Spontaneous assembly of pore complex-containing membranes ("annulate lamellae") in Xenopus egg extract in the absence of chromatin.

Extract prepared from activated Xenopus eggs is capable of reconstituting nuclei from added DNA or chromatin. We have incubated such extract in the absence of DNA and found that numerous flattened membrane cisternae containing densely spaced pore complexes (annulate lamellae) formed de novo. By electron and immunofluorescence microscopy employing a pore complex-specific antibody we followed their appearance in the extract. Annulate lamellae were first detectable at a 30-min incubation in the form of short cisternae which already contained a high pore density. At 90-120 min they were abundantly present and formed large multilamellar stacks. The kinetics of annulate lamellae assembly were identical to that of nuclear envelope formation after addition of DNA to the extract. However, in the presence of DNA or chromatin, i.e., under conditions promoting the assembly of nuclear envelopes, annulate lamellae formation was considerably reduced and, at sufficiently high chromatin concentrations, completely inhibited. Incubation of the extract with antibodies to lamin LIII did not interfere with annulate lamellae assembly, whereas in the presence of DNA formation of nuclear envelopes around chromatin was inhibited. Our data show that nuclear membrane vesicles are able to fuse spontaneously into membrane cisternae and to assemble pore complexes independently of interactions with chromatin and a lamina. We propose that nuclear envelope precursor material will assemble into a nuclear envelope when chromatin is available for binding the membrane vesicles, and into annulate lamellae when chromatin is absent or its binding sites are saturated.     

INTRANUCLEAR AND CYTOPLASMIC ANNULATE LAMELLAE IN TUNICATE OOCYTES

Electron microscope studies were made on various tunicate oocytes at different stages of growth and development. Both the inner and outer lamellae of the perforated nuclear envelope demonstrate considerable blebbing activity. The blebs of the inner lamella detach into the nucleoplasm where they undergo a special type of fusion process resulting in the formation of numerous, usually single, differentiated annulate lamellae of various lengths. The blebbing of the outer layer of the nuclear envelope contributes to the vesicular and granular endoplasmic reticulum characteristically present in the ooplasm and perhaps to the differentiation of cytoplasmic annulate lamellae as well. Cytoplasmic stacks of annulate lamellae frequently have ribosomes associated with them. In addition, granular accumulations are sometimes observed around or between the annuli. The morphological evidence suggests that, at least in many cases, the annuli in the annulate lamellae are patent.     

Structural organization and possible functional role of annulate lamellae containing cytoplasmic pores

This review is devoted to annulate lamellae, a specific compartment of endoplasmic reticulum that occurs, presumably, in actively growing and rapidly dividing cells (oocytes, embryonic and tumor cells). We summarized both earlier and recent data on the dustribution of annulate lamellae in various cell types, on their morphology, and the distribution of interaction with intracellular structures at various treatments. As the annulate lamellae contain cytoplasmic pore complexes, a special attention was paid to their relation with nuclear pores. Possible functions of the annulate lamellae in intracellular processes and, particularly, in nuclear envelope assembly, are discussed.     

Cytoplasmic annulate lamellae in human spermatogenesis

Summary Electron microscopic examination of normal human testicular tissue revealed annulate lamellae (AL) in the cytoplasm of primary spermatocytes and spermatids. AL of primary spermatocytes are encountered in the perinuclear region, parallel to the nuclear envelope and form single or multiple membranous profiles containing numerous annuli (500–600 Å in diameter) frequently associated with a fibrillar electron dense material. Spermatids contain numerous layers of AL either continuous with the nuclear envelope and caudal to the acrosome or peripherally positioned in the cytoplasm. Individual lamellae possess terminal dilations and display continuities with the endoplasmic reticulum. The interlamellar space in spermatid AL is entirely filled with a fine granular electron dense material. Additionally, the break-down of AL in spermatozoan residual bodies is indicated by a dilation of AL cisternae to form vacuoles following the dissolution of pore complexes.Supported in part by grant (AT-(40-1)-4002) from the U.S. Atomic Energy Commission     

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.     

