Intranuclear Inclusions in the Ameboid Cells of Protostelium zonatum*

SYNOPSIS. Two morphologically distinct types of intranuclear inclusions are found in ameboid cells of the protostelid mycetozoan Protostelium zonatum. One type of inclusion is a coiled tubular structure which in cross section appears as cisternae and oval to elliptical vesicles 40–60 nm in diameter. These tubular and vesicular structures are formed by a unit membrane that is connected directly with the inner nuclear membrane. The other type of inclusion is a membrane-bound structure that contains amorphous and/or fibrous material. These inclusions usually are present at several locations in a nucleus. No similar structures occur in the cytoplasm.     

Crystalline inclusions in hepatocytes and associated interhepatocytic macrophages from female Ohrid trout (Salmo letnica Kar.)

When characterizing the liver ultrastructure in Ohrid trout, we noticed that cells often displayed unusual cytoplasmic crystalline inclusions. Their morphology varied much, concerning the size, shape and electron density, showing aspects never reported in fish liver. In hepatocytes, the inclusions existed in close topographical relationship with dense bodies (often within them). Such “crystals” inclusions also existed in macrophages and in biliary passages lumina. Data revealed seasonal/breeding-related changes of the dense bodies and crystalline inclusions within hepatocytes; decreasing from the earliest to the more advanced ovary maturation stages. Additionally, based on the negative correlations between the amounts of dense bodies and of “crystals” and the ovary somatic ratio, we suggested there might be connections between the sex steroids status and the inclusions formation. A positive correlation between the quantities of “crystals” and of dense bodies further suggested that the inclusions might derive from normal processes, e.g., the females’ cyclic liver changes, that involves dense bodies’ turnover. However, a toxicological aetiology cannot be excluded. Additionally, multiple mechanisms can contribute to the “crystals” genesis. Facing literature reports and because the inclusions content seemed washed out during tissue processing, they are likely lipid in nature, but their exact composition and genesis require further analyses.     

Intranuclear crystalline inclusions in spermatozoa ofLumbricus terrestris

Summary In the nuclei of atypical spermatids ofLumbricus terrestris granular or filamentous inclusions are surrounded by dense chromatin. Aggregation and condensation of chromatin in nuclei during spermatid differentiation coincide with increase in density, granularity, and the subsequent crystallization of the intranuclear inclusion. In mature spermatozoa, the crystalline inclusion displaying an irregular shape is composed of parallel repeating units measuring 50–80 Å. The subunits sometimes possess a clear central cavity.Atypical spermatozoa, possessing inclusions that distort their normally cylindrical shape, possess typical acrosomes, middle pieces, and flagella. Spermatozoa bearing intranuclear crystals are rarely observed in the seminal receptacles ofLumbricus.These intranuclear inclusions probably represent proteinaceous material that is not eliminated during nuclear differentiation. Their sole existence in the nuclei of spermatozoa, their transformation into crystalline structures during spermiogenesis, and their similarity to crystals in virus infected plant and animal cells suggest a viral origin.Supported by a training grant (GM-00582-07) from the Public Health Service.     

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.     

ULTRASTRUCTURE OF THE THERMOPHILIC BLUE-GREEN ALGA,SYNECHOCOCCUS LIVIDUS COPELAND1

The ultrastructure of Synechococcus lividus Copeland, a thermophilic blue-green alga, was studied in thin sections. The cell envelope reveals striking similarities with that of some gram-negative bacteria. In contrast to bacteria and to many other species of blue-green algae, ribosomes are predominantly found in the central nuclear region and appear to be associated with the DNA fibrils. Thylakoids (photo-synthetic lamellae) are arranged as concentric shells, around the nuclear equivalent, lying nearly parallel to one another and to the plasma membrane. Both plasma and thylakoidal membranes, as described by other authors for different Cyanophyceae, are of the unit membrane dimension and morphology. Various types of intracellular inclusions are found: (1) Lipid inclusions, located in the cytoplasm are similar to the osmiophilic globules of higher plant chloroplasts. (2) Polyphosphate inclusions (or volutin) resembling those of other species are generally found at the cell poles but within the nuclear region. (3) Polyhedral inclusions also located in the nuclear region are clearly recognized to be different from the polyphosphate bodies, but their function remains unknown.     

