Characterization of the nuclear envelope, pore complexes, and dense lamina of mouse liver nuclei by high resolution scanning electron microscopy

We have used high resolution scanning electron microscopy (SEM) to study the nuclear envelope components of isolated mouse liver nuclei. The surfaces of intact nuclei are covered by closely packed ribosomes which are distinguishable by SEM from nuclear pore complexes. After removal of nuclear membranes with the nonionic detergent Triton X-100, the pore complexes remain attached to an underlying, peripheral nuclear lamina, as described by others. The surface of this dense lamina is composed of particulate granules, 75-150 A in diameter, which are contiguous over the entire periphery. We did not observe the pore-to-pore fibril network suggested by other investigators, but such a structure might be the framework upon which the dense lamina is formed. Morphometric analysis of pores and pore complexes shows their size, structure, and density to be similar to that of other mammalian cells. In addition, several types of pore complex-associated structures, not previously reported by other electron microscope (EM) techniques, are observed by SEM. Our studies suggest that the major role of the dense lamina is associated with the distribution, stability, and perhaps, biogenesis of nuclear pore complexes. Treatment of isolated nuclei with a combination of Triton X-100 and sodium deoxycholate removes membranes, dense lamina, and nuclear pore complexes. The resulting "chromatin nuclei" retain their integrity despite the absence of any limiting peripheral structures.     

华东安蕨卵发生的超微结构观察

核心薄囊蕨是蕨类植物中的进化类群,但对受精作用具有显著影响的卵发生研究仍较少,该文利用超微技术对其中蹄盖蕨科的华东安蕨卵发生过程进行了研究,以进一步完善薄囊蕨植物卵发生的科学资料,为理解蕨类植物的有性生殖及演化机制奠定基础。超微结构观察显示:华东安蕨的幼卵和沟细胞在颈卵器中紧密联接;随后,在卵细胞上方出现了分离腔和临时细胞壁,但在卵细胞中间孔区处卵细胞和腹沟细胞始终联接在一起;分离腔中的无定形物质沉积在卵细胞的质膜外形成了1层加厚的卵膜,而在孔区处没有形成卵膜,该位置最后形成了受精孔。在进一步的卵发生过程中,卵细胞核变得高度不规则,形成了大量的核外突和核褶皱。     

The spermatozoa of ascidians: Acrosome and nuclear envelope

Summary The spermatozoon of Ascidia callosa has a head with a wedge-shaped tip. Between the nuclear envelope and the plasmalemma, at the tip of the head, there are one or two previously undescribed vesicles, 45 to 55 nm in diameter. These vesicles have the characteristics of an acrosome. Their role in the process of fertilization has not been determined. Ultrastructural studies of sperm activation are needed, but claims that the spermatozoa of ascidians do not have an acrosome should be reconsidered.Behind the tip of the sperm there are pores in the nuclear envelope. This part of the envelope also contains a dense band of amorphous material that may have a supportive function. A nearly identical structure, associated with pores has been found in the spermatozoon of Boltenia villosa. An analysis of the nuclear envelope of Ascidia callosa indicates that the same structure has previously been misinterpreted as an acrosome in the spermatozoon of Ascidia nigra.     

Ultrastructural observations on the cuticular envelope in salt glands ofFrankenia pauciflora

Summary During five developmental stages in differentiation of salt glands in leaves ofFrankenia pauciflora, details of the deposition of incrusting material in the cuticular envelope,i.e., hydrophobic suberin and/or cutin, have been observed by means of transmission electron microscopy.Around each transfusion area in the cuticular envelope a conspicuous lamellate ring structure is found. Thin 3.5 nm lamellae associated with the plasmalemma around each ring structure appear to participate in the formation of tripartite structures showing a reversed contrast compared to the plasmalemma. Below the ring structure the cuticular envelope is divided into an outer fibrillar and an inner amorphous zone. A gradual transition between the tripartite lamellae of the ring structure and the amorphous material of the envelope is evident. Very dense material present between the lamellae appear to accumulate in the transition zone. The results are discussed in relation to basic structural features of various incrustations of lipid nature.     

