mM NaHepes, pH7.0,100 mM NaCl, 1 mM DTT. The concentration of the soluble proteinwas measured by absorbance at 280 nm. The solubility as afunction of ionic strength was determined by dialysing thesamples first into 20 mM Tris-HCl, pH 8.0, 300 mM NaCl, 1 mMDTT, then into 20 mM NaHepes, pH 7.0 or 6.5,500 mM NaCl, 1 mMDTT. The concentrated samples were diluted to the different saltconcentrations and protein concentrations were adjusted so thatall had the same starting molarity, representing protein concen-trations from 0.7 to l.Smgml"1. The absorbance of the solublematerial was measured after 3h, after centrifuging for tenminutes at ISOOOg'. Samples for electron microscopy were alsoprepared by dialysis or dilution, often using the 25 /il microdialy-sis buttons used to prepare samples for X-ray crystallography.

Electron microscopyIsolated particles were sprayed onto mica in 30 % glycerol, 250 mMNaCl, 20 mM Tris-HCl, 1 mM DTT, pH 8, as described by Stewartand Edwards (1984) and rotary-shadowed with platinum-carbonat a nominal angle of 10 degrees in a Cressington CFE-50 freeze-fracture system. Paracrystal suspensions were applied to carbon-coated 400-mesh electron microscopy grids and negatively stainedwith unbuffered 1% aqueous uranyl acetate as described byStewart (1981). Both negatively stained and shadowed specimenswere examined at 80 or 100 kV in Philips EM301, CM12, EM400and EM420 electron microscopes using standard conditions.Microscope magnification was calibrated by reference to the2.81 nm spacing of negatively stained sheaths of Methanospiril-lum hungatei (Stewart et al. 1985).

Results

Expression of lamins A and C and fragments in E. coliWe produced substantial quantities of recombinant hu-man lamins A and C by expressing cDNA in E. coli usingthe pET vector system (Studier et al. 1990). In addition tothe full-length lamins A and C, we expressed a number oflamin fragments using site-directed mutagenesis asillustrated in Fig. 1. Mutants that lacked the N- orC-terminal non-helical domains (or both) were expressedas well as material corresponding to the mature form oflamin A from which 18 amino acids have been removedfrom the C terminus (Weber et al. 1990). Mutants lackingthe N-terminal non-helical domain began at Glu31, threeresidues before the putative start of the rod domain heptadrepeat, whereas mutants lacking the C-terminal domainended at Pro393, five residues after the end of the heptad

Lamin A

Lamin AA18

Lamin C

Lamin CAN

Lamin AC

Lamin AC AN

31

CaaX

393

31 393

Fig. 1. Schematic representation of the various laminconstructs expressed. When naming the constructs A refers toa deletion of a portion of the molecule. The coiled-coil domainis shown by a box and the N- and C-non-helical terminaldomains are shown by heavy lines. The deletion of the Nterminus (AN) ends at amino acid 30 and the C-terminaldeletion (AC) begins at amino acid 394. The lamin A A18construct deletes the last 18 amino acids of lamin A as hasbeen described for the mature form (Weber et al. 1989). Thedeletion removes the CaaX motif, which is the site ofisoprenylation and methylation (see Introduction).

repeat, and corresponded to a deletion of the last 269amino acids from lamin A (Fisher et al. 1986). All theseexpressed proteins gave positive Western blots using ratanti-human lamin antiserum (data not shown).

The levels of expression obtained with the T7 vectorsystem varied for the different lamin constructs and withdifferent preparations, but was usually in the range of 5 to15mgl - 1 of culture and was comparable to the yields weobtained using the pLcEI system (Moir et al. 1990). Thelowest level of expression was obtained with lamin A, buteven in this case, the expressed protein was still visible inwhole bacterial lysates after induction with IPTG (Fig. 2,lanes B and C). The expressed proteins were isolated frombacterial pellets either as inclusion bodies (lamin A, laminC, lamin AA18, lamin AC) or as soluble material GaminCAN and lamin ACAN) and, after chromatography on ion-exchange columns, gave highly purified material asassessed by SDS-PAGE (Fig. 2, lanes D to I). In additionto being much easier to manipulate than the pLcII system,there was much less endogenous proteolysis in the pETsystem and consequently it was easier to prepare largerquantities of pure material in this way.

