Organization of the nuclear pore complex in Saccharomyces cerevisiae

Fractions enriched for nuclear pore complexes (NPCs) have been isolated from Saccharomyces cerevisiae. The sequential extraction of nuclei with detergent, nucleases, and salt reveals an organization of the yeast NPC similar to other eukaryotes. Yeast NPCs contain a 30-nm “ring” structure not previously described in other organisms. This structure appears to organize 10-nm filaments into an assembly which exhibits an eight-fold rotational symmetry. Some proteins in the NPC fraction are capable of forming intermediate-sized filaments. These studies suggest that some component of the nuclear pore complex organizes an interaction between nuclear and cytoplasmic networks of intermediate filaments.     

Yolk nucleus – The complex assemblage of cytoskeleton and ER is a site of lipid droplet formation in spider oocytes

Oocytes (future egg cells) of various animal groups often contain complex organelle assemblages (Balbiani bodies, yolk nuclei). The molecular composition and function of Balbiani bodies, such as those found in the oocytes of Xenopus laevis, have been recently recognized. In contrast, the functional significance of more complex and highly ordered yolk nuclei has not been elucidated to date. In this report we describe the structure, cytochemical content and evolution of the yolk nucleus in the oocytes of a common spider, Clubiona sp. We show that the yolk nucleus is a spherical, rather compact and persistent cytoplasmic accumulation of several different organelles. It consists predominantly of a highly elaborate cytoskeletal scaffold of condensed filamentous actin and a dense meshwork of intermediate-sized filaments. The yolk nucleus also comprises cisterns of endoplasmic reticulum, mitochondria, lipid droplets and other organelles. Nascent lipid droplets are regularly found in the cortical regions of the yolk nucleus in association with the endoplasmic reticulum. Single lipid droplets become surrounded by filamentous cages formed by intermediate filaments. Coexistence of the forming lipid droplets with the endoplasmic reticulum in the cortical zone of the yolk nucleus and their later investment by intermediate-sized filamentous cages suggest that the yolk nucleus is the birthplace of lipid droplets.     

Intermediate-sized filaments present in Sertoli cells are of the vimentin type.

The cytoplasmic structure of Sertoli cells of rat testes has been studied by electron microscopy of ultrathin sections. Sertoli cells contain numerous intermediate-sized (7-11 nm) filaments which form a meshwork extending throughout the whole cytoplasm. Often the frequency of such filaments appears especially high in juxtanuclear and cortical regions, including the apical recesses containing the spermatids. Examination of frozen sections of testes by indirect immunofluorescence microscopy using guinea pig antibodies to prekeratin and vimentin has shown the absence of intermediate-sized filaments of the cytokeratin type in all cells of the testes but the presence of filaments of the vimentin type in Sertoli cells as well as in cells of the interstitial space. These results show that the intermediate-sized filaments, abundant in Sertoli cells, are of the vimentin type. In addition we conclude that the "germ epithelium" differs from others true epithelia by the absence of cytokeratin filaments and typical desmosomes and, in Sertoli cells, the presence of vimentin filaments, suggestive of a mesenchymal character or derivation.     

Direct demonstration of the presence of two immunologically distinct intermediate-sized filament systems in the same cell by double immunofluorescence microscopy: Vimentin and cytokeratin fibers in cultured epithelial cells

The epithelial derived cell lines PtK2 and HeLa were characterized by double immunofluorescence microscopy using purified antibodies against vimentin and prekeratin. The results show that both cell types express simultaneously two immunologically distinct intermediate-sized filaments. Use of colcemid-treated cells confirms that the vimentin fibers and not the keratin-related fibers are rearranged into coils around the nucleus. In some cells staining of fibrous fragments is observed, which are perhaps involved in the synthesis or breakdown of this class of filaments. The concept that growing cells derived from differentiated cell types express not only the intermediate-sized filament system typical of the differentiated cell type but in addition contain intermediate-sized filaments of the vimentin type is discussed.     

