Identification of distinct messenger RNAs for nuclear lamin C and a putative precursor of nuclear lamin A

The lamins are the major components of the nuclear matrix and are known as lamins A, B, and C with Mr 72,000, 68,000, and 62,000 when analysed by SDS PAGE. These three polypeptides are very similar, as determined by polypeptide mapping and immunological reactivity. Lamins A and C are so homologous that a precursor-product relationship has been proposed. Using an antiserum against nuclear matrix proteins that specifically immunoprecipitates the three lamins, we examined their synthesis in the rabbit reticulocytes lysate. Four bands of Mr 62,000, 68,000, 70,000, and 74,000 were specifically immunoprecipitated when polysomes or polyadenylated RNA were translated in vitro. By two-dimensional gel electrophoresis, the 68,000- and the 62,000-mol-wt proteins were identified as lamins B and C, respectively, and the 74,000-mol-wt polypeptide had properties of a precursor of lamin A. The mRNAs of lamin C and of the putative precursor of lamin A were completely separated by gel electrophoresis under denaturing conditions, and their respective sizes were determined. These results suggest that lamin A is not a precursor of lamin C.     

Putative Arabidopsis homologues of metazoan coiled-coil cytoskeletal proteins

The Arabidopsis thaliana genome encodes about 386 proteins with coiled-coil domains of at least 50 amino acids in length. In mammalian systems, many coiled-coil proteins are part of various cytoskeletal networks including intermediate filament protein, actin-binding proteins and MAP (microtubule-associated proteins). Immunological evidence suggests that some of these cytoskeletal proteins, such as lamins, keratins and tropomyosins, may be conserved in Arabidopsis. However, coiled-coil proteins are of low complexity, and thus, traditional sequence comparison algorithms, such as BLAST may not detect homologies. Here, we use the PROPSEARCH algorithm to detect putative coiled-coil cytoskeletal protein homologues in Arabidopsis. This approach reveals putative intermediate filament protein homologues of filensin, lamin and keratin; putative actin-binding homologues of ERM (ezrin/radixin/moesin), periplakin, utrophin, tropomyosin and paramyosin, and putative MAP homologues of restin/CLIP-170 (cytoplasmic linker protein-170). We suggest that the AtFPP (Arabiopsis thaliana filament-like plant protein) and AtMAP70 (Arabidopsis microtubule-associated protein 70) families of coiled-coil proteins may, in fact, be related to lamins and function as intermediate filament proteins.     

Insulin stimulates incorporation of 32Pi into nuclear lamins A and C in quiescent BHK-21 cells

The in vivo effect of insulin upon the incorporation of 32Pi into nuclear proteins was investigated in quiescent baby hamster kidney fibroblasts (BHK-21). Insulin has previously been shown to be mitogenic in these cells (Richman, R. A., Johnson, R. A., and Friedman, D. L. (1981) Proc. Soc. Exp. Biol. Med. 168, 196-203). Incorporation into two proteins (Mr = 62,000 and 72,000) in the 0.4 M salt-resistant nuclear fraction was enhanced 2-6-fold by insulin. The effect of insulin (20 nM) was observable within 5 min of treatment, reached a maximum at 15 min, and continued for at least 90 min. The half-maximal effect of insulin was obtained at a concentration of approximately 1 nM. Analysis of nuclear matrix preparations indicated that the two insulin-sensitive proteins were prominent nuclear matrix proteins and suggested that they were lamins A and C. This was confirmed by immunostaining with lamin antibodies and by two-dimensional electrophoresis. These studies indicate that insulin rapidly stimulates the incorporation of phosphate into nuclear lamins A and C in quiescent BHK-21 fibroblasts.     

