MAN1, an inner nuclear membrane protein that shares the LEM domain with lamina-associated polypeptide 2 and emerin.

The "MAN antigens" are polypeptides recognized by autoantibodies from a patient with a collagen vascular disease and localized to the nuclear envelope. We now show that one of the human MAN antigens termed MAN1 is a 82.3-kDa protein with an amino-terminal domain followed by two hydrophobic segments and a carboxyl-terminal tail. The MAN1 gene contains seven protein-coding exons and is assigned to human chromosome 12q14. Its mRNA is approximately 5.5 kilobases and is detected in several different cell types that were examined. Cell extraction experiments show that MAN1 is an integral membrane protein. When expressed in transfected cells, MAN1 is exclusively targeted to the nuclear envelope, consistent with an inner nuclear membrane localization. Protein sequence analysis reveals that MAN1 shares a conserved globular domain of approximately 40 amino acids, which we term the LEM module, with inner nuclear membrane proteins lamina-associated polypeptide 2 and emerin. The LEM module is also present in two proteins of Caenorhabditis elegans. These results show that MAN1 is an integral protein of the inner nuclear membrane that shares the LEM module with other proteins of this subcellular localization.

Characterization of the 2A7 antigen as a 85-kDa human nucleocytoplasmic shuttling protein.

The murine monoclonal antibody 2A7 was found to react specifically with a 85-kDa human protein which is distributed throughout the nuclear interior in interphase and becomes associated with condensed chromosomes during mitosis. The 2A7 epitope was not detected in cells from other species. Two-dimensional immunoblotting analysis of HeLa cell homogenates further indicated that the 85-kDa polypeptide species recognized by the 2A7 antibody corresponds to an acidic protein which may be complexed in vivo within high-molecular-weight protein structures. Immunofluorescence monitoring of the 2A7 staining pattern during in situ preparation of nuclear matrices from HeLa cells demonstrated that the nucleoplasmic fraction of the antigen is readily solubilized by detergent and salts, whereas the nucleolar fraction resists detergent/salt extraction and DNase digestion, to be released only upon RNase activity. Mobility assays in human-mouse heterokaryons provided evidence that the 2A7 antigen is a nucleocytoplasmic shuttling protein. The nuclear distribution of this antigen remained unchanged upon drug-induced inhibition of RNA synthesis but was markedly altered by heat shock stress. All together, the data presented here suggest that the 2A7 antigen may have a function in RNA metabolism.

Molecular characterization and developmentally regulated expression of Xenopus lamina-associated polypeptide 2 (XLAP2).

Lamina-associated polypeptides 2 (LAP2alpha, beta, gamma)/thymopoietins (TPalpha, beta, gamma) are a family of proteins that are generated by alternative splicing from a single gene. These proteins have been primarily characterized in mammals. One member of this protein family, the integral membrane protein LAP2beta/TPbeta, has been localized to the inner nuclear membrane of somatic cells where it binds to chromatin and B-type lamins. By cDNA cloning we have characterized XLAP2, a Xenopus homologue of the mammalian LAP2beta. Using LAP2-specific antibodies, the Mr 68,000 XLAP2 was found to be the only member of the LAP2/TP family expressed in somatic cells and adult tissues. XLAP2 was not detected in oocytes, eggs and in early embryos up to the gastrula stage at the mRNA and protein level demonstrating that it is not synthesized from maternal mRNA. In counterpart oocytes, eggs, and embryos contained one LAP2-related integral membrane proteins of Mr 84,000. Northern blot analysis with the XLAP2 cDNA showed that a single hybridizing mRNA band of 1.8-2.0 kb was present in Xenopus somatic cells whereas two other hybridizing mRNA species of 2.8-3.0 and 0. 9-1.1 kb were present in oocytes, eggs and early embryos. All together, these results indicated that at least three distinct LAP2-related proteins might be expressed in Xenopus. The LAP2/TP protein of Mr 84,000 is present in the early embryos but its amount decreases during embryogenesis concomitant with the increase of XLAP2 in the embryo. Our results are the first description of the developmentally regulated expression of integral nuclear envelope proteins during early embryogenesis.

The 2G2 antibody recognizes an acidic 110-kDa human mitochondrial protein.

