Regulated docking of nuclear membrane vesicles to vimentin filaments during mitosis

During mitosis, several types of intermediate-sized filaments (IFs) undergo an extensive remodelling in response to phosphorylation by cdc 2 and other protein kinases. However, unlike the nuclear lamins, the cytoplasmic IFs do not seem to follow a fixed disassembly stereotype and often retain their physical continuity without depolymerizing into soluble subunits. To investigate potential interactions between mitotically modified IFs and other cellular structures, we have examined prometaphase-arrested cells expressing the IF protein vimentin. We demonstrate here that vimentin filaments associate in situ and co-fractionate with a distinct population of mitotic vesicles. These vesicles carry on their surfaces nuclear lamin B, the inner nuclear membrane protein p58, and wheat germ agglutinin (WGA)-binding proteins. Consistent with a tight interaction between the IFs and the mitotic membranes, vimentin, nuclear lamin B, and a 180-kD WGA-binding protein are co-isolated when whole mitotic homogenates are incubated with anti-vimentin or anti-lamin B antibodies immobilized on magnetic beads. The vimentin-associated vesicles are essentially depleted of ER, Golgi and endosomal membrane proteins. The interaction of vimentin with lamin B-carrying membranes depends on phosphorylation and is weakened by dephosphorylation during nuclear reassembly in vitro. These observations reveal a novel interaction between IFs and cellular membranes and further suggest that the vimentin filaments may serve as a transient docking site for inner nuclear membrane vesicles during mitosis.     

Spatial distribution of lamin A and B1 in the K562 cell nuclear matrix stabilized with metal ions.

When the nucleus is stripped of most DNA, RNA, and soluble proteins, a structure remains that has been referred to as the nuclear matrix, which acts as a framework to determine the higher order of chromatin organization. However, there is always uncertainty as to whether or not the nuclear matrix, isolated in vitro, could really represent a skeleton of the nucleus in vivo. In fact, the only nuclear framework of which the existence is universally accepted is the nuclear lamina, a continuous thin layer that underlies the inner nuclear membrane and is mainly composed of three related proteins: lamins A, B, and C. Nevertheless, a number of recent investigations performed on different cell types have suggested that nuclear lamins are also present within the nucleoplasm and could be important constituents of the nuclear matrix. In most cell types investigated, the nuclear matrix does not spontaneously resist the extraction steps, but must rather be stabilized before the application of extracting agents. In this investigation, by immunochemical and morphological analysis, we studied the effect of stabilization with different divalent cations (Zn(2+), Cu(2+), Cd(2+)) on the distribution of lamin A and B1 in the nuclear matrix obtained from K562 human erythroleukemia cells. In intact cells, antibodies to both lamin A and B1 mainly stained the nuclear periphery, although some immunoreactivity was detected in the nuclear interior. The fluorescent lamin A pattern detected in Cu(2+)- and Cd(2+)-stabilized nuclei was markedly modified, whereas Zn(2+)-incubated nuclei showed an unaltered pattern of lamin A distribution. By contrast, the distribution of lamin B1 in isolated nuclei was not modified by the stabilizing cations. When chromatin was removed by nuclease digestion and extraction with solutions of high ionic strength, a previously masked immunoreactivity for lamin A, but not for lamin B1, became evident in the internal part of the residual structures representing the nuclear matrix. Our results indicate that when metal ions are used as stabilizing agents for the recovery of the nuclear matrix, the distribution of both lamin A and lamin B1 in the final structures, corresponds to the pattern we have very recently reported using different extraction procedures. This observation strengthen the concept that intranuclear lamins may act as structural components of the nuclear matrix.     

Cutting edge: integration of human T lymphocyte cytoskeleton by the cytolinker plectin.

Chemokine-induced polarization of lymphocytes involves the rapid collapse of vimentin intermediate filaments (IFs) into an aggregate within the uropod. Little is known about the interactions of lymphocyte vimentin with other cytoskeletal elements. We demonstrate that human peripheral blood T lymphocytes express plectin, an IF-binding, cytoskeletal cross-linking protein. Plectin associates with a complex of structural proteins including vimentin, actin, fodrin, moesin, and lamin B in resting peripheral blood T lymphocytes. During chemokine-induced polarization, plectin redistributes to the uropod associated with vimentin and fodrin; their spatial distribution indicates that this vimentin-plectin-fodrin complex provides a continuous linkage from the nucleus (lamin B) to the cortical cytoskeleton. Overexpression of the plectin IF-binding domain in the T cell line Jurkat induces the perinuclear aggregation of vimentin IFs. Plectin is therefore likely to serve as an important organizer of the lymphocyte cytoskeleton and may regulate changes of lymphocyte cytoarchitecture during polarization and extravasation.     

