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.     

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.     

Determinants for intracellular sorting of cytoplasmic and nuclear intermediate filaments

The mechanism by which nuclear and cytoplasmic filaments are sorted in vivo was studied by examining which lamin sequences are required to target an otherwise cytoplasmic IF protein, the small neurofilament subunit (NF-L), to the nuclear lamina. By swapping corresponding domains between NF-L and lamin A, nuclear envelope targeting of NF-L was shown to require the presence of the "head" domain, a 42-amino acid sequence unique to lamin rod domains, a nuclear localization signal and the CAAX motif. Replacement of the entire COOH-terminal tail of lamin A with that of NF-L had no discernible effect on nuclear localization of lamin A, provided the substituted NF-L tail contained a NLS and a CAAX motif. This chimeric protein exhibited characteristics more typical of lamin B than that of the parental lamin A. With regard to cytoplasmic assembly properties, substitution of the head domain of lamin A for that of NF-L did not substantially affect the ability of NF-L to coassemble with vimentin in the cytoplasm. In contrast, insertion of a 42-amino acid sequence unique to lamin rod domains into NF-L profoundly affected NF-L coassembly with vimentin indicating that the 42-amino acid insertion in lamins may be important for sorting lamins from cytoplasmic IF proteins.     

Developmental changes in the organization of the nuclear lamina in mouse liver

We have compared the organization of the nuclear lamina in adult and fetal mouse liver. Western blot analysis of the expression of lamins with specific antibodies indicates that lamin B is expressed throughout liver development, unlike lamins A and C which are absent in fetal liver. Using [125I]lamin in blot binding assays, we have observed that lamin B binds to at least three membrane proteins (96, 54 and 34 kDa) and to lamins A and C in adult nuclear envelopes, but only to the 54 and 34 kDa proteins and lamin B itself in fetal nuclear envelopes, where lamin B appears to be hyperphosphorylated.     

The alpha-helical rod domain of human lamins A and C contains a chromatin binding site.

We examined regions of human lamins A and C involved in binding to surfaces of mitotic chromosomes. An Escherichia coli expression system was used to produce full-length lamin A and lamin C, and truncated lamins retaining the central alpha-helical rod domain (residues 34-388) but lacking various amounts of the amino-terminal 'head' and carboxy-terminal 'tail' domains. We found that lamin A, lamin C and lamin fragments lacking the head domain and tail sequences distal to residue 431 efficiently assembled into paracrystals and strongly associated with mitotic chromosomes. Furthermore, the lamin rod domain also associated with chromosomes, although efficient chromosome coating required the pH 5-6 conditions needed to assemble the rod into higher order structures. Biochemical assays showed that chromosomes substantially reduced the critical concentration for assembly of lamin polypeptides into pelletable structures. Association of the lamin rod with chromosomes was abolished by pretrypsinization of chromosomes, and was not seen for vimentin (which possesses a similar rod domain). These data demonstrate that the alpha-helical rod of lamins A and C contains a specific chromosome binding site. Hence, the central rod domain of intermediate filament proteins can be involved in interactions with other cellular structures as well as in filament assembly.     

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.     

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.     

Nuclear envelope proteins: Identification of lamin B subtypes

The lamins are a group of nuclear envelope proteins thought to form a structural layer at the nuclear periphery. Lamins A, B and C occur in many cell types although lamin C, a subtype of lamin A, is relatively decreased in chicken cells. A subtype of lamin B has been found in chicken cells. This subtype, called lamin B1, is slightly larger and more acidic than the quantitatively major subtype now called lamin B2. The lamin B subtypes have very similar primary sequences and share a distinctive topology. Two lamin B subtypes have not been observed in mammalian tissues but have been found in three avian tissues, erythrocytes of mature chickens and liver and erythrocytes of 11- to 13-day-old embryos. As these tissues differ widely in metabolic activity, both subtypes appear to be constitutive nuclear proteins.     

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.     