Using fluorescence microscopy, the mouse monoclonal antibody 2G2 was found to label mitochondria in human cells, as assessed by double staining with either Rhodamine 123 or a polyclonal antibody to mitochondrial matrix HSP-60 proteins. No reactivity to the 2G2 antibody was detected in cells from mouse, rat and chicken. Immunoblotting analysis demonstrated that the 2G2 antigen corresponds to a human protein with a relative mobility of 110kDa and an approximate isoelectric point of 6.5 that co-partitions with HSP-60 proteins during isolation of mitochondria from HeLa cells. Close examination of the 2G2 staining pattern in HeLa and Fanconi's anaemia cells revealed differences in the morphology and organization of mitochondria in these two cell types. In HeLa cells, mitochondria appear as individual tubular compartments of variable length and are closely associated with vimentin filaments, particularly at the periphery of the nucleus. In Fanconi's anaemia cells, mitochondria have a filamentous shape and form an interconnected cytoplasmic reticulum running in parallel with both vimentin filaments and microtubules. After stabilization with aldehyde- or alcohol-based fixation protocols that optimize the preservation of cytoskeletal components, the epitope targeted by the 2G2 antibody may serve as a valuable marker in the investigation of relationships between mitochondria and other cellular structures in human cells.

The MAN antigens are non-lamin constituents of the nuclear lamina in vertebrate cells.

The characterization of the human antiserum designated MAN has led to the identification of a subset of non-lamin proteins that are exclusively located at the nuclear periphery in all vertebrate cell types examined, from human to fish. Immunoreactive protein species were shown to comprise three major polypeptides of Mr 78000, 58000 and 40000. These antigens co-partitioned with the nuclear lamina during in situ isolation of nuclear matrices from lamin A/C-positive and -negative mammalian cells. Using double immunofluorescence, the spatial relationship of MAN antigens to type-A and type-B lamins was further examined throughout the cell cycle of lamin A/C-positive mammalian cells. In interphase HeLa and 3T3 cells, MAN antigens colocalized with both types of lamins at the periphery of the nucleus, but were absent from intranuclear foci of lamin B. As HeLa cells proceeded into mitosis, MAN antigens were seen to segregate from lamins A/C and coredistribute with lamin B. Lamins A/C disassembled during late prophase/early prometaphase and reassociated with chromatin in telophase/cytokinesis. In contrast, MAN antigens and lamin B dispersed late during prometaphase and reassembled on chromosomes in anaphase. Altogether, our data suggest that MAN antigens may play key functions in the maintenance of the structural integrity of the nuclear compartment in vertebrate cells.

Characterization of the 2H12 antigen as a nonshuttling human isoelectric variant of the nucleolar protein B23.

It has become obvious that a better understanding of the nucleolar compartment should encompass the elucidation of structural and functional relationships between its molecular constituents. Using a mouse monoclonal antibody referred to as 2H12, we have identified a human epitope that appears to be implicated in the regulatory events governing the elaboration and stabilization of the nucleolar architecture. By immunofluorescence and immunoblotting, the 2H12 monoclonal was shown to be directed against a nucleolar protein with a relative mobility of 38-40 kDa and an isoelectric point of 5.1 that is present in human cells, regardless of their proliferation state. No reactivity was detected in cells from other species, implying that the targeted epitope could be unique to humans. Investigation of the fate of the epitope throughout the cell cycle led to evidence that its immunoreactivity was phosphodependent and suggested that the disassembly and reassembly of the nucleolar apparatus during cell division is accompanied by dephosphorylation/phosphorylation modifications at this site. In a series of double immunofluorescence experiments and two-dimensional immunoblotting analyses, it was demonstrated that the 2H12 antigen corresponds to an isoelectric variant of the human nucleolar protein B23 that is most prominent during interphase. Tightly associated with the nuclear matrix, this human B23 isoelectric variant did not shuttle between the nucleus and the cytoplasm but remained sequestered within the human nucleolus during mobility assays in human-murine heterokaryons.

Intermediate filament proteins: many unanswered questions, few unquestioned answers.

Major constituents of the cytoskeleton and the nuclear matrix, cytoplasmic intermediate filament subunits and nuclear lamins belong to a multigene family of proteins whose function is poorly understood. It has now become a general contention that important clues to the physiological roles of these proteins may reside in their developmental and tissue-specific expression patterns, as well as their cellular organization. The present review brings into focus experimental strategies that have been developed, over the past few years, to gain insights into the cellular mechanisms regulating the molecular polymorphism and supramolecular assembly of intermediate filaments. In this context new concepts are discussed that may be pivotal for the orientation of future studies on intermediate filament proteins.

