Chromatin motion is constrained by association with nuclear compartments in human cells

BACKGROUND: In comparison with many nuclear proteins, the movement of chromatin in nuclei appears to be generally constrained. These restrictions on motion are proposed to reflect the attachment of chromatin to immobile nuclear substructures. RESULTS: To gain insight into the regulation of chromosome dynamics by nuclear architecture, we have followed the movements of different sites in the human genome in living cells. Here, we show that loci at nucleoli or the nuclear periphery are significantly less mobile than other, more nucleoplasmic loci. Disruption of nucleoli increases the mobility of nucleolar-associated loci. CONCLUSIONS: This is the first report of distinct nuclear substructures constraining the movements of chromatin. These constraints reflect the physical attachment of chromatin to nuclear compartments or steric impairment caused by local ultrastructure. Our data suggest a role for the nucleolus and nuclear periphery in maintaining the three-dimensional organization of chromatin in the human nucleus.     

Ultrastructure of the nuclear apparatus of the lower ciliateRemanella granulosa Kahl (Karyorelictida)

Summary The nuclear apparatus ofRemanella granulosa has been investigated using conventional TEM methods and Bernhard's technique of preferential RNP staining. This species has two (rarely three) macronuclei and a single micronucleus (rarely two micronuclei). The nuclei always form a single group.The macronuclei contain a fibro-granular matrix resistant to EDTA destaining, and several nucleoli and chromatin bodies. The chromatin bodies are readily bleached with EDTA and are often clustered, or even fused, forming chromocenters. The nuclei are of the compact concentric type. Some macronuclei contain nuclear bodies, as finely fibrous spheres or bundles of coarse fibers, or both. Neither type of nuclear body is destained with EDTA. The spheres are frequently associated with nucleoli. There is no evidence of any transition between the two types of nuclear bodies. The macronuclear envelope contains numerous pore complexes and is strengthened with an electron dense layer. The micronucleus is filled with spongy condensed chromatin and surrounded by an envelope with occasional pores. This nucleus lacks nucleoli and nuclear bodies.     

Fragmentation of polyploid nuclei in the giant cells of the rat trophoblast. I. The ultrastructure of the nuclear fragments

Electron microscope study of the nuclear fragments in the rat trophoblast has demonstrated that the division of the trophoblast giant nucleus results first in the formation of a multinuclear cell. Each nuclear fragment is covered with its own nuclear envelope made of two membranes with numerous pore complexes. The chromatin in these nuclear fragments is condenced with various degrees of condensation, which depends on the step of placenta development, cell differentiation and the degree of nuclear fragmentation. The nuclear ultrastructure in nuclear fragments also depends on the degree of nuclear fragmentation and on the level of chromatin condensation. The nucleolus has no granular component. On large fragments, with lower chromatin condensation the nucleolus is not homogenous being made of fragments of more and of less electron dense fibrilles. Small light lacunae are seen in the nucleolus where chromatin threads and strands pass on. With a high chromatin condensation in the nucleus, round small nucleoli look homogenous being made of moderately electron dense fibrilles. Products of chromosome activity have been found in the nuclear fragments: accumulations of minute granules (d = 15--20 nm), perichromatinous granules (d = 35--40 nm), and fibrillar nucleolus-like bodies. In the multinuclear cell, made as the result of fragmentation of the initially giant nucleus, all the small nuclei are first arranged very close to each other, so that the contours of the neighbouring nuclei coincide.     

Nuclear structures in the ovarioles of the butterfly Laspeyresia pomonella. Sex chromatin in trophocytes

The nuclear structures in the ovarioles have been studied in Laspeyresia pomonella by means of light and electron microscopy, autoradiography (RNA and DNA synthesis) and molecular hybridization in situ. The karyosphere was shown to form in oocyte nuclei at the beginning of oocyte growth. Numerous protein granules appeared in close contact with the karyosphere chromosomes; the true nucleolus was absent and the whole nucleus was inactive in RNA synthesis. A special attention was paid to studying nuclear structures in trophocytes. Numerous complex nucleoli actively synthesizing RNA formed in highly endopolyploid nuclei of trophocytes. Besides, each trophocyte had a spheroid vacuolized body of DNA which developed from one of meiotic bivalents soon after trophocyte differentiation and increased in diameter up to 10-15 mu. The DNA body in trophocytes and follicle cells was in close contact with the nucleolar material. Ribosomal DNA was present in these bodies as was shown by molecular hybridization in situ. A suggestion is put forward to the effect that the DNA bodies take part in the formation of complex nucleolar apparatus of trophocytes. On the basis of both the author's and literary data, a conclusion is drawn that DNA spheres in trophocytes and follicle cells are sex chromatin bodies formed, however, by both the X- and Y-chromosomes, rather than by one Y-chromosome.     