Nature of the nuclear inclusions formed by PQBP1, a protein linked to neurodegenerative polyglutamine diseases

PQBP1, for polyglutamine tract-binding protein-1, has been linked to progressive neurodegenerative diseases, such as spinocerebellar ataxia, that are caused by the expansion of a polyglutamine repeat in a key regulatory protein. The overexpression of PQBP1 results in the formation of nuclear inclusions, reminiscent of the protein aggregates that are detected in polyglutamine diseases. We show here that the occurrence of PQBP1-induced nuclear inclusions is dramatically increased by the co-expression of the pre-mRNA splicing factor SIPP1, a protein ligand of PQBP1. These nuclear inclusions did not co-localise with nuclear structures such as nucleoli, coiled bodies, PML bodies, speckles and stress bodies, and were not associated with (in)active chromatin or with nucleic acids. Site-directed mutagenesis showed that the facilitation in the formation of the nuclear inclusions required multiple independent interaction sites between SIPP1 and PQBP1. Moreover, the nuclear inclusions were highly dynamic and their formation did not require energy. Our data suggest that the SIPP1-PQBP1-induced nuclear inclusions are distinct from the protein aggregates that are associated with polyglutamine diseases and represent dynamic nucleoplasmic heteropolymers of SIPP1 and PQBP1.     

Intramitochondrial inclusions in various cells of a snake (Elaphae quadrivirgata)

Summary A variety of inclusion bodies occur in the mitochondria of several cell types of the snake, Elaphae quadrivirgata. These lie in the mitochondrial matrix or within the space of the cristae. The inclusions in the matrix are as follows: dense homogeneous and fine granular materials, structures with finger-print appearance and with filamentous and/or crystalloid pattern and fine ring-shaped and/or microtubular structures. The inclusions within mitochondrial cristae are glycogen particles, globular materials, and strand-like structures.These inclusions occur not only during the hibernation period of the snake, but also in the arousal period. Furthermore, some inclusions are encountered in fetal tissues. The functional significance of these inclusions is unknown; however, the present study suggests that they are related to the metabolic activity of the cells.     

Ultrastructure of nuclear condensation and localization of DNA and proteins in spermatozoa of a dasyurid marsupial,Sminthopsis crassicaudata

In the dasyurid marsupial, Sminthopsis crassicaudata, the mature spermatozoon has an inner homogeneous (C1) and a peripheral indented (C2) region. Using DNase-gold conjugates, and biotinylated genomic DNA probes, DNA was found to occur in both C1 and C2 regions. The morphogenesis of the spermatozoon nucleus was investigated using ultrastructural and cytochemical studies. Spermiogenesis was divided into 15 steps. By step 10, condensation of the C1 region was complete, and at the caudal extremity of the spermatid nucleus, the nuclear envelope enclosed an electron-lucent space. This space and the surrounding nuclear envelope became very enlarged at step 11. At this stage, a plate of approximately 70 nm in thickness was present along the caudal segment of the C1 region; this “nuclear mantle” did not bind DNase-gold conjugates but stained for lysine-rich proteins using alcoholic phosphotungstic acid. Chromatin condensation resumed at step 12 with the appearance of spherical chromatin structures peripheral to the C1 chromatin. These structures then partially coalesced and the indentations of the C2 region were observed. The expanded nuclear envelope at the caudal extremity persisted in caput epididymal spermatozoa. Spherical inclusions within it did not bind to DNase-gold conjugates but stained for lysine-rich proteins. As the sperm traveled down the epididymis, these inclusions amassed near the nuclear pores and were then removed from the nucleus. In addition, the nuclear mantle was found to have disappeared by the time the spermatozoa reached the corpus epididymidis. © 1996 Wiley-Liss, Inc.     

Tubular inclusions within polyploid nuclei ofGerris najas do not react with a monoclonal anti-β-tubulin antibody

Summary Variable aggregates, composed of tubules with a mean diameter of 19 nm, were found exclusively within polyploid nuclei of the midgut, Malpighian tubes, cyst cells, testis epithelium, and trophocytes ofGerris najas. The nuclear inclusions are always in direct contact with the nucleoplasm, and no other structures are associated with them. They appear most abundant within degenerating nuclei of the midgut surface epithelium, where they form paracrystalline bodies or spindle-shaped inclusions with tapered ends. Smaller fusiform inclusions occur in younger epithelial nuclei but not in the diploid nuclei of regenerative cells. In other tissues, mainly spindle-shaped inclusions can be observed, the longest (4.5 m) in cyst cell nuclei. The mean diameter of the tubules determined from transverse sections, resembles that of cytoplasmic microtubules and was verified statistically. The inclusions within trophocyte nuclei failed to react with monoclonal anti--tubulin antibody, although the antibodies could penetrate the nuclei after extensive lysis of the cells.     

