The 3-dimensional fine structure of the mitotic spindle in Trypanosoma cruzi

The fine structure of nuclear division in the hemoflagellate Trypanosoma cruzi has been studied with serial sections and three-dimensional reconstructions of each divisional stage. After a preliminary stage in which the chromatin becomes dispersed, there is an equatorial stage defined by the appearance of an arranged set of ten dense plaques located about the equatorial region of the nucleus. At this stage a regular microtubular spindle is formed in the nucleus. Each plaque has a symmetrical structure formed by transverse bands and the bands are formed by tightly packed fibrillar material. The wide faces of the plaques are associated with tangential microtubules coming from the poles while the front and rear edges are free to associate with chromatin. Although structural continuity between chromatin fibers and the material of the plaques is possible, this continuity has not been proved. The equatorial spindle is formed by about 120 microtubules arranged in two sets of about 60 microtubules running from each pole to the dense plaques and divided into discrete bundles which reach a single plaque. The microtubules of each bundle may pass tangential to the wide faces of the plaque and end about 0.2 m beyond it, or they may end at the pole-facing edges of the plaque. No continuous, interpolar microtubules were observed at this stage. At the beginning of the elongational stage the dense plaques split into halves and each set of half-plaques migrates to one pole. During mid-elongational stage the pole-converging microtubules and the polar bulges disappear and microtubules become rearranged between the two sets of half-plaques. During late elongational stages, continuous microtubules run between the two sets of half-plaques and maximum nuclear elongation is attained. Chromatin remains dispersed throughout nuclear division. Two main movements have been observed in these mitotic nuclei: the migration of half-plaques to the poles and the elongation of the nucleus. Both these movements are accompanied by large changes in the architecture of the microtubular spindle and are probably dependent on microtubular function. It is concluded that the dense plaques play a kinetochore-like role and thus T. cruzi would have ten chromosomal units.     

MITOSIS AND CLAMP FORMATION IN THE FUNGUS PORIA MONTICOLA

Nuclear division in P. monticola is in general similar to mitosis in higher organisms. Synchronous division of the nuclei in the dikaryon progresses with clamp development. Mitosis begins with the movement of the centriolar plaques into and under the forming clamp. The pull of the centriolar plaque on the attached nucleolus forms a long strand of nucleolar material. Chromosomes now appear as dense granules at the end of the nucleus proximal to the clamp. At this time the nucleolus moves adjacent to the centriolar plaque and contracted chromosomes. The nuclear membrane at least partially disintegrates, and the nucleolus is released into the cytoplasm where it may persist through telophase. A faintly staining spindle is often observed, and it produces a “double bridge” effect in separating chromatin. Somatic chromosomes are attached together forming strings that appear double and at least partially separated before metaphase.     

SPINDLES, SPINDLE PLAQUES, AND MEIOSIS IN THE YEAST SACCHAROMYCES CEREVISIAE (HANSEN)

The intranuclear spindle of yeast has an electron-opaque body at each pole. These spindle plaques lie on the nuclear envelope. During mitosis the spindle elongates while the nuclear membranes remain intact. After equatorial constriction there are two daughted nuclei, each with one spindle plaque. The spindle plaque then duplicates so that two side-by-side plaques are produced. These move rapidly apart and rotate so that they bracket a stable 0.8 µm spindle. Later, during mitosis, this spindle elongates, etc. Yeast cells placed on sporulation medium soon enter meiosis. After 4 hr the spindle plaques of the more mature cells duplicate, producing a stable side-by-side arrangement. Subsequently the plaques move apart to bracket a 0.8 µm spindle which immediately starts to elongate. When this meiosis I spindle reaches its maximum length of 3–5 µm, each of the plaques at the poles of the spindle duplicates and the resulting side-by-side plaques increase in size. The nucleus does not divide. The large side-by-side plaques separate and bracket a short spindle of about 1 µm which elongates gradually to 2 or 3 µm. Thus there are two spindles within one nucleus at meiosis II. To the side of each of the four plaques a bulge forms on the nucleus. The four bulges enlarge while the original nucleus shrinks. These four developing ascospore nuclei are partially surrounded by cytoplasm and by a prospore wall which originates from the cytoplasmic side of the spindle plaque. Eventually the spore nuclei pinch off and the spore wall closes. In some of the larger yeast cells this development is completed after 8 hr on sporulation medium.     