Large scale co-isolation of vimentin and nuclear lamins from ehrlich ascites tumor cells cultured in vitro

Ehrlich ascites tumor (EAT) cells propagated in mass suspension culture were used as a starting material for the simultaneous isolation and purification of large quantities of the intermediate filament protein vimentin and the nuclear lamins A/C and B. Triton cytoskeletons, obtained by repeated washing of cells with a low ionic strength buffer containing Triton X-100 and 4 mM Mg2+, were extracted with 6 M urea at low salt concentration and in the presence of EDTA. Separation of solubilized proteins from unfolded chromatin (DNA) was accomplished by recondensation of the chromatin (DNA) in the presence of Mg2+ before centrifugation. To achieve separation of vimentin from nuclear lamins, the urea extract was subjected to DEAE-Sepharose CL-6B chromatography. Single-stranded DNA-cellulose chromatography was employed for the final purification of vimentin and for the separation of lamin B from lamins A/C. Further purification of lamin B was carried out by CM-Sepharose CL-6B chromatography and of lamins A/C by chromatography on hydroxylapatite. All chromatographies were performed in the presence of 6 M urea. 500 g of pelleted EAT cells yielded approximately 700 mg of vimentin, 225 mg of lamins A/C and 21 mg of lamin B. 2D-polyacrylamide gel electrophoresis revealed great microheterogeneity of lamins A/C, which to a high extent was due to phosphorylation, whereas lamin B was much less heterogeneous. In the absence of urea and at low salt concentration, lamins A/C required pH 5 to stay in solution whereas lamin B required pH 7.5. Increasing the salt concentration to 150 or 250 mM NaCl resulted in the formation of paracrystals from a urea-free mixture of lamins A/C and B. Although the lamins could not be assembled into intermediate filaments under a variety of ionic conditions, the preparations obtained will be useful for further biochemical characterization of these nuclear proteins.     

Lamin B shares a number of distinct epitopes with lamins A and C and with intermediate filament proteins

Four monoclonal antibodies raised against rat liver nuclear lamins and an anti-intermediate filament antibody [Pruss, R. M., Mirsky, R., & Raff, M. C. (1981) Cell (Cambridge, Mass.) 27, 419-428] have been used to identify epitopes shared by lamin B with lamins A and C, and with intermediate filament proteins. The antibodies defined two major antigenic regions on the three lamins which were both homologous with mouse epidermal keratins as well as hamster vimentin and desmin. Three distinguishable epitopes shared by lamin B with lamins A and C were identified by competition studies between pairs of antibodies and by reaction against N-chlorosuccinimide and cyanogen bromide cleavage fragments. These results support the hypothesis that lamin B, despite important biochemical differences with lamins A and C, shares with them some of the structural characteristics typical of intermediate filament proteins.     

Basic proteins of the perinuclear theca of mammalian spermatozoa and spermatids: a novel class of cytoskeletal elements

The nuclei of bovine spermatids and spermatozoa are surrounded by dense cytoplasmic webs sandwiched between the nuclear envelope and the acrosome and plasma membrane, respectively, filling most of the cytoplasmic space of the sperm head. This web contains a complex structure, the perinuclear theca, which is characterized by resistance to extractions in nondenaturing detergents and high salt buffers, and can be divided into two major subcomponents, the subacrosomal layer and the postacrosomal calyx. Using calyces isolated from bull and rat spermatozoa we have identified two kinds of basic proteins as major constituents of the thecal structure and have localized them by specific antibodies at the light and electron microscopic level. These are an Mr 60,000 protein, termed calicin, localized almost exclusively to the calyx, and a group of multiple-band polypeptides (MBP; Mr 56,000-74,000), which occur in both the calyx and the subacrosomal layer. The polypeptides of the MBP group are immunologically related to each other, but unrelated, by antibody reactions and peptide maps, to calicin. We show that these basic cytoskeletal proteins are first detectable in the round spermatid stage. As we have not detected any intermediate filament proteins and proteins related to nuclear lamins of somatic cells in sperm heads, we conclude that the perinuclear theca and its constituents, calicin and MBP proteins, are the predominant cytoskeletal elements of the sperm head. Immunologically cross-reacting polypeptides with similar properties have been identified in the heads of rat and human spermatozoa. We speculate that these insoluble basic proteins contribute, during spermiogenesis, to the formation of the perinuclear theca as an architectural element involved in the shape changes and the intimate association of the nucleus with the acrosome and the plasma membrane.     

