Paired cisternae of endoplasmic reticulum in developing ovarian oocytes of the golden hamster

Paired cisternae of rough endoplasmic reticulum linked together by two parallel structures exhibiting periodic striations have been observed in the cytoplasm of small pre-antrum oocytes in the golden hamster. They are present only in oocytes from animals older than 3 weeks of age. Two or more such pairs may be associated with one another, and similar structures have been observed in contact with the nuclear envelope. The peak incidence of the paired membranes coincides with a sharp increase in rough endoplasmic reticulum as the oocyte commences rapid growth. A suggested role for the paired membranes in production of new endoplasmic reticulum is discussed.     

Is the nuclear envelope a ‘generator’ of membrane?

Summary Early diplotene oocytes from Necturus maculosus ranging from 0.2 to 0.5 mm in diameter were examined by electron microscopy. In the smallest oocytes of this range, the cytoplasm is largely devoid of membranes, but contains primarily ribosomes and mitochondria. In slightly larger oocytes, smooth-surfaced cytomembranes first appear in the perinuclear cytoplasm. At this time, the outer layer of the germinal vesicle nuclear envelope (GVNE) shows frequent connections with long membranous lamellae that extend for considerable, but variable distances into the juxtanuclear ooplasm. The number of smooth membranous lamellae increases tremendously as the oocytes increase in diameter. In such oocytes as well, frequent continuities are observed between the outer membrane of the GVNE and many of the cytoplasmic membranes. Eventually, as the ooplasm becomes populated with extensive numbers of membranous lamellae, instances of continuity between the membranous lamellae and nuclear envelope now become sparse and eventually non-existent. The frequent connections observed between membranous lamellae and the outer membrane of the GVNE during a circumscribed interval of diplotene strongly implicate the GVNE in the generation of extensive amounts of cytoplasmic membrane. The ooplasm of larger oocytes in the size range indicated contain numerous Golgi complexes and large quantities of annulate lamellae most of which are positioned in the peripheral or subcortical ooplasm, as well as extensive quantities of smooth membranes of the endoplasmic reticulum and lipid droplets.     

兔核移植胚胎起始发育的超微结构研究

对兔核移植胚胎起始发育的超微结构变化进行电镜观察,并与供体桑椹胚细胞,受体卵母细胞及同期正常受精胚胎的超微结构进行比较,“原核”期兔核移植胚胎的超微结构明显不同于供体桑椹胚细胞及受体卵母细胞的超微结构,而与同期正常受精胚胎相似,但有些核移植胚胎中皮质反应,及核仁和线粒体中出电子致密的网眼结构,与正常受精卵存在差别,分裂至２－细胞期时,与正常２－细胞胚超微结构更相似,结果提示,兔胚胎细胞核移植后,供     

Nuclear Pore Complex Structure in Birds

The nuclear envelope consists of two parallel membranes enclosing an aqueous lumen. In places there are pores in both membranes at which the two membranes are joined. Within these pores reside the nuclear pore complexes. The current structural models of the nuclear pore complex have been derived from a number of studies using different electron microscopical techniques. Recently, using surface imaging techniques such as field emission in-lens scanning electron microscopy, novel structures have been identified, particularly at the periphery of the structure, most notably the nucleoplasmic basket. One limitation of the current models is that they are based almost entirely on nuclear envelopes isolated from amphibian oocytes and a pressing question is whether this structure is the same in other organisms and tissues. Here we have studied the structure of nuclear envelopes isolated from bird oocytes. We show that the overall structure is remarkably conserved. In particular, recently discovered peripheral structures appear very similar. We see variations in basket conformation but believe that this is related to the functional states of individual pore complexes.     

Myelin-like structures as a possible source of the smooth endoplasmic reticulum in early amphibian oocytes

A comparative study of amphibian oocyte ultrastructural organization has shown a significant accumulation of elements of the smooth endoplasmic reticulum in the oocyte cytoplasm at the third stage of development. The analysis of oocytes of two frog species, Xenopus laevis and Rana temporaria, at the first and second stages of their development enabled us to recognize in the cytoplasm of the oocyte some myelin-like structures (MLs) made of 30-40 densely packaged membranous layers and shaped as dense bodies. MLs are also present in the adjacent follicular cells and in the intercellular space. In the oocyte cytoplasm these structures are located near the nuclear envelope and other intracellular organelles. At the third stage of oogenesis, which is characterized by a high functional activity of the cells, MLs are seen to unwrap sequentially into double-layer membranes similar to the smooth endoplasmic reticulum cisternae. Intermediate steps of this process being also observed. It is supposed that MLs may play the role of membrane stocks to be used eventually for the formation of nascent endoplasmic membranes in the amphibian oocytes.     