Annulate lamellae and "yolk nuclei" in oocytes of the dragonfly, Libellula pulchella

Annulated membranes in the form of single and short lamellae are present adjacent to and parallel to the nuclear envelope in oogonia and early oocyte (synaptene) stages of the dragonfly, Libellula pulchella. These solitary and short annulate lamellae are usually continuous with long, part rough- and part smooth-surfaced cisternae which extend into more distal areas of the oogonial ooplasm. These particular annulate lamellae then either disappear or decrease in number to be replaced by a much more extensive system of annulate lamellae in the cortical ooplasm of previtellogenic oocytes. The differentiation of extensive stacks of annulate lamellae is consistently observed to be restricted to large cytoplasmic areas of considerable electron density. These cytoplasmic regions consist of material which stains basophilic and contains RNA but differs structurally from the large number of ribosomes which surround the dense masses. The cytoplasmic dense masses, in terms of their formation and staining reactions, are comparable to the "yolk nuclei" or "Balbiani bodies" described in insect oocytes in earlier studies. The results of the present study thus provide evidence that the appearance of cortical ooplasmic stacks of annulate lamellae in the dragonfly oocyte is specifically limited to cytoplasmic areas of high electron density which contain RNA but which do not have a ribosomal morphology.     

Nuclear pore complex assembly studied with a biochemical assay for annulate lamellae formation

Formation of the nuclear pore is an intricate process involving membrane fusion and the ordered assembly of up to 1,000 pore proteins. As such, the study of pore assembly is not a simple one. Interestingly, annulate lamellae, a cytoplasmic organelle consisting of stacks of flattened membrane cisternae perforated by numerous pore complexes, have been found to form spontaneously in a reconstitution system derived from Xenopus egg extracts, as determined by electron microscopy (Dabauvalle et al., 1991). In this work, a biochemical assay for annulate lamellae (AL) formation was developed and used to study the mechanism of AL assembly in general and the assembly of individual nucleoporins into pore complexes in particular. Upon incubation of Xenopus egg cytosol and membrane vesicles, the nucleoporins nup58, nup60, nup97, nup153, and nup200 initially present in a disassembled form in the cytosol became associated with membranes and were pelletable. The association was time and temperature dependent and could be measured by immunoblotting. Thin-section electron microscopy as well as negative staining confirmed that annulate lamellae were forming coincident with the incorporation of pore proteins into membranes. Homogenization and subsequent flotation of the membrane fraction allowed us to separate a population of dense membranes, containing the integral membrane pore protein gp210 and all other nucleoporins tested, from the bulk of cellular membranes. Electron microscopy indicated that annulate lamellae were enriched in this dense, pore protein-containing fraction. GTP gamma S prevented incorporation of the soluble pore proteins into membranes. To address whether AL form in the absence of N-acetylglucosaminylated pore proteins, AL assembly was carried out in WGA-sepharose-depleted cytosol. Under these conditions, annulate lamellae formed but were altered in appearance. When the membrane fraction containing this altered AL was homogenized and subjected to flotation, the pore protein- containing membranes still sedimented in a distinct peak but were less dense than control annulate lamellae.     

The origin and fate of annulate lamellae in maturing sand dollar eggs

Electron micrograph evidence is presented that the nuclear envelope of the mature ovum of Dendraster excentricus is implicated in a proliferation of what appear as nuclear envelope replicas in the cytoplasm. The proliferation is associated with intranuclear vesicles which apparently coalesce to form comparatively simple replicas of the nuclear envelope closely applied to the inside of the nuclear envelope. The envelope itself may become disorganized at the time when fully formed annulate lamellae appear on the cytoplasmic side and parallel with it. The concept of interconvertibility of general cytoplasmic vesicles with most of the membrane systems of the cytoplasm is presented. The structure of the annuli in the annulate lamellae is shown to include small spheres or vesicles of variable size embedded in a dense matrix. Dense particles which are about 150 A in diameter are often found closely associated with annulate lamellae in the cytoplasm. Similar structures in other echinoderm eggs are basophilic. In this species, unlike other published examples, the association apparently takes place in the cytoplasm only after the lamellae have separated from the nucleus. If 150 A particles are synthesized by annulate lamellae, as their close physical relationship suggests, then in this species at least the necessary synthetic mechanisms and specificity must reside in the structure of annulate lamellae.     