Observations On Nuclear Division in vegetative hyphae of aquatic fungi

Summary Achlya aquatica, A. prolifera andA. racemosa are additional examples of fungi in whose vegetative mycelium, the nuclei do not divide in a manner directly comparable to ordinary mitosis. During division the entire nucleus, which consists of variously shaped chromatin and the nucleolus, becomes angular and elongates. The chromatin separates into two portions, which are situated at opposite ends of the elongating nucleus. As division proceeds the nuclei separate. Each sister nucleus sonsists of chromatin and a portion of the divided nucleolus. Spindless, chromosomal filaments or metaphase plates could not be observed during division.     

Division spindle and centrosomal plaques during mitosis and meiosis in some Ascomycetes

The behaviour of the division spindle and centrosomal plaques is described in four species of Ascomycetes (Ascobolus immersus, Ascobolus stercorarius, Podospora anserina and Podospora setosa) studied by light and electron microscopy. Two unique features of the kinetical apparatus were observed: presence of centrosomal plaques and intranuclear location of the spindle. In all types of mitoses (mycelium, crosier and postmeiotical mitosis) the apparatus is structurally identical to that found in meiosis. The centrosomal plaques, present in all divisions, are always contiguous with the nuclear envelope and never show centrioles similar to those commonly found in Metazoa and Protozoa. During metaphase and anaphase the plaque is constituted of two zones situated on each side of the nuclear envelope: an electron opaque outer zone and inner one less opaque in which most of the microtubules end. In Podospora the outer zone appears in sections as consisting of two dark layers separated by a clear one. Two dispositions of plaques are possible: either they are entirely contiguous with the nuclear envelope (Ascobolus) or only partially so, the remainder being perpendicular to the nuclear envelope (Podospora). — The localisation of the plaques in the ascus was determined by light and electron microscopy. The nuclear envelope was shown to remain intact during division. It was possible to observe that the sporal wall of each spore originated from the same unique double membrane formed in the ascus during the meiotic second division and postmeiotical mitosis. This fact is of genetical interest for the study of morphological and physiological characters of the spores.     

The ultrastructure of mitosis inChroomonas salina (Cryptophyceae)

Summary A detailed account of the ultrastructure of mitosis in a member of theCryptophyceae is given for the first time. The initial indication of mitosis is the duplication of the flagellar bases. The nucleus migrates towards the anterior of the cell and its envelope and nucleolus break down. The chromatin which at interphase is in the form of scattered clumps, condenses into a solid mass through which run narrow tunnels. Each tunnel allows the passage of one to four microtubules. At metaphase the dense plate of chromatin is situated on the equator and the spindle has a rectangular shape. Individual chromosomes cannot be recognized and no morphologically differentiated kinetochores have been observed. The flagella remain functional, their bases stay at the anterior side of the nucleus and do not move to the poles. At anaphase two plates of chromatin separate and these move apart until they come to lie against the ER sheath surrounding the chloroplasts. The new nuclear envelope starts to form on the opposite side of the daughter nucleus. Cytokinesis may commence early in mitosis and consists of a constriction of the parent cell, starting from the posterior end, followed by separation of the two daughters. The present work supports earlier views that only one chromosome is evident during the nuclear division of these organisms. The mitosis is completely different from that of theDinophyceae with which theCryptophyceae were formerly linked.     