Interphase Nuclei of Many Mammalian Cell Types Contain Deep, Dynamic, Tubular Membrane-bound Invaginations of the Nuclear Envelope

The nuclear envelope consists of a doublemembraned extension of the rough endoplasmic reticulum. In this report we describe long, dynamic tubular channels, derived from the nuclear envelope, that extend deep into the nucleoplasm. These channels show cell-type specific morphologies ranging from single short stubs to multiple, complex, branched structures. Some channels transect the nucleus entirely, opening at two separate points on the nuclear surface, while others terminate at or close to nucleoli. These channels are distinct from other topological features of the nuclear envelope, such as lobes or folds.

The channel wall consists of two membranes continuous with the nuclear envelope, studded with features indistinguishable from nuclear pore complexes, and decorated on the nucleoplasmic surface with lamins. The enclosed core is continuous with the cytoplasm, and the lumenal space between the membranes contains soluble ER-resident proteins (protein disulphide isomerase and glucose-6-phosphatase).

Nuclear channels are also found in live cells labeled with the lipophilic dye DiOC₆. Time-lapse imaging of DiOC₆-labeled cells shows that the channels undergo changes in morphology and spatial distribution within the interphase nucleus on a timescale of minutes.

The presence of a cytoplasmic core and nuclear pore complexes in the channel walls suggests a possible role for these structures in nucleo–cytoplasmic transport. The clear association of a subset of these structures with nucleoli would also be consistent with such a transport role.

     

The nuclear envelope; its structure and relation to cytoplasmic membranes

An electron microscope study of thin sections of interphase cells has revealed the following:- Circular pores are formed in the double nuclear envelope by continuities between the inner and outer membranes which permit contact between the nucleoplasm and the cytoplasm unmediated by a well defined membrane. The pores, seen in sections normal to the nuclear envelope, are profiles of the ring-shaped structures described by others and seen in tangential section. The inner and outer nuclear membranes are continuous with one another and enclose the perinuclear space. The pores contain a diffuse, faintly particulate material. A survey of cells of the rat derived from the embryonic ectoderm, mesoderm, and endoderm, and of a protozoan and an alga has revealed pores in all tissues examined, without exception. It is concluded that pores in the nuclear envelope are a fundamental feature of all resting cells. In certain cells, the outer nuclear membrane is continuous with membranes of the endoplasmic reticulum, hence the perinuclear space is continuous with cavities enclosed by those membranes. There are indications that this is true for all resting cells, at least in a transitory way. On the basis of these observations, the hypothesis is made that two pathways of exchange exist between the nucleus and the cytoplasm; by way of the perinuclear space and cavities of the endoplasmic reticulum and by way of the pores in the nuclear envelope.     

THE NUCLEAR ENVELOPE : ITS STRUCTURE AND RELATION TO CYTOPLASMIC MEMBRANES

An electron microscope study of thin sections of interphase cells has revealed the following:— Circular pores are formed in the double nuclear envelope by continuities between the inner and outer membranes which permit contact between the nucleoplasm and the cytoplasm unmediated by a well defined membrane. The pores, seen in sections normal to the nuclear envelope, are profiles of the ring-shaped structures described by others and seen in tangential section. The inner and outer nuclear membranes are continuous with one another and enclose the perinuclear space. The pores contain a diffuse, faintly particulate material. A survey of cells of the rat derived from the embryonic ectoderm, mesoderm, and endoderm, and of a protozoan and an alga has revealed pores in all tissues examined, without exception. It is concluded that pores in the nuclear envelope are a fundamental feature of all resting cells. In certain cells, the outer nuclear membrane is continuous with membranes of the endoplasmic reticulum, hence the perinuclear space is continuous with cavities enclosed by those membranes. There are indications that this is true for all resting cells, at least in a transitory way. On the basis of these observations, the hypothesis is made that two pathways of exchange exist between the nucleus and the cytoplasm; by way of the perinuclear space and cavities of the endoplasmic reticulum and by way of the pores in the nuclear envelope.     