The Origin and Fate of Annulate Lamellae in Maturing Sand Dollar Eggs

Electron micrograph evidence is presented that the nuclear envelope of the mature ovum of Dendraster excentricus is implicated in a proliferation of what appear as nuclear envelope replicas in the cytoplasm. The proliferation is associated with intranuclear vesicles which apparently coalesce to form comparatively simple replicas of the nuclear envelope closely applied to the inside of the nuclear envelope. The envelope itself may become disorganized at the time when fully formed annulate lamellae appear on the cytoplasmic side and parallel with it. The concept of interconvertibility of general cytoplasmic vesicles with most of the membrane systems of the cytoplasm is presented. The structure of the annuli in the annulate lamellae is shown to include small spheres or vesicles of variable size embedded in a dense matrix. Dense particles which are about 150 A in diameter are often found closely associated with annulate lamellae in the cytoplasm. Similar structures in other echinoderm eggs are basophilic. In this species, unlike other published examples, the association apparently takes place in the cytoplasm only after the lamellae have separated from the nucleus. If 150 A particles are synthesized by annulate lamellae, as their close physical relationship suggests, then in this species at least the necessary synthetic mechanisms and specificity must reside in the structure of annulate lamellae.     

Fibrogranular Material,Annulate Lamellae and Microtubules During Spermiogenesis in Drosophila melanogaster

During spermiogenesis in Drosophila melanogaster, a “perinuclear plasm’ accumulates between the fenestrated portion of the nuclear envelope and an adjacent lamella of ER in the young spermatid. Microtubules appear within the perinuclear plasm and become especially concentrated in a nuclear concavity. Cytoplasmic pores are present locally within the lamella of ER. In addition, localized or discrete bodies composed of fibrogranular material become closely associated with single pore complexes in the lamella of ER. A close association exists between pore complexes (annulate lamellae), the small granular and fibrillar subunits of the fibrogranular bodies, polyribosomes and the nuclear-associated microtubules during much of spermiogenesis. While the fibrogranular material becomes less concentrated during spermiogenesis, the number of pore complexes in a single section increases such that two, three or even four short annulate lamellae are intercalated within many longitudinally oriented microtubules which are present in the furrow of the spermatid nucleus. Structural relationships observed between cytoplasmic pores (annulate lamellae), fibrogranular bodies, polyribosomes and microtubules are discussed in relation to information about the timing of RNA and protein synthesis. This study extends previous observations about the distribution and structural variations of annulate lamellae elsewhere in the spermatid cytoplasm.     

Annulate lamellae in plant cells: Formation during microsporogenesis and pollen development in Canna generalis Bailey

Summary The occurrence of stacked annulate lamellae is documented for a plant cell system, namely for pollen mother cells and developing pollen grains of Canna generalis. Their structural subarchitecture and relationship to endoplasmic reticulum (ER) and nuclear envelope cisternae is described in detail. The results demonstrate structural homology between plant and animal annulate lamellae and are compatible with, though do not prove, the view that annulate lamellar cisternae may originate as a degenerative form of endoplasmic reticulum.     

Purification of the vertebrate nuclear pore complex by biochemical criteria

The nuclear pore is a large and complex biological machine, mediating all signal-directed transport between the nucleus and the cytoplasm. The vertebrate pore has a mass of ∼120 million daltons or 30 times the size of a ribosome. The large size of the pore, coupled to its tight integration in the nuclear lamina, has hampered the isolation of pore complexes from vertebrate sources. We have now developed a strategy for the purification of nuclear pores from in vitro assembled annulate lamellae (AL), a cytoplasmic mimic of the nuclear envelope that lacks a lamina, nuclear matrix, and chromatin-associated proteins. We find that purified pore complexes from annulate lamellae contain every nuclear pore protein tested. In addition, immunoblotting reveals the presence of soluble transport receptors and factors known to play important roles in the transport of macromolecules through the pore. While transport factors such as Ran and NTF2 show only transient interaction with the pores, a number of soluble transport receptors, including importin β, show a tight association with the purified pores. In summary, we report that we have purified the vertebrate pore by biochemical criteria; silver staining reveals ∼40–50 distinct protein bands.     