Manipulation of the amount of RNA in post-mitotic nuclei

Because all (or almost all) nuclear RNAs are liberated to the cytoplasm during mitosis and then return to the post-mitotic nuclei, we expected that if cytoplasm were amputated from mitotic cells the post-division nuclei would possess less than normal amounts of RNA. Experiments performed with amebae (A. proteus) show that this is in fact what happens. Furthermore, since the enucleate fragment cut from a mitotic cell possesses an “excess” of returnable nuclear RNAs, a normal interphase nucleus implanted into such mitotic cytoplasm might be expected to acquire above-normal amounts of RNA. Experiments reported here show that this expectation also is realized. Thus, the regulation of the normal nuclear concentration of these RNAs involves mechanisms other than a limited number of intranuclear “binding” sites and most likely is restricted by the rate of synthesis of these RNAs.The demonstration that nuclei can be depleted or enriched for RNAs, many of which are unique to nuclei, makes it possible to determine the consequences for cell metabolism of altered amounts of nuclear RNA. Hopefully, such studies will reveal the function(s) of these RNAs.     

External prostate of the hedgehog

The external prostate of male hedgehogs (Erinaceus europeus, Hemiechinus sp.) was studied at the beginning and at the end of the breeding seasons. Its tubular secretory units contain two types of epithelia, columnar and stratified. The columnar epithelium has abundant rough endoplasmic reticulum and forms secretory granules. The stratified epithelium undergoes cellular changes culminating in appearance of nuclear and cytoplasmic inclusions, nuclear pyknosis and desquamation of cells and nuclei into lumen. The nuclear inclusions contain protein and abundant silver-reducing substances. These inclusions may be derived from the residual protein of the nucleus.     

Ultrastructure of fat body cells of the velvetbean caterpillar infected with a nuclear polyhedrosis virus

During a study of the ultrastructure of a nuclear polyhedrosis virus of the velvetbean caterpillar, Anticarsia gemmatalis, various types of nuclear and cytoplasmic inclusions were found in fat body tissue heavily infected with the virus. Virogenic stroma was present in the nuclei of most infected cells. Bundles of fibrous material were observed in the nuclei and cytoplasm of cells containing polyhedral bodies. Other nuclear inclusions included concentric multilayered material, vacuoles, and membrane structures.     

Kinetoplast DNA in the insect trypanosomes Crithidia luciliae and Crithidia fasciculata: III. Heteroduplex analysis of the C. luciliae minicircles

We have investigated the nature of the sequence heterogeneity of the minicircles of Crithidia luciliae kinetoplast DNA by EM heteroduplex analysis of minicircles cleaved with endonuclease HindIII. Approximately 40% of the minicircles showed—after denaturation and reannealing—structures indicative of sequence rearrangements: the majority contained heteroduplex “eyes” interpreted as due to inversions; about 10% of the heteroduplexes yielded structures interpreted as due to translocations and a similar fraction showed insertions and deletions. The category of “eyed” molecules was analyzed in detail: four minicircle segments were found that displayed a high incidence of such eyes indicating that the rearrangements were not scattered at random over the minicircles. Moreover, since also “eyes” were found overlapping two or three of the four regions, we postulate that these segments are capable of recombining with each other. We conclude that specific segmental rearrangements form the main basis of the minicircle sequence heterogeneity in Crithidia.     

The ultrastructure of inclusions inVinca nuclei

Summary An electron microscopic study of leaf and petal mesophyll tissue ofVinca rosea revealed the presence of nuclear inclusions. The inclusions were found to be tubular with an outside diameter of about 100 Å. The tubular inclusions inVinca appear to be similar to those that have been described in nuclei of other plants.     

Complexes of annulate lamellae and nemaline bodies in soleplate nuclei in skeletal muscle fibers of the normal rat

Pseudointranuclear inclusions resulting from invaginations of the nuclear envelope, characteristic of myopathic muscles, were found in the soleplate nuclei of normal soleus muscles of rats. Such inclusions had annulate lamellae (AL), nemaline or rod bodies, lamellar structures, a concentric system of membranes, as well as tightly packed sarcoplasmic organelles. An accumulative origin of such included structures was suggested. The AL were sometimes connected directly to the outer membrane enclosing the inclusions, dilated into cisterns and positioned perpendicularly against the nuclear surface. AL, some of which were well-developed, were observed in the same loci as above during the course of denervation atrophy.     