Characterisation of expressed proteinsAs illustrated in Table 1, all the expressed proteins had

Expression of lamins 365

B D H

SDS-PAGE electrophoretic mobilities that correspondedclosely to those predicted from their sequence. Shadowedpreparations of dilute solutions of the different expressedproteins sprayed in glycerol onto mica showed distinctiverod-like molecular profiles consistent with the fibrousnature of lamins (Figs 3 and 4). These rods seemedgenerally to resemble similar shadowed preparations ofnative material (Aebi et al. 1986) as well as other coiled-coil proteins, such as tropomyosin and myosin rodfragments (Stewart and Edwards, 1984). The fragmentsthat retained the C-terminal non-helical domain (lamin A,lamin C, lamin AA18, lamin CAN) had a prominientglobular region at one end in addition to the rod profile,whereas the fragments in which this domain had beendeleted have only the rod (Fig. 4). The rod domain isapproximately 50 nm in length. Chemical crosslinkingwith dimethyl suberimidate produced material in whichthe apparent MT had increased by a factor of 4 (Fig. 5),indicating that all the expressed fragments aggregatedinto four-chain 'tetramer' units analogous to those ob-served with other intermediate filament proteins. As isoften observed when crosslinking fibrous proteins (Quin-lan et al. 1989), the tetramer gave a series of closely spacedbands on SDS-PAGE. Each of these bands probablycorresponded to a crosslink between different residues in

Fig. 2. SDS-PAGE showinglamin expression levels and the

^ purified fragments described inKg. 1. Lane B is total E. coliprotein from bacteria carrying

<«ta» the lamin A pET-1 expression____^ plasmid before induction, and

lane C is after induction withIPTG. The fragments are: laneD, lamin A; lane E, lamin AA18; lane F, lamin C; lane G,lamin C AN; lane H, lamin AC;lane J, lamin ACAN. Lane A,molecular weight markers, fromtop: phosphorylase b (94xl03Afr),albumin (67 xlO3Mr),ovalbumin (43xlO3Afr),carbonic anhydrase (30xl03M,.),trypsin inhibitor (20.1xl03Mr),o<-lactalbumin (14.4xlO3Mr).

the molecule and, because some secondary structure isusually retained in such fibrous proteins in the conditionsused for SDS-PAGE, the differently crosslinked speciesmigrate with slightly different mobilities even thoughthey have the same molecular weight. Because humanlamins A and C do not have a cysteine in the rod domain ofthe molecule, we were not able to employ disulphidecrosslinking to demonstrate the presence of two-chain'dimer' molecules as has been done for other intermediatefilament proteins (Quinlan et al. 1989). However, in someof the fragments (particularly those in which theN-terminal non-helical domain had been deleted: laminCAN, lamin ACAN) there was an additional crosslinkedband at close to double the Mr of the single chain, whichprobably resulted from crosslinking within two-chainmolecules (Fig. 5, arrows)

Solubility of lamin fragmentsWe measured the solubility of the different laminfragments under approximately physiological conditionsstarting with high protein concentrations in urea(Smgml"1). The different samples were dialysed intiallyinto conditions where they are very soluble (20 mMTris-HCl, pH8.0, 300 mM NaCl, lmM DTT), then into

Table 1. Determination of molecular weight and absolute solubility of the various lamin fragmentsCalculatedmolecular

weight(xlO"3)

Observedmolecular

weight(xKT3)

Solubility(mgml"f)

Solubility(MXIO"*)

Relativesolubility

Lamin ALamin AA18T.flrain CLamin CANLamin ACLamin ACAN

767464594340

747265624542

0.170.340.203.10.94.1

2.24.23.1512098

1.02.21.4249.345

The calculated molecular weights were determined from the published Bequence of the lamina (Fisher et al. 1986). The observed molecular weightswere calculated from the SDS-PAGE gel in Fig. 2. The solubility of the lamin fragments refers to the solubility in 20 mM NaHepes, pH7.0, 100 mMNaCl, 1 mM DTT. The values are the average of three experiments with two different protein preparations. Relative solubility refers the Bolubility ofthe fragments relative to lamin A.


'^*?&y^:'<Qiffi^:.*£

^••V^^^'V^'-^'v:-':;.'^::;^^';'--^1^-^^;^"

Fig. 3. Electron micrograph of a shadowed preparation of lamin A sprayed onto mica in 20 mM Tris-HCl, 300 mM NaCl, 1 mM DTTand 30 % glycerol. Some molecules in which two globular domains are visible are indicated by arrows. Bar, 260 run.

A B C D E F G

(»» • • •Lamin A

Lamin A A18

Lamin C

Lamin C AN

.] I

• • , • . • - • ; ' , ' , ! . Lamin AN AC

Lamin AC

Fig. 4. Electron micrographs of selected examples of sprayedand shadowed lamin fragments. Lamin A, lamin AA18, laminC and lamin CAN all have two obvious globular domains atone end of the rod profile whereas lamin AC and lamin ACANshow only the rod portion. Bar, 250 nm.