A modified procedure for the isolation of a pore complex-lamina fraction from rat liver nuclei

A modified procedure for the isolation of a nuclear pore complex-lamina fraction from rat liver nuclei is described. Evidence is provided that the isolated lamina, a 150-A thick, proteinaceous structure, apposes the inner nuclear envelope membrane, connecting nuclear pore complexes and surrounding the entire nucleus.     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

Integral membrane proteins and dynamic organization of the nuclear envelope

The nuclear envelope is a complex structure consisting of nuclear membranes, nuclear pore complexes and lamina. Several integral membrane proteins specific to the nuclear pore membrane and the inner nuclear membrane are known. Pore membrane proteins are probably important for organization and assembly of the nuclear pore complex, while proteins of the inner nuclear membrane are likely to play major roles in the structure and dynamics of the nuclear lamina and chromatin. Biochemical studies are now identifying potential binding partners for some of these integral membrane proteins, and analysis of nuclear envelope assembly at the end of mitosis is providing important insights into their functions.     

The subacrosomal granule and its evolution during spermiogenesis in a lizard

Summary Some aspects of spermiogenesis have been studied in the testis of the teiid lizard Cnemidophorus lemniscatus lemniscatus by electron microscopy. Shortly after the acrosomal vesicle is lodged in a nuclear concavity of the spermatid, a dense granule differentiates in the center of the subacrosomal space. It is cone-shaped and shows a longitudinal striation. Its base applies to the acrosomal membrane and, through this, to the acrosomal granule. Its rounded vertex causes a depression of the nuclear membranes which, initially juxtaposed, separates at this point to form a vesicle. The granule develops and becomes a rod when spermiogenesis is advanced and the subacrosomal space has taken the form of a secondary cap. The rod is cylindrical, retains its original striation and has a convex acrosomal end. It encloses the vesicle formed by the nuclear envelope in its base and follows the apex of the nucleus. Meanwhile, the acrosomal granule loses its identity and the acrosomal cap is filled with a dense substance, in which a fringe of translucent material differentiates. This fringe lies in the dorsal and apical margins of the acrosome and is incompletely divided by longitudinal crests of the dense acrosomal substance. A projection of the Sertoli cell forms an accessory cap which envelops the acrosome and is in turn covered by the cytoplasm of the spermatid, constituting an intricate association. Two reflex membranes underlie the plasmalemma in the outer surface of the projection of the Sertoli cell. They are continuous with one another at their ends and with the cell membrane in the edge of pores. In the peripheral cytoplasm of the spermatid facing the accessory cap, numerous microtubules run longitudinally. By means of thin membranes some are interconnected or connected with the plasmalemma, from which they seem to originate.This research forms part of project N. 31.26.S1-0244 supported by the Consejo Nacional de Investigaciones Científicas y Tecnológicas     

CYTOLOGY AND ULTRASTRUCTURE OF CHLORARACHNION REPTANS (CHLORARACHNIOPHYTA DIVISIO NOVA,CHLORARACHNIOPHYCEAE CLASSIS NOVA)1