Expression of intermediate filament proteins in TPA-induced MPC-11 and HL-60 cells.

Under normal culture conditions, the tumor cell lines MPC-11 and HL-60 exhibit high rates of proliferation and show a peculiar expression of intermediate filament proteins as they appear to synthesize only lamin B. A 48-h exposure of murine plasmacytomas MPC-11 to the phorbol ester TPA reduces their growth and induces vimentin synthesis without affecting the composition of their nuclear lamina. When applied to human leukemic promyelocytes HL-60, such treatment promotes their maturation into macrophage-like cells: their proliferative ability is suppressed, a differentiated phenotype is developed, and their content in intermediate filament proteins now includes vimentin and a full complement of lamins A, B, and C. In the present study, a kinetic analysis of vimentin and lamin A/C expression in relation to proliferation and differentiation has been performed in these two cellular systems. Proliferation rates of MPC-11 and HL-60 populations were evaluated by monitoring cell growth and measuring thymidine incorporation. Maturation of HL-60 cells was assessed by Giemsa staining and percentage of adherent cells. Expression of vimentin and lamins A/C was analyzed using immunofluorescence and immunoblotting techniques. Our data show that there is a relationship between the level of vimentin expression and the extent of growth inhibition in both systems. They also suggest that the expression of lamins A/C during the TPA-induced maturation of HL-60 promyelocytes might be part of the processes which lock these cells into the macrophage pathway.

Expression of vimentin and nuclear lamins during the in vitro differentiation of human promyelocytic leukemia cells HL-60.

We have reported previously that the human promyelocytic leukemia cell line HL-60, in its undifferentiated state, is devoid of cytoplasmic intermediate filament proteins and nuclear lamins A and C, but does express lamin B. Using immunofluorescence and immunoblotting techniques, we have further investigated the expression of vimentin and lamins A and C during differentiation of these tumor cells along the macrophage or granulocytic pathway in response to the inducing effects of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) or dimethyl sulfoxide. Our results show that, while the expression of lamin B remains largely unchanged, the synthesis of vimentin and lamins A and C is dramatically enhanced during the maturation of HL-60 cells along both hemopoietic pathways. Northern blot analysis of cellular RNAs isolated from untreated and TPA-treated HL-60 cell populations as well as from control HeLa cells was performed using two oligonucleotides, one complementary to the 5' region common to human lamin A/C mRNAs and the other to the 5' region of hamster vimentin mRNA. Very low but still detectable amounts of vimentin and lamin A/C mRNAs were found in untreated HL-60 cell population, in accordance with the detection of small quantities of vimentin and lamins A and C in these populations. This is probably due to the presence of a small number of spontaneously differentiating cells. On the other hand, strong signals comparable to those obtained with RNA from control HeLa cells were detected for the three mRNA species from TPA-treated cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Ultrastructure of Fanconi anemia fibroblasts.

Employing indirect immunofluorescence and conventional electron microscopy, gross nuclear aberrations were observed in cultured interphase fibroblasts derived from a patient suffering from Fanconi's anemia (FA). Such aberrations were predominantly expressed in cells at high passages between 28 and 34. The structure of the nuclei appeared compound in nature, often consisting of two to three nuclear fragments connected to each other by thin nuclear bridges containing chromatin and nuclear lamin material. In other cases, the nuclei appeared lobed or budded but the cells did not contain distinct nuclear fragments. Chromatin was conspicuously absent from some nuclear lobes, revealing empty, cage-like structures comprising nuclear lamin material. Micronuclei were often abundant in the perinuclear cytoplasm but in some instances they appeared to be composed of chromatin lacking a delineating nuclear lamin matrix. Residual cytoskeletons examined by whole-mount electron microscopy revealed a network of intermediate filaments (IFs) within FA fibroblasts forming a bridge between the plasma membrane and the nucleus or its major fragments. In addition, there were thinner, 3-4 nm filaments connecting individual IFs with the surface of the nucleus. Micronuclei that were not connected to the main nuclear body, but which were delineated by a distinct lamina and possessed nuclear pores, did not appear to be anchored to the IF network. Multinuclearity, nuclear fragmentation, irregular chromatin distribution and inter-nuclear chromatin/lamin bridges might result from a failure in the redistribution of chromatin to sister nuclei, incomplete cytokinesis and proliferation of nuclear envelope material. These phenomena point to precocious aging of FA fibroblasts and may occur as a consequence of spontaneous damage to the sister chromatids or through the action of DNA-toxic agents.