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.     

Intranuclear and cytoplasmic annulate lamellae in grasshopper spermatocytes (Genus Melanoplus)

Intranuclear and cytoplasmic annulate lamellae were studied in grasshopper spermatocytes (Melanoplus) with the electron microscope. Although cytoplasmic annulate lamellae were observed in all three species examined, intranuclear annulate lamellae were found in only one species. The intranuclear annulate lamellae encompass certain nuclear material adjacent to the nuclear envelope forming a vesicle that is extruded into the spermatocyte cytoplasm. In this same species, cytoplasmic annulate lamellae are seen contiguous with granular masses of varying size. These structures were noted as being morphologically indistinguishable from the "yolk nuclei" of dragonfly oocytes (Kessel and Beams, 1969; Kessel, 1973).     

非细胞体系核重建过程中两类膜泡在环形片层及核被膜形成中的作用

非洲爪蟾卵经钙离子载体A 23187激活后,在10,000g下离心得到爪蟾卵提取物。Lambda DNA加入上述提取物可构建出染色质结构,并在染色质表面重建核被膜,同时在染色质外的区域形成环形片层。核被膜与环形片层有相似的发生途径,它们都是由两类在形态、大小、膜结构上有明显差别的膜泡融合而来。首先是直径200nm的圆形小膜泡相互融合成双层膜片层,同时核孔复合体在双层膜上大量装配,以这些双层膜片层为基础,光滑的大膜泡与之融合导致环形片层的扩张与核被膜的成熟。     

Ultrastructural observations on the oogenesis of Triops cancriformis (Crustacea,Notostraca)

Summary The first stages of the oogenesis of Triops cancriformis have been studied. At the outset the oocyte is smaller than the nurse cells. Meiosis begins with typical synaptonemal complexes. The significance of these complexes and of some other peculiar structures of germ cells, i.e., pore complexes and annuli within the nucleus, and annulate lamellae within the cytoplasm are discussed. The morphofunctional uniformity of some cytoplasmic structures (annulate lamellae, concentrically arranged ER, and yolk globules) in the oocyte as well as its nurse cells is also discussed.     

ELECTRON MICROSCOPE STUDIES ON THE ORIGIN OF ANNULATE LAMELLAE IN OOCYTES OF NECTURUS

Developing oocytes, ranging from approximately 0.1 to 1.0 mm in diameter, in Necturus were studied with the electron microscope. The outer layer of the nuclear envelope is actively engaged in the formation of vesicular elements along most of its surface, especially in smaller oocytes. Groups of vesicles appear to be released into the ooplasm at about the same time, resulting in long chains of individual vesicles immediately adjacent to the nuclear membrane. This process is repeated so that chains of vesicles grouped in rather ordered ranks extend progressively into the surrounding cytoplasm. Eventually, the cytoplasm becomes more concentrated with chains of vesicles and the distance between the individual rows becomes less. Very soon after a chain of vesicles has been budded off from the nuclear membrane, fine intervesicular connections appear between certain of the vesicles comprising the rows. Several of the vesicles in a row may then fuse, forming short, flattened cisternae. Fusion of vesicles continues, individual rows of vesicles become more closely packed and, finally, regions appear in the cytoplasm which have the appearance of annulate lamellae. Further growth of the lamellae appears to occur by the progressive fusion of vesicles at the ends of those lamellae already present, as well as by the addition of other fusing rows of vesicles.     