A study on the different types of spermatogonia in buffalo (Bubalus bubalis).

As a first step to understanding spermatogenesis in the buffalo bull the cytological details of different types of spermatogonia were determined in adult buffalo bulls. Morphological changes in the nuclear details were used as a basis for classifying the different types of spermatogonia. The type A spermatogonia had a spherical to ovoid nucleus with finely granulated chromatin, homogeneously dispersed in the nucleoplasm and having one to two nucleoli adhering to the nuclear membrane. The type A0 spermatogonia were characterized by nuclei containing moderately stained, finely granulated chromatin and a nucleolus attached to the nuclear envelope. The A1 type spermatogonia, on the other hand, have pale stained, finely granulated chromatin with the nucleolus adhering to the nuclear membrane. The nuclei of A2 type spermatogonia resembled those of type A1, but contained coarse granular chromatin dispersed in the pale nucleoplasm. The intermediate type of spermatogonia acquired a central position of the nucleolus, but the chromatin remained coarsely granulated and non-clumped. Three classes of type B (B1-B3) spermatogonia were determined on the degree of clumping of the chromatin and the central position of the nucleolus. The type B1 cells were characterized by nuclei containing a few flakes of lightly stained chromatin and a centrally located nucleolus. The type B2 cells showed comparatively more clumping of chromatin than type B1 spermatogonia, which was dispersed at random in the pale nucleoplasm and along the nuclear envelope. The type B3 spermatogonia demonstrated chromophilic chromatin dispersed in the slightly grey nucleoplasm and adhering along the nuclear membrane. Since there seems to be a succession of events following differentiation of type A1 spermatogonia till the last type B cell differentiates into resting primary spermatocytes, may intermediate stages between the presently described classes of type A (A0-A2) and type B (B1-B3) could also be located in sections of the seminiferous tubules.     

四膜虫(Tetrahymena shanghaiensis)大核核仁与去膜大核能诱导非细胞体系核重建

外源DNA或染色质在非洲爪蟾卵提取物中可以诱导细胞核样结构的重建。重建核除不具有核仁样结构外,在其它形态结构上与真核细胞核十分相似。前人的工作表明在重建核中具有核仁前体结构。但可能是由于缺少活性核仁组织者的缘故,这些核仁前体不能相互融合形成新生核仁。那么活性核仁组织者在重建核中是否能发挥其功能呢?为了研究这一问题,我们提取纯化了四膜虫的大核与大核的周边核仁。进一步去除大核的核被膜,并将去除核被膜的大核与大核核仁分别加入非洲爪赡卵非细胞体系中。通过电镜超薄切片观察,我们发现无论是与大核染色质相连的周边核仁还是分离纯化的核仁结构在非洲爪赡卵非细胞体系中都不能保持其原有结构特征,而是发生了典型核重建变化,并且在诱导形成的重建核中也看不到核仁样结构。这些结果说明具有活性的核仁组织者在加入非洲爪蟾卵提取物后既不能继续保持其原有的RNA转录功能也不能诱导新的核仁的出现。     

Electron microscopic identification of sex chromatin bodies of tissue culture cells

Summary A method is described for the unequivocal identification of sex chromatin bodies in electron micrographs of thin sections of tissue culture cells derived from human skin. Fibers, rodlets, and circular profiles having a similar diameter of about 200 Å appear to be the only components of the sex chromatin bodies. The fibers and rodlets are sometimes resolvable into two similar subunits and the circular profiles often have a less electron dense center. The overall density of sex chromatin bodies is found to be considerably less than that of metaphase chromosomes. The fibers of the sex chromatin bodies found lying away from the nuclear periphery appear to be organized more compactly than the fibers of the sex chromatin bodies found lying at the nuclear periphery.This paper is based on investigations supported by a research grant No. GM-04738 from the National Institutes of Health, Public Health Service, to Dr. H. Ris, Department of Zoology, University of Wisconsin.     

