Differentiation of Acmaea digitalis oocytes with special reference to lipid-endoplasmic reticulum-annulate lamellae-polyribosome relationships

During initial stages of oogenesis, many nucleoli are adpressed to the inner membrane of the nuclear envelope. Small nucleolar fragments appear to traverse the pores of the nuclear envelope and accumulate in the perinuclear ooplasm as fibrogranular bodies. Mitochondria become closely associated with some of the fibrogranular bodies. In addition to ribosomes and polyribosomes that are present in small oocytes, lamellae of rough-surfaced endoplasmic reticulum (rER) increase greatly in number during early stages of differentiation. Some individual lamellae are attached at their ends to the outer membrane of the nuclear envelope. Many parallel lamellae of rER are then encountered as well as numerous circular profiles consisting of concentric loops of rER. Soon after the differentiation of the extensive system of rER, lipid droplets or spheres appear in the ooplasm and they are initially surrounded by many circular, concentric lamellae of rER. Initially, the number of concentric lamellae of rER surrounding a lipid droplet may vary from less than a dozen to more than two dozen. During middle and late phases of vitellogenesis, most of the lipid spheres that comprise the most numerous and significant component of the yolk are surrounded by only one or two concentric lamellae of rER (in some cases the lamellae are part rough-surfaced and part smooth-surfaced). In addition, annulate lamellae are then observed to be associated with a portion of the lipid droplet surface. The number of annulate lamellae that extend focally from the lipid sphere distally into the cytoplasm is variable; often two or three to more than a dozen lamellae. Small granules, many of which range from 6 to 12 nm and thin fibrils (approximately 2–3 nm in width) may be associated with the annulate lamellae. In addition, polyribosomes frequently appear to be continuous with the pore-associated material of the annulate lamellae. The ends of some annulate lamellae may extend as lamellae of the rER. The morphologic relationships and relationships and variations observed between the lipid droplets, rER, annulate lamellae, and polyribosomes during lipidogenesis in this oocyte are interpreted to support a recent hypothesis (Kessel, 1981a,b) that the pores of annulate lamellae may be involved in some manner with the processing of ribosomal subunits or precursors into functioning polyribosomes, and that their appearance in specific association with the surface of many lipid spheres and rER in the oocyte late in vitellogenesis may be related to the formation of additional functional polyribosomes necessary to complete the final synthesis of many lipid droplets that are present in the ooplasm of the full-grown oocyte.     

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     

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.     

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.     

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.     

Enrichment of primary pachytene spermatocytes from the human testis

Normal adult human testis has been separated using a combination of mechanical and enzymatic procedures to yield a suspension of viable single cells. The predominant cell types comprising this suspension are as follows: primary pachytene spermatocytes (7% of total cells), round spermatids (17%), residual bodies and condensing spermatids (31%), and Leydig cells (15%). Separated human germ cells viewed by Nomarski differential interference microscopy closely resemble mouse spermatogenic cells in relative size and appearance. Isolation of an enriched population of human pachytene spermatocytes has been achieved using unit gravity sedimentation (STA-PUT) according to protocols originally developed for murine tissue. Pachytene cells are enriched to 75% and are contaminated only with Leydig cells and binucleated spermatid symplasts. Ultrastructural examination of isolated human pachytene spermatocytes indicates that these cells, as well as isolated round spermatids, exhibit a normal in situ morphology. Spermatocytes, for example, show numerous synaptonemal complexes, nuclear pores, annulate lamellae, and dictyosome-like saccules. Round spermatids after isolation exhibit peripheral mitochondria, annulate lamellae, developing acrosomes, and other morphological features characteristic of early spermiogenesis. Therefore, enriched populations of human spermatogenic cells seem suitable for analysis using immunofluorescent, autoradiographic, or serological methods. In particular, isolated human spermatocytes should be useful for the analysis of molecular events involved in meiosis and should facilitate investigations concerning the pathophysiology of certain human infertility conditions.     

Are annulate lamellae in the Drosophila embryo the result of overproduction of nuclear pore components?