Defects in lamin B1 expression or processing affect interphase chromosome position and gene expression

Radial organization of nuclei with peripheral gene-poor chromosomes and central gene-rich chromosomes is common and could depend on the nuclear boundary as a scaffold or position marker. To test this, we studied the role of the ubiquitous nuclear envelope (NE) component lamin B1 in NE stability, chromosome territory position, and gene expression. The stability of the lamin B1 lamina is dependent on lamin endoproteolysis (by Rce1) but not carboxymethylation (by Icmt), whereas lamin C lamina stability is not affected by the loss of full-length lamin B1 or its processing. Comparison of wild-type murine fibroblasts with fibroblasts lacking full-length lamin B1, or defective in CAAX processing, identified genes that depend on a stable processed lamin B1 lamina for normal expression. We also demonstrate that the position of mouse chromosome 18 but not 19 is dependent on such a stable nuclear lamina. The results implicate processed lamin B1 in the control of gene expression as well as chromosome position.     

Detection of dense intra- and perinuclear 10 nm filament systems by whole mount and embedment-free electron microscopy in several species of the green algal order Dasycladales

Summary In contrast to the immense body of evidence supporting the occurrence of intermediate filament (IF) proteins in the animal kingdom, there is only limited information on their distribution in plants. Nevertheless, a number of immunocytochemical and electron microscopical observations indicate that particularly in higher plant cells IFs contribute to the construction of the cyto- and karyoskeleton. Here we show by whole mount electron microscopy of the giant nuclei extruded together with adhering cytoplasm from the rhizoids of some species of the algal order Dasycladales that cytoplasmic 10 nm filament networks also occur in unicellular, mononucleated green organisms of early evolutionary origin. The filament systems were associated with the residual nuclear envelope which consisted of a dense arrangement of pore complexes suspended by a meshwork of short 5 to 6 nm filaments; structurally it was very similar to the nuclear envelopes obtained from mammalian cells. When the Dasycladales nuclei were processed side by side with mouse skin fibroblasts, the algal filament systems were physically almost indistinguishable from the mammalian vimentin filament network. Embedment-free thin sections of rhizoids have not only confirmed the existence of the perinculear 10 nm filaments and their seamless association with the nuclear envelope, but have demonstrated the existence of an extensive intranuclear meshwork of 10 nm filaments. The latter were morphologically indistinguishable from the perinuclear 10 nm filaments and seem to be connected to these via the nuclear envelope to form a continuum. Among a variety of antibodies directed against mammalian IF proteins, only polyclonal anti-mouse lamin B antibodies decorated the cytoplasmic filaments of the Dasycladales cells. Surprisingly, none of the antibodies decorated the thinner filaments of the nuclear envelope, which possibly represent the nuclear lamina. In accord with this observation, one anti-lamin B antibody recognized in Western blot analysis of a urea extract ofAcetabularia acetabulum rhizoids three polypeptides with M_rs of approximately 47,000, 64,000, and 76,000. The proteins did not react with the -IFA antibody. Since the Dasycladales have a fossil record of nearly 600 million years — an extant genus, Acicularia, also investigated here, evolved about 170 million years ago -, the molecular characterization of the subunit proteins of their cytoplasmic filament systems might throw further light on the evolution and biological role of IFs.Dedicated to Professor Sir Henry Harris on the occasion of his 70th birthday     

TPA induces apoptosis in MPC-11 mouse plasmacytoma cells grown in serum-free medium

Apoptotic-like events could be rapidly induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) in cells of the mouse plasmacytoma cell line MPC-11 grown in serum-free medium. Indicators for apoptosis were morphological changes visualized by light and electron microscopy, such as chromatin condensation and the formation of cellular buds and fragments, as well as biochemical indices like the appearance of the so-called ‘DNA ladder’. Additionally, in these cells which are usually devoid of significant amounts of cytoplasmic intermediate filament (cIF) proteins, synthesis and accumulation of the cIF protein vimentin was rapidly induced by TPA treatment and almost all cells became vimentin-positive. Later on, substantial amounts of vimentin and lamin B degradation products appeared, and an increasing fraction of cells displayed low or even undetectable quantities of intact vimentin. This subpopulation was characterized via microscopy to be in the late stages of apoptosis. We suggest that in MPC-11 cells undergoing apoptosis in response to TPA treatment vimentin as well as lamin B are degraded, leading to a rearrangement and eventual loss of their respective filament networks.     