Study of Dientamoeba fragilis Jepps & Dobell I. Electronmicroscopic Observations of the Binucleate Stages II. Taxonomic Position and Revision of the Genus*

Light- and electronmicroscopic observations on Dientamoeba fragilis strain A (Bi) 1 dealing primarily with the binucleate (arrested telophase) stages, predominant in all populations, revealed the microtubular nature of the extranuclear spindle which extends between the 2 polar complexes each adjacent to one of the nuclei. The spindle microtubules originate in paired, nonperiodic structures apparently homologous to the “atractophores” described from hypermastigotes. To the external surface of the atractophores are applied periodic elements, which extend laterally as the parabasal filaments. Extensive Golgi complexes overlie the filaments, these structures corresponding to the components of the parabasal apparatus known from trichomonads and hypermastigotes. The 2-layered structures, consisting of the atractophores and periodic layers, together with the proximal parts of the Golgi complex and the spindle microtubules constitute the polar complex. No kinetosome- or centriole-like organelles have been found in the polar complexes or elsewhere in the organism. The extranuclear spindle is composed of 2 microtubule bundles, each with ～30-40 microtubules. One of the bundles always appears at some distance from the nucleus; the other is juxtanuclear and is seen often to course within a groove of the nuclear envelope. A 3rd bundle of ～35-45 microtubules is seen on occasion to arise from the atractophores and to pass toward the nucleus at a wide angle to the other parts of the spindle. In some instances these microtubules traverse the nucleus within channels delimited by the nuclear envelope. The double-layered nuclear envelope contains numerous pores. Two morphologic types of rounded inclusions, one microbody-like, and the other with a more electron-translucent matrix, as well as digestive vacuoles containing rice starch, bacteria, and/or myelin configurations are distributed in the cytoplasm, which abounds also in glycogen granules. The fine structure of Dientamoeba is compared with those of trichomonads and of Entamoeba spp. The taxonomic position of Dientamoeba is discussed and emended; in view of its affinities, this genus is placed among trichomonads in the family Dientamoebidae Grassé, emend.     

Ultrastructure of crystalline inclusions in the thylakoids of dodder<Emphasis Type="Italic">(cuscuta japonica)</Emphasis> plastids

The plastids from seedlings of the parasitic angiospermCuscuta japonica were ultrastructurally investigated. In shoot subapical cells from 3-d-old seedlings grown in the dark, the etioplasts contained prolamellar bodies and amorphous and dense inclusions. In the shoot subapical cells obtained from 6-d-old seedlings grown under light conditions for the last 3 d, the underdeveloped chloroplasts contained phytoferritin within the stroma as well as amorphous and dense inclusions that were limited by the thylakoid membranes. In the developing chloroplasts, electron-dense materials were detected within the transversely sectioned thylakoid lumens. This dense material presented two different images, depending upon the sectional plane. When transversely prepared, the materials appeared as somewhat thick, linear structures, whereas longitudinally sectioned thylakoids revealed very large crystalline inclusions. In the developed chloroplasts, the amounts of electron-dense material or crystalline inclusions were remarkably reduced in the thylakoid lumens, which were electron-translucent. Far fewer crystalline inclusions were observed in the developed chloroplasts of seedlings than in the developing chloroplasts. These results suggest that the crystalline inclusions may be temporarily reserved within the thylakoid lumens of chloroplasts in the Gjaponica seedlings.     

A survey on the intraclear inclusions in the Schrophulariaceae and their systematic significance

A survey of nuclear inclusions in chlorenchyma cells is given for 290 taxa of the Scrophulariaceae, covering 72 genera; 172 are new data and the remainder are from previous reports by the author. In addition to the 5 types of nuclear inclusions already determined by their different ultrastructural morphology — amorphous (A), lamellar (L), fibrillar (F), tubular (T), and crystalline (C) — and to subtypes C2, three new subtypes — A2, L2, T2 - specific to certain genera or small groups of genera are reported. These new subtypes are described in detail and their distribution in the family discussed. Although the proteinic nature was unquestionably demonstrated, the origin and function of plant nuclear inclusions is far from being understood. In the Scrophulariaceae they have been recognized in 242 out of 290 taxa studied (83.4%), in a total of 60 out of 72 genera (83.3%). On the basis of the occurrence and qualitative distribution of the nuclear inclusions, the family can be subdivided into four main group: 1) with lamellar inclusions, 2) with amorphous inclusions, 3) with inclusions other than those in the two former groups, and 4) with no nuclear inclusions. The relationship between the distribution on the nuclear inclusions throughout the family and the current systematic arrangement into subfamilies are analyzed and the transfer of some genera to different tribes or to a tribe of their own is proposed, e.g., in the case of Mimulus, Leucocarpus, Halleria, Russelia, Angelonia, Rehmannia , and Campylanthus . Finally, a possible evolutionary trend of the different types of nuclear inclusions based on their structural complexity and on the occurrence in allied families is offered.