Electron microscopic study of endopolyploid nuclei in rat trophoblast giant cells. IV. Changes in nucleolar ultrastructure during cell differentiation

The ultrastructure of the nucleolus of highly differentiated trophoblast giant cells has been studied on the 17th day of the foetus development. Changes in its morphology have been followed in relation to the degree of nuclear chromatin condensation and to the cell differentiation level. The nucleoli have a reticular structure in the nuclei with dispersed and condensed chromatin. In both the cases the nucleoli involve the four components: fibro-granular, fibrillar (of moderate and normal density) and lacunar regions; fibrillar centres are distinguished within the regions. In the nucleoli with condensed chromatin, unlike those with dispersed chromatin, the perinuclear chromatin is clearly seen, and the penetration of nucleolus-organizer threads along lacunae and deep into the nucleolus can be easily followed. The fibrillar centres are more obvious. With the run of a progressive differentiation of the trophoblast cells, the number of granules is reduced; first, the fibro-granular component covers a significant part of the nucleolus, then granules become visible only in the cortical zone of the nucleolus; in the nuclei with strongly condensed chromatin no granules are seen in the nucleolus.     

Nuclear bodies in the oocyte nucleus of ground beetles are enriched in snRNPs

Within the oocyte nucleus of many insect species, a variable number of intensely stained spherical bodies occur. These nuclear bodies differ significantly from nucleoli and their precise role in nuclei has not been elucidated yet. I have examined some of the histochemical properties as well as the molecular composition of these structures in a representative of ground (carabid) beetles. I demonstrate, using molecular markers, that the nuclear bodies are composed of small nuclear RNAs and associated proteins, including p80 coilin. Hence, they correspond to Cajal bodies (= coiled bodies) described in somatic cell nuclei as well as oocyte germinal vesicles in plant and animal organisms. It is suggested that Cajal bodies in the carabid germinal vesicle serve as a storage site for splicing factors.     

Dynamics of structural changes of Paramecium caudatum and Bursaria truncatella macronuclear chromatin and nucleoli under hypotonic conditions

Dynamics of structural changes of nucleoli, complex nucleolar aggregates and chromatin bodies in macronuclei (Ma) of ciliates Paramecium candatum and Bursaria truncatella under hypotonic conditions was studied. It was shown that after a 3 min hypotonic treatment nuclei swelled and became highly vacuolated. 3D-reconstruction showed that such nucleoli were formed by nucleolonema-like threads about 100-200 nm in thickness. Intranucleolar chromatin bodies decompacted, but remained bound with fibrillar component of the nucleolus by thin fibres about 10 nm thick. After 6 min hypotonic treatment the nucleolar material loosened and had a "gauze", or network-like appearence. After 10 min hypotonic treatment nucleoli dissociated completely. It was shown that a transition of chromatin bodies from completely compact to partially and fully decompacted state occurred cooperatively in different regions of Ma. In particular, chromatin bodies in the central part of complex nucleolar aggregates decompacted much faster than those in the Ma karyoplasm. It evidences for a specific, well-ordered chromatin organization in Ma. Prolonged hypotonic treatment led to a complete dissociation of Ma components; fibres 6-10 nm thick were solely observed in such preparations. Such fibres may represent remnant structures of the nuclear matrix. Dynamics of Ma chromatin bodies decompaction correlates well with that of chromomeres in the nuclei of higher eukaryotes. Our data confirm that chromatin 100-200 nm bodies in the ciliate Ma are analogues of chromomeres--looped discrete chromatin domains, observed in the nuclei of higher eukaryotes.     

Cytochemical and ultrastructural studies of ribonucleoprotein containing structures in oocytes of Acheta domesticus

Summary Two distinct types of ribonucleoprotein containing structures are found in oocytes of the house cricket, Acheta domesticus, a large secondary or accessory nucleolus and many small primary nucleoli. The secondary nucleolus increases in size during oocyte development and is similar in appearance to the nucleolus of somatic cells. The primary nucleoli are intimately associated with a large, extrachromosomal DNA containing body. The DNA body is no longer visible in nuclei of late diplotene stage cells when the primary nucleoli are dispersed within the nucleoplasm. Both types of nucleoli contain cytochemically detectable RNA and acid protein, little or no DNA and basic protein, and particulate structures similar to but smaller than cytoplasmic ribosomes.The authors acknowledge the technical assistance of Miss Celeste Malinoski and Mrs. Marcia Andrews. This work was supported by a U.S.P.H.S. grant, number GM-16440-01 and grants number L-16 and J-1 from the Health Research Services Foundation.     