A- and B-type lamins are differentially expressed in normal human tissues

 A selection of normal human tissues was investigated for the presence of lamins B1, B2, and A-type lamins, using a panel of antibodies specific for the individual lamin subtypes. By use of immunoprecipitation and two-dimensional immunoblotting techniques we demonstrated that these antibodies do not cross-react with other lamin subtypes and that a range of different phosphorylation isoforms is recognized by each antibody. The lamin B2 antibodies appeared to decorate the nuclear lamina in all tissues examined, except hepatocytes, in which very little lamin B2 expression was observed. In contrast to previous studies, which suggested the ubiquitous expression of lamin B1 in mammalian tissues, we show that lamin B1 is not as universally distributed throughout normal human tissues as was to be expected from previous studies. Muscle and connective tissues are negative, while in epithelial cells lamin B1 seemed to be preferentially detected in proliferating cells. These results correspond well with those obtained for lamin B1 in chicken tissues. The expression of A-type lamins is most prominent in well-differentiated epithelial cells. Relatively undifferentiated and proliferating cells in epithelia showed a clearly reduced expression of A-type lamins. Furthermore, most cells of neuroendocrine origin as well as most hematopoietic cells were negative for A-type lamin antibodies. Accepted: 4 February 1997     

Structural organization of nuclear lamins A,C, B1, and B2 revealed by superresolution microscopy

The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural organization of the major proteins composing the lamina is poorly defined. Using three-dimensional structured illumination microscopy and computational image analysis, we characterized the supramolecular structures of lamin A, C, B1, and B2 in mouse embryo fibroblast nuclei. Each isoform forms a distinct fiber meshwork, with comparable physical characteristics with respect to mesh edge length, mesh face area and shape, and edge connectivity to form faces. Some differences were found in face areas among isoforms due to variation in the edge lengths and number of edges per face, suggesting that each meshwork has somewhat unique assembly characteristics. In fibroblasts null for the expression of either lamins A/C or lamin B1, the remaining lamin meshworks are altered compared with the lamin meshworks in wild-type nuclei or nuclei lacking lamin B2. Nuclei lacking LA/C exhibit slightly enlarged meshwork faces and some shape changes, whereas LB1-deficient nuclei exhibit primarily a substantial increase in face area. These studies demonstrate that individual lamin isoforms assemble into complex networks within the nuclear lamina and that A- and B-type lamins have distinct roles in maintaining the organization 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)     

The nucleoporin Nup88 is interacting with nuclear lamin A

Nuclear pore complexes (NPCs) are embedded in the nuclear envelope (NE) and mediate bidirectional nucleocytoplasmic transport. Their spatial distribution in the NE is organized by the nuclear lamina, a meshwork of nuclear intermediate filament proteins. Major constituents of the nuclear lamina are A- and B-type lamins. In this work we show that the nuclear pore protein Nup88 binds lamin A in vitro and in vivo. The interaction is mediated by the N-terminus of Nup88, and Nup88 specifically binds the tail domain of lamin A but not of lamins B1 and B2. Expression of green fluorescent protein-tagged lamin A in cells causes a masking of binding sites for Nup88 antibodies in immunofluorescence assays, supporting the interaction of lamin A with Nup88 in a cellular context. The epitope masking disappears in cells expressing mutants of lamin A that are associated with laminopathic diseases. Consistently, an interaction of Nup88 with these mutants is disrupted in vitro. Immunoelectron microscopy using Xenopus laevis oocyte nuclei further revealed that Nup88 localizes to the cytoplasmic and nuclear face of the NPC. Together our data suggest that a pool of Nup88 on the nuclear side of the NPC provides a novel, unexpected binding site for nuclear lamin A.     