Fig. 5. SDS—PAGE of lamin fragments crosslinked withdimethyl suberimidate. The bracketed sets of bands havemolecular weights corresponding to approximate tetramericspecies. The arrows indicate dimeric species in lanes E and G.Lane B, lamin A; lane C, lamin AA18; lane D, lamin C; laneE, lamin CAN; lane F, lamin AC; lane G, lamin ACAN. LaneA, molecular weight markers, from top: myosin heavy chain(220xl03Mr), phosphorylase b (94x10^Mr), albumin(67xlO3Mr), ovalbumin (43xlO3Mr), carbonic anhydrase(30xl03Mr).


approximate physiological ionic strength (20 HIMNaHepes, pH7.0 100 mM NaCl, lmM DTT). The insolublematerial was removed by centrifugation and the concen-tration of the soluble protein measured by its absorbanceat 280 nm. We used SDS-PAGE of the soluble fractions toconfirm that this solubility was due to the fragmentsthemselves and not contamination (data not shown). Theresults are shown in Table 1, on both a mass and a molarbasis. The solubility of lamin A and lamin C was roughlysimilar and was slightly less than that of the lamin AA18.Deletion of either the N-terminal or C-terminal non-helical domain had a profound effect on lamin solubilityunder these conditions. The lamin C fragment lacking the30 residues at the N terminus was more than 20 timesmore soluble on a molar basis than full-length lamin C,whereas the fragment lacking the 179 residues of theC-terminal non-helical domain was almost 10 times moresoluble than lamin A. The rod domain, lamin ACAN, wasthe most soluble. We further investigated the solubility ofthe lamin fragments by measuring solubility as a functionof ionic strength. For these experiments the startingprotein concentration was adjusted so each sample wasapproximately 1.5X10~6M and the samples were dilutedto the appropriate conditions. Fig. 6 shows the resultsobtained at pH7.0. The solubility of full-length lamins Aand C and lamin A A18 mutant showed a strongdependence of solubility on ionic strength. However, theother three fragments, lacking the N or C terminus orboth, did not precipitate at any salt concentration at thispH. We again confirmed these results by SDS-PAGE.Similar results were obtained at pH6.5 (not shown).

Paracrystal formationThe lamin fragments formed a range of ordered aggre-gates when the pH of the buffer was below 6.5 and the ionicstrength below 200 mM. Fig. 7 is a low-magnificationmicrograph of negatively stained paracrystals formedfrom lamin A. Lamin A, lamin C and lamin AA18 readilyformed these long, thin paracrystals without the additionof divalent cations, over a range of temperatures down to4°C, and at protein concentrations above 50 jigml"1. Fig. 8shows an enlargement of areas of negatively stainedparacrystals formed from different lamin fragments. Theaxial repeat for lamin A, lamin C and lamin A A18 (Fig- 8)paracrystals was 21 nm, close to the value obtained for a

100 200 300 400NaCl concentration (ITIM)

Fig. 6. Graph of the solubility of the lamin fragments as afunction of ionic strength at pH 7.0. The samples were dialysedstepwise into 20 mM Tris-HCl, pH8.0, 300 mM NaCl, lmMDTT, then into 20mM NaHepes, pH7.0, 500mM NaCl, lmMDTT. The concentration of the six different fragments wasadjusted to the same starting molarity and the ionic strengthwas changed by dilution. After 3 h the absorbance at 280 nm ofthe soluble material was measured. (O) lamin A; (D) AA18;(•) lamin C; (A) lamin CAN; (•) lamin ACAN; (A) lamin AC.

mixture of lamins A and C isolated from rat liver nuclearenvelopes (Aebi et al. 1986). The fragment that lacked theN terminus of lamin C, lamin CAN, also formedparacrystals with a similar axial repeat but they were lesswell ordered and always aggregated in clumps (Fig. 8),Moreover, lamin CAN paracrystals only formed when thepH of the buffer was 6 or below.

The lamin AC and lamin ACAN fragments formeddifferent aggregates from those observed with intactlamina. Lamin AC formed networks of filaments with nodiscernible banding pattern (Fig. 9). The diameter of thefilaments varied and was usually in the range 15-30 nm.Lamin ACAN, the lamin rod domain, formed very well-ordered paracrystals with a 45 nm axial repeat, in whichthere were two 10 nm wide light bands separated by darkbands alternately of about 12 and 13 nm (Fig. 10). Inaddition, the 13 nm dark band was more dense than the12 nm band and so, although this pattern was superfically

Fig. 7. Electron micrograph of lamin A paracrystals stained with uranyl acetate. Lamin AA18 and lamin C formed paracrystalswith a similar appearance. Bar, 250 nm.