Constitutive aggregates of intermediate-sized filaments of the vimentin and cytokeratin type in cultured hepatoma cells and their dispersal by butyrate

Cloned hepatoma cells ($\text{72}\text{22}$) derived from the liver of a rat treated with the carcinogen, diethylnitrosamine, exhibit a genetically stable, large, acentric, juxtanuclear, hyaline aggregate of loosely packed intermediate-sized (7–11 nm) filaments, interspersed with variable but minor amounts of microtubules, polyribosomes and membranous structures. Immunofluorescence microscopy shows that the these filaments react specifically with antibodies to bovine prekeratin and to murine vimentin. The aggregates contain aster-like foci common to the arrangement of both tonofilament-like and vimentin-containing intermediate-sized filaments, although both filament systems show different fibrillar patterns in other cytoplasmic regions. While the cytokeratin filament system is not significantly altered during exposure to colcemid, the vimentin in the abnormal aggregate is rearranged during such treatment into extensive and complex perinuclear ‘whorls’ of filaments. Treatment of the cells with butyrate results in a markedly flattened, hepatocyte-like morphology, a reappearance of typical actin-containing ‘cables’, and a progressive disintegration of the filament aggregate concomitant with a normal display of filaments of both the cytokeratin and vimentin type. The results show that (i) some cells contain aggregates consisting of two different types of intermediate-sized filaments oriented onto a common focal center; (ii) such an abnormal filament arrangement is clonally stable; (iii) the vimentin-type filaments contained in such aggregates are still susceptible to the action of antimitotic drugs and can be rearranged into characteristic perinuclear whorls; and (iv) this abnormal aggregate of intermediate filaments can be reverted to normal patterns upon treatment of the cells with butyrate.     

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.     

Simultaneous expression of two different types of intermediate sized filaments in mouse keratinocytes proliferating in vitro.

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.     

Simultaneous Expression of Two Different Types of Intermediate Sized Filaments in Mouse Keratinocytes Proliferating in vitro

The intermediate-sized filaments present in epidermal keratinocytes derived from mouse skin and in an established cell line (HEL) derived from spontaneous transformation of murine keratinocytes grown in vitro, have been examined by immunofluorescence microscopy, using antibodies directed against subunit proteins of different classes of intermediate-sized filaments, as well as by electron microscopy and gel electrophoresis of cytoskeletal preparations highly enriched in intermediate-sized filaments. The keratinocytes derived from neonatal skin, which are capable of only limited replication in vitro, show only a single type of intermediate-sized filaments, i.e., the tonofibril-like arrays of filaments containing prekeratin. HEL cells, which proliferate indefinitely in vitro, retain the tonofilament-like structures typical of differentiated epidermal cells but in addition display intermediate-sized filaments of the vimentin type, i.e., the filament system typically found in mesenchymal and mesenchyme-derived cells. We discuss the possibility that (i) the advent of vimentin-type filaments in epidermal cells in culture is related either to the transformed state or the in vitro growth conditions as such and (ii) other differentiated epithelial cells proliferating in vitro may have more than one system of intermediate-sized filaments.     

Autonomous circular and radial motions of the nucleus inPleurenterium tumidum and their relation to cytoskeletal elements and the plasma membrane

Summary InPleurenterium tumidum the nucleus leaves its central position at the end of cell development and moves centrifugally towards the cortical cytoplasm of the isthmus area. It passes between the chloroplast lobes and starts to perform circular motions along the cell wall ring of the isthmus independently from other cell organelles and cytoplasmic streaming. This autonomous nuclear motion is a unique phenomenon in plant cells which is reported here for the first time. One turn of the nucleus which may occur either clockwise or counter-clockwise lasts an average of 60 minutes. The velocity of circular nuclear motion lies between 0.03 and 0.08 m per second and increases with increasing number of nuclear turns. The nucleus undergoes at least 12 but sometimes up to 70 turns and may change its direction of motion several times. When circular nuclear motion is finished the nucleus migrates centripetally towards the cell center where the next mitosis takes place.Ultrastructural studies demonstrate that a distinct arrangement of the plasma membrane forming a ring-shaped fold together with the adjacent isthmus system of microtubules (IS) serves as a hoop-like track for the nucleus during the stage of circular motion. The nucleus moves along this track by surrounding it in a deep furrow which develops parallel to its longitudinal axis at its cell wall facing side. The spatial arrangement of the plasma membrane fold and the nuclear furrow are only present during the stage of circular nuclear motion. No actin filaments seem to be involved in the nuclear circulations since the nucleus continues its circular motions after cytochalasin B (CB) treatment even at concentrations which arrest cytoplasmic streaming. Amiprophos-methyl (APM) leads to an inhibition of circular nuclear motion which resumes when the APM solution is removed. Microtubules appear to be primarily responsible also for both the radial nuclear motions as well as the anchoring of the nucleus in its central position. The meaning of circular and radial nuclear motions for thePleurenterium cell is not yet clear, a relation between the nuclear behavior and the inner cell architecture is discussed and compared to that of other desmids.     