Ultrastructural features of mitosis inLeishmania adleri

Summary The interphase nucleus ofLeishmania adleri has clumps of chromatin associated with the nuclear envelope and a large centrally located nucleolus. Prior to mitosis the basal bodies replicate at the cell anterior. Subsequently, dense plaques appear in the equatorial region of the nucleus at the time of spindle development. Microtubules appear in the nucleus adjacent to the nuclear envelope and embedded in the matrix of the plaques. A central spindle composed of a single bundle of microtubules develops and spans the nucleus. Plaques and nucleolar components laterally associate with the spindle and migrate towards the poles. The central spindle elongates to three to four times its original length separating the forming daughter nuclei and producing an interzonal spindle. A remnant of the interzonal spindle remains attached to each of the daughter nuclei until late into cytokinesis. The kinetoplast does not divide until after the completion of mitosis.     

Electron Microscopic Observations of the Bloodstream Form of Cryptobia salmositica Katz, 1951 (Kinetoplastida: Bodonina)1

The ultrastructure of the bloodstream form of Cryptobia salmositica in rainbow trout was examined during the acute phase of experimental infection. The arrangement of the major groupings of cytoplasmic microtubules originating near the basal bodies is similar to that in other bodonids. The cytostome is reinforced both by pellicular microtubules and an electron-dense plaque. Certain microtubules associated with the flagellar pocket serve as nucleating sites for pellicular microtubules. A flagellar rootlet, consisting of two parallel fibers which are bound together intermittently by electron-dense plaques, curves posteriorly from the basal body of the recurrent flagellum towards the kinetoplast. The basal body associated plaque on the kinetoplast membranes is duplicated at the same time as the basal bodies. Cytoplasmic microtubules are found in association with the plaque and the outer kinetoplastic membrane. A pulsatile vacuole, described for the first time in a hemoparasitic cryptobiid, lies adjacent to the post-flagellar pit. Smaller, interconnected vesicles of the spongiome are continuous with the pulsatile vacuole. Since a pulsatile vacuole occurs not only in free-living and ectoparasitic cryptobiids but in the hemoparasitic (=trypanoplasm) forms as well, this is no longer a character by which the genus Trypanoplasma may be separated from the genus Cryptobia. Possession of this osmoregulatory complex may allow the organism to survive outside of a host and fulfill a monoxenous life cycle, in addition to the usual heteroxenous cycle involving a leech as vector.     

Is Cadmium Exposure Associated with the Burden,Vulnerability and Rupture of Human Atherosclerotic Plaques?

The general population is exposed to cadmium from food and smoking. Cadmium is a widely spread toxic pollutant that seems to be associated with cardiovascular diseases, although little is known if it contributes to the occurrence of atherosclerotic plaques and the process whereby plaques become vulnerable and are prone to rupture. We tested the hypotheses that cadmium exposure is associated not only with an increased subclinical burden of atherosclerotic plaques in different vascular territories and early signs of plaque vulnerability, but also with cadmium content and plaque-rupture in the clinical phase of the disease. Ultrasound technique was used to measure plaque prevalence and echogenicity in the carotid and femoral arteries in a population sample of women (n = 599) in whom blood cadmium was measured. In addition cadmium was measured in snap-frozen endarterectomies and whole blood obtained from patients who were referred to surgery because of symptomatic carotid plaques (n = 37). Sixteen endarterectomies were divided into three parts corresponding to different flow conditions and plaque vulnerability. In the population sample blood cadmium was associated with the number of vascular territories with plaques (p = 0.003 after adjustment for potential confounders). The cadmium concentrations in symptomatic plaques were 50-fold higher in plaque tissue than in blood. Cadmium levels in blood and plaque correlated, also after adjustment for smoking and other cardiovascular risk factors (p<0.001). Compared with the other parts of the plaque, the cadmium content was double as high in the part where plaque rupture usually occurs. In conclusion, the results show that cadmium exposure is associated with the burden of subclinical atherosclerosis in middle-aged women with different degrees of glucose tolerance, and that the content of cadmium in symptomatic plaques in patients is related to that in blood, but much higher, and preferentially located in the part of plaque where rupture often occurs.     