Nuclear surface complex as observed with the high resolution scanning electron microscope. Visualization of the membrane surfaces of the neclear envelope and the nuclear cortex from Xenopus laevis oocytes

The nuclear envelope and associated structures from Xenopus laevis oocytes (stage VI) have been examined with the high resolution scanning electron microscope (SEM). The features of the inner and outer surfaces of the nuclear surface complex were revealed by manual isolation , whereas the membranes facing the perinuclear space (the space between the inner and outer nuclear membranes) were observed by fracturing the nuclear envelope in this plane and splaying the corresponding regions apart. Pore complexes were observed on all four membrane surfaces of this double-membraned structure. The densely packed pore complexes (55/micron2) are often clustered into triplets with shared walls (outer diameter = 90 nm; inner diameter = 25 nm; wall thickness = aproximately 30 nm), and project aproximately 20 nm above each membrane except where they are flush with the innermost surface. The pore complex appears to be an aggregate of four 30-nm subunits. The nuclear cortex, a fibrous layer (300 nm thickness) associated with the inner surface of the nuclear envelope, has been revealed by rapid fixation. This cortical layer is interrupted by funnel-shaped intranuclear channels (120-640 nm diam) which narrow towards the pore complexes. Chains of particles, arranged in spirals, are inserted into these intranuclear channels. The fibers associated with the innermost face of the nuclear envelope can be extraced with 0.6 MKI to reveal the pore complexes. A model of the nuclear surface complex, compiled from the visualization of all the membrane faces and the nuclear cortex, demonstrates relations between the intranuclear channels (3.2/micron2) and the numerous pore complexes, and the possibility of their role in nucleocytoplasmic interactions.     

NEGATIVE STAINING AND ADENOSINE TRIPHOSPHATASE ACTIVITY OF ANNULATE LAMELLAE OF NEWT OOCYTES

Semi-isolated annulate lamellae were prepared from single newt oocytes (Triturus alpestris) by a modified Callan-Tomlin technique. Such preparations were examined with the electron microscope, and the negative staining appearance of the annulate lamellae is described. The annulate lamellae can be detected either adhering to the nuclear envelope or being detached from it. Sometimes they are observed to be connected with slender tubular-like structures interpreted as parts of the endoplasmic reticulum. The results obtained from negative staining are combined with those from sections. Especially, the structural data on the annulate lamellae and the nuclear envelope of the very same cell were compared. Evidence is presented that in the oocytes studied the two kinds of porous cisternae, namely annulate lamellae and nuclear envelope, are markedly distinguished in that the annulate lamellae exhibit a much higher pore frequency (generally about twice that found for the corresponding nuclear envelope) and have also a relative pore area occupying as much as 32% to 55% of the cisternal surface (compared with 13% to 22% in the nuclear envelopes). The pore diameter and all other ultrastructural details of the pore complexes, however, are equivalent in both kinds of porous cisternae. Like the annuli of the nuclear pore complexes of various animal and plant cells, the annuli of the annulate lamellae pores reveal also an eightfold symmetry of their subunits in negatively stained as well as in sectioned material. Furthermore, the annulate lamellae are shown to be a site of activity of the Mg-Na-K-stimulated ATPase.     

An electron microscopic study of the human infundibulum

Summary For a limited period during the oogenesis of Protopterus, blebs of the perinuclear cistern contain, in addition to other inclusions, a special kind of microtubular elements. Most of these blebs face parts of multiple nucleolar bodies that extend toward and make contact with the inner nuclear membrane. The microtubular lumen contains a finely dispersed material of moderate electron density which seems to be in contact with this nucleolar material.Aside from these intracisternal structures there are, within both the perinuclear cytoplasm and the nucleoplasm, similar microtubular arrays without apparent connection with the nuclear envelope. These are either enclosed by membranes derived from those of the envelope or unconfined, having escaped through breaks in their respective bounding membranes. Extracisternal tubules are presumed to have passed their period of putative functional activity and to be undergoing a process of regression and subsequent disintegration.Among possible roles attributable to the intracisternal microtubular apparatus are the following: (1) It may serve for the transport of special nucleolar components to the cytoplasm, possibly to be incorporated in the matrix of developing perinuclear mitochondria; (2) it may provide openings in the nuclear membranes for the direct passage of particulate elements between nucleus and cytoplasm; (3) it may be instrumental in the breakdown of parts of the nuclear envelope prior to its restitution during the subsequent phase of oogenesis.Supported by grants NB-00840 and NB-05219 from the U.S.P.H.S.     