Annulate lamellae are cytoplasmic organelles composed of stacked sheets of membrane containing pores that are structurally indistinguishable from nuclear pores. The functions of annulate lamellae are not well understood. Although they may be found in virtually any eucaryotic cell, they occur most commonly in transformed and embryonic tissues. In Drosophila, annulate lamellae are found in the syncytial blastoderm embryo as it is cleaved to form the cellular blastoderm. The cytological events of the cellularization process are well documented, and may be used as temporal landmarks when studying changes in annulate lamellae. By using morphometric techniques to analyze electron micrographs of embryos, we are able to calculate the number of pores per nucleus in nuclear envelopes and annulate lamellae during progressive stages of cellularization. We find that annulate lamellae pores remain at a low level while nuclear envelopes are expanding and acquiring pores in early interphase. Once nuclear envelopes are saturated with pores, however, the number of annulate lamellae pores increases more than 10-fold in 9 min. Over the next 30 min it gradually declines to the initial low level. On the basis of these results, we propose (a) that pore synthesis and assembly continues after nuclear envelopes have been saturated with pores; (b) that these supernumerary pores accumulate transiently in cytoplasmic annulate lamellae; and (c) that because these pores are not needed by the embryo they are subsequently degraded.     

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.     

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.     

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.     

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.     

Fine structure of the pore-annulus complex in the nuclear envelope and annulate lamellae of germ cells

Summary Octagonal symmetry in the pore margin has been demonstratedin situ in annulate lamellae and the nuclear envelope of germ cells. The annular material is located to variable extent within the pore and also extends beyond the pore margin; in the latter case it may be continuous with extra-pore annular material of some adjacent pores. In thin sections of fixed material, the annular material of both the nuclear envelope and annulate lamellae appears to be composed of a matrix within which are embedded thin filaments and small granules, the disposition and interrelationship of which are described and discussed. The so-called intra-annular granule is described as consisting of a number of smaller units (similar to the granular component of the annular material) which become aggregated in the center of some pores in both the nuclear envelope and annulate lamellae. The possible significance of intra-annular granules is discussed in terms of binding and movement of macromolecules.This investigation was supported by research grants (HD-00699, GM-09229) and a Career Development Award from the National Institutes of Health, U. S. Public Health Service. The author acknowledges the skillful technical assistance of Mrs.Robert Decker.     

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.     

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis

Sperm nucleomorphogenesis in the cephalopod Sepia officinalis is the product of the interaction between perinuclear microtubules and condensing chromatin. This interaction occurs during spermiogenesis and is established through the nuclear membrane. As in other cephalopod species, the perinuclear microtubules are transient structures. In the case of S. officinalis, they begin to appear in the basal area of the early spermatid and progress from there, establishing contact with the external nuclear membrane and follow a defined, but not symmetric, geometry. Thus, the microtubules accumulate preferentially in one area of the nuclear membrane which we refer to here as the "dorsal zone". Later, the microtubules will be eliminated before the mature spermatid migrates to the epidydimis. The chromatin is condensed within the nucleus following a complex pattern, beginning as fibro-granular structures until forming fibres of approximately 45 nm diameter (patterning phases). From this stage on, an increase in the chemical basicity of DNA-interacting proteins is produced, and chromatin fibres coalesce together, being recruited to the dorsal zone of the membrane, where there is a higher density of microtubules. This last step (condensation phases) allows the chromatin fibres to be arranged parallel to the axis of the elongating nucleus, and more importantly, is deduced to cause a lateral compression of the nucleus. This lateral compression is in fact a recruitment of the ventral zone toward the dorsal zone, which brings about an important reduction in nuclear volume. The detailed observations which comprise this work complement previous studies of spermiogenesis of Sepia and other cephalopods, and will help to better understand the process of cellular morphology implicated in the evolution of sperm nuclear shape in this taxonomic group.     