The green amoeboid cells of Chlorarachnion reptans Geitler are completely naked and each contains a central nucleus, several bilobed chloroplasts each with a central projecting pyrenoid enveloped by a capping vesicle, several Golgi bodies, mitochondria with tubular cristae, extensive rough ER, and a distinct layer of peripheral vesicles. Complex extrusome-like organelles occur rarely in both the amoeboid and flagellate stages. The only organelles entering the reticulopodia are mitochondria, but microtubules are also present. The chloroplasts contain chlorophylls a and b, but histochemical tests suggest that the carbohydrate storage product probably is not a starch. The chloroplast lamellae are composed of one to three thylakoids or form deep stacks. A girdle lamella and interlamellar partitions are absent. Each chloroplast is bounded by either four separate membranes, a pair of membranes with vesicular profiles between them, or three membranes; all three arrangements may occur in the same chloroplast. A periplastidal compartment occurs near the base of the pyrenoid where there are always four surrounding membranes. The compartment has a relatively dense matrix and contains ribosome-like particles and small dense spheres; it extends over and into a deep invagination in the pyrenoid where its contents are enclosed in a double-membraned envelope which is penetrated by wide pores. The zoospores are ovoid and each bears a single laterally inserted flagellum which appears to be wrapped helically around the cell body during swimming. The flagellum lies in a groove in the cell surface and bears fine lateral hairs. Neither a second flagellum or vestige of one, nor an eyespot, is present. A single microtubular root and a larger homogeneous root run from the flagellar base parallel to the emerging flagellum, between the nuclear envelope and the plasmalemma. In the simple flagellar transition region, fine filaments connect adjacent axonemal doublets. A detailed comparison of C. reptans with all other algal taxa results in the conclusion that it must be segregated in the new class Chlorarachniophyceae, the only class in the new division Chlorarachniophyta. The possibility that C. reptans evolved from a symbiosis between a colorless amoeboid cell and a chlorophyll b- containing eukaryote is considered, but the possible affinities of the symbiont remain enigmatic. The implications of the unique chloroplast structure of C. reptans for current hypotheses concerning the origin of chloroplasts are discussed.     

Wall and membrane biogenesis in the unusual labyrinthulid-like organismSorodiplophrys stercorea

Summary Electron microscopic investigations show the scale subunits of the walls ofSorodiplophrys stercorea to be produced by the Golgi apparatus. Their chief monosaccharide subunit is identified tentatively as arabinose. Membrane flow involving the nuclear envelope, endoplasmic reticulum, Golgi apparatus, Golgi vesicles, plasmalemma, and possibly prominent refractive bodies composed of membranes is suggested. Cytochemical data supporting ultrastructural evidence concerning the sites of scale and membrane biogenesis are presented.     

Synthesis, transport and incorporation into the nuclear envelope of A-type lamins and inner nuclear membrane proteins

The mammalian NE (nuclear envelope), which separates the nucleus from the cytoplasm, is a complex structure composed of nuclear pore complexes, the outer and inner nuclear membranes, the perinuclear space and the nuclear lamina (A- and B-type lamins). The NE is completely disassembled and reassembled at each cell division. In the present paper, we review recent advances in the understanding of the mechanisms implicated in the transport of inner nuclear membrane and nuclear lamina proteins from the endoplasmic reticulum to the nucleus in interphase cells and mitosis, with special attention to A-type lamins.     

Spermatology of Some Pycnogonida (Arthropoda), with Special Reference to a Microtubule-Nuclear Envelope Complex

The sperm cells of Nymphon leptocheles and N. rubrum are of the primitive type, which is a remarkable condition among arthropods. The motile sperm consist of a somewhat elongated head, a kind of midpiece and a long tail. An acrosome is absent. The nucleus is surrounded by longitudinally oriented microtubules running in furrows in the nuclear envelope. These microtubules are not interconnected by links or connected to the nuclear envelope; they persist in the mature sperm. No appreciable chromatin condensation takes place. The midpiece contains some unmodified mitochondria and a centriole. The tail is a simple, free flagellum. The results are in particular discussed in relation to other known microtubule-nuclear envelope complexes in sperm cells. The sperm cells of Pycnogonum littorale are, on the other hand, highly aberrant. They are unmotile, elongated cells containing a very high number (often more than 1000) of longitudinal microtubules arranged in complex patterns. Some folded membranes may represent the nuclear envelope. Other organelles are unidentificable or may be absent.     