Biochemical and immunological characterization of pea nuclear intermediate filament proteins

In immunoblot assays, at least three putative nuclear intermediate filament (NIF) proteins were detected in nuclear envelope-matrix (NEM) and lamin (L1) fractions of nuclei from plumules of dark-grown pea (Pisum sativum L.) seedlings. These NIF proteins had apparent molecular masses of ca. 65, 60, and 54 kDa (also referred to as p65, p60, and p54), and appeared as multiple isoelectric forms, with pIs ranging from ca. 4.8 to 6.0. Polyclonal and monoclonal antibodies were raised to the 65-kDa NIF protein bands excised from gels after electrophoresis. These anti-pea antibodies were specifically cross-reactive with the pea nuclear p65, p60, and p54 proteins and also with chicken lamins. Sequence alignment of peptide fragments obtained from the 65- and 60-kDa pea NIF proteins showed similarity with animal intermediate filament proteins such as lamins and keratins and with certain plant proteins predicted to have long coiled-coil domains. These pea NIF proteins were further purified and enriched from the NEM fraction using methods similar to those used for isolating animal lamins. When negatively stained and viewed by transmission electron microscopy, the filaments in the pea lamin (L1) fraction appeared to be 6–12 nm in diameter. As assayed by immunofluorescence cytochemistry using a confocal laser-scanning microscope, fixed pea plumule cells displayed uniform as opposed to peripheral nuclear staining by several of the antibody preparations, both polyclonal and monoclonal. This report describes the biochemical and immunological properties of these pea NIF proteins.Abbreviations IF Intermediate filament - L Lamin fraction - LM Lamina-matrix fraction - MAb JLA20 Anti-chicken actin monoclonal antibody - MAb LN43 Anti-human lamin B2 monoclonal antibody - MAb PL19 Anti-pea lamin #19 monoclonal antibody - MAb TIB 131 Anti-intermediate filament monoclonal antibody - N Nuclei fraction - NEM Nuclear envelope-matrix fraction - NIF Nuclear intermediate filament - PAb PL3 Anti-pea lamin #3 polyclonal antibody     

Intermediate filaments in hyaline material in alcoholic liver disease (Mallory bodies) and in cultured hepatoma cells.

Antibodies specific for cytoskeletal intermediate (10 nm) filaments reacted both with the hyaline deposits in alcoholic liver disease (Mallory bodies) and with perinuclear hyaline material of cultured hepatoma cells used as a model for hyaline formation in vitro. Our results suggest that disorganization and accumulation of intermediate filaments is an important step in the pathogenesis of alcoholic liver injury.     

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.     

Evolution of homologous domains of cytoplasmic intermediate filament proteins and lamins

The earliest gene duplications in the evolution of the intermediate filament proteins created the ancestors of acidic keratins, basic keratins, nonepithelial intermediate filament proteins, and lamins. Biochemistry and function of cytoplasmic intermediate filaments differ greatly from those of lamins. Cytoplasmic intermediate filament proteins have a different cellular location than lamins, form different types of supramolecular structures, and are missing a protein segment found in lamins; but the data presented here indicate that the cytoplasmic intermediate filaments do not have a common ancestor separate from the ancestor of lamins. In the non-epithelial intermediate filament branch, the ancestor of neurofilament proteins and the common ancestor of desmin, vimentin, and glial fibrillary acidic protein (GFAP) diverged first. By evolutionary criteria, the intermediate filament protein recently discovered in neuronal cells does not belong to the neurofilament family but is more closely related to desmin, vimentin, and GFAP. Sequences of different sub-domains yield different evolutionary trees, possibly indicating existence of sub- domain-specific functions.      