"t

• » ' * < » • * . ,

Vfttvi:.'

Lamin LaminAA18

Lamin LaminCAN

Fig. 8. Electron micrographs of areas of paracrystal preparations from the four different lamin fragments with a similar axialrepeat of approximately 21 nm. Bar, 100 nm.

m9

Fig. 9. Electron micrographs of the filament network formedby lamin AC negatively stained with uranyl acetate. Thediameter of the filaments varies from 15 to 30 nm. Bar, 250 nm.

similar to that observed with intact lamins, it wasdistinctly different in detail.

Discussion

Lamin expression and characterisationWe have expressed in E. coli cDNAs for human lamins Aand C and a number of fragments derived from them.Substantial quantities of material can be produced in thisway and a range of biochemical and structural criteriaindicated that all these molecules had refolded correctly.Shadowing of isolated molecules (Figs 3 and 4) showedrod-like profiles about 50 nm long with two globular headsat one end for those constructs in which the C-terminalnon-helical domain had not been deleted. The absence ofthese globular heads from constructs lacking theC-terminal non-helical domain confirms the structuralassignment made from sequence data (McKeon et al. 1986;

Fisher et al. 1986) of shadowed native lamin molecules(Aebi et al. 1986). Chemical crosslinking (Fig. 5) indicatedthat all the constructs assembled into 4-chain units,indicative of the dimerisation of two 2-chain molecules in amanner analogous to that seen with other intermediatefilament proteins (reviewed by Stewart, 1990).

Paracrystal structureThe formation of ordered aggregates (Figs 7-10) alsoindicated that molecular structure had been preserved, atleast to the extent necessary for the usual molecularinteractions to occur. Moreover, four of the fragments,lamin A, lamin C, lamin AA18 and lamin CAN, formedparacrystals with a characteristic 21 nm axial repeat(Figs 7 and 8) that were very similar to those obtainedusing a mixture of lamins A and C prepared from rat livernuclear envelopes (Aebi et al. 1986). The contrast in thestaining pattern seen in the micrographs of negativelystained paracrystals probably derived from two sources:because the molecular length (50 nm) was greater thantwice the axial repeat of the paracrystal, there would be agap between successive molecules that would allowgreater stain penetration and so give rise to a band ofincreased density across the paracrystal at this point asproposed by Moir et al. (1990). The contrast deriving fromsuch a gap-overlap structure would be coupled with stainexclusion from the globular C terminus to accentuate thealternation between high and low stain density along theparacrystal. The paracrystals derived from the lamin roddomain (lamin ACAN) fragment had a different stainingpattern that may indicate an alternative set of molecularinteractions (Fig. 10) when both N- and C-terminal non-helical domains are absent. In the lamin rod paracrystals,there were still alternate dark and light bands indicatinga gap-overlap structure and there was clear dyadsymmetry, suggesting an antiparallel arrangement ofmolecules. However, the axial repeat distance haddoubled. Some caution may be warranted in making adetailed comparison between the staining pattern seenwith rod paracrystals and that from the other paracrys-tals, since the resolution in electron micrographs of thelatter was rather low as a result of disorder in theaggregates, and it was conceivable that it may not havebeen possible to detect the difference between successive


A -r

10

Fig. 10. Electron micrograph of a paracrystal made from the lamin ACAN, the rod domain. The axial repeat is 46 nm, with two10 nm light bands separated by dark bands of 12 and 13 nm. Bar, 260 nm.

21-nm repeats in this case. In other words, it may be thatthe true axial repeat of the lamin paracrystals was twicethat observed but that this was obscured by stronginternal pseudo-halving of the pattern. We were unable toform paracrystals with the fragment that lacked theC-terminal non-helical domain but retained the N ter-minus (lamin AC). This was probably a consequence of itsreadily forming filament networks or alternatively thepresence of the large C-terminal globular domain in-hibited filament formation in the other constructs. Theactual filaments formed by this fragment were somewhatwider than intermediate filaments and may not necess-arily represent precisely the same structure as that takenup by the lamins in the fibrous lamina.