Self-organization of endoplasmic reticulum aggregates in cells with overexpression of nucleoporin POM121

The nuclear pore complexes are complex protein structures located in the nuclear envelope, where they control the nuclear-cytoplasmic transport, and inside the stacks of endoplasmic reticulum cisternae, annulate lamellae. After overexpression of some nucleoporins, numerous granules are visible in the cytoplasm. According to the published data, these granules are the annulate lamellae. In the current paper, the structural organization of POM121-containing granules was analyzed using correlative light and electron microscopy. The ultrastructural study demonstrates that POM121-containing granules are not annulate lamellae but aggregates of endoplasmic reticulum membranes. Thus, overexpressed POM121 is not able to induce the annulate lamella formation. The mechanisms of self-organization of non-functional structures (such as the aggregates of endoplasmic reticulum membranes described here) and possible involvement of these mechanisms in the formation of cellular structures are discussed.     

Changes in Xenopus oocyte nucleus and cytoplasm organization after actin filaments depolymerization by latrunculin

Actin-containing filaments have been visualized inside the Xenopus oocyte nuclei due to combination of fluorescence and transmission electron microscopy. It has been shown that these filaments contact with nucleoli, spherical bodies and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing latrunculin causes membrane vesiculation in the cytoplasm, and disruption of the nucleoplasm and nuclear envelope integrity. We suppose that actin-containing filaments belong to crucial cell components which are involved in coordination of nuclear-cytoplasmic interactions as well as distribution and transport of intranuclear components in growing Xenopus oocytes.     

Oogenesis in the terrestrial hermit crab Coenobita clypeatus (decapoda,anomura). I. Previtellogenic oocytes

Ultrastructural study of previtellogenic oocytes found in cystlike clusters scattered throughout the length of the bilobed ovary of the hermit crab Coenobita clypeatus shows a high nuclear:cytoplasm ratio. Large, round nuclei containing synaptinemal complexes serve as good temporal markers for identification of previtellogenic oocytes. The cytoplasm contains many smooth-membraned vesicles filled with granules and probably of nuclear origin. In addition to its complement of Golgi complexes, endoplasmic reticulum, mitochondria, and free ribosomes, the cytoplasm also contains stacks of annulate lamellae, a feature not previously described for decapod oocytes. Typically, the previtellogenic oocyte with its accumulation of ribosomes has the appearance of a nonsynthetic cell preparing to go through a metabolic transition.     

Ultrastructure of cell fusion and premature chromosome condensation (PCC) of thymocyte nuclei in metaphase II mouse oocytes

Following PEG (polyethylene glycol) treatment of ovulated metaphase II mouse oocytes aggregated with thymocytes, fusion of cell membranes occurs. Prerequisites for cell fusion are: close apposition of lectin-agglutinated (phytohemagglutinin-treated) membranes of both cells, formation of firm punctual adhesion sites, and expansion of adhesion sites over a certain area. Establishment of the firm cell-cell contact is associated with development of actin-like filaments along both of the adhering plasma membranes. Membrane fusion occurs at single or multiple sites, and is followed by internalization of thymocyte-oocyte membrane complexes decorated with actin filaments into the hybrid cell cytoplasm. A filamentous actin layer forms also along the inner surface of newly formed hybrid oocyte-thymocyte plasma membrane. Thymocyte nuclei incorporated into oocyte cytoplasm undergo nuclear envelope breakdown and premature chromosome condensation (PCC) leading, eventually, to formation of single chromatids complete with kinetochores. Concomitantly with chromatin condensation an extensive polymerization of microtubules starts in the center of the chromatin mass which leads to the formation of an apparently non-functional spindle-like structure.     