Expression of NPP1 is regulated during atheromatous plaque calcification

Mutations of the ENPP1 gene encoding ecto‐nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1) are associated with medial calcification in infancy. While the inhibitory role of matrix proteins such as osteopontin (OPN) with respect to atherosclerotic plaque calcification has been established, the role of NPP1 in plaque calcification is not known. We assessed the degree of plaque calcification (computed tomography), NPP1 and OPN localization (immunohistochemistry) and expression (RT‐PCR) in a cohort of 45 patients undergoing carotid endatherectomy for significant stenosis of the internal carotid artery and in normal arteries (N= 50). We correlated NPP1 and OPN expression levels to the degree of plaque calcification, to pro‐atherogenic factors and statin therapy. NPP1 was demonstrated in the base and in the shoulder of atherosclerotic plaques. Compared to normal arteries and non‐calcified plaques, in calcified plaques NPP1 mRNA was decreased (P < 0.0001). OPN mRNA levels were up‐regulated in carotid atheroma. NPP1 and OPN expression levels positively correlated with the degree of plaque calcification (R= 0.54, P= 0.00019 and R= 0.46, P= 0.017, respectively) and with risk factors of atherosclerosis. Expression of the calcification inhibitor NPP1 is down‐regulated in calcified atherosclerotic plaques. Our correlation data point to a counter‐active mechanism, which in the end turns out to be insufficient to prevent further progression of calcification.     

Convolution Neural Networks and Support Vector Machines for Automatic Segmentation of Intracoronary Optical Coherence Tomography

Cardiovascular diseases are closely associated with deteriorating atherosclerotic plaques. Optical coherence tomography (OCT) is a recently developed intravascular imaging technique with high resolution approximately 10 microns and could provide accurate quantification of coronary plaque morphology. However, tissue segmentation of OCT images in clinic is still mainly performed manually by physicians which is time consuming and subjective. To overcome these limitations, two automatic segmentation methods for intracoronary OCT image based on support vector machine (SVM) and convolutional neural network (CNN) were performed to identify the plaque region and characterize plaque components. In vivo IVUS and OCT coronary plaque data from 5 patients were acquired at Emory University with patient’s consent obtained. Seventy-seven matched IVUS and OCT slices with good image quality and lipid cores were selected for this study. Manual OCT segmentation was performed by experts using virtual histology IVUS as guidance, and used as gold standard in the automatic segmentations. The overall classification accuracy based on CNN method achieved 95.8%, and the accuracy based on SVM was 71.9%. The CNN-based segmentation method can better characterize plaque compositions on OCT images and greatly reduce the time spent by doctors in segmenting and identifying plaques.     

NUCLEAR STRUCTURE AND DIVISION IN THE VEGETATIVE MYCELIUM OF THE SAPROLEGNIACEAE

Bakerspigel , Alexander . (The New Mount Sinai Hospital, Toronto.) Nuclear structure and division in the vegetative mycelium of the Saprolegniaceae. Amer. Jour. Bot. 47(2): 94—100. Illus. 1960.–Saprolegnia parasitica, S. ferax and Achlya racemosa are additional examples of fungi in whose vegetative mycelium the nuclei do not divide in a manner directly comparable to ordinary mitosis. During division the entire nucleus, which consists of a crescent, ring or cap of chromatin and the nucleolus, becomes angular and elongates. The chromatin separates into 2 portions which are situated at opposite ends of the elongating nucleus. As division proceeds the elongated nucleus constricts at the midregion and the sister nuclei separate. Each sister nucleus consists of a crescent of chromatin and a portion of the divided nucleolus. Spindles, chromosomal filaments or metaphase plates could not be demonstrated during division.     