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.     

Lamin B constitutes an intermediate filament attachment site at the nuclear envelope

We found that urea extraction of turkey erythrocyte nuclear envelopes abolished their ability to bind exogenous 125I-vimentin, while, at the same time, it removed the nuclear lamins from the membranes. After purification of the lamins from such urea extracts, a specific binding between isolated vimentin and lamin B, or a lamin A + B hetero- oligomer, was detected by affinity chromatography. Similar analysis revealed that the 6.6-kD vimentin tail piece was involved in this interaction. By other approaches (quantitative immunoprecipitation, rate zonal sedimentation, turbidometric assays) a substoichiometric lamin B-vimentin binding was determined under in vitro conditions. It was also observed that anti-lamin B antibodies but not other sera (anti- lamin A, anti-ankyrin, preimmune) were able to block 70% of the binding of 125I-vimentin to native, vimentin-depleted, nuclear envelopes. These data, which were confirmed by using rat liver nuclear lamins, indicate that intermediate filaments may be anchored directly to the nuclear lamina, providing a continuous network connecting the plasma membrane skeleton with the karyoskeleton of eukaryotic cells.     

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.     

Mechanics in human fibroblasts and progeria: Lamin A mutation E145K results in stiffening of nuclei

The lamina is a filamentous meshwork beneath the inner nuclear membrane that confers mechanical stability to nuclei. The E145K mutation in lamin A causes Hutchinson‐Gilford progeria syndrome (HGPS). It affects lamin filament assembly and induces profound changes in the nuclear architecture. Expression of wild‐type and E145K lamin A in Xenopus oocytes followed by atomic force microscopy (AFM) probing of isolated oocyte nuclei has shown significant changes in the mechanical properties of the lamina. Nuclei of oocytes expressing E145K lamin A are stiffer than those expressing wild‐type lamin A. Here we present mechanical measurements by AFM on dermal fibroblasts obtained from a 4‐year‐old progeria patient bearing the E145K lamin A mutation and compared it to fibroblasts obtained from 2 healthy donors of 10 and 61 years of age, respectively. The abnormal shape of nuclei expressing E145K lamin A was analyzed by fluorescence microscopy. Lamina thickness was measured using electron micrographs. Fluorescence microscopy showed alterations in the actin network of progeria cells. AFM probing of whole dermal fibroblasts did not demonstrate significant differences in the elastic moduli of nuclear and cytoplasmic cell regions. In contrast, AFM measurements of isolated nuclei showed that nuclei of progeria and old person's cells are significantly stiffer than those of the young person, indicating that the process of aging, be it natural or abnormal, increases nuclear stiffness. Our results corroborate AFM data obtained using Xenopus oocyte nuclei and prove that the presence of E145K lamin A abnormally increases nuclear stiffness.     

Binding of matrix attachment regions to lamin B1.

Eukaryotic chromatin is organized into topologically constrained loops that are attached to the nuclear matrix. The regions of DNA that interact with the matrix are called matrix attachment regions (MARs). We studied the spatial distribution of MAR-binding sites in the nuclear matrix from rat liver cells, following a combined biochemical and ultrastructural approach. We found that MAR-binding sites are distributed equally over the internal fibrogranular network and the peripheral nuclear lamina. Internal and peripheral binding sites have similar binding characteristics: both sets of binding sites show specific and saturable binding of MARs from different organisms. By means of a DNA-binding protein blot assay and in vitro binding studies, we identified lamin B1 as a MAR-binding protein, which provides evidence for a specific interaction of DNA with the nuclear lamina.     

