Lamins A and C bind and assemble at the surface of mitotic chromosomes

To study a possible interaction of nuclear lamins with chromatin, we examined assembly of lamins A and C at mitotic chromosome surfaces in vitro. When a postmicrosomal supernatant of metaphase CHO cells containing disassembled lamins A and C is incubated with chromosomes isolated from mitotic Chinese hamster ovary cells, lamins A and C undergo dephosphorylation and uniformly coat the chromosome surfaces. Furthermore, when purified rat liver lamins A and C are dialyzed with mitotic chromosomes into a buffer of physiological ionic strength and pH, lamins A and C coat chromosomes in a similar fashion. In both cases a lamin-containing supramolecular structure is formed that remains intact when the chromatin is removed by digestion with micrococcal nuclease and extraction with 0.5 M KCl. Lamins associate with chromosomes at concentrations approximately eightfold lower than the critical concentration at which they self-assemble into insoluble structures in the absence of chromosomes, indicating that chromosome surfaces contain binding sites that promote lamin assembly. These binding sites are destroyed by brief treatment of chromosomes with trypsin or micrococcal nuclease. Together, these data suggest the existence of a specific lamin-chromatin interaction in cells that may be important for nuclear envelope reassembly and interphase chromosome structure.     

The last twenty residues in the head domain of mouse lamin A contain important structural elements for formation of head-to-tail polymers in vitro

Nuclear lamins are a type of intermediate filament (IF) proteins. They have a characteristic tripartite domain structure with a alpha-helical rod domain flanked by non-alpha-helical N-terminal head and C-terminal tail domains. While the head domain has been shown to be important for the formation of head-to-tail polymers that are critical assembly intermediates for lamin IFs, essential structural elements in this domain have remained obscure. As a first step to remedy this, a series of mouse lamin A mutants in which the head domain (30 amino acid residues) was deleted stepwise from the N-terminus at intervals of 10 residues were bacterially expressed. The assembly properties in vitro of the purified recombinant proteins were explored by electron microscopy. We observed that while a lamin A mutant lacking N-terminal 10 residues formed head-to-tail polymers, a mutant lacking N-terminal 20 residues or the whole head domain (30 residues) showed significantly decreased potency to form head-to-tail polymers. These results suggest that the last 20 residues (from Arg-11 to Gln-30) of the head domain of mouse lamin A contain essential structures for the formation of head-to-tail polymers. The last 20 residues of the head domain include several conserved residues between A- and B-type lamins and also the phosphorylation site for cdc2 kinase, which affects lamin IF organization in vivo and in vitro. Our results provide clues to the molecular mechanism by which the head domain plays a crucial role in lamin polymerization.     

The role of the head and tail domain in lamin structure and assembly: analysis of bacterially expressed chicken lamin A and truncated B2 lamins.

Nuclear lamins like cytoplasmic intermediate filament proteins exhibit a characteristic tripartite domain structure with a segmented alpha-helical rod domain flanked by an N-terminal head and a C-terminal tail domain. To examine the influence of the head and tail domains on the structure and assembly properties of nuclear lamins, we have engineered "headless," "tailless," and "rod" chicken lamin B2 cDNAs and expressed them in Escherichia coli. A full-length chicken lamin A cDNA was also expressed in E. coli, and the recombinant protein compared with the structure and assembly properties of full-length chicken lamin B2 (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495). As with lamin B2, at their first level of structural organization, lamin A and the headless lamin B2 formed myosin-like dimers consisting of a 51- to 52-nm-long tail flanked by two globular heads at one end. Similarly, the tailless and rod lamin B2 fragments formed tropomyosin-like dimers consisting of a 51 to 52-nm-long rod. In contrast to the lateral mode of association of cytoplasmic IF dimers into four-chain tetramers, at their second level of structural organization, lamin A dimers, just as lamin B2 dimers (E. Heitlinger et al. (1991) J. Cell Biol. 113, 485-495), associated longitudinally to form polar head-to-tail polymers. Whereas dimers made of the truncated B2 headless and rod lamins had lost their propensity to associate head-to-tail, tailless lamin B2 dimers revealed an enhanced head-to-tail association. Finally, at their third level of structural organization, rather than assembling into stable 10-nm filaments, both lamin A and the three truncated B2 lamins formed paracrystalline arrays exhibiting distinct transverse banding patterns with axial repeats of either 24 or 48-49 nm depending on the species.     