Because in vivo lamins A and C are always expressedtogether and in equal amounts, it was not immediatelyclear if it was necessary for both to be present in a manneranalogous to that observed for the two classes ofcytokeratin (reviewed by Steinert and Roop, 1988; Stew-art, 1990). However, either lamin A or lamin C aloneformed paracrystals, implying that heterodimer or hetero-tetramer formation between the A and C isoforms was notrequired, at least for this level of assembly. Furthermore,the post-translational modifications of the lamins isolatedfrom nuclear envelopes (such as phosphorylation, isopre-nylation and proteolysis as described in the Introduction)also appear not to be an absolute requirement for this levelof assembly, although they could possibly modulate theprocess in vivo. The mature form of lamin A, that lackedthe last 18 amino acids at its C terminus, formedparacrystals identical to lamins A and C under the sameconditions (Fig. 8) and had similar solubility properties,indicating that this post-translational modification wasunlikely to result in a dramatic change in the interactionsbetween this isoform and other lamins in vivo.

Influence of domains on solubility and aggregateformationThe influence of the N-terminal and C-terminal globular

domains on lamin solubility and aggregation propertieswere somewhat different from that seen with otherintermediate filament proteins. Results with proteolyticfragments of the intermediate filament proteins desmin,vimentin (Kaufmann et al. 1985; Traub and Vorgias, 1984)and expressed GFAP (Quinlan et al. 1989) have shown thatthe N terminus is required for filament formation. The roleof the C-terminal domain in intermediate filamentformation is less clear. GFAP fragments expressed in E.coli that completely lack this domain could not formfilaments (Quinlan et al. 1989) unless extra material wasattached to their N terminus. However, proteolyticfragments of desmin that lack half of the C-terminaldomain still make filaments (Kaufmann et al. 1985) andcytokeratin 8 that retained only the first six amino acids ofthis domain also made filaments (Hatzfeld and Weber,1990). The role of the C-terminal domain appearsmarginal in these intermediate filament proteins. Bycontrast, the solubility data that we obtained indicate thatboth the N- and C-terminal domains play a role in laminassembly. Although deletion of the N-terminal domainincreased lamin solubility most dramatically, deletion ofthe C-terminal domain still produced an almost tenfoldincrease (Table 1). The differences in solubility indicatethat the strength or nature of the interactions at low ionicstrength between these mutant molecules was differentfrom that between the whole lamins. The appearance ofthe aggregates formed when the C terminus was absent(Fig. 8) confirms that this domain has an importantinfluence on lamin assembly. It is interesting to note that,although the aggregation properties of both lamins andintermediate filaments are modulated by phosphorylation,the lamins are phosphorylated in both N and C globulardomains (Ward and Kirschner, 1990; Peter et al. 1990;Heald and McKeon, 1990), whereas the intermediatefilament proteins are phosphorylated only in theN-terminal domain (Inagaki et al. 1988, 1989; Geisler andWeber, 1988). The presence of additional phosphorylationsites in the lamin C-terminal domain would be consistentwith the greater influence of this domain on solubility


compared to cytoplasmic intermediate filaments. Theaggregation of the lamins probably involves a hierarchy ofinteractions analogous to those proposed for intermediatefilaments (Aebi et al. 1986; Stewart, 1990). All thefragments apparently formed tetramers in crosslinkingassays including the rod domain (Fig. 5) so that the effectsof the N and C terminus (and possibly phosphorylation)may be to modulate assembly after this point.

It is also significant that the influence of ionic strengthon the solubility of whole lamins was the opposite to thatobserved with other intermediate filament proteins.Lamin solubility increased rapidly with increasing ionicstrength (Fig. 6) whereas the solubility of GFAP (andprobably other cytoplasmic intermediate filaments) de-creases with increasing ionic strength (Yang and Babitch,1988). In this respect, the solubility properties of thelamins more closely resembled myosin. This solubilitybehaviour further indicates that the molecular interac-tions involved in lamin aggregates are not precisely thesame as those in other intermediate filaments, whichcould reflect the different nature of the lamina andcytoplasmic filament networks. The different interactionsmay also account for the apparent failure of lamins toincorporate into cytoplasmic assemblies whereas differentcytoplasmic proteins (GFAP, vimentin, desmin) can befound in the same filament (Quinlan and Franke, 1982,1983).

We are now using the large quantities of specific laminsproduced using the pET expression to explore in greaterdetail the molecular interactions involved in laminassembly and to examine the interaction of lamins withother components of the nucleus during the cell cycle.

We particularly thank Sue Whytock for invaluable assistanceand advice with the figures. We thank Simon Atkinson forsuggestions on protocols and for helpful discussions, and ourcolleagues, in particular Rob Cross, Roy Quinlan and NigelUnwin, for their comments and criticisms. Claudio Villa providedtechnical assistance. R.D.M. was supported by the AlbertaHeritage Foundation for Medical Research and A.D.D. holds anMRC studentship.

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(Received 1 February 1991 - Accepted 12 March 1991)


expression in escherichia coli of human lamin as and c...

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