Ultrastructural analysis of cytoplasmic intermediate filaments and the nuclear lamina in the mouse plasmacytoma cell line MPC-11 after the induction of vimentin synthesis

We examined cytoplasmic intermediate filaments (IFs) and the nuclear lamina in cells of the mouse plasmacytoma cell line MPC-11 (lacking both IF proteins and lamins A and C) after induction of vimentin synthesis with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) by means of whole-mount immunogold electron microscopy (IEM). The technique of IEM was modified to allow analysis of the cytoskeleton and nuclear lamina of cells grown in suspension culture employing antibodies against vimentin and lamin B. IEM showed that newly synthesized vimentin assembled into IFs which formed anastomosing networks throughout the cytoplasm, radiating primarily from the nucleus. The filaments decorated by gold-conjugated antibodies appeared to make contact with the lipid-depleted nuclear envelope residue either by directly terminating on it or through an indirect link via short fibers of varying diameter. Some filaments terminated on the subunits of the nuclear pore complexes but they did not pass through the pores. In the absence of lamins A and C, lamin B formed a nuclear lamina consisting of a globular-filamentous network anchoring the nuclear pore complexes.     

Isolation and characterization of nuclear lamina from Ehrlich ascites tumor cells

We have developed a simple and rapid method for isolation of purified nuclear lamina from Ehrlich ascites tumor cells. The procedure employs chromatin structures prepared from whole cells at low ionic strength and is carried out under conditions that minimize the formation of artifactual protein-DNA complexes. When the isolation is performed in the presence of EDTA, nuclear lamina without distinct pore complexes is obtained. In the absence of EDTA, intact pore complexes and a large amount of vimentin 100 A filaments are seen associated with nuclear lamina. The main nuclear lamina proteins are characterized using gel electrophoresis, immunoblotting, and two-dimensional peptide mapping. An extensive structural homology is found between lamin A and lamin C, whose peptide maps differ by only one major spot, whereas lamin B has apparently unrelated pattern.     

Increased solubility of lamins and redistribution of lamin C in X-linked Emery-Dreifuss muscular dystrophy fibroblasts

Emery-Dreifuss muscular dystrophy (EDMD) is caused by mutations in the gene encoding the nuclear membrane protein emerin (X-linked EDMD) or in the gene encoding lamins A/C (autosomal dominant EDMD). One hypothesis explaining the disease suggests that the mutations lead to weakness of the nuclear lamina. To test this hypothesis we investigated lamin solubility and distribution in skin fibroblasts from X-EDMD patients. Using in situ extraction of cells and immunofluorescence microscopy or biochemical fractionation and immunoblotting, we found that all lamin subtypes displayed increased solubility properties in fibroblasts from X-EDMD patients compared to normal individuals. Lamin and emerin solubility was mildly increased in fibroblasts from an X-EDMD carrier. Biochemical fractionation and immunoblotting also indicated that lamin C but no other lamin became redistributed from the nuclear lamina to the nucleoplasm in X-EDMD fibroblasts. Indirect immunofluorescence and confocal microscopy studies using lamin A- and lamin C-specific antibodies confirmed that lamin C but not lamin A became redistributed to the nucleoplasm. Interestingly, the lamin A/C binding protein LAP2alpha was also mislocalized in X-EDMD fibroblasts.     

Age-related changes in lamin A/C expression in cardiomyocytes

Lamin A and C (A/C) are type V intermediate filaments that form the nuclear lamina. Lamin A/C mutations lead to reduced expression of lamin A/C and diverse phenotypes such as familial cardiomyopathies and accelerated aging syndromes. Normal aging is associated with reduced expression of lamin A/C in osteoblasts and dermal fibroblasts but has never been assessed in cardiomyocytes. Our objective was to compare the expression of lamin A/C in cardiomyocytes of old (24 mo) versus young (4 mo) C57Bl/6J mice using a well-validated mouse model of aging. Lamin B1 was used as a control. Immunohistochemical and immunofluorescence analyses showed reduced expression of lamin A/C in cardiomyocyte nuclei of old mice (proportion of nuclei expressing lamin A/C, 9% vs. 62%, P < 0.001). Lamin A/C distribution was scattered peripherally and perinuclear in old mice, whereas it was homogeneous throughout the nuclei in young mice. Western blot analyses confirmed reduced expression of lamin A/C in nuclear extracts of old mice (ratio of lamin A/C to B1, 0.6 vs. 1.2, P < 0.01). Echocardiographic studies showed increased left ventricular wall thickness with preserved cavity size (concentric remodeling), increased left ventricular mass, and a slight reduction in fractional shortening in old mice. This is the first study to show that normal aging is associated with reduced expression and altered distribution of lamin A/C in nuclei of cardiomyocytes.     