Nuclear and cytoplasmic organization in Xenopus oocytes after disruption of actin filaments by latrunculin

Actin-containing filaments have been visualized inside Xenopus oocyte nuclei by a combination of fluorescence and transmission electron microscopy. It was shown that these filaments contact nucleoli, spherical bodies, and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing agent, latrunculin, caused membrane vesiculation in cytoplasm and the disruption of nucleoplasm and the integrity of the nuclear envelope. We suggest that actin-containing filaments are important cell components involved in the regulation of nucleus-cytoplasm interactions, as well as of cellular transport of components during the growth of Xenopus oocytes.     

Mediators of Nuclear Protein Import Target Karyophilic Proteins to Pore Complexes of Cytoplasmic Annulate Lamellae

Nuclear pore complexes are constitutive structures of the nuclear envelope in eukaryotic cells and represent the sites where transport of molecules between nucleus and cytoplasm takes place. However, pore complexes of similar structure, but with largely unknown functional properties, are long known to occur also in certain cytoplasmic cisternae that have been termed annulate lamellae (AL). To analyze the capability of the AL pore complex to interact with the soluble mediators of nuclear protein import and their karyophilic protein substrates, we have performed a microinjection study in stage VI oocytes ofXenopus laevis.In these cells AL are especially abundant and can easily be identified by light and electron microscopy. Following injection into the cytoplasm, fluorochrome-labeled mediators of two different nuclear import pathways, importin β and transportin, not only associate with the nuclear envelope but also with AL. Likewise, nuclear localization signals (NLS) of the basic and M9 type, but not nuclear export signals, confer targeting and transient binding of fluorochrome-labeled proteins to cytoplasmic AL. Mutation or deletion of the NLS signals prevents these interactions. Furthermore, binding to AL is abolished by dominant negative inhibitors of nuclear protein import. Microinjections of gold-coupled NLS-bearing proteins reveal specific gold decoration at distinct sites within the AL pore complex. These include such at the peripheral pore complex-attached fibrils and at the central “transporter” and closely resemble those of “transport intermediates” found in electron microscopic studies of the nuclear pore complex (NPC). These data demonstrate that AL can represent distinct sites within the cytoplasm of transient accumulation of nuclear proteins and that the AL pore complex shares functional binding properties with the NPC.     

Reconstitution of the Golgi reassembly process in semi-intact MDCK cells

The Golgi apparatus, which consists of stacks of cisternae during interphase, is fragmented or dispersed throughout the cytoplasm at the onset of mitosis. A sea sponge metabolite, ilimaquinone (IQ), causes Golgi membranes to vesiculate. And after its removal, the vesiculated membranes reassemble into stacks of cisternae in the perinuclear region. To study the mechanism of Golgi membrane dynamics during mitosis, we have reconstituted the reassembly process of IQ-induced vesiculated Golgi membranes in streptolysin O-permeabilized Mardin-Darby canine kidney (MDCK) cells. Monitoring the dynamics of Golgi membranes labeled with a green fluorescence protein (GFP)-tagged protein, we dissected the process into two elementary components: the reassembly of vesiculated Golgi membranes into punctate structures; and the subsequent reformation of these structures into stacks of cisternae near the nucleus. Using morphometric analysis, we studied the kinetics and biochemical requirements for the process, and revealed that an NEM-sensitive factor, cytoplasmic dynein, and GTP binding protein were involved in the Golgi reassembly.     

Isolation and fractionation of rat liver nuclear envelopes and nuclear pore complexes

The nuclear envelope is a double lipid bilayer that physically separates the functions of the nucleus and the cytoplasm of eukaryotic cells. Regulated transport of molecules between the nucleus and the cytoplasm is essential for normal cell metabolism and is mediated by large protein complexes, termed nuclear pore complexes (NPCs), which span the inner and outer membranes of the nuclear envelope. Significant progress has been made in the past 10 years in identifying the protein composition of NPCs and the basic molecular mechanisms by which these complexes facilitate the selective exchange of molecules between the nucleus and the cytoplasm. However, many fundamentally important questions about the functions of NPCs, the specific functions of individual NPC-associated proteins, and the assembly and disassembly of NPCs, remain unanswered. This review describes approaches for isolating and characterizing nuclear envelopes and NPC-associated proteins from mammalian cells. It is anticipated that these procedures can be used as a starting point for further molecular and biochemical analysis of the mammalian nuclear envelope, NPCs, and NPC-associated proteins.