A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques.

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.     

Patterns of nuclear lamins in diverse animal and plant cells and in germ cells as revealed by immunofluorescence microscopy with polyclonal and monoclonal antibodies

Polyclonal and monoclonal antibodies against rat liver nuclear lamins have been used to evaluate the immunological cross-reactivity of lamins with a given antibody in a variety of animal and plant cells. The results indicated that lamins of all vertebrate cells but not invertebrate cells share at least one antigenic determinant, resulting in immunological cross-reaction with polyclonal antisera to lamina from rat liver. The range of cross-reaction with monoclonal antibody to rat lamins includes all mammalian cells tested but we observed no reaction with other vertebrate and invertebrate cells. Thus, by means of immunological cross-reaction a less stringently conserved pattern is observed for lamins than, for example, cytoskeletal proteins. We have also investigated the fate of the nuclear lamins during meiosis in testes and ovaries of the mouse. Lamins are absent from male meiotic cells and during oogenesis in meiotic prophases.     

Differences of expression of cytoskeletal proteins in cultured rat hepatocytes and hepatoma cells

Cytoskeletal elements, enriched in intermediate-sized filaments and insoluble in buffers of high salt concentrations and Triton X-100, were isolated from various cultures of rat hepatocytes and hepatoma cells, and their proteins were studied by one- and two-dimensional gel electrophoresis and immunofluorescence microscopy. The cells examined included several permanent cell lines (MH₁C₁, HTC, hepatoma 72/22, clone 12 from Gunn rat hepatocytes, and cell clones from normal rat hepatocytes), as well as freshly dissociated hepatocytes that were cultured and allowed to attach to substratum for increasing periods of time, beginning at 24 h after removal of the liver from the animal. Filaments containing vimentin, which were not found in hepatocytes grown in liver tissue, were detected in most of the cultured hepatocytes and hepatoma cells, except in MH₁C₁ cells, and were shown to be newly synthesized during the first days of primary culture. Maintenance of expression of filaments containing proteins immunologically related to epidermal prekeratin (‘cytokeratins’) was observed in all cells examined but HTC cells. Detailed comparison of the cytokeratin polypeptides present in various hepatocyte and hepatoma cell cultures showed that, in some of the cultured epithelial liver cells, cytokeratins are expressed which are identical with, or similar to, those of normal hepatocytes grown in the liver. On the other hand, differences in cytokeratin polypeptides were also found among different hepatocyte-derived cell cultures. Changes of expression of cytoskeletal proteins were found to occur even in cloned cell populations, and cells positive for certain cytokeratins could be seen next to other cells that were negative.The results demonstrate that profound changes of cytoskeletal composition, especially concerning intermediate filament protein patterns, can occur during culturing in vitro. Moreover, we show that different intermediate filament proteins can be expressed in different hepatocyte-derived cell cultures and that changes of cytoskeletal composition can occur in a given cell population, without obvious effects on cell growth rate and cell morphology. During culturing of hepatocytes and hepatoma cells, there seems to be a general tendency to induce the production of vimentin filaments as well as to maintain the production of cytokeratins similar to the hepatocyte-specific cytokeratins in liver tissue. However, the demonstrated exceptions speak against a role of these filament proteins as prerequisites for the growth of an epithelial cell in vitro. Rather, the presence of filaments containing certain cytokeratins and of desmosomes in epithelial cells growing in vitro seems to reflect the synthesis of specific differentiation markers which may be lost, independently, in some cells during culturing.     