Involvement of the lamin rod domain in heterotypic lamin interactions important for nuclear organization

The nuclear lamina is a meshwork of intermediate-type filament proteins (lamins) that lines the inner nuclear membrane. The lamina is proposed to be an important determinant of nuclear structure, but there has been little direct testing of this idea. To investigate lamina functions, we have characterized a novel lamin B1 mutant lacking the middle approximately 4/5 of its alpha-helical rod domain. Though retaining only 10 heptads of the rod, this mutant assembles into intermediate filament-like structures in vitro. When expressed in cultured cells, it concentrates in patches at the nuclear envelope. Concurrently, endogenous lamins shift from a uniform to a patchy distribution and lose their complete colocalization, and nuclei become highly lobulated. In vitro binding studies suggest that the internal rod region is important for heterotypic associations of lamin B1, which in turn are required for proper organization of the lamina. Accompanying the changes in lamina structure induced by expression of the mutant, nuclear pore complexes and integral membrane proteins of the inner membrane cluster, principally at the patches of endogenous lamins. Considered together, these data indicate that lamins play a major role in organizing other proteins in the nuclear envelope and in determining nuclear shape.     

The single nuclear lamin of Caenorhabditis elegans forms in vitro stable intermediate filaments and paracrystals with a reduced axial periodicity

The lamins of the tunicate Ciona intestinalis and the nematode Caenorhabditis elegans show unusual sequence features when compared to the more than 35 metazoan lamin sequences currently known. We therefore analyzed the in vitro assembly of these two lamins by electron microscopy using chicken lamin B2 as a control. While lamin dimers usually appear as a rod carrying two globules at one end, these globules are absent from Ciona lamin, which lacks the central 105-residue region of the tail domain. The deletion of 14 residues or two heptads from the coiled coil rod domain of the single C.elegans lamin results in a 1.5-nm shortening of the dimer rod. Similarly, the paracrystals assembled from the C.elegans lamin exhibit a 3.1-nm reduction of the true axial repeat compared to that of chicken lamin B2 paracrystals. We speculate that the banding pattern in the C.elegans lamin paracrystals arises from a relative stagger between dimers and/or a positioning of the globular tail domain relative to the central rod that is distinct from that observed in chicken lamin B2 paracrystals. Here we show that a nuclear lamin can assemble in vitro into 10-nm intermediate filaments (IFs). C.elegans lamin in low ionic strength Tris-buffers at a pH of 7.2-7.4 provides a stable population of lamin IFs. Some implications of this filament formation are discussed.     

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.     

Amino acid sequence and molecular characterization of murine lamin B as deduced from cDNA clones

The nuclear lamina is the karyoskeletal structure, intimately associated with the nuclear envelope, that is widespread among the diverse types of eukaryotic cells. A family of proteins, termed lamins, has been shown to be a prominent component of this lamina, and various members of this family are differentially expressed in different cell types. In mammals, three major lamins (A, B, C) have been identified, and in all cells so far examined lamin B is constitutively expressed while lamins A and C are not, suggesting that lamin B is sufficient to form a functional lamina. Because of this key importance of lamin B, cDNA clones encoding mammalian lamin B were isolated by screening murine cDNA libraries, representing F9 teratocarcinoma cells and fetal liver, with the corresponding cDNA probe of lamin LI of Xenopus laevis. The nucleotide sequence of the murine lamin B mRNA (approximately 2.9 kb) was determined. The deduced amino acid sequence of the encoded polypeptide (587 amino acids; mol. wt. 66760) is highly homologous to X. laevis lamin LI (72.9% identical residues) but displays lower similarity to A-type lamins (53.8% identical amino acid residues with human lamin A). Lamin B also conforms to the general molecular organization principle of the members of the intermediate filament (IF) protein family, i.e., an extended alpha-helical rod domain that is interrupted by two non alpha-helical linkers and flanked by non-alpha-helical head (amino-terminal) and tail (carboxy-terminal) domains. The tail domain, which does not reveal a hydrophobic region of considerable length, contains a typical karyophilic signal sequence and an uninterrupted stretch of eight negatively charged amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Last Twenty Residues in the Head Domain of Mouse Lamin A Contain Important Structural Elements for Formation of Head-to-Tail Polymers in Vitro