Organization of the vimentin system and its spatial relationship to the microtubule complex during the division of mammalian cells growing attached and in suspension.

We have used immunofluorescence staining with antibodies that detect vimentin, tubulin and the centrioles to compare the distributions of these respective antigens during the division of several suspension and attached cultured cells. Our observations demonstrate that 1) from distinct interphase organizations in suspension and attached cells, the vimentin system consistently rearranges with the onset of mitosis into a filamentous cage-like structure enclosing the spindle, 2) during cytokinesis, the polar centrosomes relocalize near the midbody in suspension cells while they remain at the pole opposite to it in attached cells, and 3) the vimentin cage is disintegrated and aggregated on each side of the midbody during cytokinesis in lymphoid cells but may be retained in other suspension cells.

Expression of nuclear lamins in mammalian somatic cells lacking cytoplasmic intermediate filament proteins.

Using immunofluorescence and immunoblotting techniques, we have examined the composition of the nuclear lamina in murine plasmacytoma cells, MPC-11, exposed to the phorbol ester TPA as well as in two cell lines devoid of cytoplasmic intermediate filament proteins, the human adrenal cortex carcinoma-derived cells SW-13 and the clone C6-M-D4 derived from the rat glial cell line C6. Our results show that the inhibition of proliferation and the induction of vimentin synthesis observed in TPA-treated MPC-11 populations are not paralleled by changes in the lamin complement of these cells, which contain lamin B but lack lamins A and C. Furthermore, the analysis performed on SW-13 and C6-M-D4 cell lines clearly demonstrates that mammalian somatic cells display considerable variations in lamin expression and indicates that lamin B may be the only lamin species constitutively expressed in mammalian cells.

Lack of lamins A and C in mammalian hemopoietic cell lines devoid of intermediate filament proteins.

Using immunofluorescence microscopy and immunoblot analysis, we have examined the composition of the nuclear lamina in several murine and human cell lines. Whereas it was shown that intermediate filament-positive Ehrlich ascites tumor and HeLa-S3 cells contain the three major mammalian lamin subspecies, only lamin B could be detected in several myeloid- and lymphoid-derived cell lines representative of distinct stages in hemopoietic differentiation but all devoid of cytoplasmic intermediate filament proteins. These included the murine plasmacytoma cell types MPC-11 and MOPC-31C, murine myeloma cells X63-Ag8.6.5.3 and human promyelocytic leukemia cells HL-60. Our results provide the first evidence that mammalian somatic cells capable of normal proliferation may lack both cytoplasmic intermediate filament proteins and a normal complement of nuclear lamins.

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)

Organizational fate of vimentin during redistribution of surface immunoglobulin in mouse splenic lymphocytes.

We have used immunofluorescence to examine the organizational fate of vimentin and its spatial relationship to the microtubule system during antibody-induced redistribution of surface immunoglobulin (sIg) in control and drug-treated mouse splenic lymphocytes. In control cells, vimentin is relocalized as a diffuse accumulation underneath the site of the cap during sIg redistribution. Observations on cells that were treated with colcemid or taxol prior to induction of sIg redistribution have further shown that vimentin accumulation corresponds to a dynamic rearrangement of this filamentous system which is related to, but is not required for, the energy-dependent translocation of sIg.

Vimentin dynamics during the mitogenic stimulation of mouse splenic lymphocytes.