Accumulation of Annulate Lamellae in the Subcortical Layer during Progesterone-induced Oocyte Maturation in Xenopus Laevis

Changes in the fine structure, the location and the number of stacks of annulate lamellae during progesterone-induced maturation of oocytes of Xenopus were determined by electron microscopy. longitudinal sections of full-grown oocytes, about 260 stacks of annulate lamellae were observed with marked concentration in the subcortical layer, particularly in the vegetal hemisphere. After exposure to. progesterone, annulate lamellae increased and accumulated further in the subcortical layer. A significant increase of annulate lamellae around the vegetal side of the germinal vesicle seen 3 h after progesterone application. In oocytes 6 h after progesterone application, an average of 320 stacks of annulate lamellae were seen in longitudinal sections and more than two-thirds of the pore complexes of annulate lamellae were localized in the subcortical layer less than 50 from the oocyte surface, the rest being distributed in the deeper ooplasm. At the time of ger- minal vesicle breakdown, all the annulate lamellae underwent complete decomposition. The results were discussed from the view point of comparative developmental biology.     

Microtubule-dependent assembly of the nuclear envelope in Xenopus laevis egg extract

Microtubules take part in several mechanisms of intracellular motility, including organelle transport and mitosis. We have studied the ability of Xenopus egg extract to support nuclear membrane and pore complex formation when microtubule dynamics are manipulated. In this report we show that the formation of a nuclear envelope surrounding sperm chromatin requires polymerized microtubules. We have observed that microtubule-depolymerizing reagents, and AS-2, a known inhibitor of the microtubule motor protein kinesin, do not inhibit the formation of a double nuclear membrane. However these double membranes contain no morphologically identifiable nuclear pore complexes and do not support the accumulation of karyophilic proteins. In contrast, the assembly of annulate lamellae, cytoplasmic structures containing a subset of pore complex proteins, was not affected. Our data show that not only polymerized microtubules, but also the microtubule motor protein kinesin, are involved in the formation of the nuclear envelope. These results support the conclusion that multiple nuclear envelope-forming mitotic vesicle populations exist, that microtubules play an essential and selective role in the transport of nuclear envelope-forming vesicle population(s), and that separate mechanisms are involved in nuclear envelope and annulate lamellae formation.     

The origin of annulate lamellae in the oocyte of the ascidian,Boltenia villosa stimpson

Summary The formation of the extranuclear annulate lamellae has been revealed to be connected with a process of nuclear emission which is very active during the previtellogenetic stages of the Boltenia oocyte development. This process involves both of the nuclear membranes. At many spots on the surface of the nuclear envelope, the outer membrane pulls away from the inner membrane, thus forming what has been designated as blisters of various sizes and shapes. Masses of nuclear content, apparently not from the nucleolus, are pushed into the blisters. These blisters may become detached from the nuclear envelope and lie free in the cytoplasm. But in many cases, the detachment seems delayed, and in each blister many emission masses are squeezed tightly together and flat one on top of the other. These masses, in sections, may present the appearance of a stack of elongated outlines. The membrane, limiting any two adjacent masses in close contact, develop annuli. It is thus that an annulate lamella is formed. Whether an annulate lamella is formed between a pair of neighboring masses depends on their proximity. So the production of the annulate lamellae is incidental to, but not a necessary part of the process of nuclear emission. After the original outer nuclear membrane forming the blister has disintegrated, the annulate lamellae are left exposed in the cytoplasm.It is clear that, 1. both membranes of an annulate lamella are of inner nuclear membrane origin, 2. they hold between them some of the content of the enlarged perinuclear space resulting from the raising of the outer nuclear membrane when the blister is formed, and 3. the material held between any two lamellae is from the nucleus.The intranuclear annulate lamellae simply arise from the narrow pouches formed by the inner nuclear membrane towards the interior of the nucleus, and on these narrow pouches annuli are developed. So the intranuclear annulate lamellae is also composed of two membranes of an inner nuclear membrane origin holding between them a quantity of the content of the perinuclear space.Supported by Grant GM-11858 of National Institute of Health. The author is indebted to Dr. Richard Cloney of the Department of Zoology, University of Washington, for the use of the electron microscope.