Nuclear lamins are a type of intermediate filament (IF) proteins. They have a characteristic tripartite domain structure with a α-helical rod domain flanked by non-α-helical N-terminal head and C-terminal tail domains. While the head domain has been shown to be important for the formation of head-to-tail polymers that are critical assembly intermediates for lamin IFs, essential structural elements in this domain have remained obscure. As a first step to remedy this, a series of mouse lamin A mutants in which the head domain (30 amino acid residues) was deleted stepwise from the N-terminus at intervals of 10 residues were bacterially expressed. The assembly properties in vitro of the purified recombinant proteins were explored by electron microscopy. We observed that while a lamin A mutant lacking N-terminal 10 residues formed head-to-tail polymers, a mutant lacking N-terminal 20 residues or the whole head domain (30 residues) showed significantly decreased potency to form head-to-tail polymers. These results suggest that the last 20 residues (from Arg-11 to Gln-30) of the head domain of mouse lamin A contain essential structures for the formation of head-to-tail polymers. The last 20 residues of the head domain include several conserved residues between A- and B-type lamins and also the phosphorylation site for cdc2 kinase, which affects lamin IF organization in vivo and in vitro. Our results provide clues to the molecular mechanism by which the head domain plays a crucial role in lamin polymerization.     

In vitro assembly properties of mutant and chimeric intermediate filament proteins: insight into the function of sequences in the rod and end domains of IF

The factors and mechanisms regulating assembly of intermediate filament (IF) proteins to produce filaments with their characteristic 10 nm diameter are not fully understood. All IF proteins contain a central rod domain flanked by variable head and tail domains. To elucidate the role that different domains of IF proteins play in filament assembly, we used negative staining and electron microscopy (EM) to study the in vitro assembly properties of purified bacterially expressed IF proteins, in which specific domains of the proteins were either mutated or swapped between a cytoplasmic (mouse neurofilament-light (NF-L) subunit) and nuclear intermediate filament protein (human lamin A). Our results indicate that filament formation is profoundly influenced by the composition of the assembly buffer. Wild type (wt) mouse NF-L formed 10 nm filaments in assembly buffer containing 175 mM NaCl, whereas a mutant deleted of 18 NH2-terminal amino acids failed to assemble under similar conditions. Instead, the mutant assembled efficiently in buffers containing CaCl2 > or = 6 mM forming filaments that were 10 times longer than those formed by wt NF-L, although their diameter was significantly smaller (6-7 nm). These results suggest that the 18 NH2-terminal sequence of NF-L might serve two functions, to inhibit filament elongation and to promote lateral association of NF-L subunits. We also demonstrate that lengthening of the NF-L rod domain, by inserting a 42 aa sequence unique to nuclear IF proteins, does not compromise filament assembly in any noticeable way. Our results suggests that the known inability of nuclear lamin proteins to assemble into 10 nm filaments in vitro cannot derive solely from their longer rod domain. Finally, we demonstrate that the head domain of lamin A can substitute for that of NF-L in filament assembly, whereas substitution of both the head and tail domains of lamins for those of NF-L compromises assembly. Therefore, the effect of lamin A "tail" domain alone, or the synergistic effect of lamin "head" and the "tail" domains together, interferes with assembly into 10-nm filaments.     

Nuclear lamin LI of Xenopus laevis: cDNA cloning, amino acid sequence and binding specificity of a member of the lamin B subfamily.

Lamins are karyoskeletal proteins associated with the nuclear envelope which can be divided into two groups, i.e. the type A lamins of near neutral pI and the more acidic lamins, including mammalian lamin B. We have isolated cDNA clones encoding a representative of the type B subfamily from Xenopus laevis, and have deduced its amino acid sequence from the coding portion of the approximately 2.9 kb mRNA. The polypeptide (mol. wt 66,433) is identified as a typical lamin by its homology to Xenopus human type A lamins, but detailed sequence comparison shows that LI is less related to Xenopus lamin A than the latter is to human lamin A. The conformation predicted for LI conforms to the general model of lamins and intermediate filament proteins and is characterized by an extended central alpha-helical coiled coil domain, flanked by non-alpha-helical domains, i.e. a relatively short N-terminal head and a long C-terminal tail. As in lamins A and C, the head of lamin LI is positively charged and the tail presents a similar C-terminal pentapeptide, a putative nuclear accumulation signal, a very negatively charged region and a number of short regions that are highly homologous in all lamins. However, LI differs from the type A lamins by the absence of the oligo-histidine stretch and a di-proline motif in the tail region and by a significantly lower number of identical amino acid positions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations of phosphorylation sites in lamin A that prevent nuclear lamina disassembly in mitosis