Fine structure and assembly in vitro of nuclear lamina in plant cells

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Fine structure and assembly in vitro of nuclear lamina in plant cells

The fine structure of the nuclear lamina (NL) in sperm cells ofGinkgo biloba was visualised using high resolution low-voltage scanning electron microscopy (LVSEM). It was shown that the nuclear lamina was composed of 10 nm filaments which formed a fine network. Lamins were purified from cultured carrot suspension cells and assembledin vitro. Long 8–12 nm diameter filaments were seen and sometimes subfilaments could be distinguished. Western blot of filament preparations showed that these contained the 66 and 84 ku lamins. These data demonstrate that plant lamins are capable of assembling into filamentsin vitro. Project supported by the National Natural Science Foundation of China (Grant No. 3500073).     

Fine structure and assemblyin vitro of nuclear lamina in plant cells

The fine structure of the nuclear lamina (NL) in sperm cells ofGinkgo biloba was visualised using high resolution low-voltage scanning electron microscopy (LVSEM). It was shown that the nuclear lamina was composed of 10 nm filaments which formed a fine network. Lamins were purified from cultured carrot suspension cells and assembledin vitro. Long 8–12 nm diameter filaments were seen and sometimes subfilaments could be distinguished. Western blot of filament preparations showed that these contained the 66 and 84 ku lamins. These data demonstrate that plant lamins are capable of assembling into filamentsin vitro.     

Biochemical and immunological identification of cytokeratin proteins present in hepatocytes of mammalian liver tissue

Hepatocytes of mammalian liver are known to contain intermediate-sized filaments of tonofilament morphology. Unlike many other epithelial cells, including cultured hepatocytes and hepatoma cells, hepatocytes present in normal liver tissue have been reported not to react, in significant intensity, with various preparations of antibodies to human and bovine epidermal prekeratin [2,6]. We have therefore examined, by biochemical and immunological methods, the cytoskeletal composition of hepatocytes grown in the body.Cytoskeletal preparations from hepatocytes of mouse and rat liver tissue resistant to high salt buffer and Triton X-100 are enriched in tangles of intermediate filaments and contain, besides some residual microfilamentous actin, a characteristic set of polypeptides. One- and two-dimensional gel electrophoresis reveals the presence of two major cytokeratin components, which appear as ‘pairs’ of isoelectric variants (component A, M_r 55 000, apparent pI values, 6.40 and 6.45; component D, M_r 49000, apparent pI values 5.43 and 5.38), and five minor components (M_r range from 41000 to 53 000), most of them also as ‘pairs’ of polypeptides slightly different in isoelectric pH value. These polypeptide patterns are very similar in mouse and rat liver although some minor but significant differences have been noted between the two species. The polypeptide patterns of liver cytoskeletons are also similar to—but clearly not identical with—the cytoskeletal protein patterns observed in other epithelial tissues and cells, including various lines of cultured rat hepatocytes and hepatoma cells.Guinea pig antibodies raised against individual cytokeratin proteins of mouse liver and against certain prekeratin polypeptides present in desmosome-attached tonofilaments of bovine muzzle are described which differ from previously described prekeratin antibodies. These prekeratin antibodies not only react with filament bundles of the prekeratin type present in many cultured epithelial cells (e.g. murine HEL, human HeLa, rat kangaroo PtK2) and various epithelial tissues, but also allow the detection of the cytokeratin components present in parenchymal cells of liver and pancreas of various species, man included. Immunofluorescence microscopy on frozen sections of liver using these antibodies reveals a novel structure, i.e. a three-dimensional filament meshwork extending throughout the whole cytoplasm of the hepatocyte, with higher intensity of staining in pericanalicular regions.The results show that parenchymal cells of normal liver and pancreas contain filaments of the cytokeratin type that are related to but not identical with epidermal prekeratin. The hepatocyte filaments appear to be different from prekeratin-type filaments present in epidermis and several other epithelial cells, both in some antigenic determinants exposed and in polypeptide composition. Our findings support the concept of the existence of a family of intermediate filament proteins, cytokeratins, containing many different polypeptides that are expressed in different epithelial cells in certain characteristic subsets in a cell type-specific mode.