The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells

Recent evidence shows that the COOH-terminal CaaX motif of lamins is necessary to target newly synthesized proteins to the nuclear envelope membranes. Isoprenylation at the CaaX-cysteine has been taken to explain the different fates of A- and B-type lamins during cell division. A-type lamins, which loose their isoprenylation shortly after incorporation into the lamina structure, become freely soluble upon mitotic nuclear envelope breakdown. Somatic B-type lamins, in contrast, are permanently isoprenylated and, although depolymerized during mitosis, remain associated with remnants of nuclear envelope membranes. However, Xenopus lamin B3, the major B-type lamin of amphibian oocytes and eggs, becomes soluble after nuclear envelope breakdown in meiotic metaphase. Here we show that Xenopus lamin B3 is permanently isoprenylated and carboxyl methylated in oocytes (interphase) and eggs (meiotic metaphase). When transfected into mouse L cells Xenopus lamin B3 is integrated into the host lamina and responds to cell cycle signals in a normal fashion. Notably, the ectopically expressed Xenopus lamin does not form heterooligomers with the endogenous lamins as revealed by a coprecipitation experiment with mitotic lamins. In contrast to the situation in amphibian eggs, a significant portion of lamin B3 remains associated with membranes during mitosis. We conclude from these data that the CaaX motif-mediated modifications, although necessary, are not sufficient for a stable association of lamins with membranes and that additional factors are involved in lamin-membrane binding.     

Differential transport and integration into the nuclear lamina for lamins A, B, and C

Lamins A, B, and C are the major proteins of the mammalian nuclear lamina and have been well studied in BHK-21 cells. Using in vivo labelling, cell fractionation, and immunoprecipitation, we have found that lamins have different patterns of nuclear transport and solubility. Newly synthesized lamin A is translocated to the nucleus faster than lamin C or B. It is the most tightly bound lamin and cannot be extracted from the lamina by nonionic detergent or high-salt buffers. Lamins B and C migrate more slowly to the nucleus. Partitioning between cytoskeleton and detergent-soluble fractions shows that integration of lamins B and C is not completed before a 1-h chase. For lamin C this process is dependent upon protein synthesis and can be inhibited with cycloheximide. Even though lamins A and C are almost identical, lamin C is never firmly bound to the lamina and can be partially solubilized upon high-salt treatment.     

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)     

Nuclear lamins A and B1: different pathways of assembly during nuclear envelope formation in living cells

At the end of mitosis, the nuclear lamins assemble to form the nuclear lamina during nuclear envelope formation in daughter cells. We have fused A- and B-type nuclear lamins to the green fluorescent protein to study this process in living cells. The results reveal that the A- and B-type lamins exhibit different pathways of assembly. In the early stages of mitosis, both lamins are distributed throughout the cytoplasm in a diffusible (nonpolymerized) state, as demonstrated by fluorescence recovery after photobleaching (FRAP). During the anaphase-telophase transition, lamin B1 begins to become concentrated at the surface of the chromosomes. As the chromosomes reach the spindle poles, virtually all of the detectable lamin B1 has accumulated at their surfaces. Subsequently, this lamin rapidly encloses the entire perimeter of the region containing decondensing chromosomes in each daughter cell. By this time, lamin B1 has assembled into a relatively stable polymer, as indicated by FRAP analyses and insolubility in detergent/high ionic strength solutions. In contrast, the association of lamin A with the nucleus begins only after the major components of the nuclear envelope including pore complexes are assembled in daughter cells. Initially, lamin A is found in an unpolymerized state throughout the nucleoplasm of daughter cell nuclei in early G1 and only gradually becomes incorporated into the peripheral lamina during the first few hours of this stage of the cell cycle. In later stages of G1, FRAP analyses suggest that both green fluorescent protein lamins A and B1 form higher order polymers throughout interphase nuclei.     

Lamins A and C are not expressed at early stages of human lymphocyte differentiation

Lamins are major proteins of the nuclear envelope that are members of the intermediate filament protein family. In vertebrates, nuclei from differentiated tissues usually contain both lamins of the A and B subtypes, while embryonic tissues contain the B-type lamin only. We have examined the composition of the nuclear lamina in human B and T lymphocytes representative of distinct stages of lymphoid differentiation. We show here that, in both cell lineages, while lamin B is constitutively expressed at all stages of differentiation, A-type lamin expression is restricted to later developmental stages.