The role of CaaX-dependent modifications in membrane association of Xenopus nuclear lamin B3 during meiosis and the fate of B3 in transfected mitotic cells

Recent evidence shows that the COOH-terminal CaaX motif of lamins is necessary to target newly synthesized proteins to the nuclear envelope membranes. Isoprenylation at the CaaX-cysteine has been taken to explain the different fates of A- and B-type lamins during cell division. A-type lamins, which loose their isoprenylation shortly after incorporation into the lamina structure, become freely soluble upon mitotic nuclear envelope breakdown. Somatic B-type lamins, in contrast, are permanently isoprenylated and, although depolymerized during mitosis, remain associated with remnants of nuclear envelope membranes. However, Xenopus lamin B3, the major B-type lamin of amphibian oocytes and eggs, becomes soluble after nuclear envelope breakdown in meiotic metaphase. Here we show that Xenopus lamin B3 is permanently isoprenylated and carboxyl methylated in oocytes (interphase) and eggs (meiotic metaphase). When transfected into mouse L cells Xenopus lamin B3 is integrated into the host lamina and responds to cell cycle signals in a normal fashion. Notably, the ectopically expressed Xenopus lamin does not form heterooligomers with the endogenous lamins as revealed by a coprecipitation experiment with mitotic lamins. In contrast to the situation in amphibian eggs, a significant portion of lamin B3 remains associated with membranes during mitosis. We conclude from these data that the CaaX motif-mediated modifications, although necessary, are not sufficient for a stable association of lamins with membranes and that additional factors are involved in lamin-membrane binding.     

Formation and function of accessory nuclei in the oocytes of the bird louse,Eomenacanthus stramineus (Insecta,Mallophaga)

In the oocytes of Eomenacanthus stramineus accessory nuclei arise by budding from the nuclear envelope. It is suggested that microtubules and the thick layer of the nuclear lamina are involved in this process. Newly formed accessory nuclei contain aggregations of fibrillogranular material. These aggregations are slightly Feulgen positive, RNA negative and stain positively with the AgNOR method. During later developmental stages one dense, RNA-positive inclusion appears in each accessory nucleus. These inclusions consist of on Ag-NOR-positive cortical layer and an Ag-NOR-negative core. The function of accessory nuclei in the species investigated is discussed in the light of these results.     

Fine structure distribution of non-specific acid phosphatase in the head region of mouse spermatozoa from various regions of the male reproductive tract.

The fine structure distribution of non-specific acid phosphatase was determined in the head region of mouse spermatozoa from the testes, the caput, corpus and cauda epididymidis and the ductus deferens. Enzymatic localization was achieved by the Gomori technique. The postacrosomal dense lamina, the nuclear side of the inner acrosomal membrane and the space between the plasmalemma and the outer acrosomal membrane showed reaction product in spermatozoa from the testis and caput epididymidis. Spermatozoa from the cauda epididymidis exhibited reaction product only between the plasmalemma and the outer acrosomal membrane. Spermatozoa from the corpus epididymidis and from the ductus deferens showed no reaction product in the head region. The changes observed in the distribution of acid phosphatase in the sperm head during epididymal transport may reflect maturational events.     

A lamin-independent pathway for nuclear envelope assembly

The nuclear envelope is composed of membranes, nuclear pores, and a nuclear lamina. Using a cell-free nuclear assembly extract derived from Xenopus eggs, we have investigated how these three components interact during nuclear assembly. We find that the Xenopus embryonic lamin protein LIII cannot bind directly to chromatin or membranes when each is present alone, but is readily incorporated into nuclei when both of the components are present together in an assembly extract. We find that depleting lamin LIII from an extract does not prevent formation of an envelope consisting of membranes and nuclear pores. However, these lamin-depleted envelopes are extremely fragile and fail to grow beyond a limited extent. This suggests that lamin assembly is not required during the initial steps of nuclear envelope formation, but is required for later growth and for maintaining the structural integrity of the envelope. We also present results showing that lamins may only be incorporated into nuclei after DNA has been encapsulated within an envelope and nuclear transport has been activated. With respect to nuclear function, our results show that the presence of a nuclear lamina is required for DNA synthesis to occur within assembled nuclei.     