We have used double immunofluorescence and electron microscopy to examine the distribution of tubulin and vimentin during the stimulation of mouse splenic lymphocytes by the mitogen concanavalin A. In unstimulated cells, vimentin forms a filamentous network partially coincident with the radial pattern of microtubules. In stimulated cells, the numbers of microtubules assembled from the centrosome have increased and vimentin is organized as an aggregate located near the centrosome. When these cells enter mitosis, vimentin is arranged into a filamentous cage enclosing the mitotic apparatus. During cytokinesis, the polar centrosomes are observed at a position adjacent to the midbody and vimentin is detected as an aggregate, similar to that seen prior to mitosis, close to the centrosome in each daughter cell. Using several agents, such as colchicine, colcemid, nocodazole, and taxol, which affect microtubule assembly, we have observed that the vimentin system, although closely related spatially to the microtubule complex in lymphocytes, can still reorganize independently as these cells progress through the cell cycle. Throughout mitogenic stimulation in the continued presence of taxol, microtubules are reorganized into a few thick bundles while the vimentin system undergoes a sequence of rearrangements similar to those observed during normal stimulation. These data suggest that vimentin dynamics may be important in the progression of lymphocytes through the cell cycle in response to mitogen.

Effects of taxol on microtubule organization in mouse splenic lymphocytes and on response to mitogenic stimulation.

We have used immunofluorescence staining with antibodies to tubulin and electron microscopy to examine the effects of microtubule assembly-promoting drug taxol on the organization of microtubules in unstimulated and in mitogen stimulated mouse splenic lymphocytes. A 4-h exposure to 10 microM taxol of unstimulated or stimulated cells results in an extensive reorganization of the microtubule system to form one to a few large bundles of microtubules which extend from the centrosome. Concomitantly, the centrosome is displaced from the normal position near the nucleus to a position near the plasma membrane. The Golgi apparatus is not disrupted by this treatment and also appears to be displaced in association with the centrosome. We then examined the capacity of lymphocytes with taxol-reorganized microtubules to respond to stimulation by the mitogen concanavalin A. The taxol-induced reorganization of microtubules has no effect on the increase in cell size (blastogenesis), the changes in structure of the nucleus and cytoplasm, or on the DNA replication that occurs in response to mitogen. The stimulated cells, however, do not proliferate and appear to accumulate in mitosis with the appearance of multiple asters. We conclude that all of the major events of mitogenic stimulation up to the first mitosis can occur in the presence of a highly reorganized microtubule system. Our results suggest that taxol will be a useful drug in determining those lymphocyte functions that are dependent on the presence of normal microtubule organization.

[Observation of filaments of 10nm in the demembraned cytoplasm of Amoeba proteus]. / Observation de filaments de 10 nm dans le cytoplasme démembrané d'Amoeba proteus.

We report here the first observation of 10 nm filaments in a protozoan, Amoeba proteus. These intermediate sized filaments were observed in spread cytoplasmic preparations of amoeba as stable cytoplasmic components over a wide range of pH (5.0-9.0). Although their morphology is grossly similar to the vertebrate intermediate filaments by negative staining, the filaments of amoeba show a characteristic helical structure with a 25 nm axial periodicity and do not display fibrillar projection along their length or at their extremity.

[Effects of pH and ATP on the cytoplasm of Amoeba proteus: relation between cytoplasm motility and actin polymerization]. / Effets du pH et de l'ATP sur le cytoplasme d'Amoeba proteus: relation entre la motilité du cytoplasme et l'état de polymerisation de l'actine.

The effect of pH and ATP was studied on isolated cytoplasm of Amoeba proteus. These two parameters were shown to influence both the motility and the organization of actin filaments in the isolated cytoplasm. Furthermore, our results demonstrate that there is a relationship between the motility and the polymeric state of actin. When the isolated cytoplasm is non-motile, actin is highly polymerized into long filaments arranged parallel in bundles. When this cytoplasm is motile, however, actin can either be weakly polymerized, i.e. observed as few short filaments, or can be polymerized in long branched filaments forming a loose network.

[Effect of phalloidin on the contractile structures in cytoplasmic preparations of Amoeba proteus]. / Observation des structures contractiles dans le cytoplasme démembranè d'Amoeba proteus après traitement à phalloïdine.

The effect of phalloidin on ultrastructural components involved in movement have been studied in spread cytoplasmic preparations of Amoeba proteus. In absence of phalloidin, actin filaments are usually rare and only myosin rods are observed. With concentrations of phalloidin between 2 X 10(-6) M and 5 X 10(-6) M, numerous F-actin filaments are present in the preparations. Most of these actin filaments are straight, however some appeared branched and interconnected. Higher concentrations of phalloidin inhibit the movement of naked cytoplasm. Fibrils composed by aggregation of F-actin filaments are present in these preparations. Myosin rods are unaffected by phalloidin.