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.     

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

Ｔｈｅｎｕｃｌｅａｒｌａｍｉｎａ(ＮＬ)ｉｎａｎｉｍａｌｃｅｌｌｓｉｓａｍｅｓｈｗｏｒｋｓｔｒｕｃｔｕｒｅｃｏｍｐｏｓｅｄｏｆｉｎｔｅｒｍｅｄｉａｔｅｆｉｌａｍｅｎｔｐｒｏｔｅｉｎｓ,ｔｅｒｍｅｄｌａｍｉｎｓ.Ｉｔｕｎｄｅｒｌｉｅｓｔｈｅｉｎｎｅｒｎｕｃｌｅａｒｍｅｍｂｒａｎｅａｎｄｃｏｎｆｅｒｓｍｅｃｈａｎｉｃａｌｓｔａｂｉｌｉｔｙｔｏｔｈｅｎｕｃｌｅａｒｅｎｖｅｌｏｐｅ[1].Ｉｎａｄｄｉｔｉｏｎ,ａｎｕｍｂｅｒｏｆｐｕｔａｔｉｖｅｒｏｌｅｓｈａｖｅｂｅｅｎｓｕｇｇｅｓｔｅｄｆｏｒｌａｍｉｎｓｂｏｔ…     

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.     

衣藻(Chlamydomonas sp)中间纤维及核纤层存在的证实(简报)

衣藻(Chlamydomonas sp)是属于绿藻门的最低等单细胞植物,为典型的真核生物。迄今以衣藻为材料所作的有关细胞骨架方面的研究多集中在微管蛋白(tubulin)。C.J.Miller等曾以衣藻(Chlamydomonas reinhardtii)全蛋白与几种中间纤维抗体进行免疫印迹实验有阳性反应,但是衣藻中是否存在中间纤维与核纤层是不清楚的问题。衣藻中间纤维与核纤层的形态研究更未见报道。目前认为中间纤维-核纤     

The nuclear lamina in male generative cells ofGinkgo biloba

Using selective extraction and diethylene glycol distearate embedment and embedment-free electron microscopy, we demonstrated nuclear lamina-like structures in sperm cells ofGinkgo biloba. A well-organized nuclear matrix network was also observed. Further studies were undertaken to determine whether or not lamin-like components exist in the pollen and sperm cells. Immunofluorescence staining using monoclonal antibodies against different animal lamins revealed lamins localized in the nuclear compartment of the sperm cells. Western blotting showed that in pollen grains there are two positive crossreaction bands at 66 kDa and 86 kDa, recognized by antibodies specific to animal lamins; in sperm cells there was only one, at 66 kDa. These results indicate that nuclear lamina containing both A-type and B-type lamins was present in male generative cells ofG. biloba. The data imply that plant lamins share some homology with animal lamins and may be conserved during evolution.     

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)     

Immunoaffinity purification and functional characterization of interphase and meiotic Drosophila nuclear lamin isoforms

Three isoforms of a single nuclear lamin have been identified in Drosophila. Two, lamins Dm1 and Dm2, are present during interphase and are apparently in equilibrium with each other in vivo. The third, lamin Dmmit, is found in cells that have undergone nuclear envelope breakdown, either during meiosis or mitosis. All three isoforms were purified under nondenaturing conditions using a novel technique of immunoaffinity chromatography and their in vitro activities were examined. Interphase lamins Dm1 and Dm2 can assemble into filaments at physiologic ionic strength; assembly is reversible upon addition of concentrated NaCl. Negative staining of filaments formed in vitro shows long, unbranched bundles approximately 20 nm in diameter. Addition of specific antilamin antibodies blocks in vitro assembly completely. In contrast with lamins Dm1 and Dm2, lamin Dmmit remains soluble at physiologic ionic strength. These observations are consistent with the notion that lamina disassembly in vivo is due, at least in part, to changes in properties of the lamins themselves.     

Fate of the inner nuclear membrane protein lamin B receptor and nuclear lamins in herpes simplex virus type 1 infection