Cytoskeletal proteins connecting intermediate filaments to cytoplasmic and nuclear periphery

Intermediate filaments (IFs), together with microtubules and microfilaments build up the cytoskeleton of most eukaryotic cells. Cytoplasmic IFs form a dense filament network radiating from the nucleus and extending to the plasma membrane. The association between the cytoplasmic and nuclear surfaces appears to provide a continuous link important for the organisation of the cytoplasm, for cellular communication, and possibly for the transport into and out of the nucleus. Cytoplasmic IFs approach the nuclear surface, thin fibrils seem to connect the IFs with the nuclear pore complexes and a direct interaction of cytoplasmic IFs with the nuclear lamin B has been observed by in vitro binding studies. However, none of the components that cross-link IFs to the nucleus has been unambiguously identified. Furthermore, if a direct interaction between cytoplasmic IFs and the nuclear lamin B occurs in vivo, the question of how cytoplasmic IFs get access to the nuclear interior remains to be resolved. The association of IFs with the plasma membranes involves different components, some of which are cell type specific. Two specialised complexes in epithelial cells: the desmosome and the hemidesmosome, serve as attachment sites for keratin filaments. Desmoplakin is considered as the cross-linking component of IFs to the desmosomal plaque, whereas BPAG1 (bullous pemphigoid antigen) would cross-link IFs at the hemidesmosomal plaque. In other cell types the modality of how IFs are anchored to the plasma membrane is less well understood. It involves different components such as the spectrin based membrane skeleton, ankyrin, myosin, plectin and certainly many other still unravelled partners. Association between the IFs and cellular membranes plays an important role in determining cell shape and tissue integrity. Thus, the identification and characterisation of the components involved in these interactions will be crucial for understanding the function of intermediate filaments.     

Ultrastructural Observation on the Intra- and Intercellular Microtrabecular Network of the Pollen Mother Cells in Onion (Allium cepa)

Using DGD embedment-free electron microscopy, ultrastructural observation on the intra- and intercellular microtrabecular network (MN) of the pollen mother cells (PMC) of the whole meiotic prophase Ⅰ in onion ( Allium cepa L.) was performed. Complex nuclear MN was observed in the nucleus of PMCs, spreading throughout the nuclear region. The nucleolus and chromosomes were connected with the MN filament network. The uniformity of nuclear MN changed with the development of the PMCs. A lamina-like structure surrounded the nucleus and joined the MN in nucleus and in cytoplasm, but it disassembled at the end of prophase Ⅰ. There was also a complex cytoplasmic MN in PMCs, without obvious variation during the prophase Ⅰ. Furthermore, MN in cytoplasmic connections (plasmodesmata and cytoplasmic channels) was noticed to link the frameworks in two neighboring PMCs into one entity. Cytomixis was observed at synizesis of prophase Ⅰ in this experiment, and MN in cytoplasm and in nucleus was noticed to distribute in these granules which migrated from one PMC into its neighboring cell. At this time the nucleus moved aside from center of the PMC, but the rest of the cell was still fulfilled with MN filaments. The relationships of nuclear MN with nucleolus and chromosomes, lamina with nucleus, as well as intra- and intercellular MN with cytomixis are discussed in this paper.     

免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体－蛋白Ａ－胶体金所特异标记。本文结果表明,结合于核孔复合体上     

免疫胶体金标记显示波形纤维与核孔复合体的结合

中间纤维与细胞核的关系是一个亟待解决的重要问题。本文采用火鸡红细胞作为研究材料,首先用细胞分级抽提结合免疫印迹反应显示火鸡红细胞中间纤维蛋白为波形纤维蛋白。然后,我们采用细胞分级抽提结合包埋前免疫胶体金标记的方法显示胞质中间纤维被抗波形纤维蛋白抗体-蛋白A-胶体金特异标记。同时,我们显示结合于核孔复合体上的胞质纤维被抗波形纤维蛋白抗体-蛋白A-胶体金所特异标记。本文结果表明,结合于核孔复合体上的胞质纤维是波形纤维,并且提示波形纤维可能通过与Nup 180的结合附着在核孔复合体上,为进一步探讨中间纤维与核孔运输的关系提供了初步实验证据。     