Annulate lamellae and chromatoid bodies in the testes of a cyprinid fish (Pimephales notatus)

Summary Observations on annulate lamellae and chromatoid bodies in spermatogonia of the cyprinid fish Pimephales notatus have revealed several commonly occurring features heretofore unreported: These include (a) the presence of annulate lamellae in close association with chromatoid bodies; (b) the existence of a chromatoid band or shell between the nuclear envelope and some chromatoid bodies with connections among them; (c) the presence of annulate pore complexes in the absence of well developed membrane envelopes as well as in association with such envelopes; (d) the presence of material just outside the nucleus and contiguous with nuclear pores which is of a similar density and texture to that of the chromatoid bands and chromatoid bodies; (e) filamentous material between the cytoplasmic sides of nuclear pores and the chromatoid band, bridging a distance of approximately 1000 Å and similar threads extending a like distance between chromatoid bodies (and bands) and annulate lamellae associated with them; and (f) mitochondria closely arranged about some chromatoid bodies.     

Ultrastructure of spermatogenesis in the sea star,Asterina minor

The ultrastructural features of spermatogenesis were investigated in the hermaphroditic sea star Asterina minor. The primordial germ cells in the genital rachis contain small clusters of electron-dense material (nuage material) and a stack of annulate lamellae. They also have a flagellum and basal body complex situated close to the Golgi complex. After the development of the genital rachis into the ovotestis, spermatogenic cells increase in number and differentiation begins. Nuage material is observed in spermatogonia, but it gradually disappears in spermatocytes. The annulate lamellae do not exist beyond the early spermatogonial stage. By contrast, a flagellum and basal body complex are found throughout spermatogenesis. The Golgi-derived proacrosomal vesicles appear in the spermatocyte and coalesce to form an acrosomal vesicle in the early spermatid. The process of acrosome formation is as follows: (1) a lamella of endoplasmic reticulum (ER) continuous with the outer nuclear membrane encloses the posterior portion of the acrosomal vesicle; (2) the vesicle attaches to the cell membrane with its anterior portion; (3) periacrosomal material accumulates in the space between the acrosomal vesicle and the ER; (4) the nucleus proper changes its features to surround the acrosome; (5) amorphous, electron-dense material is deposited under the electron-dense disk; and (6) the nucleus forms a hollow opposite the electron-dense material.     

Dynamics of annulate lamellae in Drosophila syncytial embryos

The mitotic events in eukaryotic cells are controlled by a family of evolutionary conserved cyclin-dependent kinases (cdk) that phosphorylate cell proteins, which results in the structural reorganization of the entire cell. Our recent studies of Drosophila syncytial embryos have demonstrated that changes in cdk1 activity controlling the assembly and disassembly of nuclear pore complexes also affect the structure of cytoplasmic pores in annulate lamellae. Here, we report a comparative electron microscopic analysis of the dynamics of these organelles during mitosis throughout the development of a Drosophila syncytial embryo. We presume that the distribution of annulate lamellae containing mature cytoplasmic pores across the cytoplasm reflects local reductions in the mitotic kinase cdk1 activity during the development of Drosophila syncytial embryos.     

Annulate lamellae and crystalline inclusions in granular endoplasmic reticulum of the islet organ and associated tissues of a cyclostome,Myxine glutinosa

Cytoplasmic annylate lamellae were found in the islet organ of a cyclostome, the hagfish (Myxine glutinosa), predominantly in cells interpreted as young proliferating beta-cells, and also in endocrine cells and enterocytes of the bile duct and gut and in the endothelial cells of small blood vessels. A close association was observed annulate lamellae and granular endoplasmic reticulum. Both in cells with and in those without annulate lamellae, crystalline inclusions of proteinaceous nature were seen in granular endoplasmic reticulum. These inclusions were occasionally closely associated to annulate lamellae, and a direct continuity could be seen between granular endoplasmic reticulum and the outer nuclear membrane surrounding an inclusion partially situated in the perinuclear cisterna. Rod-shaped structures and rounded electron dense bodies were seen in the nuclei of some islet parenchymal cells. The presence of annulate lamellae in the islet organ and associated tissues of Myxine glutinosa is believed to be related to the very high phylogenetic age of this species. The close association observed between annulate lamellae, granular endoplasmic reticulum, crystalline inclusions, and sometimes also nuclear membranes, may be of functional significance.     