During herpesvirus egress, capsids bud through the inner nuclear membrane. Underlying this membrane is the nuclear lamina, a meshwork of intermediate filaments with which it is tightly associated. Details of alterations to the lamina and the inner nuclear membrane during infection and the mechanisms involved in capsid transport across these structures remain unclear. Here we describe the fate of key protein components of the nuclear envelope and lamina during herpes simplex virus type 1 (HSV-1) infection. We followed the distribution of the inner nuclear membrane protein lamin B receptor (LBR) and lamins A and B(2) tagged with green fluorescent protein (GFP) in live infected cells. Together with additional results from indirect immunofluorescence, our studies reveal major morphologic distortion of nuclear-rim LBR and lamins A/C, B(1), and B(2). By 8 h p.i., we also observed a significant redistribution of LBR-GFP to the endoplasmic reticulum, where it colocalized with a subpopulation of cytoplasmic glycoprotein B by immunofluorescence. In addition, analysis by fluorescence recovery after photobleaching reveals that LBR-GFP exhibited increased diffusional mobility within the nuclear membrane of infected cells. This is consistent with the disruption of interactions between LBR and the underlying lamina. In addition to studying stably expressed GFP-lamins by fluorescence microscopy, we studied endogenous A- and B-type lamins in infected cells by Western blotting. Both approaches reveal a loss of lamins associated with virus infection. These data indicate major disruption of the nuclear envelope and lamina of HSV-1-infected cells and are consistent with a virus-induced dismantling of the nuclear lamina, possibly in order to gain access to the inner nuclear membrane.     

Immunolocalization of lamins in the thick nuclear lamina of human synovial cells

While in the great majority of cells the nuclear lamina is not resolved as a distinct structure separating the chromatin from the nuclear envelope, a demonstrable nuclear lamina ("fibrous lamina") of 30 to 300 nm thickness, interposed between the inner nuclear membrane and the peripheral chromatin, is characteristic for certain types of cells of vertebrates and invertebrates. We have examined whether the thick (50-70 nm) fibrous lamina of human synovial cells from patients suffering from rheumatoid arthritis indeed contains the lamins found in the indiscernible lamina structures present in most normal cells. We have observed, by electron microscopic immunolocalization, that both the A and the B type lamins occur throughout the entire nuclear lamina of these cells and that this structure is also resistant to treatments with nucleases and high salt buffers. This shows that the thick fibrous lamina only seen in certain vertebrate cells is compositionally related to the "masked" nuclear lamina of most other cells which usually is identified only upon removal of the adjacent nuclear structures.     

北京鸭B淋巴细胞的中间纤维—核纤层—核内骨架体系

以北京鸭腔上囊为实验材料,应用细胞分级抽提方法与非树脂包埋－去包埋剂的电镜制样技术相结合,显示出Ｂ细胞中相互连结的中间纤维－核纤层－核内骨架体系的超结构及其分布,中间纤维交织成网络状,纤维直径在９－１１ｎｍ,其成份是分子量为６７ｋＤ,等电点约为６．２的波形蛋白,核纤民支呈片层状结构环绕在核区周围,其主要成份是分子量为６７ｋＤ,等电点偏酸性的Ｌａｍｉｎ Ｂ。核内骨架由粗细不一的纤维形成网络结构,其上     

Somatic lamins in germinal vesicles of goldfish (Carassius auratus) vitellogenic oocytes

In fish and amphibians, B-type lamins are divided into somatic (B1, B2) and oocyte-type (B3) lamins. In this study, we purified nuclear lamins from rainbow trout erythrocytes, raised an anti-lamin monoclonal antibody (L-200) that recognizes goldfish somatic-lamins, and isolated cDNAs encoding goldfish B-type lamins (B1 and B2) from a goldfish cell culture cDNA library. Goldfish B-type lamins are structurally similar to lamins found in other vertebrates with minor amino acid substitutions in the conserved region. Western blot analysis showed that goldfish oocytes contained mainly GV-lamin B3 as well as some somatic lamins. Laser-confocal microscope observations revealed that lamin B3 was present only in GV nuclear lamina, whereas somatic lamins were present in dense fibrillar structures throughout nuclear gels of isolated GVs. Similar nuclear filamentous structures were also observed in GVs of paraffin embedded oocytes. Epitope mapping indicated that L-200 recognized a conserved region containing a short stretch of the alpha-helix coiled-coil rod domain (Y(E/Q)(Q/E)LL). A similar motif is also present in other cytoplasmic intermediate filaments (i.e., vimentin, desmin, peripherin and GFAP). Taken together, these findings suggest that lamins or lamin-related intermediate filaments are an important component of the interior architecture of goldfish vitellogenic oocyte nuclei (GVs).     

The Supramolecular Organization of the C. elegans Nuclear Lamin Filament

Nuclear lamins are involved in most nuclear activities and are essential for retaining the mechano-elastic properties of the nucleus. They are nuclear intermediate filament (IF) proteins forming a distinct meshwork-like layer adhering to the inner nuclear membrane, called the nuclear lamina. Here, we present for the first time, the three-dimensional supramolecular organization of lamin 10 nm filaments and paracrystalline fibres. We show that Caenorhabditis elegans nuclear lamin forms 10 nm IF-like filaments, which are distinct from their cytoplasmic counterparts. The IF-like lamin filaments are composed of three and four tetrameric protofilaments, each of which contains two partially staggered anti-parallel head-to-tail polymers. The beaded appearance of the lamin filaments stems from paired globular tail domains, which are spaced regularly, alternating between 21 nm and 27 nm. A mutation in an evolutionarily conserved residue that causes Hutchison-Gilford progeria syndrome in humans alters the supramolecular structure of the lamin filaments. On the basis of our structural analysis, we propose an assembly pathway that yields the observed 10 nm IF-like lamin filaments and paracrystalline fibres. These results serve also as a platform for understanding the effect of laminopathic mutations on lamin supramolecular organization.     