The nuclear envelope is a dynamic structure that completely disassembles in response to MPF/cdc2 activity in mitosis. A key feature of this process is the hyperphosphorylation of the major structural proteins of the envelope, the nuclear lamins A, B, and C. Two highly conserved serine residues of the lamin protein (Ser-22 and Ser-392 of lamins A and C) are symmetrically positioned 5 amino acids from the ends of the large alpha-helical domain and are shown in the accompanying paper by Ward and Kirschner to be among four sites phosphorylated during nuclear envelope breakdown. Mutations in Ser-22 and Ser-392 that prevent phosphorylation at these sites block the disassembly of the nuclear lamina during mitosis. We propose a model for the regulation of lamin assembly in which phosphorylation just outside the ends of the alpha-helical domain controls the assembly dynamics of the lamin coiled-coil dimers.     

Crystal structure of the human lamin A coil 2B dimer: implications for the head-to-tail association of nuclear lamins

Nuclear intermediate filaments (IFs) are made from fibrous proteins termed lamins that assemble, in association with several transmembrane proteins of the inner nuclear membrane and an unknown number of chromatin proteins, into a filamentous scaffold called the nuclear lamina. In man, three types of lamins with significant sequence identity, i.e. lamin A/C, lamin B1 and B2, are expressed. The molecular characteristics of the filaments they form and the details of the assembly mechanism are still largely unknown. Here we report the crystal structure of the coiled-coil dimer from the second half of coil 2 from human lamin A at 2.2A resolution. Comparison to the recently solved structure of the homologous segment of human vimentin reveals a similar overall structure but a different distribution of charged residues and a different pattern of intra- and interhelical salt bridges. These features may explain, at least in part, the differences observed between the lamin and vimentin assembly pathways. Employing a modeled lamin A coil 1A dimer, we propose that the head-to-tail association of two lamin dimers involves strong electrostatic attractions of distinct clusters of negative charge located on the opposite ends of the rod domain with arginine clusters in the head domain and the first segment of the tail domain. Moreover, lamin A mutations, including several in coil 2B, have been associated with human laminopathies. Based on our data most of these mutations are unlikely to alter the structure of the dimer but may affect essential molecular interactions occurring in later stages of filament assembly and lamina formation.     

Head and/or CaaX domain deletions of lamin proteins disrupt preformed lamin A and C but not lamin B structure in mammalian cells

The nuclear lamina is an important determinant of nuclear architecture. Mutations in A-type but not B-type lamins cause a range of human genetic disorders, including muscular dystrophy. Dominant mutations in nuclear lamin proteins have been shown to disrupt a preformed lamina structure in Xenopus egg extracts. Here, a series of deletion mutations in lamins A and B1 were evaluated for their ability to disrupt lamina structure in Chinese hamster ovary cells. Deletions of either the lamin A "head" domain or the C-terminal CaaX domain formed intranuclear aggregates and resulted in the disruption of endogenous lamins A/C but not lamins B1/B2. By contrast, "head-less" lamin B1 localized to the nuclear rim with no detectable effect on endogenous lamins, whereas lamin B1 CaaX domain deletions formed intranuclear aggregates, disrupting endogenous lamins A/C but not lamins B1/B2. Filter binding assays revealed that a head/CaaX domain lamin B1 mutant interacted much more strongly with lamins A/C than with lamins B1/B2. Regulated induction of this mutant in stable cell lines resulted in the rapid elimination of all detectable lamin A protein, whereas lamin C was trapped in a soluble form within the intranuclear aggregates. In contrast to results in Xenopus egg extracts, dominant negative lamin B1 (but not lamin A) mutants trapped replication proteins involved in both the initiation and elongation phases of replication but did not effect cellular growth rates or the assembly of active replication centers. We conclude that elimination of the CaaX domain in lamin B1 and elimination of either the CaaX or head domain in lamin A constitute dominant mutations that can disrupt A-type but not B-type lamins, highlighting important differences in the way that A- and B-type lamins are integrated into the lamina.     

On the cell-free association of lamins A and C with metaphase chromosomes

Nuclear envelopes have previously been shown to assemble spontaneously around endogenous chromosomes in cell-free homogenates of mitotic Chinese hamster ovary cells. In order to further analyze the mechanisms underlying nuclear envelope reformation and the functions of the individual nuclear lamin polypeptides, a fractionated cell-free nuclear envelope reassembly system involving purified chromosomes and either a postchromosomal supernatant or a cytosol fraction from mitotic cells has been devised. Results obtained with this fractionated system show that lamins A and C will associate with the surfaces of chromosomes in the absence of lamin B and membranes, this association being inhibitable by ATP-gamma-S. However, in the absence of membranes chromatin decondensation never occurs. Using the reversible swelling of chromosomes in low ionic strength buffers lacking divalent cations as the basis of a simple assay, it is demonstrated that the association of lamins A and C with the surfaces of chromosomes has a pronounced and easily observable effect on chromatin organization.     