Advance of mitosis by histone phosphokinase

The previous observation that growth-associated histone kinase (HKG) from Ehrlich ascites cells brings forward mitosis in Physarum polycephalum has been confirmed with more step 1 histone kinase and a more purified (step 2) histone kinase and the statistical significance of the results assessed. The mitosis appears normal in the phase contrast microscope and DNA synthesis is initiated after mitosis as usual. In vitro the growth-associated histone kinase phosphorylates chromatin, the phosphate appearing in F 1 histone. The results are interpreted as providing support for the hypothesis that growth-associated histone kinase controls the initiation of mitosis through F 1 histone phosphorylation and chromosome condensation.     

The Involvement of Acidic Nucleoplasmic DNA-binding Protein (And-1) in the Regulation of Prereplicative Complex (pre-RC) Assembly in Human Cells

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2–7 onto chromatin during late mitosis of the cell cycle. MCM2–7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G₁ phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2–7 to facilitate the assembly of MCM2–7 onto chromatin at replication origins in late mitosis and G₁ phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G₁ phase cells. Thus, human And-1 facilitates loading of the MCM2–7 helicase onto chromatin during the assembly of pre-RC.     

The enhancement of histone H4 and H2A serine 1 phosphorylation during mitosis and S-phase is evolutionarily conserved

Histone phosphorylation has long been associated with condensed mitotic chromatin; however, the functional roles of these modifications are not yet understood. Histones H1 and H3 are highly phosphorylated from late G2 through telophase in many organisms, and have been implicated in chromatin condensation and sister chromatid segregation. However, mutational analyses in yeast and biochemical experiments with Xenopus extracts have demonstrated that phosphorylation of H1 and H3 is not essential for such processes. In this study, we investigated additional histone phosphorylation events that may have redundant functions to H1 and H3 phosphorylation during mitosis. We developed an antibody to H4 and H2A that are phosphorylated at their respective serine 1 (S1) residues and found that H4S1/H2AS1 are highly phosphorylated in the mitotic chromatin of worm, fly, and mammals. Mitotic H4/H2A phosphorylation has similar timing and localization as H3 phosphorylation, and closely correlates with the chromatin condensation events during mitosis. We also detected a lower level of H4/H2A phosphorylation in 5-bromo-2-deoxyuridine-positive S-phase cells, which corroborates earlier studies that identified H4S1 phosphorylation on newly synthesized histones during S-phase. The evolutionarily conserved phosphorylation of H4/H2A during the cell cycle suggests that they may have a dual purpose in chromatin condensation during mitosis and histone deposition during S-phase.Electronic Supplementary Material Supplementary material is available in the online version of this article at http://dx.doi.org/10.1007/s00412-004-0281-9Communicated by G. Almouzni     

Condensed chromatin and cell inactivation by single-hit kinetics

Mammalian cells are extremely sensitive to gamma rays at mitosis, the time at which their chromatin is maximally condensed. The radiation-induced killing of mitotic cells is well described by single-hit inactivation kinetics. To investigate if radiation hypersensitivity by single-hit inactivation correlated with chromatin condensation, Chinese hamster ovary (CHO) K1 (wild-type) and xrs-5 (radiosensitive mutant) cells were synchronized by mitotic shake-off procedures and the densities of their chromatin cross sections and their radiosensitivities were measured immediately and 2 h into G1 phase. The chromatin of G1-phase CHO K1 cells was dispersed uniformly throughout their nuclei, and its average density was at least three times less than in the chromosomes of mitotic CHO K1 cells. The alpha-inactivation co-efficient of mitotic CHO K1 cells was approximately 2.0 Gy(-1) and decreased approximately 10-fold when cells entered G1 phase. The density of chromatin in CHO xrs-5 cell chromosomes at mitosis was greater than in CHO K1 cell chromosomes, and the radiosensitivity of mitotic CHO xrs-5 cells was the greatest with alpha = 5.1 Gy(-1). In G1 phase, CHO xrs-5 cells were slightly more resistant to radiation than when in mitosis, but a significant proportion of their chromatin was found to remain in condensed form adjacent to the nuclear membrane. These studies indicate that in addition to their known defects in DNA repair and V(D)J recombination, CHO xrs-5 cells may also be defective in some process associated with the condensation and/or dispersion of chromatin at mitosis. Their radiation hypersensitivity could result, in part, from their DNA remaining in compacted form during interphase. The condensation status of DNA in other mammalian cells could define their intrinsic radiosensitivity by single-hit inactivation, the mechanism of cell killing which dominates at the dose fraction size (1.8-2.0 Gy) most commonly used in radiotherapy.     