Nuclear matrix of the most primitive eukaryote Archezoa

Accumulating evidence suggests that unicellularArchezoa are the most primitive eukaryotes and their nuclei are of significance to the study of evolution of the eukaryotic nucleus. Nuclear matrix is an ubiquitous important structure of eukaryotic nucleus; its evolution is certainly one of the most important parts of the evolution of nucleus. To study the evolution of nuclear matrix, nuclear matrices ofArchezoa are investigated.Giardia lamblia cells are extracted sequentially. Both embedment-free section EM and whole mount cell EM of the extracted cells show that, like higher eukaryotes, this species has a residual nuclear matrix in its nucleus and rich intermediate filaments in its cytoplasm, and the two networks connect with each other to form a united network. But its nuclear matrix does not have nucleolar matrix and its lamina is not as typical as that of higher eukaryotes; Western blotting shows that lamina ofGiardia and two otherArchezoa Entanzoeba invadens andTrichomonas vaginali all contain only one polypeptide each which reacts with a mammalia anti-lamin polyclonal serum and is similar to lamin B (67 ku) of rnammiia in molecular weight. According to the results and references, it is suggested that nuclear matrix is an early acquisition of the eukaryotic nucleus, and it and the “eukaryotic chromatin” as a whole must have originated very early in the process of evolution of eukaryotic cell, and their origin should be an important prerequisite of the origin of eukaryotic nucleus: in the lamin (gene) family, B-type lamins (gene) should be the ancestral typz and that A-type lamins (gene) might derive therefrom. Project supported by the National Natural Science Foundation of China (Grant No. 3870254).     

Nuclear lamina builds tissues from the stem cell niche

Recent studies show that nuclear lamins, the type V intermediate filament proteins, are required for proper building of at least some organs. As the major structural components of the nuclear lamina found underneath the inner nuclear membranes, lamins are ubiquitously expressed in all animal cells. How the broadly expressed lamins support the building of specific tissues is not understood. By studying Drosophila testis, we have uncovered a mechanism by which lamin-B functions in the cyst stem cell (CySC) and its differentiated cyst cell, the cell types known to form the niche/microenvironment for the germline stem cells (GSC) and the developing germ line, to ensure testis organogenesis ¹. In this extra view, we discuss some remaining questions and the implications of our findings in the understanding of how the ubiquitous nuclear lamina regulates tissue building in a context-dependent manner.     

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.     

Identification and Cloning of an mRNA Coding for a Germ Cell-Specific A-Type Lamin in Mice

The nuclear lamins are karyoskeletal proteins which have important functions, such as maintaining nuclear envelope integrity and organizing high order nuclear structure during mitosis in higher eukaryotes. In somatic mammalian cells, the A-type and B-type lamins, composed of lamins A and C and lamins B1 and B2, are major components of the nuclear lamina. However, A-type lamins have as yet not been identified in germ cells and undifferentiated embryonic cells. Here we report the cloning of a new 52-kDa A-type lamin from mouse pachytene spermatocytes, termed lamin C2 because of its similarities with lamin C. It has a sequence identical to that of lamin C except that the N -terminal segment, containing the head and the α-helical coil 1A domains, is replaced with a short non-α-helical stretch of amino acids. In mice, lamin C2 was found to be specifically expressed in germ cells. This specific expression and unique structure suggests a role for lamin C2 in determining the organization of nuclear and chromosomal structures during spermatogenesis.     

Characterization of a second highly conserved B-type lamin present in cells previously thought to contain only a single B-type lamin

Previous analyses of the nuclear lamina of mammalian cells have revealed three major protein components (lamins A, B and C) that have been identified by protein sequence homology as members of the intermediate filament (IF) protein family. It has been claimed that mammalian cells contain either all three lamins or lamin B alone. Using monoclonal antibodies specific for B-type lamins and cDNA cloning we identified a second major mammalian B-type lamin (murine lamin B₂), thus showing that lamin composition in mammals is more complex than previously thought. Lamin B₂ is coexpressed with lamin B₁ (formerly termed lamin B) in all somatic cells and mammalian species that we analysed, including a variety of cells currently believed to contain only a single lamin. This suggests that two B-type lamins are necessary to form a functional lamina in mammalian somatic cells. By cDNA cloning we found thatXenopus laevis lamin L_II is the amphibian homolog of mammalian lamin B₂. Lamin expression during embryogenesis of amphibians and mammals shows striking similarities. The first lamins expressed in the early embryo are the two B-type lamins, while A-type lamins are only detected much later in development. These findings indicate that the genomic differentiation into two B-type lamins occurred early in vertebrate evolution and has been maintained in both their primary structure and pattern of expression.     