Intermediate-sized filaments of the prekeratin type in myoepithelial cells

Myoepithelial cells from mammary glands, the modified sweat glands of bovine muzzle, and salivary glands have been studied by electron microscopy and by immunofluorescence microscopy in frozen sections in an attempt to further characterize the type of intermediate-sized filaments present in these cells. Electron microscopy has shown that all myoepithelial cells contain extensive meshworks of intermediate-sized (7--11-nm) filaments, many of which are anchored at typical desmosomes or hemidesmosomes. The intermediate-sized filaments are also intimately associated with masses of contractile elements, identified as bundles of typical 5--6-nm microfilaments and with characteristically spaced dense bodies. This organization resembles that described for various smooth muscle cells. In immunofluorescence microscopy, using antibodies specific for the various classes of intermediate-sized filaments, the myoepithelial cells are strongly decorated by antibodies to prekeratin. They are not specifically stained by antibodies to vimentin, which stain mesenchymal cells, nor by antibodies to chick gizzard desmin, which decorate fibrils in smooth muscle Z bands and intercalated disks in skeletal and cardiac muscle of mammals. Myoepithelial cells are also strongly stained by antibodies to actin. The observations show (a) that the epithelial character, as indicated by the presence of intermediate-sized filaments of the prekeratin type, is maintained in the differentiated contractile myoepithelial cell, and (b) that desmin and desmin-containing filaments are not generally associated with musclelike cell specialization for contraction but are specific to myogenic differentiation. The data also suggest that in myoepithelial cells prekeratin filaments are arranged--and might function--in a manner similar to the desmin filaments in smooth muscle cells.     

Toward a more complete 3-D structure of the nuclear pore complex

The nuclear pore complex (NPC) is a large supramolecular assembly embedded in the double-membraned nuclear envelope (NE) that plays a pivotal role in the exchange of macromolecules and particles between the nucleus and the cytoplasm. Applying various methods of sample preparation to Xenopus laevis whole nuclei and isolated NEs in combination with conventional transmission electron microscopy and digital image processing, we have characterized several distinct components of the NPC, including massive cytoplasmic and more tenuous nuclear rings, NPCs devoid of their cytoplasmic or both rings, and prominent "knobs" that protrude from the periphery of the NPC proper into the lumen of the NE. Moreover, by quick freezing/freeze drying/rotary metal shadowing isolated NEs, we have visualized two distinct types of NPC-associated filaments: (1) eight short, highly twisted filaments that project from the cytoplasmic ring and sometimes collapse into short cylinders; and (2) eight long, thin filaments that protrude from the nuclear ring and whose ends join to form a distal ring centered above the NPC such that the assembly resembles a "fishtrap." These nuclear fishtraps are sensitive to divalent cations: removal unfolds them and addition reforms them. The significance of these various structural components in terms of current NPC models is discussed, and the emerging asymmetry of the NPC relative to its nuclear and cytoplasmic face is stressed.     

Electron Microscopic Observations in Subacute Sclerosing Panencephalitis Brain Cell Cultures: Their Correlation with Cytochemical and Immunocytological Findings

Three cell cultures established from brain tissue obtained by biopsy of patients with subacute sclerosing panencephalitis (SSPE) were studied with the electron microscope in an attempt to correlate ultrastructural changes with those found by cytochemistry and immunocytology. These cells contained a large number of nucleocapsids resembling those of a paramyxovirus concentrated in the nuclear inclusions, but also seen free in the nucleus and occasionally in the cytoplasmic inclusions. Nuclear bodies associated with the nucleocapsids and granular filaments occupied a vast area of the cytoplasm. The nuclear inclusions containing nucleocapsids corresponded to the eosinophilic and fluorescent nuclear inclusions. The areas occupied by granular filaments corresponded to the diffuse cytoplasmic fluorescence. The ultrastructural changes were similar to those seen in the original brain biopsies. In addition papova-like virions were noted in brain cell cultures derived from a biopsy but not in the brain tissue itself. Their relationship to SSPE remains undetermined.     

Nucleus-associated actin in Amoeba proteus

The presence, spatial distribution and forms of intranuclear and nucleus-associated cytoplasmic actin were studied in Amoeba proteus with immunocytochemical approaches. Labeling with different anti-actin antibodies and staining with TRITC-phalloidin and fluorescent deoxyribonuclease I were used. We showed that actin is abundant within the nucleus as well as in the cytoplasm of A. proteus cells. According to DNase I experiments, the predominant form of intranuclear actin is G-actin which is associated with chromatin strands. Besides, unpolymerized actin was shown to participate in organization of a prominent actin layer adjacent to the outer surface of nuclear envelope. No significant amount of F-actin was found in the nucleus. At the same time, the amoeba nucleus is enclosed in a basket-like structure formed by circumnuclear actin filaments and bundles connected with global cytoplasmic actin cytoskeleton. A supposed architectural function of actin filaments was studied by treatment with actin-depolymerizing agent latrunculin A. It disassembled the circumnuclear actin system, but did not affect the intranuclear chromatin structure. The results obtained for amoeba cells support the modern concept that actin is involved in fundamental nuclear processes that have evolved in the cells of multicellular organisms.     

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.