The ultrastructure of the rabbit oocyte at the bilaminar follicle stage]

The electron microscope study of the nucleus and organoids of the rabbit oocytes cytoplasm during growth showed nucleoluslike bodies (RNP-granules) on the lampbrushen chromosomes to reach their maximal size at the stage of bilaminar follicle. The RNP-granules differ from the nucleoli by the time of their occurrence cytochemical characteristics, and by their ultrastructural pattern. Throughout the bilaminar follicle stage four components may be seen in the oocyte nucleolus: a dense fibrillar framework around the vacuoles, islets of the granular mass loosely dispersed, and electron dense fibrillar elements filling up the numberous electrontransparant vacuoles. The nucleolus-like bodies are round in shape and have no vacuoles, consisting to two components only: distinctly outlined granules, and weakly developed fibrillar component. The nuclear envelope is seen blebbing. Separation of two nuclear membranes forms a pocket-like enlargements of the perinuclear space. The pockets are limited by small regions between the adjacent nuclear pores. The outer membrane may bulge producing lacuma and large channels in the cytoplasm, which are interconnected making a closed branched network extending inside of the cytoplasm. The nuclear envelope is suggested to be involved in formation of the endoplasmic reticulum through the blebbing process.     

Relation between nuclear envelope and nuclear lamina in nuclear assembly in vitro

Xenopus laevis egg extracts cell-free nuclear assembly system was used as an experimental model to study the process of nuclear lamina assembly in nuclear reconstitutionin vitro. The experimental results showed that lamin was involved in the nuclear assemblyin vitro. The assembly of nuclear lamina was preceded by the assembly of nuclear matrix, and probably, inner nuclear matrix assembly provided the basis for nuclear lamina assembly. Inhibition of normal assembly of nuclear Iknina, by preincubating egg extracts cell-free system with anti-lamin antibodies, resulted in abnormal assembly of nuclear envelope, suggesting that nuclear envelope assembly is closely associated with nuclear lamina assembly. Project supported by the National Natural Science Foundation of China.     

The Effect of Homologous Rabbit Antiserum on the Growth of Leishmania tarentolae—a Fine Structure Study

SYNOPSIS. The fine structure of Leishmania tarentolae growing in the presence of homologous rabbit antiserum was investigated. Immediately after agglutination of the promastigotes, a dense band between pellicles of adjacent cells could be seen, and a surface coat was rendered visible. The cells did not fuse. At dilutions of antiserum from 1:100–1:1200 the promastigotes continued to grow, forming large masses. By 5 days these consisted of individual cells adhering to one another. In cross-section, any 2 cells were separated by 2 membranes with associated subpellicular fibrils and a dense band between the 2 membranes. The term syncytium generally refers to a multinucleate cell originating by fusion of several cells and has been used to mean a multinucleate cell arising from nuclear division unaccompanied by cytokinesis. Therefore, it is unlikely that the masses formed in the presence of homologous antiserum are syncytia.     

Fine structure of photo-kinetic systems in Dinobryon cylindricum var. Alpinum (chrysophyceae)

The ultrastructure of the stigma and associated flagellar-microtubular systems in Dinobryon cylindricum var. alpinum is described in detail and compared with observations on comparable photo-kinetic systems in other chrysophycean organisms. The chloroplastidic stigma of D. cylindricum var. alpinum is shown to lie in a particular positional relationship to the flagellar swelling in the anterior furrow and to several other organelles, to consist of a monolayer of c. 40 pigmented granules, each c. 250–500 nm diameter, arranged in a definite pattern, and to be overlain by several membrane systems. Other cytoplasmic pigmented bodies with dense crystalline contents surrounded by a single “unit membrane” aggregate near the anterior furrow on the side opposite the stigma. The swelling on the proximal portion of the smooth flagellum is separated from the plasmalemma of the anterior furrow by a nearly constant distance of 75–100 nm, has a multilamellate substructure that is linked by fine radiating interconnections to the axoneme doublets, and is connected to the plasmalemma by a system of fibrillar interconnections. A transitional helix in the basal body region is described as similar to structures reported in other chrysophycean flagellates. A striated rhizoplast with a periodicity of c. 90 nm extends from basal body I to the nuclear envelope. A seven-stranded microtubular root extends from the same basal body. Other fibrous and microtubular root systems are also described. The inter-relationships and possible functions of the aforementioned structures are discussed.