History and phylogeny of intermediate filaments: Now in insects

Intermediate filaments include the nuclear lamins, which are universal in metazoans, and the cytoplasmic intermediate filaments, which are much more varied and form cell type-specific networks in animal cells. Until now, it has been thought that insects harbor lamins only. This view is fundamentally challenged by the discovery, reported in BMC Biology, of an intermediate filament-like cytoplasmic protein, isomin, in the hexapod Isotomurus maculatus. Here we briefly review the history of research on intermediate filaments, and discuss the implications of this latest finding in the context of what is known of their structure and functions.     

Characterization of a 65 kDa NIF in the nuclear matrix of the monocot Allium cepa that interacts with nuclear spectrin‐like proteins

Plant cells have a well organized nucleus and nuclear matrix, but lack orthologues of the main structural components of the metazoan nuclear matrix. Although data is limited, most plant nuclear structural proteins are coiled‐coil proteins, such as the NIFs (nuclear intermediate filaments) in Pisum sativum that cross‐react with anti‐intermediate filament and anti‐lamin antibodies, form filaments 6–12 nm in diameter in vitro, and may play the role of lamins. We have investigated the conservation and features of NIFs in a monocot species, Allium cepa, and compared them with onion lamin‐like proteins. Polyclonal antisera against the pea 65 kDa NIF were used in 1D and 2D Western blots, ICM (imunofluorescence confocal microscopy) and IEM (immunoelectron microscopy). Their presence in the nuclear matrix was analysed by differential extraction of nuclei, and their association with structural spectrin‐like proteins by co‐immunoprecipitation and co‐localization in ICM. NIF is a conserved structural component of the nucleus and its matrix in monocots with M_r and pI values similar to those of pea 65 kDa NIF, which localized to the nuclear envelope, perichromatin domains and foci, and to the nuclear matrix, interacting directly with structural nuclear spectrin‐like proteins. Its similarities with some of the proteins described as onion lamin‐like proteins suggest that they are highly related or perhaps the same proteins.     

Partial cleavage of A-type lamins concurs with their total disintegration from the nuclear lamina during apoptosis

Although activated caspase 6 is capable of cleaving both A- and B-type lamins during apoptosis, the higher-order structure of the nuclear lamina may cause a differential breakdown of these two types of lamins. In order to obtain a better understanding of the dynamics and the consequences of the rapid, coordinated breakdown of the lamina complex, we applied the green fluorescent protein (GFP) technology in living cells, in which the fate of individual caspase cleavage fragments of A- and B-type lamins was examined. CHO-K1 cells were stably transfected with cDNA constructs encoding N-terminally GFP-labelled hybrids of lamin A, lamin Adelta10, lamin C or lamin B1. The course of the apoptotic process, induced by the kinase inhibitor staurosporine or by the proteasome inhibitor MG132, was monitored by digital imaging microscopy or confocal microscopy. Time-lapse recordings showed that parallel to DNA condensation N-terminally GFP-tagged A-type lamins became diffusely dispersed throughout the nucleoplasm and rapidly translocated to the cytoplasm. In contrast, the majority of GFP-lamin B1 signal remained localised at the nuclear periphery, even after extensive DNA condensation. Comparison of lamin B1-GFP signal with A-type lamin antibody staining in the same apoptotic cells confirmed the temporal differences between A- and B-type lamina dispersal. Immunoblotting revealed only a partial cleavage of A-type lamins and an almost complete cleavage of lamin B1 during apoptosis. In contrast to lamin B1 in normal cells, this cleaved lamin B1, which is apparently still associated with the nuclear membrane, can be completely extracted by methanol or ethanol. Fluorescence loss of intensity after photobleaching experiments showed that in apoptotic cells A-type lamin-GFP molecules diffuse almost freely in both nucleoplasm and cytoplasm, while the lamin B1-GFP fragments remain more stably associated with the nuclear membrane, which is confirmed by co-localisation immunofluorescence studies with a nucleoporin p62 antibody. Our results therefore clearly show a differential behaviour of A- and B-type lamins during apoptosis, suggesting not only distinct differences in the organisation of the lamina filaments, but also that caspase cleavage of only a small fraction of A-type lamins is needed for its complete disintegration.