Novel aspect of perinuclear theca assembly revealed by immunolocalization of non-nuclear somatic histones during bovine spermiogenesis

The perinuclear theca (PT) is an important accessory structure of the sperm head, yet its biogenesis is not well defined. To understand the developmental origins of PT-derived somatic histones during spermiogenesis, we used affinity-purified antibodies against somatic-type histones H3, H2B, H2A, and H4 to probe bovine testicular tissue using three different immunolocalization techniques. While undetectable in elongating spermatid nuclei, immunoperoxidase light microscopy showed all four somatic histones remained associated to the caudal head region of spermatids from steps 11 to 14 of the 14 steps in bovine spermiogenesis. Immunogold electron microscopy confirmed the localization of somatic histones on two nonnuclear structures, namely transient manchette microtubules of step-9 to step-11 spermatids and the developing postacrosomal sheath of step-13 and -14 spermatids. Immunofluorescence demonstrated somatic histone immunoreactivity in the developing postacrosomal sheath, and on anti-beta-tubulin decorated manchette microtubules of step-12 spermatids. Focal antinuclear pore complex labeling on the base of round spermatid nuclei was detected by electron microscopy and immunofluorescence, occurring before the nucleoprotein transition period during spermatid elongation. This indicated that, if nuclear histone export precedes their degradation, this process could only occur in this region, thereby questioning the proposed role of the manchette in nucleocytoplasmic trafficking. Somatic histone immunodetection on the manchette during postacrosomal sheath formation supports a role for the manchette in PT assembly, signifying that some PT components have origins in the distal spermatid cytoplasm. Furthermore, these findings suggest that somatic histones are de novo synthesized in late spermiogenesis for PT assembly.     

An integral membrane protein from the nuclear pore complex is also present in the annulate lamellae: implications for annulate lamella formation

Annulate lamellae (AL) are cytoplasmic arrays of stacked membrane cisternae containing densely packed pore complexes which are similar in structure to the nuclear pore complexes (NPCs) and thus referred to as annulate lamella pore complexes (ALPCs). We have recently shown that the integral nuclear pore membrane protein POM121 tagged with green fluorescent protein was correctly targeted to the nuclear pores (H. S?derqvist et al., 1997, Eur. J. Biochem. 250, 808-813). Here we have investigated if POM121 fused to three tandem molecules of yellow fluorescent protein (YFP) (POM121-YFP(3)) also was able to distribute in the extensive and well-characterized AL of RC37 and BMGE cells. Transfected RC37 or BMGE cells displayed YFP fluorescence around the nuclear envelope, as well as in the cytoplasmic AL structures. The YFP fluorescence colocalized perfectly with immunostaining using antibodies specific for different NPC proteins. The AL of both transfected and untransfected BMGE cells resisted extractions with Tx-100 and 250 mM NaCl, but were completely solubilized at 450 mM NaCl. Loss of YFP fluorescence and immunostaining for other NPC proteins correlated under all extraction conditions tested, suggesting that overexpressed POM121-YFP(3) had become an integrated part both of the NPCs and of the ALPCs. Furthermore, we have generated a stable BHK cell line expressing POM121-YFP(3) located exclusively at the nuclear pores. Treatment with vinblastine sulfate, which induces formation of AL in a variety of cells, resulted in distribution of POM121-YFP(3) into cytoplasmic foci colocalizing with immunostaining for peripheral NPC proteins. Taken together, the results show that YFP-tagged POM121 is able to distribute in drug-induced or naturally occurring AL, suggesting that POM121 is a natural constituent of ALPCs. In COS cells, which normally lack or have very little AL, YFP-tagged POM121 distributed in the nuclear pores when expressed at low levels. However, at high expression levels the YFP fluorescence also distributed in a number of brightly fluorescing cytoplasmic dots or foci, which were not present in untransfected cells. This was also true for untagged POM121. The cytoplasmic foci varied in size from 0. 1 to 2 microm and were distinctly located in the immediate vicinity of ER cisternae (without colocalizing) and also contained other nuclear pore proteins, indicating that they may represent cytoplasmic AL. This idea is supported by time-lapse studies of postmitotic assembly of these structures. This raises the question of the role of POM121 in ALPC and NPC biogenesis.