The differential expression of lamin epitopes during mouse spermatogenesis

The presence of lamin proteins in mouse spermatogenic cells has been examined by using an anti-lamin AC and an anti-lamin B antisera which recognize somatic lamins A and C, and somatic lamin B, respectively. Anti-lamin B binds to the nuclear periphery of all cell types examined, including Sertoli cells, primitive type A spermatogonia, preleptotene, leptotene, zygotene and pachytene spermatocytes, and round spermatids. In sperm nuclei, the antigenic determinants are localized to a narrow domain of the nucleus. However, after removing the perinuclear theca, anti-lamin B localizes to the entire nuclear periphery in a punctate pattern, suggesting that it is binding to determinants previously covered by the theca constituents. On immunoblots anti-lamin B reacts with a ～ 68 kD polypeptide in all germ cells and, to a lesser extent, with four additional polypeptides present only in meiotic and post-meiotic nuclear matrices. Anti-lamin AC also reacts with the perinuclear region of the somatic cells in the testes, in particular, those of the interstitium and also the Sertoli cells of the seminiferous epithelium. In contrast to anti-lamin B, anti-lamin AC does not bind to the germ cells at any stage of spermatogenesis. In addition, nuclear matrix proteins from isolated spermatogenic cells do not bind anti-lamin AC on immunoblots, suggesting the lack of reactivity is not due to the masking of any antigenic sites. These data demonstrate that germ cells contain lamin B throughout spermatogenesis, even during meiosis and spermiogenesis when the nuclear periphery lacks a distinct fibrous lamina. © 1993 Wiley-Liss, Inc.     

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.     

The intermediate filament-lamina-nuclear matrix system of BHK-21 cells

We have employed collodial gold immuno-labelling in whole-mount cell and 2-D gel electrophoresis to demonstrate the intermediate filament (IF)-lamina-nuclear matrix (NM) system in BHK-21 (Baby Hamster Kidney) cells. Grown on grids, cells were gently extracted with salt solutions as previously described by S. Penman to preserve intact IF-lamina-NM systems. The extracted samples were fixed, postfixed, dehydrated and dried through the CO2 critical point, then examined under high voltage electron microscope (HVEM). The results revealed that the IF-lamina-NM system is a interconnecting network throughout the cell from cytoplasma to nuclear. The IF unit is 10 nm in diameter. IFs radiate away from the nuclear region into the spreading cytoplasm and the polarity of their distributing is obvious. The IF system closely connected to lamina. Immuno-gold labelling and 2-D gel proved that vimentin, a 55 KD protein (pI 5,6), is the major component of IFs in BHK-21 cells. Lamina can be precisely and specifically labelled with anti-lamin A, C proteins and as well as 2-D gel electrophoresis indicated that there are lamin A, B, C proteins in BHK-21 cells, whose molecular weights are 68 KD, 70 KD, 62 KD respectively. Its components are more complicated, but a few dots of NM proteins can be clearly distinguished in 2-D gel map, in which actin, a 45 KD protein (pI 4.5), might be involved. The nuclear matrix network was also clearly presented under HVEM. Its filaments can be labelled with anti-NM 298 KD protein precisely.     

Presence of a novel nuclear lamina protein in Raji cells.

In mammalian tissues, the nuclear lamina is composed of the major lamins A, B, and C, and minor lamins D/E. Although lamin B is present in all cell types, lamins A and C are absent from embryonic cells and most undifferentiated cells from hematopoietic lineage. We have investigated the nuclear lamina protein composition of the Raji cell line, lymphoblast-like cells established from a Burkitt lymphoma patient. Lamins A and C were confirmed absent by immunodetection and Northern blot analysis. Besides lamins B and D/E, a protein migrating around 71 kilodaltons was recognized by a serum directed against the nuclear lamina of BHK-21 fibroblasts. Cellular localization by sequential extraction established this 71-kilodalton protein as an exclusive component of the nuclear lamina fraction. These results indicate that the nuclear lamina has a more complex composition than previously thought to be the case for cells devoid of lamins A and C.