Distinct functions of the unique C terminus of LAP2alpha in cell proliferation and nuclear assembly

The non-membrane-bound lamina-associated polypeptide 2 isoform, LAP2alpha, forms nucleoskeletal structures with A-type lamins and interacts with chromosomes in a cell cycle-dependent manner. LAP2alpha contains a LEM (LAP2, emerin, and MAN1) domain in the constant N terminus that binds to chromosomal barrier-to-autointegration factor, and a C-terminal unique region that is essential for chromosome binding. Here we show that C-terminal LAP2alpha fragment efficiently bound to mitotic chromosomes and inhibited assembly of endogenous LAP2alpha, nuclear membranes, and lamins A/C in in vitro nuclear assembly assays. Full-length recombinant LAP2alpha, which bound to chromosomes, and N-terminal fragment, which did not bind, had no effect on assembly. This suggested an essential role for the LAP2alpha C terminus in chromosome association and for the N-terminal LEM domain in subsequent assembly stages. In vivo analysis upon transient expression of GFP-tagged LAP2alpha fragments confirmed that, unlike the N-terminal fragment, the C-terminal fragment was able to bind to chromosomes during mitosis, if expressed weakly. At higher expression levels, C-terminal LAP2alpha fragment and full-length protein led to cell cycle arrest in interphase and apoptosis, as shown by fluorescence-activated cell sorter analysis, time lapse microscopy, and BrdUrd incorporation assays. These data indicated distinct functions of LAP2alpha in cell cycle progression during interphase and in nuclear reassembly during mitosis.     

A chromatin binding site in the tail domain of nuclear lamins that interacts with core histones

Interaction of chromatin with the nuclear envelope and lamina is thought to help determine higher order chromosome organization in the interphase nucleus. Previous studies have shown that nuclear lamins bind chromatin directly. Here we have localized a chromatin binding site to the carboxyl-terminal tail domains of both A- and B-type mammalian lamins, and have characterized the biochemical properties of this binding in detail. Recombinant glutathione-S-transferase fusion proteins containing the tail domains of mammalian lamins C, B1, and B2 were analyzed for their ability to associate with rat liver chromatin fragments immobilized on microtiter plate wells. We found that all three lamin tails specifically bind to chromatin with apparent KdS of 120-300 nM. By examining a series of deletion mutants, we have mapped the chromatin binding region of the lamin C tail to amino acids 396- 430, a segment immediately adjacent to the rod domain. Furthermore, by analysis of chromatin subfractions, we found that core histones constitute the principal chromatin binding component for the lamin C tail. Through cooperativity, this lamin-histone interaction could be involved in specifying the high avidity attachment of chromatin to the nuclear envelope in vivo.     

Characterization of the Head-to-Tail Overlap Complexes Formed by Human Lamin A, B1 and B2 “Half-minilamin” Dimers

Half-minilamins, representing amino- and carboxy-terminal fragments of human lamins A, B1 and B2 with a truncated central rod domain, were investigated for their ability to form distinct head-to-tail-type dimer complexes. This mode of interaction represents an essential step in the longitudinal assembly reaction exhibited by full-length lamin dimers. As determined by analytical ultracentrifugation, the amino-terminal fragments were soluble under low ionic strength conditions sedimenting with distinct profiles and s-values (1.6-1.8 S) indicating the formation of coiled-coil dimers. The smaller carboxy-terminal fragments were, except for lamin B2, largely insoluble under these conditions. However, after equimolar amounts of homotypic amino- and carboxy-terminal lamin fragments had been mixed in 4 M urea, upon subsequent renaturation the carboxy-terminal fragments were completely rescued from precipitation and distinct soluble complexes with higher s-values (2.3-2.7 S) were obtained. From this behavior, we conclude that the amino- and carboxy-terminal coiled-coil dimers interact to form distinct oligomers (i.e. tetramers). Furthermore, a corresponding interaction occurred also between heterotypic pairs of A- and B-type lamin fragments. Hence, A-type lamin dimers may interact with B-type lamin dimers head-to-tail to yield linear polymers. These findings indicate that a lamin dimer principally has the freedom for a “combinatorial” head-to-tail association with all types of lamins, a property that might be of significant importance for the assembly of the nuclear lamina. Furthermore, we suggest that the head-to-tail interaction of the rod end domains represents a principal step in the assembly of cytoplasmic intermediate filament proteins too.     