Different modes of internalization of proteins associated with adhaerens junctions and desmosomes: experimental separation of lateral contacts induces endocytosis of desmosomal plaque material.

The distribution and fate of two junctional complexes, zonula adhaerens and desmosomes, after dissociation of cell-cell contacts is described in MDBK cells. Junctions were split between adjacent cells by treatment with EGTA and proteins associated with the plaques of zonulae adhaerentes and desmosomes were localized by immunological methods. Splitting of these junctions is accompanied by the dislocation of desmosomal plaque protein from the cell periphery and its distribution in punctate arrays over the whole cytoplasm. By contrast, vinculin associated with zonulae adhaerentes is still seen at early times (0.5-1 h) in a conspicuous belt-like structure which, however, is displaced from the plasma membrane. Strong vinculin staining is maintained on leading edges of free cell surfaces. Electron microscopy of EGTA-treated cells exposed to colloidal gold particles reveals the disappearance of junctional structures from the cell periphery and the concomitant appearance of a distinct class of gold particle-containing vesicles which are coated by dense plaques. These vesicle plaques react with antibodies to desmosomal plaque proteins and are associated with filaments of the cytokeratin type. In the same cells, extended dense aggregates are seen which are most probably the membrane-detached vinculin-rich material from the zonula adhaerens . The experiments show that, upon release from their junction-mediated connections with adjacent cells, major proteins associated with the cytoplasmic side of the junctions remain, for several hours, clustered within plaques displaced from the cell surface. While plaque material of adhaerens junctions containing vinculin is recovered in large belt-like aggregates, desmosomal plaque protein remains attached to membrane structures and appears on distinct vesicles endocytotically formed from half-desmosomal equivalents.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chromatin diminution in early embryogenesis of Ascaris lumbricoides L. var. suum

The occurrence of chromatin diminution in early Ascaris lumbricoides L. embryos has been studied in detail, and it is shown that it is possible to preselect three characteristic types of mitoses: pre-diminution, diminution, and post-diminution mitosis. The first three embryonic mitotic divisions are of the pre-diminution type. Chromatin diminution occurs after the third mitosis, but there is a variation from embryo to embryo as to whether or not chromosomal diminution occurs during the fourth, fifth, and six divisions. However, the seventh embryonic division, which gives rise to an eight-cell embryo, always exhibits chromatin diminution. Subsequent mitoses of somatic cells already in the diminished state are of the post-diminution type of mitosis.     

Wapl controls the dynamic association of cohesin with chromatin

Cohesin establishes sister-chromatid cohesion from S phase until mitosis or meiosis. To allow chromosome segregation, cohesion has to be dissolved. In vertebrate cells, this process is mediated in part by the protease separase, which destroys a small amount of cohesin, but most cohesin is removed from chromosomes without proteolysis. How this is achieved is poorly understood. Here, we show that the interaction between cohesin and chromatin is controlled by Wapl, a protein implicated in heterochromatin formation and tumorigenesis. Wapl is associated with cohesin throughout the cell cycle, and its depletion blocks cohesin dissociation from chromosomes during the early stages of mitosis and prevents the resolution of sister chromatids until anaphase, which occurs after a delay. Wapl depletion also increases the residence time of cohesin on chromatin in interphase. Our data indicate that Wapl is required to unlock cohesin from a particular state in which it is stably bound to chromatin.     

Chromosome Association of Minichromosome Maintenance Proteins in Drosophila Endoreplication Cycles

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM–chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM–chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM–chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.