Ultrastructural characteristics of insect corpora allata in relation to larval diapause

Summary The ultrastructure of active and inactive corpora allata from last instar larvae of the southwestern corn borer, Diatraea grandiosella, was examined. Active glands were obtained from pre-, early, and mid-diapausing larvae; inactive ones from late and non-diapausing larvae. Each gland contains 13 to 18 cells which have the following common features: well developed smooth endoplasmic reticulum, scattered rough endoplasmic reticulum and free ribosomes, microtubules, vacuolated nucleoli, and interlocking plasma membranes. The gland contains intercellular deposits, and is supplied by regular and neurosecretory axons.Special ultrastructural features of the corpus allatum from the five groups of larvae examined were as follows: pre-diapause: extensive vesicular smooth endoplasmic reticulum, numerous cup-shaped mitochondria and Golgi bodies with stacked cisterns and vesicles, few small lipid droplets, large nuclei with dispersed chromatin, absence of lysosomes; early diapause: stacked, whorled, and vesicular smooth endoplasmic reticulum of equal abundance, numerous rod-shaped mitochondria, some Golgi bodies but without distinct stacks of cisterns, few lipid droplets and lysosomes, chromatin dispersed and also attached to the nuclear envelope; mid-diapause: similar to early diapause except for the presence of more stacked, smooth endoplasmic reticulum, chromatin in large chunks mostly attached to the nuclear envelope; late diapause: whorled smooth endoplasmic reticulum and rod-shaped mitochondria predominating, complicated Golgi bodies with stacks of cisterns and large empty sacs, few large lipid droplets, some lysosomes containing mainly whorled bodies, chromatin in large chunks attached to the nuclear envelope; non-diapause: similar to late diapause except for less extensive smooth endoplasmic reticulum, more abundant mitochondria, fewer intercellular deposits. Although these observations suggest that the smooth endoplasmic reticulum, and possibly mitochondria, and Golgi bodies are involved in juvenile hormone production, specific sites of synthesis or storage of the hormone were not revealed.Supported in part by grant no. PCM 74-18155 A01 from the National Science Foundation. Contribution from the Missouri Agricultural Experiment Station as journal series no. 8234. We thank Ms. L. Yin for her skillful assistance, and Dr. M.F. Brown of the College of Agriculture Electron Microscope Facility for his advice and the use of equipment.     

Ultrastructure of trophocyte nuclei in polytrophic ovarioles of Chrysopa perla (Neuroptera)]

The light and electron microscope and autoradiographic studies (H3-uridin incorporation) were carried out on the trophocyte nuclei of imago polytrophic ovarioles of Chrysopa perla (Neuroptera), from the trophocyte differentiation up to their degeneration. Like the oocytes, one of the seven nurse cells o every ovariole chamber contains extrachromosomal DNA bodies. This nurse cell is formed during differential mitoses in the germarium as one of two prooocytes. In contrast to extrachromosomal DNA of oocytes the trophocyte DNA bodies are less active structures. Several (2--4) complex nucleoli develop in the trophocytes of Chrysopa in the early stages of oogenesis. They consist of three main components: the chromatin mass, fibrillar bodies and granular strands. Such nucleoli grow, through increasing in number of fibrillar bodies and granular strands. They are most developed by the start of the vitellogenesis. At the middle vitellogenesis the general nucleolar structure modify due to the beginning of trophocyte degeneration. The consecutive stages of nuclear degeneration are described. The trophocyte nucleoli synthesize RNA still in germarium. The most intensive RNA synthesis is observed at the beginning of the vitellogenesis to decrease by the beginning of trophocyte degeneration.     

FORMATION OF GERM CELL-LIKE CELLS IN THE PERITONEAL EPITHELIUM OF THE TESTES OF ESTROGEN-TREATED ADULT MALES OF THE JAPANESE RED-BELLIED NEWT, CYNOPS PYRRHOGASTER PYRRHOGASTER