Expression of chicken lamin B2 in Escherichia coli: characterization of its structure, assembly, and molecular interactions

Chicken lamin B2, a nuclear member of the intermediate-type filament (IF) protein family, was expressed as a full-length protein in Escherichia coli. After purification, its structure and assembly properties were explored by EM, using both glycerol spraying/low-angle rotary metal shadowing and negative staining for preparation, as well as by analytical ultracentrifugation. At its first level of structural organization, lamin B2 formed "myosin-like" 3.1S dimers consisting of a 52-nm-long tail flanked at one end by two globular heads. These myosin-like molecules are interpreted to represent two lamin polypeptides interacting via their 45-kD central rod domains to form a segmented, parallel and unstaggered 52-nm-long two-stranded alpha-helical coiled-coil, and their COOH-terminal end domains folding into globular heads. At the second level of organization, lamin B2 dimers associated longitudinally to form polar head-to-tail polymers. This longitudinal mode of association of laminin dimers is in striking contrast to the lateral mode of association observed previously for cytoplasmic IF dimers. At the third level of organization, these polar head-to-tail polymers further associated laterally, in an approximately half-staggered fashion, to form filamentous and eventually paracrystal-like structures revealing a pronounced 24.5-nm axial repeat. Finally, following up on recent studies implicating the mitotic cdc2 kinase in the control of lamin polymerization (Peter, M., J. Nakagawa, M. Dorée, J. C. Labbé, and E. A. Nigg. 1990. Cell. 61:591-602), we have examined the effect of phosphorylation by purified cdc2 kinase on the assembly properties and molecular interactions of the bacterially expressed lamin B2. Phosphorylation of chicken lamin B2 by cdc2 kinase interferes with the head-to-tail polymerization of the lamin dimers. This finding supports the notion that cdc2 kinase plays a major, direct role in triggering mitotic disassembly of the nuclear lamina.     

Differential sensitivity of vimentin and nuclear lamins from Ehrlich ascites tumor cells toward Ca2+ -activated neutral thiol proteinase

A comparative study of the susceptibility of vimentin and nuclear lamins from cultured Ehrlich ascites tumor (EAT) cells to degradation by Ca2+ -activated neutral thiol proteinase (calpain) has been undertaken. While pure vimentin was degraded very quickly at physiological ionic strength by purified calpain, isolated lamin B was digested comparatively slowly and purified lamins A/C were fairly resistant to proteolytic degradation. Similar digestion patterns were obtained from vimentin and lamin B with intermediary breakdown products close in size to the corresponding alpha-helical rod domains. To exclude the possibility that the low susceptibility of isolated lamins to Ca2+-dependent proteolytic degradation was due to irreversible denaturation during their isolation and purification, Triton cytoskeletons were prepared and their nuclear lamina as well as vimentin filaments were exposed to relatively large quantities of purified calpain. Under these conditions, not only vimentin filaments but also lamins A and B were digested while lamin C remained intact to a high degree. The major breakdown products of vimentin and lamins were identified as polypeptides which were 35 to 45 amino acids longer than the corresponding alpha-helical rod domains. Most of the vimentin-derived material and all high molecular weight polypeptides originating from lamins remained associated with the Triton cytoskeletons as demonstrated by sodium dodecyl sulfate polyacrylamide gel electrophoresis in conjunction with immunoblotting. Indirect immunofluorescence and electron microscope analysis of the calpain-digested Triton cytoskeletons revealed that they still contained a laminalike structure around the nuclear chromatin and numerous structurally altered intermediate filaments in the cytoplasmic remnant, although all vimentin had been degraded with the formation of 40/41 kDa polypeptides as major digestion products. In untreated Triton cytoskeletons, the vimentin filaments seemed to be in direct physical contact with the nuclear lamina, whereas in digested Triton cytoskeletons there was a distinct gap between structurally altered filaments and the nuclear surface. This shows that vimentin filaments and the nuclear lamina are differentially susceptible to degradation by calpain under certain ionic conditions and suggests that both filamentous structures are intimately associated with each other.(ABSTRACT TRUNCATED AT 400 WORDS)