Columnar cells of the peritoneal epithelium in slender cords of the testes were examined in normal and estradiol benzoate-treated Japanese red-bellied newt, Cynops pyrrhogaster pyrrhogaster, by light and electron microscopy. In normal newts, the peritoneal epithelium covering the slender cord consists of columnar cells, which contain extraordinarily large, oval or spindle-shaped nuclei with conspicuous indentations. The nucleus contains chromatin granules and the cytoplasm is filled with numerous tonofilaments. The primordial germ cells are scattered throughout the slender cord, and each cell is surrounded by a few follicle cells. Between the germ cells and follicle cells there are microvilli-like processes. The nucleus of primordial germ cells is multilobate and has electron lucent areas, dispersed chromatin and several electron-dense nucleoli. In the lighter cytoplasm, the nuage material is found very near to nuclear pores, and is frequently seen among the mitochondria. The nucleolus-like body is not associated with other organelles. The primary spermatogonia have bilobate nuclei. It is remarkable that most of the cytoplasmic organelles are found in the deep nuclear indentations. The nuage material and nucleolus-like body are well developed in the cytoplasm. After treatment of newts with estradiol benzoate for one year, four types of cells can be distinguished in the peritoneal epithelium. One type is quite different from the columnar cells. These newly appeared cells are large and light in appearance. Their nucleus is highly lobate, and contains dispersed chromatin and several nucleoli with compact electron dense material in its periphery. The cells are characterized by the presence of nuage material and nucleolus-like bodies in the cytoplasm. There are microvilli-like processes between these cells and adjacent elongated cells. These ultrastructural characteristics of the light cells are very similar to those of primordial germ cells and/or primary spermatogonia in normal testes. These findings suggest that the light cells which appear in the peritoneal epithelium of the testes on administration of estrogen may be germ line cells.     

Morphology of the reproductive system of the armadillo. The spermatogonia

Spermatogonia of the nine-banded armadillo, Dasypus novemcinctus mexicanus, were studied morphologically using light and electron microscopy and examined histochemically using light microscopy. Immature flat type spermatogonia have ovoid or irregular nuclei with loosely condensed chromatin. Free ribosomes are abundant while profiles of rough endoplasmic reticulum are scarce. Smooth endoplasmic reticulum is a prominent feature occasionally taking an unusual cylindrical form. Mature spermatogonia exhibit rounder nuclei with greater degrees of chromatin clumping. Smooth endoplasmic reticulum is no longer prominent whereas profiles of rough endoplasmic reticulum are quite common. Occasional lysosomal configurations are found in mature spermatogonia. The majority of spermatogonial cells exhibit weak to moderate reactivity when stained with the periodic acid-Schiff (PAS) reaction. Certain cells in each tubular cross section stain vividly with this reaction and the PAS positivity is removable with salivary amylase. Because of nuclear characteristics, position of the cell immediately upon the basal lamina, intensity of the PAS reaction and the relative paucity of the vividly staining cells, it is suggested that they are members of the immature spermatogonial cell line, perhaps acting as stem cells. None of the several other histochemical procedures employed was capable of selectively demonstrating these cells.     

Isolation of a nuclear ribonucleoprotein network that contains heterogeneous RNA and is bound to the nuclear envelope.

Rapidly labeled polydispersed nuclear RNA is part of a ribonucleoprotein (RNP) network which in turn is tightly bound to the nuclear membrane. The membranous attachment, therefore, established a connection between chromatin and cytoplasm. The ultrastructure of the RNP network comprises fibrils and granules similar to those observed in intact nuclei. When bound to the nuclear membrane it has the composition of 63% protein, 14% RNA, 0.4% DNA, and 22.6% lipids. The proportion of lipids diminishes to 2.2% when nuclear membrane is not present. Chromatin, nucleoli, and ribosomes are minor contaminants since histones and ribosomal proteins are not detectable in polyacrylamide gel electrophoresis. Nuclear disruption at high pressure in a French pressure cell causes fragmentation of the RNP network into a series of polydispersed RNP particles. Fragmentation can be prevented by using mild pressure, or by disrupting nuclei with high salt buffer and digesting the dispersed chromatin with deoxyribonuclease. A RNP network, almost free of membrane, is also obtained if the nucleus is deprived of its envelope by treatment with Triton X-100. Since no polydispersed RNP particles are found following dissolution of the nuclear membrane, it is assumed that the particles are components of the RNP network whose fragmentation occurs as a consequence of two processes: (a) activation of nuclear nucleases and (b) shearing forces.     

Lamellate bodies in the nuclei of dragonfly (odonata) oocytes

Summary Concentrically lamellate spherical bodies have been observed in the oocyte nuclei of the anisopterous dragonflyCordulia aenea. In their ultrastructure, these bodies resemble the cortical granules reported from the cytoplasm of the sea urchin oocytes. The lamellate bodies ofCordulia are compared with other lamellate systems known to exist in nuclei. It is suggested that the nuclear lamellate spheres ofCordulia may be homologous with the somewhat less regular lamellate bodies described from maize microspores.The competent help of MissRiitta Lallukka, M. A., is acknowledged with many thanks. We are grateful to ProfessorsESko Suomalainen andAntti Telkkä for helpful suggestions. Grants for the study have been received from the National Research Council for Sciences, from the University of Helsinki and from the Emil Aaltonen Foundation.